Development of flavonoid-based inverse agonists of the key signaling receptor US28 of human cytomegalovirus

Article abstract of DOI:10.1021/jm4003457

A series of 31 chalcone- and flavonoid-based derivatives were synthesized in good overall yields and screened for their inverse agonist activity on the US28 receptor of human cytomegalovirus (HCMV). With one exception (e.g., 2-(5-bromo-2-methoxyphenyl)-3-hydroxy-4H-chromen-4-one), halogen-substituted flavonoids were typically more potent inverse agonists than their related hydro derivatives. While toxicity could be used to partially explain the inverse agonist activity of some members of the series, 5-(benzyloxy)-2-(5-bromo-2- methoxyphenyl)-4H-chromen-4-one (11b) acted on the US28 receptor as a nontoxic, inverse agonist. The full inverse agonism (efficacy, -89%) and potency (EC ₅₀ = 3.5 μM) observed with flavonoid 11b is especially important as it provides both a new tool to study US28 signaling and a potential platform for the future development of HCMV-targeting drugs.

Full text of DOI:10.1021/jm4003457

Article
pubs.acs.org/jmc
Development of Flavonoid-Based Inverse Agonists of the Key
Signaling Receptor US28 of Human Cytomegalovirus
Ana Kralj,† Mai-Thao Nguyen,‡ Nuska Tschammer,† Nicolette Ocampo,§ Quinto Gesiotto,§
Markus R. Heinrich,† and Otto Phanstiel, IV*,§
†Department of Chemistry and Pharmacy, Friedrich Alexander University, Erlangen 91052, Germany
‡College of Pharmacy, University of Florida, Orlando, Florida 32827, United States
§Department of Medical Education, University of Central Florida, 12722 Research Parkway, Orlando, Florida 32826-3227, United
States
S
* Supporting Information
ABSTRACT: A series of 31 chalcone- and flavonoid-based derivatives were synthesized in good overall
yields and screened for their inverse agonist activity on the US28 receptor of human cytomegalovirus
(HCMV). With one exception (e.g., 2-(5-bromo-2-methoxyphenyl)-3-hydroxy-4H-chromen-4-one),
halogen-substituted flavonoids were typically more potent inverse agonists than their related hydro
derivatives. While toxicity could be used to partially explain the inverse agonist activity of some members
of the series, 5-(benzyloxy)-2-(5-bromo-2-methoxyphenyl)-4H-chromen-4-one (11b) acted on the US28
receptor as a nontoxic, inverse agonist. The full inverse agonism (efficacy, −89%) and potency (EC50 = 3.5
μM) observed with flavonoid 11b is especially important as it provides both a new tool to study US28
signaling and a potential platform for the future development of HCMV-targeting drugs.
4, on the other hand, was shown to inhibit HIV-1 integrase,
which mediates the insertion of viral DNA into host cellular
DNA, and it is essential for viral replication and virion
production.10 Together, these findings provided the rationale
for the selection of both chalcones and the cyclic flavonoids for
our study.
INTRODUCTION
■
Flavonoids are abundant in edible plants and possess a wide
variety of biological activities including antioxidant, anticarcino-
genic, antibacterial, and antifungal activities and may play a role
in preventing infectious diseases.1,2 One class of flavonoids, the
chalcones, are known to inhibit enzymes such as 15-hydroxy-
prostaglandin dehydrogenase,1a 5-lipoxygenase,3 cyclooxyge-
nase,4 and protein tyrosine phosphatase 1B.5 Thus, chalcone-
based drugs are potential tools for the treatment of gastric
lesions,1 asthma, inflammation, and allergies4 as well as type 2
diabetes and obesity.5 Indeed, the multitude of biological effects
induced by flavonoid platforms, coupled with the relative ease
of their synthesis, make them an ideal platform for further
study.
We were interested in evaluating chalcone derivatives as
potential inhibitors of the viral receptor signaling associated
with the human cytomegalovirus (HCMV). For example, in
2004, xanthohumol 1 (Figure 1), a prenylated chalcone derived
from hops, showed moderate antiviral activity against several
herpes viruses, including human cytomegalovirus (HCMV).6
Indeed, both xanthohumol 1 and butein 2 were shown to down
regulate the CXCR4 chemokine receptor (Figure 1).7,8 As such,
chalcones provided a promising structure class to pursue
antiviral agents.
Chemokine receptors are heptahelical G-protein-coupled
receptors (GPCRs) that bind chemokines (i.e., small chemo-
tactic cytokines, critical for recruiting and activating cells of the
immune system during inflammation). Chalcones have been
shown to disrupt this binding interaction. For example, butein 2
prevents interaction between the CXCR4 chemokine receptor
and its endogenous ligand chemokine CXCL12, which has
been shown to mediate human immunodeficiency virus-
induced neurotoxicity, proliferative retinopathy, and chronic
inflammation.11 Butein also prevents binding of CXCL12 to
both its receptors, CXCR7 and CXCR4, but does not prevent
the binding of other CXCR4 ligands, like CCL5 and CXCL8, to
their receptor. These observations suggest that chalcone 2 may
actually bind to the CXCL12 chemokine rather than its
receptor, CXCR4.11 In contrast to the action observed with 2,
we were interested in developing flavonoids, which directly
interact with specific chemokine receptors associated with
HCMV (e.g., US28) and not with their chemokine ligands.
This strategy was preferred because, in principle, one could
selectively treat HCMV-infected cells by targeting the virus-
associated receptor (US28) displayed on the cell surface.
Other flavonoid structures derived from natural products
have shown antiviral activity. For example, baicalein 3 and
quercetin 4 (Figure 1) were shown to have effective anti-
HCMV9 and anti-HIV activity,10 respectively. Baicalein, 3, was
shown to prevent HCMV entry into cells. This was
accomplished by targeting the kinase activity of EGFR, which
is required for HCMV entry and cellular activation.9 Quercetin,
Received: March 7, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm4003457 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Structures of bioactive chalcones and flavones 1−4, known US28 inverse agonists 5a−5d, and synthetic targets.
