"... Non vogliate negar l'esperienza di retro al sol, del mondo sanza gente. Considerate la vostra semenza fatti non foste a viver come bruti ma per seguir virtute e conoscenza."
Dante Alighieri, (1265-1321) (Divina Commedia, Inferno
43 In the last thirty years, as described in the introduction many effort have been done to synthesize somatostatin analogues useful in the treatment and diagnosis of tumour. Nevertheless only few analogues of octreotide were accepted for clinical use in a small number of tumour types and for this reason somatostatin analogues not fully fulfil the old promises. The progress in SRIF biology and medical chemistry, induced by the discovery and intensive studies on sst, rekindled the interest on somatostatin analogues. Some authors move on broad spectrum analogues other to selective one to use in specific sst expressing tumour.
In this PhD thesis we tried to achieve both target trough the synthesis of conformationally restricted peptides. In mostly of the cases SRIF analogs maintained the disulphide bridge prone to be cleaved by endogenous reducing enzymes, like glutathione reductase and thioredoxin reductase, or by nucleophilic and basic agents, resulting in a diminished bio-stability of the molecule, moreover same types of labelling procedures were unable with the disulphide bridge. The substitution of the S-S bridge with a unsaturated carbon –carbon bond can be a way to obtain more stable analogues. Using the approach of Ring Closing Metathesis (RCM) on two Hag, replacing Cys in octreotide sequence, we obtained the first so called dicarba-analogue of octreotide.90
In order to obtain more stable and flexible analogues, we synthesized also the saturated dicarba-analogues of the compounds obtained from RCM procedure.
We also focus on the design of new molecules. The classical approach of synthesize lot of molecule was substitute by a rational approach supported by the in silico and the NMR studies. By the modelling approach we predicted virtual bioactive structure whereas NMR analysis supported us in understanding SAR. Finally accordingly to recent studies we modified the sequence of the dicarba–analogues and we enlarged the cycle size. Recent studies concerning the structure-activity relationships of SRIF’s ligands, which paid attention to the role of the ring size,discoveredthateight-mer cycles containing the disulfide bridge may have antagonist activity, that is more suitable for their use as radiolabeled drugs. This find prompted us to attempt to obtain the corresponding dicarba-analogues. 91
90
Carotenuto, A.; D’Addona, D.; Rivalta, E.; Chelli, M.; Papini, A. M.; Rovero, P.; Ginanneschi, M. Synthesis of a Dicarba-Analog of Octreotide Keeping the II’ β-Turn of Pharmacophore in Water Solution. Letters in Organic Chemistry, 2005, 2, 274-279.
91
Debora D’Addona, Carotenuto A., Novellino E., Piccand V., Reubi J.C., Alessandra Di Cianni, Francesca Gori, Anna Maria Papini and Mauro Ginanneschi. ‘Novel sst5-selective Somatostatin Dicarba-analogues. Synthesis and Conformational-affinity Relationship’ Journal Medicinal Chemistry, 2008, 51, 512-520.
44 In this PhD thesis we reported the procedure used for the realization of a data-base for the in silico predictions; the synthesis of twenty-six new dicarba-analogues of octreotide, optimizing this step in oil bath and then on a microwave instrument; 3D structures of nine peptides determined by 1H NMR spectroscopy and a new pharmacophore model for sst5 was proposed. 92
The stability of the first dicarba-analogue in human sera was demonstrated. Now a day just thirteen analogues were subjected to binding essay towards all sst receptors and the most actives were conjugated with two different chelating agent and labelled with radio-nuclides. Bio-distribution essay on mice, using the radio-labelled peptides, internalisation and cellular retention, concerns on cell lines expressing ssts, were performed. Here below four tables describe the final target compounds synthesized in this PhD thesis (Table 3A. 3B. 3C. 3D). In order to facilitate the reader we report here the explanation of the abbreviation contained in the table below: dhDsa-C, dehydrodiaminosuberic acid C-terminus; dhDsa-N, dehydrodiaminosuberic acid N- terminus; Dsa-N, diaminosuberic acid N-terminus; Dsa-C, diaminosuberic acid C- terminus
3A. D-Phe2-c[Xaa3-Yaa7-D-Trp8-Lys9-Zaa10-Kaa14]-Thr(ol)15-OH
Peptides Xaa Yaa Zaa Kaa
27 dh-Dsa-N L-Phe L-Phe dh-Dsa-C 28 dh-Dsa-N L-Phe L-Thr dh-Dsa-C
45 Dsa-N L-Phe L-Thr Dsa-C
29 dh-Dsa-N L-Phe L-Tyr(Bzl) dh-Dsa-C
48 Dsa-N L-Phe L-Tyr(Bzl) Dsa-C
30 dh-Dsa-N L-1-Nal L-Thr dh-Dsa-C
46 Dsa-N L-1-Nal L-Thr Dsa-C
31 dh-Dsa-N L-1-Nal L-Tyr(Bzl) dh-Dsa-C 32 dh-Dsa-N L-Phe L-Tyr dh-Dsa-C 33 dh-Dsa-N L-Tyr L-1-Nal dh-Dsa-C 34 dh-Dsa-N L-Phe L-Tyr(Me) dh-Dsa-C
92
Saveanu, A.; Gunz, G.; Dufour, H.; Caron, P.; Fina, F.; Ouafik, L.; Culler, M. D.; Moreau, J. P.; Enjalbert A.; Jaquet P. BIM-23244, a Somatostatin Receptor Subtype 2 and 5 Selective Along with Enhanced Efficiency in Suppressing Growth Hormone (GH) from Octreotide-Resistant Human GH-Secreting Adenomas. The Journal of Clinical Endocrinology & Metabolis. 2001, 86, 140-145.
45 3B. NH2-L-Gly-[Xaa3-Kaa6-Yaa7-Jaa8-Lys9-Thr10- Zaa11- dhDsa-C14]-COOH
Peptides Xaa Kaa Yaa Jaa Zaa
36 L-dh-Dsa-N D-Phe L-Phe D-2-Nal D-Phe
3C. NH2-[Xaa3-Kaa6-Yaa7-Jaa8-Lys9-Phe10- Zaa11- dhDsa-C14]-CONH2
Peptides Xaa Kaa Yaa Jaa Zaa
37 dh-Dsa-N D-Phe L-Phe D-Trp D-Phe 47 Dsa-N D-Phe L-Phe D-Trp D-Phe
3D. NH2-[Xaa3-Kaa6-Yaa7-Jaa8-Lys9-Thr10-Zaa11-dhDsa-C14]-CONH2
Peptides Xaa Kaa Yaa Jaa Zaa
38 L-dh-Dsa-N D-Phe L-Phe D-Trp D-Phe 39 L-dh-Dsa-N L-Phe L-Phe D-Trp L-Phe 40 D-dh-Dsa-N L-Phe L-Phe D-Trp L-Phe 41 L-dh-Dsa-N L-Phe L-Phe D-2-Nal L-Phe 42 D-dh-Dsa-N L-Phe L-Phe D-2-Nal L-Phe 43 D-dh-Dsa-N L-Phe L-Tyr D-2-Nal L-Phe
46 3D. (CA)-Peptide
Compounds Chelating Agents Unsaturated-Peptide
49 DOTA 29 50 DOTA 30 51 DOTA 31 52 DOTA 32 53 PN2S 28 54 PN2S 30
Table 6. Target compounds synthesized in this work.
Formulas of all compounds were given in supplementary information (figures S27- 54).
47