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The data presented in this study are available within the article and supplementary file.
The integrin family of cell surface proteins plays an important role in the development and spread of cancers. Therefore, drugs which inhibit integrins should make effective cancer treatments. Most potential drugs developed so far target a single integrin and have not proved effective at treating cancer in human studies. Our research aims to develop more effective drugs by targeting two related integrins. This paper describes how these potential drug molecules are made, allowing chemists to make better compounds in the future, and describes the anti-integrin effects of the new compounds. Together this information will lead to the future design and development of better anticancer drugs.
The Arg-Gly-Asp (RGD)-binding family of integrin receptors, and notably the β3 subfamily, are key to multiple physiological processes involved in tissue development, cancer proliferation, and metastatic dissemination. While there is compelling preclinical evidence that both αvβ3 and αIIbβ3 are important anticancer targets, most integrin antagonists developed to target the β3 integrins are highly selective for αvβ3 or αIIbβ3. We report the design, synthesis, and biological evaluation of a new structural class of ligand-mimetic β3 integrin antagonist. These new antagonists combine a high activity against αvβ3 with a moderate affinity for αIIbβ3, providing the first evidence for a new approach to integrin targeting in cancer.
Keywords:
integrin αvβ3, integrin αIIbβ3, metastasis, RGD mimetic, cyclobutane, drug discovery
The integrin family of cell surface glycoproteins control cell&#;extracellular matrix adhesion and signalling across the cell membrane. Functional integrins are present on the cell surface as heterodimers made up of an α and a β subunit; combinations of 18 α and 8 β subunits provide the 24 integrin receptors present in humans [1,2]. Integrins have a wide range of physiological functions, and a number of them have therefore gained considerable interest as drug targets [3,4]. The β3 integrin subfamily comprises two members: αIIbβ3 is normally found only on platelets where it mediates platelet cross-linking in the blood clotting process [5,6]. αvβ3 is most highly expressed on endothelial cells, controlling cell survival and signalling pathways that regulate angiogenesis [7], and thus has become an attractive target for disorders involving neoangiogenesis.
Changes in integrin expression are associated with cancer development and progression, and in particular, the abnormal expression and activity of β3 integrins in tumour cells is associated with cancer progression and metastasis [8,9]. The expression of αvβ3 is strongly associated with advancing disease and poor prognosis. It promotes cell survival [10,11,12], migration [12,13,14], and metastasis via the lymph system [15] and bloodstream [13,14] and is particularly important in the development and growth of bone metastases [16,17,18,19,20,21,22,23,24]. αvβ3 is a stem cell marker [25], and promotes resistance to a number of cytotoxic and targeted chemotherapy agents [26]. The ectopic expression of αIIbβ3 is associated with increased tumour growth and metastatic disease [27,28,29,30,31,32,33]. Haematogenous metastasis is promoted by the interaction of tumour cells and platelets mediated by tumoural β3 integrins and platelet αIIbβ3, resulting in platelet activation and aggregation, the release of growth factors, and increased cell survival in the blood stream in addition to adhesion [34] and invasion at the metastatic site [32,35,36,37,38]. αIIbβ3 antagonists have been shown to be effective in reducing the metastasis of melanoma and breast cancer cells [22,39,40,41]. In cells expressing both β3 integrins, αIIbβ3 can supplant and suppress αvβ3 function [29], suggesting these tumours will be resistant to selective αvβ3 antagonists.
Despite promising preclinical results of β3 antagonists as anticancer agents, the failure of the first-in-class αvβ3 antagonist cilengitide to meet its primary endpoint in Phase II and III clinical trials has discouraged further exploration of αvβ3-targeted anticancer agents [42,43]. A number of reasons for failure have been proposed. αvβ3 antagonists have shown partial agonism and paradoxical effects at low concentrations [44,45], so the rapid clearance of cilengitide in vivo may result in an ineffective target coverage and partial promotion of tumour growth. Closer attention to pharmacokinetics and dosing schedules may be required for successful integrin-targeted therapy [46].
The development of αIIbβ3 antagonists has been similarly challenging. Initial successes with tirofiban, eptifibatide and abciximab as antithrombotic agents in the acute hospital setting encouraged the development of other small molecules. However, multiple failures in clinical trials led to their development being discontinued. Like αvβ3, αIIbβ3 antagonists are liable to paradoxical effects [47]. As antiplatelet agents, they are also prone to bleeding side-effects [48]. However, some studies indicate this is not inevitable [49,50] or can be mitigated by an appropriate dosing strategy [48].
Small molecule β3 antagonists have traditionally been designed to be selective for either αIIbβ3 or αvβ3. For example, cilengitide has a high affinity for αvβ3 but substantially lower anti-αIIbβ3 activity [51]. We have rationalised that dual αIIbβ3/αvβ3 antagonists will have superior anticancer effects due to their ability to antagonise multiple mechanisms involved in tumour cell survival and dissemination and have specific utility in treating tumours characterised by the expression of both β3 integrins or haematogenous metastasis [8]. β3 downregulation, suppressing the expression of both αvβ3 and αIIbβ3 integrins, significantly inhibits tumour growth, invasion, recurrence, and metastasis [14,52,53,54]. Studies with monoclonal anti-αIIbβ3/αvβ3 antibodies, or combinations of selective antagonists, have shown that dual β3 antagonism is effective at blocking tumour growth and angiogenesis through targeting tumour cell interaction with platelets and endothelial cells as well as tumour tissue [55,56,57,58], and is more effective than the use of a single integrin-targeted agent [59]. As a high expression of the β3 integrins is a particular feature of melanoma, the dual antagonist approach may be particularly valuable in treating advanced or high-risk melanomas.
We have recently developed efficient and scalable routes to highly functionalised four-membered rings [60,61], structures that to date have been underexploited in drug design despite their potential for metabolic stability and predictable pharmacokinetics [62,63]. We predicted that these cyclobutanes would possess suitable conformational and pharmacokinetic properties for use as the core scaffold in Arg-Gly-Asp (RGD)-mimetic integrin antagonists controlling the orientation of Arg and Asp mimetic sidechains presented to the integrin. A range of molecules were designed to explore the effects of sidechain identity, orientation, and length on anti-integrin activity with the aim of identifying a safe and effective dual β3 integrin antagonist. This paper describes the synthesis and initial investigation of the biological activity of cyclobutane antagonists employing pyrimidine, naphthyridine, or tetrahydronaphthyridine (THN) groups as the arginine mimetic.
Chemical reagents and anhydrous solvents were obtained from Sigma-Aldrich (Poole, Dorset, UK) and used without further purification. All other solvents were supplied by VWR (Poole, UK). Unless otherwise stated, reactions were carried out in anhydrous solvent and were not air-sensitive. Petroleum ether (PE) refers to the fraction boiling between 60 and 80 °C. Flash chromatography was carried out on silica gel (Merck Kieselgel 60 (230&#;400 ASTM) (VWR) or Davisil 60 A, 40&#;63 μm (Fisher Scientific, Loughborough, UK). Analytical TLC was carried out on 0.25 mm thick aluminium plates precoated with Merck Kieselgel F254 silica gel (VWR) and visualised by UV and aqueous alkaline potassium permanganate solution. Preparative TLC was carried out on Analtech silica plates with UV245 indicator (Sigma-Aldrich). NMR spectra were recorded on a Jeol GX270 or Bruker DPX400 spectrometer (Bruker, Coventry UK). Multiplets are indicated as: s singlet; d doublet; t triplet; q quartet; qn quintet; dd double doublet; dt double triplet; m multiplet; br broad; app apparent. Melting points were determined using a Gallenkamp melting point apparatus (VWR) and are uncorrected.
Sources of biological reagents are specified in each protocol. RGDS was obtained from Sigma-Aldrich, cRGDfV from Enzo Life Sciences (Farmingdale, NY, USA), and {"type":"entrez-nucleotide","attrs":{"text":"GR","term_id":"","term_text":"GR"}}GR from Tocris Bioscience (Bristol, UK).
Human melanoma SK-Mel-2 and M14 cells (ATCC, LGC Standards, Teddington, UK) were cultured in RPMI cell culture medium supplemented with 10% FBS, 1 mM sodium pyruvate and 2 mM L-glutamine (all Sigma) at 37 °C in a humidified atmosphere containing 5% CO2. Cells were not used for more than 10 passages.
The distance between Arg and Asp mimetic sidechains was measured on the minimum energy conformation of the molecule after the molecular geometry was optimised in Arguslab using the PM3 Hamiltonian and a maximum number of steps set to 10,000. Docking studies were carried out using the standard Arguslab docking protocol [64] with Protein Data Bank crystal structures 1TY5 (αIIbβ3) and 1L5G (αvβ3). Ligand groups were created from the previously minimised structure of the compound to be docked and the original ligand (tirofiban or cilengitide, respectively) present in the PDB crystal structure, and the binding site defined by creating a binding-site group from the original ligand. After docking, the 5 lowest energy poses were reviewed.
To a stirred solution of 4-chloro-1-butanol (1.012 g, 9.32 mmol) in DMF (5 mL) was added potassium phthalate (1.727 g, 9.32 mmol) and the reaction mixture heated to 150 °C for 23 h. The reaction mixture was poured into water (50 mL) and extracted with EtOAc (5 × 20 mL). the combined organic layers were concentrated in vacuo and purified by flash column chromatography (EtOAc:PE, 3:7&#;1:1) to yield 2-(4-hydroxy-butyl)-isoindole-1,3-dione (960 mg, 47%) as oily yellow crystals. To a stirred solution of this alcohol (530 mg, 2.42 mmol) in DCM (10 mL) was added MgSO4 (10 g) and PCC (1.565 g 7.26 mmol), and the resulting suspension vigorously stirred for 1.75 h. The reaction mixture was filtered through 1 cm SiO2 washing with EtOAc and the filtrate concentrated in vacuo and purified by flash column chromatography (EtOAc:PE, 2:3&#;1:1) to yield the title compound (404 mg, 77%) as white crystals: Rf 0.20 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 9.77 (t, J = 1.0 Hz, 1H, H-1), 7.83&#;7.85 (m, 2H, ArH), 7.71&#;7.73 (m, 2H, ArH), 3.74 (t, J = 7.1 Hz, 2H, H-4), 2.54 (dt, J = 1.0, 7.1 Hz, 2H, H-2), 2.02 (qn, J = 7.1 Hz, 2H, H-3).
Method of Sall [65]: A mixture of 5-aminopentanol (759 mg, 7.37 mmol) and phthalic anhydride 1.09 g, 7.37 mmol) was heated to 138 °C for 20 h. The resulting brown oil was cooled to room temperature and dissolved in DCM (35 mL). To the resulting solution was added MgSO4 (10 g) and PCC (4.77 g, 22.11 mmol) and the resulting suspension stirred at room temperature for 1 h 10 min. The reaction mixture was filtered through Celite&#; washing with EtOAc. The filtrate was concentrated in vacuo and purified by flash column chromatography (EtOAc:PE, 2:3) to yield the title compound (1.41 g, 83%) as a colourless oil: Rf 0.22 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 9.58 (t, J = 1.5 Hz, 1H, CHO), 7.84 (dd, J = 3.0, 5.6 Hz, 2H), 7.71 (dd, J = 3.0, 5.6 Hz, 2H), 3.71 (t, J = 7.1, 2H), 2.50 (dt, J = 1.5, 7.1, 2H), 1.63&#;1.77 (m, 4H).
6-(1,3-dioxoisoindolin-2-yl)hexanal 3 was prepared from 6-amino-1-hexanol (1.011 g, 8.63 mmol) according to the procedure for 2 to yield the title compound (87 mg, 42%) as a colourless oil: Rf 0.29 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 9.74 (t, J = 1.7 Hz, 1H, CHO), 7.80&#;7.85 (m, 2H, ArH), 7.68&#;7.73 (m, 2H, ArH), 3.68 (t, J = 7.6 Hz, 2H, H-6), 2.43 (dt, J = 1.7, 7.6 Hz, 2H, H-2), 1.68 (sext, J = 7.6 Hz, 4H), 1.33&#;1.41 (m, 2H).
3-(2-methyl-1,3-dioxolan-2-yl)propanal 30 and 4-(2-methyl-1,3-dioxolan-2-yl)butanal 31 were prepared from ethyl levulinate and ethyl 4-acetylbutyrate as described by Shindo et al. [66].
