Suresh Kumar Chettua,
Rajesh Bagepalli Madhua,
Gajendrasinh Balvantsinh Raoljia,
Korupolu Raghu Babub,
N. S. Kameswara Raoa,
Srividya Gopalakrishnanc,
Ayesha Ismailc,
G. Bhanuprakash Reddyc and
Syed Shafi*d
aGVK Biosciences Private Limited, Medicinal Chemistry Laboratory, Hyderabad 500076, India
bAndhra University, Department of Engineering Chemistry, Andhra University College of Engineering (A), Vishakhapatnam 530003, India
cNational Institute of Nutrition, Hyderabad-500007, India
dDepartment of Chemistry, Jamia Hamdard, Hamdard Nagar, New Delhi-110062, India. E-mail: Syedshafi@jamiahamdard.ac.in; Tel: +91 9990806873
First published on 21st June 2016
The enantiopure synthesis of clavatustides A (1) and B (3) were accomplished by a seven step synthetic protocol starting from commercially available (R)-phenyllactic acid. As the optical rotation values of synthetic (R)-clavatustides A and B were not in agreement with the reported values, the corresponding antipodes 2 and 4 were synthesized from (S)-phenyllactic acid to address the issue. Both (R) and (S) clavatustides A and B were evaluated for their anti-proliferative activity against three human cancer cell lines using MTT assay. The cervical cancer cell line (HeLa) was found to be most sensitive to the test compounds in decreasing the cell viability. S-Isomers (2 and 4) were found to exhibit better anti-proliferative activity when compared to their enantiomers (R-isomers) with IC50s 24.5 and 26.8 μM respectively against HeLa cell lines. All the compounds tested had a minimal effect on the cell viability of normal lung cells, indicating that these compounds are not toxic to normal cells.
Renewed interest in the structural and pharmacological characterization of naturally occurring cyclic peptides and depsipeptides has recognized that this class of natural products may be useful in the development of new therapeutics.1b,c Cyclodepsipeptides are cyclic peptides in which one or more of the amide groups are replaced by the corresponding esters (cyclic peptides that has both peptide and ester linkages in proximity). Generally they are secondary metabolites of marine bacteria, fungi, sponge and plants.1a Cyclic peptides with unique biological activities are ubiquitous in nature.2 However; cyclopeptides with an anthranilic acid unit are rare in nature. Anthranilic acid dimers with micromolar affinity for the CCK1 receptors were considered to be an important molecular scaffold in medicinal chemistry.3 Very recently; two novel cyclodepsipeptides clavatustides A (1) and B (3) containing an anthranilic acid dimer and D-phenyllactic acid were isolated by Bin Wu and co-workers from cultured mycelia and broth of Aspergillus clavatus C2WU isolated from Xenograpsus testudinatus.4
Both 1 and 3 suppressed the proliferation of hepatocellular carcinoma (HCC) cell lines (HepG2, SMMC-7721 and Bel-7402) in a dose-dependent manner, induced an accumulation of HepG2 cells in G1 phase and reduction of cells in S phase.4 In particular, clavatustide B (3) efficiently suppressed cell proliferation in a dose dependent manner on human cancer cell lines, including pancreatic cancer (Panc-1), gastric cancer (MGC-803), colorectal cancer (SW-480), retinoblastoma (WERI-Rb-1) and prostate cancer (PC-3).5 The gross structure of 1 and 3 were determined using extensive spectroscopic analysis and absolute configuration of the phenyllactic acid moiety was confirmed as ‘R’. However, it is reported that both these compounds could not be isolated in pure form in spite of iterative purifications.4 Purity assessment is perhaps most critical in the case of novel compounds to which a biological activity is ascribed, because trace impurities of high potency can lead to false conclusions. Examples are the historic case of the lead compound sesbanamide “hidden” in sesbanin,6,7 the more recent findings of inactive leads such as epiquinamide containing the β2-selective nicotinic acetylcholine receptor agonist, epibatidine,8 and the lack of in vitro anti-TB potency in high-purity ursolic acid.9 Therefore, any step towards the synthesis of pure clavatustides A and B could be rewarding as it will confirm its activity and its structural simplicity would render opportunities to design and synthesize potent new analogs. In this regard we aimed at the total synthesis of cyclodepsipeptides clavatustides A and B (Fig. 1).
