Synthesis and biological evaluation of dual action cyclo-RGD/SMAC mimetic conjugates targeting α_vβ₃/α_vβ₅ integrins and IAP proteins

Abstract

The rational design, synthesis and in vitro biological evaluation of dual action conjugates 11–13, containing a tumour targeting, integrin α_vβ₃/α_vβ₅ ligand portion and a pro-apoptotic SMAC mimetic portion (cyclo-RGD/SMAC mimetic conjugates) are reported. The binding strength of the two separate units is generally maintained by these dual action conjugates. In particular, the connection between the separate units (anchor points on each unit; nature, length and stability of the linker) influences the activity of each portion against its molecular targets (integrins α_vβ₃/α_vβ₅ for cyclo-RGD, IAP proteins for SMAC mimetics). Each conjugate portion tolerates different substitutions while preserving the binding affinity for each target.

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Introduction

In the pharmaceutical industry, inhibition of a molecular target sometimes leads to a bypass of the target itself by the diseased cells or organs (e.g. penicillins and resistance to beta-lactamases¹). Thus, there is an increasing need for multi-targeted therapies.²

Multi-targeting may be achieved by administering a cocktail of active ingredients, and cocktails active against HIV are an example of clinical success.³ However, effective drug combinations may require different administration routes, or different residence times in the human body.⁴ Well tolerated drugs may become harmful in combination with other active principles, due to drug–drug interactions.⁵

A dual action compound contains the chemical elements required to interact with two molecular targets.⁶ A connection is chosen for each pharmacophore unit, and a suitable spacer separates the two units without disturbing their biological activities.

Cancer implies a huge variety of pathologically altered mechanisms in the body, and cancer research works on hundreds of putative molecular targets.⁷ Cancer cells either mutate the drug target or transport the drug outside the cancer cell (imatinib⁸ and taxol⁹ are known examples of resistance to marketed drugs). Cancer cells are less likely to develop resistance against a dual action compound simultaneously directed against two essential molecular targets. We chose integrins/angiogenesis¹⁰ and inhibition of apoptosis proteins (IAPs)/apoptosis¹¹ because our research group has accumulated significant experience on both target classes in the past.

Project rationale

Integrins α_Vβ₃, α_Vβ₅ and α₅β₁ play a key role in angiogenesis and tumour development, and are accessible as cell surface receptors interacting with extracellular ligands.¹² They are involved in angiogenesis, tumour progression and metastasis.¹³ The tripeptide sequence Arg-Gly-Asp (RGD) is the common motif used by endogenous ligands to recognize and bind such integrins.

Cyclo[Arg-Gly-Asp-Phe-N(Me)-Val] (cilengitide)¹⁴ (1, Fig. 1) reached phase III clinical trials against glioblastoma multiforme. We reported cyclic RGD-based peptidomimetics, either built on 4-substituted aza-bicyclo[4.3.0]nonanes (ABN) (2, Fig. 1),^15,16 or built on bifunctional diketopiperazines (DKP) (3, Fig. 1),¹⁷ as potent inhibitors of the purified α_Vβ₃ receptor.

Fig. 1 Cyclic RGD ligands of α_Vβ₃ integrin {Cilengitide (1, 15-membered), cyclo[ABN-RGD] (2, 15-membered) and cyclo[DKP-RGD] (3, 17-membered)} and pro-apoptotic SMAC mimetics 4 (GDC-0152), 5–7.

Apoptosis is started by stimuli received from within the cell, or from the external environment.¹⁸ Degradation of cellular protein components is carried out by initiator and executioner Cysteine ASPartic acid-specific proteASES (caspases).¹⁹ IAPs²⁰ bind through their baculovirus inhibitor repeat (BIR) domains to the initiator CASP-9 (BIR3 domain, primary binding site) and to the executioner CASP-3 and CASP-7 (linker-BIR2 domain, secondary site), block caspases in their inactive forms and antagonize apoptosis.²¹ Endogenous SMAC protein^22,23 (Second Mitochondria-derived Activator of Caspases) binds to the BIR3/linker-BIR2 domains of IAPs²⁴ and prevents CASP-3/-7/-9 inactivation, restoring caspase-dependent apoptosis.

Structural studies^25,26 showed that the AVPI N-terminal sequence of SMAC binds to IAPs with nanomolar affinity. Pro-apoptotic AVPI mimetics such as 4 (Fig. 1) are undergoing clinical evaluation as anticancer agents.²⁷ We introduced 4-substituted aza-bicyclo[5.3.0]decane (ABD) derivatives (e.g., 5–7, Fig. 1), endowed with good cell-free potency against IAPs and moderate cytotoxicity.^28,29

Since α_V integrins are overexpressed on the surface of cancer cells, their ligands can be used as tumour-homing/anti-angiogenic peptidomimetics for site-directed delivery of cytotoxic drugs.³⁰ For example, the cyclic RGD ligand–doxorubicin conjugate 8 ³¹ (Fig. 2) is highly cytotoxic against cancer cells overexpressing integrins α_Vβ₃/α_Vβ₅ on their membrane. We reported good stability and in vivo efficacy in a mouse model of human ovarian cancer for the cyclo[ABN-RGD]paclitaxel conjugate 9 ³² and the cyclo[DKP-RGD]paclitaxel conjugate 10 ³³ (Fig. 2).

Fig. 2 Cyclic RGD ligand–doxorubicin conjugate 8, cyclo[ABN-RGD]paclitaxel 9 and cyclo[DKP-RGD]paclitaxel 10.

We reasoned that, by connecting the AVPI/SMAC mimetic unit (5–7) with a cyclic RGD ligand such as 2 or 3, dual action conjugates targeting both angiogenesis and apoptosis could be created.

Herein we present the synthesis and biological evaluation of a small series of cyclic RGD ligand–ABD SMAC mimetic dual action conjugates (Fig. 3). We explored (i) the influence of 4- (11, 12) and C-terminus (13) connections on the ABD SMAC mimetic unit, (ii) the influence of the ring size of the cyclic RGD ligand unit (15-membered compounds 11, 12, 17-membered compound 13), and (iii) the presence of an ester- (11) or of an amide-containing (12, 13) linker connecting the cyclic RGD and ABD SMAC portions.

Fig. 3 Heterodimeric cyclic RGD ligand-ABD SMAC mimetic dual action conjugates: 4-connected cyclo[DKP-RGD]-ABD compounds 11–12 and C-terminus-connected cyclo[ABN-RGD]-ABD compound 13.

Results and discussion

Chemistry

Synthetic targets – rationale. Compounds 11 and 12 (Fig. 3) are dual action conjugates where the cyclo[DKP-RGD] integrin ligand unit 3 is coupled with the ABD SMAC mimetic unit 6. The former unit bears a p-aminomethyl group on the DKP-nitrogen benzyl substituent, while the latter unit is functionalised either with a p-hydroxymethyl (11, X = O) or with a p-aminomethyl group (12, X = NH) on the 4-benzylaminoethyl substituent. Ideally, both substitutions on the cyclo[DKP-RGD] ligand 3³³ and on the ABD SMAC mimetic 6²⁹ should not affect their binding affinity to integrin α_Vβ₃ and to IAPs, respectively. 1,4-Butanedioic acid is used as a relatively short linker, because the distance between the DKP and the ABD scaffolds (21 atoms, including two phenyl groups) is largely determined by the DKP-nitrogen p-aminomethylbenzyl and the ABD 4-(p-X-methylbenzylaminoethyl) substitutions.

Compound 13 (Fig. 3) was made by coupling a cyclo[ABN-RGD] integrin ligand unit 2, where the original 4-hydroxymethyl substitution was replaced by 4-aminomethyl group, with the ABD SMAC mimetic 5, where the pro-(S) phenyl of the original C-terminal diphenylmethylamide was replaced by a carboxylic group. Once again, ideally both substitutions should be well tolerated in terms of binding affinities to the respective molecular targets.^16,34 11-Aminoundecanoic acid was used as a relatively long linker, to keep a suitable distance (18 atoms) between the ABN and ABD scaffolds.

Cyclo[DKP-RGD]-ABD dual action compounds 11 and 12. Our synthetic strategy to the ester-connected conjugate 11 started from previously reported N-Boc protected amine 16 (Scheme 1).²⁸ Its C-terminal CONH–CHPh₂ amide contributes to BIR/IAP binding,^28,35 while its 4-aminoethyl function is used as a connection for the linker, which was built as shown in Scheme 1.

Scheme 1 Synthesis of cyclo[DKP-RGD]-ABD SMAC mimetic, ester-connected dual action compound 11. Reagents and conditions: (a) NaBH₄, EtOH–THF–H₂O, −5° to 0 °C, 6 h, 83%; (b) 15, DIPEA, dry MeOH, rt, 5 h; (c) NaBH₄, rt, 30 min, 80% (two steps); (d) Boc₂O, DIPEA, dry CH₂Cl₂, 0 °C to rt, 24 h, 87%; (e) succinic anhydride, DIPEA, DMAP, dry CH₂Cl₂, 0 °C to rt, 2 h, 90%; (f) 19, N-hydroxysulfosuccinimide, DIC, dry DMF, rt, 18 h; (g) 20, CH₃CN, phosphate buffer, pH 7.5, 0 °C, 18 h, 40% over two steps; (h) TFA, CH₂Cl₂, rt, 50 min, 99%.

Namely, amine 16²⁸ was submitted to a reductive alkylation (steps b, c, Scheme 1) with 4-hydroxymethyl benzaldehyde 15 (obtained by selective reduction with sodium borohydride of terephthalaldehyde 14, step a). The resulting aminoalcohol 17 was chemoselectively N-Boc protected (step d), and finally bis-N-Boc protected benzyl alcohol 18 was acylated with succinic anhydride (step e, Scheme 1) to yield the ester-connected construct 19. The overall yield for the reaction steps from 16 to 19 was a rather good 62%. A single direct phase chromatographic column (step c) was needed to purify the reaction products.

