Design and Application of a High-Throughput, High-Content Screening System for Natural Product Inhibitors of the Human Parasite Trichomonas vaginalis

Article abstract of DOI:10.1021/acsinfecdis.9b00156

It is estimated that Trichomonas vaginalis affects an astonishing 3.9% of the world's population, and while many of those infected are asymptomatic, progression of the disease can lead to serious health problems. Currently, the nitroimidazoles constitute the only drug class approved to treat trichomoniasis in the United States, which makes the spread of drug resistance a realistic concern. We developed a new image-based, high-throughput, and high-content assay for testing natural products (purified compounds and extracts) for antitrichomonal activity. Applying this assay system to a library of fungal natural product extracts led to the identification of three general classes of natural product inhibitors that exhibited moderate to strong activities against T. vaginalis: anthraquinones, xanthone-anthraquinone heterodimers, and decalin-linked tetramic-acid-containing metabolites. The tetramate natural products emerged as the most promising candidate molecules with pyrrolocin A (51) exhibiting potent activity against the parasite (EC₅₀ = 60 nM), yet this metabolite showed limited toxicity to mammalian cell lines (selectivity index values of 100 and 167 versus 3T3 fibroblast and Ect1 normal cervical cells, respectively). The imaging-based assay system is a powerful tool for the bioassay-guided purification of single-component antitrichomonal biomolecules from complex natural product mixtures.

Full text of DOI:10.1021/acsinfecdis.9b00156

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Article
Design and Application of a High-Throughput, High-Content Screening System
for Natural Product Inhibitors of the Human Parasite Trichomonas vaginalis.
Jarrod B. King, Adam Carter, Wentao Dai, Jin Woo Lee, Yun-Seo Kil, Lin
Du, Sara Helff, shengxin cai, Brandt Huddle, and Robert H. Cichewicz
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00156 • Publication Date (Web): 20 Jun 2019
Downloaded from http://pubs.acs.org on June 26, 2019
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Design and Application of a High-Throughput, High-Content Screening System for Natural
Product Inhibitors of the Human Parasite Trichomonas vaginalis.
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Jarrod B. King, Adam C. Carter, Wentao Dai, Jin Woo Lee, Yun-Seo Kil, Lin Du, Sara
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,†
K. Helff, Shengxin Cai, Brandt C. Huddle, Robert H. Cichewicz*
†Natural Products Discovery Group, Institute for Natural Products Applications and Research
Technologies, Department of Chemistry and Biochemistry, Stephenson Life Sciences Research
Center, 101 Stephenson Parkway, Room 1000, University of Oklahoma, Norman, Oklahoma,
73019, United States
EMAIL ADDRESS OF CORRESPONDING AUTHOR: rhcichewicz@ou.edu
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It is estimated that Trichomonas vaginalis affects an astonishing 3.9% of the world’s
population, and while many of those infected are asymptomatic, progression of the disease can
lead to serious health problems. Currently, the nitroimidazoles constitute the only drug class
approved to treat trichomoniasis in the United States, which makes the spread of drug resistance a
realistic concern. We developed a new image-based, high-throughput, high-content assay for
testing natural products (purified compounds and extracts) for antitrichomonal activity. Applying
this assay system to a library of fungal natural product extracts led to the identification of three
general classes of natural product inhibitors that exhibited moderate to strong activities against T.
vaginalis: anthraquinones, xanthone-anthraquinone heterodimers, and decalin-linked tetramic-
acid-containing metabolites. The tetramate natural products emerged as the most promising
candidate molecules with pyrrolocin A (51) exhibiting potent activity against the parasite (EC =
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0 nM), yet this metabolite showed limited toxicity to mammalian cell lines (selectivity index
values of 100 and 167 versus 3T3 fibroblast and Ect1 normal cervical cells, respectively). The
imaging-based assay system is a powerful tool for the bioassay-guided purification of single-
component antitrichomonal biomolecules from complex natural product mixtures.
Keywords: Trichomonas vaginalis, natural products, fungi, quinones, tetramic acid
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Trichomonas vaginalis is the most frequently encountered sexually-transmitted parasite in the
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United States. Those infected by the parasite exhibit an increased risk for (i) HIV infection, (ii)
development of cervical cancer, and (iii) infertility, as well as certain adverse pregnancy
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outcomes including (iv) miscarriage and (v) low-preterm birth weight. Current clinical treatment
options are limited to antiprotozoal nitroimidazoles (i.e., metronidazole and tinidazole), which
still offer appreciably high levels of clinical efficacy (>90% of patients are cleared of the infection
after a single course of treatment).3-5 However, concerns have arisen that in addition to several
noted side effects (e.g., nausea, vomiting, and others), the nitroimidazoles may possess
carcinogenic and mutagenic properties.6-7 This has led to worry about nitroimidazole treatment
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during pregnancy, as well as some apprehension about their use in the general population. A
rather troubling problem, which came to light based on surveillance data collected from patients
infected with T. vaginalis and who received standard courses of nitroimidazole treatment, is the
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high rate of infection recurrence (5-31% in women). While a portion of disease recurrence is
attributable to further contact with untreated sexual partners, data suggests that a yet unknown
proportion of T. vaginalis cases has the capacity to develop into persistent infectious states, which
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raises concerns about the clinical limitations of nitroimidazole therapies. Even more concerning,
nitroimidazole resistance has been detected in up to 9.6% of clinical T. vaginalis isolates, raising
alarm that nitroimidazoles might become clinically less effective.9-10 Considering the noted safety
concerns and troubling clinical observations, there is a need for new disease management options
that extend beyond the nitroimidazoles for treating T. vaginalis infections.
An examination of the research literature has revealed surprisingly sparse information
concerning efforts to identify new classes of antiprotozoal molecules active against T. vaginalis,
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with only a small fraction of those endeavors dealing with natural products.11-12 A handful of prior
studies had focused exclusively on testing plant extracts,13-16 but revealed little about the
molecules responsible for their inhibitory effects. In a few instances, chemically-driven
investigations of extracts have yielded a handful of secondary metabolites possessing activity
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against T. vaginalis including berberine, (-)-usnic acid, emodin, hamycin, hederagin, and
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mulinolic acid; however, these compounds suffer from poor potency and lack selectivity
compared to metronidazole. Although the limited scope of reports pertaining to the discovery of
new leads to fight T. vaginalis infections is worrisome, it also signals the potential that exists to
identify new bioactive compounds for development into clinically useful agents. This expectation
is reasonable, especially for natural products, which have long served as an outstanding resource
for the discovery of new antiinfectious agents including some of the topmost antiparasitic
compounds in current clinical use (e.g., avermectins, artemisinin, and more).23-24
Aside from the scarcity of natural-product-focused drug discovery efforts concentrating on
inhibiting of T. vaginalis, there are relatively few reports of vetted assay methods that are
amenable to the systematic screening and identification of compounds that inhibit this unique
parasite. Most of the reported methods had employed manual microscopy-based approaches for
cell enumeration25-26 or used metabolically-activated dyes such as resazurin as indicators of
parasite viability.25-26 With this in mind, we set about developing an assay system that (i) was able
to reliably identify samples containing antitrichomonal compound(s), (ii) was amenable to high-
throughput sample testing, (iii) could accurately distinguish between complete versus partial
inhibition of T. vaginalis, and (iv) was applicable for use involving a wide variety of sample types
including pure compounds and natural product extracts. Herein we report the results of our assay
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development program, as well as describe new natural product scaffolds that exhibit promising
inhibitory activities against T. vaginalis.
