Successful Drug Discovery -  - ebook

Successful Drug Discovery ebook

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Retaining the successful approach found in the previous volume in this series, the inventors and primary developers of drugs that successfully made it to market tell the story of the drug's discovery and development and relate the often twisted route from the first candidate molecule to the final marketed drug. 11 selected case studies describe recently introduced drugs that have not been previously covered in textbooks or general references. These range across six different therapeutic fields and provide a representative cross-section of the current drug development efforts. Backed by copious data and chemical information, the insight and experience of the contributors makes this one of the most useful training manuals that a junior medicinal chemist can hope to find and has won the support and endorsement of IUPAC.

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Table of Contents

Cover

Title

Copyright

Preface

List of Contributors

Part I: HDAC Inhibitor Anticancer Drug Discovery

Chapter 1: From DMSO to the Anticancer Compound SAHA, an Unusual Intellectual Pathway for Drug Design

1.1 Introduction

1.2 The Discovery of SAHA (vorinostat)

1.3 Clinical Trials

1.4 Follow-On Research – Selective HDAC Inhibitors

1.5 Conclusion

References

Chapter 2: Romidepsin and the Zinc-Binding Thiol Family of Natural Product HDAC Inhibitors

2.1 Histone Deacetylases as a Therapeutic Target

2.2 The Discovery and Development of Romidepsin

2.3 The Zinc-Binding Thiol Family of Natural Product HDAC Inhibitors

2.4 Synthetic Analogues of the Zinc-Binding Thiol Natural Products

2.5 Summary

References

Chapter 3: The Discovery and Development of Belinostat

3.1 Introduction

3.2 Discovery of Belinostat

3.3 Belinostat Biological Profiling

3.4 Formulation Development

3.5 Clinical Development

3.6 Conclusions

References

Chapter 4: Discovery and Development of Farydak (NVP-LBH589, Panobinostat) as an Anticancer Drug

4.1 Target Identification: From p21Waf1 Induction to HDAC Inhibition

4.2 Program Flowchart Assays for Drug Discovery

4.3 Hit-To-Lead Campaign: Trichostatin A to LAK974

4.4 Lead Optimization: LAK974 to LAQ824

4.5 Profiling LAQ824 for Cancer Therapy

4.6 Preclinical Development of LAQ824

4.7 LAQ824 Follow-Up

4.8 Discovery of LBH589

4.9 Safety Profile for LBH589

4.10 Pan-HDAC Inhibition by LBH589

4.11 Cancer Cell-Specific Cytotoxicity of LBH589

References

Chapter 5: Discovery and Development of HDAC Subtype Selective Inhibitor Chidamide: Potential Immunomodulatory Activity Against Cancers

5.1 Introduction

5.2 Discovery of Chidamide

5.3 Molecular Mechanisms of Chidamide

5.4 Animal Studies

5.5 Clinical Development

5.6 Future Perspective

References

Part II: Steroidal CYP17 Inhibitor Anticancer Drug Discovery

Chapter 6: Abiraterone Acetate (Zytiga®): An Inhibitor of CYP17 as a Therapeutic for Castration-Resistant Prostate Cancer

6.1 Introduction

6.2 Discovery and Structure–Activity Relationships (SAR)

6.3 Preclinical Characterisation of Abiraterone and Abiraterone Acetate

6.4 Physical Characterisation

6.5 Clinical Studies

6.6 Conclusion

References

Part III: Anti-Infective Drug Discoveries

Chapter 7: Discovery of Delamanid for the Treatment of Multidrug-Resistant Pulmonary Tuberculosis

7.1 Introduction

7.2 Synthesis Strategy

7.3 Synthesis Route

7.4 Screening Evaluations

7.5 Preclinical Data of Delamanid

7.6 Clinical Data of Delamanid

7.7 Future Priorities and Conclusion

References

Chapter 8: Sofosbuvir: The Discovery of a Curative Therapy for the Treatment of Hepatitis C Virus

8.1 Introduction

8.2 Discussion

8.3 Conclusion

References

Part IV: Central Nervous System (CNS) Drug Discovery

Chapter 9: The Discovery of the Antidepressant Vortioxetine and the Research that Uncovered Its Potential to Treat the Cognitive Dysfunction Associated with Depression

9.1 Introduction

9.2 The Discovery of Vortioxetine

9.3 Clinical Development of Vortioxetine for the Treatment of MDD

9.4 Uncovering Vortioxetine’s Potential to Treat Cognitive Dysfunction in Patients with MDD

9.5 Conclusion

References

Part V: Antiulcer Drug Discovery

Chapter 10: Discovery of Vonoprazan Fumarate (TAK-438) as a Novel, Potent and Long-Lasting Potassium-Competitive Acid Blocker

10.1 Introduction

10.2 Limitations of PPIs and the Possibility of P-CABs

10.3 Exploration of Seed Compounds

10.4 Lead Generation from HTS Hit Compound 1

10.5 Analysis of SAR and Structure–Toxicity Relationship for Lead Optimization

10.6 Selection of Vonoprazan Fumarate (TAK-438) as a Candidate Compound

10.7 Preclinical Study of TAK-438

10.8 Clinical Study of TAK-438

10.9 Discussion

10.10 Conclusion

References

Part VI: Cross-Therapeutic Drug Discovery (Respiratory Diseases/Anticancer)

Chapter 11: Discovery and Development of Nintedanib: A Novel Antiangiogenic and Antifibrotic Agent

11.1 Introduction

11.2 Structure–Activity Relationships of Oxindole Kinase Inhibitors and the Discovery of Nintedanib

11.3 Structural Research

11.4 Preclinical Pharmacodynamic Exploration

11.5 Nonclinical Drug Metabolism and Pharmacokinetics

11.6 Clinical Pharmacokinetics

11.7 Toxicology

11.8 Phase III Clinical Data

11.9 Other Oncology Studies

11.10 Conclusions

References

Index

End User License Agreement

Guide

Cover

Table of Contents

Begin Reading

List of Tables

Chapter 2: Romidepsin and the Zinc-Binding Thiol Family of Natural Product HDAC Inhibitors

Table 2.1 HDAC inhibitory activity of romidepsin (in the dithiol form redFK228) and vorinostat against individual human isoforms.

Table 2.2 HDAC inhibitory activity, where available, for depsipeptide natural products in their active form (dithiol for romidepsin and thailandepsin A, thiol for largazole) against individual human isoforms.

Table 2.3 Total syntheses of zinc-binding thiol HDAC inhibitor natural products.

Chapter 3: The Discovery and Development of Belinostat

Table 3.1 Representative inhibitors from the amide series.

Table 3.2 Representative inhibitors from the sulfonamide series.

Table 3.3 Representative structures of different series of inhibitors.

Table 3.4 In vitro Cytotoxicity of belinostat in Human Tumour Cell Lines.

Table 3.5 Summary of company initiated clinical development activities.

Table 3.6 Major belinostat metabolites identified in humans.

Chapter 4: Discovery and Development of Farydak (NVP-LBH589, Panobinostat) as an Anticancer Drug

Table 4.1 Early in vitro and in vivo profiling of HDAC leads.

Table 4.2 Activity data for HDAC inhibitors with different terminal aryl groups.

Table 4.3 Activity data for HDAC inhibitors with a variety of molecular spacers.

Table 4.4 Pharmacokinetic parameters of LAQ824 after single i.v. dose to athymic nude mice bearing HCT116 or MDA-MB-435 tumours.

Table 4.5 Activity data for HDAC inhibitors with differently substituted indole rings.

Table 4.6 HDAC isoform inhibition by LBH589 compared to other HDAC inhibitors in development.

Table 4.7 Potent cell death induction (LD

50

) by LBH589 in CTCL cell lines.

