pubs.acs.

org/jnp Article

NMR Structure Elucidation of Naphthoquinones from Quambalaria

cyanescens
Eliška Prochaź kova,́ * Oleksandr Kucherak, Eva Stodůlkova,́ Zdeneǩ Tošner, Ivana Císarǒ va,́
Miroslav Flieger, Miroslav Kolarí̌ k, and Ondrě j Baszczyňski*
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ABSTRACT: Naphthoquinones isolated from Quambalaria cyanescens (quambalarines) are natural pigments possessing significant
cytotoxic and antimicrobial properties. Determining the structure of naphthoquinone compounds is important for the understanding
of their biological activities and the informed synthesis of related analogues. Identifying quambalarines is challenging, because they
contain a hydroxylated naphthoquinone scaffold and have limited solubility. Here, we report a detailed structural study of
quambalarine derivatives, which form strong intramolecular hydrogen bonds (IMHBs) that enable the formation of several
tautomers; these tautomers may complicate structural investigation due to their fast interconversion. To investigate tautomeric
equilibria and identify new quambalarines, we complemented the experimental NMR spectroscopy data with density functional
theory (DFT) calculations.

T he microscopic fungus, Q. cyanescens (Basidiomycota:

Microstromatales), is an ecologically diverse species that
acts as an animal pathogen, plant saprobe or pathogen, and
correlations in 2D NMR spectra. Moreover, strong IMHBs
enable the coexistence of several tautomers in solution.
The structures of two quambalarine derivatives 1 and 3 have
insect symbiont.1 It produces naphthoquinone pigments such been previously elucidated by X-ray diffraction.1 However, X-
as mompain and the recently discovered quambalarines.1 ray diffraction does not fully cover the hydrogen positions,
Mompain (2) which can be considered to be a basic which are usually back-recalculated and, therefore, may not be
hydroxylated 1,4-naphthoquinone scaffold was discovered in defined correctly. Moreover, X-ray diffraction describes the
1962,2 and its structure was solved three years later.3 structure in the solid state, which may significantly differ from
Quambalarines display a broad spectrum of biological the structure in solution (biological systems).9−11
activities.1 While quambalarine A (1) displays antibacterial NMR spectroscopy is a powerful tool for probing the
and antifungal activities (e.g., Aspergillus), quambalarine B (3) chemical structure and physicochemical properties such as
has significant antileukemic properties.4,5 Mompain and tautomerism and hydrogen bonding in solution. Experimental
quambalarine B have been recently revealed as submicromolar NMR data complemented by quantum-chemical calculations
inhibitors of influenza endonuclease.6 Access to these provide deeper insights into the molecular structure. The
promising compounds is limited, as they are usually isolated
from the parent fungus in tiny amounts, have limited solubility,
and their total synthesis is challenging due to a presence of a Received: August 24, 2020
hydroxylated naphthoquinone scaffold.7,8 Furthermore, struc-
tural elucidation based on commonly used NMR spectroscopy
methods is complicated by the presence of exchangeable OH
hydrogens, quaternary carbons with low signal intensity, and
the low number of hydrogens usually providing the key
© XXXX American Chemical Society and
American Society of Pharmacognosy https://dx.doi.org/10.1021/acs.jnatprod.0c00930
A J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
calculated NMR parameters such as chemical shielding can be
correlated with the experimental 13C chemical shifts to confirm
the signal assignment and investigate tautomeric forms.12 The
investigation of tautomeric forms of hydroxylated naphthoqui-
nones10,13 may contribute to better understanding of their
pharmacological activities.14,15
In this work, we report on a detailed NMR study of
naphthoquinone derivatives isolated from Q. cyanescens. Using
NMR spectroscopy combined with DFT calculations allowed
us to identify two novel quambalarine species C (4) and D (5).
Understanding key structural features within the quambalarine
family is crucial for the identification and rational design of
new biologically active derivatives with desired antineoplastic,
antibacterial, or antifungal properties.
■ RESULTS AND DISCUSSION
The studied compounds were isolated according to the
previously described procedures1 from the basidiomycete Q.
cyanescens. First, we investigated the structures of three known
naphthoquinone derivatives 1−3 by NMR spectroscopy
complemented by DFT calculations. Then, we applied the
Figure 1. (a) Structures of E and Z isomers of quambalarine A (1), (b) structures of possible tautomers, and (c) structures of rotamers. In parts b
and c, the E-isomer structures are shown, and the ethyl tetrahydrofurenyl moiety at C3 was substituted by tetrahydrofurenyl only for simplicity.
Tautomer 4-8 was not found during geometry optimization in chloroform as indicated by the red cross.
B https://dx.doi.org/10.1021/acs.jnatprod.0c00930
pubs.acs.org/jnp Article
NMR/DFT approach supported by HR-MS and UV−vis
spectra for the structural determination of the isolated
derivatives 4 and 5.
Quambalarine A (1). In the NMR spectra (1H, 13C) of 1
in CDCl3, two sets of signals in an approximately 1:0.8 ratio
were detected (Figure S87 in the SI). The two sets of signals
correspond to two isomers (E and Z) arising from restricted
rotation along the C3−C2′ double bond (Figure 1a). The two
downfield shifted 1H signals at 13.7 and 12.3 ppm of the major
isomer (and 13.6 and 12.4 ppm of the minor isomer)
correspond to the hydrogens involved in two strong IMHBs.
Therefore, eight possible tautomeric forms with two IMHBs
were proposed (Figure 1b,c). However, the hypothetical five-
membered rings formed by IMHBs are less stable than their
six-membered counterparts;11,16,17 therefore, the formation of
rotamers 5-1-rot and 4-1-rot1 are less probable than the
others in Figure 1.
For all of the optimized structures, the calculated 13C (1H)
shielding constants were correlated with experimental 13C
(1H) chemical shifts. The best fit with the experimental data
was found for tautomer 5-8 (Figure S21 in the SI). Tautomer
4-8 was not found, and the geometry optimization always
provided tautomer 5-8 instead. This may indicate that the
tetrahydrofurenyl substituent on C3 induces the formation of
keto groups on C1, C2, and C4, thus stabilizing the phenolic
form with C5−OH and C8−OH groups on C5 and C8;
therefore, tautomer 5-8 is preferred. The 4-8-rot and 4-1-rot4
tautomers (Figure 1c) did not provide a good correlation (R2 <
0.8, Figures S24 and S25 in the SI) nor did tautomers 5-1-rot
and 4-1-rot1 (R2 < 0.94 and 0.91, respectively, Figures S26 and
S27 in the SI).
Both E/Z isomers of the 5-8 tautomeric pattern were
optimized, and the calculated shielding constants were
correlated with both experimental data sets separately. The
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Figure 2. Correlations between experimental 13C chemical shifts of the major data set with calculated shielding constants of the E-isomer (A) and
the minor data set with the Z-isomer (B). The ethyl group was substituted by a methyl group in both cases to simplify calculations.