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RESULTS AND DISCUSSION
Development of an Assay for Detecting T. vaginalis Inhibitors. Resazurin-based
colorimetric assays are widely used in bioactive compound screening campaigns to identify
substances that inhibit cell viability and proliferation under aerobic and anaerobic conditions.27
This dye and its H O soluble salts are readily reduced by NADH to yield the red, fluorescent
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product resorufin. Despite its reported use for the identification of T. vaginalis inhibitors, we were
surprised to discover that, in our hands, this reagent was rather insensitive for the detection of
reasonably large numbers of viable cells. We observed that under anaerobic assay conditions, a
population of ~10,000 trichomonads per well in a 96-well microtiter plate was needed as a
threshold to consistently signal a positive response for the presence of live T. vaginalis. Given
that we established an inoculum of 40,000 trichomonads per well as an optimal starting point for
assays, it meant that compounds affording modest (~75%) parasite-kill rates would be
indistinguishable from more potent and potentially better agents (Figure 1). Therefore, we set
about designing a new assay system that was more sensitive, as well as able to handle a wide-
range of complex test substances (e.g., pure compounds and extracts containing colored or UV-
active natural products).
One method deemed to be a promising alternative to the resazurin assay was high-content
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imaging. We speculated that this approach would offer a more responsive tool for evaluating the
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live/dead-status of individual trichomonads within sample populations. However, we quickly
recognized that live trichomonads were not fully compatible with imaging-based detection
methods; the rapid movements of the flagellated parasites caused the cells to severely blur even
with a reasonably fast exposure time (10 ms). Accordingly, we identified a method to fix the
trichomonads with glutaraldehyde followed by the application of a dual cell-staining system
consisting of acridine orange (cell-permeable DNA stain) and propidium iodide (DNA stain that
is not permeant to live cells) for live/dead determination (Figure 2). Initial tests conducted using
the Operetta (PerkinElmer) high-content imaging system revealed that this assay tool could detect
as few as one live or dead trichomonad per image field and was robust (Z-factor of 0.92).
Moreover, this type of detection system was amenable to high-throughput screening of T.
vaginalis viability, especially with UV/VIS-active natural products since the stored image files
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(i.e., two image fields recorded per well) could be manually inspected to identify potential false-
positive and false-negative results. Additionally, DMSO was determined to be an acceptable
vehicle for compound testing since a concentration of 1% by volume appeared to have no
detrimental impact on the viability of T. vaginalis cells.
Testing Purified Natural Products. To examine the probative capabilities of the new T.
vaginalis imaging-based assay, a focused library of chemically diverse natural products was
tested. The compound library consisted of 430 metabolites sourced by our research team from
marine organisms, plants, bacteria, and fungi. An initial high-dose concentration (100 M) was
chosen to afford the opportunity to evaluate several challenging screening scenarios, especially
those involving potential false-positives brought about by compound precipitation and UV/VIS-
interference. The imaging-based assay proved invaluable since all the data files corresponding to
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potential hits (i.e., compounds that provided inhibition exceeding that afforded by 25 M
metronidazole) were immediately available for inspection to confirm that the number of live
trichomonads had been reduced. This test revealed that 64 of the compounds we tested exhibited
activities that were equal to or better than the positive control. The large number of active samples
was not unexpected since our pure compound library was largely populated by bioactive
substances that had previously demonstrated cytotoxicity toward mammalian cells and/or
inhibited the proliferation of various microorganisms. To refine the list of natural products
further, the hit compounds were screened at 25 M, which resulted in a subset of 9 compounds
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that exhibited activities comparable to 25 M metronidazole (Figure S1, Supporting Information).
These results also reinforced the need for a mammalian cell-based cytotoxicity counter screen to
aid in the elimination of undesirable non-specific cellular toxins. Accordingly, the 9 hits were
tested over a wide-range of concentrations extending two-orders of magnitude (0.25-25 µM)
against both T. vaginalis and a normal mouse fibroblast (NIH/3T3) cell line. With these data in
hand, the EC values for all compounds against both the parasite and mammalian cells were
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estimated. The EC values were used to calculate a selectivity index (SI) value [SI = (EC for
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T3 cells)/(EC for T. vaginalis)] for each of the bioactive substances (Figure 3). While the SI
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values revealed that most of the pure natural products were equipotent inhibitors of trichomonads
and mammalian cells, one noted exception was 2-bromoascididemin, which exhibited modest
selectivity (SI3T3 = 14). Thus, we established that the new assay was amenable to the testing of
structurally diverse natural products across a wide range of concentrations. Furthermore, when
coupled with a mammalian cell cytotoxicity screen, it could reveal a substance’s ability to
selectively inhibit the target parasite.
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Testing Fungal Natural Product Extracts. The University of Oklahoma, Natural Products
Discovery Group has generated a library of over 50,000 fungal extracts. Each extract consists of
the organic residue obtained after a fungal isolate was cultured in small scale for 3 weeks on
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Cheerios breakfast cereal, extracted with EtOAc, and partitioned against H O. For testing
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purposes, a subset of 1,748 samples was selected for examination in the high-content-imaging
assay system. For the first stage of testing, we determined that it was most efficient to screen
extracts in duplicate at a single concentration (15 µg/mL). This provided 111 ‘hits’ that inhibited
the viability of T. vaginalis at levels equal to or better than the inhibition achieved using 25 M
metronidazole. These ‘hit’ extracts were subjected to a second stage of testing over a range of
concentrations (0.15-15 µg/mL), which yielded 71 samples that retained potent activity and
generated sigmoidal concentration-response curves. During this period in the assay development
process, we considered the value of introducing a second mammalian cell type for selectivity
evaluation since fibroblasts cells, although capable of vigorous in vitro growth that made them
simple to test, might not offer a level of cytotoxin sensitivity or exhibit cellular properties
typically associated with cells at the sites of T. vaginalis infections. Thus, Ect1/E6E7 normal-type
cervical cells were chosen to provide a second point of reference for selectivity testing.
Accordingly, the EC value for each extract’s parasite-inhibitory activity, as well as its cytotoxic
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effects toward Ect1/E6E7 cells were determined and the data were plotted as illustrated in Figure
4. This data visualization method provided a simple graphical means to identify samples that
offered high levels of parasite inhibition yet exhibited limited toxicity toward mammalian cells.
