Chlorinated coumarins from the polypore mushroom fomitopsis officinalis and their activity against mycobacterium tuberculosis

Article abstract of DOI:10.1021/np400497f

An EtOH extract of the polypore mushroom Fomitopsis officinalis afforded two new naturally occurring chlorinated coumarins, which were identified as the previously synthesized compounds 6-chloro-4-phenyl-2H-chromen-2-one (1) and ethyl 6-chloro-2-oxo-4-phe

Full text of DOI:10.1021/np400497f

Article
pubs.acs.org/jnp
Chlorinated Coumarins from the Polypore Mushroom Fomitopsis
officinalis and Their Activity against Mycobacterium tuberculosis
Chang Hwa Hwang,†,‡ Birgit U. Jaki,†,‡ Larry L. Klein,‡ David C. Lankin,† James B. McAlpine,†
Jose G. Napolitano,† Nicole A. Fryling,§ Scott G. Franzblau,‡ Sang Hyun Cho,‡ Paul E. Stamets,§
́
Yuehong Wang,‡ and Guido F. Pauli*,†,‡
†Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois
60612, United States
‡Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, United States
§Fungi Perfecti, P.O. Box 7634, Olympia, Washington 98507, United States
S
* Supporting Information
ABSTRACT: An EtOH extract of the polypore mushroom Fomitopsis officinalis afforded two
new naturally occurring chlorinated coumarins, which were identified as the previously
synthesized compounds 6-chloro-4-phenyl-2H-chromen-2-one (1) and ethyl 6-chloro-2-oxo-4-
phenyl-2H-chromen-3-carboxylate (2). The structures of the two isolates were deduced by ab
initio spectroscopic methods and confirmed by chemical synthesis. In addition, an analogue of
each was synthesized as 7-chloro-4-phenyl-2H-chromen-2-one (3) and ethyl 7-chloro-2-oxo-4-
phenyl-2H-chromen-3-carboxylate (4). All four compounds were characterized physicochemi-
cally, and their antimicrobial activity profiles revealed a narrow spectrum of activity with lowest
MICs against the Mycobacterium tuberculosis complex.
Tuberculosis (TB) is one of the leading causes of mortality
among infectious diseases.1 Multidrug resistance and long
treatment duration have been the primary obstacles to global
TB control.2,3 Furthermore, emergence of extensive drug-
resistant TB (XDR-TB) and TB with HIV co-infection have
become public health threats throughout the world.4−6 TB is an
ancient disease, known for almost 9000 years, and hence there
exists an abundance of ethnomedical information. Such
knowledge may facilitate the search for new anti-TB agents.
Ethnomedical usage suggests relative safety and efficacy,
although the latter may be due partially or entirely to
symptomatic relief.7
iterative full spin analysis (HiFSA) to facilitate future
dereplication, and their biological activity was extensively
profiled, focusing on Mycobacterium tuberculosis susceptibility
assays.
RESULTS AND DISCUSSION
■
Isolation and Structure Elucidation. Two new naturally
occurring coumarins, 1 (0.5 mg) and 2 (0.2 mg), were isolated
from an EtOH extract of the polypore mushroom F. officinalis.
For 1, the HRMS measurement indicated the presence of one
chlorine in the molecule with [M + H]+ molecular ion peaks for
35Cl and 37Cl isotopes at m/z 257.0402 and 259.0378,
respectively, corresponding to a molecular formula of
C15H9ClO2. The 1H, 13C DEPTQ, and HSQC NMR
(MeOH-d4) spectra at 900/225 MHz confirmed the presence
of six quaternary (δ 160.5, 155.1, 152.6, 134.6, 129.4, and 120.2
ppm) and nine methine carbons (δ 131.8, 129.8, two at 128.8,
two at 128.2, 125.9, 118.6, and 115.5 ppm). The HRMS
measurements and the 13C and DEPTQ NMR were in
agreement with the molecular formula of C15H9ClO2. The
Fomitopsis officinalis is a wood rotting polypore fungus found
in the old growth forests of Oregon and Washington in the
Pacific Northwest, United States, and in British Columbia,
Canada, as well as in the northern regions of China and Europe.