Construct 19 was then coupled to the previously reported cyclo[DKP-RGD] derivative 20³³ (steps a, b, Scheme 1), to give the bis-N-Boc protected, ester-connected conjugate 21. The multi-functional nature of both coupling partners and the presence of potentially reactive functions (e.g., the guanidine and carboxylate functions in 20) required the initial activation of the carboxylic group of 19 (step f), followed by its coupling with the amino group of 20 (step g) under carefully controlled conditions. Namely, coupling at pH 7.5 largely prevented side reactions involving the unprotected functionalities of 20, and provided an acceptable 40% overall yield. Finally, acidic hydrolysis of the N-Boc groups led to quantitative deprotection and isolation of target cyclo[DKP-RGD]-ABD, ester-connected dual action conjugate 11 (Scheme 1). HPLC purification was required to obtain pure compounds 21 and 11.

Our synthetic strategy to the amide-connected conjugate 12, and in particular to the key bis-N-Boc protected carboxylate 28, is shown in Scheme 2. Amine 16²⁸ was submitted to a reductive alkylation (steps c, d, Scheme 2) with (p-aminomethyl)benzaldehyde 24, obtained in turn by chemoselective N-Cbz protection of (p-aminomethyl)phenyl methanol 22 (step a), followed by partial oxidation with manganese dioxide of (p-Cbz-aminomethyl)phenyl methanol 23 (step b). The resulting N-Cbz, N-Boc protected diamine 25 was N-Boc protected on its free secondary amine (step e), its primary amine was deprotected by hydrogenolysis (step f), and finally bis-N-Boc protected diamine 27 was acylated with succinic anhydride (step g, Scheme 2) to yield the amide-connected construct 28. The overall yield for the reaction steps from 16 to 28 was an excellent 82%. A direct phase chromatographic column (step d) and a reverse phase HPLC purification (step g) were required to obtain pure 28.

Scheme 2 Synthesis of cyclo[DKP-RGD]-ABD SMAC mimetic, amide-connected dual action compound 12. Reagents and conditions: (a) DIPEA, Cbz-Cl, dry THF, 0 °C to rt, 18 h, 71%; (b) MnO₂, dry THF, rt, 18 h, 97%; (c) 24, DIPEA, dry MeOH, rt, 5 h; (d) NaBH₄, rt, 30 min, 83% over two steps; (e) Boc₂O, DIPEA, dry CH₂Cl₂, 0 °C to rt, 24 h, 100%; (f) H₂, Pd/C 10%, THF–H₂O, rt, 18 h, 100%; (g) succinic anhydride, DIPEA, DMAP, dry CH₂Cl₂, 0 °C to rt, 2 h, 99%; (h) N-hydroxysulfosuccinimide, DIC, dry DMF, rt, 18 h; (i) 20, CH₃CN, phosphate buffer, pH 7.5, 0 °C, 18 h, 49% over two steps; (j) TFA, CH₂Cl₂, rt, 50 min, 100%.

Construct 28 was then coupled to the previously reported cyclo[DKP-RGD] derivative 20³³ (steps h, i, Scheme 2), to give the bis-N-Boc protected, amide-connected conjugate 29. Final acidic N-Boc deprotection (step j, Scheme 2) led to target cyclo[DKP-RGD]-ABD, amide-connected dual action conjugate 12 in a good 49% overall yield.

The experimental conditions and purification protocols for each reaction step in Scheme 4 leading to conjugate 12 are identical to the corresponding steps described in Scheme 1. A comparison between the yields of corresponding steps (steps b–e, Scheme 1, and steps c–g, Scheme 2; steps a–c, Scheme 1 and 2) shows better results for amide-connected conjugate 12. It is reasonable to hypothesize a lower stability of the ester bond in ester-connected conjugate 11, and in intermediates 19 and 21, when compared with the amide bond in amide-connected conjugate 12, and in intermediates 28 and 29. This may lead to lower overall yields either under the reaction conditions, or during the work up-purification protocols.

trans-Cyclo[ABN-RGD]-ABD dual action compound 13. Our synthetic strategy to the amide-connected conjugate 13 required the synthesis of N-Boc protected carboxylic acid 30 (Scheme 3). Its 4-(2-benzoylaminoethyl) substituent contributes to BIR/IAP binding.²⁸ The C-terminal carboxylic acid is the anchoring point for the linker.

Scheme 3 Synthesis of the N-Boc protected carboxylic acid intermediate 30. Reagents and conditions: (a) HCOONH₄, Pd(OH)₂/C, THF–H₂O 4/1, 2% AcOH, 70 °C, 6 h, then rt, 18 h; (b) Boc₂O, TEA, dry CH₂Cl₂, rt, 20 h, 59% over two steps; (c) MsCl, TEA, dry CH₂Cl₂, 0 °C to rt, 2 h; (d) nBuN₄CN, dry DMF, MicroWave, 80 °C, 2 h, 84% over two steps; (e) AcCl, dry MeOH, rt, 5 min, 100%; (f) N-Boc, N-Me-(S)-2-aminobutyric acid (Abu), EDC·HCl, HOBt, DIPEA, dry CH₂Cl₂, rt, 22 h, 82%; (g) H₂, 60 bar, Ni-Ra cartridge, citric acid, EtOH–H₂O, 60 °C, 5 h, continuous flow, 0.5 mL min⁻¹, 24 h, 98%; (h) BzCl, TEA, dry CH₂Cl₂, rt, 5 h, 92%; (i) LiOH, THF–H₂O, 0 °C to rt, 5 h, 88%.

The synthesis of N-Boc protected carboxylic acid 30 started from the already reported tricyclic ester 31.²⁸ Namely, simultaneous hydrogenolytic isoxazolidine opening and benzyl deprotection (step a, Scheme 3) and chemoselective N-Boc protection (step b) provided N-protected alcohol 32. Mesylation (step c) and microwave-assisted nucleophilic substitution (step d) led to N-protected aminonitrile 33. Acidic deprotection (step e, aminonitrile 34) was followed by amidation of the 3-amino group with N-protected/methylated (S)-aminobutyric acid (step f), yielding nitrile 35. Nitrile reduction was carried out using the H-Cube™ continuous flow hydrogenation apparatus (step g), and the amino function of compound 36 was benzoylated (step h). Finally, methyl ester 37 was hydrolysed under basic conditions (step i, Scheme 3) to provide N-Boc protected carboxylic acid 30. The overall yield for the nine reaction steps from 31 to 30 was an acceptable 32% (average reaction yield = 89%). Four direct phase chromatographic separations (steps b, d, f, h) were needed to purify the reaction products.

The synthetic strategy designed to prepare the amide-connected linker construct 38 is reported in Scheme 4. 11-Aminoundecanoic acid 39 was esterified (step a, Scheme 4) and the resulting aminoester 40 was coupled with N-Boc-(S)-phenylglycine using classical peptide coupling conditions (step b). Acidic deprotection (step c) of N-Boc protected 41 led to the aminoester linker 42 as a hydrochloride salt. The linker was then coupled to N-Boc protected carboxylic acid 30 (step d) to provide ester 43. Hydrolysis of the methyl ester (step e) yielded the expected amide-connected construct 38. The ester 43 was also N-deprotected (step f, Scheme 4) to provide the standard compound 44 for biological purposes (vide infra, Table 1). The overall yield of the reaction steps involving ABD SMAC mimetic intermediates (steps d, e; Scheme 4) was a moderate 37%. Two direct phase chromatographic separations (steps b, d) were required to purify the reaction products. The carboxylic acid construct 38 was then coupled to previously reported cyclo[ABN-RGD]methylamine 45¹⁶ (step g, Scheme 4), to give the N-Boc protected, amide-connected conjugate 46. Final acidic N-Boc deprotection (step h, Scheme 4) led to target cyclo[ABN-RGD]-ABD, amide-connected dual action conjugate 13 in a low, unoptimized two-step 17% yield.

Scheme 4 Synthesis of cyclo[ABN-RGD]-ABD SMAC mimetic, amide-connected dual action compound 13. Reagents and conditions: (a) SOCl₂, MeOH, rt to 80 °C, 4 h, 98%; (b) N-Boc-(S)-PheGly, EDC·HCl, HOBt, DIPEA, dry CH₂Cl₂, rt, 18 h, 72%; (c) 3 N HCl/dry MeOH, rt, 18 h, 97%; (d) 30, EDC·HCl, HOBt, DIPEA, dry CH₂Cl₂, rt, 18 h, 63%; (e) LiOH, THF–H₂O, 0 °C to rt, 5 h, 59%; (f) 3 N HCl/dry MeOH, rt, 4 h, quantitative; (g) HATU, HOAt, DIPEA, dry DMF, rt, 10 min, then 45, rt, 72 h, 30%; (h): TFA–thioanisole–1,2-ethanedithiol–anisole 90 : 5 : 3 : 2, rt, 2 h, 57%.

Table 1 IC₅₀s of compounds 2, 3, 5–7, 11–13, 44 on BIR portions from IAPs

Compound	BIR3, XIAP (nM)	l-BIR2-BIR3, XIAP (nM)	BIR3, cIAP2 (nM)
11	52.0 ± 4.5	50.5 ± 7.8	1.33 ± 0.3
12	25.5 ± 1.7	57.5 ± 5.8	0.73 ± 0.1
13	66.0 ± 5.4	175.8 ± 27.5	1.48 ± 0.2
5	120.0 ± 18.6	55.0 ± 13.1	NT
6	110.0 ± 26.4	27.0 ± 12.4	NT
7	760.0 ± 99.2	190.0 ± 49.1	NT
44	90.4 ± 12.2	109.8 ± 27.1	1.57 ± 0.2
2	>10 000	>10 000	NT
3	>10 000	>10 000	NT

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Biology

Compound profiling – rationale. The cell-free potency of the dual action conjugates 11–13 on the BIR domains of IAPs (SMAC mimetic unit), and on integrins α_vβ₃ and α_vβ₅ (cyclic RGD ligand unit) was investigated to confirm the affinity for their molecular targets. Then, their cytotoxicity on tumour cell lines characterized by various levels of cytoplasmic IAPs and membrane integrins was investigated.

