Yes / We report the effect of low-energy thermal neutron irradiation on the antiproliferative activities of a highly hydrophobic organometallic arene ruthenium dithiolatoecarborane complex [Ru(p-cymene) (1,2- dicarba-closo-dodecarborane-1,2-dithiolato)] (1), and of its formulation in Pluronic® triblock copolymer P123 coreeshell micelles (RuMs). Complex 1 was highly active, with and without neutron irradiation, towards human ovarian cancer cells (A2780; IC50 0.14 mM and 0.17 mM, respectively) and cisplatinresistant human ovarian cancer cells (A2780cisR; IC50 0.05 and 0.13 mM, respectively). Complex 1 was particularly sensitive to neutron irradiation in A2780cisR cells (2.6 more potent after irradiation compared to non-irradiation). Although less potent, the encapsulated complex 1 as RuMs nanoparticles resulted in higher cellular accumulation (2.5 ), and was sensitive to neutron irradiation in A2780 cells (1.4 more potent upon irradiation compared to non-irradiation). / We thank the Leverhulme Trust (Early Career Fellowship No. ECF-2013-414 to NPEB), the University of Warwick (Grant No. RD14102 to NPEB), the University of Birmingham/EPSRC Follow-on- Fund (Grant No UOBFOF026 to BP), the ERC (Grant No. 247450 to PJS), EPSRC (EP/F034210/1 to PJS).

This thesis addresses the synthesis of organic compounds, some of them are derivatives of compounds with DNA binding properties, for potential use in targeted nuclide therapy. The compounds synthesized therefore also need to contain potent nuclides. Here the nuclides considered are the radionuclide 125I, and the stable isotope 10B, which becomes radioactive upon neutron activation. 125I is an Auger-electron emitter, which emits particles that can travel only about 1-2 µm through human tissue and hence has to be delivered to the cancer cell nucleus to cause DNA damage. Neutron activated 10B emits highly cell killing α-particles and 7Li3+ ions, the application of which in Boron Nuclide Capture Therapy (BNCT) has proven very promising. The thesis can be divided into three parts: i) A nido-carborate, 7-(3´-ammoniopropyl)-7,8-dicarba-nido-undecaborate(-1), has been synthesized and radioiodinated for use as a pendant group for attachment of 125I to tumor-seeking macromolecules. Radiolabeling was achieved in greater than 95% yield. ii) Both enantiomers of m-carboranylalanine, a carborane analogue of phenylalanine, have been prepared in high enantiomeric excess, and are of potential interest in BNCT. The synthesis involved amination of the N-acyl derivative formed from [3-(1,7-dicarba-closo-dodecarborane(12)-1-yl)-2-propanoic acid and Oppolzer’s camphor sultam. iii) Derivatives of the DNA intercalating compounds 9-aminoacridine, daunorubicin and doxorubicin have been synthesized and labeled with 125I. The 9-aminoacridines were synthesised with a variety of functional groups such as carboxyl, amino and hydroxyl. The anthracylines daunorubicin and doxorubicin are efficient chemotherapeutic agents; the synthesis routes of ester, amide and amine derivatives of these compounds are presented. The Chloramine T method was used for the radioiodinations, and the radioiodination precursors of both the acridine and the anthracycline derivatives, were made to contain either a trimethylstannyl group or a phenolic substituent. In the former case the trimethylstannyl group was replaced by 125I, and in the latter case, the compounds were radiolabeled directly at the o- position to the phenolic hydroxyl group. Both methods gave high radiolabeling yields.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20400号 / 工博第4337号 / 新制||工||1672(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 中條 善樹, 教授 秋吉 一成, 教授 古賀 毅 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

