Semi-synthesis and NMR spectral assignments of flavonoid and chalcone derivatives

Article abstract of DOI:10.1002/mrc.4482

Previous investigations of the aerial parts of the Australian plant Eremophila microtheca and Syzygium tierneyanum resulted in the isolation of the antimicrobial flavonoid jaceosidin (4) and 2′,6′-dihydroxy-4′-methoxy-3′,5′-dimethyl chalcone (7), respecti

Full text of DOI:10.1002/mrc.4482

Semi-Synthesis and NMR Spectral Assignments of
Flavonoid and Chalcone Derivatives
Rohitesh Kumara, Yuting Lua, Alysha G. Elliottb, Angela M. Kavanaghb, Matthew A.
Cooperb and Rohan A. Davis*a
a
Address: Eskitis Institute for Drug Discovery, Griffith University, Brisbane, QLD
4111, Australia and bInstitute for Molecular Bioscience, University of Queensland, St
Lucia, QLD, 4072, Australia
Email: Rohan A. Davis - r.davis@griffith.edu.au. Tel.: +61 7 3735 6043.
Fax: +61 7 3735 6001.
* Corresponding author
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been through the copyediting, typesetting, pagination and proofreading process which may
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doi: 10.1002/mrc.4482
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Abstract
Previous investigations of the aerial parts of the Australian plant Eremophila
microtheca and Syzygium tierneyanum resulted in the isolation of the antimicrobial
flavonoid jaceosidin (4) and 2′,6′-dihydroxy-4′-methoxy-3′,5′-dimethyl chalcone (7),
respectively. In this current study, compounds 4 and 7 were derivatized by
acetylation, pivaloylation, and methylation reactions. The final products, 5,7,4′-
triacetoxy jaceosidin (10), 5,7,4′-tripivaloyloxy jaceosidin (11), 5,7,4′-trimethoxy
jaceosidin (12), 2′,6′-diacetoxy-4′-methoxy-3′,5′-dimethyl chalcone (13), 2′-hydroxy-4′-
methoxy-6′-pivaloyloxy-3′,5′-dimethyl chalcone (14), and 2′-hydroxy-4′,6′-dimethoxy-
3′,5′-dimethyl chalcone (15) were all fully characterized by NMR and MS. Derivatives
10 and 13 have been previously reported but were only partially characterized. This
is the first reported synthesis of 11 and 14. The natural products and their derivatives
were evaluated for their antibacterial and antifungal properties, and the natural
product, jaceosidin (4) and the acetylated derivative, 5,7,4′-triacetoxy jaceosidin (10),
showed modest antibacterial activity (32–128 µg/mL) against Staphylococcus aureus
strains.
Keywords
1
NMR; H; 13C; flavonoid; chalcone; Eremophila microtheca; Syzygium tierneyanum;
jaceosidin; 2′,6′-dihydroxy-4′-methoxy-3′,5′-dimethyl chalcone; antimicrobial.
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Introduction
Natural products (NPs) have played a major role in the development of many drugs.1-
3
Some examples of NPs as commercial drugs include the antibacterial agents
daptomycin and fidaxomicin, which were isolated from Streptomyces roseosporus4
and Dactylosporangium aurantiacum,5 respectively. NPs have also served as lead
molecules in drug development programs; noteworthy examples include the sponge
metabolite halichondrin B that was developed into the anticancer drug eribulin,6 and
camptothecin, a plant NP that was developed into the oncology drugs, topotecan and
irinotecan.7 Other notable examples include the semi-synthetic antifungal drugs
caspofungin, anidulafungin, and micafungin that were based on NP lead compounds
isolated from the fermentation products of Glarea lozoyensis,8 Aspergillus nidulans,9
and Coleophoma empetri,10 respectively.
The synthesis of analogues based on a bioactive NP lead is essential to drug
development since this process generates important structure-activity relationship
(SAR) data. Rather than pursuing the total synthesis of a bioactive NP followed by
classic medicinal chemistry for analogue generation, many research groups are now
utilizing isolated NPs as scaffolds for the creation of semi-synthetic analogue
libraries.11,12 This later approach reduces timelines and resource allocation that is
typically associated with de novo multi-step syntheses of a bioactive NP, and can
generate rapid SAR data for faster evaluation of a lead molecule.
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Previous chemical investigations of the aerial parts of the Australian desert plant
Eremophila microtheca by our group resulted in the isolation and characterization of
three new serrulatane diterpenoids, 3-acetoxy-7,8-dihydroxyserrulat-14-en-19-oic
acid (1), 3,7,8-trihydroxyserrulat-14-en-19-oic acid (2), and 3,19-diacetoxy-8-
hydroxyserrulat-14-ene (3), together with the known compounds jaceosidin (4) and
verbascoside (5) (Figure 1).13
These compounds were evaluated for their antibacterial activity against a panel of
Gram-positive and Gram-negative bacterial isolates. The serrulatanes exhibited
moderate activity against Streptococcus pyogenes.13 Jaceosidin (4) was shown to
display the greatest potency (MIC 16–32 µg/mL) against most bacterial strains, while
verbascoside was inactive.13 Jaceosidin has also been shown to inhibit the growth of
the human endometrial cancer Hec1A and KLE cells14 and has been reported to
have potential for the treatment of T cell-mediated immune diseases.15 These
biological data in combination with the low MW (330 Da), available hydroxy moieties
for synthetic elaboration, and the abundant supply of this compound from the plant
source, made 4 an attractive NP scaffold for analogue generation.