To a stirred solution of 1 (1.078 g, 4.97 mmol) in MeCN (25 mL) was added diethylamine (1.03 mL, 727 mg, 9.94 mmol) and K2CO3 (1.37 g, 9.94 mmol) and the resulting suspension stirred at room temperature for 2 h. Methyl acrylate (1.13 mL, 855 mg, 9.94 mmol) was added and the reaction mixture stirred at room temperature for a further 44 h. The reaction mixture was filtered through Celite&#; and the filtrate concentrated in vacuo. The residue was redissolved in MeCN (28 mL), methyl iodide (1.55 mL, 3.53 g, 24.85 mmol) added, and the resulting solution stirred at room temperature for 2 h. The volatiles were removed in vacuo, and the residue redissolved in DCE (28 mL), DBU (743 μL, 757 mg, 4.97 mmol) added, and the resulting solution heated to 75 °C for 3 h. The reaction mixture was concentrated in vacuo and the residue purified by flash column chromatography (EtOAc:PE, 3:7) to yield the title compound (851 mg, 60% over 3 steps) as a pale yellow oil: Rf 0.29 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 7.83&#;7.86 (m, 2H, ArH), 7.71&#;7.73 (m, 2H, ArH), 6.84 (d, J = 1.3 Hz, 1H, H-2), 3.75 (t, J = 6.9 Hz, 2H, CH2N), 3.72 (s, 3H, OCH3), 2.86 (dd, J = 4.3, 13.5 Hz, 1H, H-4), 2.72&#;2.78 (m, 1H, H-3), 2.32 (dd, J = 1.8, 13.5 Hz, 1H, H-4), 1.84&#;1.94 (m, 2H, CH2). 13C NMR (100 MHz, CDCl3) δ 168.3 (C), 162.9 (C), 148.9 (CH), 137.5 (C), 134.0 (CH), 132.1 (C), 123.4 (CH), 51.4 (CH3), 37.6 (CH), 36.5 (CH2), 34.8 (CH2), 32.0 (CH2). MS (ES+) m/z 308 ([M+Na]+, 45%), 256 ([M-OMe+H]+, 100). HRMS Found 286., C16H16O4N req. 286..
Methyl 3-(3-(1,3-dioxoisoindolin-2-yl)propyl)cyclobut-1-enecarboxylate 5: white solid. mp 74&#;75 °C. Rf 0.46 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 7.79&#;7.84 (m, 2H, ArH), 7.67&#;7.72 (m, 2H, ArH), 6.80 (d, J = 1.0 Hz, 1H, H-2), 3.69 (s, 3H, OCH3), 3.68 (t, J = 7.2 Hz, 2H, CH2N), 2.80 (dd, J = 4.3, 13.3 Hz, 1H, H-4), 2.70&#;2.75 (m, 1H, H-3), 2.23 (dd, J = 1.5, 13.3 Hz, 1H, H-4), 1.65&#;1.75 (m, 2H, CH2CH2N), 1.49&#;1.55 (m, 2H, CH2CH2CH2N). 13C NMR (100 MHz, CDCl3) δ 168.4 (C), 163.0 (C), 149.6 (CH), 137.2 (C), 134.0 (CH), 132.1 (C), 123.2 (CH), 51.3 (CH3), 39.4 (CH), 37.8 (CH2), 34.7 (CH2), 30.4 (CH2), 26.8 (CH2). MS (ES+) m/z 322 ([M+Na]+, 100%), 300 ([M+H]+, 42%). HRMS Found 300., C17H18O4N1 req. 300..
Methyl 3-(4-(1,3-dioxoisoindolin-2-yl)butyl)cyclobut-1-enecarboxylate 6: yellow oil. Rf 0.40 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 7.81&#;7.86 (m, 2H, ArH), 7.68&#;7.73 (m, 2H, ArH), 6.83 (d, J = 1.0 Hz, 1H, H-2), 3.71 (s, 3H, OCH3), 3.68 (dt, 2H, J = 2.3, 7.3 Hz, CH2N), 2.80 (dd, J = 4.3, 13.4 Hz, 1H, H-4), 2.66&#;2.71 (m, 1H, H-3), 2.23 (dd, J = 1.5, 13.3 Hz, 1H, H-4), 1.64&#;1.73 (m, 2H), 1.50&#;1.57 (m, 2H), 1.34&#;1.42 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 168.4 (C), 163.1 (C), 150.1 (CH), 137.1 (C), 133.9 (CH), 132.1 (C), 123.2 (CH), 51.3 (CH3), 39.9 (CH), 37.8 (CH2), 34.8 (CH2), 32.8 (CH2), 28.6 (CH2), 25.1 (CH2). MS (ES+) m/z 314 ([M+H+], 100%). HRMS Found 314., C18H20O4N1 req. 314..
Methyl 3-((2-methyl-1,3-dioxolan-2-yl)methyl)cyclobut-1-enecarboxylate 32: pale yellow oil. Rf 0.44 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 6.85 (s, 1H, H-2), 3.89&#;3.98 m, (4H, OCH2CH2O), 3.72 (s, 3H, OCH3), 2.89 (app qd, J = 2.5, 9.1 Hz, 2H, H-3, H-4), 2.33&#;2.37 (m, 1H, H-4), 1.87 (dd, J = 7.1, 14.1 Hz, 1H, CHH&#;), 1.84 (dd, J = 7.5, 14.1 Hz, 1H, CHH&#;), 1.33 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 163.0 (C), 150.6 (CH), 136.7 (C), 109.7 (C), 64.7 (CH2), 51.3 (CH3), 42.1 (CH2), 35.7 (CH/CH3), 35.5 (CH2), 24.2 (CH/CH3). MS (AP+) m/z 213 ([M+H+], 100%). HRMS Found 235., C11H16O4Na req. 235..
Methyl 3-(2-(2-methyl-1,3-dioxolan-2-yl)ethyl)cyclobut-1-enecarboxylate 33: pale yellow oil. Rf 0.44 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 6.84 (d, J = 1.0 Hz, 1H, H-2), 3.89&#;3.97 (m, 4H, OCH2CH2O), 3.73 (s, 3H, OCH3), 2.82 (dd, J = 4.0, 13.3 Hz, 1H, H-4), 2.69&#;2.74 (m, 1H, H-3), 2.25 (dd, J = 1.5, 13.3 Hz, 1H, H-4&#;), 1.65&#;1.72 (m, 2H), 1.56&#;1.64 (m, 2H), 1.31 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 163.1 (C), 149.9 (CH), 137.2 (C), 109.8 (C), 64.7 (CH2), 51.2 (CH/CH3), 39.9 (CH/CH3), 37.1 (CH2), 34.7 (CH2), 27.6 (CH2), 23.8 (CH/CH3). MS (AP+) m/z 226.8 ([M+H]+, 100%); HRMS Found 227., C12H19O4 req. 227..
A solution of 4 (953 mg, 3.34 mmol) in ethyl acetate (40 mL) was filtered through a 1 cm silica plug. To the filtrate was added 10% Pd/C (95 mg) and the resulting suspension stirred at room temperature under 1 atm H2 for 22.5 h. The reaction mixture was filtered through Celite&#; and concentrated in vacuo to yield the title compound (949 mg, 99%) as a pale yellow oil: Rf 0.29 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 7.69&#;7.71 (m, 2H, ArH), 7.81&#;7.84 (m, 2H, ArH), 3.64 (s, 3H, OCH3), 3.61 (t, J = 6.9 Hz, 2H, CH2NPth), 2.96 (tt, J = 8.3, 9.6 Hz, 1H, H-1), 2.28&#;2.36 (m, 2H, H-2,4), 2.18&#;2.26 (m, 1H, H-3), 1.89&#;1.96 (m, 2H, H-2,4), 1.78 (q, J = 6.9 Hz, 2H, CH2CH2NPth). 13C NMR (100 MHz, CDCl3) δ 175.4 (C), 168.4 (C), 134.0 (CH), 132.1 (C), 123.3 (CH), 51.6 (CH3), 37.1 (CH2), 35.2 (CH2), 34.5 (CH), 31.3 (CH2), 29.4 (CH). MS (ES+) m/z 326 ([M+K+], 100%).
(1r,3s)-methyl 3-(3-(1,3-dioxoisoindolin-2-yl)propyl)cyclobutanecarboxylate 8: white solid. mp 34&#;35 °C. 1H NMR (400 MHz, CDCl3) δ 7.81&#;7.85 (m, 2H), 7.69&#;7.73 (m, 2H), 3.65 (t, J = 7.3 Hz, 2H, CH2N), 3.64 (s, 3H, OCH3), 2.95 (tt, J = 8.6, 9.1 Hz, 1H, H-1), 2.18&#;2.32 (m, 3H, H-2,4, H-3), 1.86 (dq, J = 2.0, 9.6 Hz, 2H, H-2,4), 1.54&#;1.61 (m, 2H), 1.40&#;1.46 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 175.7 (C), 168.5 (C), 133.9 (CH), 132.1 (C), 123.2 (CH), 51.6 (CH3), 37.9 (CH2), 34.2 (CH), 33.7 (CH2), 31.4 (CH2), 31.2 (CH), 25.6 (CH2). MS (ES+) m/z 319 (]M+H2O]+, 78%), 302 ([M+H]+, 100). HRMS Found [M+NH4]+ 319., C17H23O4N2 req. 319..
3-[4-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-butyl]-cyclobutanecarboxylic acid methyl ester 9: white solid. Rf 0.40 (EtOAc:PE, 3:7). mp 67&#;68 °C. 1H NMR (400 MHz, CDCl3) δ 7.81&#;7.85 (m, 2H, ArH), 7.68&#;7.72 (m, 2H, ArH), 3.64 (s, 3H, OCH3), 3.64 (t, J = 6.1 Hz, 2H, CH2NPth), 2.93 (tt, J = 8.3, 9.6 Hz, 1H, H-1), 2.23&#;2.30 (m, 2H, H-2,4), 2.09&#;2.22 (m, 1H, H-3), 1.79&#;1.87 m, (2H, H-2,4), 1.59&#;1.67 (m, 2H, CH2CH2NPth), 1.39&#;1.45 (m, 2H, CH2CH2CH2CH2NPth), 1.19&#;1.27 (m, 2H, CH2CH2CH2NPth). 13C NMR (100 MHz, CDCl3) δ 175.8 (C), 168.5 (C), 133.9 (CH), 132.1 (C), 123.2 (CH), 51.6 (CH3), 38.0 (CH2), 36.2 (CH2), 34.3 (CH), 31.6 (CH), 31.5 (CH2), 28.5 (CH2), 24.2 (CH2). MS (ES+) m/z 354 (100%), 316 ([M+H+], 48).
(1s,3s)-Methyl 3-((2-methyl-1,3-dioxolan-2-yl)methyl)cyclobutanecarboxylate 34: colourless liquid. Rf 0.44 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 3.86&#;3.96 (m, 4H, OCH2CH2O), 3.65 (s, 3H, OCH3), 2.97 (tt, J = 7.6, 9.6 Hz, 1H, H-1), 2.29&#;2.42 (m, H-2,4, 3H, H-3), 1.96 (dq, J = 2.0, 9.6 Hz, 2H, H-2,4), 1.76 (d, J = 6.6 Hz, 2H, CH2), 1.26 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 175.9 (C), 109.8 (C), 64.6 (CH2), 51.6 (CH3), 45.5 (CH2), 35.2 (CH/CH3), 31.3 (CH2), 27.6 (CH/CH3), 24.1 (CH/CH3). MS (AP+) m/z 215 ([M+H+], 100%). HRMS Found 215., C11H19O4 req. 215..
(1r,3s)-Methyl 3-(2-(2-methyl-1,3-dioxolan-2-yl)ethyl)cyclobutanecarboxylate 35: grey oil. Rf 0.44 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 3.87&#;3.96 (m, 4H, OCH2CH2O), 3.65 (s, 3H, OCH3), 2.95 (tt, J = 8.6, 9.6 Hz, 1H, H-1), 2.29 (dq, J = 2.0, 8,6 Hz, 2H, H-2,4), 2.12&#;2.22 (m, 1H, H-3), 1.86 (dq, J = 2.5, 9.6 Hz, 2H, H-2,4), 1.44&#;1.54 (m, 4H, CH2CH2), 1.29 (m, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 175.6 (C), 109.9 (C), 64.6 (CH2), 51.5 (CH3), 36.3 (CH2), 34.2 (CH/CH3), 31.7 (CH/CH3), 31.4 (CH2), 31.0 (CH2), 23.7 (CH/CH3). MS (AP+) m/z 229 ([M+H]+, 100%). HRMS Found 229., C12H21O4 req. 229..
