2023 Volume 71 Issue 1 Pages 52-57
Chemotherapy refers principally to the use of small molecules to treat cancer, and natural product derivatives have been main sources of clinically using anticancer drugs. While the coumarin skeleton does not inhibit cell growth, its derivatives are often active, and numerous coumarins have been examined for antiproliferative activity against human cancer cell lines. In this study, 16 novel coumarin derivatives (1, 1a–5a, 1b, 2b, 6b, 7b, 8–13) with attached N-heterocycles, including aminopyrrolidine, aminopiperidine, aminoazepane, and indoline, were prepared and ultimately esterified or amidated with alcohols or amines, respectively. All synthesized N-heterocycles containing coumarin derivatives with alcohols, amines, and carboxylic acids were assessed for antiproliferative activity against several human cancer cell lines, containing triple-negative breast cancer (TNBC) as well as a P-glycoprotein (P-gp) overexpressing multidrug-resistant (MDR) KB subline KB-VIN. Five coumarin derivatives (3a–5a, 12, 13) showed no effect (IC50 >40 µM) against all tested cell lines. In contrast, derivative 1a showed broad-spectrum activity against four cell lines, while 1b and 10 were nearly twice as selective for KB-VIN cells as the parent KB. The coumarin derivatives 1a, 1b, and 10 were optimal for antiproliferative activity in this study and could provide a new avenue for overcoming MDR tumors. Derivatives 1a, 1b, and 10 showed MDR cell-selective antiproliferative activity, indicating that N-heterocycle-coumarins exert previously unexplored bioactivity with selective action on MDR cancer cells.
Chemotherapy primarily refers to the use of cytotoxic drugs to treat cancer, and natural product derivatives have been important sources of currently using anticancer drugs. In the review of New Chemical Entities (NCE) from 1981 to 2019, nearly 75% of antitumor agents are not purely synthetic chemicals, with 47% being either natural products including derivatives or mimicking natural compounds.1) Natural and synthetically modified coumarins comprise a large family of heterocyclic compounds with benzo-α-pyrone moiety. Coumarins are widespread in plants and has been widely studied in pharmaceutical fields as antidepressants, antimicrobials, anti-oxidants, anti-inflammatories, antinociceptives, antitumor, anti-asthmatics, antivirals (including anti-human immunodeficiency virus (HIV)), and anti-coagulants.2–6) Pyrrolidine and piperidine alkaloids have been isolated from plant and sea sponge, and their alkaloids have toxicity.7–9) Pyrrolidine alkaloids have been assessed their efficacy as glycosidase inhibitors and inhibited the growth of selected Gram-positive bacteria and yeast at μg/mL concentrations.8,9) Piperidine alkaloids displayed potent muscarinic antagonist activity8) and showed antibacterial, antiinflammatory, antidiarrheal, and cytotoxic activities.10) The structure of anticancer drugs included pyrrolidine (ex. vincristine), piperidine (ex. vincristine, irinotecan, Everolimus11)), and indoline (ex. Chetomin12)) function. Also, diterpenoid alkaloids included piperidine function and several natural diterpenoid alkaloid and their derivatives displayed antiproliferative activity against several human cancer cell lines, including A549 (lung carcinoma), DU145 (prostate carcinoma), MDA-MB-231 (triple-negative breast cancer, estrogen and progesterone receptors-negative/HER2-negative), MCF-7 (estrogen receptor-positive, HER2-negative breast cancer), KB (cervical carcinoma HeLa derivative), and multidrug-resistant (MDR) KB subline KB-VIN overexpressing P-glycoprotein (P-gp).13–15) In our previous studies, the development of coumarin-containing chiral derivatizing agents for 1H-NMR enantiomeric excess determination is quite attractive, and prior studies have reported the preparation of such compounds with chiral alcohols, amines, and carboxylic acids.16–19) Also, it is known that the hybrid compound of antitumor agent with other bioactive component such as antioxidant or other antitumor agent has exhibited some benefits in improving the antitumor efficacy and/or selectivity with decreasing the systematic toxicity.20,21) As mentioned above, coumarins and N-hetero compounds (pyrrolidine, piperidine, and indoline) had an anticancer effect. Therefore, in this study for new anticancer drugs with increased efficacy, we considered conjugation of coumarin with N-heterocycle (3-aminopyrrolidine, 3-aminopiperidine, 3-aminoazepan, and indoline) and then various function, to be a feasible approach to develop new antitumor leads. New conjugates in which coumarin were coupled with a N-heterocycle were designed and synthesized (Fig. 1). In this study, previous and newly synthesized N-heterocycle conjugated coumarin derivatives were assessed for antiproliferative activity against five human cancer cell lines [A549, MDA-MB-231, MCF-7, KB, and KB-VIN].