Chapter 5: Discovery and Development of HDAC Subtype Selective Inhibitor Chidamide: Potential Immunomodulatory Activity Against Cancers

Table 5.1 Overview of regulatory agency-approved epigenetic drugs and emerging new compounds currently under clinical development for cancer indications.

Table 5.2 SAR of some representative N-(2-amino-4-fluorophenyl)-4-substituted aromatic amides.

Table 5.3 In vitro antiproliferative effects of compounds 1–7 on different human cancer cell lines.

Table 5.4 The clinical pathological subtype of patients enrolled in pivotal trials for the four PTCL drugs.

Table 5.5 Potential OS benefit of chidamide in relapsed or refractory PTCL patients.

Chapter 6: Abiraterone Acetate (Zytiga®): An Inhibitor of CYP17 as a Therapeutic for Castration-Resistant Prostate Cancer

Table 6.1 Inhibition of human testicular 17α-hydroxylase/C

17–20

lyase enzyme (CYP17) [15, 16].

Table 6.2 Inhibition of human aromatase and testosterone 5α-reductase [16].

Table 6.3 Inhibition of human testicular 17α-hydroxylase [17].

Table 6.4 Pharmaocokinetic parameters of abiraterone acetate (8) after oral dosing [22].

Table 6.5 Comparison of abiraterone (11) inhibitory potency at a series of CYP enzymes [23].

Chapter 7: Discovery of Delamanid for the Treatment of Multidrug-Resistant Pulmonary Tuberculosis

Table 7.1 In vitro antituberculosis activity of racemic compounds 2a–e and optically active compounds 2f and 2g.

Table 7.2 In vitro antituberculosis activity of (S)-piperazine carbamates 3.

Table 7.3 In vitro antituberculosis activity of (S)-benzyl piperazines 4.

Table 7.4 In vitro antituberculosis activity of (S)-aryl piperazines 5.

Table 7.5 In vitro antituberculosis activity of (S)-piperidines 6.

Table 7.6 In vitro antituberculosis activity of (S)-hydrazone compounds 7.

Table 7.7 In vitro antituberculosis activity of (R)-phenoxy compounds 8.

Table 7.8 In vitro antituberculosis activity of (R)-phenoxy compounds 9.

Table 7.9 In vitro antituberculosis activity of (R)-phenoxy compounds 10.

Table 7.10 Colony forming units (CFU) of each group of compounds 3f, 7e, 9f and RFP on the experimental chronic tuberculosis model in mice.

Chapter 9: The Discovery of the Antidepressant Vortioxetine and the Research that Uncovered Its Potential to Treat the Cognitive Dysfunction Associated with Depression

Table 9.1 Working hypothesis on links between pharmacological targets, target occupancies and clinical effects.

Table 9.2 SAR of compounds 3–8 show that minor structural changes within the series have pronounced effects on both in vitro pharmacology and DMPK parameters.

Table 9.3 SAR of compounds 1 and 7–13 show that different substitution patterns on the distal phenylsulfanyl ring led to molecules having the desired in vitro target profile on SERT, 5-HT

1A

and 5-HT

3

but with very different NET and DAT preferences. The compounds also differed on their DMPK parameters, and overall, vortioxetine stood out as the preferred compound.

Table 9.4 SAR of compounds 14–27 show that minor structural changes to vortioxetine have pronounced effect on in vitro pharmacology. Compounds 21, 23, 25 and 27 were shown to be human metabolites [16].

Table 9.5 In vitro binding affinities and functional activities of vortioxetine at human and rat targets expressed in recombinant cell lines.

Table 9.6 Overview of effects of vortioxetine and comparator antidepressants in the animal models that were used to assess cognitive performance in the programme.

Chapter 10: Discovery of Vonoprazan Fumarate (TAK-438) as a Novel, Potent and Long-Lasting Potassium-Competitive Acid Blocker

Table 10.1 SAR study process for the HTS hit compound (summarised from [15]).

Table 10.2 Optimisation of the pyrrole derivatives from a new lead compound 4 (based on [16]).

Chapter 11: Discovery and Development of Nintedanib: A Novel Antiangiogenic and Antifibrotic Agent

Table 11.1 VEGFR2/HUVEC inhibition of 6-substituted indolinones.

Table 11.2 Inhibitory profile of 6-methoxycarbonyl-substituted indolinones.

Table 11.3 Inhibitory profile of nintedanib analogues. Adapted fromRoth et al. 2015 [1].

Table 11.4 In vitro kinase inhibition profile of nintedanib. Adapted with permission from Hilberg et al. 2008 [4].

Table 11.5 Characterisation of nintedanib in cellular systems.

Table 11.6 Pharmacokinetic parameters after a single administration of nintedanib in a number of different animal species. Adapted with permission from Roth et al. 2015 [6].

List of Illustrations

Chapter 1: From DMSO to the Anticancer Compound SAHA, an Unusual Intellectual Pathway for Drug Design

Figure 1.1 1 N-methylacetamide, 2 dimethylsulfoxide (DMSO), 3 hexamethylene bisacetamide, 4 suberoyl-bis-N-methylamide.

Figure 1.2 5 suberyol-bis-hydroxamic acid (SBHA), 6 suberyolanilide hydroxamic acid (SAHA).

Figure 1.3

Figure 1.4 N-hydroxy-4-[(N(2-hydroxyethyl)-2-phenylacetamido)-methyl)benzamide] (HPB), 4-[(hydroxyamino)carbonyl]-N-(2-hydrocyethyl)-N-phenyl-benzeneacetamide (HPOB).

Chapter 2: Romidepsin and the Zinc-Binding Thiol Family of Natural Product HDAC Inhibitors

Figure 2.1 Enzyme catalysed lysine acetylation and deacetylation.

Figure 2.2 HDAC inhibitors on the market shown with the approving agency, year of approval and therapeutic indication. The atoms that coordinate to the active site zinc cation are highlighted in red and the HDAC pharmacophore comprising a zinc-binding group, spacer and cap is illustrated for vorinostat.

Figure 2.3 The first natural products to be identified as HDAC inhibitors.

Figure 2.4 FR901228 (romidepsin) (a) and its cellular activation to the HDAC inhibitor redFK228 (b). The zinc-binding β-hydroxy acid fragment common to all the natural products in this family is highlighted in red.

Figure 2.5 Three examples of the romidepsin family of natural products.

Figure 2.6 The spiruchostatin family of HDAC inhibitors.

Figure 2.7 The thailandepsin family of HDAC inhibitors.

Chapter 3: The Discovery and Development of Belinostat

Figure 3.1 Representatives of the main classes of HDAC inhibitor.

Figure 3.2 Schematic model of 1C3S crystal structure (a). The structure of TSA is shown in yellow. The zinc ion is shown as a grey sphere and can be seen at the bottom of the tubular pocket. The three standard pieces of an HDAC inhibitor are shown in (b).

Figure 3.3

Figure 3.4

Figure 3.5 Summary of HDAC inhibitory activity of the sulfonamide series.

Chapter 4: Discovery and Development of Farydak (NVP-LBH589, Panobinostat) as an Anticancer Drug

Figure 4.1 The Mammalian cell cycle as regulated by CDKs and CDK inhibitors.

Figure 4.2 Reporting gene construct for identifying p21Waf1 inducers.

Figure 4.3 Screening hits that induce expression of p21 promoter.

Figure 4.4 HDAC inhibitor hit-to-lead optimisation flow chart.

Figure 4.5 Biochemical HDAC inhibition assay for primary screening.

Figure 4.6 Hit-to-lead campaign starting with trichostatin A.

Figure 4.7 Chemical structures of known HDAC inhibitors.

Figure 4.8 Lipophilic surface representation of the HDLP-trichostatin complex.

Figure 4.9 Cell cycle alterations in response to LAQ824 treatment.

Figure 4.10 In tumour cells LAQ824 treatment results in histone acetylation.