To a stirred solution of 5 (500 mg, 1.67 mmol) in acetone (6 mL), water (4 mL), and conc. HCl (10 mL) was added Zn (326 mg, 5.02 mmol) and the reaction mixture heated to reflux. Four further portions of Zn (326 mg, 5.02 mmol) were added at hourly intervals. One hour after the final addition (total reaction time 6 h), the reaction mixture was cooled to room temperature, diluted with water (50 mL), and extracted with EtOAc (6 × 20 mL). The combined organic layers were dried (MgSO4), filtered, and concentrated in vacuo to yield crude 3-[3-(1-oxo-1,3-dihydro-isoindol-2-yl)-propyl]-cyclobutanecarboxylic acid as a yellow oil: 1H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 7.6 Hz, 1H), 7.53 (dt, J = 1.5, 7.6 Hz, 1H), 7.45 (brt, J = 7.6 Hz, 2H), 4.38 (brs, 2H), 3.62 (brt, J = 7.1 Hz, 2H, CH2NH), 3.06&#;3.13 (m, 1H, H-1), 2.36&#;2.43 (m, 3H), 1.84&#;1.93 (m, 3H), 1.56&#;1.63 (m, 2H), 1.47&#;1.52 (m, 2H); MS (ES+) m/z 274 ([M+H+], 100%). To a stirred solution of this crude product in acetone (39 mL) was added Jones&#; reagent (2.78 mL of a 2.7 M solution, 7.52 mmol) and the resulting solution stirred at room temperature for 18.33 h. The reaction was quenched by the dropwise addition of IPA and the resulting solution filtered through Celite, washing with EtOAc, and the filtrate concentrated in vacuo to yield crude 3-[3-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-propyl]-cyclobutanecarboxylic acid: Rf 0.08 (EtOAc:PE, 3:7). mp 101&#;103 °C (from CDCl3). 1H NMR (400 MHz, CDCl3) δ 7.81&#;7.86 (m, 2H, ArH), 7.68&#;7.73 (m, 2H, ArH), 3.67 (t, J = 7.1 Hz, 2H, CH2N), 3.05&#;3.12 (m, 1H, H-1), 2.35&#;2.43 (m, 3H), 1.87&#;1.95 (m, 2H), 1.54&#;1.64 (m, 2H), 1.46&#;1.51 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 181.6 (C), 168.5 (C), 133.9 (CH), 132.1 (C), 123.2 (CH), 38.0 (CH2), 34.7 (CH), 33.5 (CH2), 31.8 (CH), 30.2 (CH2), 26.2 (CH2). MS (ES+) m/z 310 ([M+Na]+, 100%). HRMS Found (M+NH4+) 305., C16H21O4N2 req. 305.. To a stirred solution of this crude product in methanol (25 mL) was added SOCl2 (131 μL, 218 mg, 1.84 mmol) and the resulting solution heated to reflux for 27.5 h. The reaction mixture was concentrated in vacuo and the residue purified by flash column chromatography (EtOAc:PE, 3:7) to yield the title compound (343 mg, 68%) as a colourless oil: Rf 0.42 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 7.82&#;7.85 (m, 2H, ArH), 7.69&#;7.72 m, (2H, ArH), 3.67 (s, 3H, OCH3), 3.64&#;3.68 m, (2H, CH2N), 3.00&#;3.11 (m, 1H, H-1), 2.32&#;2.43 (m, 3H, H-2,4, H-3), 1.84&#;1.89 (m, 2H, H-2,4), 1.55&#;1.63 (m, 2H), 1.45&#;1.53 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 176.7 (C), 168.4 (C), 133.9 (CH), 132.1 (C), 123.2 (CH), 51.7 (CH3), 38.0 (CH2), 34.7 (CH), 33.5 (CH2), 31.8 (CH), 30.2 (CH2), 26.2 (CH2). MS (ES+) m/z 324 ([M+Na]+, 100%). Found (M+NH4+) 319., C17H23O4N2 req. 319..
3-[2-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-ethyl]-cyclobutanecarboxylic acid methyl ester 10: colourless oil. Rf 0.37 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 7.81&#;7.84 (m, 2H), 7.69&#;7.71 (m, 2H), 3.66 (s, 3H, OCH3), 3.61 (t, J = 7.6 Hz, 2H), 3.05&#;3.14 (s, 1H), 2.35&#;2.50 (m, 3H), 1.90&#;1.98 (m, 2H), 1.75&#;1.86 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 176.4 (C), 168.4 (C), 134.1 (CH), 132.1 (C), 123.4 (CH), 51.9 (CH3), 36.5 (CH2), 35.8 (CH2), 35.7 (CH), 30.0 (CH2), 29.7 (CH). MS (ES+) m/z 305 ([M+H2O]+,100%), 288 ([M+H+], 65). HRMS Found 288., C16H18O4N req. 288..
To a stirred solution of 33 (308 mg, 1.36 mmol) in acetone (5.5 mL), water (3.7 mL), and concentrated HCl (8.9 mL) was added Zn (354 mg, 5.45 mmol) and the reaction mixture heated to reflux. Five further portions of Zn (5 × 354 mg, 5 × 5.45 mmol) were added at 2 hourly intervals, then the reaction mixture heated to reflux for 14 h, diluted with water (20 mL), and extracted with EtOAc (3 × 10 mL). The combined organic layers were dried (MgSO4), filtered, and concentrated in vacuo. The residue was redissolved in methanol (24 mL), thionyl chloride (106 μL, 178 mg, 1.50 mmol) added, and the reaction mixture heated to reflux for 24 h. The solvent was removed in vacuo and the residue purified by flash column chromatography (EtOAc:PE, 1:4) to yield the title compound (192 mg, 76%) as a brown oil: Rf 0.41 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 3.68 (s, 3H, OCH3), 3.03&#;3.11 (m, 1H, H-1), 2.26&#;2.42 (m, 5H, H-2,4, H-3, COCH2), 2.14 (s, 3H, CH3CO), 1.84&#;1.91 (m, 2H, H-2,4), 1.72 (q, J = 7.6 Hz, 2H, COCH2CH2). 13C NMR (100 MHz, CDCl3) δ 208.8 (C), 51.7 (CH/CH3), 41.3 (CH2), 34.6 (CH/CH3), 31.6 (CH/CH3), 30.1 (3CH2), 30.0 (CH/CH3). MS (ES+) m/z 202 ([M+H2O]+, 100%). HRMS Found [M+NH4]+ 202., C18H20O3N req. 202..
To a stirred solution of 8 (51 mg, 0.169 mmol) in MeOH (15 mL) was added methylamine (650 μL of a 40% aqueous solution, 7.8 mmol) and the reaction mixture stirred at room temperature for 2 h. The reaction mixture was concentrated in vacuo and azeotroped with toluene (10 mL). The crude residue was dissolved in nBuOH (2 mL). 2-Chloropyrimidine (40 mg, 0.254 mmol) and DIPEA (44 μL, 33 mg, 0.254 mmol) were added and the resulting solution heated to 100 °C for 21 h. The reaction mixture was concentrated in vacuo and purified by flash column chromatography (EtOAc:PE, 3:7&#;1:0) to yield the title compound (25 mg, 59%) as a colourless oil: Rf 0.11 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 4.5 Hz, 2H, H-4&#;, 6&#;), 6.50 (t, J = 4.5 Hz, 1H, H-5&#;), 5.09 (brs, 1H, NH), 3.65 (s, 3H, OCH3), 3.37 (appq, J = 7.0 Hz, 2H, CH2NH), 2.96 (appdqn, J = 1.5, 8.1 Hz, 1H, H-1), 2.26&#;2.34 (m, 2H, H-2,4), 2.14&#;2.26 (m, 1H, H-3), 1.87 (appdq, J = 2.5, 9.6 Hz, 2H, H-2,4), 1.45&#;1.56 (m, 4H, CH2CH2). 13C NMR (100 MHz, CDCl3) δ 175.7 (C), 162.4 (C), 158.0 (CH), 110.4 (CH), 51.6 (CH3), 41.3 (CH2), 34.2 (CH), 33.9 (CH2), 31.5 (CH2), 31.4 (CH), 36.9 (CH2). MS (ES+) m/z 250 ([M+H]+, 100%). HRMS Found 250., C13H20O3N2 req. 250..
(1s,3s)-methyl 3-(2-(pyrimidin-2-ylamino)ethyl)cyclobutanecarboxylate 12: pale yellow oil. Rf 0.11 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 4.8 Hz, 2H, H-4&#;, 6&#;), 6.52 (t, J = 4.8 Hz, 1H, H-5&#;), 5.42 (brs, 1H, NH), 3.65 (s, 3H, OCH3), 3.34 (~q, J = 5.8 Hz, 2H, CH2NH), 2.94&#;3.02 (m, 1H, H-1), 2.27&#;2.37 (m, 3H, H-2,4, H-3), 1.89&#;1.99 (m, 2H, H-2,4), 1.72 (q, J = 6.8 Hz, 2H, CH2CH2NH). 13C NMR (100 MHz, CDCl3) δ 175.5 (C), 161.8 (C), 157.8 (CH), 110.3 (CH), 51.6 (CH3), 39.3 (CH2), 36.4 (CH2), 34.5 (CH), 31.4 (CH2), 29.5 (CH). MS (ES+) m/z 236 ([M+H+], 100%).
3-[4-(Pyrimidin-2-ylamino)-butyl]-cyclobutanecarboxylic acid methyl ester 14: white solid. Rf 0.17 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 4.8 Hz, 2H, H-4&#;,6&#;), 6.50 (t, J = 4.8 Hz, 1H, H-5&#;), 5.18 (br, 1H, NH), 3.65 (s, 3H, OCH3), 3.37 (dt, J = 6.0, 7.0 Hz, 2H, CH2N), 2.94 (tt, J = 8.3, 9.3 Hz, 1H, H-1), 2.23&#;2.31 (m, 2H, H-2,4), 2.11&#;2.23 (m, 1H, H-3), 1.80&#;1.88 (m, 2H, H-2,4), 1.57 (qn, J = 7.3 Hz, 2H, CH2CH2N), 1.39&#;1,45 (m, 2H, CH-3CH2), 1.23&#;1.32 m, (2H, CH2CH2CH2N). 13C NMR (100 MHz, CDCl3) δ 175.8 (C), 162.4 (C), 158.0 (CH), 110.4 (CH), 51.6 (CH3), 41.4 (CH2), 36.4 (CH2), 35.0 (CH), 31.7 (CH), 31.6 (CH2), 29.5 (CH2), 24.3 (CH2). MS (ES+) m/z 264 ([M+H]+, 100%). Found 264., C14H22O2N3 req. 264..
3-[2-(Pyrimidin-2-ylamino)-ethyl]-cyclobutanecarboxylic acid methyl ester 15: pale yellow oil. Rf 0.07 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 4.5 Hz, 2H, H-4&#;,6&#;), 6.50 (t, J = 4.5 Hz, 1H, H-6&#;), 5.27 (brm, 1H, NH), 3.67 (s, 3H, OCH3), 3.33 (td, J = 6.0, 7.1 Hz, 2H, CH2N), 3.06&#;3.14 (m, 1H, H-1), 2.45&#;2.53 (m, 1H, H-3), 2.36&#;2.43 (m, 2H, H-2,4), 1.91&#;1.98 (m, 2H, H-2,4), 1.76 (q, J = 7.1 Hz, 2H, CH2CH2NH). 13C NMR (100 MHz, CDCl3) δ 176.5 (C), 162.4 (C), 158.0 (CH), 110.4 (CH), 51.7 (CH3), 39.4 (CH2), 36.0 (CH2), 34.9 (CH), 31.5 (CH2), 29.9 (CH). MS (ES+) m/z 236 ([M+H+],100%). HRMS Found 236., C12H18O2N3 req. 236..
(1s,3r)-Methyl 3-(3-(pyrimidin-2-ylamino)propyl)cyclobutanecarboxylate 16: colourless oil. Rf 0.10 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 5.1 Hz, 2H, H-4&#;,6&#;), 6.51 (t, J = 5.1 Hz, 1H, H-5&#;), 5.08 (br, 1H, NH), 3.68 (s, 3H, OCH3), 3.36&#;3.42 (m, 2H, CH2NH), 3.04&#;3.10 (m, 1H, H-1), 2.33&#;2.44 (m, 3H, H-2,4, H-3), 1&#;83-1.92 (m, 2H, H-2,4), 1.52&#;1.55 (m, 4H, CH2CH2). 13C NMR (100 MHz, CDCl3) δ 176.7 (C), 162.4 (C), 158.1 (CH), 110.5 (CH), 51.7 (CH3), 41.4 (CH2), 34.8 (CH), 33.6 (CH2), 31.9 (CH), 30.3 (CH2), 27.2 (CH2). MS (ES+) m/z 250 ([M+H+],100%). Found 250., C13H20O3N2 req. 250..
