In vitro biological evaluation of novel 7-O-dialkylaminoalkyl cytotoxic pectolinarigenin derivatives against a panel of human cancer cell lines

Article abstract of DOI:10.1016/j.bmcl.2008.09.037

The effect of novel pectolinarigenin derivatives bearing a dialkylaminoalkyl substituent at O-7 on cell proliferation was evaluated in vitro in a panel of seven human cancer cell lines including renal adenocarcinoma ACHN, amelanotic melanoma C32, colorectal adenocarcinoma Caco-2, lung large cell carcinoma COR-L23, malignant melanoma A375, lung carcinoma A549 and hepatocellular carcinoma Huh-7D12 cell lines. Pectolinarigenin (2), obtained by hydrolysis of rutinose unit of the pectolinarin (1) isolated from Linaria reflexa, exhibited cytotoxic activity against Caco-2, A549 and A375 cell lines with IC₅₀ values of 5.3-8.2 μM. The most active pectolinarigenin derivative was 3 characterized by a dimethylamino-propoxy group in O-7 with IC₅₀ values of 7.2 and 7.4 μM against COR-L23 and A549 cell lines, respectively. A structure-activity relationship analysis of synthesized compounds was performed. None of the tested compounds affected the proliferation of skin fibroblasts 142BR suggesting a selective activity against tumor cells.

Full text of DOI:10.1016/j.bmcl.2008.09.037

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5431–5434
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
In vitro biological evaluation of novel 7-O-dialkylaminoalkyl cytotoxic
pectolinarigenin derivatives against a panel of human cancer cell lines
b,
a
c
Marco Bonesi a, Rosa Tundis a, , Brigitte Deguin , Monica R. Loizzo , Federica Menichini ,
*
*
François Tillequin b, Francesco Menichini a
a Department of Pharmaceutical Sciences, Faculty of Pharmacy and Nutrition and Health Sciences, University of Calabria, I-87036 Rende (CS), Italy
b Laboratoire de Pharmacognosie, U.M.R./C.N.R.S. No. 8638, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, 4, Avenue de l’Observatoire, F-75006
Paris, France
c Pharmacognosy Research Laboratories, Department of Pharmacy, King’s College London, Franklin-Wilkins Building, 150 Stamford St., London SE1 9NN, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 July 2008
Revised 9 September 2008
Accepted 9 September 2008
Available online 12 September 2008
The effect of novel pectolinarigenin derivatives bearing a dialkylaminoalkyl substituent at O-7 on cell
proliferation was evaluated in vitro in a panel of seven human cancer cell lines including renal adenocar-
cinoma ACHN, amelanotic melanoma C32, colorectal adenocarcinoma Caco-2, lung large cell carcinoma
COR-L23, malignant melanoma A375, lung carcinoma A549 and hepatocellular carcinoma Huh-7D12 cell
lines. Pectolinarigenin (2), obtained by hydrolysis of rutinose unit of the pectolinarin (1) isolated from
Linaria reflexa, exhibited cytotoxic activity against Caco-2, A549 and A375 cell lines with IC50 values of
Keywords:
Cytotoxic activity
Pectolinarigenin
5.3–8.2
lM. The most active pectolinarigenin derivative was 3 characterized by a dimethylamino-pro-
poxy group in O-7 with IC50 values of 7.2 and 7.4
l
M against COR-L23 and A549 cell lines, respectively.
A structure–activity relationship analysis of synthesized compounds was performed. None of the tested
compounds affected the proliferation of skin fibroblasts 142BR suggesting a selective activity against
tumor cells.
Dialkylaminoalkyl side chain
Ó 2008 Elsevier Ltd. All rights reserved.