Figure 3. Possible tautomeric forms of 3 with three IMHBs: (a) C2−OH tautomers, (b) C1′−OH tautomers, and (c) rotamers of C1′−OH
tautomers. The red cross indicates that the particular tautomer was not found during geometry optimization in chloroform.

ethyl group was substituted by methyl for simplicity during
geometry optimization (to avoid additional rotamers caused by
a flexible ethyl moiety). Excellent agreement (R2 > 0.999) was
found when correlating the minor data set (44%) with the Z-
isomer and the major data set (56%) with the E-isomer (Figure
2).
Mompain (2). In the NMR spectra of sample 1, ca. 5% of a
third component was found. It had six aromatic 13C signals and
four 1H signals similar to those found in 1, indicating a
quambalarine derivative with a high degree of symmetry. Based
on HMBC, we assumed that the third component could be
mompain (2), and the 1H signals were compared with the 1H
spectrum of mompain (Figure S1 in the SI). Unfortunately, the
13
C spectra could not be compared directly because of the
limited solubility of 2 in CDCl3. The investigation of
tautomeric forms of 2 is described in the SI Section 3.2 in
detail.
C https://dx.doi.org/10.1021/acs.jnatprod.0c00930
Quambalarine B (3). The 1H NMR spectrum of 3 in
CDCl3 showed a mixture of two forms in a 1:0.1 ratio (Figure
S91 in the SI). Both forms provided three downfield shifted 1H
signals (12−19 ppm), indicating that three strong IMHBs are
present in both forms. Compared to 1, four additional
tautomers should be considered. In the major form, the OH
(16.3 ppm) is attached to C2 (Figure 3a), as the HMBC cross-
peaks to 166.1 and 167.3 ppm corresponding to C1 and C2,
respectively, were detected (Figure S4 in the SI). On the other
hand, in the minor form, HMBC showed the key cross-peak
between the downfield shifted OH (18.3 ppm) and the
aliphatic 13C signal at 38.9 ppm (Figure S4 in the SI). Thus, we
proposed the structure in which the OH is attached to C1′
(Figure 3b,c).
Correlating the major form (90%) data set with the
calculated shielding constants of the lowest-energy C2−OH
tautomers 5-8-2 and 4-1-2 separately (Figure 3a) provided a
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Figure 4. 13C correlations of the experimental data for the major form of 3 with chemical shielding constants calculated for the two lowest-energy
C2−OH tautomers 5-8-2 (A) and 4-1-2 (B). Averaging the shielding constants in a 1:1 ratio as proposed by the Boltzman distribution provided an
excellent fit (C).

Figure 5. Correlations between experimental 13C chemical shifts of the minor form of 3 in CDCl3 and the shielding constants calculated for
tautomer 5-8-1′ (A) and its rotamer 5-8-1′-rot (B).

Figure 6. Possible tautomeric forms of 4 with two strong IMHBs. The red cross indicates that the particular tautomer was not found during
geometry optimization in chloroform, and tautomeric rearrangement was observed instead.