It has been used for the treatment of TB, pneumonia, cough,
and asthma.8−11 While F. officinalis is known to be a rich source
of unusual triterpenoids, only a few chemical studies have been
reported, mostly on the isolation of terpenes.11−15 The present
study describes the isolation of two new naturally occurring
metabolites from an anti-TB mycelial culture extract (EtOH) of
F. officinalis cultivated at Fungi Perfecti, LLC. The structures of
the two metabolites were confirmed by chemical synthesis,
together with the synthesis of two analogues. For the
unambiguous chemical and biological characterization of the
1
1
1D H, 13C DEPTQ, 2D H−1H COSY, HSQC, and HMBC
NMR spectra for 1 are provided in the Supporting Information
(S1−S5). The aromatic region of the 1H NMR spectrum clearly
showed the presence of one AMX and one AA′BB′C spin
1
four compounds, their H NMR fingerprints including all J-
Received: June 22, 2013
1
couplings and chemical shifts were completely resolved by H
© XXXX American Chemical Society and
American Society of Pharmacognosy
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system, indicative of a tri- and a monosubstituted aromatic ring,
respectively, which was confirmed in the COSY spectrum
(Figure 1). In addition, the IR spectrum exhibited a strong
4 as well as H-7 to C-8a and C-5 confirmed the remaining
coumarin skeleton. Compound 1 turned out to be a new
naturally occurring chlorinated coumarin and was identified as
the previously synthesized compound20 6-chloro-4-phenyl-2H-
chromen-2-one. Since only submilligram quantities could be
isolated, the compound was chemically synthesized in order to
enable in-depth chemical and biological evaluation. The
isolated and the synthetic product resulted in identical 13C
DEPTQ spectra (Figure 2).
Figure 1. Key 2D correlations used for the structure determination of
1 and 2.
carbonyl absorption band at 1731 cm−1, which was consistent
with an ester carbonyl resonance in the DEPTQ spectrum
corresponding to the quaternary carbonyl carbon C-2 (δ
160.5). Long-range 1H, 13C couplings from both aromatic
protons H-10/H-14 (δ 1.036) on the stated phenyl ring to the
quaternary carbon C-4 (δ 155.1) confirmed the phenyl
substitution at C-4. HMBC correlations from the olefinic
proton H-3 (δ 6.405) to C-2 (δ 160.5), C-4a (δ 120.2), and C-
9 (δ 134.6) strengthened the evidence for phenyl substitution
at C-4. The presence of an aromatic AMX spin system with a
dd splitting pattern for H-7 (8.84, 2.50 Hz) was consistent with
ortho-coupling to H-8 and meta-coupling to H-5, respectively,
placing the chlorine substitution at C-6 (δ 129.4). Additional
HMBC correlations of H-5 (δ 7.433) to C-8a (δ 152.6) and C-
In order to unambiguously differentiate the structure of 1
from a possible isomer with chlorine substitution at C-7, the
compound 7-chloro-4-phenyl-2H-chromen-2-one, 3, was syn-
1
thesized as well. The H and 13C DEPTQ NMR spectra are
available in the Supporting Information (S15 and S16). In
addition, a HiFSA was carried out for both synthetic
compounds 1 and 3 using the PERCH software tool and an
1
Figure 2. Demonstration of congruence between the synthetic and isolated compounds 1 and 2 by 13C DEPTQ and H NMR, respectively (225/
900 MHz, MeOH-d4; A = image artifact from the solvent).
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Table 2. Anti-TB Activity Profiles of the Coumarins 1−4 in Comparison with Standard TB Drugs and Cytotoxicity
MIC90 (μg/mL)
IC50 (μg/mL)
a
b
c
c
c
c
c
d
compound
H37Rv
H37Rv
>100
rRMP
>100
rINH
rSM
rKAN
rCS
Vero
SI
1
>100
44.7
23.9
35.9
>100
44.7
76.5
31.7
0.04
>2.19
0.56
0.05
0.72
5.0
>100
>100
>100
>100
>100
>100
36.7
30.4
39.4
>100
2
37.1
21.9
23.2
<0.05
46.6
45.4
44.7
>3.29
0.03
0.47
0.07
0.73
4.8
49.5
46.9
46.9
0.82
1.27
1.10
3
47.4
43.9
47.3
47.3
4
rifampin
isoniazid
streptomycin
PA824
kanamycin
cycloserine
0.08
0.03
0.12
0.02
0.65
6.02
0.09
0.02
0.02
>1250
e
0.03
>9.31
0.17
0.76
4.9
0.07
1.48
0.34
0.04
0.53
0.18
1.12
>100
>3333
>833
>5000
>154
>17
e
e
1.07
0.25
>100
e
>100
e
e
e
>25
4.9
>100
e
>10.2
>100
a
b
Determined by the microplate Alamar blue assay (MABA) for replicating M. tuberculosis. Determined by the low oxygen recovery assay (LORA)
c
for nonreplicating M. tuberculosis. M. tuberculosis strains resistant to rifampin (rRMP), isoniazid (rINH), streptomycin (rSM), kanamycin (rKAN),
and cycloserine (rCS). Selectivity index = IC50/MABA MIC. Values observed in separate experiments over time in our laboratory.
d
e
approach described recently,16−19 and this resulted in the
unambiguous assignment of all H resonances including their
multiplicities, as shown in Table 2 and the Supporting
Information (S19 and S21).
