A New Iridoid Dimer and Other Constituents from the Traditional Kurdish Plant Pterocephalus nestorianus Nábělek

Article abstract of DOI:10.1002/cbdv.201600281

Accompanied by other rare compounds, a new iridoid dimer, named kurdnestorianoside (1), showing an unprecedented secologanol configuration, has been isolated for the first time from the Kurdish medicinal plant Pterocephalus nestorianus, which is used in Kurdistan for treating oral diseases and inflammation. The structure of 1 was established from 1D- and 2D-NMR spectroscopic data. Kaempferol 3-O-[3,6-di-O-(E)-p-coumaroyl]-β-d-glucopyranoside (7) showed a remarkable antiproliferative activity against several human tumor cell lines.

Full text of DOI:10.1002/cbdv.201600281

Received Date: 08-Aug-2016
Revised Date: 03-Dec-2016
Accepted Date: 05-Dec-2016
Article Type: Full Paper
A New Iridoid Dimer and Other Constituents from the
Traditional Kurdish Plant Pterocephalus nestorianus
Nab.
a
a
By Fuad O. Abdullah * ), Faiq H. S. Hussain ),
b
c
c
Marco Clericuzio ), Alessio Porta ), Giovanni Vidari* )
a
)
Department of Chemistry, College of Science, Salahaddin
University - Erbil, Iraq (F. O. A. e-mail:
fuad.abdullah@su.edu.krd)
b
)
Dipartimento di Scienze e Innovazione Tecnologica, Università
del Piemonte Orientale, Via T. Michel 11, 15121 Alessandria, Italy
c
)
Centro CEMEC and Dipartimento di Chimica, Università di
Pavia, Via Taramelli 12, 27100 Pavia, Italy (G. V. phone:
39-0382987322; fax: +39-0382987323; e-mail:
vidari@unipv.it)
+
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/cbdv.201600281
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Accompanied by other rare compounds, a new iridoid dimer, named
kurdnestorianoside (1), showing an unprecedented secologanol
stereochemistry, has been isolated for the first time from the Kurdish
medicinal plant Pterocephalus nestorianus, which is used in Kurdistan for
treating oral diseases and inflammation. The structure of 1 was established
from 1D and 2D NMR spectroscopic data. Kaempferol 3-O-(3′′,6′′-di-O-E-p-
coumaroyl) β-D-glucopyranoside (7) showed a remarkable antiproliferative
activity against several human tumor cell lines.
Keywords: Pterocephalus nestorianus; Dipsacaceae; iridoid dimers; antitumor
activity; Kurdish medicinal plant.
Introduction. – The taxonomically very complex family of Dipsacaceae
contains about 300 species of perennial and annual herbs and shrubs; most of
them are widely distributed over the Mediterranean region and the Middle
East, with about 20% growing in Asia and Africa [1]. The genus
Pterocephalus, one of the most important genera of this family, comprises
about 30 species which are usually found in sunny, dry, rocky crevices, mostly
in Europe and Western Asia (Iran, Turkey) [2-5]. Nine species are native to the
Kurdistan region of Iraq. These plants are widely used in folkloric medicines,
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all around the world, due to different biological activities such as anti-
inflammatory, analgesic, anti-hepatotoxic, anti-oxidant, antibacterial,
spasmolytic, hemostatic, antiseptic and astringent properties. Moreover,
dozens of bioactive compounds, including hydroxycinnamic acid esters,
iridoids, phenolic glycosides, monoterpenoid glucoindole alkaloids, and
lignans [6-12], triterpenoid saponins [13, 14], and flavonoid C-glycosides [15]
have been isolated and identified from different Pterocephalus species.
P. nestorianus Nab., locally known with the Kurdish name of “Lawa”,
is popularly employed in the herbal medicine for the treatment of oral
diseases and inflammation. No phytochemical work has been reported so far
on the non-volatile components of P. nestorianus.
