Constituents of Bupleurum praealtum and Bupleurum veronense with Potential Immunomodulatory Activity

Article abstract of DOI:10.1021/acs.jnatprod.0c00437

In this investigation, chromatographic separations of the diethyl ether extracts of two European annual Bupleurum taxa, B. praealtum and B. veronense, yielded nine new natural products, namely, a series of esters of stereoisomeric tetradeca-5,7,9,11-tetraen-1-ols (1-4 and 8), a tetra-unsaturated γ-tetradecalactone (5), a dibenzylbutyrolactone lignan (7-oxoarcitin, 6), a falcarinol-related 17-membered macrolide (7) possessing a conjugated diyne-system, and an acylphloroglucinol derivative (9). All these new compounds were fully characterized by NMR, IR, UV, MS, and optical rotation measurement, including 1H NMR full spin spectral simulation, whereas the absolute configurations of 1, 5, and 9 were determined via chemical correlations and NMR analysis of Mosher esters. The in vitro potential immunomodulatory activities of 1, 4, 5, and (+)-arcitin were assessed by determining their effects on the functional properties of isolated rat splenocytes and peritoneal macrophages. The results obtained support the known immunomodulatory ethnomedicinal usage of Bupleurum species.

Full text of DOI:10.1021/acs.jnatprod.0c00437

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Article
Constituents of Bupleurum praealtum and Bupleurum veronense with
Potential Immunomodulatory Activity
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Niko Radulovic,* Milica Stevanovic, Milan Nesic, Nikola Stojanovic, Pavle Ranđelovic,
́
and Vladimir Ranđelovic
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ABSTRACT: In this investigation, chromatographic separations of the diethyl ether extracts of two European annual Bupleurum
taxa, B. praealtum and B. veronense, yielded nine new natural products, namely, a series of esters of stereoisomeric tetradeca-5,7,9,11-
tetraen-1-ols (1−4 and 8), a tetra-unsaturated γ-tetradecalactone (5), a dibenzylbutyrolactone lignan (7-oxoarcitin, 6), a falcarinol-
related 17-membered macrolide (7) possessing a conjugated diyne-system, and an acylphloroglucinol derivative (9). All these new
compounds were fully characterized by NMR, IR, UV, MS, and optical rotation measurement, including 1H NMR full spin spectral
simulation, whereas the absolute configurations of 1, 5, and 9 were determined via chemical correlations and NMR analysis of
Mosher esters. The in vitro potential immunomodulatory activities of 1, 4, 5, and (+)-arcitin were assessed by determining their
effects on the functional properties of isolated rat splenocytes and peritoneal macrophages. The results obtained support the known
immunomodulatory ethnomedicinal usage of Bupleurum species.
pecies of the genus Bupleurum L. (Apiaceae) have been
ans.1 Some of the isolated constituents have been demon-
strated to affect the viability and functional properties of cells
of the immune system (macrophages), consistent with the
ethnobotanical uses of Bupleurum species.1
In Europe, 33 annual species of the genus Bupleurum belong
to two sections: Bupleurum and Aristata.7 Fifteen Bupleurum
taxa can be commonly found in Serbia, typically on mountain
slopes at elevations of up to 2000 m above sea level, whereas
some other Balkan Bupleurum taxa grow spontaneously even
on the sea coastline.8 Two annual taxa from the section
Aristata, B. praealtum L. (subsection Juncea)7 and B. veronense
Turra (subsection Aristata),7 are good representatives of these
two habitats, respectively. While the probable European
S
utilized by humans for more than two millennia,1
predominantly as medicinal plants, with the best-documented
species being B. chinense DC., which is native to East Asia. Due
to the incorporation of its dried roots in several major
multicomponent traditional Chinese medicines, targeting
conditions related to the immune system, B. chinense has
been in the focus of prior phytochemical and biological work,
which resulted in the discovery of the potent immunomodu-
latory proapoptotic saikosaponins.2 Other members of the
genus, which encompasses ca. 190 Northern Hemisphere Old
World species, have been investigated sporadically and have
yielded new structures displaying an array of biological
activities.1 Among the isolated secondary metabolites,
polyketides (polyacetylenes),3,4 terpenoids (saponins),2,5 and
shikimate metabolites (lignans and coumarins)1,6 were the
most commonly reported. Many of the polyacetylenes found
have been related to the toxic polyunsaturated alcohol
falcarinol,3 while dibenzylbutyrolactone derivatives have been
the most frequently encountered lignans, accompanied by
arylnaphthalenes, aryltetralinelactones, and tetrahydrofurofur-
Received: April 20, 2020
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Structures of compounds 1−6 isolated from B. praealtum.
endemic species, B. veronense, has never been previously
phytochemically studied, only the essential oil of B. praealtum
has been analyzed previously.9 The leaves of B. veronense were
used against toothache,10 suggesting a possible anti-inflamma-
tory action.
RESULTS AND DISCUSSION
■
The initial GC−MS analysis of the crude Et2O extract of the
dry umbels of B. praealtum revealed the presence of one major
constituent (1, Figure 1) with a molecular ion at m/z 306 in its
MS. HRMS disclosed the molecular formula of 1 to be
C19H30O3, while the IR absorptions at 3100−3500 (max at
3334) and 1727 cm−1 suggested the presence of alcohol and
ester functions, respectively. Among the 19 resonances
observable in the 13C NMR spectrum of 1 (Table 1), an
ester carbonyl at 175.0 ppm (C-1) and two oxygenated sp3-
hybridized carbon signals at 65.4 (C-1′) and 74.9 (C-2) ppm
1H NMR spectra offer a plethora of structural information;
however, the reproducibility of the data, permitting dereplica-
tion, is more than occasionally hampered by low spectral
dispersion and the incidence of higher-order effects.11,12 1H
NMR iterative full spectral simulation represents a valuable
tool for complete spectral interpretation,13 adequate precision,
and unambiguous 1H NMR-driven dereplication.12,14 In
extended unsaturated aliphatic chains, due to local pseudo-
symmetry, pairs of protons of isolated and conjugated double
bonds are almost isochronous, producing highly complex
resonances. Such slight differences in chemical shifts and
additional accidental signal isochronicity make dereplication a
significant endeavor.12,15
Table 1. 13C NMR Spectroscopic Data (100.6 MHz, CDCl3)
of Compounds 1, 3−5, and 8
1
4
3
8
5
position
δC
position
δC
1, C
175.0
74.9
a
170.0
77.0
a
175.1
75.0
a
177.3
34.2
a
19.1
19.1
a
64.2 1, CH
28.4 2, CH2
26.1 3, CH2
27.6 4, CH
134.8
129.4
128.4
133.3
132.8
128.0
128.2
132.0
a
a
a
a
a
a
a
a
a
a
a
This study aimed to investigate, chemically, the constituents
of two Balkan species, B. praealtum and B. veronense. Extracts of
these species were subjected to various chromatographic
separations, and the structures of the obtained pure
constituents were elucidated by spectroscopic means and
chemical transformations. Structural elucidation and complete
NMR spectroscopic assignments were conducted using
2, CH
2-OH
3, CH
32.2
15.9
18.8
65.4
28.1
25.9
27.3
131.1
129.50
127.1
133.3
130.7
133.5
129.53
137.3
25.9
13.5
a
30.1
17.4
18.9
65.1
28.2
26.00
27.5
131.5
129.5
127.3
133.3
130.9
133.5
129.6
137.4
26.05
13.7
32.2
16.0
19.0
65.5
28.1
25.6
32.3
133.8
129.59
132.1
131.36
131.43
132.9
129.63
136.9
26.0
13.6
a
4, CH3
5, CH3
1′, CH2
2′, CH2
3′, CH2
4′, CH2
5′, CH
6′, CH
7′, CH
8′, CH
9′, CH
10′, CH
11′, CH
12′, CH
13′, CH2
14′, CH3
1″, C
a
177.2
28.8
29.0
80.8
128.9
133.6
135.1
130.04
129.98
135.6
129.4
138.5
26.1
13.6
a
1
iterative H NMR full spectral simulations. This resulted in
the identification of nine new compounds: four polyunsatu-
rated esters (1−4), one lactone (5), and one lignan (6) from
B. praealtum and falcarinol-related 17-membered macrolide
(7), one polyunsaturated ester (8), and acylphloroglucinol
derivative (9) from B. veronense. Compounds from B.
praealtum, available in sufficient quantity (1, 4, 5, and
(+)-arcitin), were evaluated for their immunomodulatory
activity by assessing their effect on isolated rat splenocytes
and peritoneal macrophages. The absolute configurations of
three new compounds, 1 and 5 from B. praealtum and 9 from
B. veronense, were determined via the preparation of the
corresponding Mosher esters, as well as by comparison of
spectroscopic data of the synthesized enantiopure model
compounds.