The US28 receptor is one of the viral G-protein-coupled
receptors (vGPCRs) encoded in double-stranded DNA of
human cytomegalovirus (HCMV). It possesses high homology
with human chemokine receptors. For example, US28 has 30%
and 28% amino acid sequence homology with the human
CCR1 and CXCR3 receptors, respectively.12 These similarities
enable its very efficient coupling to signaling networks of the
infected host.13,14 The US28 receptor possesses the ability to
bind different human CC-chemokines (including CCL5/
Rantes) as well as the CX3C-chemokine CX3CL1/fractalkine.
Like most of the known vGPCRs, the US28 receptor also has
ligand-independent signaling. Indeed, US28 constitutive signal-
ing enables virus survival, host invasion and, in some cases,
oncogenesis or cardiovascular disease by exploiting preferred
signaling cascades.15 HCMV establishes a lifelong persistent/
latent infection in immunocompetent hosts and can lead to
severe and life-threatening diseases in patients with immature
or suppressed immune systems.16,17 Because of the potential
role of the US28 receptor in viral dissemination and persistence
as well as its role in cardiovascular disease and tumori-
genesis,17,18 inhibition of US28 constitutive activity was chosen
for our initial screens.
hydrophobic components,19 we were interested in creating
systems with fewer hydroxyl groups and greater hydro-
phobicity. To accomplish this aim, a homologous series of
monohalogenated chalcones (and flavones) were synthesized
via the crossed aldol condensation of benzaldehyde and
acetophenone derivatives.
Synthesis. While many of the required benzaldehydes and
acetophenones were commercially available, several had to be
made via O-alkylation of phenolic precursors 6a−6c. As shown
in Scheme 1, the desired alkyl groups were readily introduced
in good yields.
a
Scheme 1
Using a US28 screen described previously,13 a series of
chalcones and flavonoids (see target structures in Figure 1)
were assessed for their ability to interact with the US28
receptor. This report describes our investigation of the antiviral
activity of these systems and identified both potent agonists and
inverse agonists of the US28 receptor. The newly identified
inverse agonists are structurally distinct from the known US28
inverse agonists, 5a−5d (Figure 1) and provide a platform for
future US28 inverse agonist design.
a
Reagents: (a) MeI or EtBr, DMF, 2 days, rt.
With the required carbonyl compounds in hand, a series of
chalcones 8a−8j and flavonols 9a−9j were synthesized via
Scheme 2 in good yield. In general, the condensation of the
respective methoxybenzaldehyde and acetophenone derivatives
in strong base at 85 °C provided the desired chalcone target (8,
Scheme 2).20 These chalcone-producing reactions gave good
yields and high purities after column chromatography. In the
synthesis of 8a, prolonged reaction time (overnight stirring)
resulted in the degradation of the product, byproduct
formation, and lower yields (54%). Shorter reaction times (3
h at reflux) were shown to be more optimal in terms of
maximizing the yields of these chalcone systems. The reaction
RESULTS AND DISCUSSION
■
The target structures prepared were inspired by the scaffolds
1−4 and the known (5-halo-2-methoxy)-benzoyl motif of the
US28 allosteric modulator, S-(−)-IBMZ (see Figure 1).19 As
most of the known US28 inverse agonists, e.g., 5, contain
B
dx.doi.org/10.1021/jm4003457 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 2
a
Regeants: (a) 40% KOH/MeOH, 85 °C; (b) 35% H2O2, KOH/96% EtOH, then 1 M HCl.
rates were system dependent, however, and the couplings
could, in some cases, be conducted at 25 °C.
chloroform and slow cooling to 4 °C (albeit in 28% yield).
Overall, this approach provided rapid entry to this structure
class for bioevaluation.
In most cases, the pure product was rapidly obtained from
the crude mixture by heating and sonicating the crude solid in
hexane, followed by cooling the mixture to room temperature
and filtering of the precipitated pure compound. Because of the
simplicity of this workup, the yields were actually higher than
the isolated yields listed in Scheme 2, as some of the chalcone
product was also observed in the filtrate. The amount of
product remaining in the filtrate varied depending upon the
structure of the chalcone and the temperature at which the
reaction was performed. Lower reaction temperatures typically
gave less complex product mixtures. We also observed the
codistillation of many of the salicylaldehyde starting materials
derivatives with aqueous alcohol solvents (MeOH, EtOH),
which facilitated their removal during the workup concen-
tration step. In cases where significant amounts of the
byproducts were formed, substances were separated by column
chromatography.
Conversion of chalcone 8 to the respective 3-hydroxy-4H-
chromen-4-one derivative 9 was accomplished using 35% H2O2
in presence of strong base.21 This reaction was performed using
two conditions: first at 0 °C and then at room temperature.
The ice bath condition resulted in low conversion as observed
by TLC. The reaction was warmed to room temperature and
demonstrated higher conversion to product. Subsequent
addition of 1 M HCl typically resulted in the formation of
the desired product 9 as a white precipitate in moderate yield.
Because HCl addition resulted in precipitation of the respective
products 9a−9j, they were each filtered to facilitate the workup
and all but 9f were further purified by column chromatography.
Conveniently, due to the poor solubility of 9f in chloroform,
this substance was purified by filtration after boiling in
Synthesis of the 5-hydroxy-4H-chromen-4-ones required a
modified approach and is illustrated in Scheme 3. Because
complex mixtures were generated during the direct con-
densation of dihydroxyacetophenone 10a and benzaldehyde, a
protecting group strategy was employed. In this regard, the
mono O-benzyl derivative 10b was synthesized from
dihydroxyacetophenone, 10a, using benzyl bromide and base
in 58% yield. In subsequent steps, benzaldehyde derivatives 6d
(R4 = H) and 6a (R4 = Br) reacted cleanly with ketone 10b (R
= OBn) to provide chalcones 8k (60%) and 8l (82%),
respectively. The synthesis of flavones 11a and 11b involved
the ring closure of the respective chalcones 8 in presence of
iodine and DMSO.20 The reaction was completed in 3 h and
provided good yields for these systems and excellent purity
after column chromatography.