Figure 4.11 Increasing LAQ824 levels increased histone acetylation in tumour cells.

Figure 4.12 Increased histone acetylation in HCT116 tumours following LAQ824 treatment.

Figure 4.13 Metabolic pathways of NVP-LAQ824-CU in liver microsomal incubations from rat, dog and liver.

Figure 4.14 SAR of the cinnamoyl hydroxamate chemotype.

Figure 4.15 Drug-induced changes on series of monophasic action potentials from the Langendorff heart model.

Figure 4.16 Normal bronchial epithelial cells and SV40-telomerase-transformed bronchial epithelial cells were treated with 0.1% DMSO, 1 μM (normal) or 0.2 μM (transformed) NVPLBH589 for 48 h. Cells were stained with Alexa Fluor 488 Annexin V and propidium iodide and observed with a fluorescent microscope using a dual filter set for FITC and rhodamine. Photographs are shown under phase contrast and fluorescence microscopy. Cells undergoing apoptosis were detected by green fluorescence (marked with arrows).

Figure 4.17 Waterfall plot showing response to LBH589 by CTCL patients in a Phase II trial [27].

Figure 4.18 Single agent efficacy study of LBH589 in the HH CTCL mouse tumour model.

Figure 4.19 Dot plots of LD

50

of large scale profiling of cancer cells.

Figure 4.20 Confirmed single agent activity of LBH589 in a heavily pretreated (two ASCTs bortezomib, thalidomide, lenalidomide) myeloma patient. Decreased M-protein was measured to demonstrate LBH589 activity [31].

Chapter 5: Discovery and Development of HDAC Subtype Selective Inhibitor Chidamide: Potential Immunomodulatory Activity Against Cancers

Figure 5.1 The synthetic routes of compounds 1–7.

Figure 5.2 Gene expression profiling of chidamide or SAHA compared with HDAC1–5 SiRNA knockdown in cell. HeLa cells were transfected with HDAC subtype-specific SiRNA plasmids (HDAC1–5) or empty vector for 96 h. For compound treatment, Hela cells were treated with SAHA (1 μM), chidamide (3 μM), or vehicle solvent for 24 h. Gene expression profiling was performed for validated samples with in-house made long oligonucleotides microarrays. The genes with significant and unique expression change in each HDAC subtype knockdown models and similar change by SAHA treatment were picked as representative gene signatures.

Figure 5.3 Proposed antitumour mechanisms of chidamide. Chidamide exerts its antitumour activity via at least three mechanisms. ① Preferential induction of growth arrest and apoptosis in blood and lymphoid-derived tumour cells. ② Stimulation of innate and adaptive immune surveillance via induction of antigen representation of tumour cells and activation of cytotoxic activity of natural killer (NK) and cytotoxic T lymphocytes (CTL). ③ Exhibiting a synergistic effect with chemotherapeutic agents or other target therapeutic agents via enhancing drug sensitivity and repressing EMT of tumour cells.

Figure 5.4 Waterfall plot of tumour size change in PTCL patients. The total tumour size changes from baseline assessment from 78 patients of the pivotal phase 2 study of chidamide in patients with relapsed or refractory peripheral T-cell lymphoma.

Figure 5.5 Overall survival of PTCL patients treated with chidamide.

Figure 5.6 Hematological adverse events vs. treatment time of PTCL patients with chidamide.

Figure 5.7 Concentration-dependent molecular actions of chidamide.

Figure 5.8 Key advances in identification of immunomodulatory effects of HDAC inhibitors.

Chapter 6: Abiraterone Acetate (Zytiga®): An Inhibitor of CYP17 as a Therapeutic for Castration-Resistant Prostate Cancer

Figure 6.1 Biosynthesis of androgens catalyzed by CYP17.

Figure 6.2 Structure of antifungal agent ketoconazole (7) and abiraterone acetate (8, CB7630, Zytiga).

Figure 6.3 Overlay of cyclohexyl 4-pyridylacetate onto pregnenolone and pyridyl cyclic ester lead molecules 9 and 10[16].

Figure 6.4 Effects of 14 days treatment with abiraterone acetate (Table 6.1, compound 8) on the organ weights of mice [20].

Figure 6.5 Abiraterone suppression of LuCaP35V tumour growth (CRPC tumour xenograft) in mice [21, 22].

Figure 6.6 Overall survival: (a) COU-AA-301 (post docetaxel); (b) COU-AA-302 (Chemotherapy naïve) [28].

Chapter 7: Discovery of Delamanid for the Treatment of Multidrug-Resistant Pulmonary Tuberculosis

Figure 7.1 Structure of delamanid.

Figure 7.2 Synthetic development designed to eliminate mutagenicity and to enhance antituberculosis activity.

Figure 7.3 Structure of 6-nitro-2,3-dihydroimidazo[2,1-b]oxazole.

Figure 7.4 Reagents and conditions to synthesise the key intermadiate 16 for optically active compounds: (a) Et

3

N, AcOEt, 60–65 °C; (b) K

2

CO

3

, MeOH, rt; (c) metanesulfonyl chloride, pyridine, < 15 °C; (d) 1,8-diazabicyclo[5.4.0]-7-undecene, AcOEt, rt.

Figure 7.5 Reagents and conditions to synthesise (S)-piperazine carbamates 3: (a) acetonitrile, 60–65 °C; (b) NaH, N,N-dimethylformamide, rt; (c) trifluoroacetic acid, CH

2

Cl

2

, rt; (d) Et

3

N, CH

2

Cl

2

, rt; (e) 1,1’-carbonyldiimidazole, CH

2

Cl

2

, rt; (f) N,N-dimethylformamide, rt.

Figure 7.6 Reagents and conditions to synthesise (S)-benzyl piperazines 4: (a) trifluoroacetic acid, CH

2

Cl

2

, rt; (b) Et

3

N, CH

2

Cl

2

, rt; (c) NaBH(OAc)

3

, dichloroethane, rt.

Figure 7.7 Reagents and conditions to synthesise (S)-aryl piperazines 5: (a) acetonitrile, 60–65 °C; (b) NaH, N,N-dimethylformamide, rt.

Figure 7.8 Reagents and conditions to synthesise (S)-piperidines 6: (a) acetonitrile, 60–65°C; (b) NaH, N,N-dimethylformamide, rt.

Figure 7.9 Reagents and conditions to synthesise (S)-hydrazone compounds 7: (a) acetonitrile, 60–65 °C; (b) NaH, N,N-dimethylformamide, rt; (c) trifluoroacetic acid, CH

2

Cl

2

, rt; (d) Et

3

N, CH

2

Cl

2

, rt; (e) N,N-dimethylformamide, rt.

Figure 7.10 Reagents and conditions to synthesise (R)-phenoxy compounds 8–10: (a) phenol derivatives, NaH, 50 °C, N,N-dimethylformamide. The substituents R’ are shown in Tables 7.7–7.9.

Figure 7.11 Structure of DM-6705, the major metabolite of delamanid.

Chapter 8: Sofosbuvir: The Discovery of a Curative Therapy for the Treatment of Hepatitis C Virus

Figure 8.1 The HCV genome with genes coding for 3 structural proteins and 7 nonstructural proteins structure of the HCV NS5b RNA-dependent RNA polymerase showing the palm, thumb and finger domains.

Figure 8.2 Conversion pathways for PSI-6130 and PSI-6206.

Figure 8.3 Conceptual first-pass liver metabolism leading to 2’-F-2’-C-methyluridine 5’monophosphate being trapped within hepatocytes.

Figure 8.4 In vitro and in vivo compound assessment strategy for exploring the feasibility of using a phosphoramidate prodrug.

Figure 8.5 Multiparameter SAR optimisation around the phosphoramidate prodrug moiety (a) and data used to cull the compounds into a preferred set for further evaluation (b).

