The Mosaic of Rottlerin: The Sequel

Article abstract of DOI:10.1021/acs.jnatprod.8b00917

Rottlerin (1) is a potent protein kinase C δinhibitor that possesses a wide range of biological activities. However, the potential of this molecule to be developed as a drug has been restricted by its limited availability. We report herein a gram scale quantity synthesis of rottlerin in a five-step synthetic route that can be completed within 2 days. The methodology was extended by the reaction of the key aminochromene intermediate (15) with various electron-rich arenes, forming novel unsymmetrical methylene-bridged compounds. The X-ray crystal structure revealed the boomerang shape of this kind of molecule for the first time. The direct transformation of rottlerin (1) into the natural product, isorottlerin (35), was observed for the first time, and we named this transformation the "isorottlerin change". In addition, the antibacterial activities of rottlerin (1) and new rottlerin analogues 32-34 were examined against Staphylococcus aureus. The compounds showed MIC values as low as 2.0 μM, which were comparable to the clinically used antibiotic gentamicin.

Full text of DOI:10.1021/acs.jnatprod.8b00917

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
The Mosaic of Rottlerin: The Sequel
Kenneth K. C. Hong, Kitty K. K. Ho, Mohan Bhadbhade, Graham E. Ball, David StC. Black,
and Naresh Kumar*
School of Chemistry, UNSW Australia, Sydney, NSW 2052, Australia
S
* Supporting Information
ABSTRACT: Rottlerin (1) is a potent protein kinase C δ inhibitor that
possesses a wide range of biological activities. However, the potential of this
molecule to be developed as a drug has been restricted by its limited availability.
We report herein a gram scale quantity synthesis of rottlerin in a five-step
synthetic route that can be completed within 2 days. The methodology was
extended by the reaction of the key aminochromene intermediate (15) with
various electron-rich arenes, forming novel unsymmetrical methylene-bridged
compounds. The X-ray crystal structure revealed the boomerang shape of this
kind of molecule for the first time. The direct transformation of rottlerin (1)
into the natural product, isorottlerin (35), was observed for the first time, and
we named this transformation the “isorottlerin change”. In addition, the
antibacterial activities of rottlerin (1) and new rottlerin analogues 32−34 were
examined against Staphylococcus aureus. The compounds showed MIC values as
low as 2.0 μM, which were comparable to the clinically used antibiotic
gentamicin.
Mallotus phillipinesis is an endangered medicinal plant that is
found in India.1 The fruit of the plant is covered with a red
powder, known as kamala, within which can be found rottlerin
(1) (Figure 1). First discovered by Thomas Anderson in 1855,
rottlerin (1) has long intrigued researchers due to its broad
spectrum of pharmacological activity. Rottlerin (1) is most well
known for its inhibition of protein kinase C delta (PKCδ),2
and extensive studies on the anticancer properties have been
performed. Recently, Wang et al.3 reported that rottlerin (1)
drastically reduced the expression of Cdc20 oncoprotein in
glioma cells, highlighting a potential new mechanism for the
treatment of glioma. Yin et al.4 also discovered the ability of
rottlerin (1) to significantly inhibit S-phase kinase associated
protein 2 (Skp2) expression in breast cancer cells, revealing a
new therapeutic strategy for combating breast cancer.
While rottlerin (1) has been mostly studied for its anticancer
activities against various cancer cells, its antibacterial activity is
not as well studied despite a few recent studies showing that
rottlerin exhibits an minimum inhibitory concentration (MIC)
of 1.0 and 6.3 μM against multiple Chlamydia and Helicobacter
pylori species.5−7 It is also known that compounds similar to
rottlerin (1), including drummondins A (2), B (3), C (4), and
F (5) (Figure 1), from Hypericum drummondii possess potent
antibacterial activities against Staphylococcus aureus, Bacillus
subtilis, and Mycobacterium smegmatis.8
Although rottlerin (1) can be purchased in small quantities
(10 mg) from several chemical suppliers, its limited availability
and high price are obstacles that prevent more extensive
research of this promising compound in various areas. Our
research group has previously reported the first total synthesis
of rottlerin (1) comprising a seven-step linear synthesis.9 The
previous synthesis (Scheme 1) began by using the
commercially available trihydroxyacetophenone (6) to prepare
the methoxymethyl (MOM)-protected chromene (7) using a
three-step protocol. Subsequent aldol condensation afforded
chromene (8), followed by acid deprotection to give 5-
hydoxychromene (9) in 32% yield. Aminomethylation using
Eschenmoser’s salt gave chromene (10) in 88% yield. Finally,
rottlerin (1) was synthesized in moderate yield by thermally
reacting 10 with acetophenone (11). More recently, Wang et
al.3 described an eight-step linear synthesis of rottlerin (1).10
However, both of these reported synthetic methodologies have
drawbacks, including a long synthetic sequence, harsh and
toxic reaction conditions such as demethylation using BBr3,
and extended production time.
We report herein an improved gram-scale, five-step synthesis
of rottlerin (1) and the development of new rottlerin
analogues. In addition, the potent antibacterial activities of
rottlerin (1) are reported for the first time. It is anticipated that
the efficient synthetic methodology reported herein will permit
the preparation of large quantities of rottlerin (1) and its
analogues to study their promising biological activities.
As rottlerin (1) is structurally similar to these compounds
with the presence of a chromene moiety with a methylene
bridge connected to a hydroxyacetophenone moiety, it would
be interesting to see if rottlerin (1) and its analogues would
possess any antibacterial activity against the methicillin-
resistant S. aureus.
Received: November 7, 2018
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Structures of rottlerin (1) and drummondins A (2), B (3), C (4), and F (5).
Scheme 1. Original Synthesis of Rottlerin (1)9
Scheme 2. Identification of Products from the Acidic Deprotection of the MOM Group
This side product was purified by silica-gel chromatography,
and it was identified to be the symmetrical homodimer
rottlerone, in a yield of 14%. The formation of such
symmetrical dimer was also reported by Dubois and Yu in
their synthesis of methylenebis(chalcone)s and dimeric
chromenes during the deprotection of MOM-protected
chalcones under acidic conditions.11,12
To avoid the formation of rottlerone, the MOM protecting
group was replaced with the tert-butyldimethylsilyl (TBDMS)
protecting group, which does not produce formaldehyde upon
RESULTS AND DISCUSSION
■
Scalable Synthesis of Rottlerin (1). The inspiration
toward the improved synthesis began when investigating the
cause of the low yield during the deprotection of the MOM
group of chromene (8) (Scheme 1).9 The reaction was
reinvestigated in depth, and it was found that apart from the
target polar 5-hydroxchromene (9) being formed in 26% yield
(Scheme 2), a nonpolar compound with a similar Rf value to 8
was also formed.
B
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Scheme 3. Synthesis of TBDMS-Protected Chromene (13)
Scheme 4. An Improved Gram Scale Synthesis of Rottlerin (1)
Figure 2. ORTEP diagram of rottlerin (1) and the measured angle around the methylene bridge.
deprotection. The synthesis of rottlerin (1) therefore began
with the monoprotection of trihydroxyacetophenone mono-
hydrate (6) using TBDMSCl to give the bis-protected
acetophenone, which was not isolated. Addition of pyridinium
p-toluenesulfonate (PPTS) to the reaction mixture and heating
to reflux gave the monoprotected acetophenone (12) in 85%
yield (Scheme 3).13 Annulation was achieved by reacting
acetophenone (12) with a 3-methyl-2-butenal in the presence
of ethylenediamine diacetate (EDDA) at room temperature for
10 min, affording chromene (13) in 90% yield. Notably, this
method is scalable and much more efficient than Adler’s
synthesis14 of chromene (13) using microwave irradiation
(300 W) for 1 h, which gave the product in 30% yield.