5, CH
6, CH
7, CH
8, CH
9, CH
10, CH
11, CH
12, CH
b
b
21.4 13, CH2
14.4 14, CH3
a
a
171.0
20.8
2″, CH3
a
a
a
a
b
No carbon or no position. Assignment can be interchanged.
B
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C
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further strengthened the existence of the mentioned functional
groups. The diastereotopic hydrogens from the C-1′ methylene
group appeared at δH 4.21 (H-1′a) and 4.17 (H-1′b) in the 1H
NMR (Table 2) and correlated through three bonds with C-1
(Figure S1.5, Supporting Information), revealing the presence
of a proximal chiral center in 1, which is in agreement with the
observed optical rotation of 1 (+64.3, c 1, MeOH). The center
was positioned α to the ester group based on the gHMBC
interaction of C-1 and a doublet of doublets (J = 6.0, 3.4 Hz)
at δH 4.05 (H-2) that coupled with a methyne hydrogen (δH
2.07) from an isopropyl group and the OH proton (δH 2.81,
exchanged upon a D2O shake experiment). The coupling with
an exchangeable proton and the gHMBC interaction of OH to
C-1 demonstrated the existence of an intramolecular hydrogen
bond. Based on this, 1 was concluded to be an ester of 2-
hydroxy-3-methylbutanoic acid and a tetra-unsaturated ali-
the methoxy group (Figure S1.7, Supporting Information). An
excellent correspondence of the data for (R)-MTPA-1 and
(R)-MTPA-(R)-1a (Table S3 and Figure S1.7, Supporting
Information), and a considerable difference from the signals of
(R)-MTPA-(S)-1a (Table S3 and Figure S1.7, Supporting
Information), enabled compound 1 to be assigned as
(5Z,7E,9E,11E)-tetradeca-5,7,9,11-tetraen-1-yl (R)-2-hydroxy-
3-methylbutanoate, a new natural compound, for which the
name praealtaester A is proposed.
The alcohol moiety of 1, but with a different configuration of
double bonds (5E,7E,11Z,13E), was reported as part of a not
fully characterized ester isolated from two Centaurea species
(Asteraceae)18,19 and, later on, from C. scabiosa, an isovalerate,
but this time it was isolated with an undetermined
configuration of the double bonds.20 Low amounts of 2, an
isovalerate of tetradeca-5,7,9,11-tetraen-1-ol, were also present
in the B. praealtum extract. To elucidate the structure of 2, a
sample of the ester of isovaleric acid and (5Z,7E,9E,11E)-
tetradeca-5,7,9,11-tetraen-1-ol, obtained by a careful meth-
anolysis of 1, was prepared in a Steglich-type reaction. A GC−
MS coinjection experiment confirmed the existence of 2,
(5Z,7E,9E,11E)-tetradeca-5,7,9,11-tetraen-1-yl 3-methylbuta-
noate (praealtaester B), in B. praealtum aerial parts, but 2
appears to be absent from the ripe schizocarps.
1
phatic primary C14-alcohol. The H NMR spectrum (Figure
S1.1, Supporting Information) displayed, along with the signals
of two diastereotopic methyl groups (δH 0.86 and 1.03, Table
2) from the acid part, one additional methyl signal as a
nonvirtually coupled triplet at δH 1.02 (J = 7.5 Hz) implying
that the unsaturated functionalities were near the ω-end of the
alcohol and that 1 did not contain any further branching. All
four unsaturations were located in four double bonds, apparent
from the 8 sp2-CH 13C NMR/DEPT-90 signals (Table 1),
which were mutually conjugated based on the characteristic
absorption maxima for conjugated tetraenes noted in the UV
spectrum of 1.16 Their position in the chain was
straightforwardly determined to be at carbons C-5′, C-7′, C-
9′, and C-11′ from gHMBC (Figure S1.5, Supporting
An isomer of 1 (3) and an acetylated derivative of 1 (4,
Figure 1) were also detected in the B. praealtum extract by
GC−MS. The retention index (DB-5MS) of 3 was 38 units
higher than that of 1 (RI(3) = 2403 and RI(1) = 2365), and
their mass spectra were almost identical. These were indicative
of a relationship of cis- and trans-geometric isomers, in this case
corresponding to 1 and 3, respectively, as exemplified by the
values of retention indices of a trienic system (RI-
(trans,cis,trans-isomer) = 1598 and RI(trans,trans,trans-isomer)
= 1648).21 This means that 3 differs from 1 only in the
geometry of the C-5′−C-6′ double bond. An enriched sample
of 3 (76%) was obtained from further fractionations, while 1D
and 2D NMR spectra of this mixture (Figures S3.1, and S3.2,
Supporting Information), along with the data from the
previously simulated spectra of 1, enabled a full spectral
assignment (Tables 1 and 2) and unambiguously confirmed
that 3 is indeed the “all-trans” isomer, i.e., (5E,7E,9E,11E)-
tetradeca-5,7,9,11-tetraen-1-yl 2-hydroxy-3-methylbutanoate
(praealtaester C, Figure 1). It is possible that 3 merely
represents an artifact of 1, a thermodynamically more stable
isomer formed as a consequence of absorption of UV light.19
This, on the other hand, may suggest a possible protective
function of 1 and related compounds in the plant against
harmful UV irradiation. This gradual conversion to an “all-
trans” isomer was also actually noticed in the case of 2,
occurring spontaneously, and was most probably light-driven.
The freshly synthesized 2 contained only small amounts
1
Information) and gradient H−1H COSY spectra; i.e., there
is an ethyl group on the ω-end of the alcohol.
The geometries of C-5′−C-6′ and the C-11′−C-12′ double
bonds were concluded to be Z and E, respectively, based on
the observable proton−proton (JH‑5′−H‑6′ = 10.3 and JH‑11′−H‑12′
= 15.0 Hz) and proton−carbon coupling constant values
1
(Figure S1.6, Supporting Information). The H-coupled 13C
NMR and 1H selective homodecoupling experiments, followed
17
1
by manual H full spin spectral simulation (Figure S1.1.1,
Supporting Information), enabled the determination of the
geometry (E,E) of the two inner double bonds and the full
assignment of its NMR signals (Tables 1 and 2). A comparison
with the NMR data on the closely related methyl parinarate
isomers16 (Tables S1 and S2, Supporting Information) further
corroborated the (5Z,7E,11E,13E)-configuration of 1. It
should be stressed that the full spectral, structural, and
stereochemical assignments could not be achieved solely by a
conventional (including 2D NMR) elucidation approach but
was possible though 1H full spectral simulation that is essential
for the dereplication of polyunsaturated aliphatic chains.12 The
NMR parameters obtained during the simulations, in this case
and all others where this approach was applied, are given, to
the relevant number of decimal points (i.e., precision; Tables
S1−S5, Supporting Information).12
1
(based on H NMR, less than 2%) of all-trans-2, while after a
week (Figure S2.3, Supporting Information), the molar
percentage rose to 22%; however, the sample was stored in a
closed vial at 4 °C in the dark.
The absolute configuration of the chiral center at C-2 in 1
1
was determined via a comparison of the H NMR data of a
A comparison of the mass spectra of 4 and 1, i.e., the
presence of a dominant m/z 43 and an increment in the mass
of 42 in the M+ ion, as well as their RI values, led to an
assumption that 4 (Figure 1) is an acetylated derivative of 1.
This hypothesis was confirmed by a synthesis of the acetate of
1 and a GC−MS coinjection experiment. The prepared sample
was utilized to acquire all of the pertaining analytical data for
prepared Mosher ester of (R)-MTPA and 1 with the
corresponding data for the diastereomerically pure (R)-
MTPA-esters of two synthesized model compounds, methyl
2-hydroxy-3-methylbutanoates, (R)-1a and (S)-1a. The differ-
entiation of the diastereomeric esters was most notable in the
case of the chemical shifts of the geminal methyl groups C-4
and C-5, and ortho-hydrogens on the benzene ring, as well as
D
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this new compound (praealtaester D, 4) as well (Tables 1 and
2).
found to be mutually trans based on the pertinent spectral data
comparison.26 In this way, 6 was recognized as 7-oxoarcitin, or
O-methylconicaol B, a compound previously unknown from
natural sources but reported almost 60 years ago as an
insufficiently characterized (only UV and IR data provided)
synthetic intermediate.27 Thus, herein, for the first time, its full
NMR assignment (Figures S6.1 and S6.2, Supporting
Information) was performed, and it represents a new natural
product.
