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Synthesis of pretubulysin-derivatives via the TubUgi-approach† Judith Hoffmann, Jan Gorges, Lukas Junk and Uli Kazmaier*
Received 24th March 2015, Accepted 20th April 2015 DOI: 10.1039/c5ob00587f www.rsc.org/obc
The Ugi reaction is found to be a very powerful tool for the synthesis of ( pre)tubulysin derivatives, allowing the introduction of various functionalized side chains in only one step. While polar groups such as amides are not well tolerated, unpolar side chains such as allyl or propargyl ether are well accepted. These functionalities also allow subsequent modifications in the side chain, e.g. via ring closing metathesis or Click reaction.
Introduction The microtubule skeleton is an interesting target for the development of antitumor drugs.1 Microtubuli are polymers of α- and β-tubulin heterodimers and play an essential role in a wide range of cellular processes, such as mitosis.2 They show rather complex polymerization dynamics, which can be modulated by antimitotic drugs. In principle, these drugs can be divided into two different groups. Compounds such as taxanes (e.g. paclitaxel), discodermolide or epothilone stabilize the tubulin skeleton, while e.g. the vinca alkaloids (e.g. vinblastin) destabilize the microtubuli (Fig. 1).3 Both scenarios suppress a regular cell division, leading to apoptosis. Many of these compounds show antitumor activity in the low or subnanomolar range, and are therefore good candidates for the development of antitumor drugs. The major drawback of these compounds results from their rather complex structure, and their synthesis in general is not a trivial issue. Therefore, in many cases, substantial amounts e.g. for clinical studies have to be isolated from natural sources. In 2000, Reichenbach and Höfle et al. reported the isolation of a family of tetrapeptides, called tubulysins, from the myxobacterial strains Archangium gephyra and Angiococcus disciformis.4 Several more representatives of this class of compounds were described by Müller et al. in 20045 and 2010.6 The tubulysins show very high cytotoxicity towards a wide range of tumor cell lines,4b,7 including multi drug resistant ones,8 and the same binding mode as the vinca alkaloids.7 Depending on the cell line, this cytotoxicity is higher by factors of 20–1000 compared to taxol or epothilone. In comparison with the other natural products mentioned, the struc-
Institute of Organic Chemistry, Saarland University, P.O. Box 151150, 66041 Saarbrücken, Germany. E-mail: [email protected] † Electronic supplementary information (ESI) available: Copies of 1H and NMR spectra. See DOI: 10.1039/c5ob00587f
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ture of the tubulysins is “relatively” simple. Besides an N-methylated (R)-pipecolic acid, especially the two prolonged unusual amino acids tubuvaline (Tuv) and tubuphenylalanine (Tup) [or tubutyrosine (Tut)] catch one’s eye. From a biosynthetic point of view, the acetoxy group and the rather unusual acylal side chain are introduced after the tetrapeptide has been formed.5
13
C
Fig. 1
Antimitotic natural products.
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According to their biosynthetic studies, Müller et al. postulated pretubulysin to be the biosynthetic precursor of the tubulysins.5 This assumption initiated our efforts to synthesize pretubulysin9 as the reference sample and to investigate the cytotoxicity of this rather simple molecule.10 Pretubulysin and its derivatives show strong anti-angiogenic effects11 and antivascular properties in vitro and in vivo,12 and are therefore a new option for the treatment of metastatic cancer.13 Although the biological activity of pretubulysin is low compared to the natural tubulysins, especially tubulysin D, the most active one, it was still in the low or subnanomolar range (depending on cell line). The cytotoxicity is still higher than that of taxol or epothilone. The promising properties of the tubulysins forced the development of several protocols towards the tubulysins14 and simplified derivatives thereof.15 The by far most critical step is the introduction of the acylal side chain,14 the position in the molecule where obviously variations are well tolerated, since the natural tubulysins mainly vary in this side chain. Therefore, variations in this position, allowing the incorporation of different, preferentially functionalized side chains, are strongly desired. The incorporation of a simple N-methyl group does not cause any problems and good yields are obtained in the peptide coupling between the Ile- and N-methyl Tuv-moieties with most modern coupling reagents.15 The situation changes dramatically if the size of the N-substituent increases. The coupling of the corresponding N-ethyl or N-allyl derivatives gives yields 100 °C) under an atmosphere of nitrogen or argon. Dried solvents were distilled before use. The products were purified by flash chromatography on silica gel columns (Macherey-Nagel 60, 0.063–0.2 mm) and with a flash chromatography system [Reveleris (Grace), RediSep-columns 4 g, 12 g, 24 g and 40 g from Axel Semrau]. Mixtures of ethyl acetate and petroleum ether or dichloromethane and methanol were generally used as eluents. Analytical TLC was performed on precoated silica gel plates (Macherey-Nagel, Polygram® SIL G/UV254). Visualization was accomplished with UV-light, KMnO4 solution or ninhydrin solution. Melting points were determined with a Dr Tottoli (Büchi) melting point apparatus and are uncorrected. 1H and 13C NMR spectra were recorded with a Bruker AV400 [400 MHz (1H) and 100 MHz (13C)] or a Bruker AV500 [500 MHz (1H) and 125 MHz (13C)] spectrometer in CDCl3. Chemical shifts are reported in ppm relative to TMS and CHCl3 was used as the internal standard. Diastereomeric ratios were determined by NMR or HPLC. Mass spectra were recorded with a Finnigan MAT 95 spectrometer (quadrupole) using the CI technique. HPLC/MS analysis was performed with a Shimadzu system (LC: 10A-series + Autosampler, MS: LCMS-2020). Elemental analyses were performed at the Saarland University. N-tert-Butyloxycarbonyl-(S)-isoleucyl-N-(N′-(2-tert-butyloxydimethylsilyloxyethyl)aminocarbonylmethyl)-(S)-valin methylester 1 (S)-Valine methylester (46.0 mg, 0.351 mmol) and paraformaldehyde (13.5 mg, 0.45 mmol) were dissolved in MeOH (0.55 mL) and were stirred for 30 min. Boc-(S)-isoleucine (84.8 mg, 0.353 mmol) was added and the solution was stirred for a further 10 min, before (tert-butyldimethylsilyloxyethyl)iso-
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cyanide (64.1 mg, 0.346 mmol) was added. The solution was stirred overnight before it was diluted with CH2Cl2 (5 mL). The organic layer was washed with sat. NaHCO3 (3 mL) and KHSO4 (2 mL), dried (Na2SO4) and evaporated in vacuo. Flash chromatography (silica, hexanes/ethylacetate 1.6 : 4; 2. 1 : 1) and subsequent crystallization (Et2O) gave rise to 1 (81.2 mg, 0.145 mmol, 48%) as a pale yellow solid. Rf (1): 0.65 (hexanes/ ethyl acetate 1 : 1). Mixture of rotamers. Major rotamer: 1 H NMR (400 MHz, CDCl3): δ = 0.06 (s, 6 H), 0.85–0.90 (m, 6 H), 0.89 (s, 9 H), 0.91 (d, J = 6.8 Hz, 3 H), 1.01 (d, J = 6.8 Hz, 3 H), 1.41 (s, 9 H), 1.57 (m, 2 H), 1.75 (m, 1 H), 2.33 (m, 1 H), 3.34 (m, 2 H), 3.70 (m, 2 H), 3.71 (s, 3 H), 4.12 (d, J = 17.7 Hz, 1 H), 4.16 (m, 1 H), 4.25 (d, J = 9.3 Hz, 1 H), 4.31 (d, J = 17.7 Hz, 1 H), 4.91 (d, J = 9.5 Hz, 1 H), 7.36 (bs, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = −5.4, 11.1, 16.0, 18.3, 19.5, 20.3, 23.9, 25.9, 28.2, 37.1, 42.1, 47.8 50.1, 52.2, 55.1, 61.5, 64.4, 79.8, 156.8, 167.9, 171.1, 173.7 ppm. Minor rotamer (selected signals): 1H NMR (400 MHz, CDCl3): δ = 0.06 (s, 6 H), 0.90 (s, 9 H), 1.43 (s, 9 H), 1.83 (m, 1 H), 3.43 (m, 2 H), 3.64 (m, 2 H), 3.97 (d, J = 15.3 Hz, 1 H), 4.05 (1 H, J = 15.3 Hz, 1 H), 4.50 (m, 1 H), 5.00 (d, J = 10.2 Hz, 1 H), 6.61 (bs, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = −3.6, 11.3, 16.1, 18.9, 19.6, 25.6, 28.3, 41.7, 52.1, 54.8, 61.8, 65.5 ppm. HRMS (CI): calculated: for C27H54N3O7Si [M + H]+ 560.3731; found: 560.3726. C27H53N3O7Si (559.81): calcd C 57.93, H 9.54, N 7.51; found C 57.93, H 9.64, N 7.05. N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(2-tertbutyloxydimethylsilyloxyethyl)aminocarbonylmethyl]desacetoxy-(S)-tubuvalin methylester 3a In analogy to 1 peptide 3a was obtained from amine 2a (21.6 mg, 84.3 μmol), paraformaldehyde (3.50 mg, 117 μmol), (R)-Mep-(S)-Ile-OH·HCl (26.5 mg, 90.5 μmol) and (tert-butyldimethylsilyloxyethyl)isocyanide (18.1 mg, 98 μmol) in MeOH (0.2 mL). The reaction mixture was stirred for 2 d. After workup and flash chromatography (CH2Cl2/MeOH 99 : 1 to 9 : 1) 3a (13.9 mg, 19.0 μmol, 23%) was obtained as an orange oil. Rf (3a): 0.40 (CH2Cl2/MeOH 9 : 1). Mixture of rotamers. Major rotamer: 1H NMR (400 MHz, CDCl3): δ = 0.05 (s, 6 H), 0.71 (d, J = 6.6 Hz, 3 H), 0.85–0.92 (m, 12 H), 0.94–0.97 (m, 6 H), 1.14–1.28 (m, 3 H), 1.37 (m, 1 H), 1.38 (t, J = 7.1 Hz, 3 H), 1.47 (m, 1 H), 1.55–1.61 (m, 3 H), 1.72 (m, 1 H), 1.77 (m, 1 H), 1.87–2.05 (m, 4 H), 2.15–2.31 (m, 4 H), 2.46 (dd, J = 10.5, 3.3 Hz, 1 H), 2.89 (m, 1 H), 3.04 (m, 1 H), 3.32–3.50 (m, 3 H), 3.53 (d, J = 13.6 Hz, 1 H), 3.64 (d, J = 13.6 Hz, 1 H), 3.69–3.73 (m, 3 H), 4.31–4.41 (m, 3 H), 6.72 (bs, 1 H), 6.97 (m, 1 H), 8.01 (s, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = −5.2, 11.7, 14.3, 15.8, 19.9, 20.4, 23.1, 24.6, 24.8, 25.9, 30.5, 30.9, 31.2, 31.6, 36.5, 42.1, 44.9, 46.6, 53.7, 55.2, 61.2, 61.6, 63.8, 69.5, 77.3, 127.2, 146.5, 161.3, 169.1, 172.2, 172.8, 173.9 ppm. Minor rotamer (selected signals): 1H NMR (400 MHz, CDCl3): δ = 0.09 (s, 6 H), 0.90 (s, 9 H), 2.22 (s, 3 H), 3.12 (m, 1 H), 3.82 (m, 1 H), 3.93 (m, 1 H), 7.05 (bs, 1 H), 7.11 (m, 1 H), 8.02 (s, 1 H) ppm. 13 C NMR (100 MHz, CDCl3): δ = −5.2, 15.8, 19.6, 20.7, 25.6, 31.3, 36.4, 52.1, 55.3, 61.5, 69.1, 127.1, 146.8, 161.6, 169.5,
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171.7 ppm. HRMS (CI): calculated: for C35H63N5O6SiS [M + 2H]+ 709.4257; found: 709.4261.