correlation discrepancy in the C1, C4, C5, and C8 atoms
(Figure 4a,b). This discrepancy is probably caused by
positioning these two protons involved in resonance-assisted
hydrogen bonding (RAHB).17,18 The RAHBs usually occur in
π-conjugated rings and cause characteristic geometrical
changes such as elongation of the formally double bond and
simultaneous shortening of the formally single bond. As the
RAHBs are considered to be a linear combination of two
tautomeric forms, averaging these two data sets in a 1:1 ratio
(proposed by DFT) provided an excellent fit (R2 = 0.9976) as
shown in Figure 4c. The same trend was also found for 1H
correlations (with R2 = 0.9969 after averaging, Figure S52 in
the SI).
The minor form data set was correlated with calculated
shielding constants of the lowest-energy C1′−OH tautomer 5-
8-1′ and its rotamer 5-8-1′-rot (Figure 3b,c). The correlation
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of the 13C chemical shifts was excellent in both cases (R2 =
0.9928 and 0.9950, respectively), as shown in Figure 5.
Therefore, it was not possible to determine the particular
rotamer of 5-8-1′ unambiguously in this case. Surprisingly,
tautomers 4-8-1′ and 4-1-1′ were not found, as the geometry
optimization always converged into the more stable tautomers
5-8-1′ and 4-1-2, respectively. Contrary to the major form,
tautomeric equilibrium between the 5-8-1′ and 4-1-1′
tautomers was not found. A similar characteristic was also
observed in compound 1. It seems that the keto form on C2
(Figure 3b,c) significantly stabilizes the quinone form on its
ring; therefore, tautomer 4-1-1′ is not preferred. On the other
hand, the presence of an OH group on C2 supports the
formation of tautomer 4-1-2, which is then in equilibrium with
5-8-2 in a 1:1 ratio.
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Figure 7. Part of the HMBC spectrum of 4 in CDCl3 at room temperature. (A) The key diagnostic cross-peaks (highlighted in yellow frames)
enable the localization of the OH group in each of the tautomers (on C2 or C1′). (B) The key cross-peak determining the geometry of C5−OH in
the major form (green frame).

Figure 8. Correlations between experimental 13C chemical shifts of the major form of 4 in CDCl3 and the calculated shielding constants of
tautomer 5-2 (A) and the minor form with the calculated shielding constants of tautomer 5-1′ (B) and its rotamer 5-1′-rot (C). An unsaturated
chain of 4 was substituted by a methyl group to simplify the geometry optimization.

Quambalarine C (4). An absorption spectrum of 4 showed
a broad band at 400 nm indicating the presence of a
conjugated system, which was unlike any of the previously
investigated compounds (Figure S16 in the SI). The 1H NMR
spectrum of 4 in CDCl3 showed the same saturated pentyl
chain as in 3, but an additional signal in the aromatic region
(7.18 ppm) was also found, suggesting an alteration in the
naphthoquinone scaffold compared to 1−3. The 7.18 ppm
signal provided a COSY cross-peak to H6 (6.75 ppm, Figure
S6 in the SI) and a coupling constant of 2.4 Hz, indicating a
four-bond interaction from H8. Moreover, two sets of 1H
signals in a 1:0.2 ratio (Figure S93 in the SI) indicate
tautomeric equilibrium similar to that found in 3. Based on
that, we propose the structure and possible tautomeric forms of
4 (Figure 6).
The two downfield shifted 1H signals of the major form at
17.6 and 12.9 ppm (and 17.8 and 11.8 ppm of the minor form)
correspond to the hydrogens involved in two strong IMHBs.
Similar to 3, the major/minor forms of 4 differ in the C2/C1′
OH groups, respectively, as is clearly visible in HMBC (Figure
7).
The second OH in the major form involved in an IMHB is
attached to C5, as the key long-range HMBC cross-peak
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between C5−OH (12.9 ppm) and C7 (161.7 ppm) was found
(Figure 7b in green). The four-bond heteronuclear couplings
are detectable when there is a W-like shaped pathway between
the two interacting nuclei as highlighted in the structure of 5-2
in green (Figure 7). The NMR/DFT correlation demonstrates
an excellent fit with tautomer 5-2 (R2 = 0.9979), as shown in
Figure 8a and Figure S64 in the SI.
In the minor form, the C4 or C5 position of the second OH
hydrogen (11.8 ppm) was not clear based on the experimental
data. The best fit with the calculated data was found for C5−
OH tautomer 5-1′ and its rotamer 5-1′-rot (R2 = 0.9945 and
0.9959, respectively, Figure 8b,c). However, we could not
unambiguously decide which rotamer is present in a solution.