Our synthesis towards 5 for clavatustide B began with the acylation of known amine 6 with 2-nitrobenzoyl chloride. Metal mediated reduction of the nitro group in 7 followed by acid amine coupling with N-Boc-Sar-OH afforded intermediate 9 as a mixture of rotamers. (R)-Phenyllactic acid was coupled to the amine obtained after deprotection of the Boc group in 9 to yield compound 10. Debenzylation of compound 10 afforded intermediate 5 in good yields (Scheme 1).
However, to our disappointment, Yamaguchi lactonization10 of compound 5 under different conditions led only to trace amount of product formation along with other impurities in all the cases (Table 1).
| Entry | Reaction conditions | Remarks |
|---|---|---|
| a The conversion was found to be sluggish. LC-MS of the crude reaction mixture indicated ∼5–10% of desired product formation along with multiple unknown impurities. | ||
| 1 | Compound 5 (100 mg, 0.21 mmol), 2,4,6-trichlorobenzoyl chloride (0.036 mL, 0.23 mmol), pyridine (0.025 mL, 0.31 mmol), DMAP (38 mg, 0.31 mmol), CH2Cl2, (5 mL) room temperature to reflux, 24 h | The conversion was found to be sluggish, trace of desired product formation |
| 2 | Compound 5 (100 mg, 0.21 mmol), 2,4,6-trichlorobenzoyl chloride (0.036 mL, 0.23 mmol), pyridine (0.025 mL, 0.31 mmol), DMAP (77 mg, 0.63 mmol), THF (5 mL), RT, 48 h | The conversion was found to be sluggish. LC-MS of the crude RM indicated ∼5% of desired product formation along with multiple unknown impurities (UV/KMnO4 active) |
| 3 | Compound 5 (100 mg, 0.21 mmol), 2,4,6-trichlorobenzoyl chloride (0.036 mL, 0.23 mmol), DMAP (77 mg, 0.63 mmol), THF (10 mL), reflux, 48 h | The conversion was found to be sluggish. LC-MS of the crude RM indicated ∼5–10% of desired product formation along with multiple unknown impurities (UV/KMnO4 active) |
| 4 | Compound 5 (100 mg, 0.21 mmol), 2,4,6-trichlorobenzoyl chloride (0.036 mL, 0.23 mmol), NEt3 (0.044 mL, 0.31 mmol), toluene (5 mL), room temperature to reflux, 48 h | The conversion was found to be sluggish, no desired product formation |
| 5 | Compound 5 (100 mg, 0.21 mmol), 2,4,6-trichlorobenzoyl chloride (0.036 mL, 0.23 mmol), NEt3 (0.044 mL, 0.31 mmol), DMAP (77 mg, 0.63 mmol), toluene (10 mL), room temperature to reflux, 48 h | The conversion was found to be sluggish, trace of desired product formation |
The unsuccessful results in the final macrolactonization prompted us to look for an alternate method of ring closure and we intended to explore approach-2 (macrolactamization approach) as shown in Fig. 3.
Our synthesis commenced by coupling known (R)-tert-butyl-2-hydroxy-3-phenylpropanoate11 (13) with 2-nitrobenzoic acid using DCC/DMAP conditions to obtain compound 14 in decent yields. The nitro group was smoothly reduced to amine 15 using 10% Pd/C in EtOAc and immediately reacted with 2-nitrobenzoyl chloride to afford compound 16 in good yields. The Abz-Abz-ester 17 having the di-anthranilic acid motif was obtained in high yield by the reduction of nitro group in compound 16.
The key precursors 11 and 12 of clavatustides A and B were obtained in good yields by amide coupling of 17 with N-ethyl glycine and sarcosine respectively as a mixture of rotamers (Scheme 3). Global de-protection of Boc and tert butyl groups of 11 and 12 followed by macrolactamization under amide coupling conditions (T3P/DIPEA/THF) furnished 1 and 3 in moderate yields. It is noteworthy that cyclization was sluggish using other amide couple reagents such as EDC·HCl/HOBt/NEt3/THF/RT and HATU/DIPEA/THF/RT.