To a stirred solution of 35 (1.26 g, 5.61 mmol) in methanol (50 mL) was added 5% aqueous HCl (10 mL) and the reaction mixture stirred at room temperature for 1.3 h. The volatiles were removed in vacuo and the residue purified by flash column chromatography (EtOAc:PE, 1:4) to yield (1r,3s)-methyl 3-(3-oxobutyl)cyclobutanecarboxylate (950 mg, 93%) as a pale yellow liquid: Rf 0.38 (EtOAc:PE, 3:7). 1H NMR (400 MHz, CDCl3) δ 3.66 (s, 3H, OCH3), 2.95 (tt, J = 8.6, 9.6 Hz, 1H, H-1), 2.32 (t, J = 7.6 Hz, 2H, COCH2), 2.29 (dq, J = 2.0, 8.6 Hz, 2H, H-2,4), 2.15&#;2.23 (m, 1H, H-3), 2.12 (s, 3H, CH3CO), 1.87 (dq, J = 2.5, 9.6 Hz, 2H, H-2,4), 1.67 (q, J = 7.6 Hz, 2H, COCH2CH2). 13C NMR (100 MHz, CDCl3) δ 208.6 (C), 175.5 (C), 51.6 (CH3), 40.9 (CH2), 34.1 (CH/CH3), 31.2 (CH2), 31.0 (CH/CH3), 30.5 (CH2), 29.8 (CH/CH3). MS (AP+) m/z 185 ([M+H]+, 100%). HRMS Found [M+NH4]+ 202., C10H20O3N req. 202.. To a stirred solution of (1r,3s)-methyl 3-(3-oxobutyl)cyclobutanecarboxylate (118 mg, 0.641 mmol) in methanol (11.8 mL) was added 2-aminonicotinaldehyde (87 mg, 0.705 mmol), pyrrolidine (59 μL, 50 mg, 0.705 mmol), and conc. H2SO4 (1 drop), and the resulting solution stirred at room temperature for 24 h. The reaction mixture was concentrated in vacuo and purified by flash column chromatography (EtOAc) to yield the title compound (178 mg, 100%) as a pale yellow oil: Rf 0.05 (EtOAc:PE, 1:1). 1H NMR (400 MHz, CDCl3) δ 9.06 (dd, J = 1.5, 5.1 Hz, 1H, H-7&#;), 8.13 (dd, J = 2.0, 8.0 Hz, 1H, H-6&#;), 8.07 (d, J = 8.1 Hz, 1H, H-4&#;), 7.42 (dd, J = 4.0, 8.1 Hz, 1H, H-5&#;), 7.35 (d, J = 8.6 Hz, 1H, H-3&#;), 3.64 (s, 3H, OCH3), 2.89&#;2.98 (m, 3H, H-2,4, H-1), 2.27&#;2.34 (m, 3H, H-2,4, H-3), 1.96&#;2.02 (m, 2H), 1.89&#;1.96 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 175.6 (C), 166.4 (C), 156.0 (C), 153.3 (CH), 136.9 (CH), 136.6 (CH), 122.4 (CH), 121.4 (CH), 121.0 (C), 51.5 (CH3), 36.6 (CH2), 36.1 (CH2), 34.2 (CH), 31.5 (CH2), 31.4 (CH). MS (AP+) m/z 271 ([M+H]+, 100%); HRMS Found 271., C16H19O2N2 req. 271..
(1s,3s)-Methyl 3-((1,8-naphthyridin-2-yl)methyl)cyclobutanecarboxylate 37: pale yellow oil. Rf 0.06 (EtOAc:PE, 1:1). 1H NMR (400 MHz, CDCl3) δ 9.07 (dd, J = 2.0, 4.5 Hz, 1H, H-7&#;), 8.14 (dd, J = 2.0, 8.1 Hz, 1H, H-5&#;), 8.08 (d, J = 8.1 Hz, 1H, H-4&#;), 7.43 (dd, J = 4.0, 8.1 Hz, 1H, H-6&#;), 7.33 (d, J = 8.1 Hz, 1H, H-3&#;), 3.66 (s, 3H, OCH3), 3.15 (d, J = 7.6 Hz, 2H, ArCH2), 3.00 (tt, J = 8.6, 9.6 Hz, 1H, H-1), 2.94 (ttt, J = 7.6, 8.1, 9.6 Hz, 1H,), 2.37 (mq, J = 8.1 Hz, 2H, H-2,4), 2.12 (dq, J = 2.5, 9.6 Hz, 2H, H-2,4). 13C NMR (100 MHz, CDCl3) δ 175.6 (C), 164.7 (C), 156.1 (C), 153.4 (CH), 137.0 (CH), 136.7 (CH), 122.7 (CH), 121.5 (CH), 121.1 (C), 51.7 (CH3), 45.8 (CH2), 34.5 (CH), 31.6 (CH2), 31.4 (CH). MS (AP+) m/z 257 ([M+H+], 100%). HRMS Found 257., C15H17O2N2 req. 257..
(1s,3r)-methyl 3-(2-(1,8-naphthyridin-2-yl)ethyl)cyclobutanecarboxylate 39: yellow oil. Rf 0.03 (EtOAc:PE, 1:1). 1H NMR (400 MHz, CDCl3) δ 9.06 (br, 1H), 8.14 (dd, J = 2.0, 8.1 Hz, 1H), 8.08 (d, J = 8.6 Hz, 1H), 7.42 (dd, J = 4.5, 8.1 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 3.66 (s, 3H, OCH3), 3.05&#;3.14 (m, 1H, H-1), 2.94&#;2.97 (m, 2H, CH2Napth), 2.35&#;2.52 (m, 3H, H-2,4, H-3), 2.05 (td, J = 7.5, 9.6 Hz, 2H, NapthCH2CH2), 1.91&#;1.98 (m, 2H, H-2,4). 13C NMR (100 MHz, CDCl3) δ 176.7 (C), 166.4 (C), 156.0 (C), 153.2 (CH), 137.0 (CH), 136.8 (CH), 122.6 (CH), 121.5 (CH), 121.0 (C), 52.1 (CH3), 36.9 (CH2), 35.9 (CH2), 34.8 (CH), 33.1 (CH), 32.0 (CH2). MS (AP+) m/z 271 ([M+H]+, 100%); HRMS Found 271., C16H19O2N2 req. 271..
(R) and (S)-3-Amino-2-benzenesulfonylamino-propionic acid were prepared as described by Egbertson et al. [67]. (S)-3-amino-2-(2,4,6-trimethylphenylsulfonamido)propanoic acid was prepared as described by Pitts et al. [68].
To a stirred solution of 3-amino-2-benzenesulfonylamino-propionic acid (918 mg, 3.76 mmol) in methanol (20 mL) was added thionyl chloride (300 μL, 492 mg, 4.13 mmol) and the reaction mixture stirred at room temperature for 23.5 h. The solvent was removed in vacuo and the residue dissolved in saturated aqueous NaHCO3 solution (40 mL) and extracted with EtOAc (8 × 15 mL). The combined organic layers were dried (MgSO4) and concentrated in vacuo to yield the title compound (324 mg, 33%) as a colourless oil: Rf 0.10 (DCM:MeOH, 95:5): 1H NMR (400 MHz, CDCl3) δ 7.84 (d, J = 8.6 Hz, 2H,), 7.57 (tt, J = 2.0, 7.1 Hz, 1H,), 7.50 (dt, J = 1.5. 8.1 Hz, 2H,), 3.92 (t, J = 4.7, 1H,), 3.52 (s, 3H), 3.02 (dd, J = 4.5, 13.6 Hz, 1H), 2.98 (dd, J = 5.1, 13.1 Hz, 1H), Lit [69].
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(S)-methyl 3-amino-2-(2,4,6-trimethylphenylsulfonamido)propanoate 20: pale yellow oil. Rf 0.13 (DCM:MeOH, 95:5). 1H NMR (400 MHz, CDCl3) δ 6.94 (s, 2H, ArH), 3.83 (t, J = 4.5 Hz, 1H, H-2), 3.56 (s, 3H, OCH3), 3.00 (dd, J = 4.5, 13.1 Hz, 1H, H-3), 2.98 (dd, J = 4.5, 13.1 Hz, 1H, H-3), 2.64 (s, 6H, ArCH3), 2.28 (s, 3H, ArCH3), Lit [70].
Compound 12 (27 mg, 0.115 mmol) was dissolved in 6M HCl and stirred at room temperature overnight. The solvent was removed in vacuo and the residue dissolved in DMF (4 mL). (S)-methyl 3-amino-2-trimethylphenylsulfonamidopropanoate (34.5 mg, 0.115 mmol), EDCI hydrochloride (66 mg, 0.345 mmol), HOBt (47 mg, 0.345 mmol), and DIPEA (100 μL, 74 mg, 0.575 mmol) were added sequentially and the reaction mixture stirred at room temperature overnight. The reaction mixture was diluted with water (10 mL) and extracted with EtOAc (3 × 5 mL). The combined organic layers were washed with water (2 × 10 mL), concentrated in vacuo, and purified by PTLC (DCM:MeOH, 97:3) to yield the title compound (20 mg, 34.5%) as a colourless oil: Rf 0.37 (DCM:MeOH, 95:5). [α] D20 + 11.5 (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 4.5 Hz, 2H, H-4&#;,6&#;), 6.94 (s, 2H, ArH), 6.50 (t, J = 4.5 Hz, 1H, H-5&#;), 5.88&#;5.93 (m, 2H, 2NH), 5.14 (vbrt, 1H, NHAr), 3.86 (dt, J = 4.0, 6.6 Hz, 1H, CHCHH&#;), 3.58&#;3.65 (m, 1H, CHH&#;N), 3.58 (s, 3H, OCH3), 3.48&#;3.56 (m, 1H, CHH&#;N), 3.32 (appq, J = 7.1 Hz, 2H, CH2NHAr), 2.76&#;2.84 (m, 1H, H-1), 2.62 (s, 6H, ArCH3), 2.25&#;2.31 (m + s, 6H, ArCH3, H-3, H-2,4), 1.86&#;1.96 (m, 2H, H-2,4), 1.71 (q, J = 6.6 Hz, 2H, CH2CH2NHAr). 13C NMR (100 MHz, CDCl3) δ 175.3 (C), 170.3 (C), 162.3 (C), 158.1 (CH), 142.7 (C), 139.2 (C), 133.0 (C), 132.1 (CH), 110.4 (CH), 55.3 (CH/CH3), 53.1 (CH/CH3), 41.8 (CH2), 39.3 (CH2), 36.3 (CH), 36.2 (CH2), 31.5 (CH2), 31.3 (CH2), 29.3 (CH/CH3), 22.9 (CH/CH3), 21.0 (CH/CH3). MS (ES+) m/z 504 ([M+H]+, 100%). HRMS Found 504., C24H34O5N5S req. 504..
3-({3-[3-(Pyrimidin-2-ylamino)-propyl]-cyclobutanecarbonyl}-amino)-propionic acid methyl ester 22: white crystals. mp 94&#;95 °C. Rf 0.35 (DCM:MeOH, 95:5). 1H NMR (400 MHz, CDCl3) δ 8.24 (d, J = 5.1, 2H, H-4&#;,6&#;), 6.49 (t, J = 5.1 Hz, 1H, H-4&#;), 5.95 (vbrt, 1H, NHβ-ala), 5.13 (br, 1H, ArNH), 3.69 (s, 3H, OCH3), 3.49 (q, J = 6.1 Hz, 2H, CH2CH2CO2Me), 3.35 (q, J = 7.1 Hz, 2H, ArNHCH2), 2.74 (td, J = 8.1, 9.6 Hz, 1H, H-1), 2.52 (t, J = 6.1 Hz, 2H, CH2CO2Me), 2.14&#;2.27 (m, 3H, H-2,4, H-3), 2.14&#;2.27 (m, 2H, H-2,4), 1.43&#;1.55 (m, 4H, CH2CH2). 13C NMR (100 MHz, CDCl3) δ 174.6 (C), 173.3 (C), 162.4 (C), 158.0 (CH), 110.4 (CH), 51.8 (CH3), 41.3 (CH2), 36.2 (CH), 34.7 (CH2), 33.8 (CH2), 33.6 (CH2), 3.14 (CH2), 31.1 (CH), 27.0 (CH2). MS (ES+) m/z 321 ([M+H]+, 100%). HRMS Found 321., C16N25O3N4 req. 321..
(S)-methyl 2-(phenylsulfonamido)-3-((1r,3r)-3-(3-(pyrimidin-2-ylamino)propyl)cyclobutanecarboxamido)propanoate 23: pale yellow oil. Rf 0.05 (DCM:MeOH, 95:5). [α] D20 + 32.9 (c 0.65, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 4.6 Hz, H-4&#;,6&#;2H,), 7.82 (d, J = 7.1 Hz, 2H, ArH), 7.54 (t, J = 7.6 Hz, 1H, ArH), 7.50 (t, J = 7.6 Hz, 2H, ArH), 6.49 (t, J = 4.5 Hz, 1H, H-5&#;), 6.31 (brs, 1H, NHSO2Ph), 5.97 (t, J = 6.1 Hz, 1H, NHCO), 5.30 (brm, 1H, NHAr), 3.99 (brt, J = 5.1 Hz, 1H, CHNHSO2Ph), 3.55 (s, 3H, OCH3), 2.51&#;3.58 (m, 2H, CHH&#;CH), 3.36 (q, J = 6.1 Hz, 2H, CH2NHAr), 2.78 (qn, J = 8.6 Hz, 1H, H-1), 2.14&#;2.28 (m, 3H, H-2,4, H-3), 1.76&#;1.86 (m, 2H, H-2,4), 1.47&#;1.53 (m, 4H, CH2CH2CH2NHAr). 13C NMR (100 MHz, CDCl3) δ 176.7 (C), 170.1 (C), 162.0 (C), 158.0 (CH), 139.3 (C), 133.1 (CH), 129.2 (CH), 127.1 (CH), 110.3 (CH), 55.7 (CH/CH3), 53.0 (CH/CH3), 41.7 (CH2), 41.3 (CH2), 36.0 (CH/CH3), 33.7 (CH2), 31.4 (CH2), 31.2 (CH/CH3), 26.8 (CH2). MS (ES+) m/z 476 ([M+H]+, 100%). HRMS Found 476., C22H30O5N5S req. 476..