A significant part of drug discovery in the past few years has
been focused on agents to prevent or treat cancer. This is not sur-
prising because, in most developed countries and, to an increasing
extent, cancer is among the three most common causes of death
and morbidity. Cancer treatments may involve surgery, radiother-
apy and chemotherapy and often a combination of two or all three
is employed. Natural compounds from plants play a significant role
in cancer chemotherapy but in spite of successes there is still much
activity directed to finding novel anticancer agents.1–3
Flavonoids, a group of polyphenolic compounds derived from 2-
phenylchromane, are found in considerable quantities in fruits,
vegetables, seed, peel and tubers.4,5 Besides their physiological role
in plants, they have been shown to possess antiallergic, antiinflam-
matory, antiviral and anticarcinogenic activities.6
Flavonoids exert specific cytotoxic activity towards different
cancer cell lines which has generated large interest in developing
flavonoid-based cytostatics for anticancer therapy.7 Different
mechanisms have been linked to flavonoids mediated cytotoxicity
including proteasome, topoisomerase and fatty acids synthesis,
phosphatidyl-inositol 3-kinase inhibition, induction of cell cycle
arrest, accumulation of p53 or enhanced expression of c-fos and
c-myc genes.8–15
The cytotoxic activity of pectolinarigenin against human gastric
adenocarcinoma MK-1, human uterus carcinoma HeLa, murine
melanoma B16F10, human small lung carcinoma GLC4 and human
colorectal cancer COLO 320 cell lines has been reported.16,17 In a
previous work, we determined the antiproliferative action of sev-
eral flavones isolated from Linaria reflexa Desf. (Scrophulariaceae)
and their derivatives against the large cell lung carcinoma cell line
COR-L23, hepatocellular carcinoma cell line HepG-2, renal adeno-
carcinoma cell line ACHN, amelanotic melanoma cell line C32
and colorectal adenocarcinoma cell line Caco-2.18 We found that
pectolinarigenin and some flavonoid glycosides like pectolinarin
exhibited strong cytotoxic activity on COR-L23 cell line with an
IC50 value of 5.03 and 4.07 lM, respectively.
As part of our screening program which considers the search for
natural products with anticancer properties,19–21 we further ex-
plored the potential cytotoxic effects of novel structural analogues
of pectolinarigenin, able to give stable water-soluble salts.
The biological interest to develop structural analogues of anti-
tumor agents possessing basic nitrogen atoms seemed highly
desirable.22–26 The results obtained in several classes of polyaro-
matic antitumour agents indicate that the introduction of an amin-
oalkylamino side chain permits to increase significantly the
biological activity and the potency of the parent compounds.27
The anthracenedione mitoxanthrone, anthrapyrazole losoxan-
throne and pyridocarbazole retelliptine are interesting examples
* Corresponding authors. Tel.: +39 984 493246; fax: +39 984 493298.
E-mail addresses: tundis@unical.it, rosa.tundis@unical.it (R. Tundis).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.037

5432
M. Bonesi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5431–5434
of this modification.28–31 The distance between the amino groups
plays an important role, but the optimal number of methylene
units, generally two or three, greatly depends on the structure of
the parent chromophore.28,29 Additionally, the substituents at the
distal amino group are important to be considered.32–35 In the acr-
onycine series, the replacement of the methoxy group at C-6 by a
dialkylaminoalkylamino substituent was recently shown to give
an interesting entry toward stable and antitumor candidates. In-
deed, 6-dialkylaminoalkylamino-3,3,12-trimethyl-3,12-dihydro-
7 H-pyrano[2,3-c]acridine-7-ones and their benzo[b]pyrano[2,3-
h]acridine-7-one homologues, exhibited increased potency when
compared with the parent compounds.36,37
On the basis of these results and following our previous work,
herein we report the synthesis of a novel series of 7-O-dial-
kylaminoalkyl pectolinarigenin derivatives and their cytotoxic po-
tential in an in vitro cell culture system against a panel of human
cancer cell lines.
The synthesis of dialkylaminoalkyl pectolinarigenin derivatives
was carried out according to the procedure described in Scheme
1.38 The reaction of pectolinarigenin (2) with different dial-
kylaminoalkylchloride hydrochlorides produced the desired com-
pounds 3–8 in good yield (63–82%). The structures of the
compounds were determined on the basis of the spectral data
(UV, IR, MS, 1H NMR, 13C NMR and 2D NMR).
The sulphorhodamine B (SRB) assay was used for measurement
of cell proliferation.39,40 Seven cancer cell lines, large cell carci-
noma COR-L23 (ECACC No. 92031919), colorectal adenocarcinoma
Caco-2 (ATCC No. HTB-37), amelanotic melanoma C32 (ATCC No.
CRL-1585), hepatocellular carcinoma HepG-2 (ECACC No.