Although 5-1′ has a slightly lower energy, the correlation is
slightly better for the rotamer 5-1′-rot.
Finally, we managed to crystallize 4 from the dichloro-
methane/methanol mixture (1:1, v/v). Single-crystal X-ray
diffraction data unambiguously confirmed the structure of 4
proposed by NMR spectroscopy. However, contrary to the
situation in solution, tautomer 5-2 represents the bulk of
molecules found in the crystal (Figure 9, see X-ray diffraction
in the Experimental Section). The chemical formula of the
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proposed structure 4 was confirmed by HR-MS (Figure S95 in
the SI).
Figure 9. Displacement ellipsoids at the 50% probability level (A),
clearly confirming the structure of 4 proposed by NMR spectroscopy
combined with DFT calculations. Crystal packing of 4 with
disordered layers of water molecules (B).
Quambalarine D (5). An absorption spectrum of 5 with a
broad absorption band in the visible region with a maximum at
485 nm resembled the spectrum of 1 (477 nm), indicating
similar aromatic systems in both compounds (Figures S12 and
S18 in the SI). Unfortunately, the low solubility of 5 in CDCl3
prevented recording acceptable S/N 13C NMR spectra, but
CD3CN gave high-quality NMR spectra with one set of signals
at room temperature. The 1H spectrum showed two aromatic
signals (8.2 and 6.7 ppm) and three downfield shifted
hydrogens (13.8, 12.3, and 12.1 ppm) indicating that all
three hydrogens are involved in IMHBs. The aliphatic region
was similar to those found in the NMR spectra of 3 and 4,
indicating the presence of a saturated pentyl chain recognized
in the COSY spectrum (Figure S8 in the SI). In the 13C
spectrum of 5, three signals at 186.0, 181.7, and 182.6 ppm
corresponding to keto forms on C1, C2, and C4, respectively,
were detected, supporting the proposed structure of the “B”
ring (similar to that found in 1, Figure 10). On the other hand,
three 13C signals at 159.2, 153.3, and 148.9 ppm corresponding
Figure 10. Part of the HMBC (A) and ROESY (B) spectra of 5 in CD3CN at room temperature showing the key cross-peaks (highlighted in yellow
frames) enabling the signal assignment and proposing the structure of 5.
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to C5, C7, and C8, respectively, are very close to the ones
found in the minor form of 3 (tautomer 5-8-1′, Figure 3).
Notably, the HMBC cross-peak pattern of H6 is comparable to
that found for the minor form of 3 (Figure 10a) and
determined the structure of the “A” ring. Contrary to
compounds 1−4, mass spectrometry analysis revealed a
nitrogen atom and possible chemical formula of C16H17NO6.
The presence of an amino group was proposed by the ROESY
experiment, where the intense NOE cross-peaks between
hydrogens resonating at 12.1 and 8.2 ppm were found (Figure
10b). This chemical shift difference also suggests that one
hydrogen of the NH2 group is involved in an IMHB (12.1
ppm), while the second is not. Moreover, the upfield shifted
hydrogen (8.2 ppm) has a ROESY cross-peak with H2′ (3.1
ppm, Figure 10b) and, therefore, is close in space to the
saturated chain. Thus, the NH2 group is attached to C1′. The
chemical formula of the proposed structure 5 was confirmed by
HR-MS (Figure S98 in the SI).
An ensemble of the tautomer analogous to that investigated
in 1-4 is depicted in Figure 11. Based on our previous
experience with 1 and the minor form of 3 (both with C2 as a
ketone), tautomer 5-8 is significantly more stable than 4−1;
thus, it should also be preferred in 5. Indeed, we obtained an
excellent correlation for tautomer 5-8-1′ (R2 = 0.9958, Figure
12a). The higher stability of 5−8 is also demonstrated by the
fact that no discrepancies in the C1/C4 and C5/C8 regions
were found; thus, no shielding constant averaging as performed
in 2 and 3 (major form) was needed. However, it was not
possible to distinguish 5-8-1′ from its rotamer 5-8-1′-rot
(Figure 12), as observed in 3 and 4 (Figures 5 and 8,
respectively).
The amino group on C1′ in 5 significantly influenced the
tautomeric forms compared to 3 (bearing an OH group
instead). In 5, amino tautomers are substantially more stable
than their imino counterparts (Figure 11a,b). Therefore, the
amino group stabilizes the tautomers with a ketone on C2
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Figure 11. Proposed tautomeric forms of quambalarine D (5) with three IMHBs: (a) C2−OH tautomers, (b) C1′−NH2 tautomers, and (c)
rotamers of C1′−NH2 tautomers. The red cross indicates that the particular tautomer was not found during geometry optimization in acetonitrile.