The 5-hydroxy-4H-chromen-4-ones (12a and 12b in Scheme
3) were then obtained via debenzylation of 11a and 11b,
respectively. The debenzylation was performed in acetic acid
and water (4:1) ratio at 110 °C for 24 h.21 Along with the
desired product (12), both benzyl alcohol and benzyl acetate
were observed as byproducts of the acetolysis reaction. These
could be removed by column chromatography to provide the
respective derivatives 12a (67%) and 12b (70%) in high purity.
As shown in Scheme 4, 8a and 8b were converted to their
4H-chromen-4-one derivatives (11c and 11d) using iodine and
DMSO at 140 °C.20 Compound 8b was also converted to the
chroman-4-one derivative 13b (11%) using a week-long reflux
in sodium acetate and n-BuOH.22a
Note: we observed significant production of the related
chroman-4-one 13h (R = Cl; 20% yield) during the synthesis of
C
dx.doi.org/10.1021/jm4003457 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
a
Scheme 3
Scheme 5
a
Reagents: (a) 40% KOH/MeOH, K2CO3, 85 °C; (b) 35% H2O2,
KOH/96% EtOH.
Surprisingly, efforts to synthesize the corresponding 3-hydroxy
system 9m, using our standard method (35% H2O2/KOH),
resulted in oxidative cleavage of the compound. Specifically, the
related cinnamic acid derivative, 15 (74%), and only a trace of
9m were obtained.
We noted in this instance that the 1-(1-hydroxy-naphthen-2-
yl)prop-2-en-1-one system (e.g., 8m) behaved as a carboxylic
acid precursor. Because this system is also highly fluorescent, it
may have utility as a carboxylic acid protecting group.
Furthermore, we noted that the naphthyl system 8m was the
only member of the chalcone series where this oxidative
cleavage reaction occurred to a major extent. We speculate that
this transformation occurred via a Baeyer−Villiger rearrange-
ment involving peroxide attack on the more electrophilic
naphthyl ketone.22b−d
a
Reagents: (a) benzyl bromide, KI, K2CO3, acetone, reflux/N2
atmosphere (58% yield); (b) 40% KOH/MeOH, 85 °C; (c) iodine/
DMSO, reflux at 140 °C; (d) CH3COOH: H2O (ratio of 4:1), 110 °C.
its precursor chalcone 8h in alcoholic KOH (Scheme 2). In this
regard, some chalcone systems like 8h after 3 h of reflux were
partially converted to the chroman-4-one system during the
chalcone synthesis step (presumably via an intramolecular
Michael addition).
1-(1-Hydroxynaphthalen-2-yl)-ethanone, 14, was used to
create a naphthyl derivative 8m for comparison to the
acetophenone derived chalcones like 8b. As shown in Scheme
5, benzaldehyde derivative 6a was condensed with ketone 14 to
give a 49% yield of the expected chalcone 8m. This reaction
was sluggish and required extensive heating at 85 °C.
In summary, a series of flavonoid structures were generated
for bioevaluation against the US28 receptor.
Bioevaluation. Having generated a homologous series of
chalcones and flavones, their ability to interact with the US28
receptor in human embryonic kidney (HEK) cells was
investigated using an Elk-1 reporter gene assay (Figure 2).
HEK cells were transfected with either an empty vector (mock
cells) or with a vector carrying US28. These matched HEK cells
provided a means to evaluate off-target effects as well as innate
drug toxicity. As many of the flavonoid drugs were not water-
soluble, all drug stock solutions were made in 100%
a
Scheme 4
a
Reagents: (a) iodine/DMSO, 140 °C; (b) CH3COONa, n-butanol, 118 °C.
D
dx.doi.org/10.1021/jm4003457 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 2. (a) PathDetect Elk1 reporter gene assay. (b) BrightGlo luminescence reaction.
dimethylsulfoxide (DMSO). The final DMSO concentration
for each cell-based experiment, however, was ≤1% DMSO. To
account for the presence of DMSO, 1% DMSO controls were
run in parallel. IC50 values of the homologous series were also
determined in Chinese hamster ovary (CHO) cells in order to
assess the relative toxicities of each member of the series in a
different cell line (see Table 1).
via the following eq 1. This equation was derived in order to
assess the net effect each compound had on non-US28
containing HEK cells transfected via the same protocol.
These control experiments were critical, as they provided a
method to address off-target effects such as compound toxicity
which could give rise to false positives in the PathDetect assay.
US28 Study. Interaction with the US28 receptor was
measured by a decrease in luminescence using a firefly
(Photinus pyralis) luciferase-based PathDetect Elk1 reporter
gene assay (Figure 2).23,24 This assay assessed the modulation
of US28-mediated activation of mitogen-activated protein
kinase (MAPK) signaling pathways (results are shown in
Table 1). As shown in Figure 2, the pathway-specific fusion
transactivator plasmid (containing GAL4 dbd and Elk1) and
the gene of interest (US28) are cotransfected with the report
plasmid containing GAL4 UAS and luciferase. After trans-
fection, the US28 is expressed and activates MAPK. The MAPK
phosphorylates the activator domain of the GAL4dbd/Elk1
fusion protein and causes its dimerization. The dimer then
binds to the GAL4 UAS region of the reporter gene and allows
for luciferase transcription. The luciferase mRNA is then
translated, and the luciferase protein is detected via
luminescence by the use of the Bright-Glo reagent.
%effect on mock cells
= (100% × (mock cell luminescence
/DMSO control luminescence)) − 100%
(1)
Compounds which had no off-target effects gave nearly
identical luminescence values in both the mock cells and the 1%
DMSO control (i.e., a mock cell luminescence/DMSO control
luminescence ratio of 1) and a net effect of 0% via eq 1 (e.g., 9j,
0% in Table 1). In contrast, compounds that were stimulatory
to the treated HEK cells gave higher mock cell luminescence
than the DMSO control, a ratio >1, and a net positive value in
eq 1 for the % effect on mock cells (e.g., 8f, 290%). On the
other hand, compounds that were intrinsically toxic gave lower
luminescence in the treated mock cells than the DMSO control,
a ratio below 1, and a net negative value for the % effect on
mock cells (e.g., 8d, −42%).