One more highly unsaturated, polyenic compound, 5
(Figure 1), was isolated from the B. praealtum aerial parts’
extract and was found to have the molecular formula C14H18O2
based on HRMS. The sharp and intense IR band at 1771 cm−1
straightforwardly revealed the presence of a γ-lactone moiety,
accounting for two of the six total unsaturations.22 The
remaining four unsaturations were allocated to four conjugated
double bonds which were initially inferred from the observed
absorption maxima in the UV spectrum of 5 and corroborated
by the expected eight methyne sp2-hybridized carbon-atom
Chromatography of the extract of B. veronense yielded
compound 7 (Figure 2), with an EIMS similar to that of
1
signals in its 13C NMR/DEPT-90 spectra. 13C NMR and H
data (Tables 1 and 2) of 5 gave further evidence of a methyl
group (a triplet at 1.02 ppm), three methylenes, one more
methyne singly bonded to an oxygen atom, and a lactone
carbon at 177.2 ppm. The signal at δH 5.00, corresponding to
H-4, provided the connectivity data of the lactone with the rest
of the molecule, via proton−proton coupling constants of 7.7,
7.0, and 6.8 Hz, and appropriate long-range C−H couplings
(Figure S5.4, Supporting Information). Hence, while the
lactone was on one side of the tetraenic system, the other was
occupied by an ethyl group, as in the cases of 1−4. The
appropriate 1H full spin spectral simulation and a concomitant
comparison with the 1H chemical shifts and coupling constant
values of 1−4 permitted the assignment of the (all-E)-
configuration of the four double bonds.
The absolute configuration of the chiral center at C-4 of 5
was deduced after a total hydrogenation experiment. The
specific optical rotation of the obtained γ-tetradecalactone
matched the sign and value of the angle of rotation of the (R)-
enantiomer.23 This means that the chiral center in 5 has the
(S)-configuration due to the formal change in the Cahn−
Ingold−Prelog priority of groups around the center upon
hydrogenation. A related doubly unsaturated lactone having
the (S)-configuration at C-4, synthesized in a stereoselective
manner,24 was also determined to have the same sign (+) of
rotation as that of 5. Herein, the new natural lactone 5 is
concluded to be (S)-5-((1E,3E,5E,7E)-deca-1,3,5,7-tetraen-1-
yl)dihydrofuran-2(3H)-one, for which the name praealtalac-
tone is proposed.
Two related dibenzylbutyrolactones were additionally
isolated from B. praealtum extracts, one of which is a known
compound, from the B. praealtum aerial parts’ extract,
straightforwardly identified as (+)-arcitin (syn. O,O-dimethyl-
matairesinol, Figure S6.5, Supporting Information), by
comparison of its NMR data with the previously reported
ones.25 The NMR, MS, and IR spectra of the other lignan (6,
Figure 1), isolated from the B. praealtum schizocarp extract,
pointed to an oxygenated derivative of arcitin, possessing a
keto function in position 7. It appears that, in deuterated
chloroform, 6 exists solely in the keto tautomeric form, as no
traces of the enolic form (Figure S6.5, Supporting
Information), expected for β-ketolactones, were visible in the
NMR spectra. These enolic tautomers were neither mentioned
in the synthetic work on closely related keto-lignans nor for the
naturally occurring conicaol B.26 The data from the mentioned
papers were also helpful in establishing the relative
configuration of the chiral centers at 8 and 8′, as the observed
NOESY interactions of 6 were inconclusive: There were both
cross-peaks noted between H-8 and H-8′ and H-8 and H-7′a/
b. As in the structure of arcitin, the two substituted benzylic
groups on the highly flexible γ-butyrolactone ring of 6 were
Figure 2. Structures of compounds 7−9 isolated from B. veronense
1
and the Mosher esters of 9 with relevant H NMR data.
falcarinol,28 a dienediyne secondary alcohol. Its molecular ion
corresponded to the formula C18H24O2, established by
HREIMS, indicating seven indices of hydrogen deficiency;
thus, there was one carbon and one oxygen atom, as well as
one degree of unsaturation more than that of falcarinol. The IR
spectrum revealed the presence of a carbonyl (1727.5 cm−1)
group, suggesting the presence of an ester functionality. The 18
carbon signals were all resolved in the 13C NMR spectrum
(Table 3), which is in accordance with the molecular formula,
and included a carbonyl (δC 173.5), a methyl (δC 9.6), nine sp3
methylenes, one oxygenated sp3 (δC 65.3), and two sp2
methyne groups, as well as four sp nonprotonated carbons
(δC 64.7, 70.4, 74.5, and 80.5). The aforementioned
functionalities accounted for six degrees of unsaturation,
indicating that 7 also contains one cycle. The ester carbonyl
C-1 (δC 173.5) was shown to be attached to C-2 by the
gHMBC correlations of H-2a and H-2b to C-1. The gradient
1H−1H COSY data established the presence of a long, proton-
bearing and spin-coupled structural unit, vicinally coupled
from C-2 to C-11 and from C-16 to C-18, while the two
moieties are mutually connected via a long-range coupling
constant over seven bonds of 0.7 Hz, i.e., over two conjugated
triple bonds (Figure S7.4, Supporting Information). The
analysis of gHMBC data of 8 corroborated the connectivity,
whereas the ester/lactone group was positioned on the
grounds of H-16−C-1 three-bond coupling, indicating that a
rare 17-membered macrolide was formed (Figure S7.4,
Supporting Information). The presence of a double bond, in
the same position as in falcarinol, was evident initially from the
chemical shifts of H-9 (δH 5.58, δC 135.2) and H-10 (δH 5.49,
δC 121.4), and it was assigned (Z)-configuration based on the
H-9−H-10 coupling constant value (J = 10.3 Hz) and
confirmed by multiple observed gHMBC correlations of H-
E
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1
Table 3. H (400 MHz, CDCl3) and 13C (100.6 MHz, CDCl3) NMR Spectroscopic Data of 7 and (Z)-Falcarinol
homodihydrofalcarinolide (7)
(Z)-falcarinol46
a
b
position
δC, type
δH , (J in Hz)
position
δC, type
δH, (J in Hz)
1
173.5, C
34.7, CH2
c
17
16
d
2a
2b
3a
3b
4
5
6
7
2.35, ddd (−13.3, 9.8, 4.1)
2.31, ddd (−13.3, 4.3, 7.5)
1.70, m
1.64, m
1.35 , m
22.6, CH2
31.9, CH2
29.2, CH2
1.22−1.31
26.6, CH2
15
14
a
28.80, CH2
29.8, CH2
30.3, CH2
28.77, CH2
28.4, CH2
overlapping peaks
1.37, m
a
1.30 , m
1.34 , m
a
13
12
11
1.43, m 1.3820, m
2.11, ddddd (−13.8, 8.4, 7.0, 6.6, −1.2)
2.06, ddddd (−13.8, 7.6, 6.7, 6.5, −1.2)
5.58, dddt (10.3, 8.4, 7.6, −1.1)
5.45, dt t (10.3, −1.2, −1.1)
2.95, ddd (−17.9, 7.4, −1.1)
2.89, ddd (−17.9, 7.3, −1.1)
c
c
e
8a
8b
9
27.2, CH2
2.02, q (7.1)
e
135.2, CH
121.4, CH
17.0, CH2
10
9
8
133.1, CH
121.9, CH
17.7, CH2
5.52, dtt (10.5, 7.1, 1.5)
5.37, dtt (10.5, 6.9, 1.5)
3.03, d (6.9)
e e
10
11a
11b
12
13
14
15
16
17
18
80.5, C
64.7, C
70.4, C
74.5, C
65.3, CH
27.3, CH2
9.6, CH3
7
6
5
4
3
2
1
80.2, C
64.0, C
71.2, C
74.3, C
63.5, CH
c
c
c
c
c
c
e
5.28, t (6.7)
1.79, dq (7.4, 6.7)
1.00, t (7.4)
4.92, d quint (5.3, 1.1)
d
a
b
1
Chemical shift and coupling constant values were inferred from H homoselective decoupling experiments and spin simulation. |J| < 0.7 values
c
d
e
are not listed for the sake of easier comparison. No protons or no position. Signals are not useful for comparison sake. Pseudo, i.e., a pseudo
multiplet. For example, a doublet of doublets but with two similar J values would likely appear as a pseudotriplet.
8−C-9 and C-10, H-10−C-9 and C-11, and H-11−C-10
(Figure S7.4, Supporting Information). Two- or three-bond
couplings, noted in the gHMBC spectrum, of H-11 to C-12
(δC 80.5) and C-13 (δC 64.7) and of H-16 to C-14 (δC 70.4)
and C-15 (δC 74.5) evidenced that a conjugated diyne moiety
(C-12 to C-15, four sp-hybridized carbons) was bridging C-11
and C-16. Hence, compound 7 was concluded to be (Z)-17-
ethyloxacycloheptadeca-10-ene-13,15-diyn-2-one, a new natu-
ral compound likely biosynthetically related to (Z)-falcarinol,
for which the name homodihydrofalcarinolide is proposed.
