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Article
Oxygenated Cyclohexene Derivatives and Other Constituents from
the Roots of Monanthotaxis trichocarpa
Gasper Maeda, Jelle van der Wal, Arvind Kumar Gupta, Joan J. E. Munissi, Andreas Orthaber,
Per Sunnerhagen, Stephen S. Nyandoro,* and Mat́ e ́ Erdeĺ yi*
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sı Supporting Information
*
ABSTRACT: Three new oxygenated cyclohexene derivatives,
trichocarpeols A (1), B (2), and C (3), along with nine known
secondary metabolites, were isolated from the methanolic root extract
of Monanthotaxis trichocarpa. They were identified by NMR
spectroscopic and mass spectrometric analyses, and the structure of
trichocarpeol A (1) was confirmed by single-crystal X-ray diffraction.
Out of the 12 isolated natural products, uvaretin (4) showed activity
against the Gram-positive bacterium Bacillus subtilis with a MIC value
of 18 μM. None of the isolated metabolites was active against the
Gram-negative Escherichia coli at a ∼5 mM (2000 μg/mL)
concentration. Whereas 4 showed cytotoxicity at EC50 10.2 μM
against the MCF-7 human breast cancer cell line, the other
compounds were inactive or not tested.
Monanthotaxis Baill. is a genus in the Uvariae tribe of the
family Annonaceae. It consists of 67 species that are confined
to tropical Africa and Madagascar, where new species of this
taxon continue to be discovered.1 Plant species of Monanthotaxis are scandent shrubs or lianas. Monanthotaxis
trichocarpa (Engl. & Diels) Verdc. is native to Kenya, Tanzania
(including Zanzibar), and Mozambique, with its distribution
being restricted to some patches of the coastal forests.2 In
Tanzania, it is locally known as “msofu dume”, with this name
also having been given to several other Annonaceae species. Its
leaves and aerial parts are used by the Digo and Giriama
communities of Kenya and Tanzania as a remedy for
headaches.3 Previous studies reported the isolation of alkaloids
from the twigs of M. trichocarpa3 and of essential oils4 and of
oxygenated cyclohexene derivatives from various Monanthotaxis species.5−8 The latter are the most common secondary
metabolites of the Annonaceae family, particularly of the tribe
Uvariae,9,10 and have also been reported to a lesser extent from
some other plant families.10,11 Oxygenated cyclohexenes
possess antimicrobial activity and have therefore been regarded
as viable candidates for drug development.5 Motivated by the
previous work on Monanthotaxis and by the current growing
need for new antibacterial agents,12 we evaluated the activity of
the constituents of the roots of M. trichocarpa against
Escherichia coli and Bacillus subtilis, as representative Grampositive and Gram-negative bacterial species, respectively.
Herein, we report the isolation, structural elucidation, and the
evaluation of the antibacterial and cytotoxicity activities of
three new (1−3) and nine known (4−12) compounds from
the methanolic root extract of M. trichocarpa.
© 2020 American Chemical Society and
American Society of Pharmacognosy
RESULTS AND DISCUSSION
Using repeated silica gel gravity column chromatography,
followed by Sephadex LH-20 gel filtration and preparative
reverse-phase HPLC, twelve secondary metabolites were
isolated from the roots of M. trichocarpa. The structures of
the isolated metabolites were established based on their NMR
spectroscopic, mass spectrometric, and single-crystal X-ray
diffractometric analyses. In addition to the three new
oxygenated secondary metabolites 1−3, nine known compounds previously isolated from other plants were identified.
The structures of these known compounds, uvaretin (4),13
diuvaretin (5), 13 1-(2-hydroxy-4-methoxyphenyl)-2-(4hydroxyphenyl)propan-1-one (6),14 uvangoletin (7),13 2methoxybenzyl benzoate (8),15 benzyl benzoate (9)13
cherrevenol H (10),16 and a mixture of stigmasterol (11)
and sitosterol (12)16 (Supporting Information, p S2), were
established by comparison of their observed and reported
spectroscopic data (Supporting Information). The structures
of compounds 1 and 4 were further confirmed by single-crystal
X-ray analyses.
