Synthesis of 2′,3′,4′-trihydroxyflavone (2-D08), an inhibitor of protein sumoylation

Article abstract of DOI:10.1016/j.bmcl.2014.01.010

Protein sumoylation is a dynamic posttranslational modification involved in diverse biological processes during cellular homeostasis and development. Recently sumoylation has been shown to play a critical role in cancer, although to date there are few small molecule probes available to inhibit enzymes involved in the SUMO conjugation process. As part of a program to identify and study inhibitors of sumoylation we recently reported the discovery that 2′,3′,4′-trihydroxyflavone (2-D08) is a cell permeable, mechanistically unique inhibitor of protein sumoylation. The work reported herein describes an efficient synthesis of 2-D08 as well as a structurally related but inactive isomer. We also report an unanticipated Wessely-Moser rearrangement that occurs under vigorous methyl ether deprotection conditions. This rearrangement likely gave rise to 2-D08 during a deprotection step, resulting in 2-D08 appearing as a contaminant in a screening well from a commercial supplier.

Full text of DOI:10.1016/j.bmcl.2014.01.010

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1094–1097
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 20,30,40-trihydroxyflavone (2-D08), an inhibitor of protein
sumoylation
⇑
Yeong Sang Kim, Samantha G. L. Keyser, John S. Schneekloth Jr.
Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, 376 Boyles St., Frederick, MD 21702, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Protein sumoylation is a dynamic posttranslational modification involved in diverse biological processes
during cellular homeostasis and development. Recently sumoylation has been shown to play a critical
role in cancer, although to date there are few small molecule probes available to inhibit enzymes involved
in the SUMO conjugation process. As part of a program to identify and study inhibitors of sumoylation we
recently reported the discovery that 20,30,40-trihydroxyflavone (2-D08) is a cell permeable, mechanisti-
cally unique inhibitor of protein sumoylation. The work reported herein describes an efficient synthesis
of 2-D08 as well as a structurally related but inactive isomer. We also report an unanticipated Wessely–
Moser rearrangement that occurs under vigorous methyl ether deprotection conditions. This rearrange-
ment likely gave rise to 2-D08 during a deprotection step, resulting in 2-D08 appearing as a contaminant
in a screening well from a commercial supplier.
Received 19 September 2013
Revised 2 January 2014
Accepted 6 January 2014
Available online 11 January 2014
Keywords:
SUMO
Synthesis
Inhibitor
Flavone
Ó 2014 Published by Elsevier Ltd.
The posttranslational modification of protein substrates with
the small ubiquitin-like modifier (SUMO) has emerged as an
important regulatory mechanism and a critical pathway in embry-
onic development and cancer.1 SUMO modification can result in a
variety of consequences that vary broadly depending on the
substrate. Documented effects of SUMO modification include al-
tered subcellular localization,2 transcriptional regulation,3 and
enzymatic activity.4 Protein sumoylation is also thought to be in-
volved with the cellular stress response, playing important roles
in recovery from heat shock,5 ischemia,6 and surviving the stress
of tumorigenesis.7 Many of the known targets of sumoylation are
transcription factors, and the modification of these targets is gen-
erally (but not always) seen as a repressive mark.1b In addition, a
number of studies have demonstrated the role of sumoylation in
cancer, as illustrated by the observation of high expression levels
of the SUMO conjugating enzyme UBC9 in ovarian tumors.7 Addi-
tionally, high levels of SUMO E2 and E3 enzymes are correlated
with decreased survival rates for multiple myeloma patients.8
While the genetic knockout mouse for UBC9 is embryonically
lethal, a recent synthetic lethal screen identified sumoylation
enzymes as required for the progression of Myc-driven cancers.9
New roles for sumoylation in cancer and developmental biology
are still emerging.
tial of sumoylation enzymes.10 However, to date little progress
has been made in this regard.11 As part of a program to identify
inhibitors of sumoylation, our laboratory recently reported the
development of a novel medium throughput microfluidic electro-
phoretic mobility shift assay to monitor substrate sumoylation
in vitro.12 This assay utilizes recombinant E1 (Aos1/Uba2) and E2
(UBC9) enzymes to effect sumoylation of a fluorescently labeled
consensus sequence-containing peptide. Additionally, we discov-
ered a synthetic oxygenated flavonoid that we named ‘2-D08’
(compound 2). This compound was found to block sumoylation
of topoisomerase I in two different cancer cell lines dosed with
camptothecin. Furthermore, our analysis indicated that 2-D08
inhibited sumoylation by preventing transfer of SUMO from the
UBC9-SUMO thioester to the substrate, a mechanism of action that
was unprecedented. Although 2 has been described in several Let-
ters,13 we were unable to find a synthetic procedure or commercial
vendor for this compound in pure form.
We identified 2 as a contaminant from a well in a commercially-
supplied screening collection. Upon completion of our pilot screen,
a well believed to contain compound 1 (Fig. 1) was identified as ac-
tive. We subsequently purchased samples of this compound from
two other vendors, and found all three samples to be equally and
reproducibly active in the microfluidic biochemical assay. LC/MS
analysis using a short gradient indicated the presence of one broad
peak, with two ions appearing at m/z 271 (presumed to be [M+H]+)
and m/z 289 (presumed to be [M+H+H2O]+). However, upon
inspection of the 1H NMR spectrum in several different solvents,
it was apparent that multiple species were present. Through
extensive HPLC analysis and purification, it was established that
A small molecule inhibitor of protein sumoylation would have
significant value in studying of the role of sumoylation in basic
and cancer biology, and would help define the therapeutic poten-
⇑
Corresponding author. Tel.: +1 301 228 4620; fax: +1 301 846 6033.
E-mail address: schneeklothjs@mail.nih.gov (J.S. Schneekloth).
0960-894X/$ - see front matter Ó 2014 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2014.01.010