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General procedure for Ugi reactions A solution of the isocyanide (0.2 mmol) in CH2Cl2 (0.5 mL) was added slowly to a solution of the amine (0.2 mmol), paraformaldehyde (0.2 mmol) and the acid component (0.23 mmol) in CH2Cl2 (2 mL). The reaction mixture was stirred for 2–4 d and was monitored by TLC and/or LC-MS. After complete consumption of the amine component, the reaction mixture was hydrolyzed with sat. NaHCO3 solution (2 mL) and the aqueous layer was washed twice with CH2Cl2 (3 mL each). The combined organic layers were dried, filtered and evaporated in vacuo. The crude product was purified by flash chromatography.
N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(2-tertbutyloxydimethylsilyloxyethyl)aminocarbonylmethyl]desacetoxy-(S)-tubuvalyl-(S)-tubuphenylalanin allylester 4b According to the general procedure for Ugi reactions 4b was obtained from amine 2b (85.0 mg, 0.186 mmol), paraformaldehyde (5.6 mg, 0.186 mmol), Mep-Ile-OH (54.8 mg, 0.214 mmol) and (tert-butyldimethylsilyloxyethyl)isocyanide (34.4 mg, 0.186 mmol) in CH2Cl2 (1 mL). After flash chromatography (CH2Cl2/MeOH 99 : 1 to 9 : 1) and freeze drying, 4b (112 mg, 0.123 mmol, 66%) was obtained as a pale yellow resin. Rf (4b): 0.19 (CH2Cl2/MeOH 95 : 5). Mixture of rotamers. Major rotamer: 1H NMR (400 MHz, CDCl3): δ = 0.03 (s, 6 H), 0.71 (d, J = 6.6 Hz, 3 H), 0.80–0.89 (m, 12 H), 0.91–0.95 (m, 6 H), 1.14–1.22 (m, 5 H), 1.33–1.48 (m, 2 H), 1.53–1.67 (m, 5 H), 1.76–2.07 (m, 6 H), 2.20 (s, 3 H), 2.49 (m, 1 H), 2.63 (m, 1 H), 2.81–3.03 (m, 4 H), 3.23–3.47 (m, 3 H), 3.57–3.68 (m, 4 H), 4.33–4.40 (m, 2 H), 4.51 (m, 2 H), 4.60 (d, J = 17.0 Hz, 1 H), 5.16 (m, 1 H), 5.23 (m, 1 H), 5.84 (ddt, J = 17.2, 10.5, 5.5 Hz, 1 H), 6.84 (bs, 1 H), 7.00 (bs, 1 H), 7.14–7.24 (m, 5 H), 7.31 (d, J = 9.2 Hz, 1 H), 7.85 (s, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = −5.4, 10.4, 15.7, 17.7, 19.6, 20.4, 23.0, 23.8, 24.7, 24.8, 25.8, 30.2, 30.3, 30.3, 30.5, 36.4, 36.5, 37.7, 41.3, 42.0, 44.6, 46.6, 48.4, 53.7, 55.2, 61.5, 63.8, 65.0, 69.3, 117.8, 122.3, 126.3, 128.3, 129.4, 132.2, 137.7, 149.3, 160.8, 169.1, 169.6, 170.9, 173.8, 175.7 ppm. Minor rotamer (selected signals): 1 H NMR (400 MHz, CDCl3): δ = 0.04 (s, 6 H), 1.03 (d, J = 6.5 Hz, 3 H), 1.15 (d, J = 7.1 Hz, 3 H), 1.95 (m, 1 H), 2.24 (s, 3 H), 2.73–3.00 (m, 2 H), 3.88 (s, 2 H), 3.61 (m, 2 H), 4.83 (dd, J = 6.8, 6.8 Hz, 1 H), 7.14 (m, 1 H), 7.24 (m, 1 H), 7.87 (s, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 11.3, 16.4, 18.2, 20.2, 20.7, 29.6, 30.7, 31.5, 37.8, 41.7, 53.0, 122.6, 126.3, 129.3, 149.6, 160.6, 175.6 ppm. MS (ESI/APCI): calculated: for C48H79N6O7SSi [M + H]+ 911.55; found: 911.45; calculated: for C48H80N6O7SSi [M + 2H]2+ 456.28; found: 456.25. HRMS (CI): calculated: for C44H69N6O7SSi [M − –tBu]+ 853.4718; found: 853.4656.
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N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(tertbutyloxycarbonylaminoethyl)aminocarbonylmethyl]desacetoxy-(S)-tubuvalyl-(S)-tubuphenylalanin allylester 6b According to the general procedure for Ugi reactions 6b was obtained from amine 2b (58.2 mg, 0.127 mmol), paraformaldehyde (3.8 mg, 0.127 mmol), Mep-Ile-OH (37.5 mg, 0.146 mmol) and (tert-butyloxycarbonylaminoethyl)isocyanide (21.7 mg, 0.127 mmol) in CH2Cl2 (1 mL). After flash chromatography (CH2Cl2/MeOH 99 : 1 to 9 : 1) and freeze drying 6b (68.2 mg, 0.076 mmol, 60%) was obtained as a pale yellow resin. Rf (6b): 0.15 (CH2Cl2/MeOH 95 : 5). Mixture of rotamers (∼1 : 1). 1H NMR (400 MHz, CDCl3): δ = 0.76 (d, J = 6.6 Hz, 1.5 H), 0.85–0.89 (m, 6 H), 0.94 (d, J = 6.4 Hz, 1.5 H), 0.98 (d, J = 6.7 Hz, 1.5 H), 1.05 (d, J = 6.4 Hz, 1.5 H), 1.16 (d, J = 7.1 Hz, 1.5 H), 1.17 (d, J = 7.1 Hz, 1.5 H), 1.21–1.24 (m, 2 H), 1.37–1.49 (m, 11 H), 1.55–1.69 (m, 4.5 H), 1.79–1.83 (m, 1.5 H), 1.88–1.94 (m, 2 H), 1.99–2.07 (m, 2.5 H), 2.22 (s, 3 H), 2.29 (m, 0.5 H), 2.51 (m, 1 H), 2.63 (m, 1 H), 2.82–2.97 (m, 4 H), 3.13–3.19 (m, 1.5 H), 3.24–3.29 (m, 2.5 H), 3.40–3.49 (m, 2 H), 3.61 (d, J = 15.6 Hz, 0.5 H), 3.82 (m, 0.5 H), 4.19–4.25 (m, 1 H), 4.35–4.45 (m, 2 H), 4.53 (dm, J = 5.5 Hz, 2 H), 5.17 (dm, J = 10.5 Hz, 1 H), 5.25 (dm, J = 17.2 Hz, 1 H), 5.56 (bs, 0.5 H), 5.75 (bs, 0.5 H), 5.85 (ddt, J = 17.3, 10.5, 5.5 Hz, 1 H), 7.06–7.08 (m, 1.5 H), 7.14 (m, 0.5 H), 7.19–7.21 (m, 3.5 H), 7.26 (m, 2 H), 7.37 (d, J = 8.7 Hz, 1 H), 7.88 (s, 0.5 H), 7.91 (s, 0.5 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 10.8, 11.6, 16.0, 16.5, 17.9, 17.9, 20.0, 20.2, 20.4, 20.6, 23.2, 24.6, 25.0, 28.4, 28.5, 29.5, 29.9, 30.2, 30.3, 30.4, 30.7, 31.5, 36.4, 36.7, 37.8, 38.0, 40.2, 40.2, 40.4, 40.4, 41.3, 41.4, 44.9, 46.6, 48.5, 48.6, 54.3, 55.3, 55.3, 63.4, 65.1, 65.2, 69.2, 79.3, 118.0, 118.0, 122.5, 122.9, 126.4, 126.5, 128.4, 129.4, 129.5, 132.3, 132.3, 137.7, 137.8, 149.6, 156.3, 160.7, 160.9, 169.5, 169.7, 170.6, 173.3, 173.4, 175.4, 175.7, 175.8 ppm. MS (ESI/APCI): calculated: for C47H74N6O8S [M + H]+ 896.53; found: 896.40; calculated: for C47H75N6O8S [M + 2H]2+ 448.77; found: 448.75. HRMS (CI): calculated: for C47H74N7O8S [M + H]+ 896.5320; found: 896.5341. N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(2-allyloxyethyl)aminocarbonylmethyl]-desacetoxy-(S)-tubuvalyl-(S)tubuphenylalanin allylester 7b According to the general procedure for Ugi reactions 7b was obtained from amine 2b (107 mg, 0.234 mmol), paraformaldehyde (7.1 mg, 0.236 mmol), Mep-Ile-OH (69.0 mg, 0.269 mmol) and (allyloxyethyl)isocyanide (26.0 mg, 0.234 mmol) in CH2Cl2 (2.5 mL). After flash chromatography (CH2Cl2/MeOH 99 : 1 to 9 : 1) 7b (159 mg, 0.190 mmol, 81%) was obtained as a white solid, m.p. 46–47 °C. Rf (7b): 0.29 (CH2Cl2/MeOH 9 : 1). [α]20 D = 33.7 (c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR (500 MHz, CDCl3): δ = 0.73 (d, J = 6.6 Hz, 3 H), 0.87 (t, J = 7.4 Hz, 3 H), 0.95 (d, J = 6.4 Hz, 3 H), 0.96 (d, J = 6.6 Hz, 3 H), 1.17 (d, J = 7.1 Hz, 3 H), 1.17–1.25 (m, 2 H), 1.23–2.13 (m, 13 H), 2.21 (s, 3 H), 2.48 (m, 1 H), 2.63 (m, 1 H), 2.82–3.14 (m, 3 H), 2.86 (dd, J = 13.7, 6.7 Hz, 1 H), 2.94 (dd, J = 13.8, 6.0 Hz, 1 H), 3.23–3.70 (m, 5 H), 3.97 (d, J = 5.7 Hz, 2 H), 4.34–4.44 (m, 2 H), 4.48–4.63 (m, 3 H),
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5.15–5.20 (m, 2 H), 5.22–5.29 (m, 2 H), 5.81–5.93 (m, 2 H), 6.94 (d, J = 8.4 Hz, 1 H), 7.10 (t, J = 4.9 Hz, 1 H), 7.16–7.28 (m, 5 H), 7.32 (d, J = 9.2 Hz, 1 H), 7.87 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.5, 15.8, 17.8, 19.7, 20.5, 23.2, 24.7, 25.0, 29.6, 30.2, 30.6, 30.7, 36.6, 37.7, 37.9, 39.6, 41.3, 44.9, 46.7, 48.5, 53.8, 55.3, 63.9, 65.1, 68.3, 69.5, 72.0, 117.3, 117.9, 122.4, 126.4, 128.3, 129.5, 132.3, 134.4, 137.7, 149.5, 160.9, 169.1, 169.7, 170.9, 174.8, 175.8 ppm. Minor rotamer (selected signals): 1H NMR (500 MHz, CDCl3): δ = 0.90 (t, J = 7.4 Hz, 3 H), 1.06 (d, J = 6.5 Hz, 3 H), 1.17 (d, J = 7.1 Hz, 3 H), 2.23 (s, 3 H), 2.48 (m, 1 H), 2.63 (m, 1 H), 2.82–3.14 (m, 3 H), 3.95 (ddd, J = 5.6, 1.4 Hz, 1.4 Hz, 2 H), 7.88 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 11.4, 16.5, 17.9, 20.3, 20.7, 23.9, 30.4, 30.7, 31.5, 36.5, 36.6, 39.4, 48.5, 52.9, 55.4, 71.9, 117.1, 118.0, 122.6, 126.4, 129.4, 132.3, 134.5, 149.7, 160.6, 169.2, 175.7 ppm. HRMS (CI): calculated: for C45H69N6O7S [M + H]+ 837.4943; found: 837.4978.