Figure 8.6 Seven compounds selected for further study based on their favourable biological profile and a desire to progress a set of compounds that were structurally diverse.

Figure 8.7 The mono-, di-and triphosphates and the intermediate di-acid metabolite, PSI-352707, of the prodrug.

Figure 8.8 X-ray structure of sofosbuvir (a) and potency of PSI-7976 and PSI-7977 (b).

Figure 8.9 A homochiral synthesis of the uridine nucleoside PSI-6206 starting with D-glyceraldehyde.

Figure 8.10 Reacting a p-nitrophenolate phosphate ester 18 with the uridine nucleoside in the presence of a strong base led to good yields of the nucleoside phosphoramidate. Using reagent 19 further optimized the reaction yield, diastereoselectivity, reagent reactivity and reaction duration.

Figure 8.11 Results of follow-ups to the Electron study. Data from PHOTON-1 (GT1, SOF + RBV), NEUTRINO (GT1, SOF + P/R), ION 1-3 (GT1, LDV/SOF), FISSION, POSITRON, FUSION, VALENCE, PHOTON-1 (GT2, SOF + RBV), VALENCE, PHOTON-1 (GT3, SOF + RBV) studies.

Chapter 9: The Discovery of the Antidepressant Vortioxetine and the Research that Uncovered Its Potential to Treat the Cognitive Dysfunction Associated with Depression

Figure 9.1 Structure of vortioxetine (1, Lu AA21004, Brintellix® or Trintellix®).

Figure 9.2 Synthesis of compound 2 as a combined SERT inhibitor and 5-HT

1A

receptor antagonist [10]. The synthesis of crude 4-(2-(4-methoxyphenyl)sulfanylphenyl)piperazine was enabled by a solid-phase synthesis strategy based on the formation of aromatic carbon-heteroatom bonds via an iron-mediated Pearson reaction [11].

Figure 9.3 Effects on extracellular 5-HT levels in the rat ventral hippocampus after acute treatment with compound 3 (a) and vortioxetine (b) relative to a high dose of escitalopram (> 80% SERT occupancy). The larger increase in extracellular 5-HT levels of compound 3 relative to escitalopram can be explained by its activity at the 5-HT

2C

receptor and SERT [12], whereas the effect of vortioxetine, at least partly, can be explained by its activity at the 5-HT

3

receptor and SERT [13]. Compound 3 and vortioxetine were tested in different laboratories, so the increase in 5-HT levels should not be compared between the two figures.

Figure 9.4 Most versatile synthetic strategy to target compounds.

Figure 9.5 Effect on extracellular 5-HT levels in the rat medial prefrontal cortex after three days of subcutaneous treatment with vehicle or vortioxetine via an osmotic minipump. The SERT occupancy at the given dose is also shown. From J. Med. Chem. with permission [14].

Figure 9.6 A schematic diagram of the hypothesised modulatory role of vortioxetine’s 5-HT receptors on glutamatergic neurotransmission. A glutamatergic pyramidal neuron and several GABA interneurons expressing the 5-HT

3

, 5-HT

1A

, 5-HT

7

and 5-HT

1B

receptors on either dendrites or axon terminals are shown. The multimodal compound vortioxetine and its possible sites of action are shown. Note that 5-HT

1A

, 5-HT

1B

and 5-HT

7

receptors may be localised on different neuronal populations. VOR, vortioxetine; Glu, Glutamate; GABA, gamma-aminobutyric acid. Slightly modified from reference [49].

Figure 9.7 SAR of the 4-(2-phenylsulfanylphenyl)piperazine series, illustrating that vortioxetine was identified relative early on in the lead optimisation phase and could not be further improved.

Chapter 10: Discovery of Vonoprazan Fumarate (TAK-438) as a Novel, Potent and Long-Lasting Potassium-Competitive Acid Blocker

Figure 10.1 Acid activation of PPIs (lansoprazole) and inhibition of H+, K+-ATPase by binding of the active form (AG-2000) to an SH group of the enzyme.

Figure 10.2 Mechanism of action of P-CABs and PPIs in inhibiting H+, K+-ATPase in the parietal cell.

Figure 10.3 Chemical structures of HTS hit compound 1 and other reported P-CABs.

Figure 10.4 Improvement of potency from a HTS hit compound 1 (based on [15]).

Figure 10.5 Correlation between measured log D value and in vitro toxic data on a series of pyrrole derivatives1).

Figure 10.6 Selection of compound 6 (TAK-438, vonoprazan fumarate) by strategic lead optimisation (based on [16]).

Figure 10.7 The effect of intravenous TAK-438 and LPZ on gastric perfusate pH during histamine stimulation in anesthetised rats (based on [17]).

Figure 10.8 Inhibitory effects of TAK-438 and LPZ on histamine-stimulated gastric acid secretion in Heidenhain pouch dogs (adapted from [18]).

Figure 10.9 Drug concentrations in plasma and the stomach at 0.25, (1, 2 and 24) h after the oral administration of 2 mg kg−1 [14C] TAK-438 in rats (adapted from [18])

Figure 10.10 Physicochemical property and pharmacokinetic behaviour of TAK-4381).

Figure 10.11 Mean intragastric pH in healthy Japanese subjects (single dose) (based on [19]).

Figure 10.12 Healing rates of reflux esophagitis in mild (a) and severe (b) ill patients as assessed by principal investigators (based on [22])

Figure 10.13 Recurrence rate of reflux esophagitis maintenance therapy, after 24 weeks in PM (a) and EM (b) patients (based on [21]).

Figure 10.14 Primary eradication rates in the H. pylori eradication study (four weeks after treatment) (based on [21]).

Chapter 11: Discovery and Development of Nintedanib: A Novel Antiangiogenic and Antifibrotic Agent

Figure 11.1 Molecular structure of nintedanib ethanesulfonate 1.

Figure 11.2 VEGFR2 hit compound 3 derived from CDK4 inhibitor 2.

Figure 11.3 Compounds with nonaromatic side chains.

Figure 11.4 Structure–activity relationships of 6-substituted oxindoles. Reproduced with permission from Roth et al. 2015 [6].

Figure 11.5 Structure of nintedanib bound to VEGFR2 (a); superimposition of the binding modes of nintedanib to VEGFR2 (b) (coloured as in (a) and FGFR1 (coloured salmon). Adapted with permission from Wollin et al. 2015 [2].

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Edited by

János Fischer and Wayne E. Childers

Successful Drug Discovery

Volume 2

Editors

János FischerGedeon Richter PlcDepartment of Medicinal Chemistry Gyömröi ut 301103 BudapestHungary

Wayne E. ChildersTemple University School of PharmacyMoulder Ctr. for Drug Discovery Res.3307 N Broad StreetPhiladelphia, PA 19140United States of America

Supported by theInternational Union of Pure and Applied Chemistry (IUPAC)Chemistry and Human Health Division PO Box 13757Research Triangle Park, NC 2770-3757USA

All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

Library of Congress Card No.:

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A catalogue record for this book is available from the British Library.

Bibliographic information published by the Deutsche Nationalbibliothek

The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at http://dnb.d-nb.de.

© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

Print ISBN 978-3-527-34115-3ePDF ISBN 978-3-527-80032-2ePub ISBN 978-3-527-80034-6Mobi ISBN 978-3-527-80033-9oBook ISBN 978-3-527-80031-5

Advisory Board Members

Magid Abou-Gharbia(Temple University, USA)

Kazumi Kondo(Otsuka, Japan)

Barry V.L. Potter(Oxford University, UK)

Anette Graven Sams(Lundbeck, Denmark)

John A. Lowe(JL3Pharma LLC, USA)

Part Editors

Helmut Buschmann(Aachen, Germany)

Juan-Miguel Jimenez(Vertex Pharmaceuticals, UK)

John Proudfoot(Boehringer Ingelheim, Ridgefield, USA)

A. Ganesan(University of East Anglia, Norwich, UK)

Stefan Laufer(University of Tübingen, Germany)

Jörg Senn-Bilfinger(Konstanz, Germany)

Preface

The first volume of Successful Drug Discovery has been well received and the International Union of Pure and Applied Chemistry (IUPAC) supported its continuation.