For discussion purposes, compounds which gave % effect
values near zero ( 10%) were considered as having little effect
on HEK cells transfected with empty vector. Using this
criterion, most of the compounds tested had % effects on mock
cells near zero or were very close to this cutoff range. An ideal
outcome was observed for 11b, which had essentially no effect
As a control, the empty pcDNA3 plasmid vector and the
components of PathDetect system were also used for mock
transfection of 293T HEK cells. Controls containing 1%
DMSO were run in parallel. The relative luminescence results
found with mock transfected 293T HEK cells treated with 10
μM drug and the untreated 1% DMSO control were compared
E
dx.doi.org/10.1021/jm4003457 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 1. Bioevaluation of 8−9 and 11−13 in Transfected Human Embryonic Kidney (HEK) 293T Cells Containing the US28
a
Receptor (Columns 2−4), Mock-Transfected HEK Cells (Column 5), and their IC50 Values (μM) in CHO Cells (Column 6)
b
b
c
d
compd
EC50 (μM)
pEC50 SEM
efficacy (%) effect (%) on mock cells with each drug at 10 μM (%)
CHO 48 h IC50 value (μM)
8a
1.4
7.8
5.0
6.7
4.9
6.0
5.5
7.8
6.4
5.84 0.29
5.11 0.03
5.30 0.03
5.18 0.10
5.31 0.07
5.22 0.06
5.26 0.05
5.11 0.03
5.19 0.02
ND
67
−44
−88
−52
−55
−95
−94
−58
−76
+52
8
25.5 2.7
8.7 0.3
7.2 0.5
7.2 0.4
16.9 1.3
6.1 0.8
6.7 0.7
8.4 0.6
6.8 0.9
14.2 2.7
ND
8b
8c
−29
−3
−42
5
8d
8e
8f
290
100
−30
33
8g
8h
8i
8j
10 (single concentration)
−4
8k
8l
toxic
toxic
2.9
toxic
toxic
35
8.1 1.0
15.6 1.0
>100
8m
9a
5.54 0.04
5.34 0.19
−92
−19
4.6
−1
9b
9c
NE
0.8
−11
−31
−28
−17
−1
88.5 10.1
13.5 1.3
>100
6.08 0.17
−41
9d
9e
NE
NE
NE
NE
1.32
0.31
1.78
7.3
18.3 2.5
17.3 1.9
17.9 1.6
25.0 2.2
>100
9f
9g
−23
2
9h
9i
5.88 0.22
6.51 0.14
5.75 0.25
5.14 0.10
5.46 0.09
−42
−41
−38
−49
−89
−17
0
9j
54.6 4.2
17.5 1.9
96.6 5.6
64.8 8.1
>100
11a
11b
11c
11d
12a
12b
13b
13h
−40
−3
3
3.5
NE
8.1
5.09 0.10
−55
−17
2
NE
NE
NE
NE
>100
−16
−5
−14
75.2 8.7
67.9 9.3
ND
a
Functional data were obtained on 293T HEK cells that transiently expressed US28 and components of the PathDetect trans Elk1 reporter gene
system.23,24 Dose response curves of 3−5 experiments (performed in triplicates) were normalized and pooled to get a mean curve from which the
EC50 value, and the maximum intrinsic activity of each compound was obtained. A dash line indicates that the analysis was not performed due to the
EC50 being greater than 10 μM. The resulting reporter gene assay data were analyzed by nonlinear regression using the algorithms in PRISM 5.0
b
(GraphPad Software, San Diego, CA). Curves were fitted to the sigmoid curve by nonlinear regression analysis in which the logEC50 values (pEC50
c
SEM) were determined. NE = no effect when the compound was tested at 10 μM; ND = not determined. The effect (%) on mock cells was
determined for selected compounds at a fixed 10 μM concentration vs an untreated control group containing 1% DMSO in order to rank the relative
nonspecific effects of the compounds in the mock-transfected HEK cell line. The substances that exerted a change in luminescence greater than
10% of the DMSO control were identified as substances which exert nonspecific effects on the cells, which were not related to US28 signaling.
d
These nonspecific, off-target effects may include compound toxicity. CHO cells were incubated for 48 h at 37 °C with the respective compound and
relative cell viability assessed with the MTS reagent.25a
on mock cells (−3% effect) and yet had dramatic negative
efficacy values in the PathDetect assay (−89% efficacy). In this
regard, 11b was confidently assigned as an inverse agonist of
the US28 receptor.
decrease in mock cell luminescence versus the 1% DMSO
control due to their toxicity.
According to the % efficacy observed in the Elk1 assay, most
of the compounds which contained a halogen within their
structure gave inverse agonist activity ranging from −19%
efficacy observed for 9a to −95% for 8f. In contrast, the two
nonhalogen-containing compounds 8a and 8j displayed
agonistic activity with efficacies of 67% and 52%, respectively.
The EC50 values ranged from 0.31 μM with 9i to no effect
(NE) observed at 10 μM with 9b, 9d−9g, 11c, 12a, 12b, 13b,
and 13h (Table 1). Compounds 8k and 8l were found to be
very toxic at 10 μM and were not tested further.