<p>A series of new methodologies to link a neurotransmitter receptor targeting vector (WAY) to carboranes and the preparation of the corresponding metallocarboranes (M = Re, ^99mTc) as a new class of organometallic CNS imaging probes is described. WAY-carboranes (<strong>5</strong>, <strong>6</strong>, <strong>16</strong>) and the corresponding metallocarboranes (M = Re (<strong>12</strong>, <strong>13</strong>, <strong>22a</strong>, <strong>22b</strong>), ^99mTc (<strong>14a</strong>, <strong>15</strong>, <strong>23</strong>)) were synthesized in yields ranging from 10-95%. The first observed 3,1,2 versus 2,1,8 rhenacarborane isomerization process was discovered for <strong>12</strong> where isomerization and complexation occurred simultaneously. Re-carboranes <strong>22a</strong> and <strong>22b</strong> had similar carbon-carbon cage configuration where electronic effects was the driving force behind isomerization.</p> <p>The lipophilicities of ^99mTc-carboranes (<strong>14a</strong>, <strong>15</strong>, <strong>23)</strong> were within the ideal range to cross the BBB (log P = 2.4-2.6). <em>In vitro</em> binding data showed that <strong>22b</strong> has high affinity for alpha-adrenergic receptors (K_i = 17-39 nM) resulting in the first organometallic complex to effectively bind to this class of receptors. SPECT images of <strong>14a</strong> in rats showed no brain uptake, while quantitative biodistribution studies indicated modest, non-negligible brain uptake in the hypothalamus region.</p> <p>The neutral [M(CO)₂(NO)(C₂B₉H₁₀R)] analogues (<strong>30</strong>, <strong>34</strong>, <strong>37</strong>) were prepared to address the limited brain uptake of the [M(CO)₃(C₂B₉H₁₀R)]^- complexes. Reactivity differences between Re and ^99mTc were noted during nitrosation conditions where the initial products from the reaction led to nitration of the phenyl group in addition to nitrosation of the metal core. The fluorescence properties of <strong>29</strong> were measured.</p> <p>Low yields and multistep syntheses associated with the preparation of substituted carborane led to the development of a carborane-alkyne platform. Alkyne-carboranes (<strong>53</strong>-<strong>55</strong>) were developed and conjugated to WAY-azide (<strong>46</strong>) using “click” chemistry. The metallocarboranes (M = Re (<strong>69</strong>-<strong>71</strong>), ^99mTc (<strong>72</strong>-<strong>74</strong>)) were generated in yields ranging from 45-71%.</p> / Doctor of Philosophy (PhD)

<p>This thesis describes the use of copper and palladium to mediate transformations of carboranes, especially <i>p</i>-carborane.</p><p>1-(1-<i>p</i>-carboranyl)-<i>N</i>-methyl-<i>N</i>-(2-butyl)-3-isoquinolinecarboxamide, a carborane containing analogue of the peripheral benzodiazepine receptor (PBR) ligand PK11195, has been synthesised. A key step in the reaction is the copper (I) mediated coupling of p-carborane with ethyl 1-bromo-isoquinoline-3-carboxylate. </p><p><i>p</i>-Carborane has been arylated on the 2-<i>B</i>-atom in high yields, using the Suzuki–Miyaura reaction. Thus the reaction between 2-I-<i>p</i>-carborane and various arylboronic acids [1-naphthyl-, phenyl-, 4-MeO-C₆H₄-, 3-CH₃CONH-C₆H₄-, 4-NC-C₆H₄-, 3-NO₂-C₆H₄-], gave the corresponding 2-aryl-<i>p</i>-carboranes in DME solution when reacted in the presence of cesium fluoride and the catalytic Pd₂(dba)₃–dppb system. Under the same conditions, the boron-boron bond forming reaction of two <i>p</i>-carboranylboronic esters (2-[(pinacolato)boron]-<i>p</i>-carborane and 2-[(neopentyl glycolato)boron]-p-carborane) was also shown feasible.</p><p><i>p</i>-Carborane has been vinylated on the 2-<i>B</i>-atom in high yields by use of the Heck reaction. The coupling between 2-I-<i>p</i>-carborane and various styrenes [4-H-, 4-C₆H₄-, 4-Cl , 4-Br-, 4-NO₂-, 4-CH3O- and 4 CH₃ ] resulted in the formation of the corresponding<i>trans</i>-β-(2-<i>B</i>-<i>p</i>-carboranyl) styrene in DMF solution when reacted in the presence of silver phosphate and the palladacycle Herrmann´s catalyst. The reaction was shown to proceed at higher rate with electron rich than with electron deficient olefins.</p><p>The feasibility of palladium-catalysed isotopic exchange of an iodinated closo-carborane with a radioisotope of iodine has been studied. 2-I-<i>p</i>-carborane was selected as a model compound. It was shown, that such isotopic exchange is possible and provides a high yield (83 ± 4.2 %) during 40 min long reaction. The reaction conditions were optimised, and it was demonstrated that presence of the tetra n-butylammonium hydrogensulphate is important in order to stabilise catalyst and provide reproducibility of labelling. In this work we have modified the methodology and extended the application to a wider range of iodinated carboranes. By the use of Herrmann’s catalyst in toluene at 100 °C this [¹²⁵I]-iodide labelling could be improved and extended. 2-I-<i>p</i>- 9-I-<i>m</i>-, 9-I-<i>o</i>-, 3-I-<i>o</i>-carborane, 1-phenyl-3-I-<i>o</i>-carborane and 1,2-diphenyl-3-I-<i>o</i>-carborane could be [¹²⁵I]-iodide labelled in high to excellent yields within 5 minutes.This reported palladium catalyzed radio-iodination of the uncharged closo-carboranes might find use in pharmacokinetic studies of carborane derivatives.</p>