(S)-methyl 3-((1r,3r)-3-(pyrimidin-2-ylamino)propyl)cyclobutane-1-carboxamido)-2-((2,4,6-trimethylphenyl)sulfonamido)propanoate 24: pale yellow oil. Rf 0.27 (DCM:MeOH, 95:5); [α] D20 + 7.8 (c 0.6, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 5.1 Hz, 2H, H-4&#;,6&#;), 6.94 (s, 2H, ArH), 6.50 (t, J = 5.1 Hz, 1H, H-5&#;), 6.06 (brd, J = 7.1 Hz, 1H, NHSO2Ar), 5.90 (brt, J = 5.7 Hz, 1H, NHCO), 5.20 (brt, J = 5.6 Hz, 1H, NHAr), 3.83&#;3.88 (m, 1H, CHCHH&#;), 3.58 (s, 3H, OCH3), 3.45&#;3.56 (m, 2H, CHCHH&#;), 3.34&#;3.40 (m, 2H, CH2NHAr), 2.78 (qn, J = 8.6 Hz, 1H, H-1), 2.62 (s, 6H, ArCH3), 21.6&#;2.30 (m + s, ArCH3, 6H, H-3, H-2,4), 1.77&#;1.87 (m, 2H, H-2,4), 1.46&#;1.55 (m, 4H, CH2CH2CH2NHAr). 13C NMR (100 MHz, CDCl3) δ 175.5 (C), 170.3 (C), 162.4 (C), 158.1 (CH), 146.8 (C), 142.7 (C), 139.2 (C), 132.1 (CH), 110.4 (CH), 55.2 (CH/CH3), 53.1 (CH/CH3), 41.7 (CH2), 41.3 (CH2), 36.1 (CH/CH3), 33.7 (CH2), 31.4 (CH/CH3), 31.3 (CH2), 26.9 (CH2), 22.9 (CH/CH3), 21.0 (CH/CH3). MS (ES+) m/z 518 ([M+H]+, 100%). HRMS Found 518., C25H36O5N5S1 req. 518..
(R)-methyl 2-(phenylsulfonamido)-3-((1r,3s)-3-(3-(pyrimidin-2-ylamino)propyl)cyclobutanecarboxamido)propanoate 25: pale yellow oil. Rf 0.05 (DCM:MeOH, 95:5); [α] D20 &#; 26.99 (c 0.715, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 4.6 Hz, 2H, H-4&#;,6&#;), 7.82 (d, J = 7.1 Hz, 2H, ArH), 7.54 (t, J = 7.6 Hz, 1H, ArH), 7.50 (t, J = 7.6 Hz, 2H, ArH), 6.49 (t, J = 4.5 Hz, 1H, H-5&#;), 6.31 (brs, 1H, NHSO2Ph), 5.97 (t, J = 6.1 Hz, 1H, NHCO), 5.30 (brm, 1H, NHAr), 3.99 (brt, J = 5.1 Hz, 1H, CHNHSO2Ph), 3.55 (s, 3H, OCH3), 2.51&#;3.58 (m, 2H, CHH&#;CH), 3.36 (q, J = 6.1 Hz, 2H, CH2NHAr), 2.78 (qn, J = 8.6 Hz, 1H, H-1), 2.14&#;2.28 (m, 3H, H-2,4, H-3), 1.76&#;1.86 (m, 2H, H-2,4), 1.47&#;1.53 (m, 4H, CH2CH2CH2NHAr). 13C NMR (100 MHz, CDCl3) δ 176.7 (C), 170.1 (C), 162.0 (C), 158.0 (CH), 139.3 (C), 133.1 (CH), 129.2 (CH), 127.1 (CH), 110.3 (CH), 55.7 (CH/CH3), 53.0 (CH/CH3), 41.7 (CH2), 41.3 (CH2), 36.0 (CH/CH3), 33.7 (CH2), 31.4 (CH2), 31.2 (CH/CH3), 26.8 (CH2). MS (ES+) m/z 498 ([M+Na]+, 63%), 476 ([M+H]+, 100). HRMS Found [M+H+] 476., C22H30N5O5S req. 476..
(S)-methyl 3-((1r,3r)-3-(4-(pyrimidin-2-ylamino)butyl)cyclobutanecarboxamido)-2-(2,4,6-trimethylphenylsulfonamido)propanoate 26: pale yellow oil. Rf 0.18 (DCM:MeOH, 95:5). [α] D20 + 25.2 (c 1.05, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 5.1 Hz, 2H, H-4&#;,6&#;), 6.94 (s, 2H, ArH), 6.50 (t, J = 5.1 Hz, 1H, H-5&#;), 5.96 (brd, J = 7.6 Hz, 1H, NHSO2Ar), 5.89 (brt, J = 5.7 Hz, 1H, NHCO), 5.19 (vbrt, J = 5.5 Hz, 1H, NHAr), 3.86 (dt, J = 4.0, 7.6 Hz, 1H, CHNHSO2Ar), 3.57 (s, 3H, CO2CH3), 3.47&#;3.66 (m, 2H, CH2NHCO), 3.37 (q, J = 7.1 Hz, 2H, CH2NHAr), 2.70 (tt, J = 8.6, 9.6 Hz, 1H, H-1), 2.61 (s, 6H, ArCH3), 2.28 (s, 3H, ArCH3), 2.11&#;2.28 (m, 3H, H-2,4, H-1), 1.76&#;1.84 (m, 2H, H-2,4), 1.57 (qn, J = 7.1 Hz, 2H, CH2CH2NHAr), 1.39&#;1.44 (m, 2H, CH2CH2CH2CH2NHAr), 1.25&#;1.32 (m, 2H, CH2CH2CH2NHAr). 13C NMR (100 MHz, CDCl3) δ 175.6 (C), 170.3 (C), 162.3 (C), 158.1 (CH), 142.7 (C), 139.2 (C), 134.3 (C), 132.1 (CH), 123.5 (C), 110.3 (CH), 55.3 (CH/CH3), 53.0 (CH/CH3), 41.7 (CH2), 41.4 (CH2), 36.2 (CH2), 36.1 (CH), 31.4 (CH2), 31.3 (CH), 29.4 (CH2), 24.2 (CH2), 22,9 (CH3), 21.0 (CH3). MS (ES+) m/z 532 ([M+H]+, 100%). HRMS Found 532., C26H38O5N5S1 req. 532..
(S)-methyl 3-((1r,3s)-3-(2-(pyrimidin-2-ylamino)ethyl)cyclobutanecarboxamido)-2-(2,4,6-trimethylphenylsulfonamido)propanoate 27: pale yellow oil. Rf 0.38 (DCM:MeOH, 95:5). [α] D20 + 26.4 (c 1.23, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 5.1 Hz, 2H, H-4&#;,6&#;), 6.94 (s, 2H, ArH), 6.50 (t, J = 5.1 Hz, 1H, H-5&#;), 5.96 (brt, J = 6.1 Hz, 2H, 2NH), 5.18 (brt, J = 5.1 Hz, 1H, ArNH), 3.88 (brm, 1H, CHNHSO2Ar), 3.57 (s, 3H, OCH3), 3.52&#;3.65 (m, 2H, CH2NHCO), 3.33 (dt, J = 6.1, 7.1 Hz, 2H, CH2NH), 2.90&#;2.97 (m, 1H, H-1), 2.61 (s, 6H, 2ArCH3), 2.28 (s, 3H, ArCH3), 2.33&#;2.48 (m, 3H, H-2,4, H-3), 1.86&#;1.95 (m, 2H, H-2,4), 1.76 (q, J = 7.1 Hz, 2H, CH2CH2NH). 13C NMR (100 MHz, CDCl3) δ 176.2 (C), 170.3 (C), 162.4 (C), 158.0 (CH), 142.6 (C), 139.2 (C), 133.1 (C), 132.1 (CH), 110.4 (CH), 55.3 (CH/CH3), 53.0 (CH/CH3), 41.8 (CH2), 39.5 (CH2), 36.3 (CH/CH3), 36.0 (CH2), 31.3 (CH2), 30.9 (CH/CH3), 22.9 (CH/CH3), 20.9 (CH/CH3). MS (ES+) m/z 504 (M+H+, 100%). HRMS Found 504., C24H34O5N5S req. 504..
(S)-methyl 3-((1s,3s)-3-(3-(pyrimidin-2-ylamino)propyl)cyclobutanecarboxamido)-2-(2,4,6-trimethylphenylsulfonamido)propanoate 28: pale yellow oil. Rf 0.30 (DCM:MeOH, 95:5). [α] D20+ 34.4 (c 1.23, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 4.5 Hz, 2H, H-4&#;,6&#;), 6.94 (s, 2H, ArH), 6.50 (t, J = 4.5 Hz, 1H, H-5), 6.05 (br, 1H, NH), 5.94 (t, J = 5.7 Hz, 1H, NHCO), 5.25 (vbrt, 1H, NHCH2), 3.89 (brt, 1H, CHNHSO2Ph), 3.57 (s, 3H, OCH3), 3.49&#;3.64 (m, 2H, CH2CHNHSO2Ph), 3.36&#;3.43 (m, 2H, NHCH2), 2.86&#;2.93 (m, 1H, H-1), 2.62 (s, 6H, ArCH3), 2.29&#;2.34 (m, 3H, H-2,4, H-3), 2.28 (s, 3H, ArCH3), 1.77&#;1.86 (m, 2H, H-2,4), 1.51&#;1.54 (m, 4H, NHCH2CH2CH2). 13C NMR (100 MHz, CDCl3) δ 176.4 (C), 170.3 (C), 162.4 (C), 158.0 (CH), 143.6 (C), 139.2 (C), 133.2 (C), 132.0 (CH), 110.4 (CH), 55.4 (CH/CH3), 53.0 (CH/CH3), 41.7 (CH2), 41.4 (CH2), 36.4 (CH/CH3), 34.1 (CH2), 31.8 (CH/CH3), 30.5 (CH2), 27.2 (CH/CH3), 22.9 (CH/CH3), 20.9 (CH/CH3). MS (ES+) m/z 518 ([M+H]+, 100%). HRMS Found 518., C25H36O5N5S req. 518..
(S)-methyl 2-(phenylsulfonamido)-3-((1s,3s)-3-(3-(pyrimidin-2-ylamino)propyl)cyclobutanecarboxamido)propanoate 29: pale yellow oil. Rf 0.22 (DCM:MeOH, 95:5). [α] D20 + 16.8 (c 1.4, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.27 (d, J = 5.1 Hz, 2H, H-4&#;,6&#;), 7.83 (td, J = 1.5, 7.5 Hz, 2H, ArH-o), 7.57 (tt, J = 1.5, 7.5 Hz, 1H, ArH-p), 7.49 (t, J = 7.6 Hz, 2H, ArH-m), 6.50 (t, J = 5.1 Hz, 1H, H-5&#;), 6.26 (vbr, 1H, NH), 5.99 (brt, J = 6.3 Hz, 1H, NHCO), 5.33 (brt, J = 5.1 Hz, 1H, NHAr), 4.02 (dd, J = 4.5, 6.7 Hz, 1H, CH2CHNHSO2Ph), 3.55 (s, 3H, OCH3), 3.52&#;3.61 (m, 2H, CH2CHNHSO2Ph), 3.38 (brq, J = 6.1 Hz, 2H, CH2NHAr), 2.85&#;2.93 (m, 1H, H-1), 2.24&#;2.37 (m, 3H, H-2,4, H-3), 1.78&#;1.85 (m, 2H, H-2,4), 1.51&#;1.53 (m, 4H, CH2CH2CH2NHAr). 13C NMR (100 MHz, CDCl3) δ 176.5 (C), 170.2 (C), 162.4 (C), 158.1 (CH), 139.4 (C), 133.2 (CH), 129.2 (CH), 127.1 (CH), 110.4 (CH), 55.8 (CH/CH3), 53.0 (CH/CH3), 41.8 (CH2), 41.4 (CH2), 36.4 (CH/CH3), 33.6 (CH2), 31.8 (CH/CH3), 30.4 (CH2), 30.3 (CH2), 27.1 (CH2). MS (ES+) m/z 476 ([M+H]+, 100%). HRMS Found 476., C22H30O5N5S req. 476..