Thus, we proceeded to prioritize samples appearing in the upper right quadrant of the graph since
those samples offered the best starting points for new bioactive compound discovery. Based on
those data, three fungi responsible for generating potent and selective extracts were selected for
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follow-up chemical investigation including: two Fusarium spp. isolates (plate 55 well F11; SIEct1
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36 and plate 72 well E6; SI3T3 ≥2.7) and a Humicola sp. isolate (plate 78 well A3; SI3T3 = 25).
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Bioassay-Guided Purification and Testing of Natural Products from Fusarium sp. Isolate
A. An extract prepared from Fusarium sp. isolate A was subjected to bioassay-guided
fractionation using silica gel vacuum liquid chromatography (VLC), HP20SS VLC, and C18
HPLC chromatography yielding three quinone-containing natural products: fusarubin (1),29
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javanicin (2), and solaniol (3) (compounds were identified based on comparisons of
experimental LC-ESIMS and NMR data to the published values)32-33 (Figure 5). The purified
metabolites exhibited a range of potencies against T. vaginalis with 1 and 2 being the most potent
(
EC values of 2.5 µM and 1.3 µM, respectively), while 3 showed greatly reduced activity (EC
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greatly exceeded the SI values determined for 2 (SIEct1 = 3.1) and 3 (SIEct1 = 0.4) (Figure 5). These
data initially suggested that simple quinone-containing scaffolds like 1 might serve as a starting
point for the identification of structural analogues that offered improved selectivity and potency.
Exploring the Bioactivities for Analogues of Compound 1. Based on the activities of natural
products 1-3, we proceeded to test 42 additional natural and synthetic compounds (compounds 4-
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5, Table S1, Supporting Information) that shared many key structural elements with the
metabolites obtained from Fusarium sp. isolate A to determine if compounds could be identified
that afforded improved potency and selectivity. Although many of the tested molecules exhibited
low-to-mid-micromolar inhibition of T. vaginalis viability, none of those compounds proved to be
as selective as metabolite 1 (other compounds exhibited SIEct1 values 4.5) (Figure 6). Results of
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the bioactivity assessment are summarized in Table S1 (Supporting Information). Notably,
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caused by Babesia spp., Plasmodium spp., and Toxoplasma spp.), exhibited only modest activity
and a poor SIEct1 value of <0.1, suggesting that this compound did not offer a reasonable-level of
crossover inhibition against T. vaginalis. Given that none of the tested compounds afforded more
favorable activity compared to 1 (i.e., no improvement in potency or SI values), as well as redox-
related concerns (e.g., potential for quinones to react with nucleophilic amino acid residues, as
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well as a notorious history of problems in a variety of assays), we became concerned that this
scaffold might not provide a suitable opportunity for further development. To examine if redox
activity might partially explain the activity of 1 and related compounds, we devised a test in
which the potencies of compound 1, 2-methoxynaphthazarin (9), and metronidazole were
assessed under three atmospheric conditions with different oxygen levels (i.e., aerobic,
microaerophillic, and anaerobic). The results of those tests revealed that the activities of 1 and 9
both decreased as oxygen was introduced into the test culture conditions (Figure 7). Curiously,
compounds 1 and 9 differed in another respect; 1 was only able to afford the partial elimination of
trichomonads under strict anaerobic condition, whereas 9 was able to provide for the complete
elimination of the parasite in an anaerobic atmosphere. In contrast, metronidazole maintained
consistent potency across all the tested atmospheric conditions (Figure 7). Given the expected
heterogeneity of oxygen levels across the various body sites infected by T. vaginalis, we
abandoned further pursuit of 1 and its analogues in favor of other, more promising compounds.
Bioassay-Guided Purification and Testing of Natural Products from a Humicola sp.
Isolate. The extract prepared from the Humicola sp. isolate was subjected to bioassay-guided
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fractionation using silica gel vacuum liquid chromatography (VLC), HP20SS VLC, and C18
HPLC purification steps to afford the xanthone-anthraquinone heterodimer xanthoquinodin A1
1
13
(46), which was identified by comparisons of its experimental H and C NMR spectra, LC-
1
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ESIMS data, and specific rotation value to the published values. Compound 46 was tested for
activity against T. vaginalis, as well as counter-screened against Ect1/E6E7 cells, which revealed
the natural product had a favorable SIEct1 of ~20. Based on our prior experiences with redox-
active compounds (vide supra), 46 was tested under aerobic, microaerophillic, and anaerobic
conditions to determine how those changes would influence its activity. To our surprise,
compound 46 maintained consistent potency under all experimental conditions (average EC = 3
50
µM) (Figure S28, Supporting Information), which suggested that this compound functioned in a
manner that was different than 1 and its analogues.
Reduction of Xanthoquinodin A1. While the activity of 46 was different compared to the
simple quinones that we had tested, we were interested in determining how this compound would
work if it were subjected to chemical reduction. For that reason, compound 46 was treated with
1
3
sodium borohydride to provide 47 (Scheme 1). Analysis of the C NMR data for 47 revealed the
loss of a carbonyl chemical shift relative to 46 with the concomitant addition of a new spin at C
7
1.9. Follow-up HSQC and HMBC experiments [e.g., correlation from H-10' ( 5.04) → C-13
H
(
 30.5)] and ROESY [e.g., cross-peak observed between H-10' ↔ H-4' ( 6.47)] confirmed
C
H
that the C-10' carbonyl in 46 had been stereospecifically reduced to an alcohol yielding a single
isomeric product. Testing revealed that the inhibitory activity of 47 against T. vaginalis (EC = 3
50
µM) was indistinguishable from 46 (Figure S8, Supporting Information). This indicated that
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unlike compounds 1-45, the activity of natural product 46 may not be as susceptible to redox-
dependent processes.
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Establishing an Antibacterial Counter-Screen with Lactobacillus acidophilus. While the
antitrichomonal activity of 46 appeared promising, it was not apparent what effect this and other
compounds we anticipated discovering would have on a person’s microflora. This was a concern
because microbiome disruption can have deleterious consequences for patients following
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8
antibiotic treatment. For that reason, Lactobacillus acidophilus was chosen as a representative
bacterium species because of its widespread occurrence in the human mouth, intestinal tract, and
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9
vagina. Unfortunately, compound 46 was determined to exhibit near equipotent activity against
T. vaginalis and L. acidophilus (Figure 8). In contrast, metronidazole showed no inhibition of the
bacterial indicator strain at the concentrations tested, although this compound does inhibit other
4
0
bacteria. Consequently, 46 was dropped from further consideration given the negative impact
that its antibacterial activity might have on the stability of a patient’s microflora.