CHO Study. In terms of the study, our goal was to identify
nontoxic potent inverse agonists of the US28 receptor. Having
assessed their effects on mock transfected HEK cells above, we
were also interested in ranking the general toxicity of the series:
8, 9, 11, 12, and 13 in another cell line. Therefore, the IC50
The most significant exceptions were compounds which at
10 μM had significantly positive % effect values on mock cells
such as 8f (290%), 8g (100%), 8i (33%), and 8m (35%), (via
eq 1) while at the same time being inverse agonists of the US28
receptor signaling pathway with −95%, −94%, −76%, and
−92% efficacies, respectively (see Table 1). Several compounds
also provided the opposite effect and at 10 μM had significant
negative % effect values on mock cells. For example, as shown
in Table 1, compounds 8b (−29% effect), 8d (−42%), 8h
(−30%), 9c (−31%), 9d (−28%), and 11a (−40%) had inverse
agonist efficacies of −44%, −52%, −58%, −41%, ND, and
−49%, respectively. These latter compounds likely caused a
F
dx.doi.org/10.1021/jm4003457 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
value of each compound was determined in CHO cells after a
48 h incubation period at 37 °C using the MTS assay.25 This
information allowed for the ranking of systems via their toxicity
profiles in the CHO line, and the 48 h CHO IC50 values are
shown in Table 1. In each case, the chalcone system 8 was more
toxic than the corresponding ring-closed 4H-chromen-4-one 9.
With the exception of 11a (CHO IC50 = 17.5 μM), compounds
11−13 were found to be relatively nontoxic to CHO cells
(CHO IC50 values >64 μM).
We noted that with the exception of 9d (CHO IC50 >100
μM), all the compounds which gave negative effects on mock
HEK cells also gave CHO IC50 values less than 18 μM: 8b (8.7
μM), 8d (7.2 μM), 8h (8.4 μM), 9c (13.5 μM), and 11a (17.5
μM). While there are clear exceptions to this trend with other
members of the series (e.g., 8f, 290% effect on mock cells,
CHO IC50 = 6.1 μM), we speculate that toxicity may explain
the observations made with 8b, 8d, 8h, 9c, and 11a.
benzoxy-substituents (analogue 11c) resulted in an inactive
ligand.
Compound 11b is a significant discovery because it is a full
inverse agonist of US28 receptor signaling (with an EC50 value
of 3.5 μM and efficacy of −89%, Table 1) and had low toxicity
in CHO cells. In this regard, compound 11b represents the
preferred balance of high potency, strong negative efficacy and
low toxicity. While several other flavonoid derivatives (9) in
Table 1 were more potent (e.g., 9c and 9h−9j have lower EC50
values in Table 1), none of these were as efficacious (11b,
−89% efficacy).
These findings are especially important because only two
other drug classes have been reported to inhibit US28
constitutive activity. The nonpeptidergic CCR 1 antagonist,26
VUF2274 5a (Figure 1), and its derivatives have been
characterized as inverse agonists for US28 in the PLCβ
signaling pathway (as measured by an accumulation of inositol
triphosphate).27,28 Compound 5a has also been evaluated via
the same Elk1 reporter assay used in Table 1 and gave an EC50
value of 4.5 μM and efficacy of −22%.13 In addition,
dihydroisoquinolinones 5b and tetrahydroisoquinolines 5c
have also been identified as promising lead scaffolds for
inhibition of US28 constitutive activity in the p42/p44
mitogen-activated protein kinase (MAPK) and p38 MAPK-
dependent pathways.13 In this regard, compound 11b identifies
new chemical space with this property and provides an
architecture which can be accessed on large scale in a limited
number of synthetic steps.
As shown in Table 1, 17 of the 31 compounds showed some
activity against the US28 receptor target. There were several
structural classes evaluated, namely chalcones 8, 4H-chromen-
4-ones (9, 11, and 12), and chroman-4-ones 13.
In general, chalcones 8 were better inverse agonists than their
corresponding cyclic flavonoids, with the exception of 4H-
chromen-4-one 11b. However, several chalcones also influ-
enced the luminescence of mock-transfected cells. The
exceptions were chalcones 8a, 8c, 8e, and 8j, which showed
% effect values near zero in Table 1. An interesting observation
was that all the halogen substituted chalcones (8b−8i, 8m)
were characterized as inverse agonists, whereas the hydro
derivative (8a) and methyl derivative (8j) showed agonist
activity on the US28 receptor. We speculate that this
observation may be due to the novel ability of halogen-
substituted drugs to interact with their protein targets.25b
Toxicity was observed for many chalcones when tested in the
10 μM concentration range with HEK cells and while
incubating them with CHO cells for 48 h. Indeed, cytotoxicity,
which is well described for other chalcone scaffolds,1a might be
the reason for the off-target (non-US28) effects observed with
several of the chalcones on the mock-transfected HEK cells.
Interestingly, the hydro- and methyl-substituted cyclic
flavonols (9a and 9j) in contrast to their chalcone analogues
(8a and 8j) inhibited US28 receptor constitutive activity. We
noted that the chloro-substituted derivative 9h was an inverse
agonist of US28 (−42% efficacy) and had little effect on mock
cells (2% effect), whereas the bromo-substituted derivative 9b
did not show any activity on the US28 receptor at 10 μM.
Flavonols 9c and 9i, bearing the ethoxy- instead of the
methoxy-substituent on their skeleton, inhibited US28 activity
as well, but at the same time showed some non-US28 related
effects on mock-transfected cells (−31 and −17% effect,
respectively).
CONCLUSION
■
A series of chalcone and flavonoid derivatives were synthesized
in good overall yields and provided new compounds which
acted as inverse agonists of the US28 receptor. While toxicity
could be used to partially explain the apparent inverse agonism
of some members of the series, 4H-chromen-4-one derivative
11b acted on the US28 receptor as a nontoxic inverse agonist.
The full inverse agonism (efficacy, −89%) observed with
flavonoid 11b is especially important as it provides a new tool
to study US28 signaling as well as a potential novel platform for
future HCMV drug development.
EXPERIMENTAL SECTION
■
Solvents were distilled prior to use and melting points are uncorrected.
Reagents were ordered from commercial sources and used without
1
further purification. H NMR spectra were obtained on a 400 MHz
NMR spectrometer, and 13C NMR spectra were obtained at 125 MHz.
Elemental analyses were determined for each compound tested, and
the purity of each tested compound was ≥95% pure with the exception
of 9f (90% pure by LC-MS).