When compiling the final characterization data of 1 and 3, a discrepancy was noted with respect to the reported optical rotations. While the 1H and 13C NMR spectra were in agreement with the reported values, the optical rotations were not only of opposite sign, but incomparable magnitudes (Table 2). Moreover, we found a discrepancy in the melting points between reported and synthesized depsipeptides. To address this discrepancy, we synthesized (S)-isomers of clavatustides A and B from advanced intermediate ent-17 (enantiomer of 17), which was in turn synthesized from (S)-tert-butyl-2-hydroxy-3-phenylpropanoate (13) through the intermediate compounds ent-14, ent-15 and ent-16 as shown in Scheme 4. The key intermediates ent-11 and ent-12 were synthesized from the compound ent-17 by coupling with corresponding amino acids (Scheme 4). Finally ent-11 and ent-12 were lactamized under macrolactamization conditions to afford the target compounds (S)-clavatustides A (2) and B (4) respectively (Scheme 4). The chemistry followed in Scheme 4 was very much similar to that adopted in Schemes 2 and 3.
| Compound | Optical rotations | Melting points | ||
|---|---|---|---|---|
| Reported values | Experimental values (present work) | Reported values | Experimental values (present work) | |
| (R)-Clavatustide A (1) | [α]24D = +22 | [α]24D = −111.68 | 187–188 °C | 246–248 °C |
| (S)-Clavatustide A (2) | [α]24D = +127.12 | 248–250 °C | ||
| (R)-Clavatustide B (3) | [α]24D = +65 | [α]24D = −129.59 | 193–194 °C | 213–216 °C |
| (S)-Clavatustide B (4) | [α]24D = +111.02 | 210–214 °C | ||
A comparison of optical rotations and melting points of the naturally occurring clavatustides, synthetic clavatustides and their enantiomers are given in Table 2.
| Compound | IC50s (in μM) | ||
|---|---|---|---|
| PC3 | MDA-MB-231 | HeLa | |
| (R)-Clavatustide-A [1] | 100 | 74.9 | 39.6 |
| (S)-Clavatustide-A [2] | 98.8 | 93.4 | 24.5 |
| (R)-Clavatustide-B [3] | 84.7 | 69.7 | 43.2 |
| (S)-Clavatustide-B [4] | 80.0 | 68.8 | 26.8 |
Among the three cell lines tested, the cervical cancer cell line (HeLa) was found to be most sensitive to the compounds. The potency of the compounds in inducing cell death was in the order: HeLa > MDA-MB-231 > PC3. Further S-isomers of clavatustides A (2) and B (4) were found to be more active when compared to their enantiomers i.e. R-isomers.
All the compounds tested had a minimal effect on the cell viability of the normal lung cells, indicating that these compounds are not toxic to normal cells (Fig. 4).
:
1), were added NH4Cl (5.12 g, 95.74 mmol), iron powder (3.46 g, 63.82 mmol) at room temperature and heated to 80 °C for 12 h. The catalyst was filtered over celite and washed with EtOAc (100 mL). The combined organic layer was concentrated under reduced pressure and the residue was purified by silica gel column (hexane–EtOAc 9/1) to obtain compound 8 (4.21 g, 76%) as a white solid. Mp: 87–91 °C (hexane