Bioassay-Guided Purification and Testing of Natural Products from Fusarium sp. Isolate
B. A crude extract prepared from Fusarium sp. isolate B was subjected to bioassay-guided
fractionation using silica gel vacuum liquid chromatography (VLC), HP20SS VLC, and C18
HPLC to afford the compound equisetin (48). In the process of obtaining 48, 5'-epiequisetin (49)
was also purified from the extract since its LC-ESIMS data had provided evidence that it was
likely a structural analogue of the bioactive constituent. The purified compounds were identified
1
13
41
by comparisons of their H and C NMR spectra and HRESIMS data to published values. The
absolute configurations of the tetramic acid portions of both compounds were determined using a
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previously described oxidative bond-cleavage and LC-ESIMS-facilitated analysis methodology.42-
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3
Testing of the purified compounds revealed a stark difference in their respective
antitrichomonal activities; whereas 48 exhibited rather potent inhibitory effects (EC = 3 µM)
5
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with good selectivity (SI3T3 = 33), 49 was inactive at concentrations up to 25 M. An examination
of both metabolites revealed that the remarkable difference in their biological activities could be
traced to the epimerization of a single stereocenter (5'S in 48 and 5'R in 49). Taking these data
into consideration, as well as noting that 48 was non-toxic to L. acidophilus at concentrations up
to 50 M (Table 3), there was ample reason to believe that further exploration of this family of
natural products had the potential to provide additional bioactive analogues.
To facilitate the identification of other natural products containing a tetramate linked to a
trans-decalin system, we turned to our extensive library of fungal isolates and their associated
chemical data. This family of natural products has been reported from a taxonomically diverse
assemblage of fungi41-42, 44-46 and our records revealed that we had previously encountered these
compounds from several fungal isolates in our collection. After considering the range of structural
variation that we could anticipate from the fungal isolates available to us, 4 fungi were selected to
serve as the subjects of a targeted (chemically-guided) purification process: (i) Fusarium sp.
isolate C, (ii) Penicillium sp., (iii) Alternaria sp., and (iv) Phoma sp. P34E5. Those efforts led to
the purification of 7 additional tetramic-acid-containing metabolites including trichosetin (50),45
42
46
pyrrolocin A (51), 5'-epipyrrolocin A (52) (new), beauversetin (53), 5'-epibeauversetin (54)
4
4
44
(
new), phomasetin (55), and 5'-epiphomasetin (56) (Figure 9 and Table 3). Whereas the
previously reported tetramic acid metabolites were identified based on comparisons of their
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experimental versus published spectrometry and spectroscopy data, the new analogues became the
foci of our structure determination efforts.
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Compound 52 (purified from the Penicillium sp. isolate) was obtained as a pale yellow solid.
-
Its molecular formula was deduced from HRESIMS data to be C H NO ([M-H] ion at m/z
2
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39
5
1
13
4
56.2747, calcd for 456.2755). The H and C NMR data (Tables 1 and 2) revealed the presence
of eight olefinic carbons, an oxygenated methylene, an oxygenated methine, and 5 methyl groups.
Comparison of the 1D NMR data of 52 with compound 51 (isolated from the same fungus)
1
3
indicated that the only noteworthy difference in the C NMR data for these two molecules was
that the carbon spin resonating at δ 68.1 in compound 51 now appeared at δ 67.4 in metabolite
C
C
5
2. Combining this information with the bond-line structure established for 52 (based on analyses
1
1
of its H- H COSY, HSQC, and HMBC data, Figure 10A), it was proposed that the new natural
product was the 5'-epimer of 51.
Upon probing the stereochemical properties of compound 52, the relative configuration of the
1
1
decalin system was affirmed based on H- H ROESY correlations detected between the following
sets of protons: Me-12 ↔ H-6 and H-3; H-10ax ↔ H-6, H-7eq, and H-8; as well as H-7ax ↔ H-
1
0eq and H-11 (Figure 10A). The absolute configuration of this moiety was determined by
comparing its CD spectrum with data obtained for similar metabolites. Specifically, the positive
Cotton effects at 234 and 291 nm indicated that the absolute configuration of the decalin-ring
portion of this structure was 2R,3S,6R,8S,11S (Figure 12, Figures S31 and S32, Supporting
4
2
Information). The absolute configuration of the hydroxy group on the olefinic side chain was
determined to be 18R based on an analysis employing the modified Mosher reaction (Figure
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1).42, 47 The absolute configuration of C-5' was determined by oxidative bond cleavage of the
tetramic acid ring followed by acid hydrolysis to yield N-Me-serine.42-43 Marfey derivatization of
the N-Me-serine was performed and the product was analyzed by LC-ESIMS together with the
derivatized products generated from both N-Me-L- and D-serine standards. The N-Me-D-serine
was made by racemization of commercially available N-Me-L-serine using acetic acid and
salicylaldehyde as described previously.48-49 The resulting amino acid residue was determined to
be N-Me-L-serine, thus, the absolute configuration of 52 was assigned as
1
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R,3S,6R,8S,11S,18R,5'S.
Compound 54 was purified as a pale yellow solid. Its molecular formula was deduced as
1
C H NO based on HRESIMS data. The H NMR spectrum of 54 (Table 1) showed thirty-one
2
4
33
4
1
3
proton signals including 5 aromatic protons and 4 methyl spins. The C NMR spectrum (Table 2)
contained 24 carbon spins, which were later classified according to their multiplicities using an
HSQC experiment. Those data indicated that 54 was structurally similar to 52. Based on 2D NMR
(1
H- H COSY, HSQC, and HMBC) data (Figure 10B), the bond-line structure of 54 was
1
confirmed to be the same as metabolite 53, which indicated that this compound was likely to be
another 5'-epimer.
The relative configuration of the decalin moiety was deduced as 2R*,3S*,6R*,8S*,11S* based
1
1
on the key H- H ROESY correlations between Me-12 ↔ H-3 and H-6, H-6 ↔ H-9eq, H-6 ↔ H-
, as well as H-11 ↔ H-7ax and H-9ax (Figure 10B). The positive Cotton effects recorded for 54
8
at 234 and 289 nm indicated that the absolute configuration of the decalin moiety in 54 was the
same as that in compound 53 (Figure 13). Further analysis of key ROESY correlations including
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13
15
H-15 ↔ H-13 and Me-17 revealed both exocyclic olefins (∆ and ∆ ) had trans geometries.
Using the same oxidative bond cleavage and LC-ESIMS analysis method that had been applied to
the tetramic acid moiety in metabolite 52, the absolute configurations of C-5' stereocenters were
determined as 5'R (53) and 5'S (54), respectively.42-43, 49 Thus, the absolute configuration of 54
was determined to be 2R,3S,6R,8S,11S,5'S.
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Assessing the Biological Activities of the Tetramic-Acid-Containing Natural Products.
The antitrichomonal effects of the tetramic-acid-containing natural products were evaluated
revealing an intriguing trend; whereas several of the compounds exhibited potent activities with
EC values at or below single digit micromolar concentrations (i.e., 48, 50, 51, 53, and 55) their
5
0
5
'-epimers were inactive even at concentrations as high as 25 M (Table 3). The three most potent
molecules, 51 (EC = 0.060 M), 53 (EC = 0.80 M), and 55 (EC = 0.35 M), revealed that
5
0
50
50
the tetramic-acid-containing molecules were among the most potent T. vaginalis inhibitors we
have observed to date. To determine if the Trichomonas-inhibitory effects of the tetramic acid
natural products were affected by the presence of molecular oxygen, metabolite 53 was tested
under aerobic, microaerophilic, and anaerobic conditions (Figure S29, Supporting Information).