Insert Chemical structures 1-10 about here
Results and Discussion. – Phytochemical investigation. P. nestorianus
flowers were collected on Safeen Mountain of Iraqi Kurdistan [16] and
extracted
with MeOH. After solvent evaporation, repeated MPLC chromatographic
separation of the residue on reversed-phase columns afforded a new
secologanin/loganin subtype bis-iridoid compound, called kurdnestorianoside
(
1), along with known loganic acid (2) [17], 3,5-di-O-caffeoylquinic acid (3)
18], chlorogenic acid (3-O-caffeoylquinic acid) (4) [19], luteolin-7-O-
[
glucoside (5) [20], kaempferol-3-O-(3′′-O-acetyl-6′′-O-E-p-coumaroyl)-β-
glucopyranoside (6) [21] and kaempferol 3-O-(3′′,6′′-di-O-E-p-coumaroyl)-β-
D-glucopyranoside (7) [22]. Likewise, loganin (8) [23] and cantleyoside (9)
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[24] were separated from a MeOH extract of the roots (underground parts) of
the same plant. Their structures were established by extensive 1D- and 2D-
NMR experiments and, for known compounds, by comparison of their spectral
data with those reported in literature.
Kurdnestorianoside (1) was obtained as a white amorphous solid. The
+
48
molecular formula C33H O19 was deduced from the HRMS analysis (M
found at m/z 748.2799; calcd. 748.2790), as well as from carbon and proton
counting in the NMR spectra. The IR spectrum indicated the presence of OH
-1
-1
(
3400 cm ) and unsaturated C=O (1705 and 1640 cm ) groups. Accordingtly,
the UV spectrum exhibited a maximum at 235 nm. Acid hydrolysis of 1
afforded D-glucose, which was ‒identified by the positive sign of the rotatory
power and coelution with a standard sample in TLC. Moreover, the NMR
signals of 1 were fully consistent with two β-configured glucopyranose units
(Table 1), as indicated by the two anomeric protons resonating as doublets (J =
1
13
7
.9 Hz) at δ 4.63 and 4.66, resp. The H and C NMR spectroscopic data of 1
(Table 1) displayed two distinct parts, indicated in Figure 1 as moieties A and
B, resp., corresponding to a glucosylated loganin-like and a secologanin-like
iridoids, resp.. The fused dihydropyran and cyclopentane ring iridoid structure
A was defined by COSY cross-peaks (Figure 1) between the proton signals at
δ 5.25 [H‒(C1a)], 2.00 [H‒(C9a)], 3.07 [H‒(C5a)] and 7.36 [H‒(C3a)], and
between the protons at δ 2.00 [H‒ (C9a)], 1.84 [H‒(C8a)], 1.07 [CH
3
(10a)],
4
.01 [H‒(7a)], 1.59 and 2.21 (CH (6a)], and 3.07 [H‒(C5a)]. Likewise, the
2
secoiridoid proton sequences emerged from the correlations between signals at
δ 5.52 [H‒(C1b)], 2.67 [H‒(C9b)], 3.14 [H‒(C5b)] and 7.57 [H‒(C3b)],
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between signals at δ 3.14 [H‒(5b)], 1.65-1.80 [CH
CH (7b)], and between protons at δ 2.67 [H‒(C9b)], 5.52 [H‒(C8b)], 5.24 and
.28 [CH (10b)], resp. (Figure 1). DEPT and HSQC spectra allowed the
assignment of the signals of all the individual protonated carbons, including a
methoxy group at  51.6 (Table 1).
2
(6b)], 4.34 and 4.43
[
2
5
2
C
The two glucosyl units were linked, through HMBC correlations (Figure 1), to
the anomeric carbons C1a ( 97.7) and C1b ( 98.0), resp., of the iridoid and
C
C
secoiridoid aglycones, resp. The position of two ester carbonyl groups at C11a
and C11b of the iridoid A and secoiridoid B parts, resp., were firmly supported
by the long-range correlations of H‒(C3a) ( 7.36) with C4a (
C11a ( 168.5), and those of H‒(C3b) ( 7.57) with C4b ( 106.0) and C11b
169.5), resp. Moreover, the methoxy group was attached to C11b thanks
to the cross-peak in the HMBC spectrum between the OMe signal at  3.64
C
114.0) and
C
C
(
C
and the carbon resonance at 
C
169.5 (C11b). Unit A was finally identified as a
1
loganic acid ester derivative for the almost complete coincidence of its H and
1
3
C NMR data with those of loganic acid ethyl ester [25] and, more closely,
with the moiety A of the semisynthetic iridoid dimer 10 (Table 1), obtained
from depresteroside by alkaline hydrolysis [26a]. This comparison, as well as
the analysis of NMR coupling constants and NOESY experiments (Figure 2),
fully supported the relative configuration of the moiety A in 1, as shown in the
formula. As far as the second half-unit B of 1 is concerned, the 1D NMR
(Table 1) and HMBC data (Figure 1) were comparable with those of 7-
acylsecologanoside derivatives [27], such as the unit B in the iridoid dimer 10
1
13
[
26a]. However, significant differences observed for several H and C NMR
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data of 1 and 10 (Table 1) strongly suggested that these two bis-iridoids were
stereoisomers.