Ten N-heterocycle-coumarin [(S)-4-(3-aminopyrrolidin-1-yl)coumarin, (S)-4-(3-aminopiperidin-1-yl)coumarin, or (S)-4-(3-aminoazepan-1-yl)coumarin] derivatives (1, 1a–5a, 1b, 2b, 6b, 7b) were prepared by previously described methods19) (Fig. 2). Six novel coumarin derivatives (8–13) were prepared by esterification or amidation of (S)-N-coumarinoindoline-2-carboxylic acid (20) with an alcohol or amine, respectively (Chart 1). After the preparation of (S)-N-carbobenzoxyindoline-2-carboxylic acid (15) from (S)-indoline-2-carboxylic acid, the esterification of compound 15 gave (S)-N-carbobenzoxyindoline-2-carboxylic acid tert-butyl ester (16). The deprotection of compound 16 gave (S)-indoline-2-carboxylic acid tert-butyl ester (17). Condensation of compound 17 and 3-benzenesulphonyl-4-chlorocoumarin produced (S)-3-phenylsulfonylcoumarinoindoline-2-carboxylic acid tert-butyl ester (18). After the preparation of (S)-N-coumarinoindoline-2-carboxylic acid tert-butyl ester (19) from 18, the hydrolysis of compound 19 gave (S)-N-coumarinoindoline-2-carboxylic acid (20). The designed esters or amides were prepared from the reaction of compound 20 and a selected alcohol or amine in the presence of N,N′-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP). The synthesized derivatives (1, 1a–5a, 1b, 2b, 6b, 7b, 8–13) were assessed for antiproliferative activity against five human cancer cell lines (A549, MDA-MB-231, MCF-7, KB, and KB-VIN). Paclitaxel was used as an experimental positive control (data shown in Table 1).
| Compounds | Cell line/IC50 (μM)a) | ||||
|---|---|---|---|---|---|
| A549 | MDA-MB-231 | MCF-7 | KB | KB-VIN | |
| 1 | 17.2 | >40 | 15.4 | 23.0 | 17.3 |
| 1a | 7.8 | 39.7 | 8.0 | 8.6 | 5.0 |
| 1b | 13.8 | >40 | 15.7 | 17.3 | 9.5 |
| 2a | 28.0 | >40 | 30.5 | 32.3 | 22.9 |
| 2b | 23.9 | >40 | 22.3 | 24.8 | 24.9 |
| 3a | >40 | >40 | >40 | >40 | >40 |
| 4a | >40 | >40 | >40 | >40 | >40 |
| 5a | >40 | >40 | >40 | >40 | >40 |
| 6b | 26.0 | 32.9 | 25.6 | 22.6 | 19.4 |
| 7b | 25.9 | 35.3 | 23.4 | 20.4 | 23.2 |
| 8 | 31.8 | 37.0 | >40 | 30.0 | 21.8 |
| 9 | 21.5 | 35.6 | 23.7 | 18.4 | 11.8 |
| 10 | 13.6 | 29.8 | 19.7 | 12.0 | 7.4 |
| 11 | 21.3 | 29.3 | 27.2 | 20.6 | 17.4 |
| 12 | >40 | >40 | >40 | >40 | >40 |
| 13 | >40 | >40 | >40 | >40 | >40 |
| Paclitaxelb) | 0.0065 | 0.0082 | 0.0136 | 0.0069 | 2.7 |
a) Antiproliferative activity as IC50 values for each cell line, the concentration of compound that caused 50% reduction relative to untreated cells determined by the SRB assay; b) Paclitaxel was used as an experimental positive control.