Figure 12. Correlations between experimental 13C chemical shifts of 5 measured in CD3CN and calculated shielding constants of tautomer 5-8-1′
(A) and its rotamer 5-8-1′-rot (B) differing in the orientation of C1′−NH2. The alkyl chain was substituted by a methyl group to simplify the
calculations.

(Figure 11b,c) and thus stabilizes tautomer 4-1-1′, which
could not have been found in 3. On the other hand, tautomer
5-8-1′ has the lowest energy, similar to 3 (minor form).
Tautomer 4-8-1′ was not found.
7-O-Methylquambalarine B (6). Concerning the already
published anticancer properties within isolated species,
quambalarine B (3) was the most promising candidate.4,5
The 7-OH group of 3 is the only one that is not involved in
IMHBs; it has a phenolic character and as the most reactive
OH can be modified. Indeed, the methylation of 3 yielded a 7-
O-methyl derivative 6. Interestingly, further methylation of 3
led to unstable species formation, which could not be isolated
in our experimental setup. Unfortunately, 6 was devoid of any
antineoplastic properties. However, we investigated its
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tautomers to extend our knowledge of the effect of substitution
on tautomeric equilibria.
In the 1H NMR spectrum of 6 in CDCl3, two sets of signals
in a 1:0.2 ratio were found (Figure S99 in the SI), indicating
similar tautomeric equilibrium as observed in 3; a mixture of 5-
8-2/4-1-2 tautomers corresponds to the major form of 6, and
the 5-8-1′ tautomer corresponds to the minor form (Figure 3
and Figure S79 and S80 in the SI, details in the SI Section 3.6).
In conclusion, we investigated the tautomerism of
naphthoquinones isolated from Q. cyanescens in order to
interpret the experimentally obtained NMR data correctly. It
was found that tautomerism within quambalarine compounds
is sensitive to the substitution on the naphthoquinone scaffold
and the solvent used. The substitution on C2 (=O and −OH,
respectively) significantly affects the equilibria of particular
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tautomeric forms. Carbon C2 in its keto form (in 1) induces
the keto form on C4, and tautomer 5-8 is preferred. On the
other hand, C2 in its hydroxy form stabilizes OH on C4;
therefore, a mixture of 5-8 and 4-1 was found in equilibrium.
Two stable forms of 3, 4, and 6 differing in tautomerism at the
C2 position (5-8-2 and 5-8-1′) were stable enough to be
clearly distinguished in the NMR spectra. Interestingly, 3 was
readily methylated at C7−OH. This modification increased the
aromaticity of ring A and stabilized tautomer 5-8. Con-
sequently, tautomer 5-8 is more stable than 4-1, and they
coexist in a ca. 4:1 ratio; meanwhile, in the case of 3, they exist
in a ca. 1:1 ratio. On the other hand, in 5, amino tautomers are
significantly more stable than their imino counterparts, and the
C1′−NH2 stabilizes C2-keto tautomers. This systematic NMR
structural study is a basis for the structure elucidation of new
natural compounds containing a hydroxylated naphthoquinone
skeleton, and it could be used for the rational design of
quambalarine derivatives with desired medicinal properties.
■
EXPERIMENTAL SECTION
General Experimental Procedures. Quambalarine A (1),
quambalarine B (3), and mompain (2) were isolated according to
the previously described protocol including strain isolation,
identification, and fermentation conditions.1
NMR spectra were recorded on a Bruker Avance III spectrometer
with a cryo-probe (5 mm TCI 1H/13C/15N/D Z/GRD) operating at
600 MHz for 1H and 150 MHz for 13C and on a Bruker Avance III
spectrometer with a broad-band cryo-probe with ATM module (5
mm CPBBO BB−1H/19F/15N/D Z/GRD) operating at 500 MHz for
1
H and 125 MHz for 13C. Low-temperature measurements were
performed on a Bruker Avance II spectrometer with a triple resonance
broad-band probe with ATM (5 mm PATBO BB−1H/19F/D Z-
GRD) operating at 500 MHz for 1H and 125 MHz for 13C. For NMR
signal assignment, standard Bruker pulse programs for commonly
used experiments (1H, 13C, COSY, HSQC, HMBC, NOESY, and
EXSY) were used. The selective excitation 1D EXSY experiment was
used to follow the exchange processes of 1H signals.
All of the structures were optimized at the DFT level of theory
using the B3LYP19,20 functional and a standard 6-31+g(d,p) basis set
with the polarizable continuum model (PCM) used for implicit
chloroform or acetonitrile solvation.21,22 The NMR parameters were