Reaction of chromene (13) with benzaldehyde under
classical aldol conditions with base in aqueous EtOH did not
afford the expected TBDMS-protected chalcone. Instead,
cleavage of the TBDMS protecting group was observed,
which could be due to the lability of TBDMS to NaOH.
Therefore, the aldol condensation was performed using NaH
as a base, and upon aqueous workup NaOH was generated in
situ, which promoted the cleavage of TBDMS, forming the
target chalcone (9) in a one-pot process in 69% yield (Scheme
4). Aminomethylation of chromene (9) using Eschenmoser’s
salt gave aminochromene (10) in quantitative yield. Heating
aminochromene (10) at high temperature generated the
putative ortho-quinone methide intermediate (14), which
could be trapped with the acetophenone (11) in a 1,4-Michael
addition reaction to provide rottlerin (1) in 31% yield after
column chromatography and recrystallization from MeCN.
While toluene was used as the solvent in the previous synthesis
of rottlerin,9 it was found that MeCN was a better solvent for
the large-scale synthesis. Comparable yields were obtained
after chromatography; however the drop in yield compared
with the previous synthesis is mainly due to the product loss
during recrystallization. Overall, the synthesis of rottlerin (1)
was reduced from a seven-9 to a five-step, gram-scale synthesis
that could be completed in 2 days.
Crystallization of rottlerin (1) from EtOAc gave single
crystals suitable for X-ray diffraction studies, thus allowing
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unambiguous structure determination (Figure 2). This is the
first X-ray crystal structure of rottlerin (1).
Table 1. List of Rottlerin-Type Analogues
In the solid state, rottlerin (1) exhibits an overall
“boomerang-like” structure, with a 46.86° angle between the
planes of the phloroglucinol and chromene moieties (Figure
2). Interestingly, two sets of intramolecular hydrogen bonds
are observed between the phloroglucinol core and the
chromene moiety. The hydrogen bonds of the 4-OH and 6-
OH across the methylene bridge were shown to have distances
of 1.878 and 1.891 Å, respectively. This could restrict rotation
around the methylene bridge, thus accounting for the presence
of only a single rotamer in solution, as observed by NMR
spectroscopy. We postulate that by altering the substituents on
the acetophenone moiety, it could be possible to engineer
molecules with various angles to tailor their interactions with
biological systems.
Synthetic Methodology and Substrate Scope Inves-
tigation. The scope of the 1,4-Michael addition reaction
methodology was investigated by reacting the key chromene
intermediate (13) with various arenes, to simpler rottlerin-type
analogues lacking the chalcone moiety but bearing different
substituents attached to the methylene group. The chromene
intermediate (13) was treated with Eschenmoser’s salt and
tetrabutylammonium fluoride (TBAF) in CHCl3 at room
temperature for 5 min, allowing aminomethylation and
deprotection of the TBDMS group in a one-pot reaction
(Scheme 5) to give 15 in 90% yield.
Scheme 5. Synthesis of Aminochromene (15) and Novel
Rottlerin-Type Analogues
The reaction of aminochromene (15) with various electron-
rich phenolic substrates (Scheme 5) such as acetophenone
derivative 11, orcinol (17), and dimethoxyphenol (19)
provided new methylene-bridged compounds 16, 18, and 20,
respectively (Table 1, entries 1−3).
However, phenol (21) and the electron-deficient 3,5-
difluorophenol (22) did not generate any products, and only
starting material was recovered (Table 1, entries 4, 5).
Probably the electron-deficient ortho-quinone methide inter-
mediate15 is favored to react with electron-rich arenes. Using
the electron-rich 3,5-dimethoxyaniline (23) as substrate, two
regioisomers, 24 and 25, were produced in 14% and 20% yield,
respectively (Table 1, entry 6).
The use of indole (26) as substrate afforded the new
compounds 27 and 28 in 21% and 8% yield (Table 1, entry 7).
Compound 27 was formed from the reaction of indole (26) at
C-3 to one equivalent of chromene (15), whereas in
compound 28, indole 26 reacted at both C-2 and C-3 with
two molecules of chromene (15) to form a dimeric species.
The behavior of C-2 of indole acting as a nucleophilic center
has also been reported by Sengul et al.16 Recently, many
research groups have reported that the structural modification
of resveratrol (29) provides pronounced biological activ-
ities.17−19 The reaction of resveratrol (29) using our
methodology generated the highly oxygenated analogue 30
in 8% yield.
After multiple attempts of recrystallization, a single crystal
was obtained for the orcinol-derived analogue (18) using
MeCN as the solvent (Figure 3). The solid-state structure of
18 revealed only one intramolecular hydrogen bond across the
methylene bridge, as opposed to the two hydrogen bonds
observed for rottlerin (1). The measured distance of the
hydrogen bond across the methylene bridge was 1.819 Å. The
measured angle between the chromene and orcinol core of
compound 18 was measured to be 69.96°, which is
significantly greater than for rottlerin (1) (46.86°). As
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Figure 3. ORTEP diagram of compound 18 and the measured angle around the methylene bridge.
Scheme 6. Synthesis of New Rottlerin Analogues 32−34
1
Figure 4. Stacked plot of H NMR spectra of rottlerin (1) in DMSO-d6 over two months showing the 6.30−6.80 ppm region.
observed, changing the number of hydrogen bonds can
remarkably change the conformation of the molecule.
Overall, the methodology has shown the ability of generating
unsymmetrical methylene-bridged compounds by utilizing the
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Scheme 7. Formation of Flavanones 35 and 36 from the Decomposition of Rottlerin (1)
generation of the ortho-quinone methide intermediate.
However, due to the overall electrophilic nature of the ortho-
quinone methide intermediate, only electron-rich arenes could
undergo 1,4-addition to give methylene-bridged compounds.
Synthesis of Direct Rottlerin Analogues. With the
scope of the methodology established, the 1,4-Michael
addition reaction methodology was extended to direct rottlerin
analogues bearing different substituents attached to the
methylene bridge. Thus, the key aminochromene intermediate
(10) was reacted with various phenolic derivatives such as
phloroglucinol (31), phloroacetophenone (6), and orcinol
(17) to generate the new rottlerin analogues 32−34 in
moderate yields of 20−25% (Scheme 6). The low yields of
these analogues were mainly caused by incomplete reactions
Figure 5. Important and distinctive HMBC correlations of the two
isomers 35 and 36.
and the loss of products through chromatography.
Stability of Rottlerin and the Isorottlerin Change. The
stability of rottlerin (1) was investigated in DMSO, a
commonly used solvent to dissolve compounds for biological
testing. The stability of rottlerin (1) in DMSO-d6 was
monitored over two months at room temperature using
NMR spectroscopy (Figure 4). The characteristic H-10 at 6.63
ppm (d, J = 10 Hz) of rottlerin (1) disappeared over time,
while two new doublets at 6.46 and 6.42 ppm with the same
coupling constant of 10 Hz appeared, suggesting that rottlerin
(1) slowly reacted to form two distinct products in an
approximately 7:1 ratio.
