Journal of Natural Products
Article
(δ) are expressed in ppm relative to TMS, and the coupling constants
(J) in Hz. EIMS and HRMS (FAB+) spectra were recorded on a JEOL
SX102A mass spectrometer, and the accurate mass was calculated
using polyethylene glycol 400 as standard. The (R)-(+)- and (S)-
(−)-α-methylbenzylamine Chiraselect ≥99.0% (sum of enantiomers,
GC) were purchased from Fluka Sigma-Aldrich.
Derivatization of rac-Tokinolide B (rac-3) with (R)-(+)-α-
Methylbenzylamine. To a solution of rac-tokinolide B (rac-3, 100.8
mg, 0.26 mmol) in anhydrous toluene (5 mL) placed in a stainless
steel reactor (100 mL) was added (R)-(+)-α-methylbenzylamine (0.06
mL, 57.12 mg, 0.47 mmol) under a nitrogen atmosphere. The reactor
was sealed and heated (130 °C) for 20 h (pressure: 50 psi). After
cooling to room temperature, the reaction mixture was concentrated at
reduced pressure. EtOAc (10 mL) was added, and the organic phase
was washed with HCl (10%), which subsequently was extracted with
EtOAc (3 × 10 mL). The combined organic layer was washed with
brine, dried with Na2SO4, and concentrated under reduced pressure.
The residue was purified by column chromatography (n-hexane/
EtOAc, gradient) to afford two products (76.6 mg, 70.2%):
spectra of (+)-8 and (−)-9, respectively, were consequently in
complete agreement with the experimental CD curves.
Encouraged by the previously reported biological activities of
the natural phthalides, and taking into consideration their
pharmacological importance,16 it was decided to investigate the
cytotoxic activity of these enantiomerically pure compounds
toward three human cancer cell lines, following standard
protocols.26 The IC50 values determined are shown in Table 1.
Table 1. Evaluation of the IC50 (μM) of the Natural Products
and Derivatives of L. porteri
a
b
c
compound
K562
HCT-15
SKLU-1
rac-3
26.6 1.4
46.1 3.8
5.7 0.9
10.5 0.9
44.8 1.3
5.4 0.5
8.5 0.6
7.5 0.5
5.2 0.2
0.29 0.02
7.1 0.6
13.2 1.3
4.1 0.1
5.9 0.5
4.9 0.3
4.3 0.4
0.21 0.02
rac-5
(−)-8
(+)-9
(+)-8
(−)-9
21.7 1.3
13.9 1.6
5.2 0.3
(−)-8 (44.6 mg, 40.9%) as a pale oil; Rf 0.47 (n-hexane/EtOAc,
65:35); [α]25D −54.8 (c 1.35 × 10−3, MeOH); UV (MeOH) λmax (log
ε) 211 (4.4) nm; CD (c 9.6 × 10−6, MeOH) 246 nm (Δε +9.11), 224
nm (Δε −20.29); IR (CHCl3) νmax 3349, 3061, 2959, 2872, 1766,
d
helenalin
0.28 0.02
a
b
c
d
Leukaemia. Colon. Lung. Positive control. Results are means
1
1689, 1494, 1454, 1351, 1214, 1166, 1037, 924, 752 cm−1; H NMR
SEM for three replicates.