:
EtOAc = 9
:
1); IR (KBr): 3455, 3325, 3030, 1686, 1663, 1532, 1254 cm−1; 1H NMR (400 MHz, DMSO-d6): δ 11.40 (s, 1H, D2O exchangeable), 8.5 (d, J = 8.0 Hz, 1H), 8.03 (dd, J = 7.6, 1.2 Hz, 1H), 7.65 (q, J = 1.6 Hz, 2H), 7.48 (t, J = 1.6 Hz, 2H), 7.42–7.34 (m, 3H), 7.26 (m, 1H), 7.19 (m, 1H), 6.84 (dd, J = 8.4, 0.8 Hz, 1H), 6.67–6.63 (m, 3H), 5.38 (s, 2H): 13C NMR (100 MHz, DMSO-d6): δ 167.4, 150.5, 140.6, 135.5, 134.2, 132.7, 130.6, 128.4, 128.1, 128.0, 127.3, 122.8, 120.7, 117.0, 116.6, 115.1, 113.7, 66.7: HRMS (ESI): calcd for C21H18N2NaO3 [M + Na]+ 369.1210; found 369.1227.In another round bottomed flask was taken D-phenyllactic acid (0.96 g, 5.8 mmol) in THF (30 mL), added DIPEA (2.88 mL, 17.38 mmol), HATU (3.3 g, 8.70 mmol) at 0 °C and stirred for 30 min. A solution of above obtained TFA salt in THF (20 mL) was added to the reaction mixture at 0 °C and stirred for 24 h at room temperature. The reaction mixture was quenched with cold water (45 mL) and extracted with EtOAc (3 × 45 mL). The combined organic layer was washed with water (50 mL), brine (25 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column (hexane–EtOAc 6/4) to afford compound 10 (2.51 g, 76% overall yield for two steps) as a brown semi solid (mixture of rotamers); mp: 121–125 °C IR (KBr): 3402, 3264, 2925, 1691, 1647, 1583, 1259 cm−1. 1H NMR (400 MHz, DMSO-d6); δ 11.18 (br.s, 1H), 10.79 (br.s, 1H), 8.36–8.31 (m, 2H), 8.01 (d, J = 7.5 Hz, 1H), 7.82 (d, J = 7.5 Hz, 1H), 7.56 (t, J = 8 Hz, 2H), 7.42 (d, J = 7.5 Hz, 2H), 7.36–7.30 (m, 3H), 7.24 (t, J = 7.5 Hz, 2H), 7.23–7.15 (m, 5H), 5.36 (s, 2H), 4.68 (d, J = 0.9 Hz, 2H), 4.19 (d, J = 16.4 Hz, 1H), 4.04 (br.s, 1H), 3.02 (br.s, 4H), 2.76–2.72 (m, 1H); HRMS (ESI): calcd for C33H31N3NaO6 [M + Na]+ 588.2105; found 588.2106.
:
EtOAc = 9
:
1); [α]25D = +9.20 (c 1.0, CHCl3); SFC: Chiralpak AD-H (4.6 × 250) mm 5 μ, CO2-0.5% DEA in MeOH, 90
:
10 flow rate = 3 g min−1, tR = 2.41 min; IR (KBr): 3498, 2979, 1728, 1157 cm−1; 1H NMR (400 MHz, CDCl3): δ 7.29–7.22 (m, 5H), 4.33–4.30 (t, J = 5.6 Hz, 1H), 3.08 (dd, J = 14.0, 4.8 Hz, 1H), 2.94 (dd, J = 14.0, 6.4 Hz, 1H), 2.82 (br.s, 1H), 1.40 (s, 9H); 13C NMR (100 MHz, CDCl3): 173.40, 136.67, 129.58, 128.17, 126.64, 82.54, 71.24, 40.51, 27.93.
:
EtOAc = 9
:
1); [α]25D = −9.09 (c 1.0, CHCl3); SFC: Chiralpak AD-H (4.6 × 250) mm 5 μ, CO2-0.5% DEA in MeOH, 90
:
10 flow rate = 3 g min−1, tR = 2.13 min; IR (KBr): 3500, 2979, 1728, 1158 cm−1; 1H NMR (400 MHz, CDCl3): δ 7.30–7.20 (m, 5H), 4.32 (br.s, 1H), 3.08 (dd, J = 14.0 Hz and J = 4.8 Hz, 1H), 2.94 (dd, J = 14.0, 6.4 Hz, 1H), 2.82 (br.s, 1H), 1.43 (s, 9H); 13C NMR (100 MHz, CDCl3): 173.29, 136.62, 129.48, 128.03, 126.49, 82.29, 71.17, 40.39, 27.80.