Unlike many of the other compounds that we tested, 53 maintained potency under all the test
atmospheric conditions exhibiting no statistically significant change in EC value, as well as
50
affording the complete elimination of the parasite. Although some tetramic-acid-containing
compounds have been reported to possess antibacterial activities,50-51 none of the compounds that
inhibited T. vaginalis inhibited L. acidophilus at 50 M (Figure 8). The selectivity of the tetramic-
acid metabolites for T. vaginalis versus mammalian cells also appeared favorable (Table 3) with
relatively large SI values for compound 51 (SI3T3 = 100, SIEct1 = 167). Based on these data, the
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tetramic-acid-containing natural products exhibited many promising characteristics that highlight
their potential for further development as inhibitors of T. vaginalis.
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CONCLUSIONS
A rapid and sensitive assay for the detection of T. vaginalis inhibitors was developed and used
to test a variety of substances varying from pure compounds to complex fungal extracts. In the
process of screening a portion of our fungal extract library, we detected and went on to purify
several natural products that exhibited inhibitory activities against T. vaginalis and were relatively
non-cytotoxic to human Ect1/E6E7 cells. In parallel to their PAINS-like qualities, quinone-
containing compounds36, 52 were generally found to be problematical starting points for the
development of new antitrichomonal scaffolds since their inhibitory properties were significantly
influenced by the relative amounts of oxygen the trichomonads were exposed to throughout the
assay period. Moreover, these types of rather promiscuous compounds exhibited antibacterial
activities against L. acidophilus suggesting that their non-specific antibiosis effects could upset
the microbiome structures of patients, thus creating new potential problems.
Based on the lessons learned from the quinones, we proceeded to test a second group of more
promising tetramic-acid-containing natural products as inhibitors of T. vaginalis. A striking
feature of their antitrichomonal effects was the decided difference in the biological activities of
the 5'-epi-isomers, which generally showed no inhibitory effects against T. vaginalis. Compound
5
1 emerged as a rather promising metabolite based on its potent activity against the parasite (EC50
= 0.060 M) and limited toxicity against both the human Ect1/E6E7 cervical cell line (SI = 167)
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and L. acidophilus (EC > 50 M). Although our testing was limited to 9 tetramic-acid-
5
0
containing natural products, the data suggested that modifications to the C-3 group of the trans-
decalin system may provide additional opportunities for generating new analogues that offer even
further improvements in potency and selectivity.
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EXPERIMENTAL SECTION
General Experimental Procedures. UV data were recorded on a Hewlett Packard 8452A
®
diode array spectrophotometer. Optical rotation measurements were made on an AUTOPOL III
automatic polarimeter. The LC-ESIMS analyses were performed on a Shimadzu UFLC system
with a quadrupole mass spectrometer using a Phenomenex Kinetex C column (3.0 mm × 75 mm,
1
8
2
.6 μm) and MeCN-H O (0.1% HCOOH) gradient solvent system. HRESIMS spectra were
2
measured using an Agilent 6538 Ultra High Definition (UHD) Accurate-Mass Q-TOF system.
1
NMR spectra were obtained on Varian spectrometers (500 MHz and 400 MHz for H, and 100
1
3
MHz for C) using DMSO-d and CDCl as solvents. Column chromatography was conducted
6
3
using silica gel and HP20SS. HPLC was performed on a Waters System equipped with a 1525
binary HPLC pump coupled to a 2998 PDA detector with a Phenomenex Gemini C column
1
8
(21.2 × 250 mm or 10 × 250 mm, 5 μm particle size).
Culture of Organisms. Trichomonas vaginalis Donne (PRA-98) was purchased from the
American Type Culture Collection (ATCC, Bethesda, MD). After some investigation, Keister’s
5
3
Modified TYI-S33 medium (adjusted to pH 6.0 and without Diamond’s vitamin solution) was
determined to be the optimum medium to support the growth of T. vaginalis. The medium was
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filter sterilized and aliquots stored frozen for later use. A notable difference in the growth of T.
vaginalis was observed dependent upon the source from which the bovine bile obtained. The best
growth of T. vaginalis was observed when Sigma B8381 bovine bile was used and it was adopted
as the sole bovine bile source for all experiments. For culture maintenance and propagation,
trichomonads were grown in a 37 °C incubator in sealed screw cap tubes. For microtiter plate
assays, trichomonads were tested under several atmospheric conditions including standard
atmospheric condition (aerobic atmosphere: ~21% O , 0.04% CO ), candle jar (microaerophilic
1
1
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atmosphere: ~10% O , 5% CO ), BD GasPak EZ Campy sachets (microaerophilic atmosphere: 6-
2
2
1
6% O , 2-10% CO ), and BD GasPak EZ Anaerobe sachets (anaerobic atmosphere: O at
2 2 2
nondetectable level, >13% CO ). Unless specified, assays were carried out in conditions
2
employing reduced levels of oxygen and increased levels of carbon dioxide (microaerophilic),
which stimulates Trichomonas growth.54
NIH/3T3 (CRL-1658) mouse fibroblast and Ect1/E6E7 (CRL-2614) normal-type human
ectocervical cell lines were purchased from ATCC. NIH/3T3 cells were maintained in Dulbecco’s
Modified Eagle’s medium with 5% FetalClone III (Hyclone) and Ect1/E6E7 cells in keratinocyte-
serum free medium (K-SFM, GIBCO-BRL 17005-042) or RPMI 1640 medium with 5%
FetalClone III and supplemented with EGF (10 ng/mL Novoprotein C029).
Lactobacillus acidophilus (ATCC 4356) was purchased from ATCC. The bacterium was
maintained and tested on MRS agar and broth at 37 °C under anaerobic conditions (BD GasPak
EZ Anaerobe sachets).
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Trichomonas vaginalis Assays. For assays, a 250 µL aliquot of T. vaginalis cryopreserved in
liquid N with 5% dimethyl sulfoxide (DMSO) of was thawed rapidly at 37 °C and put into a
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screw cap tube containing 12 mL of pre-warmed modified TYI-S-33 medium. The sample was
incubated for 24 h at which point confluent growth of the parasite had occurred and live
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trichomonads were counted. A typical count yielded > 3.010 cells per tube. Stock cultures were
diluted in fresh medium to achieve 40,000 trichomonads per 100 µL of medium in each well of a
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6-well microtiter plate. Pin tools and pipets were used to dispense extracts and pure compounds
dissolved in DMSO. Throughout the assay setup process, trichomonads were exposed to normal
atmospheric conditions for not more than 30 min before experiments commenced. The
trichomonads were determined to tolerate 1% DMSO with no detectable negative effects (no
changes to the number of live trichomonads). Accordingly, all experiments were conducted so
that the amount of DMSO did not exceed 1% by volume. All plates contained vehicle-only
(DMSO) and positive (25 µM metronidazole) controls to ensure that the biological response of
the trichomonads were consistent across assays using identical test conditions. For candle jar
assays, plates were placed in a humidified candle jar, the candle was lit, and the jar was sealed
before being placed in a 37 °C incubator for a 17 h incubation period. For assays using BD
GasPak, the manufacturer’s instructions were followed as provided. After 17 h of incubation,
microtiter plates were removed and 100 µL of room temperature fixation solution added to each
well. The fixing solution was a PBS based solution developed in our lab that contained 1%
glutaraldehyde, 5 M propidium iodide, and 5 M acridine orange (HCl salt). This solution was
found to be stable at room temperature for several months and it lasted even longer when stored in
a refrigerator. Plates treated with fixing solution were placed on an orbital shaker for 30 s to
disperse clumps of cells and then moved to an incubator for 3 h for staining and fixation to occur.