Five derivatives [(S)-4-(3-aminopiperidin-1-yl)coumarin derivatives 3a, 4a, 5a, containing a phenylmalonic acid monobenzyl amide, flurbiprofen amide, or N-trifluoroacetylproline amide, respectively, and coumarinoindoline derivatives 12, 13, containing a 3-methylpiperidine amide or ethyl piperidine-3-carboxylate amide, respectively] were inactive (IC50 >40 µM) against all tested cells. While the remaining 11 coumarin derivatives exerted varying degrees of antiproliferative activity, none showed significant efficacy against the MDA-MB-231 cell line. In fact, in addition to 3a, 4a, 5a, 12, and 13, coumarin derivatives 1, 1b, 2a, and 2b, were inactive (IC50 >40 µM) against MDA-MB-231 cell line. Furthermore, four coumarin derivatives 1a, 7b, 8, and 9 showed limited efficacy (1a, 7b, 8, 9: IC50 39.7, 35.3, 37.0, 35.6 µM, respectively) and three coumarin derivatives 6b, 10, and 11 exhibited weak efficacy (IC50 32.9, 29.8, 29.3 µM, respectively) against MDA-MB-231 cell line.
In four human cell lines (A549, MCF-7, KB, and KB-VIN), derivatives 1a, 1b, and 10 exhibited significant efficacy (average IC50 7.4, 14.1, 13.2 µM, respectively) and particularly, 1a had significantly better efficacy (IC50 A549: 7.8, MCF-7: 8.0, KB: 8.6, KB-VIN: 5.0 µM). Moreover, in KB-VIN cell line, derivatives 1b and 10 indicated significant antiproliferative activity (IC50 9.5 and 7.4 µM, respectively). Derivatives 1, 2b, 6b, 7b, 9, and 11 displayed moderate efficacy against four cell lines (average IC50 18.2, 24.0, 23.4, 23.2, 18.9, and 21.6 µM, respectively). However, while derivative 9 exhibited pronounced antiproliferative activity (IC50 11.8 µM) against KB-VIN cells, it was greatly less potent against A549, MCF-7, and KB cells. Although derivatives 2a and 8 exerted moderate antiproliferative activity against KB-VIN cells (IC50 22.9 and 21.8 µM, respectively), they exhibited less activity against A549, MCF-7, and KB cells, leading to only minor average efficacy against four cell lines (average IC50 28.4 and 30.9 µM, respectively).
Among all tested cell lines, 11 active derivatives (1, 1a, 1b, 2a, 2b, 6b, 7b, 8–11) showed reasonable efficacy against KB-VIN cells (average IC50 16.4 µM). In particular, derivatives 1a, 1b, 9, and 10 exhibited significant efficacy against KB-VIN cells (IC50 5.0, 9.5, 11.8, and 7.4 µM, respectively), and 1, 2a, 2b, 6b, 7b, 8, and 11 displayed moderate efficacy against KB-VIN cells (IC50 17.3, 22.9, 24.9, 19.4, 23.2, 21.8, and 17.4 µM, respectively). The same 11 active derivatives (1, 1a, 1b, 2a, 2b, 6b, 7b, 8–11) exhibited moderate efficacy against A549, MCF-7, and KB cells (average IC50 20.9, 21.2, and 20.9 µM, respectively), but derivative 8 was inactive against MCF-7 cells. Particular, derivative 1a displayed significant efficacy against A549, MCF-7, and KB cell lines (IC50 7.8, 8.0, and 8.6 µM, respectively), and three derivatives (1, 1b, and 10) indicated moderate efficacy against A549 cells (IC50 17.2, 13.8, and 13.6 µM, respectively). Three derivatives (1, 1b, and 10) displayed moderate efficacy against MCF-7 cells (IC50 15.4, 15.7, and 19.7 µM, respectively) and three active derivatives (1b, 9, and 10) showed moderate efficacy against KB cells (IC50 17.3, 18.4, and 12.0 µM, respectively). Seven active derivatives (1a, 6b, 7b, 8–11) exhibited weak efficacy against MDA-MB-231 cells (average IC50 34.2 µM).
The most marked fact from the data in Table 1 were the degree and relative ratio of selectivity to MDR compared to non-MDR cells (KB/KB-VIN). Among the five human cancer cell lines tested, significant efficacy against the KB-VIN cell line was exhibited by derivatives 1a, 1b, and 10 (IC50 5.0, 9.5, and 7.4 µM, respectively), followed by 1, 2a, 2b, 6b, 7b, 8, 9, and 11 (IC50 17.3, 22.9, 24.9, 19.4, 23.2, 21.8, 11.8, and 17.4 µM, respectively). Commonly, all active derivatives displayed significant efficacy against the KB-VIN cell line compared to the other four human cancer cell lines tested. Furthermore, derivatives 1, 2a, 2b, 6b, 7b, 8, 9, and 11 showed similar efficacy against KB and KB-VIN cell lines (ratio of IC50 KB/IC50 KB-VIN: 1.33, 1.41, 1.16, 0.88, 1.38, 1.56, and 1.18, respectively). Even more particularly, derivatives 1a, 1b, and 10 showed over 1.6-fold selectivity between the two cell lines, with significant antiproliferative activity against the KB-VIN cell line (ratio of IC50 KB/IC50 KB-VIN: 1.72, 1.82, and 1.62, respectively).