Y. S. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1094–1097
1095
understand the potential origin of the mixture of compounds, we
exposed trimethyl ether 8 to more vigorous deprotection condi-
tions (Fig. 2C). Upon treatment with 48% aqueous HBr and acetic
acid and heating to 130 °C in a microwave reactor, a mixture of
products was observed containing 1, 2, and 3. The yields of this
reaction were highly variable and appear to depend on a number
of factors. This latter finding provides speculative evidence that
the mixture of compounds identified from three commercial sup-
pliers arose from a vigorous deprotection (e.g. exposure to hot
HBr) during synthetic preparation by the vendors. The mechanism
of this isomerization is complex, however 3 presumably arises by
an acid-promoted ring opening reaction of 1, and 3 may then
undergo an acid-promoted ring closure to form either 1 or 2
(Fig. S5). This unanticipated reaction is a variant of the Wessely–
Moser rearrangement.15
Each of the three compounds isolated from the mixture was
evaluated in a biochemical sumoylation assay. In this assay, a
microfluidic electrophoretic mobility shift protocol was used to
monitor the conjugation of SUMO-1 to a fluorescent peptide sub-
strate.12 Compounds were evaluated in the assay at a concentra-
tion of 30 lM, and inhibition relative to a DMSO control was
measured at ꢀ30% conversion after quenching with EDTA
(Fig. 3A). Only compound 2 showed complete inhibition at this
concentration, with synthetic and purified commercial samples
having roughly equal inhibitory potency. Compound
3 was
comparatively weak, showing modest inhibitory activity at this
concentration. Furthermore, we evaluated compounds 1 and 2
(synthetic and purified commercial) in a kinetic assay (Fig. 3B).
Again, only samples of compound 2 (both synthetic and purified
commercial) showed substantial inhibitory activity, while com-
pound 1 was inactive. Although compound 2 was the major
product in all three commercial mixtures, neither its structure
nor the structure of contaminant 3 was reported by any of the
vendors.
Figure 1. (A) Partial HPLC chromatogram of
containing three components. (B) Structures of components identified from
commercial samples.
a commercial sample of 2-D08
In conclusion, herein we report an efficient synthetic route to
2-D08, an inhibitor of protein sumoylation. Furthermore, the struc-
tural identification of the active component from a mixture of three
compounds (provided by multiple separate commercial vendors) is
described. Historically, the purity of commercial screening libraries
has been problematic16 and there have been many instances of
unanticipated structures being identified from screening collec-
tions.17 In this particular case, we were able to show that an
impurity in a screening collection likely arose from a vigorous
methyl ether deprotection protocol, resulting in an unanticipated
ring opening/ring closing Wessely–Moser-type rearrangement.
This rearrangement ultimately gave rise to 2 (an isomer of the ex-
pected product 1), and 2 was identified as the active component
(2-D08). The Wessely–Moser reaction was also shown to occur on
synthetic material such as 8 upon heating with aqueous HBr to pro-
vide several products in variable yield. We found that more mild
deprotection conditions with BBr3 effectively accomplished depro-
tection and completely suppressed isomerization, albeit over a
longer reaction time. Flavone 2 and the biologically inactive isomer
1 are distinguishable, but not easily assignable, by one-dimensional
1H and 13C NMR experiments. However, HMBC analysis clearly
establishes the two structures, both of which were also confirmed
by synthesis. Once structurally confirmed, the flavonoids were eval-
uated in a biochemical assay. Compound 2 inhibited sumoylation in
both kinetic and endpoint assays, while 1 was completely inactive
under identical conditions. Another contaminant, 3, showed only
modest activity. The combination of structural, synthetic, and
biochemical studies allowed us to assign the structure 2 as 2-D08.
Finally, the synthetic procedure reported herein will be useful to
provide quantities of pure 2-D08 for further studies, and efforts
to evaluate 2-D08 in a variety of other biological contexts are
ongoing.
three distinct species were present within all three commercial
samples.
In order to accurately characterize the components of the mix-
ture, purification by preparative HPLC was pursued (Fig. 1). Each of
the three peaks was collected and analyzed by NMR and LC/MS.
Peak C was identified as b-diketone 3 (present as a mixture of
tautomers in multiple solvents). However, peaks A and B were
not easily assignable by inspection of one-dimensional 1H or 13C
spectra. HMBC experiments enabled an assignment of peak A as
flavone 1, the vendor-provided structure. Peak B, however, ap-
peared to be a different flavonoid that was assigned the structure
2, on the basis of HMBC experiments. Each of the three commercial
samples contained roughly the same ratio of these three compo-
nents (A/B/C = 1.9:2.5:1).
An independent synthetic route to both compounds 1 and 2 was
developed so as to unambiguously confirm both structures
(Fig. 2).14 The preparation of 1 began with exposure of 3,4-dimeth-
oxygalleacetophenone dimethyl ether 4 to 2-methoxybenzoyl
chloride 5 in pyridine to afford ester 6 in 80% yield. Treatment of
6 with powdered KOH in pyridine under mild heating provided 7
in 81% yield. Flavone ring formation was accomplished by brief
exposure to 1% H2SO4 in acetic acid. Finally, treatment with excess
BBr3 for three days at room temperature afforded 1 in 62% yield
(shorter reaction times afforded only partially deprotected
products). In parallel, a similar route was employed to access 2
with comparable reaction conditions and yields (Fig. 2B).
We found that a sample of synthetic 1 and had identical 1H and
13C NMR spectra in comparison to material isolated from the
commercial mixture (peak A). Similarly, synthetic 2 and peak B
had identical 1H and 13C NMR spectra. These analyses unambigu-
ously confirmed the structures of the two flavones. In an effort to

