INTRODUCTION TO EXPERIMENTAL SECTION

INTRODUCTION

TO EXPERIMENTAL

70

N,N-dialkyl-2-phenylindol-3-ylglyoxylamide derivatives, designed in 2004 by Primofiore et al.(35), have shown high affinity for TSPO from nanomolar to subnanomolar range and high selectivity. The same research group has successively refined the TSPO pharmacophore/topological model through the synthesis and the biological evaluation of a library of novel 2-phenylindol-3-ylglyoxylamide derivatives of general formula (I).(65)

N H R3 O N O R1 R2 H1 L₁ L4 L₃ R4

Figure 24. N,N-dialkyl-2-phenylindol-3-ylglyoxylamides (I)

As earlier mentioned, rationalizing the SARs in the light of the previously reported pharmacophore/receptor model made up of three lipophilic pockets (L1, L3, L4) and an H-bond donor group (H1), these authors have

individuated three fundamental interactions:(4,68)

I) The second carbonyl group of the oxalyl bridge engages an H-bond with the donor site H₁;

II) The two lipophilic substituents R1 and R2 (linear or ramified alkyl,

arylalkyl group) on the amide nitrogen interact hydrophobically with the L₃ or L₄ lipophilic pockets;

III) The 2-phenyl group establishes π-stacking interactions within the L₁ pocket.

71

The basal expression of TSPO, as referred above in “TSPO Distribution in Healthy and Pathological Tissue”, is up-regulated in a number of neuropathologies, including gliomas and neurodegenerative diseases, in various forms of brain injury and inflammation, as well as in a variety of tumors.

Consequently, TSPO has been suggested as a promising diagnostic marker to image and measure the TSPO expression and distribution levels, and thus for evaluation of disease progression by means of specific fluorescent or radiolabeled ligands. In particular, PET imaging using TSPO ligands to label activated microglia offers quantitative measures of inflammation, and then can help to understand the regional brain distribution, stage and severity of neuroinflammation.

In this thesis work, we design, synthesized and evaluated an irreversible-NBD fluorescent probe (14). This compound is characterized by the presence of a chemoreactive isothiocyanate group, able to bind the receptor protein irreversibly and covalently and by the presence of the NBD-fluorescent moiety. Furthermore, we synthesized compounds bearing an hydrophilic chelating agent NOTA, ((1,4,7-triazonane-1,4,7-triyl)triacetic acid) on the glyoxylyl bridge (21-22).

These compounds were designed following the concept of “bifunctional approach”, where we used a biomolecule (ligand) with high recettorial affinity to which is bound, means a linker spacer and a bifunctional chelating for ligand coniugation and radiometal chelation. The chelating group should be linked at a biomolecule irrilevant point to the binding, so as to preserve its biological properties (Figure 25).

72

Figure 25. Diagram of a generic radiolabeled probe: ligand acts as a carrier for specific

biological target, thanks to its selective affinity in receptor binding (biological target). A linker spacer (linker) was inserted between ligand and the chelator bifunctional.

The polydentate chelator (NOTA) was used for increase the thermodynamic stability and inertia kinetic of the metal complex (with Ga-68), for prevent the radioactive metal available toxicity. The Ga-68 was added into NOTA cycle, to form an high yield complex at low concentrations of bifunctional chelator-ligand coniugate. These compounds were synthesized to prepare and evaluate Ga-68 complexes as potential PET agents for measurement of TSPO. N H N COCONH N O N NO2 (CH2)6 NH C S 14 N H COCONH N N N OH O O OH O 21 (CH2)4 N H Radionuclide Bifunctional chelator Linker spacer

73 N H COCONH N N N OH O O OH O 22 (CH2)6 N H Figure 26.

The synthesis of the irreversible fluorescent probe 14 was achieved by the convergent procedure outlined in Scheme 1 and in Scheme 2. The first key intermediate 5-(N-BOC-amino)-2-phenylindol-3-ylgyloxyl chloride 5 was obtained in three steps starting from the 5-nitro-2-phenylindole 2(49) which was firstly catalytically hydrogenated to yield the corresponding amine 3, subsequently protected with the Boc group to give 4, and then reacted with oxalyl chloride. Scheme 1(69)

74 Scheme 1 N H NaNO3 H2SO4 N H O2N N H H2N H2/Pd-C abs. EtOH N H H N [(Boc)2O] ClCOCOCl anh. THF N H H N COCOCl 1 2 3 4 5 Boc Boc The N-Boc-N′-(7-nitro-2,1,3-benzoxadiazol-4-yl)hexamethylendiamine hydrochloride 8, prepared as previously described from Da Settimo and co-workers(70), was condensed with compound 5 in anhydrous dimethylformamide and in the presence of triethylamine at room temperature, yielding the fluorescent compound 12. Simple deprotection of the Boc group with trifluoroacetic acid in anhydrous CH₂Cl₂ gave the 5-amino derivative 13, which was then reacted with thiophosgene, yielding the desired irreversible fluorescent compound 14.(69)

75 Scheme 2 + NH2 (CH2)6 NHBoc 1) DMF; Et3N N O N Cl O₂N 6 7 2) HCl 3M /AcOEt N O N NH O₂N (CH₂)₆ NH₂ 8 N H H N COCOCl 5 + anh. DMF Et₃N N H H N COCONH N O N NO2 (CH₂)₆ NH CF₃COOH anh. CH₂Cl₂ N H H2N COCONH N O N NO₂ (CH2)6 NH Tiophosgene CH2Cl2 N H N COCONH N O N NO₂ (CH2)6 NH C S 14 12 13 Boc Boc

The synthesis of the radioligand probes (21-22) was reported in the Scheme

3. The indole was acylated with oxalyl chloride in anhydrous ethyl ether at

0 °C to give the corresponding indolylglyoxylyl chloride 16, which was allowed to react in dry toluene solution and under nitrogen atmosphere with N-Boc-1,6-diaminohexane or N-Boc-1,4-diaminobutane in presence of triethylamine, in equimolar quantity so as neutralize the hydrochloric acid formed during the reaction of condensation, yielding compounds 17 and

76 Scheme 3 N H ClCOCOCl N H COCOCl anh. Toluene; Et₃N H₂N (CH₂)_n Boc n = 6; 4 1 16 anh. Et2O 0oC H N N H COCONH Boc HCl AcOEt N N N HO OH O O OH O (NOTA) N N N OH O O OH O 21-22 17-18 19-20 (CH₂)n H N N H COCONH (CH2)n NH2 N H COCONH (CH₂)n N H CDI; anh. DMF

The protected amino group has been removed with an hydrolysis, adding HCl 3M to a stirred solution of derivates 17-18 in AcOEt. Then compounds

19 and 20 were reacted with NOTA (1,4,7-triazonane-1,4,7-triyl)triacetic

acid), in the presence of CDI (carbonyl imidazole) in anhydrous DMF. The reaction yields, chemical-physical characteristics, spectroscopic properties and more details of chemical procedure are shown in next “Experimental section” .