:
15 flow rate = 3 g min−1, tR = 4.41 min; IR (KBr): 3472, 2981, 1738, 1537 cm−1; 1H NMR (400 MHz, CDCl3): δ 7.92–7.89 (m, 1H), 7.72–7.67 (m, 1H), 7.66–7.60 (m, 2H), 7.32–7.23 (m, 5H), 5.38 (dd, J = 7.6, 5.6 Hz, 1H), 3.23–3.19 (m, 2H), 1.42 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 167.86, 164.49, 148.14, 135.57, 132.79, 131.87, 130.16, 129.44, 128.40, 127.00, 123.90, 82.73, 74.83, 37.15, 27.85; HRMS (ESI): calcd for C20H22NO6 [M + H]+ 372.1447; found 372.1446.
:
15 flow rate = 3 g min−1, tR = 5.15 min; IR (KBr): 3460, 2979, 1739, 1538 cm−1; 1H NMR (400 MHz, CDCl3): δ 7.91–7.89 (m, 1H), 7.72–7.62 (m, 3H), 7.31–7.23 (m, 5H), 5.40–5.36 (m, 1H), 3.23–3.19 (m, 2H), 1.42 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 167.88, 164.51, 148.13, 135.57, 132.81, 131.88, 130.17, 129.45, 128.41, 127.01, 123.92, 82.75, 74.83, 37.16, 27.86; HRMS (ESI): calcd for C20H22NO6 [M + H]+ 372.1447; found 372.1444.
:
EtOAc 9/1) to obtain compound 15 as an off white solid (3.41 g, 86%). TLC (hexane
:
EtOAc 8/2); [α]25D = +23.20 (c 0.5, CHCl3); SFC: Chiralcel OJ-H (4.6 × 250) mm 5 μ, CO2-0.5% DEA in MeOH, 80
:
20 flow rate = 3 g min−1, tR = 2.58 min; IR (KBr): 3469, 3356, 2982, 1719, 1692 cm−1; 1H NMR (400 MHz, CDCl3): δ 7.89 (dd, J = 8.0, 1.2 Hz, 1H), 7.31–7.27 (m, 4H), 7.24–7.22 (m, 2H), 6.66–6.61 (m, 2H), 5.60 (br.s, 2H, D2O exchangeable), 5.28 (t, J = 6.4 Hz, 1H), 3.23 (d, J = 6.4 Hz, 2H), 1.40 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 168.88, 167.28, 150.58, 136.28, 134.29, 131.41, 129.48, 128.38, 126.88, 116.54, 116.31, 110.27, 82.15, 73.39, 37.54, 27.89; HRMS (ESI): calcd for C20H24NO4 [M + H]+ 342.1627; found 342.1630; HRMS (ESI): calcd for C20H24NO4 [M + H]+ 342.1705; found 342.1701.
:
20 flow rate = 3 g min−1, tR = 3.39 min; IR (KBr): 3468, 3355, 2976, 1721, 1691 cm−1; 1H NMR (400 MHz, CDCl3): δ 7.88 (d, J = 8.0, 1.6 Hz, 1H), 7.30–7.21 (m, 6H), 6.65–6.60 (m, 2H), 5.61 (br.s, 2H, D2O exchangeable), 5.28 (t, J = 6.81 Hz, 1H), 3.24 (d, J = 6.4 Hz, 2H), 1.40 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 168.88, 167.28, 150.58, 136.27, 134.29, 131.40, 129.47, 128.37, 126.88, 116.53, 116.29, 110.24, 82.15, 73.80, 37.53, 27.88; HRMS (ESI): calcd for C20H24NO4 [M + H]+ 342.1705; found 342.1705.
:
30 flow rate = 3 g min−1, tR = 4.06 min; IR (KBr): 3297, 3268, 2981, 1743, 1530, 1275, 1259 cm−1; 1H NMR (400 MHz, CDCl3): δ 11.17 (br.s, 1H, D2O exchangeable), 8.78 (d, J = 8.4 Hz, 1H), 8.08–8.06 (m, 2H), 7.71–7.59 (m, 4H), 7.32–7.17 (m, 6H), 5.29 (dd, J = 7.6, 5.2 Hz, 1H), 3.25–3.22 (m, 2H), 1.37 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 168.10, 167.53, 164.39, 146.92, 140.89, 135.69, 135.07, 133.60, 132.98, 131.05, 130.75, 129.40, 128.50, 128.36, 127.13, 124.67, 123.52, 120.90, 115.40, 82.78, 74.22, 37.34, 27.83; HRMS: calcd for C27H27N2O7 [M + H]+ 491.1818 found 491.1812.