85011430), renal cell adenocarcinoma ACHN (ATCC No. CRL-
1611), malignant melanoma A375 (ECACC No. 88113005), lung
carcinoma A549 (ECACC No. 86012804), hepatocellular carcinoma
Huh-7D12 (ECACC No. 01042712) and one normal cell line such as
skin fibroblasts 142BR (ECACC No. 90011806) were used in our
experiments. The COR-L23, C32 and ACHN cells were cultured in
RPMI 1640medium while MRC-5, 142BR, Caco-2, A549, Huh-
7D12, Caco-2, A375 and HepG-2 cells were cultured in DMEM. Both
The hydrolysis of the rutinose unit of pectolinarin (1), isolated
from L. reflexa, was performed adding to a solution of HCl 5%
(prepared using
a
mixture H2O/MeOH 7:3)
1
(100 mg;
0.161 mmol) overnight at reflux. The reaction mixture was neu-
tralized with an aqueous solution of NaOH and extracted with
ethyl acetate. The extract was purified with column chromatog-
media were supplemented with 10% fetal bovine serum, 1% L-glu-
tamine, and 1% penicillin/streptomycin. The cytotoxic assay was
performed following a published protocol.41,42
raphy over silica gel 20–45
ane and methanol as eluent to afford pectolinarigenin (2)
(42 mg, 0.134 mmol; 83%).
l
m using a mixture of dichlorometh-
All tested compounds 1–8 were able to inhibit the in vitro pro-
liferation of seven human tumour cell lines in a concentration-
dependent manner although marked differences in the degree of
OMe
OMe
4'
OMe
HO
O
Rha - Glc - O
MeO
O
O
8a
7
RO
O
O
1'
a
b
3
MeO
4a
MeO
5
OH
O
OH
OH
2
1
3'''
2'''
3''
2''
1''
2''
1''
2''
1''
3
4
R =
R =
N CH2CH2CH2
5 R =
6 R =
7 R = 4'''
N— CH2CH2
N CH2CH2
5'''
3'''
6'''
2'''
2'''
3'''
4'''
1''
2''
2''
1''
2''
1''
N CH2CH2
O
N CH2CH2
N
CH2CH2
8 R =
5'''
6'''
5'''
Scheme 1. Synthesis of pectolinarigenin derivatives 3–8. Reagents: (a) HCl 5% in H2O/MeOH (7:3); (b) K2CO3, CH3OH, argon.
Table 1
Cytotoxic profile and logP values of pectolinarin (1), pectolinarigenin (2) and pectolinarigenin derivatives 3-8 against selected human cancer cell lines
a
Compound
logP
IC50
(l
M)a
ACHN
142 BR
Huh-7D12
Caco-2
COR-L23
C32
A549
A375
1
2
3
4
5
6
7
8
ꢀ0.7 0.1
2.0 0.3
2.4 0.2
2.3 0.1
3.0 0.2
2.6 0.2
3.0 0.4
1.9 0.1
>100
>100
>100
>100
>100
>100
>100
>100
50.8 2.8
>100
>100
>100
>100
>100
>100
>100
6.2 2.2°
5.3 1.8
>100
>100
>100
>100
78.6 1.8
>100
5.0 2.3°
4.1 2.4°
7.2 2.2
9.2 1.7
41.9 2.1
>100
17.2 3.1°
15.2 1.8°
10.8 1.9
10.5 2.6
54.1 1.3
12.7 2.7
92.6 2.5
55.7 3.2
7.2 3.2°
7.0 4.1°
8.9 2.5
>100
38.8 1.2
5.6 0.9
7.4 1.4
13.2 1.0
59.9 1.8
>100
32.9 2.9
8.2 1.3
7.7 1.1
>100
>100
>100
54.7 2.9
63.6 1.3
47.6 2.1
38.6 1.3
52.3 1.5
58.8 2.2
34.1 1.7
39.5 2.5
44.9 1.9
85.1 3.4
Cell lines: human malignant melanoma A375, human amelanotic melanoma C32, human Caucasian lung carcinoma A549, human hepatocellular carcinoma Huh-7D12,
human Caucasian lung large cell carcinoma COR-L23, human Caucasian colon adenocarcinoma Caco-2, human renal cell adenocarcinoma ACHN, human skin fibroblast 142BR.
Vinblastine sulfate salt was used as positive control for 142BR, Huh-7D12, Caco-2, COR-L23, A375 and C32 cell lines, while taxol was used for ACHN cell line.
a
Values are means of three experiments (n = 3). °Previously published.16

M. Bonesi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5431–5434
5433
cytotoxicity have been observed (Table 1). The flavone pectolinar-
References and notes
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Caco-2, A549 and A375 cell lines with IC50 values ranging from
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acterized by a pyrrolidin-1-yl-ethoxy group in 7-position, was
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group. In particular, compound 6 showed an IC50 value of 12.7
against ACHN cell line unlike compound 7 that showed an IC50 va-
lue of 92.6 M.