The identity of the functional group on the ester or amide group influenced the cytotoxic efficacy. For example, coumarin derivative 1a showed significant efficacy against four tested cancer cell lines (A549, MCF-7, KB, and KB-VIN) with IC50 values ranging from 5.0 to 8.6 µM. The same range of efficacy was found with derivatives 1b, 9, and 10 against KB-VIN cells (9.5, 11.8, and 7.4 µM, respectively). Derivatives 1, 6b, and 11 showed moderate efficacy against KB-VIN (17.3, 19.4, and 17.4 µM, respectively). The efficacies of 1, 1b, and 10 (IC50 12.0–19.7 µM) generally ranked partially below those of the most effective derivatives against A549, MCF-7, and KB cell lines, except derivative 1 was even less active against KB cell lines. Among the amidated derivatives (1, 1a, 1b), derivatives 1 and 1b exhibited moderated efficacy against four cell lines (average IC50 18.2 and 14.1 µM, respectively). Particularly, derivative 1a showed significant efficacy against A549, MCF-7, KB, and KB-VIN cells (IC50 7.8, 8.0, 8.6, and 5.0 µM, respectively), but was much less potent against MDA-MB-231 (IC50 39.7 µM). Compound 1a showed significantly better efficacy compared with corresponding derivatives 1 and 1b. Thus, amidation of (S)-4-(3-aminopiperidin-1-yl)coumarin was critical for enhanced antiproliferative activity of coumarin derivatives.
The designed esters or amides were prepared from the reaction of (S)-4-(3-aminopyrrolidin-1-yl)coumarin, (S)-4-(3-aminopiperidin-1-yl)coumarin, (S)-4-(3-aminoazepan-1-yl)coumarin, or (S)-N-coumarinoindoline-2-carboxylic acid (20) and an alcohol or amine in the presence of DCC and DMAP to provide 16 new coumarin derivatives (1, 1a–5a, 1b, 2b, 6b, 7b, 8–13). All synthesized derivatives (1, 1a–5a, 1b, 2b, 6b, 7b, 8–13) were assessed for antiproliferative activity against five human cancer cell lines including A549, KB, KB-VIN, MDA-MB-231, and MCF-7. Several novel coumarin derivatives (particularly, 1a, 1b, 10) exhibited considerable antiproliferative activities against these cell lines, except MDA-MB-231 cell lines. In contrast, coumarin derivatives 3a, 4a, 5a, 12, and 13 showed no effect. While derivatives 1, 2a, 2b, 6b, 7b, 8, and 11 displayed comparable efficacy against both KB and KB-VIN cell lines, some derivatives exhibited tumor type-selective activity. Derivatives 1a, 1b, and 10 showed greater inhibitory activity against multidrug-resistant KB-VIN cell line (1.62–1.82-fold) than the parental KB cells. Continued researches are deserved to develop these capable new leads as anticancer agents, particularly with enhanced cancer selectivity.
Melting points were measured using a Yanaco micro-melting point apparatus. Optical rotations were measured on a JASCO Model DIP-4 polarimeter (JASCO, Tokyo, Japan). IR spectra were recorded using JASCO FT-7000 (JASCO) and Perkin-Elmer Model Spectrum100 (Perkin-Elmer, Yokohama, Japan). NMR spectra were recorded in CDCl3 on a JEOL Model AL-400 and ECZ400 spectrometer (JEOL, Tokyo, Japan) with TMS as an internal standard. MS and high resolution (HR)MS were recorded on a Hitachi M-2000 mass spectrometer (Hitachi, Tokyo, Japan) and a JEOL Model JMS-700 mass spectrometer (JEOL).