The 1H and 13C DEPTQ NMR spectra for 4 are available in the
Supporting Information (S17 and S18). In addition, HiFSA was
carried out for the 900 MHz spectra of both 2 and 4, which
resulted in the full assignment of the 1H resonances and
multiplicities. The results are summarized in Table 1 and in the
Supporting Information (S20 and S22).
1
Compound 2, ethyl 6-chloro-2-oxo-4-phenyl-2H-chromen-3-
1
carboxylate, displayed very similar H and 13C DEPTQ NMR
peak patterns compared to those of 1, including the presence of
AMX and AA′BB′C spin systems. The HRMS measurement
also indicated the presence of one chlorine atom in the
molecule by affording a molecular ion peak at m/z 329.0614
[M + H]+ for the 35Cl isotope, representing a 72 amu difference
from that of 1. Although the 13C DEPTQ, HSQC, and HMBC
spectra were obtained at high field with a 5 mm inverse
cryoprobe (225/900 MHz, 3 and 1.7 mm samples, in MeOH-
d4) and even with the long accumulation time (over 24 h), they
Biological Evaluation. The anti-TB activities of the four
coumarins, 1−4, were assessed by the microplate Alamar blue
assay (MABA) and the low oxygen recovery assay (LORA)
(Table 2). MABA is used to measure the activity against
replicating M. tuberculosis, while LORA is designed to assess the
activity against nonreplicating M. tuberculosis. MABA and
LORA MICs indicated similar anti-TB activities, with MICs
ranging from 20 to 50 μg/mL, except for compound 1 (>100
μg/mL). The 7-chloro congener 4, containing an ethyl ester,
had superior anti-TB activity. The olefinic proton substitution
(in 3) of the two 7-chloro variations (in 3 and 4) seemed to
have slightly greater potency than the ethyl ester variation, but
the difference in the MIC values was insignificant (less than 2-
fold). On the other hand, in the 6-chloro variation (in 1 and 2),
only the ethyl ester substitution (in 2) led to detectable anti-TB
activity. On the basis of these observations, it may be concluded
that the structural variation containing an ethyl ester has
superior anti-TB activity if it contains chlorine in position 6,
while coumarins with a 7-chlorine substitution are less active. In
summary, of the four structural variations in 1−4, compound 3
was the most active against both replicating and nonreplicating
M. tuberculosis, with MICs of 23.9 and 21.9 μg/mL,
respectively. Furthermore, activity in the LORA indicated that
these coumarins might have the potential for shortening the
duration of therapy via efficient killing of the nonreplicating
persistor cells.
showed relatively low signal-to-noise. The 1D H, 2D H−1H
COSY, 1D 13C DEPTQ, HSQC, and HMBC NMR spectra for
the isolated compound 2 are provided in the Supporting
Information (S6−S10). Despite these limitations, the data still
confirmed that 2 possesses a coumarin skeleton with a chlorine
at C-6 (δ 131.4) and phenyl substitution at C-4 (δ 153.7). In
addition, 2 clearly did not have an olefinic proton at C-3, but
instead contained an ethoxycarbonyl moiety with H-17
methylene (2H, δ 4.052) and H-17 methyl protons (3H, δ
1
1
1
0.966). The H−1H COSY confirmed the correlation between
H2-16 and H3-17. Deshielding of both the carbon and the
proton of C-16/H-16 (δ 61.5/δ 4.052) indicated close
proximity to an electronegative oxygen atom. Long-range
couplings from H-17 (δ 4.052) to quaternary carbon C-15 (δ
165.3) in the HMBC confirmed the presence of the ethyl ester
moiety, which also accounted for the 72 amu difference. Thus,
it was concluded that 2 contained an ethyl ester moiety
attached to C-3 (δ 121.8) of 1. Overall, 2 was deduced to be
another new naturally occurring chlorinated coumarin, ethyl 6-
chloro-2-oxo-4-phenyl-2H-chromen-3-carboxylate, which also
had previously been synthesized.21 In order to confirm the
NMR-based structural assignment, 2 was synthesized and
Activity against a panel of mono drug-resistant isolates was
determined by the MABA and resulted in similar susceptibility
profiles to those obtained against wild-type M. tuberculosis. This
indicated that there is no significant difference in the anti-TB
activity for an ethyl ester with 7-chloro substitution (Table 2).