The closest-related compound to 7 is an immunosuppressive
macrolide ivorenolide B, isolated from the African mahogany
Khaya ivorensis belonging to Meliaceae.29 Ivorenolide B
possesses two additional oxygens: Instead of the C-9−C-10
double bond, there is a cis-epoxide ring, and in the allylic-
propargylic position C-11, an alcoholic group is located. It
seems that (Z)-falcarinol and homodihydrofalcarinolide (7)
share the same biosynthetic background; it is believed that a
17,18-didehydro acid corresponding to the nonlactonized 7 is
the precursor that undergoes decarboxylation to falcarinol.30
Compound 7 might represent either a missing link in the
biosynthetic pathway from crepenynic acid to falcarinol
derivatives or a diverging pathway.
immediately conveyed that the configuration(s) of the double
bonds differed from those in both 1−2 and 4 (5Z,7E,9E,11E)
and 3 (5E,7E,9E,11E). The geometry of the C-5′−C-6′ and C-
11′−C-12′ double bonds in 8 were both assigned to be (Z)
from the corresponding cis-proton−proton (JH‑5′−H‑6′ = 11.2
and JH‑11′−H‑12′ = 11.3 Hz) and trans-proton−carbon (JH‑6′−C‑4′
= 12.0, JH‑5′−C‑7′ = 11.3, and JH‑12′−C‑10′ = 11.0 Hz) coupling
constant values, either directly mined from the spectra or
derived from an appropriate spectral simulation; the latter gave
all other coupling constant values (Figure S1.6, Supporting
Information). Similarly, the configurations of the two inner
double bonds were both determined to be (E), as in 1−4. The
comparison of 13C and 1H NMR spectra of 8 with those of the
corresponding methyl parinarate isomer16 (Tables S1 and S2,
Supporting Information) confirmed the (5Z,7E,11E,13Z)-
configuration. Thus, (5Z,7E,11E,13Z)-tetradeca-5,7,9,11-tet-
raen-1-yl 2-methylpropanoate (8, in analogy to 1−4,
veronaester) is a new natural compound from B. veronense,
related to the ones from B. praealtum, differing in the identity
of the esterifying acids and the configuration of the tetraenic
alcohol. Speaking in chemotaxonomical terms, their existence
provides support for the infrageneric placement of both taxa
within the same section of the genus.
Compound 8, isolated from B. veronense, displayed in its MS,
as observed for praealtaester A (1), the same dominant
fragmentation ions (m/z 91, 105, 117, etc.), indicative of a
high degree of unsaturation. The molecular ion (at m/z 276)
suggested that 8 might be a related compound to 1, an ester of
a tetradecatetraenic alcohol and a saturated four-carbon
carboxylic acid. As described above for 1−4, the analyses of
1H and 13C NMR spectra led to the conclusion that 8 is indeed
tetradeca-5,7,9,11-tetraen-1-yl 2-methylpropanoate (Figure 2).
Compound 9 (Figure 2), located in one of the most polar
chromatographic fractions of the B. veronense extract, was
subjected to silylation. A multiply trimethylsilylated derivative
of 9 was obtained, whose MS, after the loss of a C7H14O2Si
fragment, was highly similar to that of phloroglucinol-
triTMS,31 indicating the presence of three phenolic and one
alcohol OH groups. Its IR spectrum contained an absorption
band at 1630 cm−1, suggesting the presence of an intra-
molecularly hydrogen-bonded carbonyl function conjugated
with a highly electron-donating group, and a broad ∼3241
1
The appearance of olefin proton signals in the H NMR of 8
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1
Table 4. H (400 MHz) and 13C (100.6 MHz) NMR Data of Veroglucinol (9) in DMSO-d6 and Methanol-d4
DMSO-d6
δH, (J in Hz)
methanol-d4
δH, (J in Hz)
a
b
position
δC, type
δC, type
gHMBC
NOESY
1
203.6, C
c
205.2, C
c
c
c
2a
52.9, CH2
3.13, dd (−15.8, 7.1)
53.9, CH2
3.22, dd (−16.0, 5.3)
1, 3
4
2b
3.09, dd (−15.8, 5.6)
3.18, dd (−16.0, 7.5)
3
4
1′
63.4, CH
23.8, CH3
104.3, C
164.3, C
94.8, CH
164.7, C
c
4.20, dqd (7.1, 6.1, 5.6)
1.13, d (6.1)
c
c
5.83, s
c
12.22, s
10.32, s
65.7, CH
23.7, CH3
104.3, C
166.0, C
95.9, CH
166.4, C
c
4.35, dqd (7.5, 6.1, 5.3)
1.23, d (6.1)
c
c
5.81, s
c
c
2, 4
2, 3
c
2, 4
3
c
c
c
c
c
c
2′, 6′
3′, 5′
4′
OH-2′, 6′
OH-4′
c
1′, (5′, 3′)
c
2′, 6′
4′
c
c
c
a
b
Long-range coupling between the proton in the position column with the carbon in the HMBC column. NOE observed between the proton in
the position column with the proton in the NOESY column. No protons or no position.
c
cm−1 band corresponding to several hydroxyl functions; the
occurrence of a conjugated ketone was additionally confirmed
by the 13C NMR (DMSO-d6) signal noted at 203.6 ppm
(Table 4). The number, integrals, and singlet nature of the 1H
NMR signals at 12.22 ppm (OH-2′ and 6′) and 10.32 (OH-
4′), corresponding to exchangeable phenolic hydrogens, and
5.83 ppm (H-3′ and H-5′), corresponding to protons on an
electron-rich aromatic ring, indicate that the core in 9 is
symmetrically substituted. The symmetry in the aromatic ring
was also evident from the meta coupling (2.3 Hz) of the
chemically equivalent H-3′ and H-5′ visible in the splitting of
13C-satellites in its 1H NMR (Figures S9.1 and S9.3,
Supporting Information). Chemical shifts of diastereotopic
H-2a (3.13 ppm) and H-2b (3.09 ppm), along with their
gHMBC interactions with C-1 and C-3 (Figure S9.7,
Supporting Information), positioned them α to the carbonyl
and suggested the presence of a secondary alcohol chiral center
in an aldol-type moiety (C-3, 63.4 ppm). It was concluded that
9 is 3-hydroxy-1-(2,4,6-trihydroxyphenyl)butan-1-one, a rela-
tively simple acylphloroglucinol derivative and a new natural
product (Figure 2).
Michael addition, yielding a chromanone derivative (9b), is the
most likely course of these transformations. After we keep in
mind that only traces of 9a and 9b were isolated and that they
were practically coeluting with 9, more arguments lay in favor
of the artifactual nature of the two additional phloroglucinols.
Two more lignans were isolated from the extract of B.
veronense, isoyatein,36 and lariciresinol37 (Figure S6.5, Support-
ing Information). Isoyatein is a rare dibenzylbutyrolactone
derivative previously isolated from Piper cubeba36 and detected
in Callitris preissii.37 Lariciresinol is widely occurring in plants38
but has not been previously known to be a secondary
metabolite of Bupleurum spp.
Compounds 1, 4, 5, and (+)-arcitin, isolated in sufficient
quantity, along with cisplatin (0.1 mM), were assessed for their
effects in the concentration range 10−5−10−1 mM on
splenocyte (SPC) and macrophage (Mφ) viability and
functional characteristics. All compounds altered, in a statisti-
cally significant manner (increased and/or decreased),
mitochondrial and lysosomal functions of SPCs and Mφs,
based on their ability to metabolize MTT (Figure 3) and
accumulate NR (Figure S10, Supporting Information).
Compounds 1, 4, and 5 share a common structural motif, a
deca-1,3,5,7-tetraen-1-yl chain, which is also a characteristic of
parinaric acids (Figure S10, Supporting Information). The
latter acids were established to affect the viability of human
leukemia cells (THP-1 monocytic leukemia and HL-60 human
promyelocytic leukemia) at concentrations of 5 μM or less.39
In comparison to the cytotoxic effect of parinaric acids,
praealtalactone (5) significantly reduced the viability of both
SPCs and Mφs in vitro only at 100 μM. However, both 1 and
4 in all tested concentrations and the <10 μM concentrations
of 5 induced an opposite effect, potentiating the mitochondrial
reductase ability, as well as their lysosomal function. This
selective transformed-to-normal cell cytotoxicity was previ-
ously also demonstrated for α-parinaric acid in the case of
nontumorous astrocytes.40 Parinaric acids are believed to
increase free radical production and, thus, sensitize trans-
formed cells to lipid peroxidation, by means of the enhanced
take-up of the acids by the rapidly growing malignant cells.39,41
The treatment of SPCs with (+)-arcitin (100 μM) decreased
their NR-accumulating ability (Figure S10, Supporting
Information); however, this effect was not found to be
significant. Interestingly, when Mφs were exposed to the two
highest concentrations of arcitin (100 and 10 μM, Figure S10,
Supporting Information), the opposite effect to the one on
The determination of the configuration of the chiral center
at C-3 required preparation of diastereomeric Mosher esters
1
using the commercially available enantiopure acids. The H
NMR data of the prepared esters permitted us to assign the
configuration (S) to the center (Figure 2), which was
1
additionally confirmed by comparing the relevant H NMR
chemical shifts with literature values for related compounds
obtained in an enantiospecific Baker’s yeast reduction step.32
The name veroglucinol is proposed for 9, (S)-3-hydroxy-1-
(2,4,6-trihydroxyphenyl)butan-1-one, due to its very restricted
occurrence in nature.
