*4.4. Synthesis of N25-Fmoc-8,11-dioxo-16,19,22-trioxa-7,12-diazapentacosane-1,25-diamine (4)*

A solution of **3** (0.3 g, 0.5 mmol) in DCM (3.5 mL) was cooled to 0 ◦C by an ice bath for 20 min, and then TFA (1 mL) was added dropwise. The mixture was stirred for 1 h at room temperature (TLC: CHCl3/MeOH 9:1) and then evaporated to dryness to obtain **4** (0.2 g, 0.5 mmol, quantitative yield, chemical structure in Figure 10).

**Figure 10.** Chemical structure of **4**.

<sup>1</sup>H-NMR (400 MHz, CDCl3-d)δ ppm 7.76 (d, *J* = 7.6, 2H); 7.58 (d, *J* = 7.6, 2H); 7.40 (t, *J* = 7.6, 2H); 7.31 (t, *J* = 7.6, 2H); 4.56–4.36 (m, 2H); 4.26–4.16 (m, 1H); 3.66–3.44 (m, 12H); 3.36–3.16 (m, 6H); 3.08–2.94 (m, 2H); 2.63 (s, 4H); 1.82–1.60 (m, 6H); 1.54–1.24 (m, 6H).

*4.5. Synthesis of N<sup>1</sup> -{3-[3-(1-pivaloylmethyl-5-cyclohexyl-2,3,4,5-tetrahydro-1H-benzo[b][1,4] diazepin-2-one-3-yl)]ureido}benzoyl-N25-Fmoc-8,11-dioxo-16,19,22-trioxa-7,12 diazapentacosane-1,25-diamine (6)*

CDI (68.0 mg, 0.4 mmol) was added to a 10 mL MeCN suspension of Z-360 (**5**; 0.2 g, 0.3 mmol), and the mixture was stirred and refluxed for 1 h (TLC: CHCl3/MeOH 95:5). After cooling, TEA (42 mg, 0.4 mmol) was added dropwise to the mixture, followed, after 10 min, by a solution of **4** (0.2 g, 0.3 mmol) in MeCN (10 mL; dropwise addition). The solution was stirred at room temperature for 1 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 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: EtOAc/EtOH 95:5, followed byCHCl3/MeOH 95:5) to yield **6** (0.1 g, 0.1 mmol, 32% yield, chemical structure in Figure 11).

**Figure 11.** Chemical structure of **6**.

HRMS *m*/*z* = 1143.6494 [M+H<sup>+</sup> ] (calculated); 1143.6495 [M+H<sup>+</sup> ] (found). <sup>1</sup>H-NMR (400 MHz, CDCl3-d)δ ppm 7.75 (d, *J* = 7.6, 2H); 7.67–7.52 (m, 3H); 7.38 (t, *J* = 7.6, 2H); 7.30 (t, *J* = 7.6, 2H); 7.25–7.19 (m, 2H); 7.17–7.01 (m, 4H); 6.98–6.93 (m, 1H); 5.24 (d, *J* = 17.9, 1H); 4.80–4.78 (m, 1H); 4.46–4.32 (m, 2H); 4.24–4.16 (m, 1H); 4.11 (d, *J* = 17.9, 1H); 3.66–3.10 (m, 23H); 2.54–2.40 (m, 4H); 2.10–2.00 (m, 2H); 1.88–1.26 (m, 20H); 1.15 (s, 9H).

*4.6. Synthesis of N<sup>1</sup> -{3-[3-(1-pivaloylmethyl-5-cyclohexyl-2,3,4,5-tetrahydro-1H-benzo[b][1,4] diazepin-2-one-3-yl)]ureido}benzoyl-8,11-dioxo-16,19,22-trioxa-7,12-diazapentacosane-1,25 diamine (7)*

A DMF solution (2 mL) of **6** (0.1 g, 0.1 mmol) was cooled and stirred in an ice bath for 10 min. Then, under constant cooling and stirring, morpholine (2 mL) was added, and the mixture was reacted for 1.5 h at room temperature. The completion of reaction was assessed by TLC (CHCl3/MeOH 9:1). The mixture was brought to dryness through an azeotropic distillation with toluene, and the residue was purified by column chromatography (eluent: CHCl3/MeOH 9:1) to obtain **7** (0.1 g, 97 µmol, 97% yield, chemical structure in Figure 12).

**Figure 12.** Chemical structure of **7**.

HRMS *m*/*z* = 921.5814 [M+H<sup>+</sup> ] (calculated); 921.5806 [M+H<sup>+</sup> ] (found). <sup>1</sup>H-NMR (400 MHz, MeOD-d4)δ ppm 7.76–7.73 (m, 1H); 7.48–7.43 (m, 1H); 7.40–7.35 (m, 1H); 7.34–7.25 (m, 3H); 7.11–7.03 (m, 2H); 5.19 (d, *J* = 17.9, 1H); 4.61–4.53 (d, *J* = 7.0, 1H); 4.40 (d, *J* = 17.9, 1H); 3.65–3.34 (m, 18H); 3.25–3.18 (m, 3H); 3.10–3.05 (m, 2H); 2.45 (s, 4H); 2.12–2.02 (m, 1H); 1.95–1.26 (m, 21H); 1.23 (s, 9H).