None of the tested compounds affected the proliferation of skin
lM
l
fibroblasts at the maximum concentration tested (100
gesting a selective activity against tumor cells.
lM) sug-
Moreover, logP values for pectolinarin (1), pectolinarigenin (2)
and pectolinarigenin derivatives (3–8) were evaluated. Data were
reported in Table 1. It has been reported that for good absorption
after oral administration a logP P 2 is required.43 Compounds 3–
7 showed logP values ranging from 2.3 to 3.0. These findings, taken
together with the ability to give stable water-soluble salts, suggest
that pectolinarigenin derivatives might be candidate cytotoxic
drugs with good absorption after oral administration.
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Perusal of the literature revealed a promising agreement be-
tween our results and the reported activities of a number of flavo-
noids. Indeed, an impressive body of information exists on the
antitumor action of plant flavonoids. Many works have concen-
trated on the direct and indirect actions of flavonoids on tumor
cells and have found a variety of anticancer effects such as cell
growth and kinase activity inhibition, apoptosis induction, sup-
pression of the secretion of matrix metallo-proteinases and tumor
invasive behaviour.8,10,44 In recent years, many studies revealed
that flavones, a class of flavonoids, exhibited a potent cytotoxic
activity.16,45–47
In summary, a series of novel 7-O-dialkylaminoalkyl cytotoxic
pectolinarigenin derivatives was prepared. Our results supported
published data about flavones as interesting cytotoxic secondary
metabolites. Certainly, the presence of methoxy groups in 6- and
40-positions of pectolinarigenin (2) appear to play a non-negligible
part in the antiproliferative potency of tested compounds. In our
previous work we reported the cytotoxic activity profile of some
natural pectolinarigenin glycosides, pectolinarin, linarin, isolinarin
A, isolinarin B and pectolinarigenin-7-O-b-glucoside. In the newly
synthesized series of amino-alkyloxy derivatives, the highest
potency is obtained when a dimethylamino-propoxy group or a
dimethylamino-ethoxy group is present in position 7.
27. Cholody, W. M.; Martelli, S.; Konopa, J. J. Med. Chem. 1990, 33, 2852.
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38. General procedure for synthesis of pectolinarigenin derivatives. To a solution of
pectolinarigenin (2) (1 equiv) in DMF anhydrous was added a quantity of
K2CO3 (5 equiv) and the different amino-alkylchlorides hydrochlorides
(2.5 equiv) [2-diethylaminoethyl chloride; 3-dimethylaminopropyl chloride;
2-dimethylaminoethyl chloride; N-(2-chloethyl)piperidine; 4-(2-chloroethyl)
morpholine; 1-(2-chloroethyl)pyrrolidine)]. The mixture was stirred at
temperature ranged from 80 to 120 °C under argon for 24–48 h. The mixture
was evaporated in vacuo and the residue was purified by column flash
chromatography using the mixture dichloromethane/methanol/(CH3CH2)3 N as
eluent to afford the desired pectolinarigenin derivatives.
Reactions were monitored by thin-layer chromatography (TLC) using silica gel
plates (VWR, Italy, Si gel 60F254), zones were detected visually under
ultraviolet irradiation (254 and 365 nm) and read after spraying with sulfuric
acid 50% (v/v) followed by heating. Flash column chromatography was
performed with silica gel 230–400 mesh (VWR, Milan, Italy). NMR spectra
were recorded with a Bruker AC 300 using CDCl3 as solvent. 1H NMR and 13C
NMR signals were unambiguously attributed by 2D NMR experiments. Mass
spectra (MS) were recorded with a ZQ2000 Waters mass spectrometer (ESI).
Infrared spectra have been performed with FT-IR Jasco 4200 spectrometer. The
These results contribute to further understanding the critical
molecular requirements that lead to antiproliferative properties
in the flavones series. Further in vivo studies are warranted to
confirm the biological activity of the newly synthesized flavones
and to investigate the molecular mechanisms responsible for the
antiproliferative activity of the most active compounds with a
potential pharmaceutical use.

5434
M. Bonesi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5431–5434
UV spectra were recorded on Jasco V-530 spectrometer using 1 cm quartz cells.