(CDC13, 500 MHz; assignments by COSY, NOESY, and HMQC) δ
7.58 (2 H, dd, J = 8.5, 1.5 Hz, H -4″, H-8″), 7.27 (2H, ddd, J = 8.5, 7.5,
1.5 Hz, H-5″, H-7″), 7.20 (1H, dddd, J = 8.5, 7.5, 1.5, 1.5, H-6″), 7.15
(1H, d, J = 6.5 Hz, H-7′), 6.87 (1H, dd, J = 7.0, 4.0 Hz, H-7), 4.85
(1H, q, J = 7.0 Hz, H-2″), 2.90 (1H, dddd, J = 10.5, 6.5, 4.0, 4.0 Hz, H-
6′), 2.42 (1H, ddd, J = 9.5, 7.0, 2.0 Hz, H-4′a), 2.20 (1H, dt, J = 10.5,
4.5 Hz, H-6a), 1.98−1.92 (3H, m, H-6b, H-5′a, H-8′a), 1.90 (1H, d, J =
7.5 Hz, H-9″), 1.76 (1H, ddd, J = 9.5, 6.0, 6.0 Hz, H-8′b), 1.59 (1H,
dd, J = 10.5, 4.0, Hz, H-8), 1.45 (1H, dd, J = 10.5, 4.0 Hz, H-4a),
1.35−1.32 (3H, m, H-5a, H-10a, H-5′b), 1.30−1.28 (1H, m, H-9a),
1.28−1.25 (4H, m, H-9′a, H-9′b, H-10′a, H-10′b), 1.19 (1H, ddd, J =
11.5, 6.0, 6.0 Hz, H-4′b), 1.12−1.05 (2H, m, H-5b, H-10b), 0.99 (1H,
dd, J = 10.0, 4.0 Hz, H-9b), 0.90 (1H, ddd, J = 10.0, 6.0, 6.0 Hz, H-4b),
0.86 (3H, t, J = 7.0 Hz, CH3-11), 0.83 (3 H, t, J = 7.5 Hz, CH3-11′);
13C NMR (CDCl3, 125 MHz, assignments by DEPT, HSQC, and
HMBC) δ 168.2 (CO, C-1), 165.2 (CO, C-1′), 143.4 (C, C-3″), 140.2
(CH, C-7), 139.2 (C, C-7′a), 137.4 (CH, C-7′), 130.8 (C, C-7a), 128.2
(CH, C-5″, C-7″), 128.0 (CH, C-4″, C-8″), 127.1 (CH, C-6″), 101.8
(C, C-3′), 95.3 (C, C-3), 85.2 (C, C-3a), 57.0 (C, C-3′a), 52.7 (CH, C-
2″), 45.5 (CH, C-8), 36.2 (CH, C-6′), 35.0 (CH2, C-8′), 29.0 (CH2, C-
4), 28.8 (CH2, C-9), 25.7 (CH2, C-9′), 25.0 (CH2, C-6), 23.9 (CH2,
C-4′), 23.0 (CH2, C-10′), 20.9 (CH2, C-10), 19.5 (CH3, C-9″), 17.4
(CH2, C-5′), 16.8 (CH2, C-5), 14.1 (CH3, C-11), 13.7 (CH3, C-11′);
EIMS m/z 501 [M]+ (15), 378 (17), 310 (8), 274 (33), 191 (100),
149 (10), 120 (32), 105 (28), 55 (5); HRMS (FAB+) m/z 502.2959
(calcd for C32H39O4N+H+ 502.2957).
(+)-9 (31.9 mg, 29.3%) as a pale oil; Rf 0.44 (n-hexane/EtOAc,
65:35); [α]25D +7.8 (c 1.15 × 10−3, MeOH); UV (MeOH) λmax (log ε)
210 (4.5) nm; CD (c 7.2 × 10−6, MeOH) 245 (Δε −7.89), 223 (Δε
+12.62); IR (CHCl3) νmax 3347, 3060, 2958, 2872, 1765, 1688, 1526,
1495, 1452, 1352, 1214, 1036, 1013, 924, 751 cm−1; 1H NMR
(CDC13, 500 MHz; assignments by COSY, NOESY, and HMQC) δ
7.47 (2H, dd, J = 7.5, 1.5 Hz, H-4″, H-8″), 7.29 (2H, ddd, J = 7.5, 7.5,
1.5 Hz, H-5″, H-7″), 7.22 (1H, dddd, J = 7.5, 7.5, 1.5, 1.5, H-6″), 7.22
(1H, d, J = 6.5 Hz, H-7′), 6.98 (1H, dd, J = 7.0, 4.0 Hz, H-7), 4.69
(1H, q, J = 7.0 Hz, H-2″), 2.93 (1H, dddd, J = 10.0, 6.5, 4.0, 4.0 Hz, H-
6′), 2.46−2.39 (2H, ddd, J = 11.0, 4.0, 4.0 Hz, H-6a, H-4′a), 2.19−2.16
(1H, m, H-6b), 1.99−1.89 (3H, m, H-5a, H-5′a, H-8′a), 1.85 (3H, d, J
= 7.0 Hz, H-9″), 1.63 (1H, ddd, J = 11.0, 4.5, 4.5 Hz, H-8′b), 1.65 (1H,
ddd, J = 10.0, 3.0, 3.0 Hz, H-8), 1.51 (1H, ddd, J = 10.0, 4.5, 4.5 Hz,
H-4a), 1.37−1.33 (3H, m, H-5b, H-10a, H-5′b), 1.73 (1H, dt, J = 6.5,