:
30 flow rate = 3 g min−1, tR = 3.68 min; IR (KBr): 3447, 3347, 1743, 1691, 1530, 1252 cm−1; 1H NMR (400 MHz, CDCl3): δ 11.17 (br.s, 1H, D2O exchangeable), 8.78 (d, J = 8.4 Hz, 1H), 8.07 (t, J = 8.2 Hz, 2H), 7.70–7.58 (m, 4H), 7.32–7.16 (m, 6H), 5.29 (dd, J = 7.6, 5.6 Hz, 1H), 3.25–3.22 (m, 2H), 1.37 (s, 9H) ppm; 13C NMR (100 MHz, CDCl3): δ 168.09, 167.51, 164.37, 146.91, 140.88, 135.68, 135.05, 133.59, 132.96, 131.04, 130.75, 129.39, 128.49, 128.34, 127.11, 124.66, 123.50, 120.87, 115.38, 82.77, 74.21, 37.33, 27.81 ppm; HRMS: calcd for C27H27N2O7 [M + H]+ 491.1818 found 491.1813.
:
40 flow rate = 4 g min−1, tR = 5.17 min; IR (KBr): 3445, 3347, 2978, 1736, 1671 cm−1; 1H NMR (400 MHz, CDCl3): δ 11.53 (br.s, 1H, D2O exchangeable), 8.79 (d, J = 8.8 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.60–7.56 (m, 2H), 7.33–7.22 (m, 6H), 7.10 (t, J = 7.6 Hz, 1H), 6.72–6.68 (m, 2H), 5.74 (br.s, 2H, D2O exchangeable), 5.36–5.33 (m, 1H), 3.30–3.22 (m, 2H), 1.41 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 168.27, 168.07, 167.81, 149.80, 141.96, 135.87, 134.79, 132.81, 131.06, 129.41, 128.48, 127.69, 127.07, 122.41, 120.41, 117.41, 116.75, 115.63, 115.00, 82.62, 74.23, 37.39, 27.87; HRMS (ESI): calcd for C27H29N2O5 [M + H]+ 461.2076; found 461.2079.
:
40 flow rate = 4 g min−1, tR = 4.5 min; IR (KBr): 3447, 3346, 2976, 1721, 1691 cm−1; 1H NMR (400 MHz, CDCl3): δ 11.53 (br.s, 1H, D2O exchangeable), 8.80–8.78 (m, 1H), 8.09–8.07 (m, 1H), 7.63–7.55 (m, 2H), 7.33–7.24 (m, 4H), 7.23–7.20 (m, 2H), 7.12–7.08 (m, 1H), 6.71–6.67 (m, 2H), 5.74 (br.s, 2H, D2O exchangeable), 5.36 (dd, J = 7.6, 5.2 Hz, 1H), 3.30–3.23 (m, 2H), 1.41 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 168.25, 168.05, 167.79, 149.79, 141.95, 135.85, 134.77, 132.79, 131.04, 129.40, 128.46, 127.67, 127.05, 122.39, 120.40, 117.42, 116.72, 115.60, 114.98, 82.60, 74.21, 37.37, 27.85; HRMS (ESI): calcd for C27H29N2O5 [M + H]+ 461.2076; found 461.2072.
:
40 flow rate = 3 g min−1, tR = 5.32 min; IR (KBr): 3447, 2978, 1748, 1741, 1694 cm−1; 1H NMR (400 MHz, DMSO-d6, VT NMR, 90 °C): δ 11.0 (br.s, 1H, D2O exchangeable), 10.8 (bs, 1H, D2O exchangeable), 8.44–8.39 (m, 2H), 8.0 (d, J = 6.8 Hz, 1H), 7.77 (d, J = 6.8 Hz, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.30–7.19 (m, 7H), 5.34 (t, J = 6.4 Hz, 1H), 3.97 (s, 2H), 3.30 (q, J = 7.0 Hz, 2H), 3.22 (d, J = 6.4 Hz, 2H), 1.32 (s, 18H), 1.06 (t, J = 7.2 Hz, 3H); HRMS (ESI): calcd for C36H44N3O8 [M + H]+ 646.3128; found 646.3122.