The mixture of the two products could not be separated by
silica-gel chromatography due to their close Rf values.
Therefore, preparative RP-HPLC was used to separate the
two products. The products were identified as isorottlerin (35)
and flavanone (36), which were isolated in yields of 20% and
2%, respectively, as racemic mixtures (Scheme 7).
The identities of compounds 35 and 36 were confirmed by
phase-sensitive HSQC experiments. The presence of diaster-
eotopic C-8 protons in the aliphatic region suggested the
flavanone core in both molecules. Moreover, the appearance of
a deshielded H-9 with three-bond correlations to both C-7 and
C-6 in the HMBC spectrum (Figure 5) provided further
evidence for the core structure of the molecules. The
distinctive peak to distinguish between the two compounds
was the presence of a four-bond HMBC correlation from the
HO-2 to C-7 of isorottlerin (37), which is absent in compound
38, as the OH group is at the 4-position and hence too far
away from C-7.
As seen from the NMR stacked plot, the major isomer after
incubating rottlerin (1) in DMSO-d6 at room temperature for
two months was isorottlerin (35), whereas the minor isomer
was 36. The conversion of rottlerin (1) into the two products
could be accelerated by heating in DMSO-d6 at 100 °C for 2 h,
which led to full conversion of rottlerin (1) into the two
products in an approximately 1:1 ratio. The formation of the
two products required the presence of DMSO, as the use of
MeCN, toluene, acetone, and CHCl3 as solvents did not lead
to any changes in the NMR spectrum of rottlerin (1) even with
intense heating. However, the exact mechanism of this reaction
is yet to be determined. Nevertheless, the long-term storage of
rottlerin (1) as a solid is recommended to prevent the
conversion of rottlerin (1) to isorottlerin (35) and flavanone
36 in DMSO solution. If DMSO stock solutions are required,
storage at low temperature, preferably at −20 °C or below, is
recommended, along with regular NMR spectroscopic
measurements to ensure that rottlerin (1) has not decom-
posed.
Isorottlerin (35) has not been synthesized before, and the
transformation of rottlerin (1) to isorottlerin (35) was both
unexpected and unprecedented. We named this transformation
the “isorottlerin change”, in analogy with the “rottlerone
change”.20 The “rottlerone change” describing the conversion
of rottlerin (1) to rottlerone under basic or acidic conditions
(Scheme 8) has been reported and involves a disproportiona-
tion reaction.21
Antibacterial Activities of Compounds. Rottlerin (1),
its analogues 32−34, and its isomerization products 35 and 36
were tested for their antibacterial activities against S. aureus.
The antimicrobial activity of the synthesized compounds was
evaluated by determination of their MIC values. The clinically
used antibiotic gentamicin was used as a positive control. The
MIC values were also compared with drummondins A (2), B
(3), C (4), and F (5), as they are structurally similar to
rottlerin.8 The results showed that synthesized rottlerin (1)
showed excellent antibacterial activity against S. aureus with an
F
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Scheme 8. “Rottlerone Change” and “Isorottlerin Change” of Rottlerin (1)
MIC value of 2.0 μM, making it comparable in potency with
gentamicin and drummondins A (2), B (3), C (4), and F (5)
(Table 2). On the other hand, the two isomers 35 and 36
allowed rottlerin (1) to be synthesized in gram scale. The
general substrate scope for the synthesis of methylene-bridged
type compounds and rottlerin analogues was also investigated,
revealing that electron-rich arenes were favored for 1,4-Michael
addition reaction with the putative ortho-quinone methide
intermediate. X-ray crystallography analysis of rottlerin (1) and
its analogue also suggested that the conformation of the
molecules in the solid state could be carefully tuned by altering
the substituents on the arene group. Rottlerin (1) was shown
to react with DMSO to undergo an unprecedented
“isorottlerin change” to give the natural product isorottlerin
(35). The mechanism for this transformation remains to be
elucidated. The antibacterial activity of rottlerin and novel
rottlerin analogues 32−36 was also tested against S. aureus.
The results showed that rottlerin (1) was similarly potent to
the clinically used antibiotic gentamicin, outlining the potential
for its development as an antimicrobial agent.
Table 2. MIC Values of Synthesized and Known
Compounds against S. aureus
compound
gentamicin
MIC (μM)
2.0
rottlerin (1)
2.0
32
33
15.6
7.8
34
7.8
isorottlerin (35)
36
drummondins A (2)
drummondins B (3)
drummondins C (4)
drummondins F (5)
>125
>125
1.56
3.12
3.12
0.78
EXPERIMENTAL SECTION
■
1-{4-[(tert-Butyldimethylsilyl)oxy]-2,6-dihydroxyphenyl}-
ethan-1-one (12). To a solution of acetophenone 6 (4.0 g, 21.87
mmol) in dimethylformamide (DMF) (40 mL) at 0 °C were added
imidazole (3.7 g, 53.72 mmol) and TBDMSCl (8.1 g, 53.72 mmol).
The mixture was allowed to stir at room temperature for 15 min, then
poured into water, and extracted with EtOAc. The organic layer was
dried over Na2SO4 and evaporated in vacuo. The residue was
dissolved in MeOH (40 mL), and PPTS (6.7 g, 21.87 mmol) was
added. The mixture was heated at reflux for 2 h, the solvent was
removed in vacuo, water was added, and the mixture was extracted
with EtOAc. The organic layer was dried over Na2SO4 and evaporated
in vacuo. The crude product was purified by flash column
chromatography on silica gel using n-hexane/EtOAc (19:1) to give
12 (6.5 g, 85%) as a white solid: mp 110−114 °C; lit.14 111−113 °C;
1H NMR (400 MHz, CDCl3) δ 11.1 (s, 2H, 2 × OH), 5.93 (s, 2H,
exhibited no antibacterial activity below 125 μM, suggesting
that the chalcone moiety is essential for activity. Analogues
32−34 exhibited good antibacterial activity against S. aureus,
with MICs of 15.6, 7.8, and 7.8 μM, respectively. The lower
antibacterial activity of the analogues 32−34 compared to
rottlerin (1) suggests that the nature of the arene attached to
the methylene bridge is an important determinant of activity.
The underlying mechanism of rottlerin (1) against S. aureus
is currently being investigated by our research group. Recent
studies have suggested some possible mechanisms of the
antiparasitic and antibacterial effects of rottlerin. For instance,
rottlerin (1) has antiparasitic activity against Toxoplasma gondii
through the inhibition of mTOR and elF2α phosphorylation,
which accounts for their autophagy induction and inhibition of
protein synthesis.5 Rottlerin (1) also blocks ceramide
trafficking from host Golgi apparatus to inhibit chlamydial
growth by suppressing the ability of Chlamydia to acquire
lipids.6 The fact that rottlerin (1) targets various signaling
pathways is of interest, and we are currently attempting to
pinpoint the possible mechanism of its antibacterial activity
against S. aureus.
H3, H5), 2.72 (s, 3H, COCH3), 0.93 (s, 9H, C(CH3)3), 0.18 (s, 6H,
Si(CH3)2).
1-{5-[(tert-Butyldimethylsilyl)oxy]-7-hydroxy-2,2-dimethyl-
2H-chromen-8-yl}ethan-1-one (13). To a solution of acetophe-
none 12 (6.4 g, 22.66 mmol) in CHCl3 (50 mL) were added EDDA
(4.1 g, 2.27 mmol) and 3-methyl-2-butenal (2.4 mL, 24.93 mmol).