The relative configuration of unit B in 1 was eventually determined by
analysis of the NMR coupling constants and confirmed by NOESY
experiments (Figure 2). Specifically, a J (1b,9b) value of 1.6 Hz suggested a
cis-relationship between H‒(C1b) and H‒(C9b). Actually, according to the
1
literature, in the H NMR spectra, recorded in CD
3
OD, of several secologanol
derivatives, such as iridoid 10, the vicinal coupling constant for trans-oriented
H‒(C1) and H‒(C9) assumes a typical value in the range of 5.4 ‒ 8.5 Hz [6, 8,
9
3
5
a, 26-30]. Moreover NOESY cross-peaks (Figure 2) between H‒(C5b) (
.14) and H‒(C9b) ( 2.67), as well as between H‒(C9b) and H‒(C1b) (
.52), revealed that these hydrogens were on the same side () of the
molecular plane of 1, while the absence of a NOESY correlation between
H‒(C8b) and H‒(C1b) strongly suggested they were trans-oriented on the
dihydropyran ring. The downfield chemical shift of the CH
2
(7b) group
(
 69.7) indicated that the moieties A and B of 1 were linked
C
together through an ester bond formed by the carboxylic group at C11a and the
hydroxylic group at C7b. From the above data, the structure 1 was assigned to
the new bis-iridoid kurdnestorianoside isolated from P. nestorianus.
Contrary to kurdnestorianoside (1), cantleyoside (9) [24], contained the
secoiridoid moiety of the dimeric structure as the acylating group of the OH-
(C7) group of the loganin unit. This bis-iridoid has been found in other
extracts of Pterocephalus species, from which the corresponding alkyl
hemiacetal and acetal artifacts have also been isolated, due to addition of the
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alcohol used for extraction to the formyl group [6, 8-10]. We found that the
NMR spectra of 9 were quite different in D O and in CD OD, although the
2
3
occurrence of multiple signals in both solvents revealed the presence of
1
different chemical entities engaged in chemical equilibria ). In D
2
O, the
presence of about 28% hydrated form 9a in equilibrium with about 72% free
1
aldehyde 9 was indicated by the co-occurrence, in the H NMR spectrum, of a
broad singlet at  9.57 assigned to CHO of 9 and a singlet at  7.43 attributable
1
3
to H‒(C3b) of 9a, having intensities in a ratio of about 2.6:1. Likewise, the C
NMR spectrum showed a series of low-intensity signals, attributed to 9a,
resonating near stronger signals assignable to 9 (Table 1). In particular, the
C
occurrence of a CH signal at  90.2 was strongly indicative of the presence of
the hydrated form 9a
3
Similarly, analysis of the spectral data of 9 in CD OD (Table 1)
revealed an equilibrium between about 25% free aldehyde and a 75% mixture
of diastereomeric hemiacetals at C7b (9b).
1
)
Cantleyoside (9) and its equilibria in D
2 3
O and in CD OD have not yet been
fully characterized spectroscopically [24].
Actually, the ESIMS (positive ion mode) spectrum of 9, dissolved in MeOH,
showed two pseudomolecolar ion peaks at m/z 769 and 801, attributed to the
+
[
M+Na] ion peaks of free aldehyde 9 and methyl hemiacetals 9b, resp.