Coumarin Derivatives (1, 1a–5a, 1b, 2b, 6b, 7b)Ten coumarin derivatives 1, 1a–5a, 1b, 2b, 6b, and 7b were prepared by methods described previously.19)
Synthesis of Coumarin Analogues (8–13)(S)-N-Carbobenzoxyindoline-2-carboxylic acid (15): (S)-Indoline-2-carboxylic acid (15.0 g, 92 mmol) dissolved in 2N NaOH (50 mL) and stirred at 0 °C, benzyl chloroformate (15 mL, 106 mmol) and 2N NaOH (50 mL) were added dropwise over 10 min at 0 °C. After the mixture was stirred for 30 min at 0 °C, the reaction solution was washed with diethyl ether (100 mL) and then acidified with 10% HCl. The solution was extracted with EtOAc (50 mL, 3 times) and then the organic solution was washed with brine. The organic solution was dried over anhydrous MgSO4. The solvent was removed in vacuo to give the product (S)-N-carbobenzoxyindoline-2-carboxylic acid (15: 20.2 g, yield 74%). 15: Amorphous; 1H-NMR (δ): 3.20 and 3.52 (each 1H, m), 4.96 (1H, m), 5.22 (2H, s), 6.98 (2H, m, Ar-H), 7.11 (2H, m, Ar-H), 7.31 (5H, m, Ar-H).
(S)-N-Carbobenzoxyindoline-2-carboxylic acid tert-butyl ester (16): Compound 15 (20.2 g, 68 mmol) dissolved in CH2Cl2 (200 mL) was stirred at room temperature (r.t.) and O-tert-butyl-N,N′-diisopropylurea (65 mL, 272 mmol) was added at r.t. Then the reaction solution was refluxed for 90 min at 50 °C. After filtration, the solvent was removed in vacuo. The crude product was purified by silica gel column chromatography eluting with EtOAc-hexane (1 : 2) to give (S)-N-carbobenzoxyindoline-2-carboxylic acid tert-butyl ester (16: 21.8 g, yield 91%). 16: Colorless oil; [α]D20 −71.7 (c 1.1, CHCl3); HR-secondary ion (SI)MS m/z: [M + H]+ 354.1721 (Calcd. for C21H24NO4: 354.1705); IR (neat) νmax cm−1: 2980, 1740, 1720, 1490, 1410, 1150; 1H-NMR (δ): 1.34 (9H, s, t-Bu), 3.07 (1H, m), 3.52 (1H, m), 4.83 (1H, m, H-2), 5.23 (2H, s), 6.94–7.95 (9H, m, Ar-H); 13C-NMR (δ): 27.9, 33.0, 60.6, 67.2, 81.8, 114.7, 122.8, 124.3, 127.8, 127.9, 128.0, 128.4, 135.9, 170.4.
O-tert-Butyl-N,N′-diisopropylurea: N,N′-Diisopropylcarbodiimide (77 mL, 492 mmol) and CuCl (0.486 g, 4.92 mmol) dissolved in tert-butyl alcohol (54 mL) were stirred at 0 °C under Ar, and then the reaction solution was stirred for 18 h at r.t. under Ar. After the solvent was removed in vacuo, and the crude product was purified by distillation at 44 °C in vacuo (1 mmHg) to give O-tert-butyl-N,N′-diisopropylurea (87 g, yield 89%).
(S)-Indoline-2-carboxylic acid tert-butyl ester (17): Compound 16 (23.0 g, 65 mmol) and ammonium formate (6.0 g, 97.5 mmol) dissolved in MeOH (150 mL) were stirred at r.t. and 10% Pd-C (12.0 g) was added at r.t. The reaction solution was stirred for 1 h at r.t. After filtration, the solvent was removed in vacuo. The residue was dissolved in diethyl ether (100 mL), washed with water (100 mL) and brine (100 mL), and then dried over anhydrous MgSO4. The solvent was removed in vacuo to give (S)-indoline-2-carboxylic acid tert-butyl ester (17: 13.6 g, yield 96%). 17: Colorless oil; [α]D20 +25.0 (c 1.2, CHCl3); HR-SIMS m/z: [M + H]+ 220.1311 (Calcd. for C17H18NO2: 220.1338); IR (neat) νmax cm−1: 2980, 1730, 1610, 1240, 1150; 1H-NMR (δ): 1.41 (9H, s, t-Bu), 3.21–3.52 (2H, m), 4.26 (1H, q, J = 5.7 Hz, H-2), 4.39 (1H, bs, NH), 6.69–7.16 (4H, m, Ar-H); 13C-NMR (δ): 28.0, 28.3, 33.9, 60.2, 81.6, 109.9, 119.1, 124.2, 126.7, 127.4, 150.1, 173.1.