Cell-free assays – BIR domains/IAPs. Three BIR domain-based IAP portions were used to test the affinity of the dual action conjugates 11–13. The primary BIR3 binding site/domain from XIAP and cIAP2 was selected to measure the affinity for two of the most therapeutically relevant IAPs.^28,29,36 The bi-functional linker-BIR2-BIR3 portion from XIAP was selected to determine any SMAC dimer-like behaviour of the dual action conjugates 11–13.^37,38 The standard monomeric SMAC mimetic amides 5 and 6 (Fig. 1), and esters 7 (Fig. 1) and 44 (Scheme 4) were tested for comparison. Compound 5 bears a 11, 12-like 4-benzylaminoethyl substituent, compound 6 bears a 13-like 4-benzoylaminoethyl substituent, compound 7 bears a C-terminus phenylglycinamide ester, while compound 44 bears a C-terminus phenylglycinamido-linker ester substituent. Their IC₅₀ values are reported in Table 1. The results clearly show that the attachment of a linker and a cyclic RGD ligand onto the SMAC mimetic unit does not significantly affect its affinity for IAPs. Indeed, 4-connected dual action conjugates 11 and 12 and reference compounds 5 and 6 show comparable affinity for mono- and bi-functional domains from XIAP. The same is true for C-terminus connected dual action conjugate 13 and reference compounds 7 and 44. As expected, cyclic RGD-based reference compounds 2 and 3 did not show affinity for any BIR construct from IAPs.

The higher affinity of dual action conjugates 11–13 for cIAP proteins is consistent with what is generally observed for monomeric SMAC mimetics.²⁹

Cell-free assays – α_vβ₃/α_vβ₅ integrins. The dual action conjugates 11–13 were tested in vitro for their ability to inhibit biotinylated vitronectin binding to the purified α_vβ₃ and α_vβ₅ receptors.^32,33 The standard monomeric, integrin ligands 2 and 3 (Fig. 1), built respectively on a cyclo[ABN-RGD], 13-like scaffold, and on a cyclo[DKP-RGD], 11, 12-like scaffold, were tested for comparison. Their IC₅₀ values are reported in Table 2. The three dual action conjugates 11–13 show nanomolar affinity for both α_vβ₃ (stronger interaction) and α_vβ₅ integrin (weaker interaction), with a limited loss in binding strength with respect to the reference compounds 2 and 3. However, their residual affinity (nanomolar) should be sufficient to target integrin-expressing tumour cells. As expected, reference SMAC mimetics 5–7 and 44 did not show affinity for the α_vβ₃ and α_vβ₅ receptors. Based on these promising cell-free results, we decided to examine the cytotoxic activity of the three dual action conjugates 11–13 on two different tumour cell lines.

Table 2 Inhibition of biotinylated vitronectin binding to α_vβ₃ and α_vβ₅ receptors by compounds 2, 3, 5–7, 11–13, 44

Compound	αvβ3 IC50 (nM)	αvβ5 IC50 (nM)
11	70.1 ± 0.1	900 ± 380
12	36.5 ± 0.6	1500 ± 700
13	105.2 ± 3.4	649 ± 25
2	20.2 ± 1.9	205 ± 34
3	4.5 ± 1.1	149 ± 25
5	>10 000	>10 000
6	>10 000	>10 000
7	>10 000	>10 000
44	>10 000	>10 000

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Cytotoxicity – tumour cells. The dual action conjugates 11–13 were tested in cellular assays for their ability to kill tumour cells, either expressing integrin receptors on their surface or relying on IAP proteins to impair the physiological apoptotic process. We selected ovarian IGROV-1 carcinoma cells as representatives for the former, and breast cancer MDA-MB-231 cells for the latter group. The standard monomeric, integrin ligands 2 and 3 (Fig. 1) and IAP inhibitors 5–7 and 44 (Fig. 1 and Scheme 4), were tested for comparison. Their IC₅₀ values are reported in Table 3. The results clearly show that dual action conjugates 11–13 are endowed with moderate cytotoxic activity. Namely, 4-ester-connected conjugate 11 and reference SMAC mimetic 5 are similarly potent against MDA-MB-231 cells. The same is true for C-terminus-connected conjugate 13 and reference SMAC mimetic 6. The loss of cytotoxicity for 4-amide-connected conjugate 12 with respect to 4-ester-connected conjugate 11 may be due to ester hydrolysis and SMAC mimetic unit release for the latter compound, as observed by us with cyclic RGD ligand–paclitaxel conjugates.³³ Surprisingly, C-terminus-amide connected conjugate 13 shows moderate cytotoxicity on MDA-MB-231 cells, although no hydrolysis or SMAC unit release can easily take place.

Table 3 Cytotoxicity of compounds 11–13, 2–3, 5–7, 44

Compound	MDA-MB-231 (μM)	IGROV-1 (μM)
11	9.7 ± 1.6	>25
12	>25	>25
13	20.5 ± 2.2	11.5 ± 2.5
2	>25	>25
3	>25	>25
5	13.5 ± 1.6	>25
6	8.1 ± 0.9	>25
7	>25	>25
44	>25	>25

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As expected, reference cyclic RGD ligands 2 and 3per se do not show any cytotoxicity against integrin-rich IGROV-1 cells.^32,33 The same is true for reference SMAC mimetics 5–7 and 44. Thus, we assumed that dual action conjugates 11–13 should also be inactive against IGROV-1 cells. Surprisingly, C-terminus-amide-connected conjugate 13 shows cytotoxicity on IGROV-1 cells, possibly suggesting an advantage for C-terminus connected dual action compounds in terms of cellular activity.

Conclusions

In this paper we reported the rational design, synthesis and in vitro evaluation of cyclic RGD ligand–SMAC mimetic conjugates 11–13. We successfully confirmed their affinity for the molecular target of each monomeric unit, and we ascertained that the moderate cytotoxicity of their monomeric precursors can be maintained. Some preliminary results (e.g., the cytotoxic activity for C-terminus-amide connected conjugate 13 on IGROV-1 cells) indicate that the combination of a cyclic RGD ligand-based tumour targeting/anti-angiogenic unit with a SMAC mimetic-based pro-apoptotic unit may lead to synergistic/enhanced anticancer effects.

More generally, we plan to investigate the influence of linker composition (e.g., PEG-based or amides) and hindrance (e.g., rigid/constrained linkers, short linkers), and of overall physico-chemical (e.g., lipophilicity) and electronic properties (e.g., 4-substituents on the ABD SMAC mimetic unit) on biological activity. We aim to establish a detailed SAR, in order to evaluate the potential for cyclic RGD ligand–SMAC mimetic conjugates as anticancer agents in vitro and in vivo.

Experimental

Materials and methods

Reactions requiring anhydrous conditions were carried out in flame-dried glassware, with magnetic stirring and under a nitrogen atmosphere. Commercially available reagents were used as received. Anhydrous solvents were purchased from commercial sources and withdrawn from the container by syringe, under a slight positive pressure of nitrogen. N-Boc, N-Me-(S)-2-aminobutyric acid,²⁸ SMAC amine 16,²⁸cyclo[DKP-RGD]benzylamine 20,³³ SMAC tricyclic ester 31,²⁸ and cyclo[ABN-RGD]methylamine 45¹⁶ were prepared according to literature procedures. Their analytical data were in agreement with those already published. Reactions were monitored by analytical thin-layer chromatography (TLC) using silica gel 60 F₂₅₄ pre-coated glass plates (0.25 mm thickness). Visualization was accomplished by irradiation with a UV lamp and/or staining with a potassium permanganate alkaline solution or ninhydrin. Flash column chromatography was performed according to the method of Still and co-workers³⁹ using Chromagel 60 ACC (40–63 μm) silica gel. Proton NMR spectra were recorded on a spectrometer operating at 400.16 MHz. Proton chemical shifts are reported in ppm (δ) with the solvent reference relative to tetramethylsilane (TMS) employed as the internal standard. The following abbreviations are used to describe spin multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad signal, dd = doublet of doublets. Carbon NMR spectra were recorded on a spectrometer operating at 100.63 MHz, with complete proton decoupling. Carbon chemical shifts are reported in ppm (δ) relative to TMS with the respective solvent resonance as the internal standard. HPLC purifications were performed on a Dionex Ultimate 3000 instrument equipped with a Dionex RS Variable Wavelength Detector (column: Atlantis® Prep T3 OBD™ 5 μm 19 × 100 mm). High resolution mass spectra (HRMS) were performed on a Fourier Transform Ion Cyclotron Resonance (FT-ICR) Mass Spectrometer APEX II & Xmass software (Bruker Daltonics) – 4.7 T Magnet (Magnex) equipped with ESI source, available at CIGA (Centro Interdipartimentale Grandi Apparecchiature) c/o Università degli Studi di Milano. Low resolution mass spectra (MS) were recorded on a Waters Acquity™ UPLC-MS instrument (ESI source).

Chemistry

General procedure for the synthesis of compound 11.
4-Hydroxymethylbenzaldehyde 15. Terephthalaldehyde 14 (1.0 g, 7.45 mmol, 1 eq.) was dissolved in 12.5 mL of EtOH–H₂O (95 : 5), and then 17.5 mL of THF were added. To the resulting stirred solution, NaBH₄ (70 mg, 1.86 mmol, 0.25 eq.) was added at −5 °C in small portions over 30 minutes. The mixture was stirred for 6 h, while the temperature was maintained at 0 °C. The reaction mixture was then adjusted to pH = 5 with 2 M HCl. The solvent was evaporated at reduced pressure, then H₂O (20 mL) was added and the solution was extracted three times with AcOEt. The resulting organic phase was dried over Na₂SO₄ and the solvent was evaporated at reduced pressure. The crude product was purified by flash chromatography on silica gel (n-hexane–AcOEt, 1 : 1 as eluent) to afford the pure desired product 15 as a white solid (840 mg, 6.18 mmol, 83%).