1
Moreover, the H NMR spectrum of 9 in CD
3
OD displayed a triplet (J = 1.4
Hz) at  9.70, attributable to the aldehydic proton of 9, and a double doublet (J
6.5, 5.3 Hz) at assignable to the hemiacetalic proton of 9b [8]. The
=
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1
3
C NMR spectrum showed a series of low intensity peaks, including a
quaternary carbon for a formyl group ( 200.0), attributable to the free
C
aldehyde 9, and several pairs of higher peaks, in a ratio of about 1:1, due to the
presence of diastereomeric hemiacetals 9b (Table 1). In addition, the NMR
spectra were fully consistent with that of cantleyoside methyl-hemiacetal
reported in the literature [8].
Tumor cell antiproliferative activity of kaempferol derivatives 6 and 7.
The effects of kaempferol glucosides 6 and 7 on the proliferation of human
cells were evaluated in comparison to the well-known chemotherapeutic agent
cis-diamminedichloroplatinum (II) (cisplatin) by MTT assay. In particular,
MCF7 and SkBr3 breast, IST-MES1 mesothelioma, A549 lung, BG-1 ovarian
and Ishikawa endometrial cancer cells and human mammary MCF-10A
epithelial cells were treated for 48 h with increasing concentrations of tested
samples. The resulting IC50 values (M) (Table 2) were calculated by probit
analysis (P<0.05, χ2 test). These data clearly indicated that the two flavonoids
exhibited an antitumor activity from moderate, in case of 6, to excellent for the
dicoumaroyl derivative 7. The latter activity was comparable, or even higher
than that of cisplatin on several tumor cell lines. The remarkable higher
activity of compound 7, compared to 6, was clearly due to the presence of two
coumaroyl units instead of the only one present in 6.
Conclusions. – Bis-iridoids characterize the Dipsacaceae family and
may be considered chemotaxonomic markers also for the genus
Pterocephalus. However, to our knowledge, this is the first report of a
secoiridoid having a glycosylated anomeric OH‒(C1) group which is trans-
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oriented with respect to H‒(C9). Moreover, all the mixed iridoid-secoiridoids
previously isolated from Pterocephalus species [6, 8-10], have the secoiridoid
moiety as the acylating group; in contrast, the iridoid moiety is the acylating
unit in kurdnestorianoside 1, which is characteristic of a few bis-iridoids
isolated from Gentiana species [26, 30]. To the best of our knowledge
luteolin-7-O-glucoside (5), kaempferol-3-O-(3′′-O-acetyl-6′′-O-p-coumaroyl)-
β-glucopyranoside (6), and kaempferol-3-O-(3′′,6′′-di-O-E-p-coumaroyl)-β-D-
glucopyranoside (7) have been isolated for the first time from a Pterocephalus
species. The presence of flavonoids and caffeoyl derivatives may well support
the use of P. nestorianus as anti-inflammatory herbal remedy in Kurdish
traditional medicine. In addition, kaempferol 3-O-(3′′,6′′-di-O-E-p-coumaroyl)
β-D-glucopyranoside (7) showed a remarkable antiproliferative activity against
several human tumor cell lines.
This study of the secondary metabolites of P. nestorianus is one of the
first ones on medicinal plants growing on Safeen mountain [16] and represents
an important step for more intensive investigations on the phytochemistry and
pharmacology of the principles of the rather unexplored Kurdish traditional
medicine [31].
Acknowledgement. - This work was supported by a generous grant
from the Salahaddin University/Erbil-Iraq and the University of Pavia-Italy, in
the framework of a split-side Ph.D. program between the two universities. The
authors are grateful to Abdullah Shakur Sardar for plant identification.
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Experimental Part
General. For most general experimental techniques and procedures see
1
13
reference [32]. H-NMR and C_NMR chemical shifts (δ, ppm) are relative to
deuterated MeOH signals at δ 3.27 (central line of a quintuplet) and at δ 49.0
central line of a septuplet), resp. Preparative MPLC separations were carried
out on a Biotage Isolera instrument.
C
(
Plant material. Flowers and roots of P. nestorianus Nab., collected in
June 2014 from Safeen Mountain [16] located about 50 kilometers northeast of
ꢀ
ꢀ
Erbil-Iraq (GPS coordinates: latitude 36ꢁ 22' 24; longitude 44ꢁ 18' 25"), were
authenticated by Al-Khayat (Department of Biology, Salahaddin University).
o
A voucher specimen (n 6253) has been deposited at the Herbarium of the
Department of Biology, Salahaddin Salahaddin at Erbil.