60), 122.6 (C-10), 114.3 (C-30, C-50), 111.6 (C-4a), 105.7 (C-3), 98.6 (C-8), 67.3 (C-
100), 60.7 (C6–OCH3), 57.0 (C-200), 54.7 (C40–OCH3), 54.0 (C-2000), 53.6 (C-6000),
22.8 (C-3000, C-4000, C-5000); IR (KBr) mmax cmꢀ1: 3435, 2934, 2656, 1625, 1605,
1584, 1509, 1463, 1363, 1305, 1263, 1187, 1132, 1033, 993, 830, 575; ESIMS m/
z: 426 [MH]+, 448 [MNa]+, 464 [MK]+; Anal. calcd (C24H27NO6) C = 67.72%,
H = 6.40%. Found C = 67.72%, H = 6.38%.
All solvents were dried according to standard procedures. All reagents were
used as purchased without further treatment unless otherwise stated.
Compound 3: a yellow solid (yield 63%). mp 99–100 °C. 1H NMR (400 MHz,
CDCl3) d 12.74 (1H, s, C5–OH), 7.83 (2H, d, J = 9.0 Hz, H-20, H-60), 7.01 (2H, d,
J = 9.0 Hz, H-30, H-50) 6.58 (1H, s, H-3), 6.57 (1H, s, H-8), 4.15 (2H, t, J = 7.5 Hz,
H-100), 3.90 (3H, s, C6–OCH3), 3.89 (3H, s, C40-OCH3), 2.51 (2H, t, J = 7.5 Hz, H-
300), 2.27 (6H, s, (N-(CH3)2), 2.09 (2H, m, H-200); 13C NMR (75 MHz, CDCl3) d
182.7 (C-4), 163.9 (C-2), 162.5 (C-40), 158.3 (C-7), 153.2 (C-5), 153.1 (C-8a),
132.7 (C-6), 128.1 (C-20, C-60), 123.6 (C-10), 114.4 (C-30, C-50), 106.0 (C-4a),
Compound 8: amorphous (yield 82%). 1H NMR (300 MHz, CD3OD) d 7.93 (2 H,
d, J = 8.9 Hz, H-20, H-60), 7.10 (1H, s, H-3), 7.06 (2H, d, J = 8.9 Hz, H-30, H-50),
6.59 (1H, s, H-8), 4.32 (2H, t, H-100), 3.88 (6H, s, C6–OCH3, C40–OCH3), 3.74
(4H, m, H-3000, H-5000), 2.93 (2H, m, H-200), 2.68 (4H, m, H-2000, H-6000); 13C
NMR (75 MHz, CD3OD) d 178.0 (C-4), 162.7 (C-2), 162.3 (C-40), 157.5 (C-7),
154.6 (C-5), 150.9 (C-8a), 140.6 (C-6), 127.6 (C-20, C-60), 123.0 (C-10), 114.2
(C-30, C-50), 112.1 (C-4a), 105.3 (C-3), 97.4 (C-8), 67.0 (C-100), 66.4 (C-3000, C-
0
104.1 (C-3), 91.4 (C-8), 67.4 (C-100), 60.9 (C6–OCH3), 56.1 (C-300), 55.5 (C4
-
OCH3), 45.5 (N-(CH3)2), 27.1 (C-200); IR (KBr) mmax cmꢀ1: 3433, 2923, 2853,
1606, 1586, 1495, 1463, 1419, 1363, 1298, 1243, 1178, 1118, 1032, 833; ESIMS
m/z: 400 [MH]+, 422 [MNa]+; Anal. calcd (C22H25NO6) C = 66.15%, H = 6.31%.
Found C = 66.22%, H = 6.37%.
5
000), 60.5 (C6–OCH3), 58.0 (C-200), 54.6 (C40–OCH3), 53.8 (C-2000 C-6000); IR
,
(KBr) mmax cmꢀ1: 3433, 2954, 2853, 1636, 1603, 1512, 1455, 1426, 1351,
1302, 1260, 1182, 1116, 1025, 948, 915, 835, 607, 564; ESIMS m/z: 428
[MH]+, 450 [MNa]+, 466 [M]+; Anal. calcd (C23H25NO7) C = 64.63%, H = 5.90%.
Found C = 64.58%, H = 5.88%.