R _f = 0.5 (Hex–AcOEt, 4 : 6); ¹H NMR (400 MHz, CDCl₃) δ 9.82 (s, 1H), 7.71 (d, J = 7.8 Hz, 2H), 7.39 (d, J = 7.9 Hz, 2H), 4.65 (s, 2H), 2.07 (s, 1H); ¹³C NMR (101 MHz, CD₂Cl₂) δ 192.6, 148.8, 135.9, 130.2, 127.3, 64.5.

tert-Butyl ((S)-1-(((3S,6S,7R,9aS)-3-(benzhydrylcarbamoyl)-7-(2-((4 (hydroxymethyl)benzyl)amino)ethyl)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepin-6-yl)amino)-1-oxobutan-2-yl)(methyl)carbamate 17. A solution of SMAC amine 16²⁸ (50 mg, 0.074 mmol, 1.05 eq.), DIPEA (18 μL, 0.105 mmol, 1.5 eq.) in dry MeOH (0.5 mL) was added to a flask containing compound 15 (9.5 mg, 0.074 mmol, 1 eq.) under nitrogen atmosphere. The mixture was left stirring at room temperature for 5 h. NaBH₄ (5.3 mg, 0.14 mmol, 2 eq.) was then added stepwise (5 additions within 3 min), and the mixture was stirred at room temperature under nitrogen for 30 minutes. The mixture was then concentrated under vacuum. 5 mL of a saturated solution of NaHCO₃ were added, then the mixture was extracted with AcOEt (7 mL, three times). The organic phase was dried over Na₂SO₄ and the solvent was evaporated at reduced pressure. The crude product was purified by flash chromatography on silica gel (CH₂Cl₂–MeOH, 9 : 1 as eluent) to afford the pure desired product 17 as a white foam (41 mg, 0.059 mmol, 80% yield).

R _f = 0.3 (CH₂Cl₂–MeOH, 9 : 1); ¹H NMR (400 MHz, THF-d₈) δ 8.27 (bs, 1H), 7.54 (bs, 1H), 7.40–7.00 (m, 14H), 6.21 (d, J = 8.81 Hz, 1H) 4.62 (d, J = 6.53 Hz, 1H), 4.60–4.35 (m, 4H), 4.00–3.70 (m, 4H), 2.84 (bs, 3H), 2.66 (bs, 1H), 2.52 (bs, 1H), 2.15 (m, 4H), 1.91 (m, 2H), 1.85–2.57 (m, 4H), 1.46 (s, 9H), 1.47–1.31 (m, 4H), 0.87 (t, J = 7.32 Hz, 3H); ¹³C NMR (400 MHz, THF-d₈) δ172.3, 171.5, 170.6, 157.0, 143.5, 143.3, 142.7, 129.2, 129.0, 128.9, 128.2, 128.1, 127.6, 127.5, 127.0, 80.2, 80.1, 79.7, 67.9, 67.7, 67.4, 67.2, 66.9, 66.7, 66.6, 64.6, 64.4, 53.4, 53.1, 49.3, 48.4, 47.1, 35.0, 34.9, 34.6, 34.4, 34.3, 33.9, 33.7, 33.5, 31.8, 27.7, 27.4, 27.1, 25.5, 25.3, 25.1, 24.9, 24.7, 22.4, 22.2, 22.2; MS (ESI)m/z calcd for [C₄₃H₅₈N₅O₆]⁺: 740.44 [M + H]⁺; found: 739.75.

tert-Butyl ((S)-1-(((3S,6S,7R,9aS)-3-(benzhydrylcarbamoyl)-7-(2-((tert-butoxycarbonyl)(4-(hydroxymethyl)benzyl)amino)ethyl)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepin-6-yl)amino)-1-oxobutan-2-yl)(methyl)carbamate 18. A solution of compound 17 (41 mg, 0.056 mmol, 1 eq.) and DIPEA (19 μL, 0.112 mmol, 2 eq.) in dry CH₂Cl₂ (0.6 mL) was stirred in an ice bath under nitrogen atmosphere. A solution of Boc₂O (17 mg, 0.079 mmol, 1.4 eq.) in dry CH₂Cl₂ (0.2 mL) was added. The mixture was stirred at 0 °C for 1 h under nitrogen atmosphere, then stirring was continued overnight at room temperature. Then, additional Boc₂O (6.7 mg, 0.030 mmol, 0.5 eq.) was dissolved in CH₂Cl₂ (0.2 mL) and added, and stirring continued for 4 h. The solvent was removed at reduced pressure, and the crude product was passed through a plug of silica gel (CH₂Cl₂–MeOH, 95 : 5 as eluent) to afford the desired pure product 18 as a white foam (41 mg, 0.049 mmol, 87% yield).

R _f = 0.7 (CH₂Cl₂–MeOH, 9 : 1); ¹H NMR (400 MHz, acetone-d₆) δ 8.18 (d, J = 8.70 Hz, 1H), 7.40–7.08 (m, 14H), 6.19 (d, J = 8.66 Hz, 1H), 4.69 (d, J = 7.92 Hz, 1H), 4.61 (d, J = 5.76 Hz, 2H), 4.58–4.30 (m, 4H), 4.13 (t, J = 5.76 Hz, 1H), 3.99 (m, 1H), 3.25 (m, 1H), 2.25 (m, 1H), 2.17 (bs, 1H), 2.00–1.58 (m, 7H), 1.50 (s, 18H), 1.46 (m, 4H), 1.32 (m, 2H), 0.90 (t, J = 7.38 Hz, 3H); MS (ESI)m/z calcd for [C₄₈H₆₆N₅O₈]⁺: 840.49 [M + H]⁺; found: 840.45.

4-((4-(((2-((3S,6S,7R,9aS)-3-(Benzhydrylcarbamoyl)-6-((S)-2-((tert-butoxycarbonyl)(methyl)amino)butanamido)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepin-7-yl)ethyl)(tert-butoxycarbonyl)amino)methyl)benzyl)oxy)-4-oxobutanoic acid 19. Compound 18 (17 mg, 0.020 mmol, 1 eq.) was dissolved in dry CH₂Cl₂ (0.12 mL). Then DIPEA (4.1 μL, 0.024 mmol, 1.2 eq.) was added and the mixture was stirred in an ice bath. DMAP (1.46 mg, 0.012 mmol, 0.6 eq.) and succinic anhydride (3.6 mg, 0.036 mmol, 1.8 eq.) were added. The reaction was stirred at room temperature under nitrogen atmosphere for 2 h. CH₂Cl₂ (15 mL) was then added and the solution was washed with a solution of 1 M KHSO₄ (7 mL, twice). The aqueous phase was extracted with CH₂Cl₂ (7 mL, once), then the collected organic phases were dried over Na₂SO₄. The solvent was evaporated at reduced pressure to afford the desired product 19 as a white foam that was used without further purification (17 mg, 0.018 mmol, 90% yield).

R _f = 0.6 (CH₂Cl₂–MeOH, 9 : 1); ¹H NMR (400 MHz, acetone-d₆) δ 10.72 (bs, 1H), 8.19 (d, J = 8.4 Hz, 1H), 7.48–6.98 (m, 15H), 6.19 (d, J = 8.4 Hz, 1H), 5.12 (s, 2H), 4.69 (d, J = 7.1 Hz, 1H), 4.63–4.26 (m, 3H), 3.99 (d, J = 7.1 Hz, 1H), 3.27 (s, 1H), 3.03–2.93 (m, 2H), 2.80 (s, 3H), 2.70–2.54 (m, 2H), 2.36–2.21 (m, 1H), 2.16 (s, 1H), 2.02–1.57 (m, 6H), 1.49 (s, 18H), 1.45–1.35 (m, 6H), 0.88 (t, J = 7.4 Hz, 3H). ¹³C NMR (400 MHz, acetone-d₆) δ 173.6, 172.7, 170.8, 143.6, 143.3, 139.7, 136.3, 129.4, 129.3, 129.1, 128.1, 128., 127.9, 79.8, 66.3, 62.1, 60.7, 59.2, 57.3, 55.8, 49.6, 45.4, 34.6, 34.0, 33.6, 30.1 (overlapping with solvent signal), 29.8 (overlapping with solvent signal), 29.2, 28.6, 27.3, 22.2, 11.0; MS (ESI)m/z calcd for [C₅₂H₇₀N₅O₁₁]⁺: 940.51 [M + H]⁺; found: 940.33.

2-((1S,5S,11S,15R)-18-(4-((4-((4-(((2-((3S,6S,7R,9aS)-3-(Benzhydrylcarbamoyl)-6-((S)-2-((tert-butoxycarbonyl)(methyl)amino)butanamido)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepin-7-yl)ethyl)(tert-butoxycarbonyl)amino)methyl)benzyl)oxy)-4-oxobutanamido)methyl)benzyl)-11-(3-guanidinopropyl)-4,7,10,13,17,19-hexaoxo-3,6,9,12,16,18-hexaazabicyclo[13.2.2]nonadecan-5-yl)acetic acid 21. Compound 19 (17 mg, 0.018 mmol, 1 eq.) was dissolved in 1 mL of DMF under nitrogen atmosphere in a flame dried Schlenk tube. N-Hydroxysulfosuccinimide (5 mg, 0.023 mmol, 1.24 eq.) and N,N′-diisopropylcarbodiimide (4.18 μL, 0.027 mmol, 1.52 eq.) were then added at room temperature. The solution was stirred overnight, then DMF was removed under vacuum. The residue was then dissolved within the same Schlenk tube in 1.2 mL of CH₃CN, and a solution of compound 20³³ (12.4 mg, 0.0144 mmol, 0.8 eq.) in 1 mL of phosphate buffer solution (the pH was adjusted to 7.5 with 0.2 M NaOH) was then added. The resulting mixture was cooled to 0 °C, adjusting the pH to 7.5 with 0.2 M NaOH, and stirring was continued overnight at room temperature. The reaction mixture was then concentrated at reduced pressure and purified by HPLC (gradient: from 90% H₂O + 0.2% HCOOH–10% acetonitrile + 0.2% HCOOH to 30% H₂O + 0.2% HCOOH–70% acetonitrile + 0.2% HCOOH in 15 minutes). The purified product was then freeze-dried to give the desired pure compound 21 as a white foam (9 mg, 0.072 mmol, 40% yield).

t _R = 12.7 min; ¹H NMR (400 MHz, CD₃OD) δ 8.80 (d, J = 8.3 Hz, 1H), 8.40 (t, J = 5.5 Hz, 1H), 8.34–8.15 (br m, 1H), 7.39–7.12 (m, 18H), 6.12 (d, J = 8.3 Hz, 1H), 5.21–5.03 (m, 3H), 4.84–4.73 (m, 2H), 4.61 (t, J = 5.8 Hz, 1H), 4.58–4.27 (m, 7H), 4.24–4.15 (m, 1H), 4.07–3.91 (m, 3H), 3.88 (d, J = 6.3 Hz, 1H), 3.52 (d, J = 17.2 Hz, 1H), 3.45 (dd, J = 14.7, 6.8 Hz, 1H), 3.29–3.14 (m, 3H), 3.14–2.89 (m, 1H), 2.80 (s, 3H), 2.74–2.57 (m, 6H), 2.54 (t, J = 6.7 Hz, 2H), 2.30–2.16 (m, 1H), 2.15–1.33 (m, 33H), 0.92 (t, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 174.2, 174.0, 173.7, 173.6, 173.0, 172.9, 172.3, 172.2, 172.2, 171.3, 171.2, 171.1, 158.6, 143.1, 140.2, 136.7, 135.9, 135.9, 129.7, 129.5, 129.3, 128.7, 128.6, 128.5, 128.3, 81.4, 67.1, 62.8, 62.2, 60.6, 59.7, 58.3, 56.9, 54.3, 53.2, 51.3, 50.6, 48.1, 46.0, 43.9, 43.8, 42.2, 40.1, 40.1, 39.9, 37.8, 34.0, 33.6, 31.4, 31.1, 31.0, 30.8, 30.4, 28.9, 28.8, 27.7, 26.5, 23.0, 11.1; MS (ESI)m/z calcd for [C₇₉H₁₀₆N₁₅O₁₈]⁺: 1552.78 [M + H]⁺; found: 1552.85.