In order to further explore the chemical and biological potential of jaceosidin (4) this
compound was acetylated, pivaloylated, and methylated. To supplement this small
series of analogues, a search of the in-house Davis compound library for molecules
possessing the 1,3-diphenylpropan-1-one (6) substructure was conducted (Figure 2).
This library currently contains 472 compounds isolated from Australian biota that
includes endophytic fungi,16 macrofungi,17 plants18 and marine invertebrates.19 The
substructure search resulted in the identification of three related compounds, 2′,6′-
dihydroxy-4′-methoxy-3′,5′-dimethyl chalcone (7),20 5-hydroxy-7-methoxy-6,8-
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dimethyl flavanone (8),21,22 and 2′,6′-dihydroxy-4′-methoxy-3′-methyl chalcone (9)20
(Figure 2).
All these compounds had been previously isolated from the Australian rainforest
plant, Syzygium tierneyanum, which has the common name river cherry.20
Chalcones are secondary metabolite precursors of flavonoids and isoflavonoids in
plants.23 Since chalcone 7 had the potential to be isolated in large quantities (>500
mg) from the fast-growing plant source, this molecule was selected for re-isolation
and subsequent semi-synthesis studies, which included per-acetylation, per-
pivaloylation and per-methylation. It has been reported that chalcone derivatives
show a wide range of biological activities such as inhibition of two influenza viral
strains, H1N1 and H9N222,23 and also antimicrobial, anticancer, anti-HIV, and
antifungal activities.23
Herein, the large scale re-isolation of jaceosidin (4) and 2′,6′-dihydroxy-4′-methoxy-
3′,5′-dimethyl chalcone (7) together with the structural modifications of 4 and 7 are
described, along with the full characterization of all semi-synthetic compounds. The
biological evaluation of the NPs and all derivatives against a key panel of susceptible
and drug-resistant bacteria, and fungi is also reported.
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Results and Discussion
The CH2Cl2 extract of E. microtheca was subjected to silica gel VLC using a n-
hexane/EtOAc stepwise elution, which yielded several flavonoid enriched fractions
that were further purified by C18 HPLC (MeOH/H2O/0.1% TFA) to yield jaceosidin (4,
160 mg, 0.64% dry wt). NMR and MS data of compound 4 were consistent with
literature values.24
For the SAR studies of this flavonoid, a total of three derivatives were synthesized.
This was achieved by reacting 4 with acetic anhydride, trimethylacetyl chloride
(pivaloyl chloride), and TMS-diazomethane. Subsequent purification of each reaction
product by C18 HPLC (MeOH/H2O/0.1% TFA) yielded 5,7,4′-triacetoxy jaceosidin (10,
18.8 mg, 27%), 5,7,4′-tripivaloyloxy jaceosidin (11, 24.2 mg, 27%), and 5,7,4′-
trimethoxy jaceosidin (12, 9.3 mg, 24%).
The CH2Cl2 extract of S. tierneyanum was subjected to silica gel column
chromatography (n-hexane/EtOAc), which yielded 2′,6′-dihydroxy-4′-methoxy-3′,5′-
dimethyl chalcone (7, 1300 mg, 0.43% dry wt). NMR and MS data of compound 7
were consistent with literature values.20 In an identical fashion to the jaceosidin
analogue generation, chalcone 7 was also reacted with acetic anhydride, pivaloyl
chloride, and TMS-diazomethane followed by C18 HPLC (MeOH/H2O/0.1% TFA)
purification and yielded 2′,6′-diacetoxy-4′-methoxy-3′,5′-dimethyl chalcone (13, 35.0
mg, 53%), 2′-hydroxy-4′-methoxy-6′-pivaloyloxy-3′,5′-dimethyl chalcone (14, 21.0 mg,
32%), and 2′-hydroxy-4′,6′-dimethoxy-3′,5′-dimethyl chalcone (15, 7.7 mg, 13%)
(Figure 3).