Compound 1 was obtained as white crystals from a MeOH−
CH2Cl2 (1:1) solution. Its specific optical rotation, [α]24D
−72.1 (c 0.14, MeOH), indicated it to be chiral. HRESIMS
■
Received: April 19, 2019
Published: January 27, 2020
210
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peaks (Figure S5, Supporting Information) of H-4 (δH 6.00) to
C-2 (δC 75.6), C-3 (δC 58.1), and C-6 (δC 71.6) suggested 1 to
possess a cyclohexene skeleton. This was confirmed by the
TOCSY (Figure S6, Supporting Information) correlations of
H-1 (δH 4.05), H-3 (δH 4.73), H-4 (δH 6.00), H-5 (δH 5.88),
and H-6 (δH 5.82). The HMBC cross-peak of OCH3-1 (δH
3.64) to C-1 (δC 79.2) indicated the position proposed for this
methoxy group. A hydroxy group (δH 2.96) was located at C-2
(δC 75.6), based on its HMBC cross-peak to C-1′ (δC 66.6)
and the cross-peak of the oxymethylene protons CH2-1′ (δH
4.77) to C-2 (δC 75.6). Furthermore, HMBC cross-peaks of
CH2-1′ and H-2″/6″ (δH 7.91) to the benzoyl carbonyl C-7″
(δC 166.8) revealed the linkage of one of the benzoyloxy
moieties to the cyclohexene skeleton through an oxymethylene
unit. The substitution of another benzoyloxy functionality to
the cyclohexene core was established based on the HMBC
cross-peak of H-6 (δH 5.82) to C-7‴ (δC 160.0). NOESY
cross-peaks (Figure S7, Supporting Information) of δH 4.05
(H-1) and δH 4.77 (H-1′) indicated their suprafacial
orientation. Similarly, the NOEs of H-3 (δH 4.73) to OH-2
(δH 2.96) and H-6 (δH 5.82) and of OH-2 to OCH3-1 (δH
3.64) revealed their syn orientation. The absolute configuration
of 1 has been determined by single-crystal X-ray analysis
(Figure 1) and was in agreement with the above NMR
observations. It indicated that the cyclohexene ring of 1 exists
in a half-chair conformation, with an unexpectedly low transaxial 3JH1,H6 = 5.5 Hz. The above spectroscopic and
crystallographic evidence revealed compound 1 to be the
new chlorinated secondary metabolite trichocarpeol A, which
was characterized as ((1S,2S,3S,6R)-6-(benzoyloxy)-3-chloro2-hydroxy-1-methoxycyclohex-4-en-2-yl)methyl benzoate. Similar chlorinated cyclohexene derivatives have been previously
reported from Piper hookeri,17 P. nigrum,18 Dasymaschalon
sootepense,19 Cleistochlamys kirkii,10 and some Uvaria species.20−22
Compound 2 was isolated as a white solid. It showed the
specific rotation [α]24D −88.2 (c 0.14, MeOH) and was
assigned the molecular formula C22H22O7 based on HRESIMS
([M + H]+ m/z 399.1453, calcd 399.1444, Figure S16,
Supporting Information) and NMR data analyses. Its IR
absorbance at 3464 cm−1 suggested the presence of a hydroxy
group, and that at 1715 cm−1 a conjugated carbonyl
functionality and those at 1584 and 1601 cm−1 an aromatic
moiety. UV absorptions at 274, 228, and 206 nm were in
agreement with an aromatic system. Compound 2 showed
similar spectroscopic features to compound 1 (Figures S9−
S15, Supporting Information), suggesting it to be a cyclohexene derivative, with the only difference being at C-3, which
was substituted by a hydroxy group instead of a chlorine atom.