The reaction mixture was stirred for 10 min, and the solvent was
removed in vacuo. The residue was purified by flash column
chromatography on silica gel using n-hexane/EtOAc (40:1) to give
13 as a yellow solid (7.1 g, 90%) 1H NMR (400 MHz, CDCl3) δ 6.50
(d, J = 10.0 Hz, 1H, H4), 5.91 (s, 1H, H6), 5.39 (d, J = 10.0 Hz, 1H,
H3), 2.64 (s, 3H, COCH3), 1.47 (s, 6H, 2 × CH3), 0.98 (s, 9H,
C(CH3)3), 0.24 (s, 6H, Si(CH3)2).
(E)-1-(5,7-Dihydroxy-2,2-dimethyl-2H-chromen-8-yl)-3-phe-
nylprop-2-en-1-one (9). Chromene 13 (7.0 g, 20.09 mmol) was
dissolved in anhydrous tetrahydrofuran (THF) (40 mL) under an
argon atmosphere. The solution was cooled to 0 °C, and 60% NaH in
mineral oil (4.0 g, 100.45 mmol) was added in small portions over 5
In summary, the synthesis of rottlerin (1) has been
simplified from the previous seven-step synthesis to a practical
five-step gram-scale synthesis that can be achieved in 2 days.
The key to the successful scalable synthesis of rottlerin (1)
involves the use of the TBDMS protecting group to avoid the
formation of rottlerone due to the deprotection of the MOM
group under acidic conditions. The subsequent one-pot aldol
condensation and deprotection is de novo and ultimately
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min. The mixture was allowed to stir for 5 min, and benzaldehyde (4.1
mL, 40.18 mmol) was added. The resulting mixture was allowed to
stir for 2 h, poured into water (50 mL), and stirred for an additional
30 min. The mixture was then extracted with EtOAc (3 × 100 mL),
and the combined organic extracts were washed with brine, dried
using Na2SO4, and evaporated in vacuo. Flash chromatography on
silica gel using n-hexane/EtOAc (8:2) gave 9 (4.5 g, 69%) as a red oil:
1H NMR (400 MHz, CDCl3) δ 14.23 (s, 1H, OH), 8.11 (d, J = 16
Hz, 1H, Hβ), 7.76 (d, J = 16 Hz, 1H, Hα), 7.58−7.62 (m, 2H, ArH),
7.24−7.43 (m, 3H, ArH), 6.59 (d, J = 10 Hz, 1H, H4), 6.01 (s, 1H,
H6), 5.48 (d, J = 10 Hz, 1H, H3), 1.55 (s, 6H, 2 × CH3); 13C NMR
(100 MHz, CDCl3) δ 193.2 (CO), 166.5 (ArC), 158.8 (ArC),
156.9 (ArC), 142.6 (Cβ), 135.7 (ArC), 130.3 (ArH), 129.1 (ArC),
128.4 (Cα), 127.5 (ArH), 124.9 (H3), 116.7 (H4), 106.6 (C8), 102.7
(C4a), 96.5 (C6), 78.4 (C2), 28.1 (2 × CH3); HRMS (ESI) m/z
calcd for C20H18O4 (M + H)+ 323.1278, found 323.1276.
CDCl3) δ 204.2 (C7″), 193.0 (C7), 163.0 (C6), 160.8 (C6″), 159.7
(C2″), 158.9 (C4), 156.6 (C4″), 155.6 (C2), 143.5 (C9), 135.6
(C1′), 130.5 (C4′), 129.2 (C3′,C5′), 128.5 (C2′,C6′), 126.9 (C8),
125.2 (C11), 117.4 (C10), 106.6 (C5), 106.1 (C1″), 105.4 (C1),
104.6 (C3″), 103.9 (C3), 102.0 (C5″), 78.3 (C12), 32.7 (C8″), 28.2
(C13, C14), 16.0 (C15), 7.6 (C9″); HRMS (ESI) m/z calcd for
C30H28O8 (M + H)+ 517.1857, found 517.1851.
1-[6-(3-Acetyl-2,4,6-trihydroxy-5-methylbenzyl)-5,7-dihy-
droxy-2,2-dimethyl-2H-chromen-8-yl]ethan-1-one (16). The
product was obtained as a white solid (25 mg, 34%): mp 186−190
°C; IR (neat) νmax 3324, 2974, 2825, 1611, 1508, 1336, 1117 cm−1;
1H NMR (600 MHz, acetone-d6) δ 6.63 (d, J = 10.0 Hz, 1H, H4),
5.56 (d, J = 10.0 Hz, 1H, H3), 3.78 (s, 2H, CH2), 2.69 (s, 3H,
COCH3), 2.68 (s, 3H, COCH3), 2.07 (s, 3H, CH3), 1.50 (s, 6H, 2 ×
CH3); 13C NMR (150 MHz, acetone-d6) δ 205.1 (CO), 204.6
(CO), 162.2 (C7), 161.0 (C6′), 160.3 (C4′), 159.6 (C2′), 156.8
(C8a), 126.4 (C3), 117.6 (C4), 107.2 (C6), 106.0 (C1′), 105.4
(C5′), 104.2 (C4a), 79.0 (C2), 33.0 (COCH3), 32.8 (COCH3), 27.9
(2 × CH3), 16.4 (CH2), 8.4 (CH3); HRMS (ESI) m/z calcd for
C23H24O8 (M + Na)+ 451.1363, found 451.1359.
1-[6-(2,4-Dihydroxy-6-methylbenzyl)-5,7-dihydroxy-2,2-di-
methyl-2H-chromen-8-yl]ethan-1-one (18). The product was
obtained as a white solid (15 mg, 26%): mp 172−176 °C; IR (neat)
νmax 3252, 2917, 1638, 1561, 1424, 1361, 1210, 1109 cm−1; 1H NMR
(600 MHz, CD3CN) δ 14.49 (s, 1H, OH), 6.55 (d, J = 10.0 Hz, 1H,
H4), 6.25 (d, J = 2.4 Hz, 1H, H3′), 6.24 (d, J = 2.4 Hz, 1H, H5′),
5.48 (d, J = 10.0 Hz, 1H, H3), 3.79 (s, 2H, CH2), 2.64 (s, 3H,
COCH3), 2.23 (s, 3H, CH3), 1.45 (s, 6H, 2 × CH3); 13C NMR (100
MHz, CD3CN) δ 204.7 (CO), 164.0 (C7), 158.9 (C5), 156.4
(C8a), 154.5 (C2′), 126.2 (C3), 117.4 (C4), 116.9 (C1′), 111.5
(C5′), 107.0 (C6), 106.0 (C8), 103.1 (C4a), 100.6 (C6′), 100.4
(C3′), 78.8 (C2), 33.6 (COCH3), 27.9 (2 × CH3), 20.4 (CH3), 18.5
(CH2); HRMS (ESI) m/z calcd for C21H22O6 (M + Na)+ 393.1309,
found 393.1306.