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The structures of all the analogues (10–15) were determined following 1D (1H and
13C) and 2D NMR (COSY, HSQC, HMBC) and HRESIMS data analysis. For
1
example, the H NMR spectrum for 5,7,4′-tripivaloyloxy jaceosidin (11) in DMSO-d6
showed four aromatic methine signals at δH 7.76 (1H, s), 7.75 (1H, d, J = 2.0 Hz),
7.70 (1H, dd, J = 8.0, 2.0 Hz), and 7.28 (1H, d, J = 8.0 Hz), one olefinic singlet at δH
7.02 (1H, s), two methoxy singlets at δH 3.89 (3H, s), and δH 3.73 (3H, s), and three
singlets at δH 1.40 (9H, s), 1.37 (9H, s), and 1.32 (9H, s), which were all consistent
for a per-pivaloylated jaceosidin product. The aromatic methine and olefinic signals
were assigned following comparison of the chemical shifts with jaceosidin24,25 and
analysis of the 2D NMR data. For instance, the methoxy groups were positioned at
C-3′ and C-6 based on HMBC correlations from the methoxy protons at δH 3.89 and
3.73 to the aromatic carbons at δC 151.4 (C-3′) and 141.6 (C-6), respectively. The
three pivaloyl units were also assigned based on the HMBC correlations from the
pivaloyl protons to the relevant quaternary and carbonyl carbons (Figure 4). ROESY
correlations (Figure 4) from H-8 to 7-OPv and H-5′ to 4′-OPv, and from 6-OMe to 5-
OPv enabled the three pivaloyl groups to be positioned about the flavonoid skeleton
and thus the chemical structure of 11 was fully assigned.
This derivatization chemistry resulted in the synthesis of two new (11 and 14) and
four known (10,26 12,27 13,28 and 1529) analogues. Although the four known
compounds had been previously reported, compounds 1026 and 1328 were only
partially characterized. This paper reports the full NMR assignments for these
compounds. (Tables 1 and 2 and Supplementary Material Tables S14–S17).
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Due to our interest in the discovery and development of anti-infective compounds,
and the previously identified antimicrobial data for jaceosidin,13 all analogues
together with the natural products were screened for antibacterial activity against a
panel of Gram-positive and Gram-negative bacterial isolates (Supplementary
Material Table S18), and for antifungal activity against two fungi (Supplementary
Material Table S18). Compounds 11–15 did not exhibit antibacterial activity against
the Gram-positive (Streptococcus pneumoniae and Staphylococcus aureus MRSA,
VISA, GISA), or Gram-negative (Escherichia coli, Klebsiella pneumoniae MDR,
Acinetobacter baumannii, Pseudomonas aeruginosa) bacteria tested (Table 3 and
Supplementary Material Table S19), and were also inactive against fungi (Candida
albicans and Cryptococcus neoformans) at 128 µg/mL (Supplementary Material
Table S19). However, jaceosidin (4) and its acetylated derivative (10) displayed mild
antibacterial activities against glycopeptide-intermediate and vancomycin resistant S.
aureus strains (GISA and VRSA, respectively) (Table 3).
Conclusion
In conclusion, this paper reports the large-scale extraction and isolation of the
flavonoid jaceosidin (4) and 2′,6′-dihydroxy-4′-methoxy-3′,5′-dimethyl chalcone (7)
from E. microtheca and S. tierneyanum, respectively. The natural products, 4 and 7
were chemically modified to generate the acetylated, pivaloylated and methylated
analogues resulting in a total of six derivatives. The structures of the acetoxy-,
pivaloyloxy-, and methoxy- derivatives of compounds 4 and 7 were determined using
NMR and MS data. This is the first report of pivaloylation of 4 and 7 and the full NMR
assignment of all the analogues (10–15) is reported here. Compounds 4 and 10
displayed modest antibacterial activity (MIC = 32–128 µg/mL) against glycopeptide-
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intermediate and vancomycin resistant S. aureus strains (GISA and VRSA,
respectively).
Experimental
General
NMR spectra were recorded at 30 °C on a Varian 600 MHz Unity INOVA
spectrometer or on a Bruker 500 MHz AVANCE III HD NMR spectrometer at 25 °C.
Samples analyzed with the Varian 600 MHz NMR were dissolved in 200 µL DMSO-
d6 in 3 mm NMR tubes. For the 1H NMR analyses, 8 transients were acquired with a
1 s relaxation delay using 16K data points. The 90° pulse duration was 9.0 μs, and
the spectral width was 10000 Hz. The 13C NMR spectra were obtained with a
spectral width of 37878 Hz using 32K data points. The 90° pulse duration was 10.0
μs. For the two-dimensional experiments including COSY, HSQC and HMBC, all
data were acquired with 801 × 128 (t2 × t1), 1442 × 96 (t2 × t1), 1442 ×200 (t2 × t1)
data points, respectively. Samples analyzed with the Bruker 500 MHz NMR were
1
dissolved in 500 µL DMSO-d6 in 5 mm NMR tubes. For the H NMR analyses, 16
transients were acquired with a 1 s relaxation delay using 65K data points. The 90°
pulse duration was 9.1 μs, and the spectral width was 10000 Hz. The 13C NMR
spectra were obtained with a spectral width of 25000 Hz using 65K data points. The
90° pulse duration was 10.0 μs. For the two-dimensional experiments including
COSY and HMBC, all data were acquired with 2K × 128 data points (t2 × t1), and
HSQC and ROESY were acquired with 2K × 256 data points (t2 × t1). The mixing
1
time for the ROESY experiment was 200 ms. The H and 13C chemical shifts were
referenced to the solvent peaks for DMSO-d6 at δH 2.50 and δC 39.5, respectively. All
the data were analyzed using MestReNova (2013) 8.1.4-12489. LRESIMS were
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recorded on a Waters ZQ mass spectrometer using a Phenomenex Luna C18 (2) 3
µm 100Å (50 × 4.60 mm) column. HRESIMS data was acquired on a 12 T SolariX
XR FT-ICR-MS. For vacuum liquid chromatography (VLC), a sintered glass funnel
(100 × 9.5 mm) and Alltech Davisil silica (97 g, 30-40 µm, 40Å) was used. A Fritsch
Pulverisette 19 Universal Cutting Mill was used to grind plant material. An Edwards
Instrument company Bio-line orbital shaker was used for the extraction of S.