This thesis describes the use of copper and palladium to mediate transformations of carboranes, especially p-carborane. 1-(1-p-carboranyl)-N-methyl-N-(2-butyl)-3-isoquinolinecarboxamide, a carborane containing analogue of the peripheral benzodiazepine receptor (PBR) ligand PK11195, has been synthesised. A key step in the reaction is the copper (I) mediated coupling of p-carborane with ethyl 1-bromo-isoquinoline-3-carboxylate. p-Carborane has been arylated on the 2-B-atom in high yields, using the Suzuki–Miyaura reaction. Thus the reaction between 2-I-p-carborane and various arylboronic acids [1-naphthyl-, phenyl-, 4-MeO-C6H4-, 3-CH3CONH-C6H4-, 4-NC-C6H4-, 3-NO2-C6H4-], gave the corresponding 2-aryl-p-carboranes in DME solution when reacted in the presence of cesium fluoride and the catalytic Pd2(dba)3–dppb system. Under the same conditions, the boron-boron bond forming reaction of two p-carboranylboronic esters (2-[(pinacolato)boron]-p-carborane and 2-[(neopentyl glycolato)boron]-p-carborane) was also shown feasible. p-Carborane has been vinylated on the 2-B-atom in high yields by use of the Heck reaction. The coupling between 2-I-p-carborane and various styrenes [4-H-, 4-C6H4-, 4-Cl , 4-Br-, 4-NO2-, 4-CH3O- and 4 CH3 ] resulted in the formation of the correspondingtrans-β-(2-B-p-carboranyl) styrene in DMF solution when reacted in the presence of silver phosphate and the palladacycle Herrmann´s catalyst. The reaction was shown to proceed at higher rate with electron rich than with electron deficient olefins. The feasibility of palladium-catalysed isotopic exchange of an iodinated closo-carborane with a radioisotope of iodine has been studied. 2-I-p-carborane was selected as a model compound. It was shown, that such isotopic exchange is possible and provides a high yield (83 ± 4.2 %) during 40 min long reaction. The reaction conditions were optimised, and it was demonstrated that presence of the tetra n-butylammonium hydrogensulphate is important in order to stabilise catalyst and provide reproducibility of labelling. In this work we have modified the methodology and extended the application to a wider range of iodinated carboranes. By the use of Herrmann’s catalyst in toluene at 100 °C this [125I]-iodide labelling could be improved and extended. 2-I-p- 9-I-m-, 9-I-o-, 3-I-o-carborane, 1-phenyl-3-I-o-carborane and 1,2-diphenyl-3-I-o-carborane could be [125I]-iodide labelled in high to excellent yields within 5 minutes.This reported palladium catalyzed radio-iodination of the uncharged closo-carboranes might find use in pharmacokinetic studies of carborane derivatives.

<p>This thesis describes the design and synthesis of molecules with potential roles in biomedicine, with an emphasis on molecular recognition in complex biological environments. The first chapter describes the synthesis and evaluation of compounds for use in nuclide therapy. Carboranes are frequently used in the development of drugs for Boron Neutron Capture Therapy. New routes for monohydroxylation at the B and C atoms of <i>p</i>-carborane have been developed. The Suzuki-Miyaura reaction has been applied to the cross-coupling of <i>bis</i>(neopentyl glycolato)diboron or <i>bis</i>(pinacolato)diboron and 2-I-<i>p</i>-carborane. The synthesized derivatives are important intermediates in the synthesis of a number of potentially biologically active carborane-containing molecules.</p><p>The DNA intercalator doxorubicin has been functionalized to enable ¹²⁵I labelling. The aim of combining the DNA intercalator with ¹²⁵I was to achieve high delivery of cytotoxic radiation to the nucleus. The DNA-binding ability and cellular uptake of the synthesized compounds have been evaluated. One of the compounds bound strongly to DNA and had similar cellular uptake as daunorubicin, which makes the compound very interesting for further biological evaluation.</p><p>The second chapter describes the use of polypeptide conjugates to broaden our knowledge of molecular recognition. The polypeptides consist of 42 amino acids each and are designed to fold into helix-loop-helix motifs that dimerize due to their amphiphilic character. The polypeptides are combined with a variety of small organic molecules. The incorporation of small aromatic molecules to influence the structure and dynamics of a polypeptide has been investigated. By attaching a dansyl group to the side chain of a lysine residue, the dynamics of the protein’s hydrophobic core where affected to such a degree that a native-like fold was formed. The polypeptide conjugates have also been used to study the binding and recognition of native proteins. High-affinity binders for chitinases and acetylcholine esterase have been developed and evaluated.</p>