3.0, H-9a), 1.28−1.25 (1H, m, H-9b), 1.23−1.17 (2H, m, H-10b, H-
4′b), 1.15−1.09 (2H, m, H-9′a, H-10′a), 1.02−0.96 (3H, m, H-4b, H-
9′b, H-10′b), 0.87 (3H, t, J = 7.0 Hz, CH3-11), 0.66 (3H, t, J = 7.0 Hz,
CH3-11′); 13C NMR (CDCl3, 125 MHz, assignments by DEPT,
HSQC, and HMBC) δ 168.0 (CO, C-1), 165.0 (CO, C-1′), 143.2 (C,
The two pairs of enantiomers (−)-8/(+)-8 and (+)-9/(−)-9
were more potent compared with rac-3 and rac-5, although less
potent compared to the positive control (helenalin). Moreover,
it was observed that compounds (−)-8 and (−)-9 were
significantly more active than their enantiomers (+)-8 and
(+)-9 in the cell lines K562 and HCT-15. This suggested that
the compounds might have exerted their cytotoxic effect by
interacting with a chiral target, and other derivatives of 8 and 9
would possibly shed light on this. For the SKLU-1 cell line only
small differences between the enantiomers are noted.
The reaction between the racemic mixture of rac-3 and (R)-
(+)-α-methylbenzylamine, as well as (S)-(−)-α-methylbenzyl-
amine, yielded two pairs of enantiomers, ( )-8 and ( )-9,
derived from the intramolecular cyclizations of the dimeric
phthalides. The circular dichroism method and its rules
together with the exciton chirality method were used to
determine the absolute configuration of (−)-8, (+)-8, (−)-9,
and (+)-9. The theoretical ECD curves of (+)-8 and (−)-9
were in agreement with the experimental spectra. The four
enantiomers prepared were more potent toward the three
human cancer cell lines compared to the natural products rac-3
and rac-5, and (−)-8 and (−)-9 were, in general, more potent
compared to their antipodes.
EXPERIMENTAL SECTION
■
General Experimental Procedures. rac-Tokinolide B (rac-3)
and rac-5 were isolated from the acetone extract of the rhizomes of L.
porteri by repeated column chromatography,2 carried out on silica gel
(230−400 mesh, Merck). Thin-layer chromatography analyses were
done on aluminum-backed silica gel 60 F254 plates (0.20 mm
thickness, Merck), and visualization of chromatograms was first done
under a UV lamp and then with a solution of ammonium cerium
sulfate, followed by drying and gentle heating. Infrared spectra were
recorded with an FTIR Bruker TENSOR 27 instrument. Ultraviolet
spectra were determined on a Shimadzu UV160U instrument. The
optical rotation was measured in MeOH using a Perkin-Elmer 341
polarimeter. The 1H and 13C NMR experiments were performed at 25
°C using a Varian UnityPlus 500 spectrometer (at 500/125 MHz) and
a Varian XR-300 (at 300/75 MHz); the spectra were recorded in
CDCl3, and the solvent residual signals (7.26 and 77.0 ppm for 1H and
13C NMR, respectively) were used as reference. The chemical shifts
862
dx.doi.org/10.1021/np200645p | J. Nat. Prod. 2012, 75, 859−864