(S)-3-Phenylsulfonylcoumarinoindoline-2-carboxylic acid tert-butyl ester (18): 3-Phenylsulfonyl-4-chlorocoumarin (14.1 g, 45 mmol) dissolved in acetonitrile (300 mL) was stirred at 0 °C under Ar and triethylamine (6 mL) and compound 17 (10.7 g, 50 mmol) dissolved in acetonitrile (50 mL) were added dropwise over 10 min at 0 °C. The reaction mixture was stirred for 24 h at r.t. After filtration, the solvent was removed in vacuo, and then the crude product was purified by silica gel column chromatography eluting with EtOAc–hexane (1 : 3) to give (S)-3-phenylsulfonylcoumarinoindoline-2-carboxylic acid tert-butyl ester (18: 16.2 g, yield 74%). 18: Amorphous; HR-SIMS m/z: [M + H]+ 504.1499 (Calcd. for C28H26NO6S: 504.1481); IR (KBr) νmax cm−1: 3450, 1730, 1525, 1370, 1150; 1H-NMR (δ): 1.19 (9H, s, t-Bu), 3.58–3.82 (2H, m), 5.74 (1H, m, indoline H-2), 6.30–7.94 (13H, m, Ar-H); 13C-NMR (δ): 14.1, 27.7, 33.4, 60.4, 70.0, 77.2, 82.6, 109.5, 117.2, 118.4, 121.0, 124.1, 125.3, 127.4, 128.5, 128.9, 129.0, 129.2, 133.5, 134.6, 141.5, 147.5, 153.9, 169.9.
(S)-N-Coumarinoindoline-2-carboxylic acid tert-butyl ester (19): Compound 18 (16.0 g, 32 mmol) dissolved in THF (500 mL) was stirred at 0 °C and zinc powder (125 g, 1.82 mol) was added at 0 °C. Ammonium chloride saturated solution (300 mL) was added dropwise under 30 min at 0 °C. The reaction mixture was stirred for 1 h at 0 °C. After filtration, the solvent was removed. The residue was dissolved in diethyl ether (100 mL), washed with the water (100 mL) and brine (100 mL), and then dried over anhydrous MgSO4. Then the solvent was removed in vacuo. The crude product was purified by silica gel column chromatography eluting with EtOAc–hexane (1 : 3) to give (S)-N-coumarinoindoline-2-carboxylic acid tert-butyl ester (19: 10.8 g, yield 94%). 19: Amorphous; [α]D20 +95.4 (c 1, CHCl3); HR-SIMS m/z: [M + H]+ 364.1568 (Calcd. for C22H22NO4: 364.1549); IR (KBr) νmax cm−1: 1740, 1715, 1610, 1490, 1380, 1150; 1H-NMR (δ): 1.37 (9H, s, t-Bu), 3.29–3.60 (2H, m), 4.92 (1H, t, J = 9.3 Hz, indoline H-2), 6.01 (1H, s, coumarin H-3), 6.58–7.75 (8H, m, Ar-H); 13C-NMR (δ): 27.8, 33.9, 66.8, 101.3, 112.4, 116.1, 117.4, 121.9, 123.2, 125.0, 126.1, 127.3, 129.1, 132.2, 146.1, 153.97, 154.02, 162.1, 169.5.
(S)-N-Coumarinoindoline-2-carboxylic acid (20): Compound 19 (10.8 g, 30 mmol) dissolved in CH2Cl2 (20 mL) was stirred at r.t. and trifluoroacetic acid (100 mL) was added dropwise at r.t. The reaction mixture was stirred for 1 h at r.t. After removal of the solvent in vacuo, isopropyl alcohol (50 mL) was added. After filtration, the solvent was removed in vacuo. The crude product was purified by silica gel column chromatography eluting with EtOAc–hexane (1 : 1) to give (S)-N-coumarinoindoline-2-carboxylic acid (20: 7.4 g, yield 87%). 20: mp 105–107.5 °C; [α]D20 +154.6 (c 1, CHCl3); HR-electron ionization (EI)MS m/z: 307.0819 (Calcd. for C18H13NO4: 307.0845); IR (KBr) νmax cm−1: 3418, 2976, 1719, 1686, 1607, 1553, 1483, 1402, 1195; 1H-NMR (δ): 3.43 and 3.62 (each 1H, m), 5.02 (1H, t, J = 8.0 Hz, indoline H-2), 6.06 (1H, s, coumarin 3-H), 6.59, 7.25, 7.37 and 7.71 (each 1H, d, J = 8.0 Hz, Ar-H), 6.93, 7.03, 7.17 and 7.53 (each 1H, t, J = 8.0 Hz, Ar-H); 13C-NMR (δ): 22.6, 31.5, 33.9, 65.8, 99.9, 112.8, 115.5, 117.4, 122.2, 123.1, 125.0, 126.0, 127.0, 129.4, 132.1, 145.3, 153.5, 163.7.
