M.
Mingozzi‡
a,
L.
Manzoni‡
b,
D.
Arosio
b,
A.
Dal Corso
a,
M.
Manzotti
a,
F.
Innamorati
a,
L.
Pignataro
a,
D.
Lecis
c,
D.
Delia
c,
P.
Seneci
*a and
C.
Gennari
*a
aUniversità degli Studi di Milano, Dipartimento di Chimica, Via Golgi 19, I-20133, Milan, Italy. E-mail: pierfausto.seneci@unimi.it; cesare.gennari@unimi.it; Fax: +39-02-50314072; Tel: +39-02-50314060 Tel: +39-02-50314091
bIstituto di Scienze e Tecnologie Molecolari, Consiglio Nazionale delle Ricerche, Via Golgi 19, I-20133 Milano, Italy
cFondazione IRCCS Istituto Nazionale dei Tumori, Dipartimento di Oncologia Sperimentale e Medicina Molecolare, Via Amadeo 42, I-20133 Milan, Italy
First published on 21st March 2014
The rational design, synthesis and in vitro biological evaluation of dual action conjugates 11–13, containing a tumour targeting, integrin αvβ3/αvβ5 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 αvβ3/αvβ5 for cyclo-RGD, IAP proteins for SMAC mimetics). Each conjugate portion tolerates different substitutions while preserving the binding affinity for each target.
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.
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 αVβ3 receptor.
Fig. 1 Cyclic RGD ligands of αVβ3 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 protein22,23 (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 studies25,26 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 αV 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 831 (Fig. 2) is highly cytotoxic against cancer cells overexpressing integrins αVβ3/αVβ5 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 932 and the cyclo[DKP-RGD]paclitaxel conjugate 1033 (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. |
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.
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.
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.
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.
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 | >10000 | >10000 | NT |
3 | >10000 | >10000 | NT |
The higher affinity of dual action conjugates 11–13 for cIAP proteins is consistent with what is generally observed for monomeric SMAC mimetics.29
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 | >10000 | >10000 |
6 | >10000 | >10000 |
7 | >10000 | >10000 |
44 | >10000 | >10000 |
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 |
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.
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.
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.
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.
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.
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.
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.
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.
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]+.
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]+.
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+].
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+].
MS (ESI): m/z calcd for C23H40N4O6: 468.29; found: 469.40 [M + H+].
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+].
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+].
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).
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+].
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+].
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+].
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.
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+].
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+].
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. |
This journal is © The Royal Society of Chemistry 2014 |