:
40 flow rate = 3 g min−1, tR = 7.39 min; IR (KBr): 3466, 3307, 2977, 1745, 1694 cm−1; 1H NMR (400 MHz, DMSO-d6, VT NMR, 90 °C): δ 11.0 (br.s, 1H, D2O exchangeable), 10.8 (br.s, 1H, D2O exchangeable), 8.44–8.39 (m, 2H), 7.99 (d, J = 7.6 Hz, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.55 (t, J = 7.8 Hz, 1H), 7.30–7.19 (m, 7H), 5.34 (t, J = 6.4 Hz, 1H), 3.91 (s, 2H), 3.30 (q, J = 7.2 Hz, 2H), 3.22 (d, J = 6.8 Hz, 2H), 1.32 (s, 18H), 1.06 (t, J = 7.0 Hz, 3H); HRMS (ESI): calcd for C36H44N3O8 [M + H]+ 646.3128; found 646.3127.
:
45 flow rate = 3 g min−1, tR = 4.9 min; IR (KBr): 3271, 2978, 1748, 1694, 1253 cm−1; 1H NMR (400 MHz, DMSO-d6, VT NMR, 90 °C): δ 11.03 (br.s, 1H, D2O exchangeable), 10.83 (br.s, 1H, D2O exchangeable), 8.43 (d, J = 8.0 Hz, 1H), 8.36 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 7.55 (t, J = 7.8 Hz, 1H), 7.30–7.19 (m, 7H), 5.34 (t, J = 6.4 Hz, 1H), 3.88 (s, 2H), 3.21 (d, J = 6.4 Hz, 2H), 2.88 (s, 3H), 1.25 (s, 18H); HRMS (ESI): calcd for C35H42N3O8 [M + H]+ 632.2972; found 632.2975.
:
45 flow rate = 3 g min−1, tR = 5.91 min. IR (KBr): 3305, 2978, 1744, 1695, 1264 cm−1; 1H NMR (400 MHz, DMSO-d6, VT NMR, 90 °C): δ 11.03 (br.s, 1H, D2O exchangeable), 10.8 (br.s, 1H, D2O exchangeable), 8.43 (d, J = 8.4 Hz, 1H), 8.37 (d, J = 8.0 Hz, 1H), 8.01–7.98 (m, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.70–7.65 (m, 1H), 7.57–7.53 (m, 1H), 7.30–7.19 (m, 7H), 5.35 (t, J = 6.4 Hz, 1H), 3.93 (s, 2H), 3.22 (d, J = 6.4 Hz, 2H), 2.89 (s, 3H), 1.32 (s, 18H); HRMS (ESI): calcd for C35H42N3O8 [M + H]+ 632.2972; found 632.2975.
:
40 flow rate = 4 g min−1, tR = 3.3 min; IR (KBr): 3275, 1727, 1690, 1660 cm−1; 1H NMR (400 MHz, CDCl3): δ 10.21 (br.s, 1H, D2O exchangeable), 8.58 (d, J = 8.4 Hz, 1H), 7.85 (br.s, 1H, D2O exchangeable), 7.64 (d, J = 7.2 Hz, 1H), 7.57–7.46 (m, 3H), 7.38–7.30 (m, 6H), 7.22 (d, J = 7.2 Hz, 1H), 7.12 (t, J = 7.2 Hz, 1H), 5.47 (dd, J = 8.8, 6.2 Hz, 1H), 5.25 (d, J = 14.8 Hz, 1H), 3.97–3.92 (m, 1H), 3.49–3.44 (m, 1H), 3.31–3.21 (m, 3H), 1.02 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 169.50, 167.90, 167.43, 167.09, 138.09, 135.83, 134.50, 132.42, 132.14, 129.63, 129.36, 128.69, 127.54, 127.26, 126.38, 126.30, 125.87, 123.03, 122.84, 121.71, 71.56, 51.60, 43.24, 37.62, 13.64; HRMS (ESI): calcd for C27H26N3O5 [M + H]+ 472.1872; found 472.1873.