The presence of a methoxy group at C-1 (δC 80.1) was
confirmed by its HMBC cross-peak of δH 3.62 (OCH3-1)
(Table 2 and Figure S13, Supporting Information). Similarly to
1, trichocarpeol B (2) is benzoyloxy substituted at C-6, as
confirmed by the HMBC cross-peak of H-6 (δH 5.73) to
carbonyl C-7‴ (δC 166.3). As no single crystals suitable for Xray analysis were obtained, the absolute configuration of 2
could not be determined. However, its most plausible
configuration could be established by comparison of its
NMR data to that of the structurally closely related 1. The
3
JH1,H6 = 4.5 Hz of H-1 (δH 3.92) and H-6 (δH 5.73) suggest
their cis axial−equatorial configuration. The magnitude of this
coupling is very similar to that observed for 1 (5.5 Hz, Table
1), which has a trans diaxial H-1−H-6 configuration (Figure
analysis (m/z 417.1105, calcd 417.1105 [M + H]+) suggested
the molecular formula C22H21ClO6 and indicated 12 degrees of
unsaturation. Its chlorine content was revealed by the isotope
mass peaks at m/z 419.1100 and 417.1105 with a 1:3 intensity
ratio (Figure S8, Supporting Information). The IR absorptions
at 3675 and 3499 cm−1 suggested the presence of hydroxy
groups. The IR absorption at 1707 cm−1 suggested 1 to possess
a conjugated carbonyl system, and those at 1584 and 1601
cm−1 the presence of carbon−carbon double bonds, whereas
those at 2968 and 2901 cm−1 indicated the prevalence of
aliphatic C−H bonds. An aromatic system and an α,βunsaturated carbonyl were indicated by the UV absorptions at
274 and 223 nm. 1H NMR signals were observed (Figure S1,
Supporting Information) corresponding to two pairs of
protons with integrals of two each at δH 7.91 (H-2‴/6‴)
and δH 7.98 (H-2″/6″), four protons at δH 7.37 (H-3‴/5‴, 3″/
5″), and two overlapping protons at δH 7.50 (H-4″/4‴)
assigned to two aromatic rings (Table 1). The 3JH4,H5 = 9.9 Hz
indicated δH 6.00 (H-4) and δH 5.88 (H-5) to be cis-olefinic.
The COSY correlations (Figure S3, Supporting Information)
of H-3 (δH 4.73) and H-4 (δH 6.00), H-4 and H-5 (δH 5.88),
H-5 and H-6 (δH 5.82), and H-6 and H-1 (δH 4.05) (Figure
S3, Supporting Information) along with the HMBC crossTable 1. 1H and 13C NMR Spectroscopic Data for
Compound 1 (600 MHz, CDCl3)
position
δC, type
δH
(J in Hz)
HMBC
1
79.2, CH
4.05
d (5.5)
OCH3-1
OH-2
3
60.3, CH3
75.6, C−O
58.1, CH
3.64
2.96
4.73
s
br s
d (3.6)
4
129.5, CH
6.00
5
6
126.4, CH
71.6, CH
5.88
5.82
1′
66.6, CH2
4.77
ddd (9.9,
3.6, 1.4)
dd (9.9, 3.1)
ddd (5.5,
3.1, 1.4)
d (AA′)
(12.6)
1″
2″/6″
3″/5″
4″
129.58, C
129.7, CH
128.5, CH
133.3, CH
7.91
7.37
7.50
dd (8.1, 1.3)
dd (8.1, 7.6)
tt (7.6, 1.3)
C-7, C-3″, C-4″, C-6″
C-1″, C-2″, C-4″, C-5″
C-2″, C-3′’, C-5″, C6″
7″
1‴
2‴/6‴
3‴/5‴
166.8, CO
129.63, C
129.8, CH
128.6, CH
7.98
7.37
dd (7.6, 1.6)
dd (7.6, 7.6)
4‴
133.2, CH
7.50
tt (7.6, 1.6)
C-8, C-3‴, C-4‴, C-6‴
C-1‴, C-2‴, C-4‴, C5‴
C-2‴, C-3‴, C-5‴, C6‴
7‴
166.0, CO
Article
C-1′, C-3, C-5, OCH31, C-2, C-6
C-1
C-1, C-1′, C-2, C-3
C-1, C-1′, C-2, C-4,
C-5
C-2, C-3, C-5, C-6
C-1, C-3, C-4
C-1, C-4, C-5, C-7‴
C-1, C-2, C-3, C-7″
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Article
Figure 1. Solid state structures of trichocarpeol A (1) to the left and uvaretin (4) to the right, shown as thermal ellipsoids with 50% probability
levels.