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Y. S. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1094–1097
Figure 2. (A) Synthesis of compound 1. (B) Synthesis of 2-D08 (compound 2). (C) Wessely–Moser-type isomerization observed during deprotection of 8 with aqueous HBr.
Abbreviations: pyr = pyridine, EDCI = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, DMAP = 4-dimethylaminopyridine.
Figure 3. (A) Inhibitory activity of selected compounds in an endpoint biochemical assay. Compounds were incubated with SUMO1, E1, E2 enzymes, a fluorescent substrate,
and ATP in an appropriate buffer and quenched with EDTA after 90 min. Conversion was quantified by ratiometric peak height in a microfluidic electrophoretic mobility shift
assay using a Perkin Elmer EZ Reader II. Values represent the mean of three replicates. (B) Evaluation of selected compounds in kinetic biochemical sumoylation assays.
Compounds were incubated with SUMO1, E1, E2 enzymes, a fluorescent substrate, and ATP in an appropriate buffer and monitored over the course of 110 min. Conversion
was quantified by ratiometric peak height in a microfluidic electrophoretic mobility shift assay using a Perkin Elmer EZ Reader II.

Y. S. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1094–1097
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Acknowledgments
This work was supported by the Intramural Research Program
of the NIH, Center for Cancer Research, and the National Cancer
Institute, National Institutes of Health. Vectors encoding for SAE
1/2 and UBC9 were kindly provided by Dr. Christopher Lima,
Memorial Sloan Kettering Cancer Institute. We thank Drs. Christo-
pher Lai and James Kelley for obtaining high-resolution mass
spectra for this work. We thank Drs. Martin Schnermann and Ter-
rence Burke for helpful comments during the preparation of this
manuscript.
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Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
01.010.
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