1-[5,7-Dihydroxy-6-(2-hydroxy-4,6-dimethoxybenzyl)-2,2-
dimethyl-2H-chromen-8-yl]ethan-1-one (20). The product was
obtained as a white solid (38 mg, 45%): mp 172−176 °C; UV (THF)
λmax 279 (ε 37 800 cm−1 M−1) nm; IR (neat) νmax 3289, 2924, 1636,
1599, 1516, 1467, 1365, 1268, 1137 cm−1; 1H NMR (600 MHz,
acetone-d6) δ 6.60 (d, J = 10.0 Hz, 1H, H4), 6.22 (d, J = 2.4 Hz, 1H,
H3′), 6.13 (d, J = 2.4 Hz, 1H, H5′), 5.57 (d, J = 10.0 Hz, 1H, H3),
4.00 (s, 3H, OCH3), 3.77 (s, 2H, CH2), 3.73 (s, 3H, OCH3), 2.67 (s,
3H, COCH3), 1.50 (s, 6H, 2 × CH3); 13C NMR (150 MHz, acetone-
d6) δ 203.9 (CO), 161.7 (C8), 160.2 (C4′), 157.9 (C5), 157.2
(C2′), 156.7 (C6′), 155.7 (C8a), 125.4 (C3), 116.3 (C4), 108.2
(C6), 106.0 (C1′), 102.7 (C4a), 95.2 (C3′), 91.0 (C5′), 78.2 (C2),
55.9 (OCH3) 54.3 (OCH3), 32.3 (COCH3), 27.0 (2 × CH3), 15.3
(CH2); HRMS (ESI) m/z calcd for C22H24O7 (M + Na)+ 423.1414,
found 423.1410.
1-[6-(2-Amino-4,6-dimethoxybenzyl)-5,7-dihydroxy-2,2-di-
methyl-2H-chromen-8-yl]ethan-1-one (24). The product was
obtained as a yellow solid (26 mg, 14%): mp 170−174 °C; IR (neat)
νmax 3440, 3298, 2925, 1618, 1590, 1545, 1423, 1134, cm−1; 1H NMR
(600 MHz, CD3CN) δ 15.0 (s, 1H, OH), 6.57 (d, J = 10.0 Hz, 1H,
H4), 6.02 (d, J = 2.4 Hz, 1H, H3′), 6.00 (d, J = 2.4 Hz, 1H, H5′),
5.52 (d, J = 10.0 Hz, 1H, H3), 3.99 (s, 3H, OCH3), 3.73 (s, 2H,
CH2), 3.69 (s, 3H, OCH3), 2.66 (s, 3H, COCH3), 1.48 (s, 6H, 2 ×
CH3); 13C NMR (150 MHz, CD3CN) δ 203.4 (CO), 162.3 (C7),
160.2 (C4′), 158.5 (C5), 157.0 (C6′), 155.4 (C8a), 148.5 (C2′),
125.1 (C3), 116.5 (C4), 105.8 (C6), 104.7 (C8), 103.3 (C1′), 102.3
(C4a), 94.2 (C3′), 87.8 (C5′), 77.9 (C2), 55.8 (OCH3), 54.4
(OCH3), 32.4 (COCH3), 27.0 (2 × CH3), 16.0 (CH2); HRMS (ESI)
m/z calcd for C22H25NO6 (M + H)+ 400.1755, found 400.1753.
1-[6-(4-Amino-2,6-dimethoxybenzyl)-5,7-dihydroxy-2,2-di-
methyl-2H-chromen-8-yl]ethan-1-one (25). The product was
obtained as yellow oil (26 mg, 14%): IR (neat) νmax 3438, 3320, 2970,
1610, 1589, 1464, 1342, 1173, cm−1; 1H NMR (600 MHz, CD3CN) δ
14.3 (s, 1H, OH), 8.65 (s, 1H, OH), 6.54 (d, J = 10 Hz, 1H, H4),
6.04 (s, 2H, H3′, H5′), 5.46 (d, J = 10 Hz, 1H, H3), 4.66 (bs, 2H,
NH2), 3.79 (s, 2H, CH2), 3.78 (s, 6H, 2 × OCH3), 2.63 (s, 3H,
(E)-1-{6-[(Dimethylamino)methyl]-5,7-dihydroxy-2,2-di-
methyl-2H-chromen-8-yl}-3-phenylprop-2-en-1-one (10). To a
solution of chromene 9 (4.50 g, 13.96 mmol) in CHCl3 was added
Eschenmoser’s salt (0.057 g, 0.31 mmol). The mixture was heated at
reflux for 0.5 h. After completion, water (50 mL) was added and the
mixture extracted with CHCl3 (3 × 50 mL). The combined organic
extracts were washed with brine, dried using Na2SO4, and evaporated
in vacuo. A small amount of MeCN was added, and an orange
precipitate was formed, which was filtered and dried to give 10 (4.66
g, 88%) as an orange powder: mp 110−114 °C; IR (neat) νmax 2966,
2925, 1624, 1590, 1340, 1130, 972 cm−1; 1H NMR (400 MHz,
CDCl3) δ 15.14 (s, 1H, OH), 8.11 (d, J = 15.6 Hz, 1H, Hβ), 7.81 (d, J
= 15.6 Hz, 1H, Hα), 7.59−7.63 (m, 2H, ArH), 7.39−7.46 (m, 3H,
ArH), 6.80 (d, J = 10.0 Hz, 1H, H4), 5.51 (d, J = 10.0 Hz, 1H, H3),
4.21 (s, 2H, NCH2), 2.86 (s, 6H, N(CH3)2), 1.56 (s, 6H, 2 × CH3);
13C NMR (150 MHz, CDCl3) δ 193.3 (CO), 166.4 (ArC), 160.1
(ArC), 158.2 (ArC), 143.5 (Cβ), 135.5 (ArC), 130.6 (ArCH), 129.2
(ArC), 128.5 (Cα), 127.1 (ArCH), 125.1 (C3), 117.5 (C4), 106.0
(ArC), 105.4 (ArC), 99.0 (ArC), 79.1 (C2), 51.0 (NCH2), 42.1
(N(CH3)2), 28.5 (2 × CH3); HRMS (ESI) m/z calcd for C23H25NO4
(M + H)+ 380.1856, found 380.1858.
1-{6-[(Dimethylamino)methyl]-5,7-dihydroxy-2,2-dimethyl-
2H-chromen-8-yl}ethan-1-one (15). To a solution of 13 (1.0 g,
2.87 mmol) in CHCl3 (20 mL) were added Eschenmoser’s salt (0.80
g, 4.30 mmol) and 1 M TBAF in THF (2.87 mL, 2.87 mmol). The
reaction was allowed to stir for 5 min. The solution was evaporated to
dryness to give a crude yellow-orange solid. The residue was washed
with hot n-hexane (3 × 20 mL) and filtered while hot. The filtrate was
evaporated in vacuo to give 15 as a bright yellow powder (0.75 g,
90%): mp 146−150 °C; IR (neat) νmax 3150, 2868, 1684, 1640, 1603,
1467, 1361 cm−1; 1H NMR (600 MHz, CDCl3) δ 14.6 (s, 1H, OH),
6.79 (d, J = 10.0 Hz, 1H, H4), 5.45 (d, J = 10.0 Hz, 1H, H3), 4.22 (s,
2H, NCH2) 2.84 (s, 3H, NCH3), 2.82 (s, 3H, NCH3), 1.48 (s, 6H, 2
× CH3); 13C NMR (150 MHz, CDCl3) δ 203.8 (CO), 165.4
(ArC), 159.8 (ArC), 125.0 (C3), 117.2 (C4), 104.6 (ArC), 98.1
(NCH2), 78.4 (C2), 50.1 (NCH2), 33.2 (COCH3), 28.0 (2 × CH3);
HRMS (ESI) m/z calcd for C16H22NO4 (M + H)+ 292.1543, found
292.1544.