*4.7. Synthesis of N<sup>1</sup> -{3-[3-(1-pivaloylmethyl-5-cyclohexyl-2,3,4,5-tetrahydro-1H-benzo[b] [1,4]diazepin-2-one-3-yl)]ureido}benzoyl-N25-{2-[4,7,10-tri(tertbutoxycarbonylmethyl)-1,4,7,10 tetrazacyclododecan-1-yl]acetyl}-8,11-dioxo-16,19,22-trioxa-7,12-diazapentacosane-1,25 diamine (8)*

BOP (35.0 mg, 80 µmol) and TEA (16.0 mg, 0.2 mmol) were added to a 2 mL DMF solution of DOTA(tBu)<sup>3</sup> ester (40.0 mg, 71.0 µmol). The mixture was stirred for 10 min, and then a 2 mL DMF solution of **7** (74.0 mg, 80.0 µmol) was added dropwise. The solution was reacted under stirring for 24 h at room temperature (TLC: CHCl3/MeOH 9:1). Then, it was concentrated to dryness, re-dissolved in 10 mL EtOAc, and finally washed with a saturated water solution of NH4Cl (5 mL × 2). The organic phase was concentrated to dryness, and the obtained residue was purified by column chromatography (eluent: CHCl3/MeOH 9:1) to yield **8** (30.7 mg, 21.2 mmol, 30% yield, chemical structure in Figure 13).

**Figure 13.** Chemical structure of **8**.

HRMS *m*/*z* = 1475.9493 [M+H<sup>+</sup> ] (calculated); 1475.9584 [M+H<sup>+</sup> ] (found). <sup>1</sup>H-NMR (400 MHz, CDCl3-*d*)δ ppm 8.07 (broad s, 1H); 7.68–7.56 (m, 2H); 7.40–7.28 (m, 2H); 7.24–7.18 (m, 3H); 6.84–6.78 (m, 1H); 6.60–6.52 (m, 1H); 5.25 (d, *J*=17.9, 1H); 4.78–4.66 (m, 1H); 4.13 (d,

*J* = 17.9, 1H); 3.64–3.12 (m, 35H); 2.68–2.42 (m, 16H); 2.10–2.00 (m, 2H); 1.88–1.50 (m, 16H); 1.46–1.42 (m, 27H); 1.37–1.33 (m, 4H); 1.17 (s, 9H).

*4.8. Synthesis of N<sup>1</sup> -{3-[3-(1-pivaloylmethyl-5-cyclohexyl-2,3,4,5-tetrahydro-1H-benzo[b] [1,4]diazepin-2-one-3-yl)]ureido}benzoyl-N25-{2-[4,7,10-tri(carboxymethyl)-1,4,7,10-tetrazacyclododecan-1-yl]acetyl}-8,11-dioxo-16,19,22-trioxa-7,12-diazapentacosane-1,25-diamine (IP-001)*

A solution of **8** (30.7 mg, 21.0 µmol) in 2 mL of DCM was pre-cooled to 0 ◦C for 20 min in an ice bath. Then, TFA (0.6 mL) was added dropwise, and the mixture was stirred overnight at room temperature. After the assessment of the completion of the reaction by TLC (CHCl3/MeOH 9:1), the solution was evaporated to dryness, and the product was purified by preparative HPLC. IP-001 was obtained in a quantitative yield (27 mg, 21.0 µmol, purity > 95%, chemical structure in Figure 14). Lipophilicity and other pharmacokinetic properties for the IP-001 and Z-360 were predicted by using the SwissADME web tool, as reported in [34].

**Figure 14.** Chemical structure of **IP-001**.

HRMS *m*/*z* = 1307.7615 [M+H<sup>+</sup> ] (calculated); 1307.7650 [M+H<sup>+</sup> ] (found). <sup>1</sup>H-NMR (400 MHz, MeOD-*d4*)δ ppm 7.88 (d, *J* = 8.1, 1H); 7.74 (d, *J* = 8.1, 1H); 7.60–7.53 (m, 1H); 7.52–7.46 (m, 1H); 7.44–7.25 (m, 3H); 7.15–7.02 (m, 1H); 5.20 (d, *J* = 17.9, 1H); 4.60–4.46 (m, 1H); 4.39 (d, *J* = 17.9, 1H); 3.65–3.45 (m, 14H); 3.40–3.15 (m, 16H); 2.46 (s, 4H); 2.12–2.03 (m, 1H); 1.80–160 (m, 4H); 1.64–1.56 (m, 4H); 1.54–1.44 (m, 4H); 1.40–1.30 (m, 10H); 1.24 (s, 9H).
