The carbazole compounds PK9320 (1-(9-ethyl-7-(furan-2-yl)-9H-carbazol-3-yl)-N-methylmethanamine) and PK9323 (1-(9-ethyl-7-(thiazol-4-yl)-9H-carbazol-3-yl)-N-methylmethanamine), second-generation analogues of PK083 (1-(9-ethyl-9H-carbazol-3-yl)-N-methylmethanamine), restore p53 signaling in Y220C p53-mutated cancer cells by binding to a mutation-induced surface crevice and acting as molecular chaperones. In the present paper, these three molecules have been tested for mutant p53-independent genotoxic and epigenomic effects on wild-type p53 MCF-7 breast adenocarcinoma cells, employing a combination of Western blot for phospho-γH2AX histone, Comet assay and methylation-sensitive arbitrarily primed PCR to analyze their intrinsic DNA damageinducing and DNA methylation-changing abilities. We demonstrate that small modifications in the substitution patterns of carbazoles can have profound effects on their intrinsic genotoxic and epigenetic properties, with PK9320 and PK9323 being eligible candidates as “anticancer compounds” and “anticancer epi-compounds” and PK083 a “damage-corrective” compound on human breast adenocarcinoma cells. Such different properties may be exploited for their use as anticancer agents and chemical probes.

The widely used pentafluorosulfanyl group (SF5) was deployed as a bioisosteric replacement for a chloro-group in the benzodiazepine diazepam (Valium™). Reaction of 2-amino-5-pentafluorosulfanyl-benzophenone with chloroacetyl chloride followed by hexamethylenetetramine, in the presence of ammonia, led to 7-sulfurpentafluoro-5-phenyl-1H-benzo[1,4]diazepin-2(3H)-one (2c). The latter was able to undergo a Pd-catalysed ortho-arylation, demonstrating that these highly fluorinated benzodiazepines can be further modified to form more complicated scaffolds. The replacement of Cl by the SF5 group, led to a loss of potency for potentiating GABAA receptor activation, most likely because of a lost ligand interaction with His102 in the GABAA receptor α subunit. Dedicated to Professor Jonathan Williams, an inspirational and humble pioneer, a colleague and mentor in chemistry.

Combined photochemical arylation, “nuisance effect” (S N Ar) reaction sequences have been employed in the design of small arrays for immediate deployment in medium throughput X‐ray protein‐ligand structure determination. Reactions have been deliberately let “out of control,” in terms of selectivity; for example the ortho‐arylation of 2‐phenylpyridine gave five products resulting from mono‐, bis‐ arylations combined with S N Ar processes. As a result, a number of crystallographic hits against NUDT7, a key peroxisomal CoA ester hydrolase, have been identified.

We have previously shown that the thermolabile, cavity-creating p53 cancer mutant Y220C can be reactivated by small-molecule stabilizers. In our ongoing efforts to unearth druggable variants of the p53 mutome, we have now analyzed the effects of other cancer-associated mutations at codon 220 on the structure, stability and dynamics of the p53 DNA-binding domain (DBD). We found that the oncogenic Y220H, Y220N and Y220S mutations are also highly destabilizing, suggesting that they are largely unfolded under physiological conditions.
A high-resolution crystal structure of the Y220S mutant DBD revealed a mutation-induced surface crevice similar to that of Y220C, whereas the corresponding pocket’s accessibility to small molecules was blocked in the structure of the Y220H mutant. Accordingly, a series of carbazole-based small molecules, designed for stabilizing the Y220C mutant, also bound to and stabilized the folded state of the Y220S mutant, albeit with varying affinities due to structural differences in the binding pocket of the two mutants. Some of the compounds also bound to and stabilized the Y220N mutant, but not the Y220H mutant. Our data validate the Y220S and Y220N mutant as druggable targets and provide a framework for the design of Y220S or Y220N-specific compounds as well as compounds with dual Y220C/Y220S specificity for use in personalized cancer therapy

Aim: The p53 cancer mutation Y220C creates a conformationally unstable protein with a unique elongated
surface crevice that can be targeted by molecular chaperones. We report the structure-guided optimization
of the carbazole-based stabilizer PK083. Materials & methods: Biophysical, cellular and x-ray
crystallographic techniques have been employed to elucidate the mode of action of the carbazole scaffolds.
Results: Targeting an unoccupied subsite of the surface crevice with heterocycle-substituted PK083
analogs resulted in a 70-fold affinity increase to single-digit micromolar levels, increased thermal stability
and decreased rate of aggregation of the mutant protein. PK9318, one of the most potent binders, restored
p53 signaling in the liver cancer cell line HUH-7 with homozygous Y220C mutation. Conclusion: The
p53-Y220C mutant is an excellent paradigm for the development of mutant p53 rescue drugs via protein
stabilization. Similar rescue strategies may be applicable to other cavity-creating p53 cancer mutations.