tierneyanum while a Transtek Systems Soniclean 160T was used for the extraction
of E. microtheca. A Merck Kieselgel 60 flash glass column (55 × 150 mm) with Merck
Kieselgel 60 was used for column chromatography. A Waters 600 pump equipped
with a Waters 966 PDA detector and a Gilson 715 liquid handler was used for semi-
preparative separation. Alltech C18 bonded silica (35-75 µm, 150 Å) and an Alltech
stainless steel guard cartridge (10 × 30 mm) were used for pre-adsorption work. A
ThermoElectron C18 Betasil 5 µm 143 Å (21.2 × 150 mm) column was used for semi-
preparative HPLC separations while a Phenomenex Luna C18(2) 5 µm 100 Å (250 ×
150 mm) column was used to purify the products. All solvents used for
chromatography and MS were Lab Scan HPLC grade, and the H2O was Millipore
Milli-Q PF filtered. All synthetic reagents were obtained from Sigma–Aldrich and
used without further purification.
Collection of plant material
The aerial parts of E. microtheca were collected from South Brisbane, Queensland,
Australia during April 2013. A voucher specimen (RAD039) has been deposited at
the Eskitis Institute, Griffith University, Brisbane, Australia.
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The leaves of S. tierneyanum were collected from South Brisbane, Queensland,
Australia during August 2013. A voucher specimen (RAD003) has been deposited at
the Eskitis Institute, Griffith University, Brisbane, Australia. Both the plants E.
microtheca and S. tierneyanum were cultivated, collected and identified by R. Davis
(Griffith University). All plants were air-dried, ground to a fine powder, and stored at
room temperature prior to extraction.
Extraction and isolation
E. microtheca: The air-dried and ground aerial parts of E. microtheca (194 g) were
extracted with CH2Cl2 (3 × 1 L) and sonicated (30 min × 3). The CH2Cl2 extract was
dried under reduced pressure to yield a green gum (25 g). A portion (5 g) of the
CH2Cl2 extract was pre-adsorbed to C18-bonded silica and then subjected to
preparative C18 HPLC. Isocratic conditions of 50%H2O (0.1% TFA)/50% MeOH
(0.1% TFA) were held for 10 min, followed by a linear gradient to MeOH (0.1% TFA)
over 70 min, then isocratic conditions of MeOH (0.1% TFA) for 10 min, all at a flow
rate of 20 mL/min that resulted in semi-pure jaceosidin. This was pre-adsorbed to
C18-bonded silica and then subjected to semi-preparative C18 HPLC. Isocratic
conditions of 70% H2O (0.1% TFA)/30% MeOH (0.1% TFA) were held for 10 min,
followed by a linear gradient to MeOH (0.1% TFA) over 40 min, then isocratic
conditions of MeOH (0.1% TFA) for 10 min, all at a flow rate of 9 mL/min. Sixty
fractions (60 × 1 min) were collected from the start of the HPLC run. Following
1
repeated HPLC purification, (+)-LRESIMS and H NMR spectroscopy, fraction 40
was shown to contain pure jaceosidin (4, 160 mg).
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S. tierneyanum: The air-dried and ground leaves of S. tierneyanum (300 g) were
extracted with CH2Cl2 (3 × 1 L) while being shaken at 150 rpm. The CH2Cl2 extract
was dried under reduced pressure to yield a green gum (10.2 g). This was pre-
adsorbed to Merck Kieselgel 60 (50g) overnight and was loaded onto n-hexane
equilibrated Merck Kieselgel 60 flash column (55 × 150 mm). The extract was
fractionated with a 5% stepwise gradient solvent system from 100% n-hexane to
50% n-hexane/50% EtOAc (200 mL). 180 fractions were collected and monitored by
1
TLC. Similar fractions were pooled. Based on TLC and H NMR analyses, fractions
that eluted between 15% and 30% EtOAc were combined to yield pure 2′,6′-
dihydroxy-4′-methoxy-3′,5′-dimethyl chalcone (7, 1300 mg).