2-((1S,5S,11S,15R)-18-(4-((4-((4-(((2-((3S,6S,7R,9aS)-3-(Benzhydrylcarbamoyl)-6-((S)-2-(methylamino)butanamido)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepin-7-yl)ethyl)amino)methyl)benzyl)oxy)-4-oxobutanamido)methyl)benzyl)-11-(3-guanidinopropyl)-4,7,10,13,17,19-hexaoxo-3,6,9,12,16,18-hexaazabicyclo[13.2.2]nonadecan-5-yl)acetic acid 11. Compound 21 (9 mg, 0.0058 mmol, 1 eq.) was dissolved in CH₂Cl₂ (100 μL). TFA (66 μL, 0.87 mmol, 150 eq.) was then added, and stirring was continued at room temperature for 50 minutes. The solvent and TFA were removed at reduced pressure, and the crude product was purified by HPLC (gradient: from 90% H₂O + 0.1% CF₃COOH–10% acetonitrile + 0.1% CF₃COOH to 30% H₂O + 0.1% CF₃COOH–70% acetonitrile + 0.1% CF₃COOH in 14 min). The purified product was then freeze-dried to give the trifluoroacetate salt of the desired pure compound 11 as a white foam (9.7 mg, 0.0057 mmol, 99% yield).

t _R = 8.7 min; ¹H NMR (400 MHz, CD₃CN) δ 7.97 (d, J = 8.1 Hz, 1H), 7.86 (dd, J = 8.4, 3.6 Hz, 1H), 7.44–7.13 (m, 18H), 6.05 (d, J = 8.1 Hz, 1H), 5.08 (s, 2H), 4.99 (d, J = 15.2 Hz, 1H), 4.75 (t, J = 6.8 Hz, 1H), 4.54 (d, J = 9.4 Hz, 1H), 4.50 (dd, J = 7.9, 4.2 Hz, 1H), 4.45 (dd, J = 9.0, 4.3 Hz, 1H), 4.31–4.23 (m, 3H), 4.10 (s, 2H), 4.04–3.89 (m, 3H), 3.89–3.78 (m, 3H), 3.46 (d, J = 17.1 Hz, 1H), 3.34 (dd, J = 15.0, 6.5 Hz, 1H), 3.12 (t, J = 6.5 Hz, 2H), 3.07–2.98 (m, 1H), 2.82–2.66 (m, 2H), 2.65–2.54 (m, 6H), 2.55–2.43 (m, 3H), 2.26–2.14 (m, 1H), 2.13–1.42 (m, 15H, overlapping with solvent signal), 0.92 (t, J = 7.5 Hz, 3H); ¹³C NMR (101 MHz, CD₃CN) δ 173.9, 173.2, 173.1, 173.0, 172.2, 171.2, 170.8, 170.6, 170.6, 170.0, 168.6, 168.5, 157.9, 143.1, 142.9, 140.1, 138.7, 135.4, 131.8, 131.2, 129.7, 129.3, 129.1, 128.8, 128.4, 128.3, 128.0, 66.4, 63.2, 62.4, 60.3, 59.2, 58.0, 56.5, 54.9, 52.8, 51.7, 50.0, 48.2, 46.4, 43.4, 43.2, 41.8, 39.8, 38.9, 37.3, 35.5, 33.6, 32.9, 32.5, 32.3, 31.1, 30.2, 29.0, 28.6, 26.9, 26.0, 23.9, 9.1; MS (ESI)m/z calcd for [C₆₉H₉₀N₁₅O₁₄]⁺: 1352.68 [M + H]⁺; found: 1353.41; HRMS (ESI)m/z calcd for [C₆₉H₉₀N₁₅O₁₄]⁺: 1352.67862 [M + H]⁺; found: 1352.68112.

General procedure for the synthesis of compound 12. The general procedure for the synthesis of compound 12 is reported in the ESI.†

General procedure for the synthesis of compound 13.
(3S,6S,7S,9aS)-Methyl 6-(tert-butoxycarbonylamino)-7-(hydroxymethyl)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepine-3-carboxylate 32. Compound 31²⁸ (1.06 g, 3.07 mmol, 1 eq.), ammonium formate (2.23 g, 34.36 mmol, 11.2 eq.) and palladium hydroxide on carbon (320 mg, 30%) were dissolved in a mixture of THF–H₂O 4 : 1 + 2% acetic acid (300 mL). The reaction mixture was stirred at 70 °C for 6 h, then at rt overnight. The reaction mixture was then filtered over celite, washing with THF and CH₂Cl₂. The solvent was then removed at reduced pressure, obtaining 1.12 g of crude aminoalcohol that was used without any further purification.

A solution of Boc₂O (1.73 g, 7.67 mmol, 2.5 eq.) in 10 mL of dry CH₂Cl₂ was prepared and stirred at room temperature under nitrogen atmosphere. The previously prepared crude aminoalcohol (theoretical 3.07 mmol, 1 eq.) and dry TEA (1.1 mL, 7.89 mmol, 2.6 eq.) in 20 mL of dry CH₂Cl₂ were then added, and the reaction mixture was left under stirring at room temperature for 20 h. The reaction mixture was then diluted with CH₂Cl₂ and washed with 2 × 30 mL of 5% citric acid solution, with 2 × 30 mL of saturated NaHCO₃ and 2 × 30 mL of brine. The aqueous phase was washed with 2 × 20 mL of CH₂Cl₂, the organic phases were dried with Na₂SO₄, filtered and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (n-hexane–AcOEt 6 : 4 as eluent), yielding 643 mg of pure compound 32 as a white solid (1.80 mmol, 59% yield over two steps).

¹H NMR (400 MHz, CDCl₃) δ 6.14 (bd, 1H), 4.58 (dd, 1H, J = 4.6 Hz, J = 8.1), 4.25 (dd, 1H, J = 6.2 Hz, J = 10.5 Hz), 3.95–3.82 (m, 2H), 3.73 (s, 3H), 3.38 (dd, 1H, J = 12.1 Hz, J = 2.8 Hz), 2.25 (m, 1H), 2.18–1.96 (m, 4H), 1.92–1.72 (m, 3H), 1.55 (m, 1H), 1.42 (s, 9H). ¹³C NMR (100.6 MHz, CDCl₃) δ 173.1, 171.4, 158.3, 81.3, 65.3, 61.3, 59.6, 55.9, 53.0, 42.5, 34.2, 33.5, 31.8, 28.9, 28.4. MS (ESI): m/z calcd for C₁₇H₂₈N₂O₆: 356.19; found: 257.0 [M − Boc + H]⁺.

(3S,6S,7R,9aS)-Methyl 6-((tert-butoxycarbonyl)amino)-7-(cyanomethyl)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepine-3-carboxylate 33. A 0.1 M solution of compound 32 (210 mg, 0.59 mmol, 1 eq.) in dry CH₂Cl₂ (6 mL) was prepared and stirred under nitrogen atmosphere; MsCl (101 mg, 0.88 mmol, 1.5 eq.) and dry TEA (246 μL, 1.76 mmol, 3 eq.) were added at 0 °C and the reaction mixture was then stirred for 2 h at room temperature. After reaction completion the solution was diluted in 20 mL of CH₂Cl₂, washed with a 5% citric acid aqueous solution (2 × 50 mL), a saturated solution of NaHCO₃ (2 × 50 mL) and brine (2 × 50 mL). The organic phase was dried over Na₂SO₄ and evaporated under reduced pressure. The crude mesylate (0.83 g, theoretical 1.91 mmol, quantitative yield) was used without any purification.

A 2 N solution of the crude mesylate in dry DMF (1 mL) was prepared and nBu₄⁺CN⁻ (770 mg, 2.87 mmol, 1.5 eq.) was added. The solution was stirred at 80 °C in a microwave synthesizer for 2 h. The solvent was removed at reduced pressure and the crude product was purified by flash chromatography (n-hexane–EtOAc 4 : 6 as eluent). The desired pure product 33 (0.59 g, 1.61 mmol) was obtained as a white powder (yield 84%).

¹H NMR (400 MHz, CDCl₃) δ 5.83 (bd, 1H), 4.60 (dd, 1H, J = 7.9, J = 4.3 Hz), 4.26 (m, 1H, J = 10.0 Hz, J = 7.6 Hz), 3.93 (dd, 1H, J = 15.3 Hz, 7.6 Hz), 3.76 (s, 3H), 2.76 (dd, 1H, J = 4.5 Hz, J = 17.0 Hz), 2.42–2.23 (m, 3H), 2.08 (m, 2H), 1.96 (m, 1H), 1.91–1.62 (m, 4H), 1.42 (s, 9H). ¹³C NMR (100.6 MHz, CDCl₃) δ 172.4, 169.6, 80.4, 60.6, 58.8, 56.3, 52.6, 37.8, 34.1, 33.5, 32.9, 28.4, 27.9, 21.0. MS (ESI): m/z calcd for C₁₈H₂₇N₃O₅: 365.20; found: 366.2 [M + H]⁺.