Activity against mono drug-resistant isolates suggests a
molecular target distinct from those for the corresponding
existing clinical drugs.
1
revealed identical H NMR spectra, as shown in Figure 2. The
detailed synthetic schemes are provided in the Supporting
Information (S24).
In addition to the naturally occurring compounds, the
analogue with chlorine substitution at C-7, ethyl 7-chloro-2-
oxo-4-phenyl-2H-chromen-3-carboxylate (4), was synthesized.
Considering the differences in their spectroscopic data, this also
ruled out the positional isomer 4 as a potential structure of 3.
In order to better understand the spectrum of antimicrobial
activity, MICs of these chlorinated coumarins were determined
for a panel of eight species: S. aureus, E. coli, C. albicans, E.
faecalis, P. aeruginosa, A. baumanii, S. pneumoniae, and M.
smegmatis. All four compounds were inactive at the highest test
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in September 2001. The tissue culture and stock cultures are
maintained at Fungi Perfecti Research Laboratories in Shelton, WA,
USA. The partial sequence of 18S and 28S rRNA genes established the
identification of F. officinalis. Sequence data are available from
GenBank (EU854436.1).
concentration, 100 μg/mL. In addition, activity against other
non-tuberculous mycobacteria (NTM) was assessed with M.
chelonae, M. abscessus, M. marinum, M. kansasii, M. avium, and
M. bovis. In this series, M. bovis has the closest gene homology
to M. tuberculosis and, not surprisingly, showed the most similar
susceptibility profile to that of M. tuberculosis. Compounds 2
against M. marinum and 3 against M. kansasii exhibited MICs of
97.1 and 49.3 μg/mL, respectively. Otherwise, no activity was
observed at 100 μg/mL. The activities against the eight non-
mycobacterial species and NTM are documented in S23 of the
Supporting Information.
Mammalian cell cytotoxicity using Vero cells for 1−4 was
evaluated and described as the 50% growth inhibitory
concentration (IC50), resulting in values of >100, 36.7, 30.4,
and 39.4 μg/mL, respectively. This translates into selectivity
indices (SIs; IC50/MIC) around unity for the active coumarins
2−4 (Table 2). Cytotoxicity against Vero cells was observed at
a similar concentration to that causing significant inhibition
against M. tuberculosis and M. bovis, whereas activities against
other microorganisms are essentially lacking (S23, Supporting
Information). However, it is not clear at this point whether the
chlorinated coumarins are hitting similar targets in the M.
tuberculosis complex species and Vero cells. As SIs greater than
unity are generally regarded as a requirement for progression as
TB drug leads, analogues of these coumarins with significantly
higher SIs will need to be found or synthesized in order to
provide a useful novel anti-TB pharmacophore. Although these
isolated coumarins, based on the yield and the concentration
within the crude extract and the activity of the compounds in
question, do not explain the claimed in vitro anti-TB activity of
the crude extract with respect to its ethnomedical use, the
present study led to the identification of a new compound class
for this organism and revealed structural entities that show anti-
TB potency and could represent a possible link to the
traditional use of F. officinalis as an anti-TB treatment.
Mycelial cultures were grown in sterile Petri dishes containing
sterilized antibiotic malt extract yeast agar. After three weeks of
colonization in a clean room laboratory at temperatures between 21
and 24 °C, the cultures were aseptically transferred into a 1000 mL
Eberbach stirrer containing 800 mL of sterilized water. The Eberbach
container was activated using a Waring blender base, and the mycelium
was fragmented in a process known as liquid fragmentation (the
dissociated fragmented mycelial mass allows for a multiple loci
inoculation, resulting in accelerated colonization). Approximately 50−
100 mL of myceliated broth was then transferred into a polypropylene
incubation bag containing approximately 3 kg of moistened sterilized
rice (approximately 45−50% moisture content). These bags of freshly
inoculated rice were then incubated for 60−120 days in a class 100
clean room. Once colonization was determined to be sufficient, the
mycelium-colonized rice was transferred to glass containers for
extraction. The mycelium was gently transferred and immediately
covered with an equal weight of 95% EtOH. The mixture was agitated
and then allowed to macerate at room temperature.