The main goal of this book series is to help experts of drug research and development both in academia and industry with case histories desribed by their key inventors or recognised experts whose contributions can also serve as teaching examples.

This year marks the tenth anniversary of the approval of vorinostat, the first marketed histone deacetylase inhibitor (HDAC). This event inaugurated a stream of HDAC inhibitor approvals and confirmed the validity of this drug target and of epigenetic modulation as a viable therapeutic mechanism. To celebrate this important milestone the volume presents a number of HDAC inhibitor drug discovery stories.

The editors of the second volume focused on the following six parts:

I. HDAC Inhibitor Anticancer Drug DiscoveryPart Editor: A. Ganesan (University of East Anglia, Norwich, UK)

1.

Vorinostat

Ronald Breslow (Columbia University, USA) describes the discovery of vorinostat, which is a pioneer HDAC inibitor whose discovery started from dimethylsulfoxide as a lead molecule.

2.

Romidepsin

A. Ganesan (University of East Anglia, UK) gives an overview of the discovery of romidepsin, a depsipeptide natural product. High-throughput screening led to an anticancer drug that proved to be a potent inhibitor of class I HDACs.

3.

Belinostat

Paul W. Finn and coworkers (University of Buckingham, UK) report on belinostat, which is a potent pan-inhibitor of class I and II HDACs. It was approved in 2014 for the treatment of peripheral T-cell lymphoma.

4.

Panobinostat

Peter Atadja and coworker (Novartis Institute for Biomedical Research, US & China) present the story of how a functional high-throughput screen looking for inducers of cyclin-dependent kinase 2 (CDK2) inhibitor p21 provided hits that were identified as HDAC inhibitors, ultimately resulting in the discovery of panobinostat.

5.

Chidamide

Xian-Ping Lu and coworkers (Shenzen Chipscreen Biosciences, China) describe the discovery and development of chidamide which is a novel benzamide type inhibitor of class I HDACs and class IIb HDAC10.

II. Steroidal CYP17 Inhibitor Anticancer Drug DiscoveryPart Editor: Juan-Miguel Jimenez (Vertex Pharmaceuticals, UK)

6.

Abiraterone acetate

Gabriel Martinez Botella and coworkers (SAGE Therapeutics, USA) have written a chapter on the discovery of abiraterone acetate, which is a key therapeutic in the treatment of metastatic castrate-resistant prostate cancer.

III. Anti-infective Drug DiscoveriesPart Editor: John Proudfoot (Boehringer Ingelheim, Ridgefield, USA)

7.

Delamanid

Hidetsugu Tsubouchi and coworkers (Otsuka, Japan) summarise the discovery of delamanid, which is a new drug for the treatment of multidrug-resistant pulmonary tuberculosis.

8.

Sofosbuvir

Michael J. Sofia (Arbutus Biopharma, USA) describes the discovery of sofosbuvir, which has become the backbone agent of combination curative therapy for hepatitis C virus infection.

IV. Central Nervous System (CNS) Drug DiscoveryPart Editor: Helmut Buschmann (Aachen, Germany)

9.

Vortioxetine

Benny Bang-Andersen and coworkers (Lundbeck, Denmark and USA) give an overview of the discovery of vortioxetine, a new multimodal antidepressant drug with serotonin modulator and stimulator activity.

V. Antiulcer Drug DiscoveryPart Editor: Jörg Senn-Bilfiger (Konstanz, Germany)

10.

Vonoprazan fumarate

Haruyuki Nishida (Takeda, Japan) describes the discovery of vonoprazan fumarate, which is a novel, potent and long-lasting potassiumcompetitive acid blocker showing several advantages over proton pump inhibitors.

VI. Cross-Therapeutic Drug Discovery (Respiratory Diseases/Anticancer)Part Editor: Stefan Laufer (University of Tübingen, Germany)

11.

Nintedanib

Gerald J. Roth and coworkers (Boehringer Ingelheim, Biberach, Germany) summarise the discovery and development of nintedanib, which represents a pioneer discovery of a cross-therapeutic research for the treatment of solid tumours and idiopathic pulmonary fibrosis.

The editors and part editors thank the advisory board members: Magid Abou-Gharbia (Temple University, USA), Kazumi Kondo (Otsuka, Japan), John A. Lowe (JL3Pharma LLC, USA), Barry V.L. Potter (Oxford University, UK) and Anette Graven Sams (Lundbeck, Denmark). Special thanks are due to the following reviewers who helped both the authors and the editors: Jan Heeres, Manfred Jung, Sándor Mahó, Tom Perun (Division Chemistry and Human Health of IUPAC) and Ron Weir (Interdivisional Committee on Terminology, Nomenclature and Symbols of IUPAC).

Last but not least the editors and authors thank the coworkers of Wiley-VCH, especially Dr Frank Weinreich, for their critical and most appreciated support and collaboration.

Budapest, HungaryPhiladelphia, USA31 March 2016

János Fischer andWayne E. Childers

List of Contributors

Peter Atadja

Novartis Institute for Biomedical Research

Shanghai

[email protected]

Benny Bang-Andersen

Discovery Chemistry & DMPK and Brintellix Clinical Science

H. Lundbeck A/S

2500 Valby

[email protected]

Gabriel M. Belfort

SAGE Therapeutics215 First StreetCambridge, MA 02142USA

Rudolf Binder

Boehringer Ingelheim Pharma GmbH & Co KG

Medicinal Chemistry/Research Germany

Birkendorfer Str. 65

88397 Biberach

Germany

Gabriel Martinez Botella

SAGE Therapeutics215 First StreetCambridge, MA [email protected]

Ronald Breslow

Department of Chemistry Columbia UniversityNew York, NY [email protected]

Hai-Xiang Cao

Shenzhen Chipscreen BiosciencesShenzhen Hi-Tech Industrial Park Nanshan District

Shenzhen

Guangdong 518057

China

Elisabeth Carstensen

Buckingham Institute for Translational Medicine

University of Buckingham

Hunter Street

Buckingham MK18 1EG

UK

Florian Colbatzky

Boehringer Ingelheim Pharma GmbH & Co KG

Medicinal Chemistry/Research Germany

Birkendorfer Str. 65

88397 Biberach

Germany

Claudia Dallinger

Boehringer Ingelheim Pharma GmbH & Co KG

Medicinal Chemistry/Research Germany

Birkendorfer Str. 65

88397 Biberach

Germany

Paul W. Finn

Buckingham Institute for Translational Medicine

University of Buckingham

Hunter Street

Buckingham MK18 1EG

UK

[email protected]

A. Ganesan

School of PharmacyUniversity of East AngliaNorwich Research ParkNorwich NR4 7TJ

UK

[email protected]

Xia Guo

Shenzhen Chipscreen Biosciences

Shenzhen Hi-Tech Industrial Park Nanshan District

Shenzhen

Guangdong 518057

China

Boyd L. Harrison

SAGE Therapeutics

215 First Street

Cambridge, MA 02142

USA

Frank Hilberg

Boehringer Ingelheim Pharma GmbH & Co KG

Medicinal Chemistry/Research Germany

Birkendorfer Str. 65

88397 Biberach

Germany

Hiroshi Ishikawa

Fellow Pharmaceutical Business Division

Otsuka Pharmaceutical Co., Ltd. 3-2-27 Otedori

Chuo-ku

Osaka 540-0021

Japan

Rolf Kaiser

Boehringer Ingelheim Pharma GmbH & Co KG

Medicinal Chemistry/Research Germany

Birkendorfer Str. 65

88397 Biberach

Germany

Zhi-Bin Li

Shenzhen Chipscreen Biosciences

Shenzhen Hi-Tech Industrial Park

Nanshan District

Shenzhen

Guangdong 518057

China

Einars Loza

Buckingham Institute for Translational Medicine

University of Buckingham

Hunter Street

Buckingham MK18 1EG

UK

Xian-Ping Lu

Shenzhen Chipscreen Biosciences

Shenzhen Hi-Tech Industrial Park

Nanshan District

Shenzhen

Guangdong 518057

China

[email protected]

Makoto Matsumoto

Pharmaceutical Business Division

Otsuka Pharmaceutical Co., Ltd.