(3S,6S,7R,9aS)-Methyl 6-amino-7-(cyanomethyl)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepine-3-carboxylate 34. Compound 33 (317 mg, 0.87 mmol, 1 eq.) was dissolved in 2.8 mL of dry MeOH under nitrogen atmosphere at room temperature. Acetyl chloride (930 μL, 13.03 mmol, 15 eq.) was added one pot and under vigorous stirring. After 5 minutes a white solid started to precipitate. The solvent was then removed at reduced pressure, yielding 258 mg of desired pure compound 34 as a white solid (0.87 mmol, quantitative yield).

¹H NMR (400 MHz, CDCl₃) δ 4.58 (dd, 1H, J = 4.2 Hz, J = 8.3 Hz), 4.19 (d, 1H, J = 10.2 Hz), 4.01 (m, 1H), 3.72 (s, 3H), 2.80 (dd, 1H, J = 17.0 Hz, J = 3.8 Hz), 2.61 (dd, 1H, J = 17.0 Hz, J = 9.0 Hz), 2.32 (m, 1H), 2.26–2.14 (m, 2H), 2.11–2.02 (m, 1H), 2.01–1.92 (m, 2H), 1.92–1.74 (m, 3H). ¹³C NMR (100.6 MHz, CDCl₃) δ 170.1, 120.8, 70.4, 64.7, 64.2, 63.0, 62.0, 59.5, 55.5, 43.7, 40.7, 38.0, 37.4, 36.6, 36.1, 35.6, 35.3, 31.2, 31.1, 24.2. MS (ESI): m/z calcd for C₁₃H₁₉N₃O₃: 265.14; found: 266.27 [M + H⁺].

(3S,6S,7R,9aS)-Methyl 6-((R)-2-((tert-butoxycarbonyl)(methyl)amino)butanamido)-7-(cyanomethyl)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepine-3-carboxylate 35. A solution of N-methyl-N-Boc-aminobutyric acid (270 mg, 1.24 mmol, 1.4 eq.), EDC·HCl (240.3 mg, 1.25 mmol, 1.4 eq.) and HOBt (181 mg, 1.34 mmol, 1.5 eq.) in 2.5 mL of dry CH₂Cl₂ was prepared under nitrogen atmosphere, then 300 μL of dry DIPEA (1.68 mmol, 1.9 eq.) were added. The solution was left under stirring for 10 minutes. A solution of compound 34 (258 mg, 0.87 mmol, 1 eq.) and 320 μL of dry DIPEA (1.79 mmol, 2.1 eq.) in 6.2 mL of dry CH₂Cl₂ was then added dropwise, and the reaction mixture was left under stirring at rt for 22 h. The reaction mixture was diluted with CH₂Cl₂ and washed with 20 ml of a citric acid 5% solution, 20 mL of a NaHCO₃ saturated solution and 20 mL of brine. The organic phase was then dried with Na₂SO₄, filtered and the solvent was removed at reduced pressure. The crude product was purified by flash chromatography (n-hexane–EtOAc 4 : 6 as eluent), yielding 330 mg of desired pure compound 35 as a white solid (0.71 mmol, 82% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.14 (bd, 1H), 4.59 (dd, 1H, J = 3.9 Hz, J = 7.3 Hz), 4.54 (m, 1H), 4.35 (m, 1H), 3.94 (m, 1H), 3.76 (s, 3H), 2.85 (s, 3H), 2.64 (m, 1H), 2.38 (m, 1H), 2.34–2.26 (m, 2H), 2.08 (m, 2H), 2.01 (m, 1H), 1.97–1.68 (m, 6H), 1.48 (s, 9H), 0.91 (t, 3H, J = 6.9 Hz). ¹³C NMR (100.6 MHz, CDCl₃) δ 173.1, 169.8, 79.8, 60.4, 58.7, 54.4, 52.5, 37.4, 33.8, 33.3, 32.6, 28.2, 27.6, 21.4, 10.5. MS (ESI): m/z calcd for C₂₃H₃₆N₄O₆: 464.26; found: 465.54 [M + H⁺].

(3S,6S,7R,9aS)-Methyl 7-(2-aminoethyl)-6-((R)-2-((tert-butoxycarbonyl)(methyl)amino)butanamido)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepine-3-carboxylate 36. Hydrogenation of the nitrile 35 was performed using the H-Cube™ continuous-flow hydrogenation reactor. Compound 35 (130 mg, 0.28 mmol, 1 eq.) was dissolved in 50 mL of 90 : 10 EtOH–H₂O and 1 M aqueous citric acid (0.28 mmol) was added. The reaction mixture was flowed through a Ni/Ra cartridge (hydrogen pressure: 60 bar; T = 60 °C; flow 0.5 mL min⁻¹). The reaction was completed after 24 hours. The solvent was removed at reduced pressure. The crude desired compound 36 (0.13 g, 0.277 mmol, quantitative yield) was used for the next step without any purification.

MS (ESI): m/z calcd for C₂₃H₄₀N₄O₆: 468.29; found: 469.40 [M + H⁺].

(3S,6S,7R,9aS)-Methyl 7-(2-benzamidoethyl)-6-((R)-2-((tert-butoxycarbonyl)(methyl)amino)butanamido)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepine-3-carboxylate 37. Compound 36 (234 mg, 0.50 mmol, 1 eq.) was dissolved under inert atmosphere in 5.0 mL of dry CH₂Cl₂, then 350 μL of dry TEA were added (2.50 mmol, 5 eq.). Benzoyl chloride (70 μL, 0.60 mmol, 1.2 eq.) was slowly added under vigorous stirring. The solution turned from yellow to dark red. After 2 h additional TEA (50 μL, 0.37 mmol, 0.7 eq.) and benzoyl chloride (50 μL, 0.43 mmol, 0.86 eq.) were added. After 3 h the reaction mixture was diluted with CH₂Cl₂ and washed with 30 mL of a 5% citric acid solution, 30 mL of a saturated NaHCO₃ solution and 30 mL of brine. The organic phase was then dried with Na₂SO₄, filtered and the solvent removed under reduced pressure. A brownish oil residue was purified by flash chromatography (n-hexane–EtOAc 1 : 9), yielding 264 mg of pure desired compound 37 as a white solid (0.46 mmol, 92% yield).

¹H NMR (400 MHz, [D₆]acetone) δ 7.90 (m, 2H), 7.70 (bs, 1H), 7.54–7.42 (m, 3H), 7.16 (bs, 1H), 4.58–4.50 (m, 2H), 4.47 (dd, 1H, J = 8.5 Hz, J = 3.9 Hz), 4.06 (m, 1H), 3.65 (s, 3H), 3.41 (m, 2H), 2.80 (m, 3H), 2.31 (m, 1H), 2.25–2.09 (m, 2H), 2.01–1.84 (m, 5H), 1.73 (m, 1H), 1.70–1.56 (m, 4H), 1.52 (m, 1H), 1.42 (s, 9H), 0.87 (t, 3H, J = 7.4 Hz). ¹³C NMR (100.6 MHz, [D₆]Acetone) δ 173.2, 171.4, 170.8, 167.0, 135.8, 131.3, 128.7, 127.6, 79.5, 60.2, 58.1, 55.0, 51.4, 37.4, 37.3, 33.0, 32.5, 31.7, 27.5, 27.3, 21.3, 10.0. MS (ESI): m/z calcd for C₃₀H₄₄N₄O₇: 572.32; found: 573.8 [M + H⁺].

(3S,6S,7R,9aS)-7-(2-Benzamidoethyl)-6-((R)-2-((tert-butoxycarbonyl)(methyl)amino)butanamido)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepine-3-carboxylic acid 30. A 0.1 M solution of compound 37 (35 mg, 0.061 mmol, 1 eq.) in 1 mL of THF–H₂O 3/1 was prepared at 0 °C, and 2 N aqueous LiOH (92 μL, 0.183 mmol, 3 eq.) was slowly added. The reaction mixture was stirred at 0 °C for 30 minutes, then at room temperature for 5 h. The reaction was then quenched with 1 mL of 1 M aqueous KHSO₄ and the resulting mixture was extracted with CH₂Cl₂ (3 × 5 mL). The combined organic phases were dried over Na₂SO₄ and evaporated at reduced pressure. The crude desired product 30 (0.03 g, 0.054 mmol, yield 88%) was used for the next step without any purification.

¹H NMR (400 MHz, CDCl₃) δ 7.83 (m, 2H), 7.68 (bd, 1H), 7.48–7.32 (m, 4H), 4.76 (m, 1H), 4.57 (m, 1H), 4.51 (m, 1H), 3.99 (m, 1H), 3.41 (m, 2H), 2.94 (bs, 3H), 2.26 (m, 1H), 2.14–1.99 (m, 4H), 1.91–1.71 (m, 5H), 1.65–1.51 (m, 3H), 1.37 (s, 9H), 0.94 (t, 3H, J = 6.0 Hz). ¹³C NMR (100.6 MHz, CDCl₃) δ 174.2, 173.0, 127.6, 135.3, 131.5, 128.8, 128.7, 128.1, 127.7, 81.1, 60.7, 60.1, 58.6, 54.7, 37.2, 36.7, 32.8, 32.7, 32.5, 31.4, 30.7, 29.5, 28.1, 27.4, 22.7, 10.8. MS (ESI): m/z calcd for C₂₉H₄₂N₄O₇: 558.31; found: 559.7 [M + H⁺].

11-Aminoundecanoic acid methyl ester 40. A 0.5 M solution of 11-aminoundecanoic acid 39 (5.09 g, 25.3 mmol, 1 eq.) in MeOH (50 mL) was prepared and SOCl₂ (2.93 mL, 40.4 mmol, 1.6 eq.) was slowly added. The reaction mixture was stirred at room temperature for 2 h, then refluxed at 80 °C for additional 2 h. The solvent was then evaporated at reduced pressure and the solid residue was washed with 10 mL of dry Et₂O, then filtered. The desired compound 40 (5.3 g, 24.6 mmol, quantitative yield) was obtained as a white powder and used for the next step without any purification.

¹H NMR (400 MHz, CDCl₃) δ 8.25 (bs, 3H), 3.65 (s, 3H), 2.90 (m, 2H), 2.25 (t, 2H, J = 7.6 Hz), 1.70 (m, 2H), 1.60 (m, 2H), 1.40–1.15 (m, 12H).