General Experimental Procedures for the Synthesis of
Rottlerin (1) and Methylene-Bridged Chromene Compounds
(16, 18, 20, 24, 25, 27, 28, 30, 32−34). Dimethylaminomethyl-
chromene (10 or 15, 1.0 equiv) and arene (1.0 equiv) were added to
MeCN (10 mL), and the mixture was heated at reflux for 2 h. The
reaction was monitored by NMR by taking a small aliquot from the
reaction mixture. The solvent was evaporated in vacuo, and the residue
was purified by flash column chromatography to give the methylene-
bridged chromene compound.
Rottlerin (1). Compound 1 was obtained (1.90 g, 31%) as a red-
orange solid: mp 210−214 °C; 1H NMR (600 MHz, CDCl3) δ 16.51
(s. 1H), 15.60 (bs, 1H, OH), 9.55 (bs, 1H, OH), 8.19 (d, J = 15.6 Hz,
1H, H8), 7.84 (d, J = 15.6 Hz, 1H, H9), 7.59−7.63 (m, 2H, H3′,
H5′), 7.39−7.45 (m, 3H, H2′, H4′, H6′), 6.66 (d, J = 10.1 Hz, 1H,
H10), 5.49 (d, J = 10.1 Hz, 1H, H11), 3.81 (s, 2H, H15), 2.71 (s, 3H,
H8″), 2.08 (s, 3H, H9″), 1.53 (s, 6H, H13, H14); 13C (150 MHz,
H
DOI: 10.1021/acs.jnatprod.8b00917
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
COCH3), 1.47 (s, 6H, 2 × CH3); 13C NMR (150 MHz, CD3CN) δ
203.8 (CO), 164.8 (C7), 159.3 (C1′, C6′), 158.8 (C5), 155.8
(C8a), 149.8 (C4′), 125.2 (C3), 117.7 (C4), 107.7 (C6), 105.8 (C8),
104.5 (C1′), 92.2 (C3′, C5′), 78.4 (C2), 56.0 (2 × OCH3), 33.6
(COCH3), 28.0 (2 × CH3), 16.0 (CH2); HRMS (ESI) m/z calcd for
C22H25NO6 (M + Na)+ 422.1574, found 422.1575.
1-{6-[(1H-Indol-3-yl)methyl]-5,7-dihydroxy-2,2-dimethyl-
2H-chromen-8-yl}ethan-1-one (27). The product was obtained as
a white solid (26 mg, 21%): mp 160−164 °C; IR (neat) νmax 3540,
3220, 1611, 1338, 1203, 1111 cm−1; 1H NMR (600 MHz, CD3CN) δ
14.20 (s, 1H, OH), 9.00 (s, 1H, OH), 7.61 (d, J = 7.9 Hz, 1H, H7′),
7.36 (d, J = 8.1 Hz, H4′), 7.11 (ddd, J = 1.0, 8.1, 15.1 Hz, 1H, H6′),
7.02 (ddd, J = 1.0, 8.1, 15.1 Hz, 1H, H5′), 6.89−6.91 (m, 1H, H2′),
6.54 (d, J = 9.9 Hz, 1H, H4), 5.54 (d, J = 9.9 Hz, 1H, H3), 4.00 (s,
2H, CH2), 2.66 (s, 3H, COCH3), 1.48 (s, 6H, 2 × CH3); 13C NMR
(150 MHz, CD3CN) δ 204.8 (CO), 164.3 (C7), 158.0 (C5), 156.3
(C8a), 137.7 (C7′a), 128.3 (C3′a), 126.5 (C3), 123.5 (C2′), 122.5
(C6′), 119.9 (C7′), 119.7 (C5′), 117.3 (C4), 114.4 (C3′), 112.2
(C4′), 107.6 (C6), 106.5 (C8), 103.1 (C4a), 78.7 (C2), 33.7
(COCH3), 27.8 (2 × CH3), 18.8 (CH2); HRMS (ESI) m/z calcd for
C22H21NO4 (M + Na)+ 386.1363, found 386.1361.
(CH2); HRMS (ESI) m/z calcd for C27H24O7 (M + H)+ 461.1595,
found 461.1590.
(E)-1-[6-(3-Acetyl-2,4,6-trihydroxybenzyl)-5,7-dihydroxy-
2,2-dimethyl-2H-chromen-8-yl]-3-phenylprop-2-en-1-one
(33). The product was obtained as a red solid (40 mg, 20%): mp
248−252 °C; UV (THF) λmax 284 (ε 65916 cm−1 M−1), 356 (47511)
nm; IR (neat) νmax 3224, 2978, 1649, 1539, 1481, 1345, 1217, cm−1;
1H NMR (600 MHz, acetone-d6) δ 8.28 (d, J = 15.6 Hz, 1H, Hβ),
7.82 (d, J = 15.6 Hz, 1H, Hβ), 7.73−7.75 (m, 2H, H3″, H5″), 7.45−
7.51 (m, 3H, H2″, H4″, H6″), 6.68 (d, J = 10.0 Hz, 1H, H4), 5.99 (s,
1H, H5′), 5.56 (d, J = 10.0 Hz, 1H, H3), 3.76 (s, 2H, CH2), 2.66 (s,
3H, COCH3), 1.56 (s, 6H, 2 × CH3); 13C NMR (150 MHz, acetone-
d6) δ 203.8 (CO), 191.5 (CO), 162.8 (C2′), 161.2 (C5′), 155.3
(C8a), 142.3 (Cα), 135.6 (C1″), 130.3 (C4″), 129.1 (C3″, C5″),
128.3 (C2″, C4″), 127.0 (Cβ), 124.6 (C3), 117.3 (C4), 107.1 (C6),
105.7 (C1′), 104.7 (C3′), 104.1 (C4a), 95.6 (C5′), 78.0 (C2), 31.8
(COCH3), 27.1 (2 × CH3), 15.6 (CH2); HRMS (ESI) m/z calcd for
C29H28O8 (M + H)+ 504.1700, found 504.1693.
(E)-1-[6-(2,4-Dihydroxy-6-methylbenzyl)-5,7-dihydroxy-2,2-
dimethyl-2H-chromen-8-yl]-3-phenylprop-2-en-1-one (34).
The product was obtained as a white solid (66 mg, 25%): mp
248−252 °C; UV (THF) λmax 286 (ε 70 389 cm−1 M−1), 351
(29 546) nm; IR (neat) νmax 3244, 2978, 1600, 1508, 1471, 1345,
1113 cm−1; 1H NMR (600 MHz, CD3CN) δ 14.82 (s, 1H, OH), 8.15
(d, J = 15.6 Hz, 1H, Hβ), 7.74 (d, J = 15.6 Hz, 1H, Hα), 7.66−7.70
(m, 2H, H3″, H5″), 7.43−7.47 (m, 3H, H2″, H4″, H6″), 6.58 (d, J =
10.0 Hz, 1H, H4), 6.27 (d, J = 2.4 Hz, 1H, C3′), 6.25 (d, J = 2.4 Hz,
1H, C5′), 5.53 (d, J = 10.0 Hz, 1H, H3), 3.83 (s, 2H, CH2), 2.26 (s,
3H, CH3), 1.51 (s, 6H, 2 × CH3); 13C NMR (150 MHz, CD3CN) δ
192.9 (CO), 164.0 (C7), 158.9 (C5), 155.8 (C4′), 154.8 (C8a),
153.7 (C2′), 141.5 (Cα), 135.5 (C1″), 130.3 (C4″), 129.1 (C3″,
C5″), 128.2 (C2″, C4″), 127.8 (Cα), 125.1 (C3), 116.7 (C4), 116.0
(C6′), 110.4 (C5′), 106.6 (C6), 102.6 (C4a), 99.7 (C3′), 78.1 (C2),
27.1 (2 × CH3), 19.5 (CH3), 17.7 (CH2); HRMS (ESI) m/z calcd for
C28H26O6 (M + Na)+ 481.1622, found 481.1619.