Palladacycles have many uses in catalytic chemistry. New examples including Pd(II)/Pd(IV) cycles, meta-CH activations and transannular processes are becoming more popular with increasingly interesting synthetic outputs.

This thesis focuses on developing efficient, atom-economic synthesis routes for functionalised 5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one libraries. 1,4-Benzodiazepines (BZDs) are often referred to as “privileged scaffolds” due to their important biological activities; therefore, finding new efficient methods for synthesising such analogues is highly desirable in pharmaceutical and medicinal research.

Chapter 1 introduces the project detailing the biological importance and applications of BZDs. The classical synthetic routes towards BZDs and some of the limitations for efficient and rapid BZD-based library synthesis, followed by the aims of the project.

Chapter 2 presents a late-stage C-H activation method for synthesising ortho-arylated 5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one, including a library of over twenty novel analogues. The microwave-mediated palladium catalysed arylation method is also applicable to nordazepam (7-chloro-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one), the active metabolite of diazepam. Further diversification of the compounds is achieved by N-alkylation and/or H/D exchange, which affords elaborated pharmaceuticals.

Chapter 3 describes an alternative catalytic visible light-mediated photoredox method for ortho-arylated 5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-ones. The protocol uses aryldiazonium salts in refluxing methanol and showcases an interesting phenomenon known as the “nuisance effect” with 2- or 4-fluorobenzenediazonium salts. It results in both fluoroaryl and methoxyaryl- products, the latter result from a nucleophilic aromatic substitution (SNAr) on the fluorobenzenediazonium salt (nuisance effect). The results from biological tests of the benzodiazepine libraries against GABAA receptors indicated that the C-7 substituent is vital for activities in GABA and only the 7-chloro-benzodiazepines show any reasonable activities, although ortho arylation is detrimental to bioactivity. A computational DFT analysis of the reaction mechanism from our collaborators is also discussed in the Chapter.

Chapter 4 contains a brief overview of C-H functionalisation protols. Moreover, in this Chapter, the photoredox C-H activation method combined with the nuisance effect are extended to other privileged scaffolds. This Chapter describes the synthesis of small libraries of 2-phenylpyridines and 1-phenyl-2-pyrrolidinones. The nuisance effect proves to be effective in creating small arrays of compounds from a single reaction and in X-ray screening arrays for biological testing. A number of 1-phenyl-2-pyrrolidinone analogues display promising biological activities towards NUDT7, a peroxisomal coenzyme A diphosphatase of current interest.

Chapter 5 focuses on the synthesis of a series of N1-arylated 5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-ones. The N-arylation occurs in one-step using a single 1,4-benzodiazepine precursor with unsymmetrical diaryliodonium salts in aqueous ammonia as a base.

Chapter 6 reports the scale-up synthesis of 6-(1H-indol-4-yl)-8-methoxy-1-methyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine-4-acetic acid methyl ester, TC-AC-28. This BZD derivative is a highly selective bromo and extra terminal (BET) bromodomain inhibitor and a useful epigenetic tool compound. The near gram-scale, seven-step synthesis of this key chemical probe compound enabled it to be available for researchers through Tocris, one of our industry sponsors, and where I spent 3 months as part of my CASE award.

Chapter 7 concludes the thesis and concentrates on future directions.

A library of N1-arylated 5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-ones has been synthesized starting with unsymmetrical diaryliodonium salts using aqueous ammonia as a base. This can also be applied to a similar 1,3,4-benzotriazepin-2-one derivative.

TC AC 28, 6-(1H-Indol-4-yl)-8-methoxy-1-methyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine-4-acetic acid methyl ester, has been synthesized on a near gram scale in seven steps with notable improvements in the reported poor yielding last 2 steps enabling this key chemical probe compound to be available for researchers.

5-Phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-ones react under palladium- and visible light photoredox catalysis, in refluxing methanol, with aryldiazonium salts to afford the respective 5-(2- arylphenyl) analogues. With 2- or 4-fluorobenzenediazonium derivatives, both fluoroaryl- and methoxyaryl- products were obtained, the latter resulting from a SNAr on the fluorobenzenediazonium salt (“nuisance effect”). A computational DFT analysis of the palladium-catalysed and the palladium/ruthenium-photocalysed mechanism for the functionalization of benzodiazepines indicated that in the presence of the photocatalyst the reaction proceeds via a low-energy SET pathway avoiding the high-energy oxidative addition step in the palladium-only catalysed reaction pathway.