Table 2. 1H and 13C NMR Spectroscopic Data for
Compound 2 (400 MHz, CD2Cl2)
position
δC, type
δH
1
80.1, CH
3.92
d (4.5)
OCH3-1
OH-2
3
OH-3
4
60.4, CH3
76.0, C
70.5, CH
131.7, CH
3.62
2.87
4.39
2.79
5.98
5
6
126.2, CH
71.5, CH
5.83
5.73
1′
65.9, CH2
4.79
4.57
d (1.7)
br s
d (3.4)
br s
ddd (10.2, 3.4,
1.7)
dd (10.2, 3.4)
ddd (4.8, 3.4,
1.7)
d (11.9)
d (11.9)
1″
2″/6″
133.6, C
129.9, CH
7.87
m
3″/5″
128.8, CH
7.34
m
4″
7″
(J in Hz)
7.51
1‴
2‴/6‴
130.2, CH
167.3,
CO
133.7, C
130.0, CH
m
7.94
m
3‴/5‴
128.9, CH
7.34
m
4‴
7‴
130.2, CH
166.3,
CO
7.51
m
Table 3. 1H and 13C NMR Spectroscopic Data for
Compound 3 (600 MHz, CD2Cl2)
HMBC
position
δC, type
δH
C-2, C-6, C-5, C-3,
OCH3-1, C-1′
C-1
1
OCH3-1
3.88
3.61
d (4.0)
s
C-2, C-3, C-6, OCH3-1
C-1
C-1′, C-2, C-5, C-4
2
OH-2
3
80.5, CH
60.4,
OCH3
75.9, C
2.79
3.96
br s
d (2.0)
3.52
s
C-1, C-2, C-3
C-1, C-2, C-4, C-5, C-6,
OCH3-3, C-1′
C-3
6.07
ddd (8.0, 2.0,
0.8)
dd (8.0, 3.6)
C-3, C-6, C-2
OCH3-3
C-3, C-6, C-1
C-4, C-5, C-1, C-7‴
4
79.4, CH
59.1,
OCH3
130.2,
CH
126.1,
CH
71.4, CH
C-2, C-1, C-3, C-7″
C-2, C-1, C-6, C-7″
5
C-2″/6″, C-3″/5″, C-1″,
C-7″
C-3″/5″, C-2″/6″, C-1″,
C-4″
C-3″/5″, C-2″/6″
1′
66.0,
CH2
1″
2″/6″
130.3, C
129.9,
CH
128.8,
CH
133.4,
CH
166.9,
CO
130.5, C
130.0,
CH
128.9,
CH
133.7,
CH
166.3,
CO
6
3″/5″
C-2‴/6‴, C-3‴/5‴, C1‴, C-7‴
C-2‴/6‴, C-3‴/5‴, C4‴, C-1‴
C-3‴/5‴, C-2‴/6‴
4″
7″
1‴
2‴/6‴
3‴/5‴
1). However, in contrast to 1, a strong NOE correlation
(Figure S15, Supporting Information) of H-1 (δH 3.92) and H6 (δH 5.73) was observed, indicating these protons to be synoriented. The NOEs between H-1 (δH 3.92) and H-1′ (δH 4.57
and 4.79) indicated these to be syn-oriented as well and thus
revealed the relative configuration of C-2. The NOE of H-3
(δH 4.39) and OH-2 (δH 2.87) as well as the absence of an
NOE between H-6 and H-3 suggested C-3 to have the
opposite relative configuration as compared to the C-3 of 1.
Based on the above spectroscopic evidence, this new
compound, trichocarpeol B (2), was characterized as (6β(benzoyloxy)-2β,3α-dihydroxy-1β-methoxycyclohex-4-en-2yl)methyl benzoate.