General procedure for synthesis of coumarin analogues Alcohol or amine (0.1 mmol) dissolved in CH2Cl2 (3 mL) was stirred at r.t. Compound 20 (0.15 mmol) and N,N′-dicyclohexylcarbodiimide (DCC, 0.17 mmol) dissolved in CH2Cl2 (1 mL) were added, followed by DMAP (0.3 mmol) dissolved in CH2Cl2 (1 mL). The reaction mixture was stirred for 1 h at r.t. After removal of the solvent in vacuo, EtOAc (20 mL) was added, and the reaction solution was washed with the water, saturated aq. NaHCO3 and brine, and then dried over anhydrous MgSO4. After the solvent was removed in vacuo, the crude product was purified by silica gel column chromatography eluting with EtOAc–hexane to give the desired product.
Coumarinoindoline derivative 8: 87% yield; Amorphous; HR-electrospray ionization (ESI)MS m/z: 384.1222 (Calcd. for C22H19NO4Na: 384.1212); IR (ATR) νmax cm−1: 3270, 1650, 1594, 1535, 1258; 1H-NMR (δ): 1.22 (3H, d, J = 6.3 Hz), 3.36, 3.60, 5.02, 5.35, and 5.73 (each 1H, m), 5.14 (2H, m), 6.00 (1H, s), 6.61, 7.23, 7.38, and 7.74 (each 1H, d, J = 8.1 Hz), 6.92, 7.05, 7.19, and 7.55 (each 1H, t, J = 8.1 Hz); ESIMS m/z: 384 [(M + Na)+].
Coumarinoindoline derivative 9: 78% yield; Amorphous; HR-EIMS m/z: 419.2103 (M+, Calcd for C26H29NO4: 419.2095); IR (neat) νmax cm−1: 3301, 2986, 1670, 1601, 1415, 1160; 1H-NMR (δ): 0.79 (3H, t, J = 7.1 Hz), 1.04 (3H, d, J = 6.3 Hz), 1.06–1.35 (10H, m), 3.27 and 3.50 (each 1H, m), 4.84 and 4.91 (each 1H, m), 5.93 (1H, s), 6.51, 6.54, 7.16, and 7.31 (each 1H, d, J = 7.8 Hz), 6.85, 7.11, 7.47, and 7.67 (each 1H, t, J = 7.8 Hz); EIMS m/z: 419 [M+], 305, 288, 262, 145.
Coumarinoindoline derivative 10: 94% yield; Amorphous; HR-EIMS m/z: 401.1653 (Calcd for C25H23NO4: 401.1626); IR (ATR) νmax cm−1: 3317, 2930, 1624, 1606, 1575, 1537, 1263, 1243, 1196; 1H-NMR (δ): 0.77, 0.95, 1.24, 1.91, 3.35, and 3.58 (each 1H, m), 1.29, 1.41, and 4.98 (each 2H, m), 2.12 and 2.38 (each 1H, bs), 5.98 (1H, s), 6.58, 7.21, 7.36, and 7.72 (each 1H, d, J = 7.3 Hz), 6.90, 7.03, 7.17, and 7.53 (each 1H, t, J = 7.3 Hz); EIMS m/z: 401 [M+], 305, 262, 145.