calculated using the GIAO method with the 6-311+g(d,p) basis set
with PCM. The Gaussian 09 program package23 was used throughout
this study.
The crystallographic experiment of 4 was carried on a Bruker D8
VENTURE Kappa Duo PHOTONIII by a IμS microfocus sealed tube
with Cu Kα radiation (λ = 1.54178 Å) at a temperature of 120 K. The
structures were solved by direct methods (XT)24 and refined by full
matrix least-squares based on F2 (SHELXL2018).25 The hydrogen
atoms on carbon were fixed into idealized positions (riding model)
and assigned temperature factors of either Hiso(H) = 1.2 Ueq(pivot
atom) or Hiso(H) = 1.5 Ueq (pivot atom) for methyl moieties. The
hydrogen atoms on O were found on the difference Fourier map and
refined with assumptions of a riding model. The hydrogen atoms of
the two −OH moieties forming short intramolecular bonds could be
expected to easily jump between corresponding oxygen atoms and
switch toward another tautomer in the crystal. The precision of the
structure does not allow the refinement of such disorder; however, the
majority of those hydrogens were found on the difference Fourier
map. Therefore, the corresponding tautomer represents the majority
of molecules in the crystal. The six symmetrically independent
molecules are stacked into columns via π−π interactions. The
hydrophilic parts of two columns are interconnected by a layer of
disordered solvate molecules via the hydrogen bond O(7*)−H(7*)···
O (oxygen of water molecule).
Mass spectra were measured on an LTQ Orbitrap XL (Thermo
Fisher Scientific) using electrospray ionization (ESI).
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Pigment extraction: The fermentation broth of Q. cyanescens CCM
8372 (7 l) was centrifuged, filtered, and extracted with an equal
volume of ethyl acetate containing 3% (v/v) acetic acid (three times).
The pooled extracts were dried over anhydrous Na2SO4, filtered, and
evaporated to dryness under reduced pressure to produce a dark violet
solid (3.2 g). More details about the culture are described in our
previous work.1
HPLC analysis of the extract of Q. cyanescens: The extract was
repeatedly loaded onto a Gemini 5 C18 column (Phenomenex) and
eluted by the following gradient elution, water (A) and methanol (B),
both containing 1% TFA, flow rate of 1 mL/min, and DAD UV
detection. This procedure led to the isolation of the following
standards: mompain (11.3 mg, 98%), quambalarine A (12.5 mg,
94%), quambalarine B (20.5 mg, 97%), quambalarine C (3.8 mg,
98%), and quambalarine D (3.5 mg, 99%).
Batch isolation of quambalarine B (3): The extract of 3 was
obtained from Q. cyanescens as described above. Processing of the
extract of 3 by HPLC (conditions above) gave a concentrate of 3
(240 mg) as a mixture of three compounds (three spots visible in the
iodine chamber). The main portion of 3 was obtained by treating the
concentrate of quambalarine B with diethyl ether (3 mL) and further
filtration. The filter cake was washed with diethyl ether (2 mL, three
times) and dried in air to give a pure sample of 3 as a dark violet
powder (80 mg). An additional amount of quambalarine B (30 mg)
was obtained from the filtrate. Diethyl ether was evaporated in vacuo,
and the obtained residue was purified by column chromatography on
SiO2 (eluent DCM−cyclohexane (1:1), then pure DCM, and finally
DCM−MeOH−TFA (95:5:0.1)). The three obtained fractions were
identified by NMR and HR-MS: The first was a colorless oil (methyl
oleate), and the second was a white waxlike solid (palmitic acid); the
third was a dark violet powder of 3, with Rf = 15% in the above-
mentioned system (DCM−MeOH−TFA).
Synthesis of 7-O-Methylquambalarine B (6). Initial compound 3
(30 mg, 1.0 equiv) was dissolved in dry DMF (3 mL), and then MeI
(90 μL, 15 equiv) and K2CO3 (27 mg, 2.0 equiv) were added to the
solution. The flask was closed with a septum, and the reaction mixture
was stirred at 40 °C overnight. Flash TLC (eluent DCM−MeOH =
9:1) showed complete consumption of the initial material, one minor
spot (yellow, fast) and one major spot of the product (wine-red,
slow). The reaction was quenched with TFA (30 μL, 4.0 equiv) and
concentrated under vacuum. The residue obtained was mixed with
DCM and loaded onto a SiO2 column. Elution was performed with
DCM, then with DCM−MeOH−H2O = 70:10:1, and finally with
DCM−MeOH−TFA = 95:5:0.1 to give a dark residue. It was washed
with Et2O (1 mL twice) to obtain the product 6, which was a wine-