Compound 4: a yellow solid (yield 65%). mp 140–141 °C. 1H NMR (300 MHz,
CD3OD) d 7.95 (2H, d, J = 8.3 Hz, H-20, H-60), 7.08 (2H, d, J = 8.3 Hz, H-30, H-50),
6.82 (1H, s, H-3), 6.68 (1H, s, H-8), 4.29 (2H, t, J = 5.2 Hz, H-100), 3.90 (3H, s, C6–
OCH3), 3.87 (3H, s, C40–OCH3), 2.94 (2H, t, J = 5.2 Hz, H-200), 2.46 (6H, s, (N-
39. Fricker, S. P.; Buckley, R. G. Anticancer Res. 1996, 16, 3755.
40. Loizzo, M. R.; Tundis, R.; Menichini, F.; Saab, A. M.; Statti, G. A., Menichini, F.
Cell. Proliferation 2008, 41, in press.
41. Loizzo, M. R.; Tundis, R.; Statti, G. A.; Menichini, F.; Houghton, P. J. J. Pharm.
Pharmacol. 2005, 57, 897.
13
(CH3)2); C NMR (75 MHz, CD3OD) d 182.8 (C-4), 164.8 (C-2), 163.1 (C-40),
158.1 (C-7), 153.3 (C-5), 152.5 (C-8a), 132.5 (C-6), 128.0 (C-20, C-60), 123.0 (C-
10), 114.2 (C-30, C-50), 105.5 (C-4a), 102.9 (C-3), 91.7 (C-8), 67.1 (C-100), 59.7 (C6-
OCH3), 57.3 (C-200), 54.7 (C40–OCH3), 44.6 (N-(CH3)2), IR (KBr) max cmꢀ1: 3433,
m
2956, 2931, 2360, 1601, 1496, 1464, 1426, 1367, 1254, 1186, 1128, 1116, 1024,
910, 835, 574; ESIMS m/z: 386 [MH]+; Anal. calcd (C21H23NO6) C = 65.44%,
H = 6.02%. Found C = 65.05%, H = 5.90%.
42. SRB assay. One hundred microliters per well of this cell suspension was seeded
in 96-well microtiter plates and incubated to allow for cell attachment. After
24 h, the cells were treated with serial dilutions of pure compounds. Each
compound was initially dissolved in an amount of DMSO and diluted further in
medium to produce six concentrations. Hundred microliters per well of each
concentration was added to the plates in six replicates. By these serial
dilutions, the final mixture used for treating the cells contained not more than
0.5% of the solvent (DMSO), the same as in the solvent- control wells. The final
Compound 5: a yellow solid (yield 80%). mp 92–93 °C. 1H NMR (300 MHz,
CD3OD) d7.96 (2H, d, J = 8.9 Hz, H-20, H-60), 7.09 (2H, d, J = 8.9 Hz, H-30, H-50),
6.83 (1H, s, H-3), 6.69 (1H, s, H-8), 4.34 (2H, t, J = 4.5 Hz, H-100), 3.90 (3H, s, C6–
OCH3), 3.87 (3H, s, C40–OCH3), 3.25 (2 H, t, J = 4.5 Hz, H-200), 2.96 (4H, q, (N-
(CH2–CH3)2), 1.24 (6H, t, (N-(CH2–CH3)2); 13C NMR (75 MHz, CD3OD) d 182.8
(C-4), 164.8 (C-2), 163.1 (C-40), 157.8 (C-7), 153.3 (C-5), 152.5 (C-8a), 132.4 (C-
6), 128.0 (C-20, C-60), 123.0 (C-10), 114.2 (C-30, C-50), 105.6 (C-4a), 102.9 (C-3),
91.7 (C-8), 66.4 (C-100), 59.7 (C6–OCH3), 54.7 (C40–OCH3), 54.5 (C-200), 50.7 ((N-
volume in each well was 200
lL. The plates were incubated for a select
exposure time of 48 h. At the end of exposure time, 100
l
L of ice-cold 40%
trichloroacetic acid (TCA) was added to each well, left at 4 °C for 1 h, and
washed five times with distilled water. The TCA-fixed cells were stained for
(CH2–CH3)2), 9.6 ((N-(CH2–CH3)2); IR (KBr)
m
max cmꢀ1: 3434, 2965, 2934, 2842,
1644, 1606, 1510, 1496, 1463, 1427, 1357, 1297, 1254, 1193, 1117, 1030, 826,
605; ESIMS m/z: 414 [MH]+; Anal. calcd (C23H27NO6) C = 66.81%, H = 6.58%.