(S)-methyl 3-((1r,3r)-3-(2-(1,8-naphthyridin-2-yl)ethyl)cyclobutanecarboxamido)-2-(phenylsulfonamido)propanoate 40: colourless oil. [α] D20 + 0.057 (c 1.35, CHCl3). Rf 0.15 (DCM:MeOH, 95:5). 1H NMR (400 MHz, CDCl3) δ 9.08 (dd, J = 2.0, 4.0 Hz, 1H) 8.16 (dd, J = 2.0, 8.1 Hz, 1H), 8.09 (dd, J = 8.1 Hz, 1H), 7.82 (dd, J = 1.5, 7.6 Hz, 2H, PhH-o), 7.56 (tt, J = 1.5, 7.6 Hz, 1H, PhH-p), 7.49 (tm, J = 7.6 Hz, 2H, PhH-m), 7.44 (dd, J = 4.0, 8.1 Hz, 1H), 7.38 (d, J = 8.6 Hz, 1H), 6.13 (brt, J = 6.1 Hz, 1H, NH), 6.07 (vbr, 1H, NH), 4.01 (dd, J = 4.0, 6.1 Hz, 1H, CHNHSO2Ph), 3.57 (s, 3H, OCH3), 3.49&#;3.65 (m, 2H, CHH&#;NH), 2.97 (dd, J = 7.6, 8.1 Hz, 2H, CH2Napth), 2.70 (tt, J = 8.1, 9.1 Hz, 1H, H-1), 2.20&#;2.33 (m, 3H, H-2,4, H-3), 1.98 (brq, J = 7.1 Hz, 2H, H-2,4), 1.83&#;1.89 (m, 2H, CH2). 13C NMR (100 MHz, CDCl3) δ 175.6 (C), 170.1 (C), 166.6 (C), 155.9 (C), 153.3 (CH), 139.6 (C), 137.0 (CH), 136.8 (CH), 132.9 (CH), 129.3 (CH), 127.1 (CH), 122.6 (CH), 121.4 (CH), 121.1 (C), 55.7 (CH/CH3), 52.9 (CH/CH3), 41.6 (CH2), 26.6 (CH2), 36.0 (CH), 35.8 (CH2), 31.3 (CH2), 31.2 (CH). MS (ES+) m/z 497 ([M+H]+, 77%), 241 (55), 121 (100). HRMS Found 497., C25H29O5N4S req. 497..
(S)-methyl 3-((1r,3r)-3-(2-(1,8-naphthyridin-2-yl)ethyl)cyclobutanecarboxamido)-2-(2,4,6-trimethylphenylsulfonamido)propanoate 41: colourless oil. [α] D20 + 0.24 (c 0.5, CHCl3). Rf 0.39 (DCM:MeOH, 95:5). 1H NMR (400 MHz, CDCl3) δ 9.08 (dd, J = 2.0, 4.0 Hz, 1H), 8.15 (dd, J = 2.0, 8.1 Hz, 1H), 8.09 (d, J = 8.1, 1H), 7.44 (dd, J = 4.0, 8.1 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 6.93 (s, 2H), 5.98 (t, J = 6.1 Hz, 1H, NH), 5.89 (d, J = 7.1 Hz, 1H, NH), 3.89 (dt, J = 4.0, 7.1 Hz, 1H, CHNHSO2), 3.61 (ddd, J = 4.5, 6.6, 14.1 Hz, 1H, CHH&#;NH), 3.58 s, (3H, OCH3), 3.51 (td, J = 6.1, 14.1 Hz, 1H, CHH&#;NH), 2.97 (dd, J = 7.1, 8.1 Hz, 2H, ArCH2), 2.71&#;2.81 (m, 1H, H-1), 2.61 (s, 6H, ArCH3), 2.28 (s, 3H, ArCH3), 2.21&#;2.31 (m, 3H, H-2,4, H-3), 1.99 (brq, J = 6.6 Hz, 2H, H-2,4), 1.84&#;1.90 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 175.5 (C), 170.3 (C), 166.5 (C), 157.6 (C), 153.3 (CH), 142.6 (C), 139.2 (C), 136.9 (CH), 136.7 (CH), 133.3 (C), 132.0 (CH), 122.5 (CH), 121.4 (CH), 121.0 (C), 55.4 (CH/CH3), 52.9 (CH/CH3), 41.7 (CH2), 36.7 (CH2), 36.1 (CH/CH3), 35.8 (CH2), 31.4 (CH2), 31.3 (CH2), 31.2 (CH/CH3), 22.8 (CH/CH3), 20.9 (CH/CH3). MS (ES+) m/z 539 ([M+H]+, 100%). HRMS Found 539., C28H35O5N4S req. 539..
Methyl 3-((1r,3s)-3-(2-(1,8-naphthyridin-2-yl)ethyl)cyclobutanecarboxamido)propanoate 42: white crystals. mp 101&#;102 °C. Rf 0.17 (DCM:MeOH, 95:5). 1H NMR (400 MHz, CDCl3) δ 9.06 (dd, J = 2.0, 4.0 Hz, 1H), 8.15 (dd, J = 2.0, 8.1 Hz, 1H), 8.08 (d, J = 8.6 Hz, 1H), 7.43 (dd, J = 4.5, 8.1 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 5.97 (vbrt, 1H, NH), 3.68 (s, 3H, OCH3), 3.49 (q, J = 6.1 Hz, 2H, NHCH2), 2.95 (dd, J = 6.1, 7.6 Hz, 2H, ArCH2), 2.71&#;2.79 (m, 1H, H-1), 2.52 (t, J = 6.1 Hz, 2H, NHCH2CH2), 2.21&#;2.29 (m, 3H, H-2,4, H-3), 1.97 (brq, J = 7.3 Hz, 2H, ArCH2CH2), 1.82&#;1.92 (m, 2H, H-2,4). 13C NMR (100 MHz, CDCl3) δ 174.7 (C), 173.2 (C), 166.5 (C), 155.9 (C), 153.3 (CH), 137.0 (CH), 136.8 (CH), 122.5 (CH), 121.4 (CH), 121.0 (C), 51.8 (CH3), 36.7 (CH2), 36.2 (CH), 36.1 (CH2), 34.7 (CH2), 33.9 (CH2), 31.8 (CH2), 31.4 (CH). MS (AP+) m/z 342 ([M+H]+, 100%). HRMS Found 364. (M+Na+), C19H23O3N3Na req. 364..
(S)-methyl 3-((1s,3r)-3-((1,8-naphthyridin-2-yl)methyl)cyclobutanecarboxamido)-2-(2,4,6-trimethylphenylsulfonamido)propanoate 43: colourless oil. [α] D20 + 0.127 (c 0.55, CHCl3). Rf 0.28 (DCM:MeOH, 95:5). 1H NMR (400 MHz, CDCl3) δ 9.08 (dd, J = 1.5, 4.0 Hz, 1H, H-7&#;), 8.16 (dd, J = 1.5, 8.1 Hz, 1H, H-5&#;), 8.05 (d, J = 8.6 Hz, 1H, H-4&#;), 7.45 (dd, J = 4.5, 8.1 Hz, 1H, H-6&#;), 7.34 (d, J = 8.6 Hz, 1H, H-3&#;), 6.91 (s, 2H, ArH), 6.44 (t, J = 6.1 Hz, 1H, NHSO2Mes), 6.20 (br, 1H, NH), 3.93 (dd, J = 4.0, 7.1 Hz, 1H, CHNHSO2Mes), 3.65 (dd, J = 4.5, 6.6, 14.1 Hz, 1H, CHH&#;CH), 3.56 (s, 3H, OCH3), 3.48&#;3.59 (m 1H, CHH&#;CH), 3.15 (d, J = 7.1 Hz, 2H, NapthCH2), 2.79&#;3.05 (m, 2H, H-1,3), 2.60 (s, 6H, ArCH3), 2.26 (s, 3H, ArCH3), 2.26&#;2.39 (m, 2H, H-2,4), 2.10&#;2.21 (m, 2H, H-2,4). 13C NMR (100 MHz, CDCl3) δ 175.7 (C), 170.4 (C), 164.9 (C), 155.9 (C), 153.3 (CH), 142.5 (C), 139.2 (C), 136.9 (CH), 133.3 (CH), 123.0 (CH), 121.1 (CH), 55.5 (CH/CH3), 53.4 (CH2), 52.8 (CH/CH3), 45.0 (CH2), 41.6 (CH2), 36.4 (CH/CH3), 31.4 (CH2), 30.8 (CH/CH3), 23.0 (CH/CH3), 20.9 (CH/CH3). MS (AP+) m/z 525 ([M+H]+, 9%), 121 (100). HRMS Found 525., C27H33O5N4S req. 525..
(S)-methyl 3-((1s,3s)-3-(2-(1,8-naphthyridin-2-yl)ethyl)cyclobutanecarboxamido)-2-(2,4,6-trimethylphenylsulfonamido)propanoate 44: pale brown gum. Rf 0.30 (DCM:MeOH, 95:5). [α] D20 + 0.134 (c 1.55, CHCl3). 1H NMR (400 MHz, CDCl3) δ 9.07 (dd, J = 2.0, 4.0 Hz, 1H), 8.16 (dd, J = 2.0, 8.1 Hz, 1H), 8.09 (d, J = 8.6 Hz, 1H), 7.44 (dd, J = 4.0, 8.1 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 6.92 (s, 2H, ArH), 6.16 (brt, J = 6.1 Hz, 1H, NHCH2), 6.07 (vbrd, J = 5.1 Hz, 1H, NHSO2Ar), 3.89 (br, 1H, CHNHSO2Ar), 3.56 (s, 3H, OCH3), 3.53&#;3.64 (m, 2H, NHCHH&#;), 2.90&#;2.97 (m, 3H, NapthCH2, H-1), 2.27 (s, 6H, ArCH3), 2.29&#;2.43 (m, 3H, H-2,4, H-3), 2.27 (s, 3H, ArCH3), 2.04 (td, J = 7.6, 10.6 Hz, 2H, NapthCH2CH2), 1.84&#;1.92 (m, 2H, H-2,4); 13C NMR (100 MHz, CDCl3) δ 176.4 (C), 170.4 (C), 166.5 (C), 156.0 (C), 153.5 (CH), 142.6 (C), 139.2 (C), 137.1 (CH), 136.8 (CH), 133.2 (C), 132.2 (CH), 122.7 (CH), 121.5 (CH), 121.1 (C), 55.4 (CH/CH3), 52.9 (CH/CH3), 41.7 (CH2), 37.0 (CH2), 36.3 (CH/CH3), 35.8 (CH2), 31.9 (CH/CH3), 30.3 (CH2), 30.1 (CH2), 22.9 (CH/CH3), 21.0 (CH/CH3). MS (ES+) m/z 539 ([M+H]+, 100%). HRMS Found 539., C28H35O5N4S req. 539..
To a stirred solution of 43 (24 mg, 0.046 mmol) in methanol (5 mL) was added PtO2 (4 mg) and the reaction mixture stirred under 1 atm H2 for 24 h. The reaction mixture was filtered through Celite&#; and concentrated in vacuo to yield the title compound (21 mg, 87.5%) as a colourless oil: [α] D20 + 0.038 (c 1.05, MeOH). Rf 0.28 (DCM:MeOH, 95:5). 1H NMR (400 MHz, CDCl3) δ 8.25 (br, 1H), 7.33 (d, J = 6.6 Hz, 1H), 6.92 (s, 2H), 6.37 (dd, J = 7.6 Hz, 1H), 6.27 (br, 1H), 3.96 (br, 1H), 3.46&#;3.57 (m, 5H), 3.18 (qn, J = 7.6 Hz, 1H), 2.52&#;2.95 (m, 4H), 1.23&#;2.35 (m, 8H), 1.85&#;2.05 (m, 6H), 1.40.1.47 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 172.2 (C), 170.2 (C), 168.7 (C), 148.1 (C), 142.6 (C), 140.7 (CH), 139.2 (C), 131.9 (CH), 110.4 (CH), 119.2 (C), 114.7 (C), 55.4 (CH/CH3), 52.7 (CH/CH3), 41.4 (CH2), 41.1 (CH2), 39.2 (CH2), 35.8 (CH/CH3), 30.7 (CH2), 30.4 (CH/CH3), 30.4 (CH2), 25.5 (CH2), 23.0 (CH/CH3), 20.9 (CH/CH3), 19.3 (CH2). MS (AP+) m/z 529 ([M+H]+, 8%), 246 (63), 200 (100). HRMS Found 529., C27H37O5N4S req. 529..