Compound 3 was obtained as a white solid and showed a
specific rotation of [α]24D −75.7 (c 0.14, MeOH). It was
assigned the molecular formula C23H24O7 based on the
analyses of HRESIMS ([M + H]+ m/z 413.1572, calcd
413.1600, Figure S24, Supporting Information) and NMR data
(Figures S17−S23, Supporting Information and Table 3). Its
IR spectrum suggested the presence of hydroxy (3471 cm−1),
4‴
7‴
5.85
5.70
(J in Hz)
HMBC
C-2, C-3, C-5, C-6
C-1, C-4, C-6
4.65
ddd (4.0,
3.60. 0.8)
d (11.9)
C-1, C-2, C-4, C-5, C-7‴
C-1, C-2, C-7″
4.56
d (11.9)
C-1, C-2, C-7″
7.85
m
7.33
m
C-2″/6″, C-3″/5″, C-1″,
C-7″
C-3″/5″, C-2″/6″, C-4″
7.49
m
C-3″/5″, C-2″/6″
7.92
m
7.33
m
C-2‴/6‴, C-3‴/5‴, C-1‴,
C-4‴, C-7‴
C-2‴/6‴, C-3‴/5‴, C-4‴
7.49
m
C-3‴/5‴, C-2‴/6‴
carbonyl (1716 cm−1), and aromatic (1601 and 1584 cm−1)
groups, with the latter being confirmed by the UV absorptions
at 274, 230, and 205 nm. Its NMR data (Table 3, Figures S17−
S23, Supporting Information) showed high similarities to those
of trichocarpeol B (2), with the only difference being the
alkylation of the OH-3 of the latter to a methoxy group. This
was indicated by the HMBC cross-peaks of OCH3-3 (δH 3.52)
to C-3 (δC 79.4) and of H-3 (δH 3.96) to OCH3-3 (δC 59.1).
The relative configuration of H-1 (δH 3.88) and H-6 (δH 5.70)
was established as cis axial−equatorial, based on the observed
3
JH1,H6 = 4.0 Hz, which was similar to trichocarpeol B (2) (4.5
Hz, Table 2) and corroborated by the NOE (Figure S23,
Supporting Information) of H-1 (δH 3.88) and H-6 (δH 5.70).
Overall, the NOE correlations of compound 3 resembled those
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of 2, enabling the assignment of the relative configurations of
C-1, C-2, C-3, and C-6. Based on the above spectroscopic
evidence, this new compound, trichocarpeol C (3), was
characterized as 6β-(benzoyloxy)-2β-hydroxy-1β,3β-dimethoxycyclohex-4-en-2-yl)methyl benzoate. Similar cyclohexene
derivatives possessing methoxy functionalities have previously
been reported from C. kirkii.10
The isolated natural products were evaluated against the
Gram-positive bacterium B. subtilis and Gram-negative E. coli,
and for cytotoxicity using human MCF-7 cells. Whereas B.
subtilis is nonpathogenic and can serve as a proxy for the
microbiological investigation of Gram-positive species, many E.
coli strains are human pathogens and are medically relevant.
Compound 4 was active against B. subtilis with a MIC value of
18.0 μM, and it also inhibited MCF-7 cells with an EC50 value
of 10.2 μM. All other compounds were either inactive or not
tested for their cytotoxicity. None of the isolated metabolites
were active against the Gram-negative Escherichia coli. at ∼5
mM (2000 μg/mL) concentration. The bioactivities of
compound 4 corroborate the previously reported antimicrobial
and anticancer activities of some chalcones.23,24 This is the first
report of the occurrence of polyoxygenated cyclohexenes in M.
trichocarpa, which is of chemotaxonomic importance. The
present phytochemical observations support the morphological
and phylogenetic placement of this genus to the Uvariae tribe
alongside other related genera of the family Annonaceae.5−11
0577335 UTM 8871102. The plant was identified by Mr. F. M.
Mbago, a senior taxonomist of the Herbarium, Botany Department,
University of Dar es Salaam, where a voucher specimen (FMM-3793)
was deposited.
Extraction and Isolation. The root bark of M. trichocarpa was
air-dried for 2 weeks and then powdered to obtain 581.2 g of plant
material. The ground material was then soaked in MeOH for 48 h
twice consecutively. The filtrate was concentrated in vacuo on a rotary
evaporator at 40 °C to obtain 55 g of root bark crude extract. Gravity
column chromatography of the crude extract (55 g) was performed,
by adsorbing the extract on silica gel and by gradient elution ranging
from 5% ethyl acetate−isohexane to 10% ethyl acetate−MeOH.
Altogether, 759 fractions of ca. 100 mL were collected and, based on
TLC analysis, were pooled into 102 subfractions. Fractions 21−22,
obtained with 5% ethyl acetate−isohexane, were combined and
subjected to preparative HPLC, collecting benzyl benzoate (9, 10.6
mg) with 90:10 H2O−MeOH. Fractions 51 and 52, obtained with 5%
ethyl acetate−isohexane, were combined and purified by preparative
HPLC to obtain 2-methoxybenzyl benzoate (8, 1.8 mg), collected
with 90:10 H2O−MeOH. Combined fractions 125−127, eluted with
5% ethyl acetate−isohexane, followed by precipitation from MeOH,
gave a mixture of stigmasterol (11) and sitosterol (12) (10.0 mg).