5-Bromo-2-ethoxybenzaldehyde (7a). A mixture of 5-bromo-2-
hydroxybenzaldehyde 6a (1.01 g, 5 mmol), ethyl bromide (1.09 g, 10
mmol), and potassium carbonate (1.38 g, 10 mmol) in N,N-
dimethylformamide (5 mL) was stirred at room temperature for 2
days. Workup included evaporation of DMF under reduced pressure.
The resultant solid was dissolved in chloroform, and the organic phase
was washed with 1 M HCl. The chloroform layer was separated, dried
over anhydrous Na2SO4, filtered, and evaporated under reduced
pressure to obtain a light-yellow solid 7a (1.09 g, 95%); mp 63−64 °C;
While chroman-4-ones 13b and 13h did not have any
significant effect on US28 receptor, compound 11b, which
contained a 4H-chromen-4-one structure was one of the most
efficacious US28 receptor inverse agonists of the series tested
(with −89% efficacy). Compound 11b had essentially no effect
on mock-transfected cells (% effect = −3%) and was essentially
nontoxic to CHO cells (CHO IC50 = 96.6 μM). Its analogues
without the bromo- or benzoxy-substituent (11a and 11d)
showed lower efficacy (−49% and −55%) and increased
nonspecific effects on mock cells (−40% and −17%,
respectively). The removal of both the bromo- and the
1
Rf 0.46 (15% ethyl acetate:hexane). H NMR (CDCl3): δ 10.41 (s,
1H), 7.91 (s, 1H), 7.60 (dd, 1H), 6.87 (d, 1H), 4.14 (q, 2H, CH2),
1.48 (t, 3H, CH3). 13C NMR (CDCl3): δ 188.5, 160.2, 138.2, 130.8,
126.1, 114.5, 113.3, 64.6, 14.6.
5-Chloro-2-methoxybenzaldehyde (7b). A mixture of 5-chloro-
2-hydroxybenzaldehyde 6b (940 mg, 6 mmol), iodomethane (1.14 g, 8
mmol), and potassium carbonate (1.66 g, 12 mmol) in N,N-
G
dx.doi.org/10.1021/jm4003457 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
dimethylformamide (6 mL) was stirred at room temperature for 2
days. Workup included evaporation of DMF under reduced pressure.
The resultant solid was dissolved in chloroform, and the organic phase
was washed with 1 M HCl. The chloroform layer was separated, dried
over anhydrous Na2SO4, filtered, and evaporated under reduced
pressure to obtain yellow solid 7b (1.04 g, 96% pure, 98% yield) that
was used in subsequent step without issue; mp 65−66 °C; Rf 0.34
Na2SO4, filtered, and concentrated to give a yellow solid (2.5 g). The
crude solid was heated and sonicated in hexane, then cooled down to
room temperature to obtain the enriched product. Column
chromatography was performed (20% ethyl acetate:hexane) to obtain
pure product 8b (1.78 g, 71%); mp 119−121 °C; Rf 0.41 (20% ethyl
acetate:hexane). 1H NMR (CDCl3): δ 12.83 (s, 1H, OH), 8.14 (d, 1H,
J = 15.5 Hz), 7.93 (d, 1H), 7.76 (s, 1H), 7.72 (d, 1H, J = 15.6 Hz),
7.50 (m, 2H), 7.03 (d, 1), 6.96 (m, 1H), 6.85 (d, 1H), 3.93 (s, 3H,
OMe). 13C NMR (CDCl3): δ 193.9, 163.6, 157.9, 139.2, 136.4, 134.5,
131.4, 129.8, 125.7, 121.8, 120.1, 118.9, 118.6, 113.1, 55.9. 8b Anal.
C16H13O3Br: C, H.
1
(15% ethyl acetate:hexane). H NMR (CDCl3): δ 10.41 (s, 1H), 7.78
(s, 1H), 7.49 (dd, 1H), 6.95 (d, 1H), 3.93 (s, 3H, OMe). 13C NMR
(CDCl3): δ 188.5, 160.3, 135.4, 128.0, 126.4, 125.6, 113.3, 56.0.
5-Chloro-2-ethoxybenzaldehyde (7c). A mixture of 5-chloro-2-
hydroxybenzaldehyde 6b (940 mg, 6 mmol), ethyl bromide (1.31 g, 12
mmol), and potassium carbonate (1.66 g, 12 mmol) in N,N-
dimethylformamide (6 mL) was stirred at room temperature for 2
days. Workup included evaporation of DMF under reduced pressure.
The resultant solid was dissolved in chloroform, and the organic phase
was washed with 1 M HCl. The chloroform layer was separated, dried
over anhydrous Na2SO4, filtered, and evaporated under reduced
pressure to obtain pure light-yellow solid 7c (1.06 g, 96%); mp 52−54
°C; Rf 0.48 (15% ethyl acetate:hexane). 1H NMR (CDCl3): δ 10.43 (s,
1H), 7.77 (s, 1H), 7.46 (dd, 1H), 6.93 (d, 1H), 4.14 (q, 2H, CH2),
1.48 (t, 3H, CH3). 13C NMR (CDCl3): δ 188.7, 159.8, 135.4, 127.8,
126.2, 125.7, 114.1, 64.6, 14.6.
(E)-3-(5-Bromo-2-ethoxyphenyl)-1-(2-hydroxyphenyl)prop-
2-en-1-one (8c). 2-Hydroxyacetophenone (409 mg, 3 mmol), 5-
bromo-2-ethoxybenzaldehyde 7a (687 mg, 3 mmol), and MeOH (15
mL) were combined, and KOH in methanol (40% weight/volume, 15
mL) was added at room temperature. A water condenser was attached
to the flask, and the yellow solution quickly turned orange upon
heating and was stirred at reflux in an oil bath at 85 °C for 3 h.