Coumarinoindoline derivative 11: 46% yield; Amorphous; HR-EIMS m/z: 418.2232 (Calcd for C26H30N2O3: 418.2254); IR (neat) νmax cm−1: 3294, 2910, 2842, 1671, 1594, 1555, 1249, 1191; 1H-NMR (δ): 0.82 (3H, t, J = 7.1 Hz), 0.96 (3H, d, J = 6.6 Hz), 1.06–1.29 (10H, m), 3.30, 3.58, 3.91, 4.76 and 4.82 (each 1H, m), 6.08 (1H, s), 6.71, 7.23, 7.39, and 7.67 (each 1H, d, J = 7.5 Hz), 7.00, 7.13, 7.22, and 7.27 (each 1H, t, J = 7.5 Hz); EIMS m/z: 418 [M+], 288, 262, 145.
Coumarinoindoline derivative 12: 80% yield; Amorphous; HR-EIMS m/z: 388.1807 (Calcd for C24H24N2O3: 388.1786); IR (ATR) νmax cm−1: 3315, 2930, 1664, 1626, 1540, 1448, 1245; 1H-NMR (δ): 0.88 (3H, d, J = 6.3 Hz), 1.56, 2.33, 2.60, 2.80, 3.12, 3.26, 3.46, 3.75, 3.82, 4.38, and 4.43 (each 1H, m), 5.33 (1H, t, J = 8.6 Hz), 5.83 (1H, s), 6.56, 7.19, 7.35, and 7.78 (each 1H, d, J = 8.1 Hz), 6.88, 7.01, 7.16, and 7.52 (each 1H, t, J = 8.1 Hz); EIMS m/z: 388 [M+], 288, 262, 145.
Coumarinoindoline derivative 13: 32% yield; Amorphous; HR-EIMS m/z: 446.1851 (Calcd for C26H26N2O5: 446.1841); IR (neat) νmax cm−1: 3300, 2938, 1742, 1680, 1607, 1543, 1373, 1230; 1H-NMR (δ): 1.20 (3H, t, J = 7.2 Hz), 1.77, 2.47, 2.58, 2.93, 3.16, 3.25, 3.44, 3.57, 3.96, 4.38, and 4.58 (each 1H, m), 4.11 (2H, q, J = 7.2 Hz), 5.65 (1H, t, J = 8.2 Hz), 5.86 (1H, s), 6.58, 7.19, 7.37, and 7.78 (each 1H, d, J = 8.1 Hz), 6.90, 7.03, 7.18, and 7.53 (each 1H, t, J = 8.1 Hz); EIMS m/z: 446 [M+], 288, 262, 145.
Cell Culture and Antiproliferative ActivityAll cell lines were obtained from American Type Culture Collection (ATCC, Virginia, U.S.A.) or UNC Lineberger Comprehensive Cancer Center (North Carolina, U.S.A.), except MDR subline KB-VIN (vincristine-resistant KB subline), which was a gift from Professor Y.-C. Cheng (Yale University, Connecticut, U.S.A.). Cells were cultured in medium (RPMI 1640) containing 25 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (HEPES) and 2 mM L-glutamine (Corning, New York, U.S.A.), supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, MA, U.S.A.), 100 IU penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B (Corning). KB-VIN cells were maintained in media containing 100 nM vincristine (Sigma-Aldrich). Cells were maintained at 37 °C in a humidified 5% CO2 atmosphere.
Cytotoxicity was evaluated by the sulforhodamine B (SRB) method as described before.22) In brief, all compounds were prepared at 10 mM with dimethyl sulfoxide (DMSO), and the highest concentration of DMSO in the cultures (0.4% (v/v)) used for the cytotocxicity assay had no effect on cell growth. Freshly trypsinized cell suspensions were seeded in 96-well microtiter plates at densities of 4000–11000 cells per well with compounds for 72 h followed by fixation in 10% trichloroacetic acid and then staining with 0.04% sulforhodamine B. The protein-bound dye was solubilized by 10 mM Tris base and absorbance was measured at 515 nm using a ELx800 microplate reader with Gen5 software (BioTek, Vermont, U.S.A.). Antiproliferative activity was evaluated by data obtained from at least three independent experiments performed in duplicate.
We appreciate crucial comments, suggestions, and editing of the manuscript by Dr. Susan L. Morris-Natschke (UNC-CH) and analyzing of ESIMS by Dr. Megumi Mizukami (HUS). This study was supported in part by a Grant-in-Aid (2021) provided by Hokkaido University of Science to K. Wada, and by NIH Grant CA177584 from the National Cancer Institute awarded to K.H.L. as well as IBM junior faculty development award to M.G.
The authors declare no conflict of interest.