Fractions 276−285, obtained with 20% ethyl acetate−-isohexane,
precipitated from the eluent. Washing this precipitate with isohexane
afforded colorless needles of trichocarpeol A (1, 57.5 mg).
Subsequently, the combined fractions 292−331 were separated on a
Sephadex column eluting with 1:1 MeOH−CH2Cl2, which resulted in
11 fractions of ca. 1 mL each. Upon standing, fraction 322 crystallized
from MeOH−CH2Cl2 to give uvangoletin (7,5.9 mg). Combined
fractions 390−396, obtained with 25% ethyl acetate−isohexane, were
purified by preparative HPLC, utilizing 90:10 H2O−MeOH, yielding
trichocarpeol D (3, 6.3 mg) and diuvaretin (5, 4.6 mg), with a 12.9
and 19.8 min elution time, respectively. Fraction 397, obtained with
25% ethyl acetate−isohexane, crystallized from MeOH−CH2Cl2 to
give uvaretin (4, 2.2 mg), while fraction 413, obtained with the same
gradient, precipitated from MeOH−CH2Cl2 and gave additional
uvaretin (4, 3.0 mg). The combined fractions 592−599 were
subjected to HPLC utilizing 90:10 H2O−MeOH, yielding trichocarpeol C (3, 15.5 mg). HPLC purification of fractions 489−501
yielded cherrevenol H (10, 1.1 mg).
Trichocarpeol A (1): white crystals (isohexane); [α]20D −72.1 (c
0.14, MeOH); UV (MeOH) λmax (log ε) 274 (2.92), 223 (6.40) nm;
IR νmax 3675, 3499, 2968, 2901, 1707, 1601, 1584, 1449, 1393, 1381,
1360, 1317, 1278, 1253, 1179, 1140, 1096, 1072, 1027, 950, 966, 927
cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 417.1105
[M + H]+ (calcd for C22H22ClO6 417.1105).
Trichocarpeol B (2): white solid; [α]20D −88.2 (c 0.14, MeOH);
UV (MeOH) λmax (log ε) 274 (2.32), 228 (3.36), 206 (3.28) nm; IR
νmax 3446, 3056, 1781, 1743, 1423, 1265, 110, 940, 738 cm−1; 1H and
13
C NMR data, see Table 2. HRESIMS m/z 399.1453 [M + H]+
(calcd for C22H23O7399.1444).
Trichocarpeol C (3): white solid; [α]20D −75.7 (c 0.14, MeOH);
UV (MeOH) λmax (log ε) 274 (1.96), 230 (2.93), 205 (3.11) nm; IR
νmax 3471, 2934, 1716, 1601, 1584, 1451, 1315, 1272, 1096, 955, 710
cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 413.1572
[M + H]+ (calcd for C23H25O7413.1600)
X-ray Diffraction Analysis of Trichocarpeol A (1) and
Uvaretin (4). Single crystals of 1 and 4, obtained from isohexane,
were collected on a Bruker D8 APEX-II equipped with a CCD camera
using Mo Kα radiation (λ = 0.710 73 Å). Crystals were mounted on a
fiber loop and fixated using Fomblin oil. Data reduction was
performed with SAINT,25 and absorption corrections for the area
detector were performed using SADABS.26 Structures were solved by
direct methods and refined by least-squares methods on F2 using the
SHELX and the OLEX2 software suits.27,28 The data for 1 and 4 were
collected at 123.15 and 150(2) K, respectively. Non-hydrogen atoms
were refined anisotropically. Hydrogen atoms were constrained in
geometrical positions to their parent atoms. Anomalous dispersion
was used to determine the absolute structure of 1 with a Flack
parameter of 0.06(2).29,30 One phenyl ring containing C17 was
■
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
determined using an Autopol II Rudolph Research Analytical WLG
polarimeter at 589 nm, at 24.1 °C. UV absorptions were determined
using a Shimadzu UV-1650PC ultraviolet visible (UV/vis) spectrophotometer. IR spectra were measured on a PerkinElmer Spectrum
One FTIR spectrometer. NMR spectra were acquired on Agilent
MR400-DD2 (400 MHz), Bruker Avance NEO 500 and 600 MHz, or
Bruker Avance III HD 800 MHz spectrometers and were processed
using the software MestreNova (v12.0.3). Chemical shifts were
indirectly referenced to tetramethylsilane, by referencing to the
residual solvent signal. Structural assignments were based on 1H, 13C,
COSY, TOCSY, NOESY, HSQC, and HMBC spectra. LC-ESIMS
were acquired on an API SCIEX 150 EX PerkinElmer ESIMS (30 eV)
spectrometer attached to a PerkinElmer gradient pump system and a
5 mm RP-C8 110 Å column (Gemini), utilizing acetonitrile and 1%
formic acid in Milli-Q water (gradient elution using 5−95%
acetonitrile over 4 min) as mobile phases. HRESIMS were obtained