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The plates were quantitatively imaged using the Perkin Elmer Operetta and analyzed using the
Harmony 3.5.1 software package. Quantification involved the software identifying all
trichomonads and subtracting live (green only) from dead (green and red) cells using a propidium
iodide threshold of less than 6500 units; this threshold was determined to enable the assessment of
membrane integrity and thus served as an indicator of live versus dead cells.
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A traditional resazurin based assay employing a plate reader for live cell quantification was
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also performed. Trichomonads were grown in microtiter plates and at the end of experiments, 10
µL of a 0.1 mg/mL resazurin stock in PBS was added to each well. Plates were incubated at room
temperature in the dark for 1 h. At the end of the incubation period, the plates were shaken and
analyzed on a fluorescence plate reader (Tecan Infinite M200) with a 556 nm excitation
wavelength and an emission of 590 nm.
Mammalian Cell Cytotoxicity Assays. Mammalian cell assays were performed as described
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previously and viability determined by MTT assay or by a Calcein AM and Hoechst 33342
live cell area assay using an Operetta system. Assays were performed by adding to each well of
the microtiter plate 5 µL of a 40 M Calcein AM and 160 M Hoechst 33342 stock solution
prepared in DMSO that was diluted (1:5) in PBS buffer. After treatment, plates were incubated
for 30 min before being analyzed on the Operetta system. Harmony software was used to
calculate the live cell area by finding all Hoechst-labeled nuclei and the cells were assigned live
or dead status based on their degree of green fluorescence. Live cells contain esterases that cleave
the AM group from Calcein AM causing the cells to glow bright green.
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Lactobacillus acidophilus Viability Assay. Colonies of L. acidophilus were sampled with a
loop and used to inoculate 10 mL of MRS broth and then vortexed. 100 µL aliquots were added to
each well of a 96-well microtiter plate. Test compounds were dissolved in DMSO (0.5% final
concentration DMSO) and added to the wells. Plates were read on a microplate reader at 600 nm
to establish baseline absorbance (dispersion) values for each well, the plates were incubated at
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7 °C for 18 h, and their contents analyzed on the microtiter plate reader at 600 nm to determine
changes in their optical density values.
Purification of Secondary Metabolites 1-3. A fungal isolate, Fusarium solani (internal strain
designation Tree 400 EW+PNGP-3), identified based on its ribosomal internal transcribed spacer
(ITS) sequence data (GenBank accession number MK424121), was obtained from a soil sample
collected under a tree in the Oliver Wildlife Preserve in Norman, Oklahoma. This isolate was
grown for 4 weeks on Cheerios breakfast cereal supplemented with a 0.3% sucrose solution with
0
.005% chloramphenicol in three large mycobags (Unicorn Bags, Plano, TX) prior to being
homogenized, extracted, and partitioned with EtOAc. The EtOAc soluble material (40.4 g) was
subjected to silica gel vacuum liquid chromatography with elution steps of 1:1 hexanes/DCM,
DCM, 10:1 DCM/MeOH, and MeOH, yielding 4 fractions. The 10:1 DCM/MeOH fraction (7.8 g)
was subjected to HP20SS vacuum liquid chromatography and eluted with a step gradient of
MeOH in H O (30%, 50%, 70%, 90%, and 100%) and 1:1 DCM/MeOH, yielding a total of 6
2
fractions. Bioassay analysis of the resulting fractions indicated T. vaginalis-inhibitory activity was
concentrated in the 90% MeOH fraction. This fraction was subjected to further bioassay-guided
purification using preparative C HPLC (250 mm  21.2 mm, 5 μm) with a MeOH-H O gradient
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2
(30:70 to 100:0), followed by isocratic semi-preparative C HPLC (250 mm x 10 mm, 5 μm)
1
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with MeCN-H O containing 0.1% HCOOH (50:50) to yield three known bioactive
2
naphthoquinones [1 (1.5 mg), 2 (1.8 mg), and 3 (3.0 mg)].
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Testing of Additional Commercially Available and Synthesized Quinones. The structures
of compounds 4-45 are shown in Table S1, Supporting Information. The identities of synthesized
1
compounds were confirmed through H NMR, as well as by comparisons to additional
spectroscopic data as reported in the literature.
Synthesis of Naphthopurpurin (8). Compound 8 was synthesized according to a previously
5
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reported procedure. Specifically, 153 mg of naphthazarin (0.80 mmol) was dissolved in 45 mL
of aqueous KOH (2 M). The solution was stirred vigorously and refluxed for 4 h. After cooling to
ambient temperature, glacial acetic acid was used to neutralize the reaction mixture, resulting in a
color change from purple to red. The reaction mixture was partitioned with 4  40 mL of
chloroform. The aqueous phase was acidified with 5 mL of HCl (1 M) and partitioned with 4  50
mL of EtOAc. The organic phases were combined and the solvent was removed under vacuum to
1
yield naphthopurpurin (130 mg, 78% yield). H NMR (400 MHz, CDCl ): 12.74 (s, 1H), 11.47 (s,
3
1
H), 7.34 (d, J = 9.5 Hz, 1H), 7.21 (d, J = 9.5 Hz, 1H), 6.37 (s, 1H).
Synthesis of 2-Methoxynaphthazarin (9). Compound 9 was synthesized according to a
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previously reported procedure. Specifically, 120 mg of naphthopurpurin (0.58 mmol) was
dissolved in 5 mL of absolute MeOH and 80 μL of HCl (12 M). The solution was stirred and
refluxed for 4 h, then brought back to room temperature. The product was purified from the
reaction mixture by silica gel vacuum liquid chromatography using a stepwise gradient (hexanes,
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:1 hexanes/EtOAc, 2:1 hexanes/EtOAc, 1:1 DCM/MeOH, and MeOH). From the 3:1
1
hexanes/EtOAc fraction, 2-methoxynaphthazarin was obtained (17 mg, 13% yield). H NMR (400
MHz, CDCl ): 12.63 (s, 1H), 12.16 (s, 1H), 7.27 (d, J = 9.5 Hz, 1H), 7.20 (d, J = 9.5 Hz, 1H),
3
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.16 (s, 1H), 3.93 (s, 3H).