Synthesis of 5,7,4′-triacetoxy jaceosidin
Jaecosidin (4, 50 mg, 0.152 mmol) was dissolved in dry pyridine (1 mL) and
anhydrous Ac2O (1 mL) was added dropwise and the mixture was stirred for 16 h at
room temperature. The reaction mixture was dried under N2 and the resulting crude
product was pre-adsorbed to C18 silica and packed into a guard cartridge that was
subsequently attached to a semi-preparative Phenomenex Luna C18 column for
semi-preparative C18 HPLC. Isocratic conditions of 70% H2O (0.1% TFA)/30% MeOH
(0.1% TFA) were held for 10 min, followed by a linear gradient to MeOH (0.1% TFA)
over 40 min, then isocratic conditions of MeOH (0.1% TFA) for 10 min, all at a flow
rate of 9 mL/min. Sixty fractions (60 × 1 min) were collected from the start of the
HPLC run. The acetylated product, 5,7,4′-triacetoxy jaceosidin (10, 18.8 mg, 27%),
eluted in fraction 37.
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Synthesis of 5,7,4′-tripivaloyloxy jaceosidin
Pivaloyl chloride (180 mg, 1.52 mmol) was added dropwise to the mixture of pyridine
(1 mL) and jaceosidin (4, 50 mg, 0.152 mmol) at 0 °C with constant stirring. The
mixture was allowed to warm to room temperature and then stirred for 16 h. The
solvents were evaporated to dryness and the remaining residue was pre-adsorbed to
C18 silica and packed into a guard cartridge that was subsequently attached to a
semi-preparative Phenomenex Luna C18 column for semi-preparative HPLC.
Isocratic conditions of 70% H2O (0.1% TFA)/30% MeOH (0.1% TFA) were held for
10 min, followed by a linear gradient to MeOH (0.1% TFA) over 40 min, then
isocratic conditions of MeOH (0.1% TFA) for 10 min, all at a flow rate of 9 mL/min.
Sixty fractions (60 × 1 min) were collected from the start of the HPLC run. The
pivaloylated product, 5,7,4′-tripivaloyloxy jaceosidin (11, 24.2 mg, 28%), eluted in
fraction 52.
Synthesis of 5,7,4′-trimethoxy jaceosidin
Jaecosidin (4, 34.4 mg, 0.104 mmol) was dissolved in anhydrous MeOH and CH2Cl2
1:1 (500 µL) and TMS-diazomethane (165 µL, 1.04 mmol) was added dropwise and
was stirred for 16 h. The mixture was dried under N2 and the resulting crude was
pre-adsorbed to C18 silica and packed into a guard cartridge that was subsequently
attached to a semi-preparative Phenomenex Luna C18 column for semi-preparative
C18 HPLC separation. Isocratic conditions of 70% H2O (0.1% TFA)/30% MeOH
(0.1% TFA) were held for 10 min, followed by a linear gradient to MeOH (0.1% TFA)
over 40 min, then isocratic conditions of MeOH (0.1% TFA) for 10 min, all at a flow
rate of 9 mL/min. Sixty fractions (60 × 1 min) were collected from the start of the
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HPLC run. The methylated product, 5,7,4′-trimethoxy jaceosidin (12, 9.3 mg, 24%),
eluted in fraction 37.
Synthesis of 2′,6′-diacetoxy-4′-methoxy-3′,5′-dimethyl chalcone
2′,6′-dihydroxy-4′-methoxy-3′,5′-dimethyl chalcone (7, 51.6 mg, 0.173 mmol) was
dissolved in dry pyridine (0.5 mL) and anhydrous Ac2O (0.5 mL) was added dropwise
and the mixture was stirred for 16 h at room temperature. The reaction mixture was
dried under N2 and the resulting crude product was pre-adsorbed to C18 silica and
packed into a guard cartridge that was subsequently attached to a semi-preparative
Phenomenex Luna C18 column for semi-preparative HPLC. Isocratic conditions of
50% H2O (0.1% TFA)/50% MeOH (0.1% TFA) were held for 10 min, followed by a
linear gradient to MeOH (0.1% TFA) over 40 min, then isocratic conditions of MeOH
(0.1% TFA) for 10 min, all at a flow rate of 9 mL/min. Sixty fractions (60 × 1 min)
were collected from the start of the HPLC run. The acetylated product, 2′,6′-
diacetoxy-4′-methoxy-3′,5′-dimethyl chalcone (13, 35.0 mg, 53%), eluted in fraction
36.
Synthesis of 2′-hydroxy-4′-methoxy-6′-pivaloyloxy-3′,5′-dimethyl chalcone
Pivaloyl chloride (102 mg, 0.851 mmol) was added dropwise to the mixture of
pyridine (0.5 mL) and 2′,6′-dihydroxy-4′-methoxy-3′,5′-dimethyl chalcone (7, 50.7 mg,
0.170 mmol) at 0 °C with constant stirring. The mixture was allowed to warm to room
temperature and was stirred for 16 h. The solvents were evaporated to dryness
under N2 and the remaining residue was pre-adsorbed to C18 silica and packed into a
guard cartridge that was subsequently attached to a semi-preparative Phenomenex
Luna C18 column for semi-preparative HPLC. Isocratic conditions of 50% H2O (0.1%
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TFA)/50% MeOH (0.1% TFA) were held for 10 min, followed by a linear gradient to
MeOH (0.1% TFA) over 40 min, then isocratic conditions of MeOH (0.1% TFA) for 10
min, all at a flow rate of 9 mL/min. Sixty fractions (60 × 1 min) were collected from
the start of the HPLC run. The compound, 2′-hydroxy-4′-methoxy-6′-pivaloyloxy-3′,5′-
dimethyl chalcone (14, 21 mg, 32%), eluted in fractions 53–54.