Rechromatography of the fraction preceding the one
containing 9 yielded two additional compounds, a dehydrated
and a cyclized (9a and 9b, respectively, Figure S9.6,
Supporting Information) derivative of 9. Compound 9a is
mentioned in the literature only as a synthetic intermediate33
and has never been isolated from a natural source to date. The
cyclized derivative 9b was previously isolated from a plant
species Aphanamixis grandifolia34 and a fungus Xylaria
nigripes.35 It appears that the three compounds, 9, 9a, and
9b, might be either biosynthetically or artifactually inter-
connected; because 9 exists as a single enantiomer, it seems
that a (facile) dehydration of the β-hydroxyketone, giving a
conjugated enone (9a), and a subsequent intramolecular
G
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Figure 3. Effects of 1, 4, 5, and (+)-arcitin on the abilities of splenocytes (A) and macrophages (B) to metabolize MTT. Data shown are the mean
percentages (%) and SDs calculated based on the values obtained from RPMI-treated cells. Mutual comparison of the data was performed using
#
one-way ANOVA, followed by Tukey’s posthoc test: *p < 0.001, **p < 0.01, and ***p < 0.05 vs RPMI-treated cells; p < 0.001 vs cisplatin
treatment.
SPCs was found, i.e., a statistically significant increase in Mφ
ability to accumulate NR (Figure S10, Supporting Informa-
tion). Arcitin was previously found to exhibit marked
cytotoxicity toward Jurkat T cells (immortalized human T
lymphocytes)25 and to lower the amount of IgE in cultures of
U266 cells in a concentration-dependent manner (immortal-
ized human B lymphocytes).42 The present results with
primary cell cultures of rat spleen lymphocytes (and macro-
phages) showed that (+)-arcitin does not reduce their viability
but stimulates their ability to both metabolize MTT and
accumulate NR, suggesting a functional potentiation of these
immune cells. Thus, it appears that this lignan exerts a selective
cytotoxic effect toward transformed lymphocytes, while it does
not affect the viability and/or function of normal cells.
Mφ myeloperoxidase (Figure 4). This activity can be related
either to their ability to inhibit MPO or to their potential to
react with free radicals43 generated by MPO, suggesting their
direct involvement in the oxidative cell processes caused by
reactive oxygen species similar to the α-parinaric acid.39 This
antioxidant nature of 1, 4, and 5, in cell culture, suggests a
possible protective role in plant tissues. The more pronounced
cytotoxic nature of 5 could be explained by its potentially
different metabolism in cells, leading potentially to the
formation of Michael acceptor-type cytotoxic agents. It is
known that aliphatic γ-lactones undergo hydrolysis catalyzed
by paroxonidases, followed by steps of α- or β-oxidative
transformation of fatty acids.44 In the case of 5, after hydrolysis
and initial oxidation of the newly formed alcohol group, the
resulting α,β-unsaturated conjugated ketone appears to be the
likely Michael acceptor, i.e., a possible alkylating agent. One
Praealtalactone (5), predominantly, and praealtaester A (1),
to a lesser extent, showed potential in altering the activity of
H
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Figure 4. Myeloperoxidase (MPO) activities in macrophages (Mφs) treated with 1, 4, 5, and (+)-arcitin. Data shown are the mean percentages
(%) and SD calculated based on the values obtained from RPMI-treated cells. Mutual comparison of the data was performed using one-way
#
ANOVA, followed by Tukey’s posthoc test: *p < 0.001, **p < 0.01, and ***p < 0.05 vs RPMI-treated cells; p < 0.001 vs cisplatin treatment.
ane, 30 m × 0.25 mm, film thickness 0.25 μm, Agilent Technologies,
Santa Clara, California, USA) and coupled with a 5975B mass
selective detector of the same company. Retention indices (RIs) were
determined using a mixture of n-alkanes from C8−C40. Analytical
TLC separations were done on coated Al silica gel plates (Kieselgel 60
F254, 0.2 mm, Merck, Darmstadt, Germany). TLC plates were initially
visualized by UV light (254 nm) and then sprayed with 50% (w/w)
aqueous H2SO4 followed by heating. High-resolution mass spectrom-
etry (HRMS) analysis was performed using an MStation JMS-700
mass spectrometer (JEOL, Peabody, MA, USA) with an ionization
energy of 70 eV, an ionization trap current of 300 μA, and a source
temperature of 230 °C.
can conclude that the enthopharmacologically praised
Bupleurum taxa warrant their status of immunomodulatory-
acting herbal medicines, more specifically, such herbal
medicines that boost the functional characteristics of normal
SPCs and Mφs, although specific metabolites might have a
different effect on their own.
EXPERIMENTAL SECTION
■
General Experimental Procedures. All used chemicals and
solvents were obtained from commercial sources (Sigma-Aldrich, St.
Louis, MO, USA; Merck, Darmstadt, Germany; Fisher Scientific,
Waltham, MA, USA) and used as received, except for the solvents,
which were predistilled and dried before use. Silica gel used for
preparative separations (both dry-flash and column) was silica gel 60
(0.04−0.063 mm, Merck, Darmstadt, Germany), while gel filtrations
were done on Sephadex LH-20 (GE Healthcare Bio-Sciences AB,
Uppsala, Sweden). Optical rotations were measured in MeOH at 25
°C using an Autopol IV (Rudolph Research Analytical, Hackettstown,
New Jersey, USA) polarimeter equipped with a sodium lamp (589
nm) and a 10 cm microcell. UV measurements were carried out on a
Shimadzu UV-1800 UV−vis spectrophotometer (Kyoto, Japan). IR
spectra (ATR-attenuated total reflectance) were recorded on a
Thermo Nicolet model 6700 FT-IR instrument (Waltham, MA,
1H NMR full spin analysis of compounds 1−4 and 7−9 was
performed by manually adjusting δH and J values to fit the
experimentally available values and further optimized using
MestReNova 11.0.3 software (tools/spin simulation). This procedure
led to a systematic refinement of all calculated NMR parameters until
the simulation outcome was in excellent agreement (NRMSD <
0.05%) with the experimental data of the isolated compounds.
Plant Material. The above-ground plant parts of Bupleurum
praealtum in the intermediate flowering-fruit-bearing phase were
̌
collected in August 2015 on the slopes of Suva Planina Mt. (near Nis,
southeastern Serbia, 43°11′53.1″N 22°08′33.6″E), while the aerial
parts of B. veronense, fully flowering, were collected in June 2018 on
1
USA). H and 13C NMR spectra were recorded on a Bruker Avance
̌
Peljesac Peninsula (near Mali Ston, Dalmatia, Croatia, 42°50′39.7″N
1
17°42′11.8″E), both from single populations. Voucher specimens
̈
III 400 MHz NMR spectrometer (Fallanden, Switzerland; H at 400
MHz, 13C at 100.6 MHz), equipped with a 5 mm dual 13C/1H probe
head at 20 °C. All the NMR spectra were recorded in chloroform-d,
methanol-d4, or DMSO-d6 (Sigma-Aldrich, St. Louis, MO, USA) with
tetramethylsilane as the internal standard. Chemical shifts (δ) are
reported in ppm and referenced to tetramethylsilane (δH = 0.00 ppm),
or the (residual) solvent signal (CHCl3, CHD2OD, CHD2SOCD3),
and 13CDCl3, 13CD3OD, and 13CD3SOCD3, in 1H NMR and 13C
NMR and heteronuclear 2D spectra, respectively. Scalar couplings are
reported in Hertz (Hz). The acquired NMR experiments, both 1D
and 2D, were recorded using standard Bruker built-in pulse
sequences. GC−MS analyses (three repetitions) were carried out
using a Hewlett−Packard 6890N gas chromatograph equipped with a
fused silica capillary column DB-5MS ((5%-phenyl)-methylpolysilox-
have been deposited in the Herbarium of the Faculty of Sciences and
̌
Mathematics, University of Nis (voucher nos. HMN 12112 and HMN
13669 for B. praealtum and B. veronense, respectively). The plant
material was identified by one of the authors (V.R.).
