*4.2. Instrumentations*

NMR spectra were recorded by means of a Bruker 400-AMX spectrometers using tetramethylsilane (TMS) as the internal standard. For each sample, 1–2 mg were weighed and diluted up to 0.6 mL with the deuterated solvent described in the specific synthetic paragraph into a 5 mm NMR tube. A 90◦ pulse was calibrated for each sample, and standard NMR parameters were used.

Mass spectrometry was performed through the direct injection of the sample on an Applied Biosystem Mariner System 5220 equipped with a MALDI TOF/TOF 400 Plus AB Sciex, (Framingham, MA, USA) analyzer.

The purification of the precursor (IP-001) was carried out by an Agilent 1290 Infinity II preparative HPLC equipped with a binary pump and UV–Visible detector. A semipreparative Zobrax-Eclipse (Agilent) (C18; 5µm; 250 × 21.2 mm) column was used. Eluents were H2O + 0.1% TFA (A) and ACN (B) at flow = 17mL/min with the following gradient: minutes 0–2 A: 90% B: 10%; minutes 2–23 B: 10% -> 76%; minutes 23–27 B: 76% -> 100%; and minutes 27–28 B: 100% -> 10%. The detection of reaction products was performed, evaluating absorbance at λ = 225 nm. During the purification process, IP-001 showed a retention time of 19 min.

HPLC analyses on indium-111-labelled IP-001 were performed on an S1125 HPLC pump system, and the column used was an Acclaim 120 C18, 3 × 150 mm, 3 µm 120 Å pore size; flow: 0.6 mL/min; gradient: minutes 0–2 A: 70% B: 30%; minutes 2–12 B: 30% -> 95%; and minutes 12–15 B: 95% -> 30%, where A: H2O (0.1% TFA) and B: MeCN. Detection was carried out with an S3245 UV–Vis and an S3700 gamma detector system.

TLC plates used for the assessment of the preparative reactions were silica gel 60 F245 (0.2 mm, Merck) with the eluents mentioned for the specific product in the following paragraphs. Column chromatography was carried out with silica gel 60 (0.063–0.100 mm, Merck), and elution was performed using the eluent described for the specific preparations. The radioactive incorporation and stability of [111In]In-IP-001 were assessed on ITLC-SG plates, developed in a 1 M ammonium acetate/MeOH 1:1 *v*/*v* solution, by using a flatbed-imaging scanner (Cyclone, Perkin Elmer).

#### *4.3. Synthesis of N<sup>1</sup> -Boc-N25-Fmoc-8,11-dioxo-16,19,22-trioxa-7,12-diazapentacosane-1,25-diamine (3)*

CDI (0.2 g, 1.5 mmol) was added to a 32 mL MeCN solution of N19-Fmoc-4-oxo-9,12,15 trioxa-5,19-diazanonadecanoic acid (**2**; 0.5 g, 1.0 mmol), and the mixture was stirred and heated to reflux for 1 h (TLC: CHCl3/MeOH 9:1). After cooling, triethylamine (TEA) (0.2 g, 1.5 mmol) was added to the mixture, followed, after 10 min, by a solution of N-Boc-1,6 hexanediamine (**1**; 0.3 g, 1.2 mmol) in MeCN (20 mL; dropwise addition). The solution was stirred at room temperature for 2 h (TLC: CHCl3/MeOH 9:1). The mixture was than acidified to pH 5 with a 1 M CH3COOH solution in MeCN and concentrated to dryness. The residue was dissolved in 40 mL of ethyl acetate (EtOAc) and washed with a saturated NH4Cl solution (20 mL × 2). The organic phase was then concentrated to dryness, and the product was purified by column chromatography (eluent: CHCl3/MeOH 95:5) to yield **3** (0.3 g, 0.46 mmol, 46% yield, chemical structure in Figure 9).

**Figure 9.** Chemical structure of **3**.

<sup>1</sup>H-NMR (400 MHz, CDCl3-d)δ ppm 7.75 (d, *J* = 7.6, 2H); 7.60 (d, *J* = 7.6, 2H); 7.39 (t, *J* = 7.6, 2H); 7.30 (t, *J* = 7.6, 2H); 6.66 (broad s, 1H); 6.40 (broad s, 1H); 5.67–5.59 (m, 1H); 4.62 (broad s, 1H); 4.45–4.35 (m, 2H); 4.24–4.17 (m, 1H); 3.65–3.49 (m, 12H); 3.35–3.25 (m, 4H); 3.22–3.14 (m, 2H); 3.12–3.02 (m, 2H); 2.46 (s, 4H); 1.83–1.68 (m, 4H); 1.50–1.44 (m, 4H); 1.42 (s, 9H); 1.34–1.26 (m, 4H).