Synthesis of 2′-hydroxy-4′,6′-dimethoxy-3′,5′-dimethyl chalcone
2′,6′-dihydroxy-4′-methoxy-3′,5′-dimethyl chalcone (7, 54.8 mg, 0.184 mmol) was
dissolved in anhydrous MeOH and CH2Cl2 1:1 (500 µL) and TMS-diazomethane
(145 µL, 0.919 mmol) was added dropwise and was stirred for 16 h. The mixture was
dried under N2 and the resulting crude was pre-adsorbed to C18 silica and packed
into a guard cartridge that was subsequently attached to a semi-preparative
Phenomenex Luna C18 column for semi-preparative HPLC purification. Isocratic
conditions of 50% H2O (0.1% TFA)/50% MeOH (0.1% TFA) were held for 10 min,
followed by a linear gradient to MeOH (0.1% TFA) over 40 min, then isocratic
conditions of MeOH (0.1% TFA) for 10 min, all at a flow rate of 9 mL/min. Sixty
fractions (60 × 1 min) were collected from the start of the HPLC run. The methylated
product, 2′-hydroxy-4′,6′-dimethoxy-3′,5′-dimethylchalcone (15, 7.7 mg, 13%), eluted
in fractions 33–34.
5,7,4′-Triacetoxy jaceosidin (10): white powder (18.8 mg, 27%); UV (MeOH) (log ε)
212 (5.20), 237 (4.95), 265 (4.90), 316 (4.96) nm; IR νmax 1759, 1639, 1453, 1355,
1
13
1190, 1165, 1071, 899, 857 cm–1; H NMR (600 MHz, DMSO-d6), C NMR (125
MHz, DMSO-d6) see Supplementary Material Table S14; (+)-LRESIMS m/z 457
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(100) [M + H]+ ; (+)-HRESIMS m/z 479.0949 [M + Na]+ (calcd for C23H20O10Na,
479.0948).
5,7,4′-Tripivaloyloxy jaceosidin (11): white powder (24.2 mg, 28%); UV (MeOH) (log
ε) 207 (5.00), 243 (4.68), 265 (4.65), 315 (4.74) nm; IR νmax 1756, 1647, 1466, 1271,
1096, 892 cm–1; 1H NMR (600 MHz, DMSO-d6), 13C NMR (125 MHz, DMSO-d6) see
Table 1 and Supplementary Material Table S15; (+)-LRESIMS m/z 583 (100) [M +
H]+ ; (+)-HRESIMS m/z 605.2350 [M + Na]+ (calcd for C32H38O10Na, 605.2357).
5,7,4′-Trimethoxy jaceosidin (12): white powder (9.3 mg, 24%); UV (MeOH) (log ε)
213 (4.66), 239 (4.36), 266 (4.16), 328 (4.40) nm; IR νmax 1628, 1586, 1513, 1457,
1
1323, 1258, 1115, 1018, 815 cm–1; H NMR (500 MHz, DMSO-d6), 13C NMR (125
MHz, DMSO-d6) see Supplementary Material Table S14; (+)-LRESIMS m/z 373
(100) [M + H]+; (+)-HRESIMS m/z 395.1103 [M + Na]+ (calcd for C20H20O7Na,
395.1101).
2′,6′-Diacetoxy-4′-methoxy-3′,5′-dimethyl chalcone (13): bright yellow gum (35 mg,
53%); UV (MeOH) (log ε) 205 (5.77), 219 (5.54), 299 (5.68) nm; IR νmax 1765, 1648,
1
1603, 1187, 1099, 1077, 992, 874 cm–1; H NMR (500 MHz, DMSO-d6), 13C NMR
(125 MHz, DMSO-d6) see Supplementary Material Table S16; (+)-LRESIMS m/z 383
(100) [M + H]+ 787 [2M + Na]+; (+)-HRESIMS m/z 405.1669 [M + Na]+ (calcd for
,
C22H22O6Na, 405.1672). aSignals are interchangeable.
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2′-Hydroxy-4′-methoxy-6′-pivaloyloxy-3′,5′-dimethyl chalcone (14): bright orange
powder (18.8 mg, 27%); UV (MeOH) (log ε) 204 (4.73), 226 (4.40), 323 (4.53), 316
(4.96) nm; IR νmax 3298, 1749, 1643, 1570, 1324, 1104, 994, 759 cm–1; 1H NMR (500
MHz, DMSO-d6), 13C NMR (125 MHz, DMSO-d6) see Table 2 and Supplementary
Material Table S17; (+)-LRESIMS m/z 383 (100) [M + H]+, 787 [2M + Na]+; (+)-
HRESIMS m/z 405.1316 [M + Na]+ (calcd for C23H26O5Na, 405.1309).