N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(2-allyloxyethyl)aminocarbonylmethyl]-desacetoxy-(S)-tubuvalyl-(S)tubuphenylalanin methylester 7c According to the general procedure for Ugi reactions 7c was obtained from amine 2c (150 mg, 0.347 mmol), paraformaldehyde (10.4 mg, 0.347 mmol), Mep-Ile-OH (102 mg, 0.399 mmol) and (allyloxyethyl)isocyanide (38.5 mg, 0.347 mmol) in CH2Cl2 (3.5 mL). After flash chromatography (CH2Cl2/MeOH 99 : 1 to 9 : 1) 7c (225 mg, 0.277 mmol, 80%) was obtained as a white solid, m.p. 51–52 °C. Rf (7c): 0.32 (CH2Cl2/MeOH 9 : 1). [α]20 D = 12.1 (c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR (500 MHz, CDCl3): δ = 0.73 (d, J = 6.6 Hz, 3 H), 0.87 (t, J = 7.4 Hz, 3 H), 0.95 (d, J = 6.4 Hz, 3 H), 0.96 (d, J = 6.7 Hz, 3 H), 1.15 (d, J = 7.1 Hz, 3 H), 1.17–1.28 (m, 2 H), 1.30–2.12 (m, 13 H), 2.22 (s, 3 H), 2.50 (m, 1 H), 2.61 (m, 1 H), 2.72–3.14 (m, 3 H), 2.85 (dd, J = 13.7, 6.8 Hz, 1 H), 2.94 (dd, J = 13.7, 5.8 Hz, 1 H), 3.20–3.70 (m, 6 H), 3.62 (s, 3 H), 3.97 (d, J = 5.7 Hz, 2 H), 4.34–4.43 (m, 2 H), 4.57 (d, J = 16.4 Hz, 1 H), 5.17 (m, 1 H), 5.26 (m, 1 H), 5.87 (ddt, J = 17.1, 10.4, 5.7 Hz, 1 H), 6.88 (s, 1 H), 7.10 (s, 1 H), 7.16–7.28 (m, 5 H), 7.32 (d, J = 9.2 Hz, 1 H), 7.88 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.5, 15.8, 17.8, 19.7, 20.4, 23.2, 24.7, 24.9, 29.6, 30.2, 30.5, 30.6, 36.5, 37.8, 37.9, 39.6, 41.4, 44.9, 46.7, 48.4, 51.8, 53.8, 55.3, 63.9, 68.3, 69.5, 71.9, 117.4, 122.4, 126.3, 128.3, 129.5, 134.4, 137.7, 149.4, 160.9, 169.1, 169.7, 170.9, 173.8, 176.7 ppm. Minor rotamer (selected signals): 1H NMR (500 MHz, CDCl3): δ = 0.90 (t, J = 7.4 Hz, 3 H), 1.06 (d, J = 6.4 Hz, 3 H), 1.15 (d, J = 7.1 Hz, 3 H), 3.62 (s, 3 H), 3.87 (d, J = 14.9 Hz, 1 H), 3.95 (ddd, J = 5.6, 1.4, 1.4 Hz, 2 H), 4.84 (m, 1 H), 6.88 (s, 1 H), 7.90 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 11.4, 16.4, 20.3, 20.7, 23.8, 30.4, 30.7, 31.5, 36.5, 36.6, 37.7, 39.4, 41.3, 48.5, 71.9, 117.2, 122.7, 126.4, 129.4, 134.4, 149.6, 160.7, 169.2, 170.6, 176.6 ppm. HRMS (CI): calculated: for C43H67N6O7S [M + H]+ 811.4787; found: 811.4826.
Org. Biomol. Chem.
Organic & Biomolecular Chemistry
N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(2-propargyloxyethyl)aminocarbonylmethyl]-desacetoxy-(S)-tubuvalyl-(S)tubuphenylalanin allylester 8b According to the general procedure for Ugi reactions 8b was obtained from amine 2b (144 mg, 0.314 mmol), paraformaldehyde (9.4 mg, 0.314 mmol), Mep-Ile-OH (93.0 mg, 0.361 mmol) and ( propargyloxyethyl)isocyanide (34.2 mg, 0.314 mmol) in CH2Cl2 (4 mL). After flash chromatography (CH2Cl2/MeOH 99 : 1 to 9 : 1) 8b (229 mg, 0.274 mmol, 87%) was obtained as a white solid, m.p. 50–51 °C. Rf (8b): 0.30 (CH2Cl2/MeOH 9 : 1). [α]20 D = 34.9 (c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR (500 MHz, CDCl3): δ = 0.74 (d, J = 6.6 Hz, 3 H), 0.87 (t, J = 7.5 Hz, 3 H), 0.95 (d, J = 6.7 Hz, 3 H), 0.97 (d, J = 6.5 Hz, 3 H), 1.17 (d, J = 7.1 Hz, 3 H), 1.18–1.23 (m, 2 H), 1.54–2.12 (m, 13 H), 2.21 (s, 3 H), 2.44 (t, J = 2.4 Hz, 1 H), 2.48 (m, 1 H), 2.63 (m, 1 H), 2.82–3.03 (m, 5 H), 3.22–3.69 (m, 7 H), 4.12 (m, 2 H), 4.35–4.44 (m, 2 H), 4.53 (m, 2 H), 5.18 (ddt, J = 10.5, 1.3, 1.3 Hz, 1 H), 5.25 (ddt, J = 17.2, 1.5, 1.5 Hz, 1 H), 5.86 (ddt, J = 17.3, 10.6, 5.7 Hz, 1 H), 6.95 (d, J = 9.3 Hz, 1 H), 7.09 (t, J = 5.5 Hz, 1 H), 7.16–7.29 (m, 5 H), 7.32 (d, J = 9.2 Hz, 1 H), 7.87 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.5, 15.8, 17.8, 19.7, 20.5, 23.2, 24.7, 25.0, 29.6, 30.2, 30.6, 30.7, 36.6, 37.7, 37.9, 39.4, 41.3, 44.9, 46.7, 48.5, 53.8, 55.3, 58.2, 63.9, 65.1, 68.2, 69.4, 74.8, 79.3, 117.9, 122.4, 126.4, 128.3, 129.5, 132.3, 137.7, 149.5, 160.9, 169.1, 169.7, 170.9, 174.8, 175.7 ppm. Minor rotamer (selected signals): 1H NMR (500 MHz, CDCl3): δ = 0.90 (t, J = 7.5 Hz, 3 H), 1.05 (d, J = 6.5 Hz, 3 H), 1.17 (d, J = 7.1 Hz, 3 H), 2.23 (s, 3 H), 2.41 (t, J = 2.4 Hz, 1 H), 3.85 (d, J = 14.6 Hz, 1 H), 3.96 (d, J = 14.7 Hz, 1 H), 4.12 (m, J = 2.3 Hz, 2 H), 4.83 (m, 1 H), 7.88 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 11.4, 16.4, 17.8, 20.3, 20.7, 23.9, 30.4, 31.5, 36.6, 36.6, 39.2, 41.3, 48.5, 53.0, 55.3, 58.1, 62.9, 74.7, 79.4, 118.0, 122.7, 126.4, 129.4, 132.3, 149.7, 160.6, 169.2, 173.8, 174.0, 174.5, 175.7 ppm. HRMS (CI): calculated: for C45H67N6O7S [M + H]+ 835.4787; found: 835.4825. N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(2-propargyloxyethyl)aminocarbonylmethyl]-desacetoxy-(S)-tubuvalyl-(S)tubuphenylalanin methylester 8c According to the general procedure for Ugi reactions 8c was obtained from amine 2c (177 mg, 0.410 mmol), paraformaldehyde (12.3 mg, 0.410 mmol), Mep-Ile-OH (121 mg, 0.471 mmol) and ( propargyloxyethyl)isocyanide (44.7 mg, 0.471 mmol) in CH2Cl2 (4 mL). After flash chromatography (CH2Cl2/MeOH 99 : 1 to 9 : 1) 7c (264 mg, 0.326 mmol, 80%) was obtained as a white solid, m.p. 55–56 °C. Rf (8c): 0.38 (CH2Cl2/MeOH 9 : 1). [α]20 D = 21.1 (c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR (500 MHz, CDCl3): δ = 0.73 (d, J = 6.5 Hz, 3 H), 0.86 (t, J = 7.4 Hz, 3 H), 0.95 (d, J = 6.5 Hz, 3 H), 0.96 (d, J = 6.5 Hz, 3 H), 1.14 (d, J = 7.1 Hz, 3 H), 1.16–1.24 (m, 2 H), 1.57–2.12 (m, 13 H), 2.20 (s, 3 H), 2.44 (t, J = 2.3 Hz, 1 H), 2.48 (m, 1 H), 2.60 (m, 1 H), 2.72–3.12 (m, 5 H), 3.21–3.70 (m, 6 H), 3.61 (s, 3 H), 4.13 (d, J = 2.2 Hz, 2 H), 4.30–4.42 (m, 2 H), 4.56 (d, J = 16.9 Hz, 1 H), 6.96 (d, J =
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9.4 Hz, 1 H), 7.09 (t, J = 5.4 Hz, 1 H), 7.17–7.29 (m, 5 H), 7.32 (d, J = 9.2 Hz, 1 H), 7.87 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.4, 15.8, 17.8, 19.6, 20.4, 23.2, 24.6, 25.0, 29.6, 30.1, 30.4, 30.5, 36.4, 37.7, 37.9, 39.4, 41.3, 44.9, 46.7, 48.4, 51.6, 53.7, 55.2, 58.1, 63.9, 68.1, 69.3, 74.8, 79.3, 122.4, 126.3, 128.3, 129.4, 137.7, 149.4, 160.9, 169.1, 169.7, 173.8, 174.8, 176.6 ppm. Minor rotamer (selected signals): 1H NMR (500 MHz, CDCl3): δ = 1.05 (d, J = 6.5 Hz, 3 H), 2.22 (s, 3 H), 2.40 (t, J = 2.3 Hz, 1 H), 4.11 (d, J = 2.3 Hz, 2 H), 4.82 (m, 1 H), 7.89 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 11.4, 16.4, 20.3, 20.7, 23.8, 30.6, 31.5, 36.4, 36.5, 37.7, 39.1, 41.3, 48.5, 53.0, 55.3, 58.0, 74.7, 79.3, 122.7, 126.4, 129.4, 149.6, 160.6, 169.2, 170.9, 174.0, 176.5 ppm. HRMS (CI): calculated: for C43H65N6O7S [M + H]+ 809.4630; found: 809.4660. C43H64N6O7S (809.08): calcd C 63.83, H 7.97, N 10.39; found C 63.74, H 7.34, N 10.20. N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(2-allyloxyethyl) aminocarbonylmethyl]-desacetoxy-(S)-tubuvalyl-(S)tubuphenylalanin·CF3COOH 7-OH Ester 7c (33.7 mg, 0.042 mmol) was dissolved in dioxane (0.5 mL) before a 1 M LiOH solution (90 μl, 0.090 mmol) was added and the solution was stirred overnight at room temperature. The reaction was monitored by TLC and after complete consumption of 7c the reaction mixture was diluted with H2O and acidified to pH 2 using trifluoroacetic acid. The aqueous phase was extracted twice with ethyl acetate, the organic layer was dried (Na2SO4) and evaporated in vacuo. To remove the excess trifluoroacetic acid, the residue obtained was suspended twice in ether, decanted and dried in high vacuo. The acid 7-OH (30.2 mg, 0.040 mmol, 96%) was obtained as a colorless solid, m.p. 82–83 °C. [α]20 D = −13.6 (c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR (500 MHz, CDCl3): δ = 0.71 (m, 3 H), 0.87 (t, J = 7.0 Hz, 3 H), 0.93 (d, J = 6.6 Hz, 3 H), 0.96 (d, J = 6.6 Hz, 3 H), 1.13 (d, J = 7.0 Hz, 3 