Isolation. The F. officinalis mycelium culture extract (1.75 L, 95%
EtOH) was evaporated under vacuum to give 17 g of dried crude
extract. The extract was redissolved in 1 L of 75% EtOH and
partitioned with petroleum ether, hexane, CHCl3, EtOAc, and n-
BuOH (1:1, v/v) to give 474, 30, 4270, 48, and 8569 mg of dried
extract, respectively. The CHCl3 partition fraction was further
separated on an Isolera Flash purification system (SNAP 100 g, 30
mL/min) with a linear solvent gradient of CHCl3−MeOH (100:0, v/
v) to 100% MeOH over 100 min, to afford 135 fractions (25 mL/
fraction). The recombination into 22 fractions was based on TLC
(silica; CHCl3−MeOH, 85:15) analysis. From the 22 fractions,
fractions 11, 12, and 13 were again recombined (300 mg) for further
separation on an Isolera Flash purification system (SNAP 10 g, 9 mL/
min) with a linear solvent gradient of CHCl3−MeOH (90:10, v/v) to
15% MeOH over 28 min, affording 85 subfractions (3 mL/fraction).
These were recombined to seven fractions based on TLC analysis and
dried to yield 17, 9, 6, 24, 31, 37, and 17 mg, respectively. The first
combined fraction (17 mg) was further fractionated using reversed-
phase HPLC on a Waters Delta 600 system with a Waters 996
photodiode array detector using a semipreparative column (Waters,
C18, 5 μm, 250 × 10 mm, 3 mL/min). Five separated injections with a
linear solvent gradient of MeOH−H2O (80:20, v/v) to 100% MeOH
over 25 min afforded 0.2 mg of 2 at 15 min and 0.5 mg of 1 at 17 min.
General fraction monitoring for chromatographic separation was done
by TLC analysis with precoated Alugram SIL G/UV plates (Macherey-
EXPERIMENTAL SECTION
■
General Experimental Procedures. The UV−vis spectra were
obtained with a SpectraMax Plus 384 at 25 °C. Optical rotations [α]D
were measured on a Perkin-Elmer 242 polarimeter at 25 °C. IR spectra
were measured on a Thermo Nicolet 6700 FT-IR spectrometer. All
NMR experiments were obtained at either 600 or 900 MHz and
performed on Bruker AVANCE-600 or AVANCE II-900 instruments,
each equipped with a cryogenic sensitivity-enhanced triple-resonance 5
mm inverse TCI cryoprobe. The samples were dissolved in 200 μL of
MeOH-d4, and 150 μL was transferred to 3 mm NMR tubes (or 50 μL
of MeOH-d4 and 40 μL transferred to 1.7 mm NMR tubes). All NMR
experiments were performed using standard Bruker pulse sequences,
and the temperature was maintained at 25 °C (298 K). High-
resolution ESI mass spectra were obtained using a Shimadzu IT-TOF
LC mass spectrometer. The HiFSA was performed using the PERCH
NMR software (v. 2010.1, PERCH Solutions Ltd., Kuopio, Finland).
The 1H NMR spectra were processed with NUTS (Acorn NMR Inc.),
imported into PERCH as JCAMP-DX files, and subjected to baseline
correction, peak picking, and integration. The 1H NMR parameters in
MeOH-d4 were predicted using the PERCH Molecular Modeling
System. After a manual examination of the 1H assignments, the
Nagel, Duren, Germany).
̈
Synthetic 6-Chloro-4-phenyl-2H-chromen-2-one (1) (ref 20):
amorphous, white solid; [α]25D 0 (c 0.001, MeOH); UV/vis (MeOH)
λmax (log ε) 257 (2.87), 264 (3.09), 288 nm (3.42); IR (neat) νmax
2359, 2341, 1731 cm−1; 1H/DEPTQ 13C NMR (900/225 MHz,
MeOH-d4), see Table 1; HRMS m/z 257.0372 [M + H]+ (calcd for
C15H10ClO2, 257.0369). Synthetic methodology is available in the
Supporting Information (S24).