2′-Hydroxy-4′,6′-dimethoxy-3′,5′-dimethyl chalcone (15): yellow gum (7.7 mg, 13%);
UV (MeOH) (log ε) 205 (4.56), 227 (3.37),324 (3.86) nm; IR νmax 3300, 1593, 1563,
1
1344, 1141, 1109, 992 cm–1; H NMR (500 MHz, DMSO-d6), 13C NMR (125 MHz,
DMSO-d6) see Supplementary Material Table S16; (+)-LRESIMS m/z 313 (100) [M +
H]+; (+)-HRESIMS m/z 335.1255 [M + Na]+ (calcd for C19H20O4Na, 335.1254).
Antibacterial assay
Bacteria were obtained from American Type Culture Collection (ATCC; Manassas,
VA, US), Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA)
or The University of Queensland Centre for Clinical Research (UQCCR). The tested
bacteria strains (detailed in Supplementary Material Table S18) were cultured in
Cation-adjusted Mueller Hinton broth (CAMHB) (Bacto laboratories, Cat. no. 212322)
at 37 °C overnight with shaking (200 RPM). A sample of each culture was then
diluted 40-fold in fresh CAMHB broth and incubated with shaking (200 RPM) at 37
°C for 2-3 h. A dose response MIC (Minimum Inhibitory Concentration) assay was
performed using a standard microdilution assay according to the Clinical and
Laboratory Standard Institutes (CLSI) guidelines. To prepare compound containing
assay plates, compounds were serially diluted two-fold across the wells of 384-well
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plates (Corning; 3640, non-binding surface (NBS) plates) with concentrations
ranging from 1.28 mg/mL – 1.25 µg/mL (volume 5 µL), plated in duplicate (n = 2).
The resultant mid-log phase cultures were diluted and 45 µL was added to each well
of the compound-containing plates, giving a final cell density of 5105 CFU/mL and a
final compound concentration range of 128 – 0.125 µg/mL. Plates were covered and
incubated at 37 °C for 18 h. MICs were determined visually, being defined as the
lowest concentration showing no visible growth. Vancomycin (Sigma 861987), and
colistin sulfate (Sigma C4461) were used as positive inhibitor controls for Gram-
positive and Gram-negative strains respectively, at 4 concentrations, with 2 above
and 2 below the expected MIC value. For the strain-compound combinations that
gave positive inhibition within the tested concentration range the assay was repeated
for a final data set of n = 4.
Antifungal assay
Fungi strains (as detailed in Supplementary Material Table S18) were cultured for 3
days on Yeast Extract-Peptone Dextrose (YPD) agar at 30 C. A yeast suspension of
1×106 to 5×106 CFU/mL (as determined by OD530) was prepared from five colonies.
The stock suspensions were diluted with Yeast Nitrogen Base (YNB) broth and 45
μL was added to each well of the compound-containing plates (5 µL, as prepared for
the antibacterial assay) to give a final cell density of 2.5 × 103 CFU/mL and a final
compound concentration range of 128 – 0.125 µg/mL for the tested samples. Plates
were covered and incubated at 35 C for 24 h without shaking.
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Growth inhibition of C. albicans was determined by measuring absorbance at 530
nm (OD530), while the growth inhibition of C. neoformans was determined by
measuring the difference in absorbance between 600 and 570 nm (OD600-570), after
the addition of resazurin (0.001% final concentration) and incubation at 35 C for an
additional 2 h. The absorbance was measured using a Biotek Synergy HTX plate
reader. The percentage of growth inhibition was calculated for each well, using the
negative control (media only) and positive control (fungi without inhibitors) on the
same plate. The significance of the inhibition values was determined by Z-scores,
calculated using the average and standard deviation of the sample wells (no
controls) on the same plate. Samples with inhibition value above 80% and Z-Score
above 2.5 for either replicate (n = 2 on different plates) were classed as active at 128
μg/mL. Fluconazole (Sigma, F8929) was used as a positive inhibitor control, at 4
concentrations, with 2 above and 2 below its expected MIC value.
Supplementary Material
13
1
13
1H and C NMR spectra for compounds 10–15, H, C NMR and 2D NMR data of
compounds 10–15 and a list of microbial strains tested and the antimicrobial results.