(S)-Methyl 11-(2-((tert-butoxycarbonyl)amino)-2-phenylacetamido)undecanoate 41. A solution of (S)-N-Boc-phenylglycine (573 mg, 2.28 mmol, 1 eq.), HOBt (369 mg, 2.74 mmol, 1.2 eq.), EDC·HCl (524 mg, 2.74 mmol, 1.2 eq.) and dry DIPEA (1.39 mL, 7.98 mmol, 3.5 eq.) in dry CH₂Cl₂ (12 mL) was stirred under nitrogen atmosphere for 15 minutes at 0 °C. Then a solution of compound 40 (491 mg, 2.28 mmol, 1 eq.) in dry CH₂Cl₂ (12 mL) was slowly added, and the reaction mixture was stirred at room temperature overnight. Then the reaction mixture was diluted in 50 mL of CH₂Cl₂ and washed with a 5% citric acid aqueous solution (2 × 50 mL), a saturated solution of NaHCO₃ (2 × 50 mL) and brine (2 × 50 mL). The combined organic phases were dried over Na₂SO₄ and evaporated at reduced pressure. The residue was purified by flash chromatography (n-hexane–EtOAc 6 : 4 as eluent), and the desired pure product 41 (0.74 g, 1.65 mmol) was obtained as a white foam (yield 72%).

¹H NMR (400 MHz, CDCl₃) δ 7.40–7.30 (m, 5H), 5.87 (bs, 1H), 5.76 (bs, 1H), 5.13 (bs, 1H), 3.69 (s, 3H), 3.23 (dd, 2H, J = 7.0 Hz, J = 13.1 Hz), 2.32 (t, 2H, J = 7.5 Hz), 1.63 (m, 2H), 1.43 (m, 9H), 1.38–1.10 (m, 14H). ¹³C NMR (100.6 MHz, CDCl₃) δ 174.3, 169.9, 155.2, 138.8, 129.0, 128.3, 127.2, 80.0, 58.7, 51.4, 39.8, 34.1, 29.3, 29.2, 29.1, 28.3, 26.6, 24.9. MS (ESI): m/z calcd for C₂₅H₄₀N₂O₅: 448.29; found: 471.3 [M + Na⁺].

(S)-Methyl 11-(2-amino)-2-phenylacetamido)undecanoate 42. Compound 41 (298 mg, 0.66 mmol, 1 eq.) was dissolved in a 3 N methanolic HCl solution (11 mL 33 mmol, 50 eq.) and stirred under inert atmosphere at room temperature overnight. The reaction was then quenched with 10 mL of saturated NaHCO₃ and extracted with EtOAc (3 × 10 mL). The combined organic phases were dried over Na₂SO₄ and evaporated at reduced pressure. The desired compound 42 was obtained (0.22 g, 0.64 mmol, yield 97%) and used for the next step without any purification.

¹H NMR (400 MHz, CDCl₃) δ 7.42–7.26 (m, 5H), 7.03 (bs, 1H), 4.53 (s, 1H), 3.68 (s, 3H), 3.26 (ddd, 2H, J = 13.1 Hz, J = 7.1 Hz, J = 2.2 Hz), 2.31 (t, 2H, J = 7.5 Hz), 1.35–1.23 (m, 12H). ¹³C NMR (100.6 MHz, CDCl₃) δ 175.0, 173.5, 141.7, 129.3, 128.4, 127.3, 60.0, 51.4, 39.2, 34.0, 29.4, 29.2, 29.1, 29.0, 26.7, 24.8. MS (ESI): m/z calcd for C₂₀H₃₂N₂O₃: 348.24; found: 349.6 [M + H⁺].

Methyl 11-((S)-2-((3S,6S,7R,9aS)-7-(2-benzamidoethyl)-6-((R)-2-((tert-butoxycarbonyl)(methyl)amino)butanamido)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepine-3-carboxamido)-2-phenylacetamido)undecanoate 43. Compound 30 (45.0 mg, 0.081 mmol, 1 eq.), HATU (47.0 mg, 0.124 mmol, 1.4 eq.), HOAt (24.3 mg, 0.178 mmol, 2 eq.) and dry DIPEA (50 μL, 0.28 mmol, 3 eq.) were dissolved in dry DMF (0.6 mL) under nitrogen atmosphere at room temperature. The solution was left under stirring for 10 minutes. Then a solution of compound 42 (46.8 mg, 0.122 mmol, 1.4 eq.) and dry DIPEA (100 μL, 0.56 mmol, 6 eq.) in dry DMF (0.6 mL) was prepared under nitrogen atmosphere, and was added dropwise. The resulting reaction mixture was left under stirring at rt for 43 hours. Then, the solvent was removed under reduced pressure, the crude was dissolved in EtOAc (90 mL) and washed with a 5% citric acid aqueous solution (25 mL) and a saturated NaHCO₃ solution (25 mL). The organic phase was then dried with Na₂SO₄, filtered and the solvent was removed at reduced pressure. The crude product (100 mg) was purified by flash chromatography (EtOAc 100% as eluant), and the desired pure product 43 (44.1 mg, 0.050 mmol, yield 61%) was obtained as a white solid.

¹H NMR (400 MHz, [D₆]DMSO, ≈1 : 1 mixture of two conformers) δ 8.61 (bd, 0.5H), 8.48 (d, 0.5H, J = 8 Hz), 8.33 (m, 1H), 8.21 (m, 0.5H), 7.88–7.72 (m, 3H), 7.68 (m, 0.5H), 7.52–7.21 (m, 8H), 5.36 (d, 0.5H, J = 6 Hz), 5.34 (d, 0.5H, J = 5.9 Hz), 4.59 (dd, 1H, J = 7.4 Hz, 3.7 Hz), 4.52 (m, 1H), 4.40 (m, 1H), 4.00–3.90 (m, 1H), 3.58 (s, 3H), 3.23 (m, 2H), 3.04 (m, 2H), 2.70 (s, 1.5H), 2.69 (s, 1.5H), 2.28 (td, 2H, J = 7.4 Hz, 2.8 Hz), 2.14 (m, 1H), 2.05 (m, 1H), 1.94 (m, 1H), 1.86 (m, 1H), 1.80 (m, 1H), 1.79–1.70 (m, 2H), 1.68–1.57 (m, 2H), 1.57–1.38 (m, 6H), 1.35 (m, 2H), 1.34 (s, 9H), 1.26–1.14 (m, 12H), 0.81 (bt, 3H). ¹³C NMR (100.6 MHz, [D₆]Acetone, ≈1 : 1 mixture of two conformers) δ 172.2, 170.2, 170.5, 170.1, 169.9, 167.0, 140.1, 139.5, 135.8, 131.3, 129.0, 128.8, 128.7, 128.6, 128.2, 128.1, 127.8, 127.7, 127.6, 61.2, 61.1, 58.3, 57.2, 57.0, 54.9, 39.1, 37.3, 34.1, 33.4, 33.1, 32.9, 32.7, 31.9, 27.6, 26.4, 26.3, 24.7, 10.0. MS (ESI): m/z calcd for C₄₉H₇₂N₆O₉: 889.13; found: 890.0 [M + H⁺].

11-((S)-2-((3S,6S,7R,9aS)-7-(2-Benzamidoethyl)-6-((R)-2-((tert-butoxycarbonyl)(methyl)amino)butanamido)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepine-3-carboxamido)-2-phenylacetamido)undecanoic acid 38. Compound 43 (34.1 mg, 0.038 mmol, 1 eq.) was dissolved in THF–H₂O 3 : 1 (400 μL), and 2 N aqueous LiOH (60 μL, 0.122 mmol, 3 eq.) was slowly added. The reaction mixture was stirred at room temperature for 5 h. Then the reaction mixture was quenched with 1 M aqueous KHSO₄ (1 mL), diluted with distilled water (10 mL) and extracted sequentially with CH₂Cl₂ (3 × 15 mL) and EtOAc (3 × 15 mL). The combined organic phases were dried over Na₂SO₄ and evaporated at reduced pressure. The desired compound 38 (0.0315 g, 0.036 mmol, yield 95%) was obtained as a white solid and used for the next step without any purification. MS (ESI): m/z calcd for C₄₈H₇₀N₆O₉: 874.52; found: 875.8 [M + H⁺].
Methyl 11-((S)-2-((3S,6S,7R,9aS)-7-(2-benzamidoethyl)-6-((S)-2-(methylamino)butanamido)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepine-3-carboxamido)-2-phenylacetamido)undecanoate 44. Compound 43 (5.3 mg, 0.0060 mmol, 1 eq.) was dissolved under inert atmosphere in 3 N methanolic HCl (200 μL). The reaction mixture was left under vigorous stirring at room temperature for 4 hours, then the solvent was slowly removed under reduced pressure. The pure desired acid 44 (5.0 mg, 0.0060 mmol, quantitative yield) was obtained as a white solid.

¹H NMR (400 MHz, [D₆]acetone, ≈1 : 1 mixture of two conformers) δ 8.64 (m, 0.5H), 8.57 (m, 0.5H), 8.53 8 m, 0.5H), 8.45 (m, 0.5H), 8.21 (m, 0.5H), 8.16–8.03 (m, 2.5H), 7.89 (m, 0.5H), 7.67 (m, 0.5H), 7.50–7.18 (m, 8H), 5.53 (d, 0.5H, J = 7.7 Hz), 5.50 (d, 0.5H, J = 7.8 Hz), 4.76 (m, 1H), 4.69 (m, 1H), 4.20 (m, 1H), 4.10 (m, 0.5H), 4.03 (m, 0.5H), 3.61 (s, 3H), 3.44 (m, 2H), 3.15 (m, 2H), 2.78 (s, 3H), 2.28 (td, 2H, J = 7.4 Hz, J = 1.8 Hz), 2.22 (m, 1H), 2.19–2.09 (m, 2.5H), 2.05 (m, 0.5H), 2.02–1.94 (m, 2.5H), 1.91 (m, 0.5H), 1.90–1.83 (m, 2.5H), 1.76–1.62 (m, 2.5H), 1.57 (m, 2H), 1.43 (m, 2H), 1.33 (m, 1H), 1.31–1.19 (m, 12H), 1.03 (m, 3H). ¹³C NMR (100.6 MHz, [D₆]acetone, ≈1 : 1 mixture of two conformers) δ 130.4, 128.0, 127.6, 127.3, 127.2, 126.8, 126.7, 61.0, 60.4, 57.3, 56.1, 54.6, 49.6, 38.1, 36.4, 36.1, 35.7, 35.6, 32.7, 32.6, 32.5, 32.4, 32.1, 31.9, 31.8, 31.7, 31.3, 29.3, 28.3, 28.2, 26.2, 26.1, 25.5, 25.4, 23.7, 21.1, 21.0, 7.1.