This thesis describes the design and synthesis of molecules with potential roles in biomedicine, with an emphasis on molecular recognition in complex biological environments. The first chapter describes the synthesis and evaluation of compounds for use in nuclide therapy. Carboranes are frequently used in the development of drugs for Boron Neutron Capture Therapy. New routes for monohydroxylation at the B and C atoms of p-carborane have been developed. The Suzuki-Miyaura reaction has been applied to the cross-coupling of bis(neopentyl glycolato)diboron or bis(pinacolato)diboron and 2-I-p-carborane. The synthesized derivatives are important intermediates in the synthesis of a number of potentially biologically active carborane-containing molecules. The DNA intercalator doxorubicin has been functionalized to enable 125I labelling. The aim of combining the DNA intercalator with 125I was to achieve high delivery of cytotoxic radiation to the nucleus. The DNA-binding ability and cellular uptake of the synthesized compounds have been evaluated. One of the compounds bound strongly to DNA and had similar cellular uptake as daunorubicin, which makes the compound very interesting for further biological evaluation. The second chapter describes the use of polypeptide conjugates to broaden our knowledge of molecular recognition. The polypeptides consist of 42 amino acids each and are designed to fold into helix-loop-helix motifs that dimerize due to their amphiphilic character. The polypeptides are combined with a variety of small organic molecules. The incorporation of small aromatic molecules to influence the structure and dynamics of a polypeptide has been investigated. By attaching a dansyl group to the side chain of a lysine residue, the dynamics of the protein’s hydrophobic core where affected to such a degree that a native-like fold was formed. The polypeptide conjugates have also been used to study the binding and recognition of native proteins. High-affinity binders for chitinases and acetylcholine esterase have been developed and evaluated.

This thesis describes the synthesis and radiohalogenation of compounds of potential use for tumor targeting. The first section describes the synthesis and radioiodination of DNA intercalating compounds. The compounds are derivatives of 9-aminoacridine, and the anthracyclins daunorubicin and doxorubicin. The precursor compounds were labeled with 125I (T1/2 = 60 days), which is an Auger emitting nuclide. 125I decaying in the close vicinity of DNA is known to have a much higher cell killing effect than 125I decaying in the cytoplasm and some of the labeled compounds prepared in this thesis are currently being tested for use in targeted radionuclide therapy for cancer. The second section describes the radiobromination of closo-carboranes by subjecting the corresponding iodinated compounds to palladium-catalyzed halogen exchange using [76Br]bromide. The 76Br isotope (T1/2 = 16.2 h) is a positron emitting nuclide that is suitable for PET studies. Via the halogen exchange reaction good to excellent radiochemical yields of radiobrominated closo-carboranes were obtained. The results of the present study may prove to be applicable to pharmacokinetic studies of carboranes and their derivatives. The third and final section describes the indirect radiobromination of the trastuzumab anti-HER2 monoclonal antibody and of the anti-HER2 Affibody by means of an “one-pot” procedure using N-succinimidyl-5-(tributylstannyl)-3-pyridinecarboxylate (SPC) and ((4-hydroxyphenyl)ethyl))maleimide (HPEM), respectively. It was found that SPC and HPEM can be efficiently radiobrominated and thereafter coupled to the antibody and Affibody, respectively. The labeled proteins retained their capacity to bind specifically to HER2 expressing SKOV-3 cells in vitro. Application of this method to 76Br might enable the use of PET in the detection of HER2 expression in breast, ovarian, and urinary bladder carcinomas.

The amount of boron accumulated in tumor tissue plays an important role regarding the success of the boron neutron capture therapy (BNCT). In this article, we report a modular system, combining readily available starting materials, like glycine, 1,3,5-triazine and the well-known 9-mercapto-1,7-dicarba-closo-dodecaborane(12), as well as alpha-d-galactopyranose for increased hydrophilicity, with a novel boron-rich tris-meta-carboranyl thiol.