H), 1.16–1.36 (m, 2 H), 1.36–2.26 (m, 13 H), 2.62 (m, 1 H), 2.67 (s, 3 H), 2.72–3.34 (m, 6 H), 3.34–3.72 (m, 6 H), 3.97 (d, J = 5.7 Hz, 2 H), 4.03 (m, 1 H), 4.32–4.52 (m, 2 H), 5.14–5.30 (m, 2 H), 5.85 (ddt, J = 16.1, 10.9, 5.6 Hz, 1 H), 7.15–7.26 (m, 5 H), 7.52 (s, 1 H), 7.75 (d, J = 7.8 Hz, 1 H), 7.87 (s, 1 H), 8.24 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.6, 16.0, 17.0, 19.7, 20.4, 21.5, 22.5, 24.3, 29.5, 29.9, 30.3, 30.6, 35.8, 36.8, 37.1, 39.4, 41.3, 41.9, 46.7, 48.5, 55.3, 55.6, 64.0, 68.1, 68.2, 71.9, 116.5 (q, J = 292 Hz), 117.4, 122.9, 126.5, 128.4, 129.2, 134.3, 137.9, 149.4, 161.5, 162.2 (q, J = 36 Hz), 169.6, 171.0, 172.8, 173.1 ppm. The signal of the COOH group could not be detected. Minor rotamer (selected signals): 1H NMR (500 MHz, CDCl3): δ = 1.07 (d, J = 6.4 Hz, 3 H), 3.90 (d, J = 5.0 Hz, 2 H), 4.80 (m, 1 H), 7.87 (s, 1 H), 7.91 (s, 1 H), 8.52 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 11.3, 15.5, 31.5, 39.6, 71.7, 134.4, 149.6 ppm. HRMS (CI): calculated: for C42H66N6O7S [M + 2H]2+ 399.2351; found: 399.2359. N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(2-propargyloxyethyl)aminocarbonylmethyl]-desacetoxy-(S)-tubuvalyl-(S)tubuphenylalanin·CF3COOH 8-OH According to acid 7-OH acid 8-OH was obtained from ester 8c (35.1 mg, 0.043) as a colorless solid (36.8 mg, 0.040 mmol,
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93%), m.p. 55–56 °C. [α]20 D = 21.1 (c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR (500 MHz, CDCl3): δ = 0.73 (bs, 3 H), 0.87 (m, 3 H), 0.91 (d, J = 6.4 Hz, 3 H), 0.96 (d, J = 6.8 Hz, 3 H), 1.13 (d, J = 7.1 Hz, 3 H), 1.18–1.37 (m, 2 H), 1.38–2.29 (m, 14 H), 2.45 (t, J = 2.1 Hz, 1 H), 2.50–2.77 (m, 4 H), 2.72–3.15 (m, 5 H), 3.38–3.74 (m, 6 H), 3.93 (m, 1 H), 4.14 (d, J = 2.0 Hz, 2 H), 4.28–4.50 (m, 2 H), 7.12–7.32 (m, 6 H), 7.58 (bs, 1 H), 7.74 (bs, 1 H), 7.87 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.7, 16.0, 17.6, 20.3, 20.4, 21.5, 22.6, 24.2, 29.4, 29.9, 30.4, 31.4, 35.8, 36.5, 37.3, 39.4, 41.3, 41.9, 46.7, 48.4, 55.3, 55.6, 57.9, 64.0, 68.0, 68.1, 74.8, 79.3, 116.5 (q, J = 291 Hz), 123.0, 126.5, 128.4, 129.2, 138.0, 149.3, 161.5, 162.1 (q, J = 35 Hz), 167.6, 168.3, 169.7, 172.8, 178.8 ppm. Minor rotamer (selected signals): 1H NMR (500 MHz, CDCl3): δ = 1.07 (d, J = 6.4 Hz, 3 H), 2.42 (t, J = 2.3 Hz, 1 H), 4.04 (s, 2 H), 4.79 (m, 1 H), 7.92 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 11.3, 15.5, 30.6, 39.1, 58.0, 126.6, 128.4, 137.7, 171.0, 172.8 ppm. HRMS (CI): calculated: for C42H64N6O7S [M + 2H]2+ 398.2273; found: 398.2280. Cyclopeptide 9 Allylester 7c (50.4 mg, 0.060 mmol) and a 4 M HCl solution in dioxane (75 μl, 0.3 mmol) was dissolved in CH2Cl2 (13 mL). This solution was added dropwise over 30 min to a refluxed solution of the Grubbs II catalyst (1.7 mg, 0.002 mmol) in CH2Cl2 (3 mL) under argon. After 10 and 20 min, further Grubbs II catalyst (1.7 mg, 0.002 mmol each) was added, and after complete addition of 7c the reaction mixture was refluxed for a further 10 min. After cooling to room temperature DMSO (21 μl, 0.30 mmol) was added and the solution was stirred overnight. The solvent was removed in vacuo and the crude product was purified by flash chromatography ((1) CH2Cl2/ MeOH 95 : 5; (2) reversed phase silica, MeCN/H2O/0.1% HCOOH gradient). 9 was obtained as a pale yellow solid (40.4 mg, 0.047 mmol, 79%), m.p. 190–191 °C. Rf (9): 0.36 (CH2Cl2/MeOH 9 : 1). [α]20 D = 16.8 (c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR (500 MHz, CDCl3): δ = 0.76 (d, J = 6.4 Hz, 3 H), 0.87 (t, J = 7.4 Hz, 3 H), 1.04–1.09 (m, 6 H), 1.14 (d, J = 7.0 Hz, 3 H), 1.22–1.31 (m, 2 H), 1.45–2.21 (m, 13 H), 2.25 (m, 1 H), 2.30 (s, 3 H), 2.68 (m, 1 H), 2.75–3.11 (m, 5 H), 3.22–3.97 (m, 9 H), 4.22–4.58 (m, 4 H), 5.51 (s, 1 H), 5.73 (m, 1 H), 7.15–7.31 (m, 6 H), 7.34 (d, J = 9.1 Hz, 1 H), 7.37 (d, J = 9.1 Hz, 1 H), 7.86 (s, 1 H), 8.28 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.7, 15.8, 15.9, 19.6, 20.6, 22.7, 23.7, 24.7, 29.7, 29.9, 30.6, 31.5, 36.2, 36.9, 37.5, 39.6, 40.9, 44.0, 46.3, 48.1, 53.7, 55.1, 63.9, 64.1, 68.1, 70.1, 70.3, 122.0, 126.4, 128.4, 129.4, 129.6, 130.2, 138.0, 149.8, 160.7, 168.8, 169.8, 173.1, 173.6, 176.0 ppm. Minor rotamer (selected signals): 1 H NMR (500 MHz, CDCl3): δ = 0.89 (t, J = 7.3 Hz, 3 H), 1.00 (d, J = 6.8 Hz, 3 H), 1.02 (d, J = 6.9 Hz, 3 H), 1.20 (d, J = 7.0 Hz, 3 H), 2.35 (s, 3 H), 6.68 (m, 1 H), 7.89 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 11.4, 16.3, 16.5, 20.3, 20.4, 24.1, 24.4, 30.2, 30.9, 36.2, 36.7, 39.4, 39.5, 43.6, 48.8, 54.1, 55.0, 64.0, 68.5, 71.9, 118.0, 122.5, 126.6, 127.2, 129.4, 137.8, 149.7, 160.5, 165.5, 168.6, 175.8 ppm. HRMS (CI): calculated: for C43H65N6O7S [M + H]+ 809.4630; found: 809.4656.
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N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-{N′-[2-(1-benzyl-1H-1,2,3triazol-4-yl)methoxyethyl]aminocarbonylmethyl}-desacetoxy(S)-tubuvalyl-(S)-tubuphenylalanin methylester 10 Water (0.8 mL) was added with vigorous stirring to a solution of benzyl azide (10.2 mg, 0.076 mmol) and alkyne 8c (61.8 mg, 0.076 mmol) in DMSO (2 mL). Subsequently a solution of Cu (II) sulfate (2.4 mg, 0.015 mmol) and sodium ascorbate (3.0 mg, 0.015 mmol) in water (0.15 mL each) was added dropwise. The suspension was stirred vigorously at room temperature for 3 d. Water (10 mL) was added and the aqueous layer was extracted twice with ethyl acetate (25 mL each). The combined organic layers were washed with water (3 × 5 mL) and brine (3 mL), dried (Na2SO4) and evaporated in vacuo. The crude product was purified by flash chromatography (CH2Cl2/ MeOH 95 : 5 to 9 : 1) to provide 10 (56.1 mg, 0.060 mmol, 78%) as a pale yellow solid, m.p. 44–45 °C. Rf (10): 0.22 (CH2Cl2/ MeOH 9 : 1). [α]20 D = 20.6 (c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR (500 MHz, CDCl3): δ = 0.73 (d, J = 6.6 Hz, 3 H), 0.86 (t, J = 7.4 Hz, 3 H), 0.94 (d, J = 6.9 Hz, 3 H), 0.95 (d, J = 6.6 Hz, 3 H), 1.13 (d, J = 7.1 Hz, 3 H), 1.15–1.22 (m, 2 H), 1.23–2.08 (m, 13 H), 2.21 (s, 3 H), 2.45 (m, 1 H), 2.61 (m, 1 H), 2.76–3.11 (m, 5 H), 3.20–3.70 (m, 6 H), 3.61 (s, 3 H), 4.32–4.43 (m, 2 H), 4.53 (m, 1 H), 4.60 (s, 2 H), 5.51 (s, 2 H), 6.94 (s, 1 H), 7.12–7.39 (m, 12 H), 7.52 (s, 1 H), 7.86 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 11.4, 15.8, 17.8, 19.6, 20.4, 23.2, 24.6, 25.0, 29.6, 30.1, 30.4, 30.5, 36.4, 37.8, 37.9, 39.5, 41.0, 41.3, 46.6, 48.4, 51.6, 54.1, 54.1, 55.2, 63.8, 64.4, 68.9, 69.4, 122.3, 122.6, 126.4, 126.4, 128.0, 128.3, 129.1, 129.4, 134.5, 137.7, 145.1, 149.4, 160.9, 169.1, 169.7, 173.8, 174.5, 176.6 ppm. Minor rotamer (selected signals): 1H NMR (500 MHz, CDCl3): δ = 0.88 (t, J = 7.4 Hz, 3 H), 0.92 (d, J = 6.8 Hz, 3 H), 1.04 (d, J = 6.5 Hz, 3 H), 2.19 (s, 3 H), 3.60 (s, 3 H), 4.58 (s, 2 H), 4.80 (m, 1 H), 5.50 (s, 2 H), 7.07 (s, 1 H), 7.54 (s, 1 H), 7.87 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.5, 16.4, 20.3, 20.6, 23.9, 30.7, 31.5, 36.5, 36.6, 37.7, 39.2, 41.3, 44.9, 48.5, 68.7, 69.3, 122.4, 122.5, 128.7, 128.7, 129.1, 129.4, 134.6, 149.6, 170.9, 176.5 ppm. HRMS (CI): calculated: for C50H72N9O7S [M + H]+ 942.5270; found: 942.5309.
Organic & Biomolecular Chemistry
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Acknowledgements Financial support from the Deutsche Forschungsgemeinschaft (FOR 1406, Ka 880/10-2) was gratefully acknowledged. J. Hoffmann gratefully acknowledges a fellowship of the Studienstiftung des Deutschen Volkes.