Compound 46. Compound 38 (31.5 mg, 0.036 mmol, 1 eq.), HATU (20.8 mg, 0.053 mmol, 1.5 eq.), HOAt (10.0 mg, 0.073 mmol, 2 eq.) and dry DIPEA (50 μL, 0.28 mmol, 7.8 eq.) were dissolved in dry DMF (200 μL) and stirred for 10 minutes at room temperature under nitrogen atmosphere. Then a solution of compound 45¹⁶ (40.8 mg, 0.051 mmol, 1.4 eq.) in dry DMF (300 μL) was added dropwise. The reaction mixture was left stirring at room temperature for 72 h. The solvent was thoroughly removed under reduced pressure, then the crude was re-dissolved in EtOAc (50 mL) and washed with a 5% citric acid solution (30 mL) and with a saturated NaHCO₃ solution (30 mL). The organic phase was then dried over Na₂SO₄, filtered and the solvent was removed under reduced pressure. The crude residue was purified by flash chromatography (Biotage™, reverse phase, from 5% CH₃CN–95% H₂O to 100% CH₃CN as eluent), yielding pure 46 as a white solid (18.1 mg, 0.011 mmol, 30% yield).

¹H NMR (400 MHz, [D₆]acetone, mixture of two conformers) δ 8.51–8.30 (m, 1H), 8.16–8.06 (m, 1H), 7.92–7.44 (m, 4H), 7.59 (bs, 1H), 7.51–7.17 (m, 9H), 6.96 (m, 1H), 6.68 (s, 1H), 6.51 (bs, 1H), 5.44 (m, 1H), 4.68 (m, 1H), 4.59 (m, 1H), 4.57 (m, 1H), 4.53 (m, 1H), 4.30 (m, 1H), 4.26 (m, 1H), 4.20 (m, 1H), 4.14 (m, 1H), 4.09 (m, 0.5H), 4.00 (m, 0.5H), 3.84 (s, 3H), 3.47–3.25 (m, 3H), 3.23–3.06 (m, 8H), 2.99 (m, 1H), 2.80 (s, 3H), 2.72 (m, 1H), 2.67 (s, 3H), 2.61 (s, 3H), 2.55 (m, 1H), 2.44–2.31 (m, 3H), 2.26 (m, 1H), 2.14 (m, 1H), 2.08 (m, 2H), 2.05 (m, 2H), 2.02–1.85 (m, 4H), 1.75–1.18 (m, 52H), 0.88 (m, 3H). ¹³C NMR (75.4 MHz, [D₆]acetone, mixture of two conformers) δ 174.7, 172.2, 171.0, 170.8, 158.8, 157.3, 137.0, 135.9, 131.7, 129.3, 129.0, 128.5, 128.0, 124.7, 112.4, 80.9, 63.3, 62.0, 59.1, 57.8, 56.6, 55.8, 54.3, 52.0, 43.6, 41.1, 40.0, 38.1, 37.4, 36.8, 35.8, 34.2, 33.6, 32.8, 30.5, 30.3, 29.8, 29.3, 29.0, 28.5, 28.2, 27.3, 26.8, 26.3, 24.2, 22.4, 18.6, 14.3, 12.1, 11.1, 8.0. MS (ESI): m/z calcd for C₈₄H₁₂₃N₁₅O₁₈S: 1662.89; found: 1685.9 [M + Na⁺].

Cyclo-RGD/SMAC mimetic conjugate 13. Compound 46 (16.1 mg, 0.0097 mmol, 1 eq.) was dissolved in TFA–thioanisole–1,2-ethanedithiol–anisole 90 : 5 : 3 : 2 (2 mL) and stirred at room temperature for 2 h. After reaction completion, the solvent was evaporated under reduced pressure, then the crude was dissolved in H₂O (10 mL) and washed with iPr₂O (10 mL). The aqueous phase was evaporated under reduced pressure and the crude purified by HPLC (Waters Atlantis Prep T3 OBD 5 μm, 19 × 100 mm, column; solvents: (A) H₂O + 0.1% TFA, (B) CH₃CN + 0.1% TFA gradient from 90%A–10%B to 30%A–70% B over 15 min.; flow rate 15 mL min⁻¹, λ = 210 nm, R_t = 8.8 min). The purified product was then freeze-dried to give the trifluoroacetate salt of the desired pure compound 13 as a white foam (8.4 mg, 0.0059 mmol, 57% yield).

¹H NMR (400 MHz, D₂O) δ: 7.66 (d, 2H), 7.52 (m, 1H), 7.42 (q, 2H, J = 7.8 Hz) 7.38–7.28 (m, 5H), 5.23 (m, 1H), 4.62 (dd, 1H, J = 9.3 Hz, J = 7.3 Hz), 4.55–4.40 (m, 3H), 4.33 (d, 1H, J = 8.4 Hz), 4.25–4.15 (m, 2H), 4.06 (m, 1H), 3.98–3.83 (m, 2H), 3.48–3.29 (m, 3H), 3.24–2.95 (m, 7H), 2.76–2.63 (m, 2H), 2.62 (s, 3H), 2.60 (s, 3H), 2.42–2.30 (m, 3H), 2.21 (m, 1H), 2.15–1.97 (m, 5H), 1.96–1.74 (m, 7H), 1.72–1.50 (m, 6H), 1.49–1.39 (m, 4H), 1.34 (m, 2H), 1.22–1.11 (m, 5H), 1.10–0.99 (m, 8H), 0.95 (t, 3H, J = 7.5 Hz), 0.90 (t, 3H, J = 7.5 Hz). ¹³C NMR (75.5 MHz, D₂O) δ: 178.3, 175.2, 174.6, 173.8, 172.3, 171.5, 171.0, 136.9, 132.9, 129.9, 129.6, 127.9, 127.8, 63.6, 62.9, 62.5, 59.0, 57.3, 57.1, 56.7, 53.7, 52.3, 43.2, 41.2, 40.9, 40.1, 38.3, 35.8, 33.6, 33.0, 32.3, 31.7, 30.9, 29.2, 29.1, 28.3, 27.7, 26.5, 26.1, 25.2, 24.3, 9.0. HRMS (ESI): m/z calcd for C₆₅H₉₅N₁₅O₁₃: 1294.7301; found: 1294.73243 [M + H⁺].

Biology

General procedure for the fluorescence polarization-based BIR/IAP binding assay. The affinity of each tested compound for the BIR domains of XIAP and cIAP2 was evaluated as previously described.^36–38 Recombinant XIAP (60 nM final concentration) and cIAP2 (25 nM) BIR3, or XIAP BIR23 (3 nM) domains were diluted in the assay buffer containing 100 mM potassium phosphate, pH 7.5, 100 μg mL⁻¹ bovine γ-globulin and 0.02% sodium azide. Serial dilutions of each tested compound (concentrations ranging from 2 μM to 0.2 nM) were added to displace a fluorescent probe (FITC-SMAC for BIR3 domains; SMAC-1F for BIR23) and fluorescence polarization was measured with the Ultra plate reader (Tecan), at excitation and emission wavelengths of 485 nm and 530 nm, respectively. All experiments were performed in black, flat-bottom 96 well plates (Greiner Bio-One). Three independent experiments were performed in duplicates. The analyses were performed using Graphpad Prism v.5.02.

General procedure for the solid-phase integrin receptor binding assay. Purified α_vβ₃ and α_vβ₅ receptors (Millipore Corporation, Billerica, MA, USA) were diluted to 0.5 μg mL⁻¹ in coating buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MnCl₂, 2 mM CaCl₂, and 1 mM MgCl₂. An aliquot of diluted receptors (100 μL per well) was added to 96-well microtiter plates (NUNC MW 96F Maxisorp Straight) and incubated overnight at 4 °C. The plates were then incubated with blocking solution (coating buffer plus 1% bovine serum albumin) for an additional 2 h at room temperature to block nonspecific binding, followed by 3 h incubation at room temperature with various concentrations (10⁻⁵–10⁻¹² M) of test compounds in the presence of biotinylated vitronectin (1 μg mL⁻¹). Biotinylation was performed using an EZ-Link Sulfo-NHS-Biotinylation kit (Pierce, Rockford, IL, USA). After washing, the plates were incubated for 1 h at room temperature with biotinylated streptavidin–peroxidase complex (Amersham Biosciences, Uppsala, Sweden) followed by 30 min incubation with 100 μL per well Substrate Reagent Solution (R&D Systems, Minneapolis, MN, USA) before stopping the reaction with the addition of 50 μL per well 2 N H₂SO₄. Absorbance at 415 nm was read in a Synergy™ HT Multi-Detection Microplate Reader (BioTek Instruments, Inc.). Each data point represents the average of triplicate wells; data analysis was carried out by nonlinear regression analysis with GraphPad Prism software. Each experiment was repeated in triplicate.

General procedure for the measurement of cytotoxic activity. Human breast MDA-MB231 and ovarian IGROV-1 carcinoma cell lines were cultured in vitro with RPMI plus 10% fetal calf serum. For cytotoxic experiments, 10⁴ cells were plated in 96 well plates and grown overnight before being treated with serial dilutions of the compounds (concentrations ranging from 25 μM to 10 nM). Cell viability was determined after 72 hours using the CellTiter-Glo (Promega) assay for ATP. Three independent experiments were performed in triplicates.

Acknowledgements

We thank Milan University for PhD Fellowships (to M.M. and A.D.C.). We also gratefully acknowledge “Ministero dell'Università e della Ricerca” for financial support (PRIN project 2010NRREPL: Synthesis and biomedical applications of tumour-targeting peptidomimetics).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Experimental protocols for the synthesis of cyclic RGD ligand–SMAC mimetic conjugate 17 and ¹H and ¹³C NMR spectra for all compounds. See DOI: 10.1039/c4ob00207e

‡ These authors contributed equally to the project.

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