with a Q-TOF-LC/MS spectrometer with a lockmass-ESI source
(Stenhagen Analysis Lab AB, Gothenburg, Sweden) using a 2.1 × 30
mm 1.7 μm RP-C18 column and a H2O−CH3CN gradient (5:95 to
95:5, with 0.2% HCO2H). The isolation process was monitored using
analytical thin-layer chromatography (TLC), performed on silica gel
60 F254 (Merck) precoated aluminum plates, and visualized under
UV light (254 and 365 nm). Following elution, the TLC plates were
sprayed with 4-anisaldehyde reagent followed by heating for
identification of UV-negative compounds and for detection of color
change of the UV-positive spots. The latter reagent was prepared by
mixing 3.5 mL of 4-anisaldehyde with 2.5 mL of concentrated H2SO4,
4 mL of glacial HOAc, and 90 mL of MeOH. Gravity column
chromatography was carried out using silica gel 60 (230−400 mesh).
Gel filtration was done using Sephadex LH-20 (Pharmacia)
suspended in CH2Cl2−MeOH (1:1). Preparative HPLC was
performed on a Waters 600E system using the Chromulan (Pikron
Ltd.) software and an RP-C8 Kromasil column (250 mm × 25 mm)
with a H2O−MeOH gradient (70:30 to 100:0) for 20−40 min with a
flow rate of 7 mL/min.
Plant Material. Roots of Monanthotaxis trichocarpa were collected
in March 2017 from the coastal forest at Mikindani ya Leo, in the
Lindi Rural District, Lindi Region, Tanzania, at GPS location 37L
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modeled with a positional disorder with partial occupancy of 20% to
80%. The solid-state structure of compound 4 was previously reported
from room-temperature measurements without thermal displacement
parameters.31,32 The X-ray structure (cif) data of 1 (CCDC 1906380)
and 4 (CCDC 1906381) have been deposited with the Cambridge
Crystallographic Data Centre. Copies of the data can be obtained, free
of charge, on application to the Director, CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK (fax: + 44-(0)1223-336033 or e-mail:
deposit@ccdc.cam.ac.uk).
Antibacterial Assays. The antibacterial activity of the isolated
compounds was determined against Escherichia coli and Bacillus
subtilis. The compounds were first redissolved at 10 mg/mL in 100%
DMSO, then further diluted 30× in H2O and stored at −20 °C. E. coli
and B. subtilis were cultured as previously described by Mueller and
Hinton33 and Doyle.33,34 For in vitro determination of antibacterial
activity, a culture of bacterial cells was grown to OD 600nm = 0.5. The
culture was diluted 10× with prewarmed medium, and the substances
to be tested were added to the culture medium for a final
concentration of 30 μg/mL, each at 100 μL in a 96-well microtiter
plate. Cultures with substances to be tested were then incubated at 37
°C without agitation for 18 h. To measure cell health, we used the
resazurin-based assay as described previously.35 Next, 12 μL of
AlamarBlue solution (commercial name of resazurin solution,
ThermoFisher) was added to each well, and incubation at 37 °C
was continued for 1 h. Then, fluorescence was measured using a
POLARstar Omega microplate reader from BMG Labtech with the
excitation filter set to 544 nm and emission filter to 590 nm. Cells
exposed to an equivalent concentration of DMSO only were used as
negative control. Before setting up the assay in microtiter format,
bleed-through of fluorescence from resorufin between wells in the
microtiter plate fluorescence reader was measured and found to be
<1% between adjacent wells. To check for quenching of fluorescence
by any of the investigated compounds, normally grown bacterial
cultures were mixed after 1 h of incubation with resazurin and the
compound of interest at the highest concentration to be investigated,
and the immediately measured fluorescence was compared with
samples without compound added. All tests of compound activity
were performed in three independent replicates. All compounds
where a reduction of fluorescence by at least 50% relative to the
solvent control was observed in any of the species were followed up
by additional tests for more accurate determination of the degree of
antibacterial activity.