Extraction and Isolation. Before air-drying to constant mass, the
umbels of B. praealtum were carefully detached from the rest of the
aerial parts; the dry umbels were lightly shaken to free the ripe
schizocarps which were additionally sieved. The aerial parts of B.
praealtum were cut into small pieces, whereas the B. praealtum
schizocarps were mechanically ground and extracted separately by
maceration with Et2O for 10 days with occasional shaking at room
temperature protected from light. The dried plant material of B.
veronense was treated in an analogous manner, except the entire aerial
I
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parts were extracted together. The extracts were dried with anhydrous
MgSO4 and gravity filtered to remove all insoluble material before the
removal of Et2O at room temperature in vacuo. The yields of the B.
praealtum aerial parts, B. praealtum schizocarp, and B. veronense Et2O
extracts were 2.3, 1.9, and 2.5% (w/w, based on dry plant mass),
respectively.
(37). HREIMS m/z 306.4456 [M]+ (calcd for C19H30O3, 306.4460).
RI (DB-5MS) 2403.
(5Z,7E,9E,11E)-Tetradeca-5,7,9,11-tetraen-1-yl (R)-2-Acetoxy-3-
methylbutanoate (Praealtaester D, 4). This compound is a
yellowish oil. [α]25 + 80.0 (c 1, MeOH). UV (CH3CN) λmax (log
D
ε) 288 (4.81), 301 (4.97), 316 (4.92), 342 (3.84) nm. IR (neat) νmax
∼ 3500, 2961, 2916, 2848, 1727, 1462, 1138, 996 cm−1. For 1H NMR
(CDCl3, 400 MHz) and 13C NMR (CDCl3, 100.6 MHz) data, see
Tables 2 and 1, respectively. EIMS m/z: [M]+ 348 (21), 131 (17),
117 (26), 115 (52), 105 (19), 91 (37), 79 (19), 55 (14), 43 (100), 41
(22). HREIMS m/z: [M]+ 348.4829 (calcd for C21H32O4, 348.4830).
RI (DB-5MS) 2485.
All of the extracts were subjected to silica gel dry-flash
chromatography under gradient conditions using mixtures of
increasing polarity starting with hexane−Et2O, through Et2O−
EtOAc, and ending in EtOAc−methanol mixtures. Fraction 19
(hexane−Et2O = 2:1, v/v, 97 mg) of the B. praealtum schizocarp
extract represented pure compound 1, while fraction 18 (hexane−
Et2O = 2:1, v/v) was a mixture mostly composed of 1, 3, and 4;
fraction 25 eluted with the mixture of Et2O−EtOAc (1:1, v/v, 20 mg)
was pure compound 6. Because fractions 18 and 19 of the B.
praealtum aerial parts’ extract (hexane−Et2O = 2:1, v/v) represented a
similar mixture of 1, 3, and 4, these fractions and fraction 18 from the
B. praealtum schizocarp extract were pooled and subjected to further
separation, first on Sephadex LH-20 (isocratic, CHCl3−MeOH = 1:1,
v/v) and then by silica gel-column chromatography (hexane−Et2O =
2:1, v/v). These resulted in additional quantities of pure 1 and a
mixture of 1 and 3 considerably more enriched in 3, while 4 remained
in a mixture. Compound 5 (23 mg) was isolated from the B.
praealtum aerial parts’ extract in the first silica gel dry-flash
chromatography, located in fraction 25 which was eluted with
hexane-Et2O (1:3, v/v). Fraction 30 (EtOAc−MeOH, 7:3, v/v), 12
mg, represented pure (+)-arcitin (synonym dimethylmatairesinol).25
Additionally, the initial chromatography of the B. praealtum aerial
parts’ extract resulted in fractions 11 and 12, 5, and 10 mg,
respectively, both eluted with 15% (v/v) Et2O in hexane, that
contained, based on GC−MS analyses, compound 2, unfortunately in
a complex mixture. Its identity was hinted from its mass spectrum and
corroborated by synthesis, as detailed below.
(S)-5-((1E,3E,5E,7E)-Deca-1,3,5,7-tetraen-1-yl)dihydrofuran-
2(3H)-one (Praealtalactone, 5). This compound is a colorless oil.
[α]25 + 70.1 (c 1, MeOH). UV (CH3CN) λmax (log ε) 288 (2.64),
D
301 (2.80), 316 (2.74), 342 (2.07), 410 (2.64) nm. IR (neat) νmax
2923, 2853, 2360, 2342, 1771, 1169, 1053, 1007 cm−1. For 1H NMR
(CDCl3, 400 MHz) and 13C NMR (CDCl3, 100.6 MHz) data, see
Tables 2 and 1. EIMS m/z: [M]+ 218 (30), 117 (34), 105 (52), 91
(100), 79 (65), 77 (56), 66 (38), 65 (34), 55 (37), 41 (58), 39 (56).
HREIMS m/z: [M]+ 218.2957 (calcd for C14H18O2, 218.2960). RI
(DB-5MS) 2107.
7-Oxoarcitin ((3S,4R)-3-(3,4-Dimethoxybenzoyl)-4-(3,4-
dimethoxybenzyl)dihydrofuran-2(3H)-one O-Methylconicaol B, 6).
This compound is an amorphous solid. UV (MeOH) λmax (log ε) 231
(4.08), 279 (3.90), 313 (3.69), 398 (3.41), 419 (3.42), 442 (3.42),
1
470 (3.32) nm. IR (neat) νmax 2924, 2853, 1732, 1021 cm−1. H
NMR (CDCl3, 400 MHz): δ 7.46 (1H, d, J = 2.1 Hz, H-2), 7.34 (1H,
dd, J = 8.5, 2.1 Hz, H-6), 6.84 (1H, d, J = 8.5, H-5), 6.76 (1H, d, J =
8.1, H-5′), 6.69 (1H, dd, J = 8.1, 2.1 Hz, H-6′), 6.63 (1H, d, J = 2.1
Hz, H-2′), 4.54 (1H, dd, J = 9.0, 7.0, H-9′a), 4.27 (1H, d, J = 6.0, H-
8), 4.15 (1H, dd, J = 9.0, 5.7, H-9′b), 3.95 (1H, s, OMe-4), 3.91 (1H,
s, OMe-3), 3.85 (1H, s, OMe-4′), 3.80 (1H, s, OMe-3′), 3.41 (1H, m,
H-8′), 2.84 (1H, dd, J = 13.7, 7.7, H-7′a), 2.78 (1H, dd, J = 13.7, 8.0,
H-7′b). 13C NMR (CDCl3, 100.6 MHz): δ 191.5 (C, C-7), 173.1 (C,
C-9), 154.3 (C, C-4), 149.3 (C, C-3′), 149.3 (C, C-3), 148.2 (C, C-
4′), 130.0 (C, C-1′), 128.8 (C, C-1), 124.7 (C, C-6), 121.2 (C, C-6′),
112.0 (C, C-2′), 111.4 (C, C-5′), 110.9 (C, C-2), 110.1 (C, C-5),
72.1 (CH2, C-9′), 56.3 (CH3, OMe-4), 56.1 (CH3, OMe-3), 56.0
(CH3, OMe-4′), 55.9 (CH3, OMe-3′), 53.7 (CH, C-8), 41.4 (CH, C-
8′), 38.0 (CH2, C-7′). EIMS m/z: [M]+ 400 (17), 178 (12), 177
(100), 165 (41), 151 (19), 146 (7), 137 (7), 107 (9), 79 (7), 77 (8).
HREIMS m/z: [M]+ 400.4272 (calcd for C22H24O7, 400.4270). RI
(DB-5MS) 3310.
(5Z,7E,9E,11E)-Tetradeca-5,7,9,11-tetraen-1-yl (R)-2-Hydroxy-3-
methylbutanoate (Praealtaester A, 1). This compound is a yellowish
oil. [α]25 + 64.3 (c 1, MeOH). UV (CH3CN) λmax (log ε) 289
D
(4.28), 302 (4.54), 316 (4.51), 341 (2.95) nm. IR (neat) νmax ∼ 3500,
1
2961, 2916, 2848, 1727, 1462, 1138, 996 cm−1. H NMR (CDCl3,
400 MHz) and 13C NMR (CDCl3, 100.6 MHz) data, see Tables 2
and 1. EIMS m/z: [M]+ 306 (99), 159 (42), 131 (58), 119 (36), 117
(70), 105 (49), 91 (100), 79 (51), 73 (34), 55 (39). HREIMS m/z:
[M]+ 306.4459 (calcd for C19H30O3, 306.4460). RI (DB-5MS) 2365.