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Synthesis of pretubulysin-derivatives via the TubUgi-approach† Judith Hoffmann, Jan Gorges, Lukas Junk and Uli Kazmaier*
Received 24th March 2015, Accepted 20th April 2015 DOI: 10.1039/c5ob00587f www.rsc.org/obc
The Ugi reaction is found to be a very powerful tool for the synthesis of ( pre)tubulysin derivatives, allowing the introduction of various functionalized side chains in only one step. While polar groups such as amides are not well tolerated, unpolar side chains such as allyl or propargyl ether are well accepted. These functionalities also allow subsequent modifications in the side chain, e.g. via ring closing metathesis or Click reaction.
Introduction The microtubule skeleton is an interesting target for the development of antitumor drugs.1 Microtubuli are polymers of α- and β-tubulin heterodimers and play an essential role in a wide range of cellular processes, such as mitosis.2 They show rather complex polymerization dynamics, which can be modulated by antimitotic drugs. In principle, these drugs can be divided into two different groups. Compounds such as taxanes (e.g. paclitaxel), discodermolide or epothilone stabilize the tubulin skeleton, while e.g. the vinca alkaloids (e.g. vinblastin) destabilize the microtubuli (Fig. 1).3 Both scenarios suppress a regular cell division, leading to apoptosis. Many of these compounds show antitumor activity in the low or subnanomolar range, and are therefore good candidates for the development of antitumor drugs. The major drawback of these compounds results from their rather complex structure, and their synthesis in general is not a trivial issue. Therefore, in many cases, substantial amounts e.g. for clinical studies have to be isolated from natural sources. In 2000, Reichenbach and Höfle et al. reported the isolation of a family of tetrapeptides, called tubulysins, from the myxobacterial strains Archangium gephyra and Angiococcus disciformis.4 Several more representatives of this class of compounds were described by Müller et al. in 20045 and 2010.6 The tubulysins show very high cytotoxicity towards a wide range of tumor cell lines,4b,7 including multi drug resistant ones,8 and the same binding mode as the vinca alkaloids.7 Depending on the cell line, this cytotoxicity is higher by factors of 20–1000 compared to taxol or epothilone. In comparison with the other natural products mentioned, the struc-
Institute of Organic Chemistry, Saarland University, P.O. Box 151150, 66041 Saarbrücken, Germany. E-mail: [email protected] † Electronic supplementary information (ESI) available: Copies of 1H and NMR spectra. See DOI: 10.1039/c5ob00587f
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ture of the tubulysins is “relatively” simple. Besides an N-methylated (R)-pipecolic acid, especially the two prolonged unusual amino acids tubuvaline (Tuv) and tubuphenylalanine (Tup) [or tubutyrosine (Tut)] catch one’s eye. From a biosynthetic point of view, the acetoxy group and the rather unusual acylal side chain are introduced after the tetrapeptide has been formed.5
13
C
Fig. 1
Antimitotic natural products.
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According to their biosynthetic studies, Müller et al. postulated pretubulysin to be the biosynthetic precursor of the tubulysins.5 This assumption initiated our efforts to synthesize pretubulysin9 as the reference sample and to investigate the cytotoxicity of this rather simple molecule.10 Pretubulysin and its derivatives show strong anti-angiogenic effects11 and antivascular properties in vitro and in vivo,12 and are therefore a new option for the treatment of metastatic cancer.13 Although the biological activity of pretubulysin is low compared to the natural tubulysins, especially tubulysin D, the most active one, it was still in the low or subnanomolar range (depending on cell line). The cytotoxicity is still higher than that of taxol or epothilone. The promising properties of the tubulysins forced the development of several protocols towards the tubulysins14 and simplified derivatives thereof.15 The by far most critical step is the introduction of the acylal side chain,14 the position in the molecule where obviously variations are well tolerated, since the natural tubulysins mainly vary in this side chain. Therefore, variations in this position, allowing the incorporation of different, preferentially functionalized side chains, are strongly desired. The incorporation of a simple N-methyl group does not cause any problems and good yields are obtained in the peptide coupling between the Ile- and N-methyl Tuv-moieties with most modern coupling reagents.15 The situation changes dramatically if the size of the N-substituent increases. The coupling of the corresponding N-ethyl or N-allyl derivatives gives yields 100 °C) under an atmosphere of nitrogen or argon. Dried solvents were distilled before use. The products were purified by flash chromatography on silica gel columns (Macherey-Nagel 60, 0.063–0.2 mm) and with a flash chromatography system [Reveleris (Grace), RediSep-columns 4 g, 12 g, 24 g and 40 g from Axel Semrau]. Mixtures of ethyl acetate and petroleum ether or dichloromethane and methanol were generally used as eluents. Analytical TLC was performed on precoated silica gel plates (Macherey-Nagel, Polygram® SIL G/UV254). Visualization was accomplished with UV-light, KMnO4 solution or ninhydrin solution. Melting points were determined with a Dr Tottoli (Büchi) melting point apparatus and are uncorrected. 1H and 13C NMR spectra were recorded with a Bruker AV400 [400 MHz (1H) and 100 MHz (13C)] or a Bruker AV500 [500 MHz (1H) and 125 MHz (13C)] spectrometer in CDCl3. Chemical shifts are reported in ppm relative to TMS and CHCl3 was used as the internal standard. Diastereomeric ratios were determined by NMR or HPLC. Mass spectra were recorded with a Finnigan MAT 95 spectrometer (quadrupole) using the CI technique. HPLC/MS analysis was performed with a Shimadzu system (LC: 10A-series + Autosampler, MS: LCMS-2020). Elemental analyses were performed at the Saarland University. N-tert-Butyloxycarbonyl-(S)-isoleucyl-N-(N′-(2-tert-butyloxydimethylsilyloxyethyl)aminocarbonylmethyl)-(S)-valin methylester 1 (S)-Valine methylester (46.0 mg, 0.351 mmol) and paraformaldehyde (13.5 mg, 0.45 mmol) were dissolved in MeOH (0.55 mL) and were stirred for 30 min. Boc-(S)-isoleucine (84.8 mg, 0.353 mmol) was added and the solution was stirred for a further 10 min, before (tert-butyldimethylsilyloxyethyl)iso-
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cyanide (64.1 mg, 0.346 mmol) was added. The solution was stirred overnight before it was diluted with CH2Cl2 (5 mL). The organic layer was washed with sat. NaHCO3 (3 mL) and KHSO4 (2 mL), dried (Na2SO4) and evaporated in vacuo. Flash chromatography (silica, hexanes/ethylacetate 1.6 : 4; 2. 1 : 1) and subsequent crystallization (Et2O) gave rise to 1 (81.2 mg, 0.145 mmol, 48%) as a pale yellow solid. Rf (1): 0.65 (hexanes/ ethyl acetate 1 : 1). Mixture of rotamers. Major rotamer: 1 H NMR (400 MHz, CDCl3): δ = 0.06 (s, 6 H), 0.85–0.90 (m, 6 H), 0.89 (s, 9 H), 0.91 (d, J = 6.8 Hz, 3 H), 1.01 (d, J = 6.8 Hz, 3 H), 1.41 (s, 9 H), 1.57 (m, 2 H), 1.75 (m, 1 H), 2.33 (m, 1 H), 3.34 (m, 2 H), 3.70 (m, 2 H), 3.71 (s, 3 H), 4.12 (d, J = 17.7 Hz, 1 H), 4.16 (m, 1 H), 4.25 (d, J = 9.3 Hz, 1 H), 4.31 (d, J = 17.7 Hz, 1 H), 4.91 (d, J = 9.5 Hz, 1 H), 7.36 (bs, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = −5.4, 11.1, 16.0, 18.3, 19.5, 20.3, 23.9, 25.9, 28.2, 37.1, 42.1, 47.8 50.1, 52.2, 55.1, 61.5, 64.4, 79.8, 156.8, 167.9, 171.1, 173.7 ppm. Minor rotamer (selected signals): 1H NMR (400 MHz, CDCl3): δ = 0.06 (s, 6 H), 0.90 (s, 9 H), 1.43 (s, 9 H), 1.83 (m, 1 H), 3.43 (m, 2 H), 3.64 (m, 2 H), 3.97 (d, J = 15.3 Hz, 1 H), 4.05 (1 H, J = 15.3 Hz, 1 H), 4.50 (m, 1 H), 5.00 (d, J = 10.2 Hz, 1 H), 6.61 (bs, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = −3.6, 11.3, 16.1, 18.9, 19.6, 25.6, 28.3, 41.7, 52.1, 54.8, 61.8, 65.5 ppm. HRMS (CI): calculated: for C27H54N3O7Si [M + H]+ 560.3731; found: 560.3726. C27H53N3O7Si (559.81): calcd C 57.93, H 9.54, N 7.51; found C 57.93, H 9.64, N 7.05. N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(2-tertbutyloxydimethylsilyloxyethyl)aminocarbonylmethyl]desacetoxy-(S)-tubuvalin methylester 3a In analogy to 1 peptide 3a was obtained from amine 2a (21.6 mg, 84.3 μmol), paraformaldehyde (3.50 mg, 117 μmol), (R)-Mep-(S)-Ile-OH·HCl (26.5 mg, 90.5 μmol) and (tert-butyldimethylsilyloxyethyl)isocyanide (18.1 mg, 98 μmol) in MeOH (0.2 mL). The reaction mixture was stirred for 2 d. After workup and flash chromatography (CH2Cl2/MeOH 99 : 1 to 9 : 1) 3a (13.9 mg, 19.0 μmol, 23%) was obtained as an orange oil. Rf (3a): 0.40 (CH2Cl2/MeOH 9 : 1). Mixture of rotamers. Major rotamer: 1H NMR (400 MHz, CDCl3): δ = 0.05 (s, 6 H), 0.71 (d, J = 6.6 Hz, 3 H), 0.85–0.92 (m, 12 H), 0.94–0.97 (m, 6 H), 1.14–1.28 (m, 3 H), 1.37 (m, 1 H), 1.38 (t, J = 7.1 Hz, 3 H), 1.47 (m, 1 H), 1.55–1.61 (m, 3 H), 1.72 (m, 1 H), 1.77 (m, 1 H), 1.87–2.05 (m, 4 H), 2.15–2.31 (m, 4 H), 2.46 (dd, J = 10.5, 3.3 Hz, 1 H), 2.89 (m, 1 H), 3.04 (m, 1 H), 3.32–3.50 (m, 3 H), 3.53 (d, J = 13.6 Hz, 1 H), 3.64 (d, J = 13.6 Hz, 1 H), 3.69–3.73 (m, 3 H), 4.31–4.41 (m, 3 H), 6.72 (bs, 1 H), 6.97 (m, 1 H), 8.01 (s, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = −5.2, 11.7, 14.3, 15.8, 19.9, 20.4, 23.1, 24.6, 24.8, 25.9, 30.5, 30.9, 31.2, 31.6, 36.5, 42.1, 44.9, 46.6, 53.7, 55.2, 61.2, 61.6, 63.8, 69.5, 77.3, 127.2, 146.5, 161.3, 169.1, 172.2, 172.8, 173.9 ppm. Minor rotamer (selected signals): 1H NMR (400 MHz, CDCl3): δ = 0.09 (s, 6 H), 0.90 (s, 9 H), 2.22 (s, 3 H), 3.12 (m, 1 H), 3.82 (m, 1 H), 3.93 (m, 1 H), 7.05 (bs, 1 H), 7.11 (m, 1 H), 8.02 (s, 1 H) ppm. 13 C NMR (100 MHz, CDCl3): δ = −5.2, 15.8, 19.6, 20.7, 25.6, 31.3, 36.4, 52.1, 55.3, 61.5, 69.1, 127.1, 146.8, 161.6, 169.5,
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171.7 ppm. HRMS (CI): calculated: for C35H63N5O6SiS [M + 2H]+ 709.4257; found: 709.4261.