:
40 flow rate = 4 g min−1, tR = 2.31 min; IR (KBr): 3277, 1727, 1690, 1651 cm−1; 1H NMR (400 MHz, CDCl3): δ 10.22 (br.s, 1H, D2O exchangeable), 8.55 (d, J = 8.4 Hz, 1H), 7.92 (br.s, 1H, D2O exchangeable), 7.62 (d, J = 8.0 Hz, 1H), 7.57–7.46 (m, 3H), 7.39–7.31 (m, 6H), 7.21 (d, J = 7.6 Hz, 1H), 7.11 (d, J = 7.6 Hz, 1H), 5.47 (dd, J = 8.2, 6.0 Hz, 1H), 5.24 (d, J = 14.8 Hz, 1H), 3.95–3.93 (m, 1H), 3.46 (dd, J = 14.0, 8.0 Hz, 1H), 3.31–3.20 (m, 3H) 1.01 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 169.49, 167.86, 167.47, 167.11, 138.04, 135.78, 134.53, 132.40, 132.09, 129.61, 129.26, 128.67, 127.47, 127.25, 126.46, 126.19, 125.92, 123.02, 122.81, 121.60, 71.53, 51.57, 43.22, 37.61, 13.61; HRMS (ESI): calcd for C27H26N3O5 [M + H]+ 472.1872; found 472.1876.
:
40 flow rate = 4 g min−1, tR = 4.21 min; IR (KBr): 3306, 3211, 2925, 1721, 1690 cm−1; 1H NMR (400 MHz, CDCl3): δ 10.14 (br.s, 1H, D2O exchangeable), 8.55 (d, J = 8.4 Hz, 1H), 7.86 (br.s, 1H, D2O exchangeable), 7.69 (d, J = 6.8 Hz, 1H), 7.58–7.45 (m, 3H), 7.37–7.31 (m, 6H), 7.23 (d, J = 7.6 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 5.53 (t, J = 7.6 Hz, 1H), 5.34 (d, J = 14.4 Hz, 1H), 3.43 (dd, J = 13.2, 8.0 Hz, 1H), 3.29 (dd, J = 13.6, 7.2 Hz, 1H), 3.12 (d, J = 14.4 Hz, 1H), 3.06 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 169.65, 167.45, 167.32, 167.20, 137.97, 135.46, 134.68, 132.55, 132.09, 129.52, 128.65, 127.37, 127.28, 126.37, 126.32, 126.03, 123.06, 122.86, 121.73, 71.39, 54.11, 37.15, 35.74; HRMS (ESI): calcd for C26H24N3O5 [M + H]+ 458.1716; found 458.1713.
:
40 flow rate = 4 g min−1, tR = 2.41 min; IR (KBr): 3307, 3212, 2925, 1722, 1691, 1661 cm−1; 1H NMR (400 MHz, CDCl3): δ 10.14 (br.s, 1H, D2O exchangeable), 8.55 (d, J = 8.0 Hz, 1H), 7.84 (br.s, 1H, D2O exchangeable), 7.69 (d, J = 6.8 Hz, 1H), 7.58–7.45 (m, 3H), 7.37–7.31 (m, 6H), 7.23 (d, J = 7.6 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 5.53 (t, J = 7.6 Hz, 1H), 5.34 (d, J = 14.4 Hz, 1H), 3.44–3.41 (m, 1H), 3.32–3.29 (m, 1H), 3.12 (d, J = 14.8 Hz, 1H), 3.06 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 169.65, 167.46, 167.30, 167.21, 137.95, 135.44, 134.69, 132.54, 132.07, 129.51, 128.64, 127.32, 127.28, 126.36, 126.32, 126.03, 123.05, 122.85, 121.69, 71.37, 54.10, 37.14, 35.73, HRMS (ESI): calcd for C26H24N3O5 [M + H]+ 458.1716; found 458.1716.Footnote |
| † Electronic supplementary information (ESI) available: 1H, 13C NMR and chiral HPLC spectra for all compounds 1–9. DEPT, COSY, HMBC and HSQC for both antipodes of 1 and 2. See DOI: 10.1039/c6ra08861a |
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