(5Z,7E,9E,11E)-Tetradeca-5,7,9,11-tetraen-1-yl 3-Methylbuta-
1
noate (Praealtaester B, 2). This compound is a colorless oil. H
NMR (CDCl3, 400 MHz): δ 6.44 (1H, dddd, J = 14.5, 11.3, 1.0, 0.7
Hz, H-7′), 6.224 (1H, ddt, J = 14.5, 11.2, 0.7 Hz, H-8′), 6.217 (1H,
ddd, J = 15.5, 10.8, 0.7 Hz, H-10′), 6.18 (1H, ddt, J = 15.5, 11.2, 0.7
Hz, H-9′), 6.09 (1H, ddtd, J = 15.0, 10.8, 1.4, 0.7 Hz, H-11′), 6.05
(1H, ddtd, J = 11.3, 10.3, 1.4, 0.7 Hz, H-6′), 5.76 (1H, dt, J = 15.0, 6.8
Hz, H-12′), 5.38 (1H, dtd, J = 10.3, 7.4, 1.0 Hz, H-5′), 4.07 (2H, t, J
= 6.6 Hz, H-1′), 2.33 (2H, d, J = 6.7 Hz, H-2), 2.24 (2H, tdd, J = 7.6,
7.4, 1.4 Hz, H-4′), 2.15 (2H, qdd, J = 7.5, 6.8, 1.4 Hz, H-13′), 2.08
(1H, t sept, J = 6.7, 6.6 Hz, H-3), 1.64 (2H, tt, J = 7.6, 6.6 Hz, H-2′),
1.46 (2H, pseudo quint, J = 7.6, 7.6 Hz, H-3′), 1.01 (3H, t, J = 7.5 Hz,
H-14′), 0.95 (6H, d, J = 6.6 Hz, H-4 and H-5). 13C NMR (CDCl3,
100.6 MHz): δ 173.5 (C, C-1), 137.4 (CH, C-12′), 133.5 (CH, C-
10′), 133.3 (CH, C-8′), 131.6 (CH, C-5′), 130.9 (CH, C-9′), 129.6
(CH, C-11′), 129.4 (CH, C-6′), 127.4 (CH, C-7′), 64.2 (CH2, C-1′),
43.6 (CH2, C-2), 28.4 (CH2, C-2′), 27.6 (CH2, C-4′), 26.1 (CH, C-
3), 26.0 (CH2, C-3′), 25.8 (CH2, C-13′), 22.6 (CH3, C-4 and C-5),
13.7 (CH3, C-14′). EIMS m/z: [M]+ 290 (58), 131 (53), 117 (63),
105 (45), 91 (100), 85 (55), 79 (50), 67 (34), 57 (59), 41 (52).
HREIMS m/z: [M]+ 290.2250 (calcd for C19H30O2, 290.2246). RI
(DB-5MS) 2227.
Fractions 11 and 12 of the B. veronense extract (hexane−Et2O =
6:1, v/v, 99 mg in total) were subsequently isocratically (hexane−
Et2O = 7:1, v/v) chromatographed on a longer silica gel column
giving pure 7 (19 mg) and 8 (5 mg). Fraction 25 (EtOAc−MeOH =
9:1, v/v) of the original dry-flash chromatography was pure 9 (58
mg), while fraction 24, after being rechromatographed on Sephadex
LH-20 (isocratic, CHCl3−MeOH = 1:1, v/v), yielded pure minute
samples (less than a milligram) of 9a33 and 9b,33 as well as the lignans
isoyatein (11 mg),36 and lariciresinol (3 mg).37
(Z)-17-Ethyloxacycloheptadeca-10-ene-13,15-diyn-2-one (Ho-
modihydrofalcarinolide, 7). This compound is a transparent oil.
UV (CH3CN) λmax (log ε) 216 (3.11), 252 (2.86), 266 (2.83), 281
1
(2.74) nm. IR (neat) νmax 2930, 2858, 1728 cm−1. For H NMR
(CDCl3, 400 MHz) and 13C NMR (CDCl3, 100.6 MHz) data, see
Table 3. EIMS m/z: [M]+ 272 (100), 129 (62), 128 (78), 117 (38),
115 (67), 105 (38), 91 (79), 77 (57), 55 (53), 41 (54). HREIMS m/
z: [M]+ 272.3877 (calcd for C18H24O2, 272.3880). RI (DB-5MS)
2185.
(5Z,7E,9E,11Z)-Tetradeca-5,7,9,11-tetraen-1-yl Isobutyrate (Ver-
onaester, 8). This compound is a transparent oil. For 1H NMR
(CDCl3, 400 MHz) and 13C NMR (CDCl3, 100.6 MHz) data, see
Tables 2 and 1. EIMS m/z: [M]+ 276 (59), 131 (58), 145 (35), 117
(69), 105 (46), 91 (100), 79 (48), 71 (45), 43 (66), 41 (38).
HREIMS m/z: [M]+ 276.4198 (calcd for C18H28O2, 276.4200). RI
(DB-5MS) 2127.
(5E,7E,9E,11E)-Tetradeca-5,7,9,11-tetraen-1-yl (R)-2-Hydroxy-3-
methylbutanoate (Praealtaester C, 3). This compound is a colorless
1
oil. For H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100.6
MHz) data, see Tables 2 and 1. EIMS m/z [M]+ 306 (52), 131 (47),
117 (62), 105 (42), 91 (100), 79 (51), 73 (40), 67 (34), 55 (43), 41
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3-Hydroxy-1-(2,4,6-trihydroxyphenyl)butan-1-one (Veroglucinol,
9). This compound is a colorless oil. UV (MeOH) λmax (log ε) 225
(4.25), 288 (4.35), 331 (3.59) shoulder nm. IR (neat) νmax 3311,
(R)-MTPA Ester of (R)-1a. This compound is a yellowish oil. For 1H
NMR (CDCl3, 400 MHz) data, see Table S3 of the Supporting
Information. EIMS m/z: [M]+ 348 (1), 190 (44), 189 (97), 170 (47),
126 (31), 119 (17), 105 (36), 83 (100), 77 (23), 55 (49), 41 (17). RI
(DB-5MS) 1701.
1
2943, 2832, 1021 cm−1. For H NMR (DMSO-d6 and methanol-d4,
400 MHz) and 13C NMR (DMSO-d6 and methanol-d4, 100.6 MHz)
data, see Table 4.
(R)-MTPA Ester of (S)-1a. This compound is a yellowish oil. For 1H
NMR (CDCl3, 400 MHz) data, see Table S3 of the Supporting
Information. EIMS m/z: [M]+ 348 (1), 190 (44), 189 (97), 170 (47),
126 (31), 119 (17), 105 (36), 83 (100), 77 (23), 55 (49), 41 (17). RI
(DB-5MS) 1718.
Conversion of 1 to 4. To verify the structure of 4, the compound
was obtained by conversion of 1. A solution of 1 (16 mg, 0.05 mmol),
acetic acid (9 mg, 0.15 mmol), 4-(dimethylamino)pyridine (DMAP, 4
mg, 0.03 mmol), and N,N′-dicyclohexylcarbodiimide (DCC, 31 mg,
0.15 mmol) in 1 mL of dry CH2Cl2 was stirred in a vial overnight at
room temperature, under argon. Afterward, the solvent was removed
in vacuo; then, 3 mL of cold pentane was added to the residue, and
the precipitated N,N′-dicyclohexylurea was filtered off. The filtrate
was concentrated in vacuo, and the resulting residue was purified by
silica gel column chromatography giving 7 mg (40% yield) of 4.
Methanolysis of 1 and Synthesis of 2. A solution of 1 (18 mg)
in methanol (1 mL) was added at room temperature to an excess of
freshly prepared solution of sodium methoxide (ca. 1 g of Na in an
excess of MeOH), protected from sunlight. The mixture was stirred
and heated until reflux and then immediately cooled in ice water.
Methanol was removed under reduced pressure at 30 °C, while the
residue was partitioned between Et2O and a saturated solution of
sodium bicarbonate. The ether layer was washed several times with
brine, and after the evaporation of Et2O, (5Z,7E,9E,11E)-tetradeca-
5,7,9,11-tetraen-1-ol was obtained and was used as is in the following
step. Compound 2 was prepared starting from (5Z,7E,9E,11E)-
tetradeca-5,7,9,11-tetraen-1-ol (3 mg, 0.01 mmol), isovaleric acid (3
mg, 0.03 mmol), DMAP (1 mg, 0.01 mmol), and DCC (6.2 mg, 0.03
mmol). The crude ester 2 was purified by silica gel column
chromatography, and this gave 3 mg (71% yield) of pure 2.
Hydrogenation of 6. A sample of 6 (10 mg) dissolved in EtOAc
was mixed with 5 mg of Pd (10%) on charcoal, with the vigorously
stirred reaction mixture flushed two times with hydrogen gas and kept
under 1 atm of hydrogen for 2 h at room temperature. Afterward, the
EtOAc solution was filtered through a small column of Celite, and the
catalyst and the column were washed with EtOAc. EtOAc was
removed from the pooled solutions in vacuo, and the residue was
subjected to GC−MS to confirm that the hydrogenation was
complete and that the γ-tetradecalactone obtained was pure (10
mg, 96%).