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General procedure for Ugi reactions A solution of the isocyanide (0.2 mmol) in CH2Cl2 (0.5 mL) was added slowly to a solution of the amine (0.2 mmol), paraformaldehyde (0.2 mmol) and the acid component (0.23 mmol) in CH2Cl2 (2 mL). The reaction mixture was stirred for 2–4 d and was monitored by TLC and/or LC-MS. After complete consumption of the amine component, the reaction mixture was hydrolyzed with sat. NaHCO3 solution (2 mL) and the aqueous layer was washed twice with CH2Cl2 (3 mL each). The combined organic layers were dried, filtered and evaporated in vacuo. The crude product was purified by flash chromatography.
N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(2-tertbutyloxydimethylsilyloxyethyl)aminocarbonylmethyl]desacetoxy-(S)-tubuvalyl-(S)-tubuphenylalanin allylester 4b According to the general procedure for Ugi reactions 4b was obtained from amine 2b (85.0 mg, 0.186 mmol), paraformaldehyde (5.6 mg, 0.186 mmol), Mep-Ile-OH (54.8 mg, 0.214 mmol) and (tert-butyldimethylsilyloxyethyl)isocyanide (34.4 mg, 0.186 mmol) in CH2Cl2 (1 mL). After flash chromatography (CH2Cl2/MeOH 99 : 1 to 9 : 1) and freeze drying, 4b (112 mg, 0.123 mmol, 66%) was obtained as a pale yellow resin. Rf (4b): 0.19 (CH2Cl2/MeOH 95 : 5). Mixture of rotamers. Major rotamer: 1H NMR (400 MHz, CDCl3): δ = 0.03 (s, 6 H), 0.71 (d, J = 6.6 Hz, 3 H), 0.80–0.89 (m, 12 H), 0.91–0.95 (m, 6 H), 1.14–1.22 (m, 5 H), 1.33–1.48 (m, 2 H), 1.53–1.67 (m, 5 H), 1.76–2.07 (m, 6 H), 2.20 (s, 3 H), 2.49 (m, 1 H), 2.63 (m, 1 H), 2.81–3.03 (m, 4 H), 3.23–3.47 (m, 3 H), 3.57–3.68 (m, 4 H), 4.33–4.40 (m, 2 H), 4.51 (m, 2 H), 4.60 (d, J = 17.0 Hz, 1 H), 5.16 (m, 1 H), 5.23 (m, 1 H), 5.84 (ddt, J = 17.2, 10.5, 5.5 Hz, 1 H), 6.84 (bs, 1 H), 7.00 (bs, 1 H), 7.14–7.24 (m, 5 H), 7.31 (d, J = 9.2 Hz, 1 H), 7.85 (s, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = −5.4, 10.4, 15.7, 17.7, 19.6, 20.4, 23.0, 23.8, 24.7, 24.8, 25.8, 30.2, 30.3, 30.3, 30.5, 36.4, 36.5, 37.7, 41.3, 42.0, 44.6, 46.6, 48.4, 53.7, 55.2, 61.5, 63.8, 65.0, 69.3, 117.8, 122.3, 126.3, 128.3, 129.4, 132.2, 137.7, 149.3, 160.8, 169.1, 169.6, 170.9, 173.8, 175.7 ppm. Minor rotamer (selected signals): 1 H NMR (400 MHz, CDCl3): δ = 0.04 (s, 6 H), 1.03 (d, J = 6.5 Hz, 3 H), 1.15 (d, J = 7.1 Hz, 3 H), 1.95 (m, 1 H), 2.24 (s, 3 H), 2.73–3.00 (m, 2 H), 3.88 (s, 2 H), 3.61 (m, 2 H), 4.83 (dd, J = 6.8, 6.8 Hz, 1 H), 7.14 (m, 1 H), 7.24 (m, 1 H), 7.87 (s, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 11.3, 16.4, 18.2, 20.2, 20.7, 29.6, 30.7, 31.5, 37.8, 41.7, 53.0, 122.6, 126.3, 129.3, 149.6, 160.6, 175.6 ppm. MS (ESI/APCI): calculated: for C48H79N6O7SSi [M + H]+ 911.55; found: 911.45; calculated: for C48H80N6O7SSi [M + 2H]2+ 456.28; found: 456.25. HRMS (CI): calculated: for C44H69N6O7SSi [M − –tBu]+ 853.4718; found: 853.4656.
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N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(tertbutyloxycarbonylaminoethyl)aminocarbonylmethyl]desacetoxy-(S)-tubuvalyl-(S)-tubuphenylalanin allylester 6b According to the general procedure for Ugi reactions 6b was obtained from amine 2b (58.2 mg, 0.127 mmol), paraformaldehyde (3.8 mg, 0.127 mmol), Mep-Ile-OH (37.5 mg, 0.146 mmol) and (tert-butyloxycarbonylaminoethyl)isocyanide (21.7 mg, 0.127 mmol) in CH2Cl2 (1 mL). After flash chromatography (CH2Cl2/MeOH 99 : 1 to 9 : 1) and freeze drying 6b (68.2 mg, 0.076 mmol, 60%) was obtained as a pale yellow resin. Rf (6b): 0.15 (CH2Cl2/MeOH 95 : 5). Mixture of rotamers (∼1 : 1). 1H NMR (400 MHz, CDCl3): δ = 0.76 (d, J = 6.6 Hz, 1.5 H), 0.85–0.89 (m, 6 H), 0.94 (d, J = 6.4 Hz, 1.5 H), 0.98 (d, J = 6.7 Hz, 1.5 H), 1.05 (d, J = 6.4 Hz, 1.5 H), 1.16 (d, J = 7.1 Hz, 1.5 H), 1.17 (d, J = 7.1 Hz, 1.5 H), 1.21–1.24 (m, 2 H), 1.37–1.49 (m, 11 H), 1.55–1.69 (m, 4.5 H), 1.79–1.83 (m, 1.5 H), 1.88–1.94 (m, 2 H), 1.99–2.07 (m, 2.5 H), 2.22 (s, 3 H), 2.29 (m, 0.5 H), 2.51 (m, 1 H), 2.63 (m, 1 H), 2.82–2.97 (m, 4 H), 3.13–3.19 (m, 1.5 H), 3.24–3.29 (m, 2.5 H), 3.40–3.49 (m, 2 H), 3.61 (d, J = 15.6 Hz, 0.5 H), 3.82 (m, 0.5 H), 4.19–4.25 (m, 1 H), 4.35–4.45 (m, 2 H), 4.53 (dm, J = 5.5 Hz, 2 H), 5.17 (dm, J = 10.5 Hz, 1 H), 5.25 (dm, J = 17.2 Hz, 1 H), 5.56 (bs, 0.5 H), 5.75 (bs, 0.5 H), 5.85 (ddt, J = 17.3, 10.5, 5.5 Hz, 1 H), 7.06–7.08 (m, 1.5 H), 7.14 (m, 0.5 H), 7.19–7.21 (m, 3.5 H), 7.26 (m, 2 H), 7.37 (d, J = 8.7 Hz, 1 H), 7.88 (s, 0.5 H), 7.91 (s, 0.5 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 10.8, 11.6, 16.0, 16.5, 17.9, 17.9, 20.0, 20.2, 20.4, 20.6, 23.2, 24.6, 25.0, 28.4, 28.5, 29.5, 29.9, 30.2, 30.3, 30.4, 30.7, 31.5, 36.4, 36.7, 37.8, 38.0, 40.2, 40.2, 40.4, 40.4, 41.3, 41.4, 44.9, 46.6, 48.5, 48.6, 54.3, 55.3, 55.3, 63.4, 65.1, 65.2, 69.2, 79.3, 118.0, 118.0, 122.5, 122.9, 126.4, 126.5, 128.4, 129.4, 129.5, 132.3, 132.3, 137.7, 137.8, 149.6, 156.3, 160.7, 160.9, 169.5, 169.7, 170.6, 173.3, 173.4, 175.4, 175.7, 175.8 ppm. MS (ESI/APCI): calculated: for C47H74N6O8S [M + H]+ 896.53; found: 896.40; calculated: for C47H75N6O8S [M + 2H]2+ 448.77; found: 448.75. HRMS (CI): calculated: for C47H74N7O8S [M + H]+ 896.5320; found: 896.5341. N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(2-allyloxyethyl)aminocarbonylmethyl]-desacetoxy-(S)-tubuvalyl-(S)tubuphenylalanin allylester 7b According to the general procedure for Ugi reactions 7b was obtained from amine 2b (107 mg, 0.234 mmol), paraformaldehyde (7.1 mg, 0.236 mmol), Mep-Ile-OH (69.0 mg, 0.269 mmol) and (allyloxyethyl)isocyanide (26.0 mg, 0.234 mmol) in CH2Cl2 (2.5 mL). After flash chromatography (CH2Cl2/MeOH 99 : 1 to 9 : 1) 7b (159 mg, 0.190 mmol, 81%) was obtained as a white solid, m.p. 46–47 °C. Rf (7b): 0.29 (CH2Cl2/MeOH 9 : 1). [α]20 D = 33.7 (c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR (500 MHz, CDCl3): δ = 0.73 (d, J = 6.6 Hz, 3 H), 0.87 (t, J = 7.4 Hz, 3 H), 0.95 (d, J = 6.4 Hz, 3 H), 0.96 (d, J = 6.6 Hz, 3 H), 1.17 (d, J = 7.1 Hz, 3 H), 1.17–1.25 (m, 2 H), 1.23–2.13 (m, 13 H), 2.21 (s, 3 H), 2.48 (m, 1 H), 2.63 (m, 1 H), 2.82–3.14 (m, 3 H), 2.86 (dd, J = 13.7, 6.7 Hz, 1 H), 2.94 (dd, J = 13.8, 6.0 Hz, 1 H), 3.23–3.70 (m, 5 H), 3.97 (d, J = 5.7 Hz, 2 H), 4.34–4.44 (m, 2 H), 4.48–4.63 (m, 3 H),
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5.15–5.20 (m, 2 H), 5.22–5.29 (m, 2 H), 5.81–5.93 (m, 2 H), 6.94 (d, J = 8.4 Hz, 1 H), 7.10 (t, J = 4.9 Hz, 1 H), 7.16–7.28 (m, 5 H), 7.32 (d, J = 9.2 Hz, 1 H), 7.87 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.5, 15.8, 17.8, 19.7, 20.5, 23.2, 24.7, 25.0, 29.6, 30.2, 30.6, 30.7, 36.6, 37.7, 37.9, 39.6, 41.3, 44.9, 46.7, 48.5, 53.8, 55.3, 63.9, 65.1, 68.3, 69.5, 72.0, 117.3, 117.9, 122.4, 126.4, 128.3, 129.5, 132.3, 134.4, 137.7, 149.5, 160.9, 169.1, 169.7, 170.9, 174.8, 175.8 ppm. Minor rotamer (selected signals): 1H NMR (500 MHz, CDCl3): δ = 0.90 (t, J = 7.4 Hz, 3 H), 1.06 (d, J = 6.5 Hz, 3 H), 1.17 (d, J = 7.1 Hz, 3 H), 2.23 (s, 3 H), 2.48 (m, 1 H), 2.63 (m, 1 H), 2.82–3.14 (m, 3 H), 3.95 (ddd, J = 5.6, 1.4 Hz, 1.4 Hz, 2 H), 7.88 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 11.4, 16.5, 17.9, 20.3, 20.7, 23.9, 30.4, 30.7, 31.5, 36.5, 36.6, 39.4, 48.5, 52.9, 55.4, 71.9, 117.1, 118.0, 122.6, 126.4, 129.4, 132.3, 134.5, 149.7, 160.6, 169.2, 175.7 ppm. HRMS (CI): calculated: for C45H69N6O7S [M + H]+ 837.4943; found: 837.4978.