(S)-methyl 2-(phenylsulfonamido)-3-((1r,3r)-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)cyclobutanecarboxamido)propanoate 45: yellow oil. [α] D20 + 0.113 (c 0.15, CHCl3). Rf 0.41 (DCM:MeOH, 95:5). 1H NMR (400 MHz, CDCl3) δ 7.85 (dd, J = 1.5, 8.1 Hz, 2H, ArH-o), 7.58 (tt, J = 1.5, 8.1 Hz, 1H, ArH-p), 7.50 (dt, J = 1.5, 8.1 Hz, 2H, ArH-m), 7.12 (d, J = 7.4 Hz, 1H, H-4&#;), 6.32 (d, J = 7.4 Hz, 1H, H-3&#;), 6.14 (br, 1H, NH), 5.89 (vbr, 1H, NH), 3.91 (t, J = 5.2, 1H, CHNHSO2Ph), 3.57&#;3.60 (m, 2H, CHH&#;CHNHSO2Ph), 3.57 (s, 3H, CH3), 3.42 (dt, J = 2.5, 6.0, 2H, H-7&#;), 2.81 (qn, J = 8.6, 1H, H-1), 2.70 (t, J = 6.1 Hz, 2H, H-5&#;), 2.48 (dt, J = 3.0, 8.6 Hz, 2H, ArCH2), 2.14&#;2.32 (m, 3H, H-2,4, H-3), 1.82&#;1.94 (m, 4H, H-2,4, H-6&#;), 1.77 (q, J = 7.5 Hz, 2H, ArCH2CH2). 13C NMR (100 MHz, CDCl3) δ 175.8 (C), 170.1 (C), 139.5 (C), 137.7 (C), 133.0 (CH), 129.2 (CH), 127.2 (CH), 124.1 (C), 110.8 (CH), 55.8 (CH/CH3), 53.0 (CH/CH3), 41.5 (2CH2), 36.6 (CH2), 36.1 (CH/CH3), 31.3 (CH2), 31.0 (CH2), 31.0 (CH/CH3), 26.1 (CH2), 20.9 (CH2). MS (ES+) m/z 502 ([M+H]+, 100%). HRMS Found 501., C25H33O5N4S req. 501..
(S)-methyl 3-((1r,3r)-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)cyclobutanecarboxamido)-2-(2,4,6-trimethylphenylsulfonamido)propanoate 46: colourless oil. [α] D20 + 0.113 (c 0.3, CHCl3). Rf 0.27 (DCM:MeOH, 95:5). 1H NMR (400 MHz, CDCl3) δ 7.12 (d, J = 7.3 Hz, 1H, H-4&#;), 6.94 (s, 2H, ArH), 6.33 (d, J = 7.3 Hz, 1H, H-3&#;), 6.03 (br, 1H, NH), 5.88 (vbr, 2H, 2NH), 3.89 (dd, J = 4.0, 6.6 Hz, 1H, CHNHSO2), 3.57 (s, 3H, OCH3), 3.49&#;3.64 (m, 2H, NHCHH&#;CH), 3.41&#;3.44 (m, 2H, H-7&#;), 2.79 (qn, J = 8.6 Hz, 1H, H-1), 2.70 (t, J = 6.3 Hz, 2H, H-5&#;), 2.62 (s, 6H, ArCH3), 2.48 (dd, J = 7.6, 8.1 Hz, 2H, ArCH2CH2), 2.29 (s, 3H, ArCH3), 2.16&#;2.99 (m, 3H, H-2,4, H-3), 1.83&#;1.94 (m, 4H, H-6&#;+H-2,4), 1.77 (q, J = 7.6 Hz, 2H, ArCH2CH2). 13C NMR (100 MHz, CDCl3) δ 175.6 (C), 170.3 (C), 155.1 (C), 142.6 (C), 139.5 (C), 139.2 (C), 137.2 9 (C), 133.2 (C), 132.0 (3 CH), 111.0 (CH), 55.4 (CH/CH3), 52.9 (CH/CH3), 41.6 (CH2), 41.5 (CH2), 36.5 (CH/CH3), 36.1 (CH2), 34.5 (CH2), 31.3 (CH2), 31.2 (CH2), 31.0 (CH/CH3), 26.2 (CH2), 23.1 (CH/CH3), 21.2 (CH/CH3). MS (ES+) m/z 543 ([M+H]+, 100%). HRMS Found 543., C28H39O5N4S req. 543..
Methyl 3-((1r,3s)-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)cyclobutanecarboxamido)propanoate 47: white crystals. mp 117&#;119 °C. Rf 0.31 (DCM:MeOH, 95:5). 1H NMR (400 MHz, CDCl3) δ 7.05 (d, J = 7.1 Hz, 1H, H-4&#;), 6.30 (d, J = 7.6 Hz, 1H, H-3&#;), 5.96 (brt, 1H, NHCH2), 4.95 (brs, 1H, NH-8&#;), 3.69 (s, 3H, OCH3), 3.49 (q, J = 6.0 Hz, 2H, CH2NH), 3.39 (dt, J = 2.5, 6.1 Hz, 2H, H-7&#;), 2.75 (tt, J = 8.1, 9.6 Hz, 1H, H-1), 2.68 (t, J = 6.0 Hz, 2H, H-5&#;), 2.53 (t, J = 6.0 Hz, 2H, CH2CH2NH), 2.43 (dd, J = 6.1, 7.6 Hz, 2H, CH2Ar), 2.13&#;2.33 (m, 3H, H-2,4, H-3), 1.79&#;1.92 (m, H-6&#;, 4H, H-2,4), 1.73 (td, J = 7.1, 9.6 Hz, 2H, CH2CH2Ar). 13C NMR (100 MHz, CDCl3) δ 174.8 (C), 173.3 (C), 157.7 (C), 155.5 (C), 136.8 (CH), 113.4 (C), 111.1 (CH), 51.8 (CH3), 41.6 (CH2), 36.6 (CH2), 36.3 (CH), 35.0 (CH2), 34.7 (CH2), 33.9 (CH2), 31.4 (CH2), 31.1 (CH), 26.3 (CH2), 21.4 (CH2). MS (AP+) m/z 346 ([M+H]+, 100%), 239 (67). HRMS Found 346., C19H28N3O3 req. 346..
(S)-methyl 3-((1s,3s)-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)cyclobutanecarboxamido)-2-(2,4,6-trimethylphenylsulfonamido)propanoate 49: yellow oil. Rf 0.50 (DCM:MeOH, 95:5). [α] D20 + 0.11 (c 1.05, CHCl3). 1H NMR (400 MHz, CDCl3) δ 7.20 (d, J = 7.1 Hz, 1H), 6.93 (s, 2H, ArH), 6.35 (m, 1H, NHCH), 6.32 (d, J = 7.1 Hz, 1H), 3.90 (dd, J = 4.5, 6.0 Hz, 1H, CHNHSO2Ar), 3.54 (s, 3H, OCH3), 3.51&#;3.60 (m, 2H, CHH&#;CH), 2.91&#;2.98 (m, 2H, NHCH2CH2CH2), 2.91&#;2.98 (m, 1H, H-1), 2.71 (brt, J = 6.1 Hz, 2H, NHCH2CH2CH2), 2.62 (s, 6H, ArCH3), 2.53&#;2.65 (m, 2H, PyrCH2), 2.29&#;2.39 (m, 3H, H-2,4, H-3), 2.28 (s, 3H, ArCH3), 1.82&#;1.92 (m, 6H, NHCH2CH2CH2 + H2,4 + PyrCH2CH2). 13C NMR (100 MHz, CDCl3) δ 176.5 (C), 170.3 (C), 151.7 (C), 142.5 (C), 139.2 (C), 139.0 (CH), 133.3 (C), 132.0 (CH), 127.6 (C), 122.5 (C), 110.5 (CH), 55.5 (CH/CH3), 52.78 (CH/CH3), 41.7 (CH2), 41.3 (CH2), 36.4 (CH/CH3), 36.0 (CH2), 31.6 (CH/CH3), 30.3 (CH2), 30.2 (CH2), 25.9 (CH2), 23.1 (CH/CH3), 21.0 (CH/CH3), 20.3 (CH2). MS (ES+) m/z 543 ([M+H]+, 100%). HRMS Found 543., C28H39O5N4S req. 543..
Compound purity was estimated from the integration of 1H NMR spectra: The following compounds contained no detectable organic impurities (purity > 95%): 21, 22, 27, 28, 40, 41, 42, 44, 45, 46, and 48. Other compounds had the following purities: 23, 94%; 24, 90%; 25, 95%; 26, 95%; 29, 90%; 43, 85%; 47, 90%; and 49 85%. This is a limitation of the work.
(S)-2-(phenylsulfonamido)-3-((1r,3r)-3-(3-(pyrimidin-2-ylamino)propyl) cyclobutanecarboxamido)propanoic acid 51 as a yellow oil. Compound 23 was dissolved in 6 M aqueous HCl (1 mL) and stirred at room temperature for 23.5 h. The volatiles were removed in vacuo to yield the title compound which was used immediately. MS (ES+) m/z 479 ([M+H2O]+, 100%). HRMS Found 479. ([M+H2O]+), C21H29N5O6S req. 479..
(S)-3-((1s,3r)-3-(2-(pyrimidin-2-ylamino)ethyl)cyclobutanecarboxamido)-2-(2,4,6-trimethylphenylsulfonamido)propanoic acid 50: yellow oil. MS (ES-) m/z 488 ([M-H]&#;, 100%). HRMS Found 488., C23H30O5N5S req. 488..
(S)-3-((1r,3s)-3-(2-(pyrimidin-2-ylamino)ethyl)cyclobutanecarboxamido)-2-(2,4,6-trimethylphenylsulfonamido)propanoic acid 52: yellow oil. MS (ES+) m/z 507 ([M+H2O, 100%), 490 ([M+H+], 54%). HRMS found 488.([M-H]-). C23H30O5N5S req. 488..
(S)-3-((1s,3s)-{3-[3-(Pyrimidin-2-ylamino)-propyl]-cyclobutanecarbonyl}-amino)-2-(2,4,6-trimethyl-benzenesulfonylamino)-propionic acid 53: yellow oil. MS (ES+) m/z 504 ([M+H]+ 100%). HRMS Found 504., C24H34N5O5S req. 504..
(S)-2-(phenylsulfonamido)-3-((1s,3s)-3-(3-(pyrimidin-2-ylamino)propyl) cyclobutanecarboxamido)propanoic acid 54: yellow oil. MS (ES+) m/z 479 ([M+H2O]+, 100%). HRMS Found 479. ([M+H2O]+), C21H29N5O6S req. 479..
(S)-3-{(1r,3r)-[3-(2-[1,8] Naphthyridin-2-yl-ethyl)-cyclobutanecarbonyl]-amino}-2-(2,4,6-trimethyl-benzenesulfonylamino)-propionic acid 55: yellow oil. MS (ES+) m/z 525 ([M+H]+, 100%). HRMS Found 525.; C27H33O5N4S req. 525..
(S)-3-((1r,3r)-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)cyclobutanecarboxamido)-2-(2,4,6-trimethylphenylsulfonamido)propanoic acid 56: yellow oil. MS (ES+) m/z 529.4 ([M+H]+, 100%). Found 527. C27H35O5N4S req.527..
ELISA assays were performed as previously described [71] with minor modifications. Briefly, serial dilutions of compounds were prepared in dimethyl sulfoxide (DMSO). C8 Maxi Immuno modules (Fisher Scientific) were incubated overnight at 4 °C with 0.5 μg/well of fibrinogen (Sigma) in sterile phosphate buffered saline (PBS, 0.2 g/L KCl, 8.0 g/L NaCl, 0.2 KH2PO4, 1.15 g/L Na2HPO4, pH 7.4). All subsequent wash steps were performed using 25 mM Tris, pH 7.6, 150 mM NaCl, 1 mM MnCl2, 1 mg/mL BSA, and binding/inhibition was carried out in 25 mM Tris, pH 7.6, 150 mM NaCl, 1 mM MnCl2, 1 mM MgCl2, 1 mM CaCl2, 1 mg/mL BSA.
Wells were washed and blocked with a blocking solution (PBS, 0.1% Tween 20, 3% BSA) for 1 h at 37 °C. Compounds were added to the wells at indicated concentrations (final 0.5% DMSO) in the presence of 0.5 µg/well αIIbβ3 (Sigma) and incubated at room temp for 1 h. After 3 washes, primary anti-αIIb (1:200 dilution, Santa Cruz Biotech, Heidelberg, Germany) and antigoat&#;HRP (1:500 dilution, Dako, Agilent, Santa Clara, CA, USA) were added at room temp for 1 h. The wells were washed and incubated with 0.1 mg/mL tetra-methylbenzidine (Sigma) for 25 min and the reaction was stopped with 1N H2SO4 (100 μL/well). The absorbance was measured at 450 nm using a Multiscan Spectrum reader (Thermo Scientific, Thermofisher, UK) and SkanIT RE (v2.4.4.5) software. Results were plotted as percent binding vs. log concentration and IC50 values determined.