Workup involved evaporation of the solvent under reduced pressure,
addition of 1N HCl until the solution was at pH 1, and extraction with
ethyl acetate (twice). Each organic layer was separated, pooled
together, dried over anhydrous Na2SO4, filtered, and concentrated to
give a crude yellow solid (974 mg). Two column chromatography
steps were needed to purify 8c. One column (10% ethyl
acetate:hexane) was performed to remove the ketone starting material
and another one (40% chloroform:hexane) to separate 8c from a
cyclized byproduct. 8c was obtained as a yellow solid (438 mg, 42%);
mp 84−86 °C; Rf 0.42 (10% ethyl acetate:hexane); Rf 0.26 (40%
chloroform:hexane). 1H NMR (CDCl3): δ 12.84 (s, 1H, OH), 8.11 (d,
1H, J = 15.6 Hz), 7.91 (d, 1H), 7.80 (d, 1H, J = 15.6 Hz), 7.74 (s, 1H),
7.51 (t, 1H), 7.46 (dd, 1H), 7.03 (d, 1H), 6.95 (t, 1H), 6.89 (d, 1H),
4.14 (q, 2H, CH2), 1.54 (m, 3H, CH3). 13C NMR (CDCl3): δ 194.0,
163.6, 157.4, 139.6, 136.4, 134.4, 132.0, 129.7, 125.6, 121.9, 120.1,
118.9, 118.6, 113.9, 112.9, 64.5, 14.8. 8c Anal. C17H15O3Br: C, H.
HRMS for C17H15O3Br (M + H): theory 347.0277; found 347.0354.
(E)-3-(5-Bromo-2,3-dimethoxyphenyl)-1-(2-hydroxyphenyl)-
prop-2-en-1-one (8d). 2-Hydroxyacetophenone (300 mg, 2.2
mmol), 5-bromo-2,3-dimethoxybenzaldehyde (541 mg, 2.2 mmol),
and MeOH (11 mL) were combined, and KOH in methanol (40%
weight/volume, 11 mL) was added at room temperature. A water
condenser was attached to the flask, and the yellow solution quickly
turned orange upon heating and was stirred at reflux in an oil bath at
85 °C for 2 h. Workup involved evaporation of the solvent under
reduced pressure, addition of 1N HCl until the solution was at pH 1,
and extraction with ethyl acetate (twice). Each organic layer was
separated, pooled together, dried over anhydrous Na2SO4, filtered, and
concentrated to give a yellow solid (752 mg). The crude solid was
heated and sonicated in hexane, then cooled to room temperature and
filtered to obtain the majority of the pure product (315 mg). The
hexane filtrate was concentrated under reduced pressure, and column
chromatography (20% hexane:chloroform) was performed to obtain
more of the desired product. The total yield of final product 8d was
382 mg (48%); mp 122−124 °C; Rf 0.54 (20% hexane:chloroform).
1H NMR (CDCl3): δ 12.76 (s, 1H, OH), 8.12 (d, 1H, J = 15.6 Hz),
1-(2-Hydroxy-6-methoxyphenyl)ethanone (7d). Iodomethane
(2.05 g, 14 mmol) was added dropwise to a mixture of 1-(2,6-
dihydroxyphenyl)ethanone 6c (2.00 g, 13 mmol) and potassium
carbonate (1.99 g, 14 mmol) in N,N-dimethylformamide (13 mL), and
the resulting mixture was stirred at room temperature for 2 days.
Workup included evaporation of DMF under reduced pressure. The
remaining solid was dissolved in chloroform, and the organic phase
was washed with water. The chloroform layer was separated, dried
over anhydrous Na2SO4, filtered, and evaporated under reduced
pressure to obtain a light-yellow solid (1.77 g). This crude material
was purified by column chromatography (20% ethyl acetate:hexane) to
obtain the product 7d (1.30 g, 60%); mp 50−52 °C; Rf 0.50 (20%
1
ethyl acetate:hexane). H NMR (CDCl3): δ 13.28 (s, 1H, OH), 7.36
(t, 1H), 6.59 (d, 1H, J = 8.4 Hz), 6.41 (d, 1H, J = 8.4 Hz), 3.92 (s, 3H,
OCH3), 2.69 (s, 3H, CH3). 13C NMR (CDCl3): δ 205.2, 164.7, 161.5,
136.1, 111.3, 110.7, 101.1, 55.6, 33.7.
(E)-1-(2-Hydroxyphenyl)-3-(2-methoxyphenyl)prop-2-en-1-
one (8a). 2-Hydroxyacetophenone (1.02 g, 7.5 mmol), 2-methox-
ybenzaldehyde (1.03 g, 7.5 mmol), and MeOH (38 mL) were
combined and KOH in methanol (40% weight/volume, 38 mL) added
at room temperature.20 A water condenser was attached to the flask,
and the yellow solution quickly turned orange upon heating and was
stirred at reflux in an oil bath at 85 °C overnight. Workup involved
evaporation of the solvent and addition of water and 1N HCl until the
solution was at pH 1, followed by extraction with ethyl acetate
(twice).29 The organic layer was separated, dried over anhydrous
Na2SO4, filtered, and concentrated to give a yellow oily solid. The
mixture was heated and sonicated in hexane, cooled to room
temperature, and filtered to give a crude solid (1.2 g). Column
chromatography was performed (20% ethyl acetate:hexane) to obtain
pure product 8a (1.02 g, 54%); mp 101−103 °C; Rf 0.41 (20% ethyl
acetate:hexane). 1H NMR (CDCl3): δ 12.94 (s, 1H, OH), 8.23 (d, 1H,
J = 15.7 Hz), 7.93 (d,1H), 7.79 (d, 1H, J = 15.7 Hz), 7.65 (d, 1H),
7.49 (m, 1H), 7.41 (m, 1H), 7.03 (d, 1H), 6.96 (m, 3H), 3.95 (s, 3H,
OMe). 13C NMR (CDCl3): δ 194.3, 163.6, 159.1, 141.2, 136.2, 132.2,
129.72, 129.66, 123.7, 120.8, 120.2, 118.8, 118.6, 111.3, 55.6. 8a Anal.
C16H14O3: C, H.