Synthesis of 1,4-Dihydro-5,8-dihydroxy-2-methyl-9,10-anthracenedione (10). Compound
5
9
1
0 was synthesized according to a previously reported procedure. Specifically, 157 mg of
naphthazarin (0.83 mmol) was dissolved in 5 mL glacial acetic acid. To this solution, 200 μL of
isoprene (2.0 mmol) was added, and the mixture was stirred with heating to 70 °C for 48 h. The
product was semi-purified from the reaction mixture by silica gel vacuum liquid chromatography,
eluted with 25:1 hexanes/EtOAc, yielding a crude mixture of products. The mixture was dissolved
in 5 mL of aqueous KOH (2 M) and stirred vigorously for 30 min. HCl (1 M) was added to
acidify the solution, which was gravity filtered to yield 1,4-dihydro-5,8-dihydroxy-2-methyl-9,10-
1
anthracenedione (38 mg, 18% yield). H NMR (400 MHz, CDCl ): 12.54 (s, 2H), 7.21 (s, 2H),
3
5
.55 (m, 1H), 3.25 (m, 2H), 3.14 (m, 2H), 1.82 (s, 3H).
Synthesis of 1,4-Dihydro-2-methyl-9,10-anthracenedione (11). Compound 11 was
5
9
synthesized according to a previously reported procedure. Specifically, 187 mg of 1,4-
naphthoquinone (1.2 mmol) was dissolved in 5 mL glacial acetic acid. To this solution, 200 μL of
isoprene (2.0 mmol) was added, and the mixture was stirred with heating to 70 °C for 48 h. To the
reaction mixture, 5 mL of H O were added and the mixture was cooled to 0 °C. The mixture was
2
gravity filtered to yield a crude mixture of products. The mixture was dissolved in 5 mL of
aqueous KOH (2 M) and stirred vigorously for 15 min. The solution was acidified with 1 mL of
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HCl (12 M). The resulting solution was gravity filtered to yield 1,4-dihydro-2-methyl-9,10-
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anthracenedione (95 mg, 35% yield). H NMR (400 MHz, CDCl ): 8.07 (m, 2H), 7.69 (m, 2H),
3
5.54 (m, 1H), 3.23 (m, 2H), 3.12 (m, 2H), 1.80 (s, 3H).
1
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Purification of Secondary Metabolite 46. A fungal isolate, Humicola grisea (internal strain
designation Mystery-9 SEA-2), was identified based on its ribosomal internal transcribed spacer
(
ITS) sequence data (GenBank accession number MK424122). The fungus was grown for 4
weeks on Cheerios breakfast cereal supplemented with a 0.3% sucrose solution containing
.005% chloramphenicol in three large mycobags (Unicorn Bags, Plano, TX) prior to being
0
homogenized, extracted, and partitioned with EtOAc. The EtOAc soluble material (57.5 g) was
subjected to silica gel vacuum liquid chromatography (VLC) with elution steps of 1:1
hexanes/DCM, DCM, 10:1 DCM/MeOH and MeOH, yielding 4 fractions. The 10:1 DCM/MeOH
fraction (8.0 g) was subjected to HP20SS VLC and eluted with a step gradient of MeOH in H O
2
(30%, 50%, 70%, 90%, and 100%) and 1:1 DCM/MeOH, yielding a total of 6 fractions. Bioassay
analysis of the resulting fractions revealed the T. vaginalis-inhibitory activity was concentrated in
the MeOH fraction from HP20SS VLC. The MeOH fraction was subjected to further bioassay-
guided fractionation using preparative C HPLC (250 mm  21.2 mm, 5 μm) with a MeOH-H O
1
8
2
gradient containing 0.1% HCOOH (50:50 to 100:0) to yield the known bioactive compound 46
(52.2 mg).
Borohydride Reduction of 46. To a flask, 9.0 mg of 46 (0.016 mmol) in 4 mL MeOH was
added. While stirring, 7.6 mg of sodium borohydride (0.20 mmol) dissolved in 1 mL MeOH was
added slowly over a 5 min period. The reaction mixture was stirred at room temperature for 45
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min. The reaction was quenched by adding 500 μL of 1M HCl to the flask and stirred for 10 min.
Solvent was removed from the reaction mixture under vacuum to yield compound 47 (8.8 mg,
1
96% yield). H NMR (500 MHz, CDCl ): 14.44 (s, 1H), 13.94 (s, 1H), 11.91 (s, 1H), 11.44 (s,
3
0
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H), 7.04 (s, 1H), 6.70 (s, 1H), 6.47 (m, 2H), 6.08 (s, 1H), 5.04 (s, 1H), 4.74 (dd, J = 5.5, 2.0 Hz,
H), 4.26 (dd, J = 4.0, 2.0 Hz, 1H), 3.68 (s, 3H), 3.05 (d, J = 18.0 Hz, 1H), 2.81 (m, 1H), 2.58 (d,
1
3
J = 18.0 Hz, 1H), 2.40 (s, 3H), 2.37 (m, 1H), 2.12 (m, 1H), 1.91 (m, 1H). C NMR (125 MHz,
CDCl ): 188.5, 186.7, 184.6, 179.4, 171.1, 161.4, 158.5, 155.9, 148.3, 147.6, 141.8, 134.9, 131.8,
3
1
17.5, 117.22, 111.7, 111.3, 106.5, 104.6, 100.1, 83.8, 71.9, 66.8, 53.4, 43.6, 37.6, 30.5, 24.4,
23.0, 22.4.
Purification of Tetramic Acid Derivatives 48-56. 5 fungal isolates coded Miller-1 Cz-3,
Miller-26 SEA-3, KY6863 TV8-3, Column L5 SEA-1, CA9310 TV8-3, were identified as
Fusarium sp. isolate B, Fusarium sp. isolate C, Penicillium sp., Alternaria sp., Phoma sp. P34E5,
respectively, based on their ribosomal internal transcribed spacer (ITS) sequence data (GenBank
accession numbers MK401898, MK401899, MK401895, MK401896, and MK401897,
respectively). These five fungal isolates were fermented on a solid-state medium composed of
Cheerios breakfast cereal supplemented with 0.3% sucrose solution for 4 weeks at 25 °C. The
fungal isolate designated Column L5 SEA-1 was inoculated into 16 L of PDB media (10 g/L
dried mashed potato, 5 g/L glucose) supplemented with 2 g/L NaNO and grown for 9 days with
3
shaking.
The fungal culture designated Miller-1 Cz-3 (Fusarium sp. isolate B) was homogenized and
partitioned 3 with equal volumes of EtOAc. The resulting EtOAc soluble material (25.0 g) was
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subjected to silica gel VLC with elution steps of 1:1 hexanes/DCM, DCM, 10:1 DCM/MeOH,
and MeOH, yielding 4 fractions. The 10:1 DCM/MeOH fraction was subjected to HP20SS VLC
and eluted with a step gradient of MeOH in H O (30%, 50%, 70%, 90%, and 100%), yielding 5
2
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fractions. The 90% MeOH fraction from HP20SS VLC was further fractionated by preparative
C HPLC (250 mm  21.2 mm, 5 μm) with a MeCN-H O gradient containing 0.1% HCOOH
1
8
2
(70:30 to 100:0) to yield compounds 48 (56 mg) and 49 (18.9 mg).