N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(2-allyloxyethyl)aminocarbonylmethyl]-desacetoxy-(S)-tubuvalyl-(S)tubuphenylalanin methylester 7c According to the general procedure for Ugi reactions 7c was obtained from amine 2c (150 mg, 0.347 mmol), paraformaldehyde (10.4 mg, 0.347 mmol), Mep-Ile-OH (102 mg, 0.399 mmol) and (allyloxyethyl)isocyanide (38.5 mg, 0.347 mmol) in CH2Cl2 (3.5 mL). After flash chromatography (CH2Cl2/MeOH 99 : 1 to 9 : 1) 7c (225 mg, 0.277 mmol, 80%) was obtained as a white solid, m.p. 51–52 °C. Rf (7c): 0.32 (CH2Cl2/MeOH 9 : 1). [α]20 D = 12.1 (c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR (500 MHz, CDCl3): δ = 0.73 (d, J = 6.6 Hz, 3 H), 0.87 (t, J = 7.4 Hz, 3 H), 0.95 (d, J = 6.4 Hz, 3 H), 0.96 (d, J = 6.7 Hz, 3 H), 1.15 (d, J = 7.1 Hz, 3 H), 1.17–1.28 (m, 2 H), 1.30–2.12 (m, 13 H), 2.22 (s, 3 H), 2.50 (m, 1 H), 2.61 (m, 1 H), 2.72–3.14 (m, 3 H), 2.85 (dd, J = 13.7, 6.8 Hz, 1 H), 2.94 (dd, J = 13.7, 5.8 Hz, 1 H), 3.20–3.70 (m, 6 H), 3.62 (s, 3 H), 3.97 (d, J = 5.7 Hz, 2 H), 4.34–4.43 (m, 2 H), 4.57 (d, J = 16.4 Hz, 1 H), 5.17 (m, 1 H), 5.26 (m, 1 H), 5.87 (ddt, J = 17.1, 10.4, 5.7 Hz, 1 H), 6.88 (s, 1 H), 7.10 (s, 1 H), 7.16–7.28 (m, 5 H), 7.32 (d, J = 9.2 Hz, 1 H), 7.88 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.5, 15.8, 17.8, 19.7, 20.4, 23.2, 24.7, 24.9, 29.6, 30.2, 30.5, 30.6, 36.5, 37.8, 37.9, 39.6, 41.4, 44.9, 46.7, 48.4, 51.8, 53.8, 55.3, 63.9, 68.3, 69.5, 71.9, 117.4, 122.4, 126.3, 128.3, 129.5, 134.4, 137.7, 149.4, 160.9, 169.1, 169.7, 170.9, 173.8, 176.7 ppm. Minor rotamer (selected signals): 1H NMR (500 MHz, CDCl3): δ = 0.90 (t, J = 7.4 Hz, 3 H), 1.06 (d, J = 6.4 Hz, 3 H), 1.15 (d, J = 7.1 Hz, 3 H), 3.62 (s, 3 H), 3.87 (d, J = 14.9 Hz, 1 H), 3.95 (ddd, J = 5.6, 1.4, 1.4 Hz, 2 H), 4.84 (m, 1 H), 6.88 (s, 1 H), 7.90 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 11.4, 16.4, 20.3, 20.7, 23.8, 30.4, 30.7, 31.5, 36.5, 36.6, 37.7, 39.4, 41.3, 48.5, 71.9, 117.2, 122.7, 126.4, 129.4, 134.4, 149.6, 160.7, 169.2, 170.6, 176.6 ppm. HRMS (CI): calculated: for C43H67N6O7S [M + H]+ 811.4787; found: 811.4826.
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Organic & Biomolecular Chemistry
N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(2-propargyloxyethyl)aminocarbonylmethyl]-desacetoxy-(S)-tubuvalyl-(S)tubuphenylalanin allylester 8b According to the general procedure for Ugi reactions 8b was obtained from amine 2b (144 mg, 0.314 mmol), paraformaldehyde (9.4 mg, 0.314 mmol), Mep-Ile-OH (93.0 mg, 0.361 mmol) and ( propargyloxyethyl)isocyanide (34.2 mg, 0.314 mmol) in CH2Cl2 (4 mL). After flash chromatography (CH2Cl2/MeOH 99 : 1 to 9 : 1) 8b (229 mg, 0.274 mmol, 87%) was obtained as a white solid, m.p. 50–51 °C. Rf (8b): 0.30 (CH2Cl2/MeOH 9 : 1). [α]20 D = 34.9 (c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR (500 MHz, CDCl3): δ = 0.74 (d, J = 6.6 Hz, 3 H), 0.87 (t, J = 7.5 Hz, 3 H), 0.95 (d, J = 6.7 Hz, 3 H), 0.97 (d, J = 6.5 Hz, 3 H), 1.17 (d, J = 7.1 Hz, 3 H), 1.18–1.23 (m, 2 H), 1.54–2.12 (m, 13 H), 2.21 (s, 3 H), 2.44 (t, J = 2.4 Hz, 1 H), 2.48 (m, 1 H), 2.63 (m, 1 H), 2.82–3.03 (m, 5 H), 3.22–3.69 (m, 7 H), 4.12 (m, 2 H), 4.35–4.44 (m, 2 H), 4.53 (m, 2 H), 5.18 (ddt, J = 10.5, 1.3, 1.3 Hz, 1 H), 5.25 (ddt, J = 17.2, 1.5, 1.5 Hz, 1 H), 5.86 (ddt, J = 17.3, 10.6, 5.7 Hz, 1 H), 6.95 (d, J = 9.3 Hz, 1 H), 7.09 (t, J = 5.5 Hz, 1 H), 7.16–7.29 (m, 5 H), 7.32 (d, J = 9.2 Hz, 1 H), 7.87 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.5, 15.8, 17.8, 19.7, 20.5, 23.2, 24.7, 25.0, 29.6, 30.2, 30.6, 30.7, 36.6, 37.7, 37.9, 39.4, 41.3, 44.9, 46.7, 48.5, 53.8, 55.3, 58.2, 63.9, 65.1, 68.2, 69.4, 74.8, 79.3, 117.9, 122.4, 126.4, 128.3, 129.5, 132.3, 137.7, 149.5, 160.9, 169.1, 169.7, 170.9, 174.8, 175.7 ppm. Minor rotamer (selected signals): 1H NMR (500 MHz, CDCl3): δ = 0.90 (t, J = 7.5 Hz, 3 H), 1.05 (d, J = 6.5 Hz, 3 H), 1.17 (d, J = 7.1 Hz, 3 H), 2.23 (s, 3 H), 2.41 (t, J = 2.4 Hz, 1 H), 3.85 (d, J = 14.6 Hz, 1 H), 3.96 (d, J = 14.7 Hz, 1 H), 4.12 (m, J = 2.3 Hz, 2 H), 4.83 (m, 1 H), 7.88 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 11.4, 16.4, 17.8, 20.3, 20.7, 23.9, 30.4, 31.5, 36.6, 36.6, 39.2, 41.3, 48.5, 53.0, 55.3, 58.1, 62.9, 74.7, 79.4, 118.0, 122.7, 126.4, 129.4, 132.3, 149.7, 160.6, 169.2, 173.8, 174.0, 174.5, 175.7 ppm. HRMS (CI): calculated: for C45H67N6O7S [M + H]+ 835.4787; found: 835.4825. N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(2-propargyloxyethyl)aminocarbonylmethyl]-desacetoxy-(S)-tubuvalyl-(S)tubuphenylalanin methylester 8c According to the general procedure for Ugi reactions 8c was obtained from amine 2c (177 mg, 0.410 mmol), paraformaldehyde (12.3 mg, 0.410 mmol), Mep-Ile-OH (121 mg, 0.471 mmol) and ( propargyloxyethyl)isocyanide (44.7 mg, 0.471 mmol) in CH2Cl2 (4 mL). After flash chromatography (CH2Cl2/MeOH 99 : 1 to 9 : 1) 7c (264 mg, 0.326 mmol, 80%) was obtained as a white solid, m.p. 55–56 °C. Rf (8c): 0.38 (CH2Cl2/MeOH 9 : 1). [α]20 D = 21.1 (c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR (500 MHz, CDCl3): δ = 0.73 (d, J = 6.5 Hz, 3 H), 0.86 (t, J = 7.4 Hz, 3 H), 0.95 (d, J = 6.5 Hz, 3 H), 0.96 (d, J = 6.5 Hz, 3 H), 1.14 (d, J = 7.1 Hz, 3 H), 1.16–1.24 (m, 2 H), 1.57–2.12 (m, 13 H), 2.20 (s, 3 H), 2.44 (t, J = 2.3 Hz, 1 H), 2.48 (m, 1 H), 2.60 (m, 1 H), 2.72–3.12 (m, 5 H), 3.21–3.70 (m, 6 H), 3.61 (s, 3 H), 4.13 (d, J = 2.2 Hz, 2 H), 4.30–4.42 (m, 2 H), 4.56 (d, J = 16.9 Hz, 1 H), 6.96 (d, J =
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9.4 Hz, 1 H), 7.09 (t, J = 5.4 Hz, 1 H), 7.17–7.29 (m, 5 H), 7.32 (d, J = 9.2 Hz, 1 H), 7.87 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.4, 15.8, 17.8, 19.6, 20.4, 23.2, 24.6, 25.0, 29.6, 30.1, 30.4, 30.5, 36.4, 37.7, 37.9, 39.4, 41.3, 44.9, 46.7, 48.4, 51.6, 53.7, 55.2, 58.1, 63.9, 68.1, 69.3, 74.8, 79.3, 122.4, 126.3, 128.3, 129.4, 137.7, 149.4, 160.9, 169.1, 169.7, 173.8, 174.8, 176.6 ppm. Minor rotamer (selected signals): 1H NMR (500 MHz, CDCl3): δ = 1.05 (d, J = 6.5 Hz, 3 H), 2.22 (s, 3 H), 2.40 (t, J = 2.3 Hz, 1 H), 4.11 (d, J = 2.3 Hz, 2 H), 4.82 (m, 1 H), 7.89 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 11.4, 16.4, 20.3, 20.7, 23.8, 30.6, 31.5, 36.4, 36.5, 37.7, 39.1, 41.3, 48.5, 53.0, 55.3, 58.0, 74.7, 79.3, 122.7, 126.4, 129.4, 149.6, 160.6, 169.2, 170.9, 174.0, 176.5 ppm. HRMS (CI): calculated: for C43H65N6O7S [M + H]+ 809.4630; found: 809.4660. C43H64N6O7S (809.08): calcd C 63.83, H 7.97, N 10.39; found C 63.74, H 7.34, N 10.20. N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(2-allyloxyethyl) aminocarbonylmethyl]-desacetoxy-(S)-tubuvalyl-(S)tubuphenylalanin·CF3COOH 7-OH Ester 7c (33.7 mg, 0.042 mmol) was dissolved in dioxane (0.5 mL) before a 1 M LiOH solution (90 μl, 0.090 mmol) was added and the solution was stirred overnight at room temperature. The reaction was monitored by TLC and after complete consumption of 7c the reaction mixture was diluted with H2O and acidified to pH 2 using trifluoroacetic acid. The aqueous phase was extracted twice with ethyl acetate, the organic layer was dried (Na2SO4) and evaporated in vacuo. To remove the excess trifluoroacetic acid, the residue obtained was suspended twice in ether, decanted and dried in high vacuo. The acid 7-OH (30.2 mg, 0.040 mmol, 96%) was obtained as a colorless solid, m.p. 82–83 °C. [α]20 D = −13.6 (c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR (500 MHz, CDCl3): δ = 0.71 (m, 3 H), 0.87 (t, J = 7.0 Hz, 3 H), 0.93 (d, J = 6.6 Hz, 3 H), 0.96 (d, J = 6.6 Hz, 3 H), 1.13 (d, J = 7.0 Hz, 3 H), 1.16–1.36 (m, 2 H), 1.36–2.26 (m, 13 H), 2.62 (m, 1 H), 2.67 (s, 3 H), 2.72–3.34 (m, 6 H), 3.34–3.72 (m, 6 H), 3.97 (d, J = 5.7 Hz, 2 H), 4.03 (m, 1 H), 4.32–4.52 (m, 2 H), 5.14–5.30 (m, 2 H), 5.85 (ddt, J = 16.1, 10.9, 5.6 Hz, 1 H), 7.15–7.26 (m, 5 H), 7.52 (s, 1 H), 7.75 (d, J = 7.8 Hz, 1 H), 7.87 (s, 1 H), 8.24 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.6, 16.0, 17.0, 19.7, 20.4, 21.5, 22.5, 24.3, 29.5, 29.9, 30.3, 30.6, 35.8, 36.8, 37.1, 39.4, 41.3, 41.9, 46.7, 48.5, 55.3, 55.6, 64.0, 68.1, 68.2, 71.9, 116.5 (q, J = 292 Hz), 117.4, 122.9, 126.5, 128.4, 129.2, 134.3, 137.9, 149.4, 161.5, 162.2 (q, J = 36 Hz), 169.6, 171.0, 172.8, 173.1 ppm. The signal of the COOH group could not be detected. Minor rotamer (selected signals): 1H NMR (500 MHz, CDCl3): δ = 1.07 (d, J = 6.4 Hz, 3 H), 3.90 (d, J = 5.0 Hz, 2 H), 4.80 (m, 1 H), 7.87 (s, 1 H), 7.91 (s, 1 H), 8.52 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 11.3, 15.5, 31.5, 39.6, 71.7, 134.4, 149.6 ppm. HRMS (CI): calculated: for C42H66N6O7S [M + 2H]2+ 399.2351; found: 399.2359. N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-[N′-(2-propargyloxyethyl)aminocarbonylmethyl]-desacetoxy-(S)-tubuvalyl-(S)tubuphenylalanin·CF3COOH 8-OH According to acid 7-OH acid 8-OH was obtained from ester 8c (35.1 mg, 0.043) as a colorless solid (36.8 mg, 0.040 mmol,
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93%), m.p. 55–56 °C. [α]20 D = 21.1 (c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR (500 MHz, CDCl3): δ = 0.73 (bs, 3 H), 0.87 (m, 3 H), 0.91 (d, J = 6.4 Hz, 3 H), 0.96 (d, J = 6.8 Hz, 3 H), 1.13 (d, J = 7.1 Hz, 3 H), 1.18–1.37 (m, 2 H), 1.38–2.29 (m, 14 H), 2.45 (t, J = 2.1 Hz, 1 H), 2.50–2.77 (m, 4 H), 2.72–3.15 (m, 5 H), 3.38–3.74 (m, 6 H), 3.93 (m, 1 H), 4.14 (d, J = 2.0 Hz, 2 H), 4.28–4.50 (m, 2 H), 7.12–7.32 (m, 6 H), 7.58 (bs, 1 H), 7.74 (bs, 1 H), 7.87 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.7, 16.0, 17.6, 20.3, 20.4, 21.5, 22.6, 24.2, 29.4, 29.9, 30.4, 31.4, 35.8, 36.5, 37.3, 39.4, 41.3, 41.9, 46.7, 48.4, 55.3, 55.6, 57.9, 64.0, 68.0, 68.1, 74.8, 79.3, 116.5 (q, J = 291 Hz), 123.0, 126.5, 128.4, 129.2, 138.0, 149.3, 161.5, 162.1 (q, J = 35 Hz), 167.6, 168.3, 169.7, 172.8, 178.8 ppm. Minor rotamer (selected signals): 1H NMR (500 MHz, CDCl3): δ = 1.07 (d, J = 6.4 Hz, 3 H), 2.42 (t, J = 2.3 Hz, 1 H), 4.04 (s, 2 H), 4.79 (m, 1 H), 7.92 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 11.3, 15.5, 30.6, 39.1, 58.0, 126.6, 128.4, 137.7, 171.0, 172.8 ppm. HRMS (CI): calculated: for C42H64N6O7S [M + 2H]2+ 398.2273; found: 398.2280. Cyclopeptide 9 Allylester 7c (50.4 mg, 0.060 mmol) and a 4 M HCl solution in dioxane (75 μl, 0.3 mmol) was dissolved in CH2Cl2 (13 mL). This solution was added dropwise over 30 min to a refluxed solution of the Grubbs II catalyst (1.7 mg, 0.002 mmol) in CH2Cl2 (3 mL) under argon. After 10 and 20 min, further Grubbs II catalyst (1.7 mg, 0.002 mmol each) was added, and after complete addition of 7c the reaction mixture was refluxed for a further 10 min. After cooling to room temperature DMSO (21 μl, 0.30 mmol) was added and the solution was stirred overnight. The solvent was removed in vacuo and the crude product was purified by flash chromatography ((1) CH2Cl2/ MeOH 95 : 5; (2) reversed phase silica, MeCN/H2O/0.1% HCOOH gradient). 9 was obtained as a pale yellow solid (40.4 mg, 0.047 mmol, 79%), m.p. 190–191 °C. Rf (9): 0.36 (CH2Cl2/MeOH 9 : 1). [α]20 D = 16.8 (c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR (500 MHz, CDCl3): δ = 0.76 (d, J = 6.4 Hz, 3 H), 0.87 (t, J = 7.4 Hz, 3 H), 1.04–1.09 (m, 6 H), 1.14 (d, J = 7.0 Hz, 3 H), 1.22–1.31 (m, 2 H), 1.45–2.21 (m, 13 H), 2.25 (m, 1 H), 2.30 (s, 3 H), 2.68 (m, 1 H), 2.75–3.11 (m, 5 H), 3.22–3.97 (m, 9 H), 4.22–4.58 (m, 4 H), 5.51 (s, 1 H), 5.73 (m, 1 H), 7.15–7.31 (m, 6 H), 7.34 (d, J = 9.1 Hz, 1 H), 7.37 (d, J = 9.1 Hz, 1 H), 7.86 (s, 1 H), 8.28 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.7, 15.8, 15.9, 19.6, 20.6, 22.7, 23.7, 24.7, 29.7, 29.9, 30.6, 31.5, 36.2, 36.9, 37.5, 39.6, 40.9, 44.0, 46.3, 48.1, 53.7, 55.1, 63.9, 64.1, 68.1, 70.1, 70.3, 122.0, 126.4, 128.4, 129.4, 129.6, 130.2, 138.0, 149.8, 160.7, 168.8, 169.8, 173.1, 173.6, 176.0 ppm. Minor rotamer (selected signals): 1 H NMR (500 MHz, CDCl3): δ = 0.89 (t, J = 7.3 Hz, 3 H), 1.00 (d, J = 6.8 Hz, 3 H), 1.02 (d, J = 6.9 Hz, 3 H), 1.20 (d, J = 7.0 Hz, 3 H), 2.35 (s, 3 H), 6.68 (m, 1 H), 7.89 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 11.4, 16.3, 16.5, 20.3, 20.4, 24.1, 24.4, 30.2, 30.9, 36.2, 36.7, 39.4, 39.5, 43.6, 48.8, 54.1, 55.0, 64.0, 68.5, 71.9, 118.0, 122.5, 126.6, 127.2, 129.4, 137.8, 149.7, 160.5, 165.5, 168.6, 175.8 ppm. HRMS (CI): calculated: for C43H65N6O7S [M + H]+ 809.4630; found: 809.4656.
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N-Methyl-(R)-pipecolyl-(S)-isoleucyl-N-{N′-[2-(1-benzyl-1H-1,2,3triazol-4-yl)methoxyethyl]aminocarbonylmethyl}-desacetoxy(S)-tubuvalyl-(S)-tubuphenylalanin methylester 10 Water (0.8 mL) was added with vigorous stirring to a solution of benzyl azide (10.2 mg, 0.076 mmol) and alkyne 8c (61.8 mg, 0.076 mmol) in DMSO (2 mL). Subsequently a solution of Cu (II) sulfate (2.4 mg, 0.015 mmol) and sodium ascorbate (3.0 mg, 0.015 mmol) in water (0.15 mL each) was added dropwise. The suspension was stirred vigorously at room temperature for 3 d. Water (10 mL) was added and the aqueous layer was extracted twice with ethyl acetate (25 mL each). The combined organic layers were washed with water (3 × 5 mL) and brine (3 mL), dried (Na2SO4) and evaporated in vacuo. The crude product was purified by flash chromatography (CH2Cl2/ MeOH 95 : 5 to 9 : 1) to provide 10 (56.1 mg, 0.060 mmol, 78%) as a pale yellow solid, m.p. 44–45 °C. Rf (10): 0.22 (CH2Cl2/ MeOH 9 : 1). [α]20 D = 20.6 (c = 1.0, CHCl3). Mixture of rotamers. Major rotamer: 1H NMR (500 MHz, CDCl3): δ = 0.73 (d, J = 6.6 Hz, 3 H), 0.86 (t, J = 7.4 Hz, 3 H), 0.94 (d, J = 6.9 Hz, 3 H), 0.95 (d, J = 6.6 Hz, 3 H), 1.13 (d, J = 7.1 Hz, 3 H), 1.15–1.22 (m, 2 H), 1.23–2.08 (m, 13 H), 2.21 (s, 3 H), 2.45 (m, 1 H), 2.61 (m, 1 H), 2.76–3.11 (m, 5 H), 3.20–3.70 (m, 6 H), 3.61 (s, 3 H), 4.32–4.43 (m, 2 H), 4.53 (m, 1 H), 4.60 (s, 2 H), 5.51 (s, 2 H), 6.94 (s, 1 H), 7.12–7.39 (m, 12 H), 7.52 (s, 1 H), 7.86 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 11.4, 15.8, 17.8, 19.6, 20.4, 23.2, 24.6, 25.0, 29.6, 30.1, 30.4, 30.5, 36.4, 37.8, 37.9, 39.5, 41.0, 41.3, 46.6, 48.4, 51.6, 54.1, 54.1, 55.2, 63.8, 64.4, 68.9, 69.4, 122.3, 122.6, 126.4, 126.4, 128.0, 128.3, 129.1, 129.4, 134.5, 137.7, 145.1, 149.4, 160.9, 169.1, 169.7, 173.8, 174.5, 176.6 ppm. Minor rotamer (selected signals): 1H NMR (500 MHz, CDCl3): δ = 0.88 (t, J = 7.4 Hz, 3 H), 0.92 (d, J = 6.8 Hz, 3 H), 1.04 (d, J = 6.5 Hz, 3 H), 2.19 (s, 3 H), 3.60 (s, 3 H), 4.58 (s, 2 H), 4.80 (m, 1 H), 5.50 (s, 2 H), 7.07 (s, 1 H), 7.54 (s, 1 H), 7.87 (s, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 10.5, 16.4, 20.3, 20.6, 23.9, 30.7, 31.5, 36.5, 36.6, 37.7, 39.2, 41.3, 44.9, 48.5, 68.7, 69.3, 122.4, 122.5, 128.7, 128.7, 129.1, 129.4, 134.6, 149.6, 170.9, 176.5 ppm. HRMS (CI): calculated: for C50H72N9O7S [M + H]+ 942.5270; found: 942.5309.
Organic & Biomolecular Chemistry
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Acknowledgements Financial support from the Deutsche Forschungsgemeinschaft (FOR 1406, Ka 880/10-2) was gratefully acknowledged. J. Hoffmann gratefully acknowledges a fellowship of the Studienstiftung des Deutschen Volkes.
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