First, 96-well plates (Corning, VWR) were coated overnight at 4 °C with 0.5 μg/well fibronectin (Sigma). SK-Mel-2 cells were trypsinised, washed and resuspended in RPMI medium only at 105 cells/mL. Cells (100 µL/well) were treated with compound (DMSO final concentration 0.1%) at the indicated concentration for 4 hrs on a rotary shaker. Plates were washed (3 × PBS) and blocked with PBS/5% BSA for 2 hrs at 37 °C prior to adding the cells. Cells were incubated on the plates at 37 °C in a humidified chamber with 5% CO2 for 1 hr. The plates were washed 3× with PBS and a 200 µL/well RPMI medium containing 10% FCS added. The plates were incubated overnight at 37 °C as above. Finally, 0.5 mg/mL of 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was added to each well and the plates incubated for a further 4 h. The medium was removed, and the insoluble formazan dissolved in 150 µL of DMSO. Absorbance was measured at 540 nm using a Thermo Multiskan EX (Thermofisher, UK)and Ascent Software (v2.6). Total binding was determined based on controls lacking any compounds (100% binding) in fibronectin-coated wells and uncoated wells blocked with BSA (0% binding) and corrected for background (no cells, fibronectin-coated).
M14 cells (4 × 105/mL in RPMI medium) were seeded in six-well plates and incubated at 37 °C in a 5% CO2 humidified atmosphere for 48 h. The resulting confluent monolayer was scratched with a sterile P200 pipette tip to create a gap (approximately 2 cm in length and 650 μm in width) in the centre of the well. The medium was removed, and the cells washed with Hank&#;s balanced salt solution (HBSS) (1 mL) and replaced with a medium containing the test antagonist. After 24 h, the cells were washed twice with HBSS and fixed with ice-pre-cooled methanol for thirty minutes at &#;20 °C. Following hydration with two washes in PBS, the monolayers were counterstained with Harris&#;s haematoxylin solution for two minutes, then washed in tap water for one minute and left to dry at room temperature. The plates were observed using an inverted microscope, and digital images captured. The scratch width was measured at ten positions throughout the scratch area and the % inhibition calculated by comparing the average migration into the scratch at 24 h to that of the untreated control. Immunolabelling for Ki-67 (AB, Chemicon Millipore Watford, Watford, UK) showed low levels of nuclear expression after 24 h, confirming the migration rather than proliferation.
Platelet aggregation was measured in hirudin-anticoagulated whole blood from a healthy donor with ADP as agonist (end concentration 6.4 μM) using a Multiplate® impedance aggregometer (Dynabyte Informationssysteme GmbH, Munich, Germany). At 37 °C, cells were charged with 300 μL of saline, 300 μL of blood, 1 μL of compound (0.1% DMSO) and incubated for 3 min. The agonist was added and the increase in electrical impedance measured from 2 electrode pairs/cell for 6 min, transformed into arbitrary units (AU), and the area under the curve was calculated.
Metastatic dissemination of melanoma, including haematogenous metastasis, is still a clinically relevant problem despite the introduction of new targeted therapies. The high expression of the β3 subunit [90,91,92,93] and αvβ3 have been reported to be a characteristic of melanoma [94] and the ectopic expression of αIIbβ3 also occurs [29,30]. Therefore, melanoma cell lines were chosen for use in assays of integrin-mediated adhesion and migration as a first step to developing β3 integrin antagonists which could be developed as potential treatments for advanced melanoma. Our results confirmed a high αv and β3 expression in the M14 and Sk-Mel-2 cell lines used for adhesion and migration studies. However, these lines did not express detectable levels of αIIb mRNA or protein; Kopatz and Selzer [95] have also reported αIIb was unquantifiable in a wider panel of melanoma cell lines. It is known that integrin expression alters in response to the extracellular matrix [96], and, in prostate cancer, αIIb expression is present in vivo but reduced by in vitro culture [32].
Biological investigation of this small library of compounds provided structure activity relationship information on the role of the cyclobutane geometry, linker length, and the identity and stereochemistry of the Asp mimetic. THN-containing compounds had higher anti-αvβ3 antiadhesive activity than those containing naphthyridine or pyrimidine Arg mimetics, for example, pyrimidine ICT 24 19.1% vs. naphthyridine ICT 41 27.5% and THN ICT 46 98.4% inhibition of adhesion at 5 μM. Compounds without a sulfonamide exosite-binding group [97,98] showed little activity; the most active example was THN ICT 47, which inhibited 22.5% of the adhesion at 50 μM. A more lipophilic group increased the activity, for example, phenylsulfonamide ICT 29 was essentially inactive and ICT 45 gave a 59.5% inhibition at 5 μM vs. mesitylsulfonamides ICT 24 19.1% and ICT 46&#;s 98.4% inhibition at 5 μM
The length of the linker had little effect in the pyrimidine series (n = 1 ICT 21 35.2%; n = 2 ICT 24 19.1%; n = 3 ICT 26 20.9% adhesion inhibition at 5 μM), but the shorter THN ICT 48 (61.5%) was less active than ICT 46 (98.4% adhesion inhibition at 5 μM). There was no clear relationship between cis- vs. trans-substituted cyclobutane rings and antiadhesive activity; three out of five pairs (21/27; 23/29; 41/44) had no significant difference in activity, and in the other two (24/28 and 46/49), the cis was slightly more effective.
Activity in the wound healing assay was less sensitive to the identity of the exosite-binding group; phenylsulfonamide ICT 45 (IC50 1.0 ± 0.09 μM) and ICT 22 (IC50 4.8 ± 0.2 μM) with no exosite-binding group were both significantly more active in this assay than anticipated from their effects on cell adhesion. Apart from this, trends in anti-αvβ3 activity were consistent between assays: THN was the most active Arg mimetic (ICT 46 IC50 < 0.1 μM and its free acid ICT 56 IC50 0.2 ± 0.06 μM) and configuration of the cyclobutane had little impact on activity (trans THN ICT 49 IC50 0.15 ± 0.03 μM; slightly less active than cis ICT 46). In general, active compounds showed lower IC50s for the inhibition of the M14 cell migration than they did for the Sk-Mel-2 adhesion despite the very similar integrin expression profile of the two cell lines. These melanoma cell lines contain different mutations in genes such as BRAF and NRAS [99,100] which activate the same cell signalling pathways as that of the integrin ligation, so this could affect their responsiveness to integrin antagonists. There is a need for further work to establish the effects of nonintegrin receptors and signalling pathways dysregulated in cancer on the response to integrin antagonists; we are working to establish the interactions between integrin and nonintegrin receptors and identify effective combination therapies using integrin antagonists with other targeted therapeutics.
Cell adhesion and migration are both essential processes in the metastatic pathway. β3 integrin function is known to be required for adhesive cell&#;cell and cell&#;matrix interactions facilitating the formation of new tumours [101,102,103]. The identification of ICT 46 and the corresponding acid ICT 56 as more effective than positive control cRGDfV in both cell-based assays identified it as a compound of interest, and we progressed to assess its compound activity against αIIbβ3.
As expected, esters were less active (all IC50 50 μM or greater) than the corresponding free acids in inhibiting the binding of αIIbβ3 to fibrinogen since they are unable to form ionic bonds to the MIDAS metal ion. Several free acids had a moderate ability to block the αIIbβ3/Fg interaction in vitro (IC50 ranging from 0.39 ± 0.19 μM (ICT 53) to 7.8 ± 2.8 μM (ICT 51)), however their ability to prevent ex vivo platelet aggregation was much lower (11.1&#;31.4% inhibition at 100 μM with ICT 56 as the most effective inhibitor). To rationalize the lower anti-αIIbβ3 activity of ICT, the compound was docked into the binding sites of αvβ3 (PDB crystal structure IL5G) and αIIbβ3 (PDB crystal structure ITY5) using Arguslab. The lowest energy poses ( ) suggested that ICT could adopt both an extended and a curved conformation; the extended conformation effectively bridges the distance between the αv Asp218 residue and allows for a two-point interaction between this residue and the two THN nitrogen atoms, and the β3 metal ion effectively fills the αv RGD-binding site. However, docking with αIIbβ3 showed binding to the β3 metal ion only with the molecule in a more curved conformation which did not place the THN nitrogen atoms near the αIIb Asp224 residue. This is consistent with the compounds&#; observed profile of higher inhibition of functional αvβ3 activity, although it should be interpreted with some caution given the limitations of Arguslab [104]. Additionally, the minimum energy conformation of some compounds was investigated using the inbuilt MM2 function in Chem3D v15. This supported the existence of a low energy curved conformation, giving a distance of only 8.4 Å between the Arg mimetic nitrogen and Asp mimetic carboxylate in ICT. However, 8.4 Å is shorter than the αvβ3 binding site as well, which supports other more extended conformations being available for binding.
Open in a separate windowDespite the lower than anticipated anti-αIIbβ3 activity, the effectiveness of ICT and its prodrug ester ICT identify the 1,3-substituted cyclobutane structure as a starting point for modifying the flexibility of the molecular skeleton to develop higher-affinity compounds which will be investigated in models of melanoma dissemination.
During this project, cyclobutane-containing αv integrin antagonists developed by Bristol Myer Squibb (BMS) were reported in the patent literature. This included the independent synthesis of ICT 56 and related compounds. The IC50s of 56 were reported to be αvβ3 2.08 nM; αvβ5 0.2 nM; αvβ6 0.37 nM; αvβ1 3.1 nM and αvβ8 15 nM in a cell-free homogeneous time-resolved fluorescence assay [105]. This activity in a cell-free assay is consistent with the effects on integrin function we observed in high-αvβ3-expressing cells, and taken together, these results indicate the THN arginine sidechain mimetic and mesityl exosite-binding group are important in controlling the binding affinity to αv-subfamily integrins. The BMS methods for synthesising cyclobutane integrin antagonists involve adding sidechains to a small cyclobutane building block, so they are limited by the existing building blocks available. Since our method allows the incorporation of functional groups at all positions of the cyclobutane ring, either by the choice of reactants in the cyclisation step or by a later functionalisation of the cyclobutene, it gives a more flexible approach and will be more suited for synthesizing and refining further potential antagonists for investigation.
In summary, we have developed a telescoped synthesis of functionalised cyclobutenes from aldehydes, which facilitates the versatile and efficient large-scale synthesis of novel molecules. This methodology can be used to generate cyclobutanes bearing protected amine, alcohol, or carboxylic acid sidechains with both 1,3-cis and 1,3-trans geometry, thus providing diverse building blocks for further elaboration to integrin antagonists, small molecules targeting other receptors or enzymes, or natural products. We have synthesised a small library of cyclobutane-based RGD-mimetic anti-integrin agents and report the first assessment of these compounds in β3 integrin functional assays. This is the first demonstration that cyclobutanes are effective β3 integrin inhibitors in cancer cell lines and lays the foundation for future development of dual- or singly targeted anti-integrin agents as effective cancer therapeutics.
UK patent application No. .2 avb3 integrin antagonists filed 24 January .
We thank the EPSRC National Mass Spectrometry Facility, Swansea for HRMS measurements.
The following supporting information can be downloaded at: https://www.mdpi.com/article/10./cancers/s1, Figure S1: Expression levels of RGD-binding integrin subunit mRNA in Sk-Mel-2 and M14 cell lines; Figure S2: Expression of integrin subunits in in Sk-Mel-2 and M14 cell lines by Western blot; Figure S3: Quantification of αV and β3 integrin subunits in a panel of human tumour cell lines; Figure S4: Immunocytochemical analysis of M14 cells; Figure S5: Example of analysis of scratch assay; Table S1. Cytotoxicity of compounds on the cell lines used in the functional assays.
Click here for additional data file.(989K, zip)This work was supported by the EPSRC (RCUK Academic Fellowship and Grant EP/H/1 to H.M.S.) and Prostate Cancer UK (Pilot Grant PA10-01). F.O.F.O.A-S.. was funded by the Public Authority for Applied Education and Training, Kuwait (PhD studentship).
Conceptualization, H.M.S.; methodology, H.M.S., M.S. and S.D.S.; investigation, H.M.S., M.S., A.G., F.O.F.O.A.-S. and A.T.; resources, H.M.S., A.C.L.C., H.P., S.D.S. and L.H.P.; writing&#;original draft preparation, H.M.S.; writing&#;review and editing, H.M.S., M.S., S.D.S., A.T. and L.H.P.; supervision, H.M.S. and S.D.S.; project administration, H.M.S.; funding acquisition, H.M.S., S.D.S., M.S. and L.H.P. All authors have read and agreed to the published version of the manuscript.
Use of human blood samples was approved by the Independent Scientific Advisory Committee of Ethical Tissue, University of Bradford (protocol code Application 13/046 approved 28 March . Ethical Tissue operates under ethical approval from the NHS Leeds (East) REC reference 07/H/98+5).
Informed consent was obtained from all anonymous donors of blood samples involved in the study.
The data presented in this study are available within the article and supplementary file.
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
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