Extraction and isolation from flowers. Freshly collected flowers were
air-dried in the shade at room temperature for one week, and then ground,
using an electrical mill. The powder (100 g) was defatted and freed of most
chlorophyll by soaking in n-hexane (3× 350 ml) with occasional shaking in an
ultrasonic bath for 30 min; subsequently, the mixture was left in the same
solvent at rt for 24 h, and then it was filtered. The residue was macerated in
EtOAc (3×400 ml) with occasionally stirring in an ultrasonic bath for 30 min,
then it was left in the same solvent at rt for 24 h. The combined EtOAc
extracts were filtered from some insoluble material and then evaporated in
o
vacuo at 25-30 C to give a yellowish residue (A, 3.75 g). The material
insoluble in EtOAc was then taken on in MeOH (3×450 ml); filtration and
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evaporation of the combined alcoholic solutions afforded a dark-brown residue
o
(
B, 17.5 g). A and B were stored at -20 C. Chlorophyll was then completely
removed from A and B by dissolving separate batches (1 g each) of the two
residues in 50 ml of MeOH-H O, 90:10, and passing the solution through a
SPE column, fitted with RP-18 phase (100 g). Elution was carried out with 50
ml of MeOH-H O, 90:10, and then with 100 ml of Me CO before the
2
2
2
successive separation. A total of 3.26 g and 16.5 g of chlorophyll-free extracts
A1 and B1 were recovered. Subsequently, a sample of A1 (1 g) was separated
2
(column A) by MPLC on a RP-18 column (100 g), eluted with a MeOH-H O
gradient, from 40:60 to 100:0 in 67.4 min; flow rate: 30 ml/min; volume of
each collected fraction: 15 ml. After TLC analysis, original fractions were
collected in 15 main fractions (EF1-EF15). Purification of fraction EF4 by
exposing the residue to MeOH, followed by Me
glucoside (5) (8 mg), identical with the literature [20]. A sample (10 g) of
residue B1 was initially fractionated by repartition between H O and, in the
order, CH Cl , EtOAc, and n-BuOH to afford, after evaporation of the organic
solutions, three residues B1 (0.2 g), B1 (1.44 g), and B1 (2.98 g), resp. B1
was further repartitioned between H O and CH Cl , followed by Et O, to give,
after evaporation, residues B1 -1 (48 mg) and B1 -2 (165 mg), resp. MPLC
separation of B1 -2 on a RP-18 column (25 g), eluted with a MeCN-H
2
CO, gave luteolin-7-O-
2
2
2
A
B
C
B
2
2
2
2
B
B
B
2
O
gradient, from 0:100 to 100:0, afforded fifteen fractions (Et1- Et15). Separate
crystallisations of Et12 and Et12 from MeOH gave kaempferol 3-O-(3’’-O-
acetyl-6’’-O-E-p-coumaroyl)-β-D-glucopyranoside (6) (7.9 mg) [22] and
kaempferol 3-O-(3′′, 6′′-di-O-E-p-coumaroyl)-β-D-glucopyranoside (7) (13.3
mg) [23], resp., identical with the literature. MPLC chromatography of Et6
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(
27.3 mg on a RP-18 column (12 g), eluted with a MeOH-H
afforded 3, 5-di-O-caffeoylquinic acid (3) (19 mg), identical with the literature
18]. B1 was further partitioned between H O and, in the order, CH Cl
EtOAc, and n-BuOH to afford, after evaporation of the organic solvents,
residues B1 -1 (30 mg), B1 -2 (557 mg) and B1 -3 (1.203 g), resp. MPLC
separation of a sample of B1 -2 (90 mg) on a RP-18 column (12 g), eluted
with a MeOH gradient in H O afforded chlorogenic acid (4) (10 mg), identical
with the literature [19]. MPLC separation of a sample (620 mg) of B1 -3 on a
RP-18 column (100 g), eluted with a MeOH-H O gradient, from 10:90 to
-3/13). MPLC separations of B1
-3/10 (20 mg) on two different RP-18 columns (12 g
O gradient, from 05:95- to 100:0, gave loganic
2
O gradient
[
C
2
2
2
,
C
C
C
C
2
C
2
1
3
00:0, afforded 13 fractions (B1
/7 (36.3 mg) and B1
C
-3/1 ‒ B1
C
C
-
3
each), eluted with a MeOH-H
2
acid (2) (3 mg), identical with the literature [17] and kurdnestorianoside (1) (5
mg), resp.