Synthetic Ethyl 6-chloro-2-oxo-4-phenyl-2H-chromene-3-
carboxylate (2) (ref 21): amorphous, white solid; [α]25 0 (c
D
0.001, MeOH); UV/vis (MeOH) λmax (log ε) 257 (3.16), 265 (3.52),
289 (3.95), 294 nm (3.94); IR (neat) νmax 1745, 1724, 1599, 1244,
1
1039 cm−1; H/DEPTQ 13C NMR (900/225 MHz, MeOH-d4), see
1
calculated H chemical shifts, signal line widths, and J-couplings were
Table 1; HRMS m/z 329.0594 [M + H]+ (calcd for C18H14ClO4,
329.0581). Synthetic methodology is available in the Supporting
Information (S24).
refined by using the integral-transform (D) and total-line-fitting (T)
modes until an excellent agreement between the observed and
simulated spectra was attained (total rms ≤0.5%). Cellular viability was
assessed by the measurement of fluorescence, luminescence, or
absorbance with the Victor3 multilabel reader (PerkinElmer Life
Sciences).
Synthetic 7-Chloro-4-phenyl-2H-chromen-2-one (3) (refs 20,
22): amorphous, white solid; [α]25 0 (c 0.001, MeOH); UV/vis
D
(MeOH) λmax (log ε) 257 (3.17), 265 (3.55), 289 (3.89), 294 nm
(3.89); IR (neat) νmax 1743, 1725, 1601, 1279, 1037 cm−1; 1H/
DEPTQ 13C NMR (900/225 MHz, MeOH-d4), see Table 1; HRMS
Organism Collection, Identification, Culture, and Extraction.
Fomitopsis officinalis was collected from Morton, Washington (USA),
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m/z 257.0377 [M + H]+ (calcd for C15H10ClO2, 257.0369). Synthetic
methodology is available in the Supporting Information (S24).
Synthetic Ethyl 7-chloro-2-oxo-4-phenyl-2H-chromene-3-
incubation after adding 12 μL of 20% Tween80 and 20 μL of Alamar
blue dye. M. avium and M. kansasii were incubated with 7H9 media at
37 °C for 6 days, plus an additional day of incubation after adding 12
μL of 20% Tween80 and 20 μL of Alamar blue dye. Viability was
assessed by measuring fluorescence at 530 nm excitation/590 nm
emission with a Victor3 multilabel reader (PerkinElmer).
Spectrum of Activity. Staphylococcus aureus (ATCC29213),
Candida albicans (ATCC90028), Escherichia coli (ATCC25922), M.
smegmatis MC2155, Streptococcus pneumoniae (ATCC49619), Enter-
ococcus faecalis (ATCC29212), and Pseudomonas aeruginosa
(ATCC27853) were purchased from ATCC. The relative activity
against S. aureus, C. albicans, S. pneumoniae (with 2% HBL), E. faecalis,
P. aeruginosa, and E. coli was determined by broth microdilution with a
spectrophotometric readout at 570 nm (S. pneumonia at 490 nm) as
described in the National Committee on Clinical Laboratory
Standards.28,29 The activity against M. smegmatis was determined by
the MABA, incubating 3 days at 37 °C plus an additional 4 h of
incubation after adding 12 μL of 20% Tween80 and 20 μL of Alamar
blue dye. Viability was assessed by the measurement of fluorescence
with the Victor 3.
carboxylate (4) (refs 21, 22): amorphous, white solid; [α]25 0 (c
D
0.001, MeOH); UV/vis (MeOH) λmax (log ε) 257 (3.17), 265 (3.55),
289 (3.89), 294 nm (3.89); IR (neat) νmax 1743, 1725, 1601, 1279,
1
1037 cm−1; H/DEPTQ 13C NMR (900/225 MHz, MeOH-d4), see
Table 1; HRESIMS m/z 329.0597 [M + H]+ (calcd for C18H14ClO4,
329.0581). Synthetic methodology is available in the Supporting
Information (S24).
Bacterial Strain Preparation for Anti-TB Bioassay and
Minimum Inhibitory Concentration (MIC). M. tuberculosis H37Rv
ATCC 27294 was purchased from American Type Culture Collection
(ATCC) and cultured to late log phase in the 7H12 media,
Middlebrook 7H9 broth supplemented with 0.2% (v/v) glycerol,
0.05% Tween80, and 10% (v/v) oleic acid-albumin-dextrose-catalase.