Preparation of (R)-MTPA Ester of 1. Under argon, a solution of
1 (23 mg, 0.07 mmol), Et3N (28 μL, 0.35 mmol), and DMAP (a small
crystal, ca. 1 mg) in CHCl3 (2 mL) was added to the previously
prepared crude chloride (R)-MTPA (35 mg, ca. 0.15 mmol). After 72
h of stirring, 10 mL of CHCl3 were added, and the resulting solution
was washed with brine (3 × 10 mL). The organic layer was dried with
anhydrous MgSO4 and concentrated in vacuo. After column
chromatography on silica gel (10% of Et2O in hexane, v/v), pure
(R)-MTPA ester of 1 was isolated in 31% yield (12 mg).
1
(R)-MTPA Ester of 1. This compound is a yellowish oil. For H
NMR (CDCl3, 400 MHz) data, see Table S3 of the Supporting
Information. EIMS m/z: [M]+ 522 (4), 189 (96), 186 (20), 159 (18),
145 (49), 133 (25), 131 (34), 119 (16), 117 (30), 105 (29), 91
(100). RI (DB-5MS) 2968.
Preparation of (R)- and (S)-MTPA Esters of 9. The previously
prepared crude (R)- or (S)-MTPA chloride (ca. 15 mg, 0.06 mmol)
was treated with 9 (7 mg, 0.03 mmol) in a similar manner to that
described for 1. Pure MTPA esters (14 mg (40%) and 12 mg (35%)
for (R) and (S), respectively) were obtained by silica gel column
chromatography (25% of Et2O in hexane, v/v).
(R)-MTPA Ester of 9. 1H NMR (CDCl3, 400 MHz): δ 5.93 (s, H-3′
and H-5′), 5.26 (1H, ddd, J = 7.0, 6.3, 5.5 Hz, H-3), 2.94 (1H, dd, J =
−18.6, 5.5 Hz, H-2a), 2.74 (1H, dd, J = −18.6, 7.0 Hz, H-2b), 1.09
(3H, d, J = 6.3 Hz, H-4). 13C NMR (CDCl3, 100.6 MHz): δ 195.5 (C,
C-1), 164.3 (C, C-4′), 163.6 (C, C-2′ and C-6′), 104.6 (C, C-1′),
95.7 (CH, C-3′ and C-5′), 67.0 (CH, C-3), 49.0 (CH2, C-2), 16.9
(CH3, C-4).
(S)-MTPA Ester of 9. 1H NMR (CDCl3, 400 MHz): δ 6.02 (2H, s,
H-3′ and H-5′), 4.79 (1H, td, J = 7.8, 6.3 Hz, H-3), 2.79 (2H, d, J =
7.8 Hz, H-2), 1.61 (3H, d, J = 6.3 Hz, H-4). 13C NMR (CDCl3, 100.6
MHz): δ 195.9 (C, C-1), 163.5 (C, C-4′), 163.4 (C, C-2′ and C-6′),
105.2 (C, C-1′), 97.1 (CH, C-3′ and C-5′), 76.4 (CH, C-3), 42.6
(CH2, C-2), 25.3 (CH3, C-4).
(R)-γ-Tetradecalactone. [α]20 + 30.1 (c 1, CH3Cl), literature
D
value [α]20 + 32 (c 1.1, CHCl3).23
D
Synthesis of (+)- and (−)-Mosher’s Acid Chlorides. Oxalyl
chloride (131 μL, 1.5 mmol) was added to a solution of (R)-(+)- or
(S)-(−)-α-methoxy-α-trifluoromethylphenylacetic acid (MTPA, 70
mg, 0.3 mmol) and DMF (25 μL, 0.3 mmol) in hexane (2 mL) at
room temperature, under argon. After 1 h, the mixture was
concentrated under a vacuum, and the resulting residue was used as
such in the esterification step.
Preparation of (R)- and (S)-1a. Starting from 1.93 g of DL-valine
and 1.00 g of L-valine (R/S- and S-, respectively), the corresponding
(R/S)- and (S)-2-hydroxy-3-methylbutanoic acids were separately
prepared, as described previously.45 The crude acids were converted
into methyl esters by the reaction with diazomethane and subjected to
gradient column silica gel chromatography (0−100% Et2O in hexane).
In this way, pure methyl (S)-2-hydroxy-3-methylbutanoate, (S)-1a,
was obtained in 20% yield (216 mg), while the racemic mixture of (R/
S)-2-hydroxy-3-methylbutanoate, (R)- and (S)-1a, was isolated in
16% yield (182 mg). The purity of the esters was checked by TLC
and GC−MS.
Preparation of (R)-MTPA Esters of (R)- and (S)-1a. (R)-MTPA
and the prepared (R)- and (S)-1a were subjected to Steglich-type
esterification, as described for 4. The DCC-driven esterification was
effectuated in 79% yield (28 mg) in the case of (R)-MTPA ester of
(S)-1a, while the mixture of esters of (R)-MTPA and (R)- and (S)-1a
was further separated by gradient silica gel chromatography (0−40%,
v/v, Et2O in hexane), to afford a pure sample of (R)-MTPA ester of
(R)-1a (30 mg, 85%).
Silylation Procedure of 9. Compound 9 (20 mg, 0.084 mmol) was
dissolved in 10 mL of dry Et2O. After the addition of a large excess of
chlorotrimethylsilane (200 μL, 1.6 mmol), followed by 120 μL of dry
triethylamine and one drop of DMSO, the reaction mixture was
stirred for 2 h at room temperature under N2. Then, water (10 mL)
was added, and the resulting mixture was extracted thrice with Et2O.
The organic layers were combined, dried with anhydrous MgSO4, and
concentrated in vacuo to yield a crude mixture containing a
tetrasilylated derivative of 9 (84%) which was subjected directly to
GC−MS.
Silylated 9. EIMS m/z: [C22H44O5Si4]+ 500 (2), [M-Me]+ 485
(14), 395 (32), 370 (27), [M-C6H15OSi]+ 369 (59), [M-2Me3Si−H]+
353 (17), 344 (20), 343 (48), [M-C7H14O2Si]+ 342 (100),
+
[C5H13OSi] 117 (42), 75 (25), [Me3Si]+ 73 (100). RI (DB-5MS)
2111.
In Vitro Cell Assays. Determination of Splenocyte and
Macrophage Viability. See refs 46 and 47 and the Supporting
Information.
Cell Lysosomal Function. See ref 47 and the Supporting
Information.
Macrophage Myeloperoxidase Activity Determination. See ref 48
and the Supporting Information.
Statistical Analysis. See the Supporting Information.
K
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00437.
■
sı
(11) Napolitano, J. G.; Lankin, D. L.; Graf, T. N.; Friesen, B. J.;
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1H and 13C NMR spectra of compounds 1−9 and
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of compounds 1−8 and silylated 9; observed HMBC
and NOESY interaction of compounds 1−9; NMR data
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AUTHOR INFORMATION
Corresponding Author
■
́
Niko Radulovic − Department of Chemistry, Faculty of Sciences
̌
̌
and Mathematics, University of Nis, 18000 Nis, Serbia;
orcid.org/0000-0003-1342-7567; Phone: +381 62
8049210; Email: nikoradulovic@yahoo.com; Fax: +381 18
533014
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Blagojevic, P. J. Nat. Prod. 2019, 82, 1874−1885.
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Authors
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Sciences and Mathematics, University of Nis, 18000 Nis, Serbia
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Milan Nesic − Department of Chemistry, Faculty of Sciences and
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Mathematics, University of Nis, 18000 Nis, Serbia
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Nikola Stojanovic − Department of Physiology, Faculty of
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Medicine, University of Nis, 18000 Nis, Serbia
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Pavle Ranđelovic − Department of Physiology, Faculty of
Medicine, University of Nis, 18000 Nis, Serbia
Vladimir Ranđelovic − Department of Biology and Ecology,
Faculty of Sciences and Mathematics, University of Nis, 18000
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̌
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Nis, Serbia
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M.; Tang, S.; Wainwright, T.; Whitehead, K.; Barker, D. Molecules
2018, 23, 3057−3083.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00437
(26) (a) Fan, C. Q.; Zhu, X. Z.; Zhan, Z. J.; Ji, X. Q.; Li, H.; Yue, J.
M. Planta Med. 2006, 72, 590−595. (b) He, H.; Dai, L. X.; You, S. L.
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Notes
The authors declare no competing financial interest.
This work is a part of the Ph.D. dissertation of M.S.
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ACKNOWLEDGMENTS
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(28) Radulovic, N.; Đorđevic, M.; Dekic, M.; Blagojevic, P. Chem.
Biodiversity 2016, 13, 403−415.
This work was supported by the Ministry of Education,
Science and Technological Development of Serbia [project no.
172061; grant no. 451-03-68/2020-14/200124].
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https://dx.doi.org/10.1021/acs.jnatprod.0c00437
J. Nat. Prod. XXXX, XXX, XXX−XXX