2
3
Kurdnestorianoside (1). Pale-yellow powder; [α]
D
= ‒ 57 (c, 0.002,
MeOH
MeOH); R
f
0.61 (MeOH: H
2
O, 1:1); max
nm (log ) 235 (4.15); IR (KBr)
-1
3
400, 1705, 1640, 1435, 1305, 1070, 1035, 855 cm . Dark reddish brown spot
on TLC plate under UV light; positive reaction to vanillin-sulphuric acid
1
13
reagent. H NMR (300 MHz, CD OD) and C NMR (75 MHz CD OD)
3
3
+
spectra: Table 1. MS (ESI, positive ion mode): m/z 771.17 [M + Na ]. HRMS:
+
[
M ] calcd for C33
H
48
O19: m/z 748.2790; found: m/z 748.2799.
Acidic hydrolysis of 1. Compound 1 (1.5 mg) was dissolved in 2 M
HCl in a sealed vial and heated at 100°C for 45 min. After cooling to rt and
extraction with CHCl , the aqueous layer was repeatedly evaporated to dryness
3
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with the aid of MeCN. The residue, which showed positive optical rotation,
was identified as glucose by TLC [33], upon comparison with an authentic
sample of D-(+)-glucose.
Extraction and isolation from underground parts. Powdered roots
(underground parts) of P. nestorianus (100 g) were defatted by soaking in n-
hexane (3×400 ml) with occasional shaking in an ultrasonic bath for 30 min;
then the biomass was left in the same solvent at rt for 24 h. After filtration, the
biomass was macerated in EtOAc (3×500 ml) with occasional stirring in an
ultrasonic bath for 30 min, then it was left at rt for 24 h. Filtration of the
combined EtOAc extracts from some insoluble material and evaporation in
o
vacuo at 25-30 C gave a yellowish residue (C, 0.63 g). The fraction insoluble
in EtOAc was then taken on in MeOH (3×600 ml); filtration and evaporation
of the alcoholic solution afforded a dark-brown residue (D, 9.25 g). C and D
o
were stored at -20 C. Subsequently, residue D was fractionated by repartition
between H
evaporation of the organic solvents and H
(4.45 g), and D (4.1 g), resp. MPLC separation of a sample of D
a RP-18 column (100 g), eluted with a MeOH-H
00:0, gave 12 main fractions (D /1‒D /12), after TLC analysis of initially
collected fractions (12 ml each). MPLC separation of D /7 (150 mg) on a RP-
8 column (12 g), eluted with a MeOH-H O gradient, from 0:100 to 100:0,
afforded 7 subfractions (D /7-1 ‒ D /7-7). D /7-2 was constituted by loganin
8) (5 mg), identical with the literature [23]. Preparative TLC of a sample of
/7-6 (30 mg) on an analytical C-18 plate, eluted with MeOH-H O, 1:1,
2 2
O and, in the order, Et O, EtOAc, and n-BuOH to afford, after
2
O, residues D
A
(0.4 g), D
B
(0.28 g),
D
C
D
C
(1 g) on
2
O gradient, from 0:100 to
1
C
C
C
1
2
C
C
C
(
D
C
2
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afforded cantleyoside (9) (20 mg), identical with the literature [24].
Antiproliferative activity. Cell cultures. MCF-7 breast and A549 lung
cancer cells were maintained in DMEM/F-12 supplemented with 10% fetal
bovine serum (FBS), 100 μg/ml penicillin/streptomycin (Life Technologies,
Milan, Italy). SkBr3 breast and BG-1 ovarian cancer cells were cultured in
RPMI-1640 and DMEM medium resp., without phenol red supplemented with
1
0% FBS and 100 μg/ml penicillin/streptomycin (Life Technologies, Milan,
Italy). Ishikawa endometrial cancer cells were maintained in MEM
supplemented with 10% FBS, 100 μg/ml penicillin/streptomycin, 2mM L-
glutamine and 1% Non-Essential Amino Acids Solution (Life Technologies,
Milan, Italy). IST-MES1 malignant mesothelioma cells were grown in
Nutrient Mixture F-10 Ham (Ham's F-10) medium supplemented with 10%
FBS and 100 μg/ml penicillin/streptomycin. Human mammary MCF-10A
epithelial cells were cultured in DMEM/F-12 medium supplemented with 5%
Horse Serum (Eurobio, Les Ullis, Cedex, France) and 0.1 nmol/l nonessential
amino acid, 2 mmol/l L-glutamine, and 50 units/ml penicillin/streptomycin.