red powder. The yield was 11 mg (35%).
Quambalarine A. (1, E/Z isomer ratio 1:0.8). 1H NMR (600
MHz, CDCl3, 25 °C) major isomer (56%): δ = 13.64 (s, 1H, 8-OH),
12.32 (s, 1H, 5-OH), 6.91 (s, 1H, H6), 6.38 (s, 1H, 7-OH), 5.07 (m,
1H, H5′), 3.78 (ddd, 1H, H3′a, Jgem = 20.8, J3′‑4′a = 9.6, J3′‑4′b = 4.8
Hz), 3.53−3.62 (m, 1H, H3′b), 2.37−2.43 (m, 1H, H4′a), 1.80−2.05
(m, 3H, H4′b, H1″), 1.13 ppm (t, 3H, H2″, J2″‑1″ = 7.5 Hz). Minor
isomer (44%): δ = 13.55 (s, 1H, 8-OH), 12.34 (s, 1H, 5-OH), 6.90 (s,
1H, H6), 6.39 (s, 1H 7-OH), 5.01 (m, 1H, H5′), 3.86 (ddd, 1H,
H3′a, Jgem = 20.6, J3′‑4′a = 9.5, J3′‑4′b = 4.2 Hz), 3.53−3.62 (m, 1H,
H3′b), 2.37−2.43 (m, 1H, H4′a), 1.80−2.05 (m, 3H, H4′b, H1″),
1.13 ppm (t, 3H, H2″, J2″‑1″ = 7.5 Hz). 13C NMR (151 MHz, CDCl3,
25 °C) major isomer (56%): δ = 193.59 (C2′), 185.27 (C4), 183.48
(C1), 178.79 (C2), 159.43 (C5), 151.82 (C7), 146.86 (C8), 113.28
(C8a), 112.99 (C6), 111.45 (C3), 106.38 (C4a), 93.22 (C5′), 38.51
(C3′), 27.96 (C1″), 26.19 (C4′), 9.56 ppm (C2″). Minor isomer
(44%): δ = 192.64 (C2′), 187.50 (C4), 183.47 (C1), 175.65 (C2),
159.06 (C5), 151.98 (C7), 146.83 (C8), 113.31 (C8a), 112.57 (C6),
111.63 (C3), 106.76 (C4a), 92.58 (C5′), 38.09 (C3′), 28.10 (C1″),
26.60 (C4′), 9.67 ppm (C2″).
HR-MS (m/z) calcd for C16H15O7 [M + H]+ 319.08123, found
319.08125. Absorption maxima at 329 and 477 nm were found.
Mompain. (2, as 5% component in the sample of 1). 1H NMR
(600 MHz, CDCl3, 25 °C) δ = 12.90 (s, 1H, 5-OH), 11.66 (s, 1H, 8-
OH), 6.77 (s, 2H, 2-OH, 7-OH), 6.60 ppm (s, 2H, H3, H6). 13C
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
NMR (151 MHz, CDCl3, 25 °C) δ = 173.52 (C5, C4), 165.32 (C8,
C1), 154.47 (C7, C2), 112.40 (C6, C3), 110.62 (C8a), 104.80 ppm
(C4a).
HR-MS (m/z) calcd for C10H5O6 [M − H]− 221.00916, found
221.00921. Absorption maxima at 314 and 515 nm were found.
Quambalarine B. (3, tautomer ratio 1:0.1). 1H NMR (600 MHz,
CDCl3, 25 °C) major form (90%): δ = 16.32 (s, 1H, 2-OH), 14.37 (s,
1H, 5-OH), 12.10 (s, 1H, 8-OH), 6.85 (bs, 1H, 7-OH), 6.68 (s, 1H,
H6), 3.30 (m, 2H, H2′), 1.73−1.78 (m, 2H, H3′), 1.37−1.47 (m, 4H,
H4′, H5′), 0.95 (t, 3H, H6′, J6′‑5′ = 7.0 Hz). Minor form (10%): δ =
18.34 (s, 1H, 1′-OH), 12.59 (s, 1H, 8-OH), 11.76 (s, 1H, 5-OH),
6.91 (s, 1H, H6), 6.53 (bs, 1H, 7-OH), 3.18 (m, 2H, H2′), 1.73−1.78
(m, 2H, H3′), 1.37−1.47 (m, 4H, H4′, H5′), 0.94 (t, 3H, H6′, J6′‑5′ =
7.1 Hz). 13C NMR (151 MHz, CDCl3, 25 °C) major form (90%): δ =
209.09 (C1′), 173.78 (C5), 173.01 (C4), 167.30 (C2), 166.12 (C1),
165.80 (C8), 154.56 (C7), 114.47 (C3), 113.16 (C6), 112.18 (C8a),
102.98 (C4a), 43.91 (C2′), 31.34 (C4′), 23.81 (C3′), 22.46 (C5′),
13.92 ppm (C6′). Minor form (10%): δ = 205.17 (C1′), 189.41 (C4),
183.11 (C2), 175.60 (C1), 159.49 (C5), 153.64 (C7), 148.76 (C8),
112.67 (C8a), 112.50 (C6), 110.37 (C3), 103.65 (C4a), 38.71 (C2′),
31.60 (C4′), 25.20 (C3′), 22.34 (C5′), 13.89 ppm (C6′).
HR-MS (m/z) calcd for C16H17O7 [M + H]+ 321.09688, found
321.09684. Absorption maxima at 316 and 515 nm were found.
Quambalarine C. (4, tautomer ratio 1:0.2). 1H NMR (600 MHz,
CDCl3, 25 °C) major form (80%): δ = 17.58 (s, 1H, 2-OH), 12.58 (s,
1H, 5-OH), 7.18 (d, 1H, H8, 4J8−6 = 2.4 Hz), 6.75 (d, 1H, H6, 4J6−8 =
2.4 Hz), 5.94 (s, 1H, 7-OH), 3.27 (m, 2H, H2′), 1.71−1.76 (m, 2H,
H3′), 1.36−1.46 (m, 4H, H4′, H5′), 0.94 ppm (t, 3H, H6′, J6′‑5′ = 7.1
Hz). Minor form (20%): δ = 17.84 (s, 1H, 1′-OH), 11.75 (s, 1H, 5-
OH), 7.26 (s, 1H, H8), 6.74 (s, 1H, H6), 6.21 (s, 1H, 7-OH), 3.15
(m, 2H, H2′), 1.71−1.76 (m, 2H, H3′), 1.36−1.46 (m, 4H, H4′,
H5′), 0.93 ppm (t, 3H, H6′, J6′‑5′ = 7.1 Hz). 13C NMR (151 MHz,
CDCl3, 25 °C) major form (80%): δ = 209.18 (C1′), 186.81 (C4),
179.52 (C1), 172.25 (C2), 164.85 (C5), 161.68 (C7), 132.33 (C8a),
113.47 (C3), 110.92 (C6), 109.12 (C4a), 108.14 (C8), 42.32 (C2′),
31.98 (C4′), 24.11 (C3′), 22.43 (C5′), 13.91 ppm (C6′). Minor form
(20%): δ = 204.17 (C1′), 191.49 (C4), 179.11 (C1), 176.29 (C2),
164.92 (C5), 162.85 (C7), 134.44 (C8a), 110.92 (C6), 110.39 (C3),
109.16 (C4a), 110.40 (C8), 38.32 (C2′), 31.60 (C4′), 25.37 (C3′),
22.32 (C5′), 13.89 ppm (C6′).
HR-MS (m/z) calcd for C16H18O6 [M + H]+ 305.10196, found
305.10186. Absorption maxima at 312 and 399 nm were found.
Crystal data for 4: C16H16O6. H2O, Mr = 322.29; Triclinic, P −1
(No. 2), a = 10.4098 (5) Å, b = 21.2446 (11) Å, c = 21.6607 (12) Å,
α = 68.899 (3)°, β = 86.272 (3)°, γ = 81.935 (3)°, V = 4424.4 (4) Å3,
Z = 12, Dx = 1.443 Mg m−3, temperature of sample 120 (2) K, red-