463-10 Kagasuno kawauchi-cho

Tokushima 771-0192

Japan

Zhi-Qiang Ning

Shenzhen Chipscreen Biosciences

Shenzhen Hi-Tech Industrial Park Nanshan District

Shenzhen

Guangdong 518057

China

Haruyuki Nishida

Medicinal Chemistry Research Laboratories

Pharmaceutical Research Division

Takeda Pharmaceutical Company Limited

26-1, Muraokahigashi 2-chome Fujisawa

Kanagawa 251-8555

Japan

[email protected]

Christina Kurre Olsen

Discovery Chemistry & DMPK and Brintellix Clinical Science

H. Lundbeck A/S

2500 Valby

Denmark

De-Si Pan

Shenzhen Chipscreen Biosciences

Shenzhen Hi-Tech Industrial Park

Nanshan District

Shenzhen

Guangdong 518057

China

John Park

Boehringer Ingelheim Pharma

GmbH & Co KG

Medicinal Chemistry/Research Germany

Birkendorfer Str. 65

88397 Biberach

Germany

Alexander Pautsch

Boehringer Ingelheim Pharma

GmbH & Co KG

Medicinal Chemistry/Research Germany

Birkendorfer Str. 65

88397 Biberach

Germany

Lawrence Perez

Novartis Institute for Biomedical Research

US & China

Gerald J. Roth

Boehringer Ingelheim Pharma GmbH & Co KG

Medicinal Chemistry/Research Germany

Birkendorfer Str. 65

88397 Biberach

Germany [email protected]

Connie Sanchéz

Lundbeck USA Paramus, NJ 07652 USA

Hirofumi Sasaki

Medicinal Chemistry Research Laboratories

Otsuka Pharmaceutical Co., Ltd.

463-10 Kagasuno kawauchi-cho

Tokushima 771-0192

Japan

Rozsa Schlenker-Herceg

Boehringer Ingelheim Pharma GmbH & Co KG

Medicinal Chemistry/Research Germany

Birkendorfer Str. 65

88397 Biberach

Germany

Song Shan

Shenzhen Chipscreen Biosciences

Shenzhen Hi-Tech Industrial Park

Nanshan District

Shenzhen

Guangdong 518057

China

Michael J. Sofia

Arbutus Biopharma, Inc.

3805 Old Easton Road

Doylestown, PA 18902,

[email protected]

Hidetsugu Tsubouchi

Compliance & Ethics Department

Otsuka Pharmaceutical Co., Ltd. 3-2-27 Otedori

Chuo-ku

Osaka 540-0021

[email protected]

Lutz Wollin

Boehringer Ingelheim Pharma GmbH & Co KG

Medicinal Chemistry/Research Germany

Birkendorfer Str. 65

88397 Biberach

Germany

Qian-Jiao Yang

Shenzhen Chipscreen Biosciences

Shenzhen Hi-Tech Industrial Park Nanshan District

Shenzhen

Guangdong 518057

China

Jin-Di Yu

Shenzhen Chipscreen Biosciences

Shenzhen Hi-Tech Industrial Park

Nanshan District

Shenzhen

Guangdong 518057

China

Part IHDAC Inhibitor Anticancer Drug Discovery

Chapter 1From DMSO to the Anticancer Compound SAHA, an Unusual Intellectual Pathway for Drug Design

Ronald Breslow

1.1 Introduction

This is an account of aspects of a collaboration between Ronald Breslow (originally Professor of Chemistry at Columbia University, also a member of the Biological Sciences Department, now University Professor at Columbia) and Paul Marks (originally Professor of Human Genetics and Medicine, Dean of the Faculty of Medicine, then Vice President for Health Sciences and Director of the Comprehensive Cancer Center at Columbia University, then President and Chief Executive Officer at Memorial Sloan Kettering Cancer Center, now President Emeritus and Member of the Sloan Kettering Institute) in the invention and development of suberoylanilide hydroxamic acid (SAHA), an effective anticancer agent that has been in human use for years after approval in the United States, Canada and more recently Japan. The Breslow group designed new potential molecules and carried out their syntheses in the Columbia University chemistry department, and submitted them to Paul Marks and Richard Rifkind at the Columbia Cancer Center, and later at the Sloan Kettering Institute for Cancer Research, for biological evaluation. Paul Marks instituted the collaboration, based on some work by Charlotte Friend of Mount Sinai School of Medicine.

This is the way most modern pharmaceuticals are created in pharmaceutical companies or in academic medicinal departments. Biologists may be aware of a promising area for drug development, medicinal chemists then design and create candidate molecules and send them to the biologists, who then evaluate them. With promising results, the chemists continue to create new, perhaps better, candidates while the biologists extend testing to animals and then to humans. Successful medicines are then approved for human use.

Normally the chemists are aware of compounds that have some promise, based on binding studies, and they can design around those structures. In the case of SAHA, the initial lead, dimethylsulfoxide (DMSO) 1, was very far from a potential medicine so the design was based on a series of hypotheses. Even so, the eventual structure of SAHA proved to be ideal as a binder to the biological target, although this is not how it was discovered. Thus the editors of this volume have invited me to describe the unusual intellectual history that led to its structure. I am a physical organic chemist who had designed and created new molecules for novel properties, such as unusual conjugative stability or instability, or effective catalytic enzyme mimics, but not medicinal properties. However, I have a Master’s degree in Medical Science from Harvard University in addition to my Ph.D. in Chemistry, and I had been a consultant with pharmaceutical companies for many years. There I proposed both new synthetic approaches to their target compounds and also possible alternative medicinal targets themselves.

A few years ago, Paul Marks and I wrote a short review describing the work of both our labs in the development of SAHA [1], but the present chapter will concentrate only on the chemical approach that led to drug development. Thus it does not describe in detail the brilliant biological work done by Paul Marks and Richard Rifkind. The references are only those in which Paul Marks and I are both authors, and it will not cover the many papers and a book produced by the Marks lab alone and several papers from only our lab that related the SAHA story to our other work.

1.2 The Discovery of SAHA (vorinostat)

Stem cells have two functions. They multiply to form additional stem cells, and they differentiate to adult tissue cells with specialised functions. In 1966 Paul Marks approached me with the information that Charlotte Friend had seen something remarkable [2, 3]. When a suspension of murine erythroleukemia cells (MELC) was treated with dimethylsulfoxide (DMSO) (1) at 280 mmolar approximately 60% of the cells underwent cytodifferentiation to normal erythrocytes. This was the first example in which such a process occurred, and it suggested a new approach to cancer treatment generally. Of course such a required concentration was totally impractical for a medicine, so it was important to find more potent analogs of DMSO. Marks and I agreed to collaborate and build a research programme based on this finding. The Breslow lab with my students and postdocs would conceive and create new compounds that would be tested by Marks and his associates for cytodifferentiation of erythroleukemia cells, as DMSO had done, but with more practical doses. Marks would also further evaluate promising leads with biological testing. This led to the discovery of SAHA. In time Marks and Breslow and Richard Rifkind formed a company, ATON Pharma Inc. It received the patent rights from Columbia University and Sloan Kettering and funded the Phase I human trials for SAHA.