All cell lines were obtained by ATCC and used less than 6 months after
resuscitation, except IST-MES1 cells which were kindly provided by Dr.
Orengo (Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy).
Inhibtion of cell proliferation. The effects of kaempferol derivatives 6
and 7 on cell viability were determined by the MTT [3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide] assay, which is based on the conversion
of MTT to MTT-formazan by mitochondrial enzyme [34]. Cisplatin was used
as the reference compound. Cells were seeded in quadruplicate in 96-well
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plates in regular growth medium and grown until 70% confluence. Cells were
washed once they had attached and then treated with increasing concentrations
of each compound for 48 h in regular medium supplemented with 1% FBS.
Relative cell viability was determined by MTT assay according to the
manufacturer’s protocol (Sigma-Aldrich, Milan, Italy). Mean absorbance for
each sample dose was expressed as percentage of the cells treated with vehicle
absorbance and plotted versus sample concentration. IC50 values were
calculated by probit analysis (P < 0.05, χ2 test) and are expressed in μM.
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Table 1. NMR data of compounds 1, 9, 9a, 9b and 10
H/C
1
10*
1
10*
9
9a
9b
1
†,‡
13
¶,ɸ
13
§,ɸ
13
¶,ɸ,±
H-NMR
C-NMR
C-NMR
C-NMR
Iridoid moiety A
Aglycone
a
1
3
4
5
a
a
a
a
5.25 d (4.5)
7.36 d (1.1)
‒‒
5.29 d (4)
7.38 d (1.5)
‒‒
97.7
97.7
97.2
152.0
113.2
97.2
97.5
152.6
113.3
152.1
114.0
152.0
114.3
152.0
113.3
3.07 dddd (9.2,
3.10 m (9.5, 8,
7.7, 1.5)
7
.9, 7.6, 1.1)
 2.21 ddd (13.9,
.9, 1.3)
1.59 ddd (13.9,
.6, 5)
32.2
42.7
32.0
42.8
31.0
39.1
30.9
37.5
32.6
40.3
6
a
7
2.23 m (14, 8, 1.5)
1
.66 m (14, 7.5, 5)

7
a
7
8
9
a
a
a
4.01 br t (4.6, 1.3)
1.84 m (9.2, 6.9,
4.04 ddd (5, 5,
75.1
42,2
46.5
75.1
42.1
47.7
79.1
40.0
46.1
79.1
40.0
46.1
78.3
1
.5)
1.86 m (9, 7, 5)
40.8
4
.6)
2.00 td (9.2, 4.5)
2.04 ddd (9.5, 9,
47.1 ; 47.2
4
)
1
1
0a
1a
1.07 d (6.9)
1.09 d (7)
13.4
168.5
‒‒
13.2
169.1
‒‒
12.9
170.7
52.6
12.9
170.7
52.6
13.8 ; 13.9
169.4
‒‒
‒‒
‒‒
‒‒
OMe
51.8
Sugar
a
a
a
b
1
2
3
4
5
6
a’
a’
a’
a’
a’
a’
4.63 d (7.9)
4.66 d (8)
99.7
74.7
100.2
74.9
79.1
71.8
79.4
99.4
99.4
100.2
b
b
c
3.16 dd (8.9 7.9)
73.3
73.3
74.7
a
c
c
77.9
76.3
76.3
77.9
71.6
b
3.25-3.40 m
ca 3.20-3.40 m
71.5
70.3
77.1
70.3
77.1
a
78.4
78.4
b
3.88 dd (12, 1.7)
3.91 dd (12, 2)
a
c
c
d
3
.66 dd (12, 5.7)
3.69 dd (12, 6)
62.8
62.8
61.4
61.4
62.7
Secoiridoid moiety B
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Aglycone
a
1
3
4
5
b
5.52 d (1.6)
7,57 d (2,5)
‒‒
5.54 d (6)
7.46 d (1)
‒‒
98.0
153.9
106.0
97.8
97.7
154.3
109.7
98.1
153.4
111.4
97.8 ; 97.9
153.1 ; 153.3
112.2
b
153.6
111.5
b
b*
3.14 m
2.92 m (7.5, 6.5,
5
.5, 1)
28.4
25.9
30.8
30.0
28.0
45.0
29.3
45.0
29.6 ; 29.8
37.5 : 37.8
R
b
6
2.09 m (14, 6.5.