Found C = 66.70%, H = 6.51%.
30 min with 50 lL of 0.4% (w/v) SRB in 1% HOAc. The plates were washed five
times with 1% HOAc and air-dried overnight. Vinblastine sulfate salt was used
as positive control for 142BR, Huh-7D12, Caco-2, COR-L23, A375, and C32 cell
lines, while taxol was used for ACHN cell line. On the day of reading the plates,
Compound 6: a yellow solid (yield 76%). mp 86–87 °C. 1H NMR (300 MHz,
CDCl3) d 7.85 (2H, d, J = 8.7 Hz, H-20, H-60), 7.03 (2H, d, J = 8.7 Hz, H-30, H-50),
6.59 (1H, s, H-3), 6.56 (1H, s, H-8), 4.26 (2H, t, J = 6.1 Hz, H-100), 3.92 (3H, s, C6–
bound dye was solubilized with 100
l
L
of 10 mM TRIS base
(tris[hydroxymethyl] aminomethane). The absorbance of each well was read
on an ELISA reader at 564 nm. Cell survival was measured as the percentage
absorbance compared to the control (non-treated cells). All values are
expressed as means standard deviation of the mean (SD). All products are
purchased from Sigma, Italy. The inhibitory concentration 50% (IC50) was
calculated from a dose–response curve obtained by plotting the percentage of
OCH3), 3.90 (3H, s, C40–OCH3), 3.04 (2 H, t, J = 6.1 Hz, H-200), 2.71 (4H, m, H-2000
,
H-5000), 1.86 (4H, m, H-3000, H-5000); 13C NMR (75 MHz, CDCl3) d 182.7 (C-4),
164.0 (C-2), 162.6 (C-40), 158.0 (C-7), 153.2 (C-5), 153.1 (C-8a), 132.8 (C-6),
128.0 (C-20, C-60), 123.6 (C-10), 114.5 (C-30, C-50), 106.2 (C-4a), 104.1 (C-3), 91.4
(C-8), 68.5 (C-100), 60.8 (C6–OCH3), 55.5 (C40–OCH3), 54.8 (C-2000, C-5000), 54.4 (C-
200), 23.6 (C-3000, C-5000); IR (KBr)
m
max cmꢀ1: 3425, 3080, 2931, 1654, 1606, 1511,
inhibition versus the concentrations with the use of GraphPad Prism
software.
4
1496, 1463, 1428, 1363, 1299, 1254, 1195, 1118, 1031,830, 606; ESIMS m/z:
412 [MH]+, 434 [MNa]+, 450 [MK]+; Anal. calcd (C23H25NO6) C = 67.14%,
H = 6.12%. Found C = 67.12%, H = 6.08%.
43. Wakita, K.; Yoshimoto, M.; Miyamoto, S.; Watanabe, H. Chem. Pharm. Bull.
1986, 34, 4663.
44. Kandaswami, C.; Lee, L. T.; Lee, P. P.; Hwang, J. J.; Ke, F. C.; Huang, Y. T.; Lee, M.
T. In Vivo 2007, 21, 553.
45. Cushman, M.; Nagarathnam, D. J. Nat. Prod. 1991, 54, 1656.
46. Zhang, Q.; Zhao, X. H.; Wang, Z. J. Food Chem. Toxicol. 2008, 46, 2042.
47. Dauzonne, D.; Folléas, B.; Martinez, L.; Chabot, G. G. Eur. J. Med. Chem. 1997, 32,
71.
Compound 7: a yellow solid (yield 65%). mp 148–149 °C. 1H NMR (300 MHz,
CD3OD) d 8.03 (2H, d, J = 9.0 Hz, H-20, H-60), 7.36 (1H, s, H-3), 7.12 (2H, d,
J = 9.0 Hz, H-30, H-50), 6.82 (1H, s, H-8), 4.60 (2H, t, H-100), 3.95 (3H, s, C6–OCH3),
3.90 (3H, s, C40–OCH3), 3.58 (2H, t, H-200), 3.17 (4H, m, H-3000, H-7000), 2.00 (6H,
m, H-4000, H-5000, H-6000); 13C NMR (75 MHz, CD3OD) d 178.9 (C-4), 163.4 (C-2),
163.1 (C-40), 156.6 (C-7), 154.6 (C-5), 149.8 (C-8a), 140.2 (C-6), 128.0 (C-20, C-