yellow prism of dimensions 0.16 × 0.06 × 0.04 mm3, multiscan
absorption correction (μ = 0.97 mm−1) Tmin = 0.78, Tmax = 0.96; a
total of 52 063 measured reflections (θmax = 65.0°), from which
14 994 were unique (Rint = 0.128), and 5384 were observed according
to the I > 2σ(I) criterion. The refinement converged (Δ/σmax =
0.001) to R = 0.109 for observed reflections and wR(F2) = 0.312,
GOF = 1.24 for 1269 parameters and all 14 994 reflections. The final
difference map displayed no peaks of chemical significance (Δρmax =
0.68, Δρmin −0.31 e.Å−3).
The X-ray crystallographic data have been deposited with the
Cambridge Crystallographic Data Center under deposition number
CCDC 2022130 for 4 and can be obtained free of charge from the
center via its Web site (www.ccdc.cam.ac.uk/getstructures).
Quambalarine D. (5). 1H NMR (500 MHz, CD3CN, 25 °C): δ =
13.78 (s, 1H, 5-OH), 12.32 (bs, 1H, 8-OH), 12.09 (s, 1H, NHa), 8.16
(s, 1H, NHb), 7.97 (bs, 1H, 7-OH), 6.73 (s, 1H, H6), 3.04 (m, 2H,
H2′), 1.65 (m, 2H, H3′), 1.34−1.46 (m, 4H, H4′, H5′), 0.92 (t, 3H,
H6′, J6′‑5′ = 7.0 Hz). 13C NMR (125 MHz, CD3CN, 25 °C): δ =
186.01 (C1), 182.62 (C4), 181.68 (C2), 159.23 (C5), 153.28 (C7),
148.85 (C8), 114.33 (C8a), 113.25 (C6), 108.36 (C3), 106.68 (C4a),
38.17 (C2′), 32.49 (C4′), 28.79 (C3′), 23.07 (C5′), 14.30 ppm
(C6′). C1′ was not detected.
HR-MS (m/z) calcd for C16H19O6N [M + H]+ 320.11286, found
320.11296. Absorption maxima at 326 and 485 nm were found.
I https://dx.doi.org/10.1021/acs.jnatprod.0c00930
pubs.acs.org/jnp Article
7-O-Methylquambalarine B. (6, tautomer ratio 1:0.2)1H NMR
(500 MHz, CDCl3, 25 °C) major form (80%): δ = 16.67 (s, 1H, 2-
OH), 14.18 (s, 1H, 5-OH), 12.70 (s, 1H, 8-OH), 6.61 (s, 1H, H6),
4.00 (s, 3H, OCH3), 3.31 (m, 2H, H2′), 1.76 (m, 2H, H3′), 1.21−
1.50 (m, 4H, H4′, H5′), 0.96 ppm (m, 3H, H6′). Minor form (20%):
δ = 18.43 (s, 1H, 1′-OH), 12.88 (s, 1H, 8-OH), 11.92 (s, 1H, 5-OH),
6.61 (s, 1H, H6), 4.03 (s, 3H, OCH3), 3.20 (m, 2H, H2′), 1.76 (m,
2H, H3′), 1.21−1.50 (m, 4H, H4′, H5′), 0.91 ppm (m, 3H, H6′). 13C
NMR (125 MHz, CDCl3, 25 °C) major form (80%): δ = 209.28
(C1′), 176.92 (C4), 172.01 (C1), 169.00 (C2), 168.78 (C5), 161.84
(C8), 157.54 (C7), 114.30 (C3), 112.30 (C8a), 110.15 (C6), 103.01
(C4a), 56.75 (7-OCH3), 43.64 (C2′), 29.71 (C4′), 23.87 (C3′),
22.49 (C5′), 13.96 ppm (C6′).
HR-MS (m/z) calcd for C17H16O7 [M − H]− 333.09798, found
333.09759. Absorption maxima at 313 and 511 nm were found.
■
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00930.
Spectroscopic data of the new compounds, including
HPLC chromatograms, NMR, and HR-MS spectra, and
computational details including x,y,z coordinates of the
optimized structures and the NMR/DFT correlations
(PDF)
Quambalarine species C (CIF)
■
AUTHOR INFORMATION
Corresponding Authors
Eliška Procházková − Institute of Organic Chemistry and
Biochemistry, Czech Academy of Sciences, 166 10 Prague,
Czech Republic; orcid.org/0000-0002-4768-3422;
Email: prochazkova@uochb.cas.cz
Ondřej Baszczyňski − Faculty of Science, Charles University,
128 43 Prague, Czech Republic;
Email: ondrej.baszczynski@natur.cuni.cz
Authors
Oleksandr Kucherak − Faculty of Science, Charles University,
128 43 Prague, Czech Republic
Eva Stodůlková − Institute of Microbiology, Czech Academy of
Sciences, 142 20 Prague, Czech Republic
Zdeněk Tošner − Faculty of Science, Charles University, 128
43 Prague, Czech Republic
Ivana Císařová − Faculty of Science, Charles University, 128
43 Prague, Czech Republic
Miroslav Flieger − Institute of Microbiology, Czech Academy
of Sciences, 142 20 Prague, Czech Republic
Miroslav Kolařík − Institute of Microbiology, Czech Academy
of Sciences, 142 20 Prague, Czech Republic
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00930
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was financially supported by the Czech Science
Foundation (O.B., Grant No. 20-25137Y), by the Experientia
Foundation (O.B., Start-Up Grant No. SG-2018-1), and
BIOCEVBiotechnology and Biomedicine Center of the
Academy of Sciences and Charles University (M.F. and M.K.,
Grant No. CZ.1.05/1.1.00/02.0109). We would like to
acknowledge Květoslava Kertisová from the Mass Spectrom-
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products pubs.acs.org/jnp Article

etry Department at IOCB for the HR-MS analysis and Tomás ̌

Belloň for the design of the graphical abstract.

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