6,.5)
e
1
.65-1.80 m
p
o
1.77 m (14, 7.5,
7
.5, 6)
7
b
4.43 ddd (11.1,
.0, 2.4)
4.20 ddd (11, 6, 5)
4.09 ddd (11, 7.5,
6)
r
4
69.7
63.4
207.3
90.2
98.2 ; 98.5
t
4.34 td (11,1, 3.0)
5.52 ddd (17, 10,
8
9
b
5.76 ddd (16,
10.5, 8.5)
e
9
.5)
2.67 ddd (9.5, 5.5,
.6)
5.28 dd (17, 1.7)
133.3
43.8
135.6
45.2
133.7
44.4
134.4
44.1
135.8 ; 135.9
45.4 ; 45.5
d
b
2.68 ddd (8.5, 6,
5.5)
1
1
0b
5.27 dd (16, 1.5)
f
5
.24 dd (10, 1.7)
5.25 dd (10.5. 1.5) 120.8
119.6
121.5
120.7
119.7
r
o
1
1b
‒‒
3.64 s
‒‒
‒‒
3.67 s
‒‒
169.5
51.6
‒‒
169.0
51.8
‒‒
169.2
‒‒
169.7
‒‒
168.4
‒‒
MeO
m
CD
3
O
‒‒
‒‒
49.6 ; 49.9
Sugar
r
a
a
a
b
1
2
3
4
5
6
b’
4.66 d (7.9)
4.69 d (8)
100.0
74.7
100.2
74.9
79.1
71.8
79.4
99.5
99.5
100.1
b
b
c ;
c
be’
3.16 dd (8.9 7.9)
73.4
73.4
74.4 74.8
a
c
c
b’
f
b’
78.0
76.4
76.4
77.9
71.6
b
3.25-3.40 m
ca 3.20-3.40 m
71.6
70.3
77.1
70.3
77.1
e
a
b’
78.4
78.4
b
br’
3.88 dd (12, 1.7)
3.88 dd (12, 1.5)
a
c
c
d
3
.64 dd (12, 5.7)
3.64 dd (12, 5.5)
62.7
62.8
61.4
61.4
62.8
e
†
‡
¶
nce [26a]; in CD
3
OD ( 3.27); at 300 MHz for 1; at 400 MHz for 10; in
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§
ɸ
CD
3
OD ( 49.0); in D
2
O and 1,4-dioxane-d8; at 75 MHz for 1, 9, 9a, 9b; at
a-d
5
0 MHz for 10; values with the same superscript in the same column are
±
interchangeable; doubled signals are due to the presence of two
diastereomeric hemiacetals at C-7b.
a
Table 2. Antiproliferative activity (IC50) of kaempferol derivatives 6 and 7 against human cell
lines.
Tested
sample
MCF-7
SkBr3
IST-MES1
20 (±2)
A-549
BG-1
Ishikawa
15 (±3)
MCF-10A
>50
Compound 6
2
0 (±4)
(±1)
19 (±3)
22 (±3)
27 (±3)
25 (±5)
Compound 7
2
4 (±1)
4 (±3)
3 (±2)
7 (±3)
6 (±2)
3 (±1)
4 (±2)
8 (±1)
5 (±2)
Cisplatin
10 (±2)
15 (±4)
39 (±4)
a
IC50 ± SD values are expressed in M.
Legends to Figures
Figure 1. COSY and HMBC correlations of kurdnestorianoside (1).
Figure 2. Significant NOESY correlations of kurdnestorianoside (1).
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Chemical structures 1-10
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Figure 1
Figure 2
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