1,1′-{[(1H-Indole-2,3-diyl)bis(methylene)]bis(5,7-dihydroxy-
2,2-dimethyl-2H-chromene-6,8-diyl)}bis(ethan-1-one) (28).
The product was obtained as a white solid (17 mg, 8%): mp 156−
160 °C; IR (neat) νmax 3552, 3260, 1609, 1554, 1460, 1209, 1105
1
cm−1; H NMR (600 MHz, DMSO-d6) δ 14.4 (s, 1H, OH), 8.88 (s,
1H, OH), 8.05 (s, 1H, OH), 7.33 (d, J = 8.0 Hz, 1H, H7″), 7.20 (d, J
= 8.0 Hz, 1H, H4″), 6.95 (ddd, J = 1.1, 8.1, 15.1 Hz, 1H, H6″), 6.85
(ddd, J = 1.1, 8.1, 15.1 Hz, 1H, H5″), 6.58 (d, J = 9.9 Hz, 1H, H4),
6.49 (d, J = 9.9 Hz, 1H, H4′), 5.50 (d, J = 9.9 Hz, 1H, H3), 5.44 (d, J
= 9.9 Hz, 1H, H3′), 4.12 (s, 2H, CH2), 4.09 (s, 2H, CH2), 2.65 (s,
6H, 2 × COCH3), 1.47 (s, 6H, 2 × CH3), 1.44 (s, 6H, 2 × CH3); 13
C
NMR (150 MHz, DMSO-d6) δ 203.0 (2 × CO), 163.5 (ArC),
159.9 (ArC), 155.8 (ArC), 155.2 (ArC), 135.5 (ArC), 128.4 (ArC),
124.7 (ArCH), 124.6 (ArCH), 120.5 (ArCH), 120.4 (ArCH), 118.6
(ArCH), 118.4 (ArCH), 117.0 (ArCH), 116.9 (ArCH), 110.4
(ArCH), 108.0 (ArC), 105.9 (ArC), 104.8 (ArC), 103.2 (ArC),
102.8 (ArC), 77.5 (C2), 77.3 (C2′), 32.4 (2 × COCH3), 26.8 (2 ×
CH3), 26.7 (2 × CH3), 19.1 (CH2), 17.3 (CH2); HRMS (ESI) m/z
calcd for C36H35NO8 (M + Na)+ 632.2255, found 632.2257.
Isorottlerin (35). Rottlerin 1 (50 mg, 0.097 mmol) was dissolved
in DMSO (1 mL) and heated at 100 °C for 2 h. The solution was
cooled and the mixture purified directly by preparative HPLC using a
5 μM column (150 × 10 mm i.d.) at a flow rate of 7 mL min−1 with a
gradient elution from 0 to 100% B (solvent A: 0.1% TFA/H2O and
solvent B: 0.1% TFA/CH3CN) over 60 min, to afford isorottlerin
(35) (10 mg, 20%) as a yellow solid: mp 118−122 °C; IR (neat) νmax
(E)-1-{6-[2,4-Dihydroxy-6-(4-hydroxystyryl)benzyl]-5,7-di-
hydroxy-2,2-dimethyl-2H-chromen-8-yl}ethan-1-one (30). The
product was obtained as a white solid (16 mg, 8%): mp 178−182 °C;
1
3347, 2974, 1616, 1581, 1516, 1438, 1358, 1244, 1103 cm−1; H
1
IR (neat) νmax 3243, 2979, 1594, 1433, 1304, 1170 cm−1; H NMR
NMR (600 MHz, DMSO-d6) δ 12.5 (s, 1H, OH), 11.8 (s, 1H, OH),
11.0 (s, 1H, OH), 9.00 (s, 1H, OH), 7.50−7.54 (m, 2H, H3′, H5′),
7.39−7.44 (m, 2H, H6′, H2′), 7.36−7.39 (m, 1H, H4′), 6.46 (d, J =
9.9 Hz, 1H, H4), 5.58 (dd, J = 2.9, 13.0 Hz, 1H, H8), 5.57 (d, J = 9.9
Hz, 1H, H3), 3.76 (d, J = 15.0 Hz, 1H, CH2), 3.69 (d, J = 15.0 Hz,
1H, CH2), 3.20 (dd, J = 13.0, 17.2 Hz, 1H, H7α), 2.89 (dd, J = 2.9,
17.2 Hz, 1H, H7β), 2.55 (s, 3H, COCH3), 1.93 (s, 3H, CH3), 1.22 (s,
3H, CH3), 1.05 (s, 3H, CH3); 13C NMR (150 MHz, DMSO-d6) δ
203.5 (CO), 197.7 (CO), 161.3 (C6″), 160.0 (C2″), 159.9
(C9a), 159.8 (C10a), 158.8 (C4″), 155.9 (C5), 139.5 (C1′), 129.0
(C2′, C6′), 128.8 (C4′), 127.3 (C3), 126.8 (C3′, C5′), 115.3 (C4),
108.5 (C10), 106.6 (C1″), 102.7 (C5″), 102.6 (C5a, C4a), 78.8
(C8), 78.1 (C2), 42.8 (C7), 33.3 (COCH3), 27.8 (CH3), 27.2 (CH3),
16.9 (CH2), 9.2 (CH3); HRMS (ESI) m/z calcd for C30H28O8 (M +
Na)+ 539.1676, found 539.1674.
6-(3-Acetyl-2,4,6-trihydroxy-5-methylbenzyl)-5-hydroxy-
2,2-dimethyl-8-phenyl-8,9-dihydro-2H,10H-pyrano[2,3-f ]-
chromen-10-one (36). Rottlerin 1 (50 mg, 0.097 mmol) was
dissolved in DMSO (1 mL) and heated at 100 °C for 2 h. The
solution was cooled, and the mixture purified directly by preparative
HPLC using a 5 μM column (150 × 10 mm i.d.) at a flow rate of 7
mL min−1 with a gradient elution from 0 to 100% B (solvent A: 0.1%
TFA/H2O and solvent B: 0.1% TFA/CH3CN) over 60 min, to afford
38 (1.0 mg, 2%) as a yellow solid: mp 127−131 °C; IR (neat) νmax
3262, 2973, 1606, 1553, 1471, 1344, 1284, 1129 cm−1; 1H NMR (600
MHz, DMSO-d6) δ 12.8 (s, 1H, OH), 11.6 (s, 1H, OH), 11.3 (s, 1H,
OH), 9.03 (s, 1H, OH), 7.51−7.54 (m, 2H, H2′, H6′), 7.41−7.46
(600 MHz, CD3CN) δ 14.89 (s, 1H, OH), 7.62 (d, J = 16.0 Hz, 1H,
Hα), 7.38 (d, J = 8.6 Hz, 2H, H2″, H6″), 6.85 (d, J = 16.0 Hz, 1H,
Hβ), 6.78 (d, J = 8.6 Hz, 2H, H3″, H5″), 6.71 (d, J = 2.1 Hz, H5′),
6.54 (d, J = 10.0 Hz, H4), 6.36 (d, J = 2.1 Hz, 1H, H3′), 5.46 (d, J =
10.0 Hz, 1H, H3), 3.94 (s, 2H, CH2), 2.61 (s, 3H, COCH3), 1.42 (s,
6H, 2 × CH3); 13C NMR (150 MHz, CD3CN) δ 204.5 (CO),
163.4 (C7), 159.7 (C5), 157.7 (C4″), 157.1 (C4′), 156.4 (C8a),
155.2 (C2′), 140.8 (C6′), 130.7 (Cβ), 129.1 (C2″, C6″), 126.1 (C3),
125.2 (Cα), 117.5 (C4), 117.0 (C1′), 116.4 (C3″, C5″), 107.8 (C6),
105.8 (C8), 105.6 (C5′), 103.2 (C4a), 102.4 (C3′), 78.8 (C2), 33.5
(COCH3), 27.9 (2 × CH3), 18.0 (CH2); HRMS (ESI) m/z calcd for
C28H26O7 (M + Na)+ 497.1571, found 497.1567.