The fungal culture designated Miller-26 SEA-3 (Fusarium sp. isolate C) was homogenized and
partitioned 3 with equal volumes of EtOAc. The resulting EtOAc soluble material (14.0 g) was
subjected to silica gel VLC with elution steps of 1:1 hexanes/DCM, DCM, 10:1 DCM/MeOH,
and MeOH, yielding 4 fractions. The 10:1 DCM/MeOH fraction was subjected to HP20SS VLC
and eluted with a step gradient of MeOH in H O (30%, 50%, 70%, 90%, and 100%), yielding 5
2
fractions. The 70% MeOH fraction from HP20SS VLC was further fractionated by isocratic
preparative C HPLC (250 mm  21.2 mm, 5 µm) with MeOH-H O (75:25) to yield compound
1
8
2
50 (50 mg).
The fungal culture designated KY6863 TV8-3 (Penicillium sp.) was homogenized and
partitioned 3 with equal volumes of EtOAc. The resulting EtOAc soluble material (31.0 g) was
subjected to silica gel VLC with elution steps of 1:1 hexanes/DCM, DCM, 10:1 DCM/MeOH,
and MeOH, yielding 4 fractions. The 10:1 DCM/MeOH fraction was subjected to HP20SS VLC
and eluted with a step gradient of MeOH in H O (30%, 50%, 70%, 90%, and 100%), yielding 5
2
fractions. The 90% MeOH fraction from HP20SS VLC was further fractionated by preparative
C HPLC (250 mm  21.2 mm, 5 µm) with a MeOH-H O gradient containing 0.1% HCOOH
1
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(
85:15 to 100:0) to yield compounds 51 (50 mg) and 52 (15 mg). 5'-epipyrrolocin A (52): pale
yellow plates; [α]20 +93 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 238 (2.37), 265 (0.42), 295
D
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(0.77); CD (MeOH) λmax (Δε) 234 (8.1), 291 (6.1); H and C NMR data, see Tables 1 and 2;
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6
7
8
9
0
-
HRESIMS m/z 456.2747 [M - H] (calcd for 456.2755).
Fungal culture broth prepared from the fungus designated Column L5 SEA-1 (Alternaria sp.)
was partitioned 3 with equal volumes of EtOAc to provide 3.0 g of EtOAc-soluble residue. The
organic residue was subjected to HP20SS VLC and eluted with a step gradient of MeOH in H O
2
(30%, 50%, 70%, 90%, and 100%), yielding 5 fractions. The 90% MeOH fraction was further
fractionated by preparative C HPLC (250 mm  21.2 mm, 5 µm) with a MeOH-H O gradient
1
8
2
containing 0.1% HCOOH (90:10 to 100:0) yielding 6 fractions (Fr.1-6). Bioactive compounds in
fractions 2 and 4 were further purified by isocratic semi-preparative C HPLC (250 mm  10
1
8
mm, 5 µm) with MeCN-H O containing 0.1% HCOOH (80:20) to yield compounds 53 (67 mg)
2
and 54 (7.5 mg). 5'-epibeauversetin (54): pale yellow plate; [α]20 +409 (c 0.17, MeOH); UV
D
1
(MeOH) λmax (log ε) 236 (4.39), 287 (4.11); CD (MeOH) λmax (Δε) 232 (8.2), 285 (9.8); H and
13
-
C NMR data, see Tables 1 and 2; HRESIMS m/z 398.2337, [M - H] (calcd for C H NO ,
2
4
32
4
3
98.2337).
The fungal culture designated CA9310 TV8-3 (Phoma sp. P34E5) was homogenized and
partitioned 3 with equal volumes of EtOAc. The resulting EtOAc soluble material (38.0 g) was
subjected to silica gel VLC with elution steps of 1:1 hexanes/DCM, DCM, 10:1 DCM/MeOH,
and MeOH, yielding 4 fractions. The 10:1 DCM/MeOH fraction was subjected to HP20SS VLC
and eluted with a step gradient of MeOH in H O (30%, 50%, 70%, 90%, and 100%) yielding 5
2
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ACS Infectious Diseases
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fractions. The 70% MeOH fraction from HP20SS VLC was further fractionated by isocratic
preparative C HPLC (250 mm  21.2 mm, 5 µm) with MeOH-H O containing 0.1% HCOOH
18
2
(85:15) to yield compounds 55 (140 mg) and 56 (4 mg).
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Preparation of MTPA Esters. Compound 52 (0.5 mg) was dissolved in pyridine-d and equal
5
4
7
portions transferred to two NMR tubes. The (R)-α-methoxy-α-trifluoromethyl-phenylacetyl
chloride [(R)-MTPA-Cl] (5 μL) was added into one sample under an N gas stream. After mixing,
2
1
it was incubated at 40 °C in a H O bath and monitored every 2 h by H NMR. The same method
2
was used to treat the second sample using (S)-α-methoxy-α-trifluoromethyl-phenylacetyl chloride
[(S)-MTPA-Cl] as the reagent to yield the (R)-Mosher ester derivative. (S)-Mosher ester
1
derivative: H NMR (500 MHz, pyridine-d ): 6.38 (1H, m, H-14), 6.07 (1H, m, H-15), 5.90 (1H,
5
m, H-16), 5.69 (1H, m, H-13), 3.87 (1H, m, H-12), 2.37 (1H, m, H-17), 2.10 (1H, m, H-17), 1.20
1
(3H, d, J = 6.5 Hz, CH -19). (R)-Mosher ester derivative: H NMR (500 MHz, pyridine-d ) 6.39
3
5
(1H, m, H-14), 6.07 (1H, m, H-15), 5.92 (1H, m, H-16), 5.72 (1H, m, H-13), 3.94 (1H, m, H-12),
2
.37 (1H, m, H-17), 2.10 (1H, m, H-17), 1.19 (3H, d, J = 6.5 Hz, CH -19).
3
Cleavage of the Tetramic Acid Ring and Marfey’s Reaction. Samples of compounds (20
mg) were dissolved in MeOH (0.6 mL) followed by addition of 1 M NaOH (75 L) and a NaOCl
solution (available chlorine 10−15%, 0.35 mL) as per prior reports.42-43 The mixture was stirred at
room temperature for 8 h and then it was quenched by adding 1 M aqueous Na SO (3 mL) and
2
2
neutralized by the addition of 1 M HCl. After removal of the solvent, the residue was diluted with
H O, and the resulting mixture was partitioned against EtOAc. The organic layer was retained and
2
the solvent removed in vacuo. The residue was dissolved in 6 M HCl (500 µL) and held overnight
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