Many small molecule linear and cyclic amides were examined. N-Methylacetamide (2) was fivefold more effective than DMSO, but still not effective enough to be a practical drug [4]. Thus the chemists decided to create linked dimers of acetamide, to take advantage of the well-known chelate effect that leads to stronger binding, and thus should require lower doses for anticancer effectiveness. Double binders have entropy advantages over single ligands if both ends contribute to the binding. This involved the hope that there were more binding sites than a single one for the initial compounds, and thus linking them together could be useful. The first compound, hexamethylene bis-acetamide (HMBA, linked at the nitrogen atoms) (3), was indeed one order of magnitude (tenfold) more potent than simple acetamide, and changing the linking groups from three methylenes up to nine made it clear that a six methylene chain – the first one we tried – was the optimum [5–7]. This preference will eventually be seen and understood when we describe SAHA. We also prepared a dimer of acetamide linked at the methyl groups, suberoyl-bis-N-methylamide (4), and it also showed tenfold stronger binding than simple acetamide [8]. Various dimers including dimers of DMSO were also examined [8, 9]. HMBA had extensive biological study, and indeed some human trials were performed with HMBA [10–13]. There were some useful responses in cancer patients, but the doses required were too high to be well tolerated in human patients. When even trimers and tetramers of acetamide were not more effective [14, 15], we concluded that simple amides were not bound strongly enough.

Figure 1.11N-methylacetamide, 2 dimethylsulfoxide (DMSO), 3 hexamethylene bisacetamide, 4 suberoyl-bis-N-methylamide.

We were already thinking that the target could be an enzyme, perhaps a metalloenzyme, to explain the strong preference for particular lengths of our compounds. Since DMSO and the amides had polar groups that could be metal ligands, we decided to go to even better metal ion binders. We synthesised a bis-amide like 4 but with hydroxyl groups instead of methyl groups, creating compound 5 that we called suberoyl-bis-hydroxamic acid, SBHA [14]. Hydroxamic acids were known to be strong binders to metal ions. Compound 5 was more effective than was HMBA, compound 3, suggesting that indeed there was a metal ion in the biological target. Again the six-methylene chain length was optimal. However, the chance that a receptor protein would have two metal ions that distance apart seemed unlikely, so we decided to replace the hydroxyl of one hydroxamic group with a hydrophobic phenyl group to see if it could make an even better binder. This would bind to a metal ion with its hydroxamic group while binding to a hydrophobic region of a protein with the phenyl group. This was speculation, but it turned out to be correct.

Figure 1.25 suberyol-bis-hydroxamic acid (SBHA), 6 suberyolanilide hydroxamic acid (SAHA).

We created SAHA, suberoylanilide hydroxamic acid 6 [14]. It inhibited histone deacetylases was approximately sixfold more potent than was SBHA in the MELC assay and also in various other tests [15–17]. Again we varied the chain length, and the six-methylene linker was optimal. We and others have replaced the phenyl group with many other larger hydrophobic units, which made compounds much more strongly bound, but in animal studies the more strongly bound analogs showed increased toxicity. This represents a fundamental problem not always recognised by medicinal chemists.

A binding constant is a ratio of two rate constants, the second-order rate constant for binding over the first-order rate constant for dissociation. It is often difficult to increase the rate of binding, which is limited by the collision rate. Strong binding instead often reflects slower dissociation, the first-order process, as the attractive interactions must be broken. Thus strong binders are often bound to biological receptors for a longer time. Putting it another way, for effectiveness a drug must normally be 50% or so bound to the receptor, and with strong binders a smaller dose is needed for 50% binding. If the strong binding reflects slower dissociation, the drug will be present on the biological targets for a long time. In the case of SAHA, physicians have found that unpleasant or dangerous side effects are minimised in human patients if the drug is present for only 8 h or so before excretion, so SAHA is administered once a day. With tenfold slower dissociation the drug would be present for 80 h, and side effects could be serious. With any SAHA analog significantly more strongly bound – and we looked at several with subnanomolar dissociation constants – adverse toxic side effects appeared in animal tests that could not be overcome by cutting back the dose.

SAHA proved to be an effective drug against a variety of cancers, as Paul Marks and our other collaborators established. In some cases the cancer cells differentiated into normal cells, as had happened with DMSO in the Charlotte Friend experiments. Examples included human colon (HT-29) and adult leukemia (HL-60) cells. The National Cancer Institute (NCI) then examined SAHA in sixty different human cancer cell types and saw stasis (lack of growth) with all, and about equal occurrences of either cytodifferention to normal cells or apoptosis (programmed cell death, not simple toxicity). SAHA also caused cytodifferention of MCF-7 breast adenocarcinoma cells into normal functioning breast milk cells. Very many cancers have been examined with SAHA.

The scientific question is, of course, how does SAHA cause these effects? A strong clue came from the work of Yoshida with two other cytodifferentiating agents, trichostatin A and trapoxin B. He showed that they induced cytodifferentiation by inhibiting the enzyme histone deacetylase (HDAC) [18]. The structure of trichostatin A 7 is similar to that of SAHA, although it is a less attractive drug. We saw that SAHA was also an inhibitor of HDAC and that the potency of various SAHA derivatives as HDAC inhibitors ran parallel to their biological anticancer effectiveness. We created a derivative 8 of SAHA with an azido group on the phenyl para position and tritium labeling in the phenyl, and irradiated it with HDAC in solution. The azido group lost nitrogen to form a reactive nitrene that then attached it to HDAC, so it was clear that HDAC was the binding target [19]. Finally, X-ray crystal structures were obtained in the lab of Pavletich that showed the detailed structure of the complex of SAHA and of trichostatin A with HDAC [20]. SAHA bound into HDAC by inserting into a pore with the phenyl group bound to a surface hydrophobic face of the protein while the hydroxamic acid group bound to a Zn2+ metal ion that was part of the HDAC protein. The six methylenes were the perfect length to reach between these two binding sites. We also synthesised a compound called pyroxamide 9 in which a pyridine ring replaced the phenyl ring of SAHA, and it had similar properties to SAHA [21].

Figure 1.3

The enzyme histone deacetylase binds an acetylated lysine from the protein histone at the zinc of HDAC, which catalyzes the hydrolysis of the acetyl group – hence histone deacetylase. The structure of SAHA bound to HDAC almost perfectly matches the structure of an acetylated lysine group of histone bound into the pore of the protein, with the six-methylene chain mimicking the side chain of an acetylated lysine. Although SAHA was not invented this way, it is ideal as a mimic of the transition state for zinc-catalyzed hydrolysis of an acetylated lysine group from histone. Other work not detailed here shows that particular lysines, when acetylated, can induce differentiation of stem cells or cancer cells, so blocking the deacetylation as SAHA does upregulate (increase) the acetylation level of the histone [22, 23]. Other studies suggest how apoptosis is also triggered by SAHA.

1.3 Clinical Trials

Phase I trials of SAHA in human cancer patients showed that it was well tolerated and that it had useful clinical results. At this point more extensive trials were needed, and several companies were interested in buying ATON for SAHA and its patents and data. Merck and Co bought ATON in 2004, and performed trials that were successful, so Merck obtained approval for the human use of SAHA against disease, first in the United States in 2006, then in Canada in 2009 and more recently in 2011 in Japan. SAHA has been used in clinical trials against many cancers, and it is still in active use in chemotherapy treatment of cancer patients.