(E)-1-[5,7-Dihydroxy-2,2-dimethyl-6-(2,4,6-trihydroxyben-
zyl)-2H-chromen-8-yl]-3-phenylprop-2-en-1-one (32). The
product was obtained as a red solid (20 mg, 21%): mp 248−252
°C; UV (THF) λmax 285 (ε 121184 cm−1 M−1), 349 (28376) nm; IR
(neat) νmax 3252, 2972, 1594, 1609, 1545, 1419, 1340, 1223, 1151
1
cm−1; H NMR (600 MHz, acetone-d6) δ 8.28 (d, J = 15.6 Hz, 1H,
Hβ), 7.80 (d, J = 15.6 Hz, 1H, Hα), 7.71−7.73 (m, 2H, H3″, H5″),
7.43−7.51 (m, 3H, H2″, H4″, H6″), 6.67 (d, J = 10.0 Hz, 1H, H4),
5.98 (s, 2H, H3′, H5′), 5.53 (d, J = 10.0 Hz, 1H, H3), 3.74 (s, 2H,
CH2), 1.55 (s, 6H, 2 × CH3); 13C NMR (150 MHz, acetone-d6) δ
192.2 (CO), 163.3 (C7), 158.3 (C4′), 156.9 (C2′, C6′), 156.1
(C8a), 142.8 (Cα), 136.6 (C1″), 131.1 (4″), 130.0 (C3″, C5″), 129.1
(C2″, 6″), 128.1 (Cβ), 125.3 (C3), 118.4 (C4), 108.9 (C6), 106.1
(C1′), 105.1 (C4a), 96.3 (C3′, C5′), 78.7 (C2), 28.2 (2 × CH3), 16.9
I
DOI: 10.1021/acs.jnatprod.8b00917
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(m, 2H, H3′, H5′), 7.37−7.40 (m, 1H, H4′), 6.40 (d, J = 10.0 Hz,
1H, H4), 5.63 (dd, J = 3.0, 12.6 Hz, 1H, H8), 5.54 (d, J = 10.0 Hz,
1H, H3), 3.73 (d, J = 15.0 Hz, 1H, CH), 3.71 (d, J = 15.0 Hz, 1H,
CH), 3.27−3.31 (m, 1H, H9α), 2.87 (dd, J = 3.0, 17.2 Hz, 1H, H9β),
2.61 (s, 3H, COCH3), 1.94 (s, 3H, CH3), 1.13 (s, 3H, CH3), 1.08 (s,
3H, CH3); 13C NMR (150 MHz, DMSO-d6) δ 203.6 (CO), 197.4
(C10), 161.5 (C6″), 160.0 (C2″), 159.9 (C6a), 159.8 (C5), 159.0
(C4″), 154.9 (C10b), 139.2 (C1′), 129.1 (C3′, C5′), 129.0 (C4′),
127.5 (C3), 126.9 (C2′, C6′), 115.5 (C4), 109.1 (C6), 106.4 (C1″),
102.7 (C5″), 101.7 (C4a), 101.0 (C10a), 78.8 (C8), 78.0 (C2), 42.5
(C9), 33.3 (COCH3), 27.6 (CH3), 27.3 (CH3), 16.2 (CH2), 9.1
(CH3); HRMS (ESI) m/z calcd for C30H28O8 (M + Na)+ 539.1676,
found 539.1674.
Antibacterial Assay. The MIC of the active compounds was
determined by following a previously published protocol.22 The
compounds were dissolved in sterilized DMSO and diluted in
autoclaved Millipore water. A single colony of S. aureus was cultured
overnight in tryptone soya broth (TSB) at 37 °C. The resulting
bacteria were collected by centrifugation and resuspended in the same
volume of TSB twice. The optical density (OD) of the resulting
culture was adjusted to 0.1 at 600 nm (equivalent to 108 CFU/mL) in
TSB, and the adjusted culture was further diluted to 105 CFU/mL in
TSB. Then, 150 μL of the bacterial solution was added to wells of a
96-well plate containing 50 μL of serially diluted compound. Two
controls were also prepared: one containing 150 μL of TSB media and
50 μL of sterile water and the other containing 150 μL of bacterial
solution and 50 μL of sterilized water. The plates were incubated at
37 °C for 24 h, and the MIC was recorded by measuring the OD
value at 600 nm using a Wallac Victor (PerkinElmer) microplate
reader. The MIC value was determined as the lowest concentration of
compound that inhibited the complete growth of the bacteria (i.e.,
OD similar to that of the control having no bacteria). Each
experiment was performed in triplicate and was repeated in three
independent experiments.
Facility in the Mark Wainwright Analytical Centre at UNSW
Australia for the use of the instruments.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.8b00917.
■
S
(18) Madadi, N. R.; Zong, H.; Ketkar, A.; Zheng, C.; Penthala, N.
R.; Janganati, V.; Bommagani, S.; Eoff, R. L.; Guzman, M. L.; Crooks,
P. A. MedChemComm 2015, 6, 788−794.
Crystallographic data for rottlerin (1) and compound 18
(CIF)
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AUTHOR INFORMATION
Corresponding Author
*Tel: +61 2 9385 4698. Fax: +61 2 9385 6141. E-mail: n.
kumar@unsw.edu.au.
■
ORCID
Graham E. Ball: 0000-0002-0716-2286
David StC. Black: 0000-0001-9300-4737
Naresh Kumar: 0000-0002-0951-9621
Notes
The authors declare no competing financial interest.
CCDC-1406109 contains supplementary crystallographic data
for rottlerin (1). CCDC-1406110 contains supplementary
crystallographic data for compound 18. These data can be
obtained free of charge from the Cambridge Crystallographic
Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.
ACKNOWLEDGMENTS
■
This work was supported by a Discovery project grant
(DP180100845) from the Australian Research Council.
K.K.C.H. is grateful to UNSW Australia for a Postgraduate
Scholarship. The authors are grateful to the Nuclear Magnetic
Resonance Facility and the Bioanalytical Mass Spectrometry
J
DOI: 10.1021/acs.jnatprod.8b00917
J. Nat. Prod. XXXX, XXX, XXX−XXX