The culture was harvested and resuspended in phosphate-buffered
saline. Suspensions were then filtered through 8 μm filter membranes
and frozen at −80 °C. Prior to use of bacterial stocks for the anti-TB
assay, colony-forming units (CFUs) were determined by plating on
7H11 agar media. The MIC is defined here as the lowest
concentration resulting in ≥90% growth inhibition of the bacteria
relative to untreated controls. MIC against replicating M. tuberculosis
was measured by the MABA.23,24
Low Oxygen Recovery Assay. The luciferase reporter gene
luxAB recombinant M. tuberculosis was prepared as previously
reported.25 The bacteria were adapted to low oxygen during culture
in a BioStatQ fermenter. The low-oxygen-adapted culture was exposed
to the test samples in 96-well microplates for 10 days at 37 °C in a
hypoxic environment created with an Anoxomat (WS-8080, MART
Microbiology). The cultures were then transferred to a normoxic
environment at 37 °C for 28 h. Viability was assessed by the
measurement of luciferase-mediated luminescence. The LORA MIC
was defined as the lowest concentration effecting a reduction of
luminescence of ≥90% relative to untreated cultures.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H, 13C DEPTQ, COSY, HSQC, and HMBC NMR spectra of
1
1
isolated 1 and 2. H and 13C DEPTQ NMR spectra, full H
NMR spin analysis, and synthesis of 1−4. These materials are
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +1 (312) 355-1949. Fax: +1 (312) 355-2693. E-mail:
gfp@uic.edu.
■
Notes
Cytotoxicity. Cytotoxicity26,27 was assessed using Vero (ATCC
CRL-1586) cells. Vero cells were cultured in 10% fetal bovine serum in
Eagle’s minimum essential medium. The culture was incubated at 37
°C under 5% CO2 in air and then diluted with phosphate-buffered
saline to 106 cells/mL. In a transparent 96-well plate (Falcon Microtest
96), 2-fold serial dilutions of testing samples with a final volume of 200
μL cell culture suspension were prepared. After 72 h incubation at 37
°C, the medium was removed and monolayers were washed twice with
100 μL of warm Hanks’ balanced salt solution. A 100 μL amount of
medium and 20 μL of MTS-PMS (Promega) were added to each well.
Plates were then incubated for 3 h, and cytotoxicity was determined by
the measurement of absorbance at 490 nm.
MIC against Drug-Resistant M. tuberculosis Isolates. M.
tuberculosis strains individually resistant to rifampin (RMP, ATCC
35838), isoniazid (INH, ATCC 35822), streptomycin (SM, ATCC
35820), cycloserine (CS, ATCC 35826), and kanamycin (KAN,
ATCC 35827) were obtained from ATCC. Cultures were prepared,
and MICs against drug-resistant M. tuberculosis isolates were
determined by the MABA as described above for M. tuberculosis
H37Rv.
MIC against Non-tuberculous Mycobacteria. M. abscessus
(ATCC19977), M. chelonae (ATCC35752), M. avium
(ATCC15769), M. marinum (ATCC927), M. kansasii
(ATCC12478), and M. bovis BCG (ATCC35734) were purchased
from ATCC. Cultures were prepared, and MICs against these
mycobacteria were determined by the MABA as described above for
M. tuberculosis H37Rv. M. abscessus was incubated with 7H12 medium
at 37 °C for 3 days and for an additional 4 h after adding 12 μL of 20%
Tween80 and 20 μL of Alamar blue dye. M. bovis was incubated with
7H12 medium at 37 °C for 7 days and an additional 1 day of
incubation after adding 12 μL of 20% Tween80 and 20 μL of Alamar
blue dye. M. chelonae was incubated with 7H9 medium at 30 °C for 3
days, plus an additional 6 days of incubation after adding 12 μL of 20%
Tween80 and 20 μL of Alamar blue dye. M. marinum was incubated
with 7H9 medium at 30 °C for 5 days and an additional 1 day of
The authors declare the following competing financial
interest(s): Paul E. Stamets is the founder of Fungi Perfecti
LLC. Nicole A. Fryling is affiliated with Fungi Perfecti LLC.
ACKNOWLEDGMENTS
■
We are grateful to Dr. R. Nally from Fungi Perfecti LLC,
Olympia, for the preparation of the extracts. The construction
of the UIC CSB and the 900 MHz (21.1 T) NMR
spectrometer was funded by NIGMS grant P41 GM068944.
REFERENCES
(1) Dye, C. Lancet 2006, 367, 938−940.
(2) Dmitriev, V. A. Antibiot. Khimioter. 2008, 53, 3−6.
(3) Ginsberg, A. M.; Spigelman, M. Nat. Med. 2007, 13, 290−294.