Acknowledgements
The authors acknowledge the National Health and Medical Research Council
(NHMRC) for financial support (Grant APP1024314 to RAD, and AF511105 to MAC)
and thank the Australian Research Council (ARC) for support towards NMR and MS
equipment (Grant LE0668477 and LE0237908) and financial support (Grant
LP120200339 to RAD). We acknowledge W. Loa (Griffith University) for acquiring
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the HRESIMS measurements. RK would like to thank Griffith University for the
Griffith University Postgraduate Research Scholarship (GUPRS) and Griffith
University International Postgraduate Research Scholarship (GUIPRS). Antimicrobial
screening was performed by CO-ADD (The Community for Antimicrobial Drug
Discovery), funded by the Wellcome Trust (UK) and The University of Queensland
(Australia). Strains were sourced from either ATCC (America Type Culture
Collection, http://www.atcc.org/) or NARSA (Network on Antimicrobial Resistance in
Staphylococcus aureus) via BEI Resources (www.beiresources.org), and S. aureus
Clinical isolate mMRSA was kindly provided by David Patterson of The University of
Queensland Centre for Clinical Research. (UQCCR).
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Table 1: NMR data of 5,7,4′-tripivaloyloxy jaceosidin (11)a
Position 1H (int., mult., J in Hz)
13C, type
1
2
160.9, C
3
7.02 (1H, s)
107.5, CH
4
175.60, C
5
142.4, C
6
141.6, C
7
148.4, C
8
7.76 (1H, s)
110.9, CH
9
152.0, C
10
115.6, C
1′
129.5, C
2′
7.75 (1H, d, 2.0)
110.5, CH
3′
151.4, C
4′
142.6, C
5′
6′
7.28 (1H, d, 8.0)
7.70 (1H, dd, 8.0, 2.0)
1.40 (9H, s)
3.73 (3H, s)
1.37 (9H, s)
123.3, CH
118.9, CH
175.29, C; 38.6, C; 26.9, CH3
61.8, CH3
175.26, C; 38.7, C; 26.6, CH3
56.3, CH3
5-OPv
6-OMe
7-OPv
3′-OMe
4′-OPv
3.89 (3H, s)
1.32 (9H, s)
175.45, C; 38.5, C; 26.8, CH3
a Spectra were recorded on 600 MHz (for 1H) and 150 MHz (for 13C) NMR in DMSO-d6 at 30 °C.
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Table 2: NMR data of 2′-hydroxy-4′-methoxy-6′-pivaloyloxy-3′,5′-dimethylchalcone
(14)a
Position
1
2
3
4
5
6
7
8
9
1′
2′
3′
4′
5′
6′
1H (int., mult., J in Hz)
13C, type
134.4, C
128.6, CH
129.1, CH
130.8, CH
129.1, CH
128.6, CH
144.1, CH
127.2, CH
194.0, C
116.6, C
155.7, C
114.8, C
156.0, C
115.1, C
151.9, C
7.74 (1H, dd, 7.0, 2.0)
7.45 (1H, m)
7.46 (1H, m)
7.45 (1H, m)
7.74 (1H, dd, 7.0, 2.0)
7.62 (1H, d, 15.9)
7.55 (1H, d, 15.9)
2′-OH
3′-Me
4′-OMe
5′-Me
6′-OPv
11.24 (1H, s)
1.95 (3H, s)
3.61 (3H, s)
1.92 (3H, s)
1.37 (9H, s)
8.9, CH3
62.2, CH3
8.9, CH3
175.0, C; 38.9, C; 26.9, CH3
a Spectra were recorded on 500 MHz (for 1H) and 125 MHz (for 13C) NMR in DMSO-d6 at 25 °C
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Table 3: Antimicrobial activity, MIC (Minimum Inhibitory Concentration) of 4, 7–15
against Gram-positive bacteria.
S. aureus
S. aureus
S. aureus
S. aureus S. aureus
S. pneumoniae
ATCC 7000677
MDR
ATCC 43300 Clinical isolate VRS 10
NRS 17
GISA
NRS 1
GISA, MRSA
MRSA
mMRSA
VRS
MIC [µg/mL] n = 2, n = 4*
Van
4
1
1
>64
8
8
1
>128
>128
>128
>128
>128
128
>128
>128
>128
64
128
128
>128
>128
>128
128
128
>128
>128
>128
>128
>128
7
>128
>128
>128
32/64
>128
>128
>128
32/64
8
9
10
11
12
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
13
14
15
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
* those deemed to be active at ≤128 µg/mL were repeated for n = 4; Vancomycin (Van) inhibitory
values within the expected range.
NB negative inhibitory data against Gram-negative bacteria and fungi is displayed in Table S19.
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Figure 1: Chemical structures of compounds isolated from E. microtheca.
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Figure 2: Chemical structures of 1,3-diphenylpropan-1-one (6), and the natural
products 2′,6′-dihydroxy- 4′-methoxy-3′,5′-dimethyl chalcone (7), 5-hydroxy-7-
methoxy-6,8-dimethyl flavanone (8), and 2′,6′- dihydroxy-4′-methoxy-3′-methyl
chalcone (9).
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Figure 3: Chemical structure of the natural products jaceosidin (4) and 2′,6′-
dihydroxy-4′-methoxy-3′,5′- dimethyl chalcone (7) and their semi-synthetic derivatives
10–15.
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Figure 4: Key HMBC and ROESY correlations for 11.
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