1.4 Follow-On Research – Selective HDAC Inhibitors

Humans have eleven different zinc-dependent HDACs, with different structures and different selectivities for the cleavage of acetyl groups from various proteins. SAHA has rather broad selectivity among them, but a particular enzyme called HDAC6 is selective for the removal of acetyl groups from the protein tubulin; it is less effective against acetylated histone, for example. Thus the name histone deacetylase (HDAC) is a misnomer, since some of these deacetylases have other protein targets. The first example of an inhibitor of HDAC6 was a compound called tubacin, created by Schreiber in 2003 [24]. Kozikowski has been studying enzymes with such selectivity, and has made some compounds that are selective in blocking hydrolytic removal of the acetyl group from acetylated tubulin. He has suggested that HDAC6 has a shorter distance from the surface of the protein to the hydroxamic acid site. We have two very promising compounds that are quite selective for HDAC6 alone among the HDACs. They were designed to have a shorter distance between the surface and the HDAC group than SAHA has, and both have a branching group that prevents the compound from penetrating further into the protein cavity.

One compound we called HPOB, 10, selectively inhibits HDAC6 catalytic activity in vivo and in vitro [25]. Paul Marks compared it with Schreiber’s tubacin and saw that HPOB was 51.8-fold selective for HDAC6 versus HDAC1 while tubacin was only 4.3-fold selective. As we described, HPOB has very good biological properties, and is very promising in combination therapy to enhance the potency of various anticancer drugs. A second compound we call HPB, 11, we described in a paper just published in Proceedings of the National Academy of Sciences [26]. It is a little less selective than HPOB but it has even better anticancer properties, including lack of side effects, in animal studies. Both compounds need human evaluation before they can be seen as true improvements over SAHA. We are planning such studies.

Figure 1.4N-hydroxy-4-[(N(2-hydroxyethyl)-2-phenylacetamido)-methyl)benzamide] (HPB), 4-[(hydroxyamino)carbonyl]-N-(2-hydrocyethyl)-N-phenyl-benzeneacetamide (HPOB).

1.5 Conclusion

It has been over 40 years since Charlotte Friend placed transfected MELC cells in the presence of DMSO and started the field down the path of using HDAC inhibition to treat cancer. Since the approval of vorinostat for the treatment of persistent cutaneous T-cell lymphoma in 2006 (currently marketed under the trade name Zolinza®), a number of HDAC inhibitors from multiple chemical classes have followed suit. Some of these are discussed in following chapters. I am told by medicinal chemistry colleagues from Memorial Sloan–Kettering Cancer Center who are actively engaged in cancer drug discovery that SAHA is still used by physicians and continues to be a useful medicine for the treatment of human cancers. All things considered, this is a gratifying destiny for a drug whose origins began with such a simple molecule as DMSO.

List of Abbreviations

AIDS

autoimmune deficiency syndrome

DMSO

dimethylsulfoxide

HDAC

histone deacetylase

HMBH

hexamethylene bis-acetamide

HPB

N

-hydroxy-4-[(

N

(2-hydroxyethyl)-2-phenylacetamido)-methyl)-benzamide]

HPOB

4-[(hydroxyamino)carbonyl]-

N

-(2-hydroxyethyl)-

N

-phenyl-benzene-acetamide

MELC

murine erythroleukemia cells

PNAS

Proceedings of the National Academy of Sciences, USA

SAHA

suberoylanilide hydroxamic acid

SBHA

suberoyl-bis-hydroxamic acid

References

1 Marks, P.A. and Breslow, R. (2007) Dimethylsulfoxide to vorinostat: Development of this histone deacetylase inhibitor as an anticancer drug. Nat. Biotechnol., 25, 84–90.

2 Friend, C., Patuleia, M.C. and DeHarven, E. (1966) Erythrocytic maturation in vitro of virus-induced leukemic cells. Cells. Natl. Cancer Inst. Monogr., 22, 505–520.

3 Friend, C., Scher, W., Holland, J.G. and Sato, T. (1971) Hemoglobin synthesis in murine virus-induced leukemic cells in vitro: Stimulation of erythroid differentiation by dimethyl sulfoxide. Proc. Natl. Acad. Sci. USA, 68, 378–382.

4 Tanaka, M., Levy, J., Terada, M., Breslow, R., Rifkind, R.A. and Marks, P.A. (1975) Induction of erythroid differentiation in murine virus infected erythroleukemia cells by highly polar compounds. Proc. Natl. Acad. Sci. USA, 72, 1003–1006.

5 Reuben, R.C., Wife, R.L., Breslow, R., Rifkind, R.A., Marks, P.A. (1976) A new group of potent inducers of differentiation in murine erythroleukemia cells. Proc. Natl. Acad. Sci. USA, 73, 862–866.

6 Reuben, R.C., Wife, R.L., Breslow, R., Rifkind, R.A. and Marks, P.A. (1976) Identification of a new group of potent inducers of differentiation in murine erythroleukemia cells. Proc. Am. Assoc. Cancer Res., 17, 76.

7 Marks, P.A., Reuben, R., Epner, E., Breslow, R., Cobb, W., Bogden, A.E. and Rifkind, R.A. (1978) Induction of murine erythroleukemia cells to differentiate: A model for the detection of new anti-tumor drugs. Antibiot. Chemother. (Basel), 23, 33.

8 Reuben, R.C., Khanna, P.L., Gazitt, Y., Breslow, R., Rifkind, R.A. and Marks, P.A. (1978) Inducers of erythroleukemic differentiation; relationship of structure to activity among planar–polar compounds. J. Biol. Chem., 253, 4214–4218.

9 Marks, P.A., Breslow, R., Rifkind, R.A., Ngo, L. and Singh, R. (1989) Polar/apolar chemical inducers of differentiation of transformed cells: Strategies to improve therapeutic potential. Proc. Natl. Acad. Sci. USA, 86, 6358–6362.

10 Marks, P.A., Breslow, R. and Rifkind, R.A. (1989) Induced cytodifferentiation of transformed cells: An approach to cancer treatment. J. Cell. Pharmacol., 262, 7–11.

11 Marks, P.A., Rifkind, R.A. and Breslow, R. (1990) Induced differentiation of transformed cells: Mechanism of action and application in cancer therapy, in Molecular Basis of Haematopoiesis, (eds L. Sachs, N.G. Abraham, C. Weidemann, A.S. Levineand and G. Konwalinka), Intercept, Ltd., Andover, pp. 579–586.

12 Breslow, R., Jursic, B., Yan, Z.F., Friedman, E., Leng, L., Ngo, L., Rifkind, R.A. and Marks, P.A. (1991) Potent cytodifferentiating agents related to hexamethylenebisacetamide. Proc. Natl. Acad. Sci. USA, 88, 5542–5546.

13 Marks, P.A., Rifkind, R.A., Richon, V., Powell, T., Busquets, X., Leng, L., Kiyokawa, H., Michaeli, J., Jursic, B. and Breslow, R. (1992) Hexamethylene bisacetamide and related agents as inducers of differentiation of transformed cells: Mechanism of action and potential for cancer therapy. in Concise Reviews in Experimental and Clinical Hematology, (ed. M.J. Murphy), AlphaMed Press, Dayton, pp. 91–99.

14 Richon, V.M., Webb, Y., Merger, R., Sheppard, T., Jursic, B., Ngo, L., Civoli, F., Breslow, R., Rifkind, R.A. and Marks, P.A. (1996) Second generation hybrid polar compounds are potent inducers of transformed cell differentiation. Proc. Natl. Acad. Sci. USA, 93, 5705–5708.

15 Richon, V.M., Emiliani, S., Verdin, E., Webb, Y., Breslow, R., Rifkind, R.A. and Marks, P.A. (1998) A class of hybrid polar inducers of transformed cell differentiation inhibits histone deacetylases. Proc. Natl. Acad. Sci. USA, 95, 3003–3007.

16 Webb, Y., Ngo, L., Richon, V.M., Breslow, R., Rifkind, R. and Marks, P.A. (1998) Identification of a potential target for hybrid polar cytodifferentiation agents. Proc. Am. Assoc. Cancer Res., 39, 108.

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