(4) Abdool Karim, S. S.; Churchyard, G. J.; Abdool Karim, Q.; Lawn,
S. D. Lancet 2009, 374, 921−933.
■
(5) Abubakar, I.; Moore, J.; Drobniewski, F.; Kruijshaar, M.; Brown,
T.; Yates, M.; Anderson, C.; Smith, E. G.; Magee, J.; Lipman, M.;
McMenamin, J.; Ruddy, M.; Watson, J. M. Thorax 2009, 64, 512−515.
(6) Donald, P. R.; van Helden, P. D. N. Engl. J. Med. 2009, 360,
2393−2395.
(7) Farnsworth, N. R.; Akerele, O.; Bingel, A. S.; Soejarto, D. D.;
Guo, Z. Bull. W. H. O. 1985, 63, 965−981.
(8) Stamets, P. Int. J. Med. Mushr. 2005, 7, 495−506.
(9) Stamets, P. Int. J. Med. Mushr. 2002, 4, 179−192.
(10) Grazywnowicz, K. Int. J. Med. Mushr. 2001, 3, 2−3.
(11) Wu, X.; Yang, J. S.; Yan, M. Chem. Pharm. Bull. (Tokyo) 2009,
57, 195−197.
(12) Anderson, C. Phytochemistry 1971, 10, 2713−2717.
(13) Anderson, C. Phytochemistry 1972, 11, 2847−2852.
(14) Epstein, W. J. Am. Chem. Soc. 1979, 101, 2748−2750.
(15) Wu, X.; Yang, J.-S.; Zhou, L.; Dong, Y.-S. Chem. Pharm. Bull.
2004, 52, 1375−1377.
F
dx.doi.org/10.1021/np400497f | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(16) Napolitano, J. G.; Godecke, T.; Rodriguez-Brasco, M. F.; Jaki, B.
̈
U.; Chen, S. N.; Lankin, D. C.; Pauli, G. F. J. Nat. Prod. 2012, 75,
238−248.
(17) Molina-Salinas, G. M.; Rivas-Galindo, V. M.; Said-Fernandez, S.;
Lankin, D. C.; Munoz, M. A.; Joseph-Nathan, P.; Pauli, G. F.;
Waksman, N. J. Nat. Prod. 2011, 74, 1842−1850.
(18) Scher, J. M.; Schinkovitz, A.; Zapp, J.; Wang, Y.; Franzblau, S.
G.; Becker, H.; Lankin, D. C.; Pauli, G. F. J. Nat. Prod. 2010, 73, 656−
663.
(19) Inui, T.; Wang, Y.; Nikolic, D.; Smith, D. C.; Franzblau, S. G.;
Pauli, G. F. J. Nat. Prod. 2010, 73, 563−567.
(20) Cresente-Campo, J. Eur. J. Org. Chem. 2010, 2010, 4130−4135.
(21) Tawada, H. Chem. Pharm. Bull. 1995, 26, 616−625.
(22) Gabbutt, C. D.; Heron, B. M.; Instone, A. C. Tetrahedron 2006,
62, 737−745.
(23) Collins, L.; Franzblau, S. G. Antimicrob. Agents Chemother. 1997,
41, 1004−1009.
(24) Franzblau, S. G.; Witzig, R. S.; McLaughlin, J. C.; Torres, P.;
Madico, G.; Hernandez, A.; Degnan, M. T.; Cook, M. B.; Quenzer, V.
K.; Ferguson, R. M.; Gilman, R. H. J. Clin. Microbiol. 1998, 36, 362−
366.
(25) Cho, S. H.; Warit, S.; Wan, B.; Hwang, C. H.; Pauli, G. F.;
Franzblau, S. G. Antimicrob. Agents Chemother. 2007, 51, 1380−1385.
(26) Cantrell, C. L.; Lu, T.; Fronczek, F. R.; Fischer, N. H.; Adams,
L. B.; Franzblau, S. G. J. Nat. Prod. 1996, 59, 1131−1136.
(27) Falzari, K.; Zhu, Z.; Pan, D.; Liu, H.; Hongmanee, P.; Franzblau,
S. G. Antimicrob. Agents Chemother. 2005, 49, 1447−1454.
(28) Approved standard M7-A2; National Committee for Clinical
Laboratory Standards (NCCLS): Villanova, PA, 1990.
(29) Informational Supplement M100-S3; National Committee for
Clinical Laboratory Standards (NCCLS): Villanova, PA, 1991.
G
dx.doi.org/10.1021/np400497f | J. Nat. Prod. XXXX, XXX, XXX−XXX