Cytotoxicity Assay. The cytotoxicity of the isolated compounds
was evaluated against human MCF-7 cells grown in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal calf serum and
kept in exponential growth as previously reported.23,24 Before the
assay, cells were reseeded into 96-well microtiter plates at a density
allowing continued exponential growth and allowed to settle for 24 h.
The isolated compounds were added from a stock solution in DMSO,
for a final concentration of 0.3% v/v of the solvent in the culture
medium. After 24 h of incubation in the presence of the compound,
cell viability was assayed using PrestoBlue cell viability reagent
(ThermoFisher) according to the manufacturer’s instructions. A Polar
Star Omega plate reader (BMG Lab Tech) was used to measure
resorufin fluorescence at 544 nm excitation/590 nm emission.
Survival was expressed as percentage of the solvent-only control.
EC50 values for each compound were calculated, from three
independent replicate experiments, using 2-fold dilution intervals.
The original NMR spectra along with the corresponding
NMReDATA36 for the new compounds 1−3 are freely available on
Zenodo as DOI: 10.5281/zenodo.3592334.
■
■
Article
AUTHOR INFORMATION
Corresponding Authors
Stephen S. Nyandoro − Chemistry Department, College of
Natural and Applied Sciences, University of Dar es Salaam, Dar
es Salaam, Tanzania; Phone: +255-754-206560;
Email: nyandoro@udsm.ac.tz
Máté Erdélyi − Department of Chemistry - BMC, Uppsala
University, SE-751 23 Uppsala, Sweden; Department of
Chemistry and Molecular Biology, University of Gothenburg,
SE-412 96 Gothenburg, Sweden; Center for Antibiotic
Resistance Research (CARe) at the University of Gothenburg,
405 30 Gotheburg, Sweden; orcid.org/0000-0003-03595970; Phone: +46-72-9999166; Email: mate.erdelyi@
kemi.uu.se
Authors
Gasper Maeda − Chemistry Department, College of Natural and
Applied Sciences, University of Dar es Salaam, Dar es Salaam,
Tanzania; Department of Chemistry - BMC, Uppsala
University, SE-751 23 Uppsala, Sweden
Jelle van der Wal − Department of Chemistry and Molecular
Biology, University of Gothenburg, SE-412 96 Gothenburg,
Sweden; Center for Antibiotic Resistance Research (CARe) at
the University of Gothenburg, 405 30 Gotheburg, Sweden
Arvind Kumar Gupta − Department of Chemistry - BMC,
Uppsala University, SE-751 23 Uppsala, Sweden
Joan J. E. Munissi − Chemistry Department, College of Natural
and Applied Sciences, University of Dar es Salaam, Dar es
Salaam, Tanzania
Andreas Orthaber − Department of Chemistry - Ångström,
Uppsala University, SE-751 20 Uppsala, Sweden; orcid.org/
0000-0001-5403-9902
Per Sunnerhagen − Department of Chemistry and Molecular
Biology, University of Gothenburg, SE-412 96 Gothenburg,
Sweden; Center for Antibiotic Resistance Research (CARe) at
the University of Gothenburg, 405 30 Gotheburg, Sweden;
orcid.org/0000-0002-0967-8729
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.9b00363
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The Swedish Research Council (Swedish Research Links,
2016-05857) and the Center for Antibiotic Resistance
Research (CARe) are gratefully acknowledged for financial
support. We thank Mr. F. M. Mbago, the curator at the
Herbarium of the Department of Botany, University of Dar es
Salaam, for locating and identifying the investigated plant
species. This study made use of the NMR Uppsala infrastructure, which is funded by the Department of Chemistry BMC and the Disciplinary Domain of Medicine and Pharmacy.
■
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ASSOCIATED CONTENT
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