7.92 (d, 1H), 7.69 (d, 1H, J = 15.6 Hz), 7.52 (t, 1H), 7.41 (s, 1), 7.09
(s, 1H), 7.04 (d, 1H), 6.96 (t, 1H), 3.89 (s, 6H, 2 × OMe). 13C NMR
(CDCl3): δ 193.7, 163.7, 153.9, 148.3, 138.7, 136.6, 130.2, 129.8,
122.5, 122.0, 120.0, 118.9, 118.7, 117.6, 116.8, 61.5, 56.2. 8d Anal.
C17H15O4Br·0.05H2O: C, H. HRMS for C17H15O4Br (M + H): theory
363.0226; found 363.0316.
(E)-3-(5-Bromo-2-methoxyphenyl)-1-(2-hydroxyphenyl)-
prop-2-en-1-one (8b). 2-Hydroxy-acetophenone (1.02 g, 7.5 mmol),
5-bromo-2-anisaldehyde (1.61 g, 7.5 mmol), and MeOH (38 mL)
were combined, and KOH in methanol (40% weight/volume, 38 mL)
was added at room temperature. A water condenser was attached to
the flask, and the yellow solution quickly turned orange upon heating
and was stirred at reflux in oil bath at 85 °C for 2 h. Workup involved
evaporation of the solvent under reduced pressure, addition of 1N HCl
until the solution was at pH 1, and extraction with ethyl acetate
(twice). The organic layer was separated, dried over anhydrous
(E)-3-(5-Bromo-2,4-dimethoxyphenyl)-1-(2-hydroxyphenyl)-
prop-2-en-1-one (8e). 2-Hydroxyacetophenone (409 mg, 3 mmol),
5-bromo-2,4-dimethoxybenzaldehyde (735 mg, 3 mmol), and MeOH
(15 mL) were combined, and KOH in methanol (40% weight/volume,
15 mL) was added at room temperature. The reaction mixture was
stirred at rt for 5 h. Workup involved evaporation of the solvent under
reduced pressure, addition of 1N HCl until the solution was at pH 1,
and extraction with ethyl acetate (twice). Each organic layer was
separated, pooled together, dried over anhydrous Na2SO4, filtered, and
concentrated to give a yellow solid 8e (1.02 g, 94%); mp 153−157 °C;
H
dx.doi.org/10.1021/jm4003457 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
Rf 0.33 (40% ethyl acetate:hexane). 1H NMR (CDCl3): δ 12.97 (s, 1H,
OH), 8.15 (d, 1H, J = 15.6 Hz), 7.93 (d, 1H), 7.84 (s, 1H), 7.62 (d,
1H, J = 15.6 Hz), 7.49 (t, 1H), 7.02 (d, 1H), 6.95 (t, 1H), 6.49 (s,
1H), 3.98 (s, 3H, OMe), 3.96 (s, 3H, OMe). 13C NMR (CDCl3): δ
193.9, 163.6, 159.9, 158.9, 139.3, 136.1, 133.0, 129.6, 120.2, 118.8 (2 ×
C), 118.6, 117.8, 102.8, 96.1, 56.4, 56.0. 8e Anal. C17H15O4Br: C, H.
HRMS for C17H15O4Br (M + H): theory 363.0226; found 363.0222.
(E)-3-(5-Bromo-2-methoxyphenyl)-1-(2-hydroxy-6-
methoxyphenyl)prop-2-en-1-one (8f). 1-(2-Hydroxy-6-
methoxyphenyl)ethanone (499 mg, 3 mmol), 5-bromo-2-methox-
ybenzaldehyde (645 mg, 3 mmol), and MeOH (15 mL) were
combined, and KOH in methanol (40% weight/volume, 15 mL) was
added at room temperature. The reaction mixture was stirred at rt for
5 h. Workup involved evaporation of the solvent under reduced
pressure, addition of 1N HCl until the solution was at pH 1, and
extraction with ethyl acetate (twice). Each organic layer was separated,
pooled together, dried over anhydrous Na2SO4, filtered, and
concentrated to give an orange solid (1.11 g). The crude solid was
heated and sonicated in hexane, then cooled down to room
temperature and filtered to obtain a pure orange solid 8f (560 mg,
51%). The hexane filtrate was concentrated under reduced pressure to
obtain 60% pure product 8f (472 mg) that was washed with hexane
again as described above and used in further reaction step without
(10% ethyl acetate:hexane). H NMR (CDCl3): δ 12.83 (s, 1H, OH),
8.15 (d, 1H, J = 15.6 Hz), 7.92 (d, 1H), 7.72 (d, 1H, J = 15.6 Hz), 7.61
(s, 1H), 7.51 (t, 1H), 7.35 (dd, 1H), 7.03 (d, 1H), 6.95 (t, 1H), 6.89
(d, 1H), 3.93 (s, 3H, OMe). 13C NMR (CDCl3): δ 193.9, 163.6, 157.4,
139.3, 136.4, 131.5, 129.7, 128.5, 125.9, 125.1, 121.7, 120.1, 118.9,
118.6, 112.6, 56.0. 8h Anal. C16H13O3Cl: C, H. HRMS for C16H14O3Cl
(M + H): theory 289.0626; found 289.0680.
(E)-3-(5-Chloro-2-ethoxyphenyl)-1-(2-hydroxyphenyl)prop-
2-en-1-one (8i). 2-Hydroxyacetophenone (544 mg, 4 mmol), 5-
chloro-2-ethoxybenzaldehyde 7c (738 mg, 4 mmol), and MeOH (20
mL) were combined, and KOH in methanol (40% weight/volume, 20
mL) was added at room temperature. The reaction mixture was stirred
at rt for 5 h. Workup involved evaporation of the solvent under
reduced pressure, addition of 1N HCl until the solution was at pH 1,
and extraction with ethyl acetate (twice). Each organic layer was
separated, pooled together, dried over anhydrous Na2SO4, filtered, and
concentrated to give a yellow solid (1.20 g). Column chromatography
(30% chloroform:hexane) was performed to obtain the total yield of
final product 8i (359 mg, 30%); mp 61−65 °C; Rf 0.25 (30%
chloroform:hexane). 1H NMR (CDCl3): δ 12.85 (s, 1H, OH), 8.10 (d,
1H, J = 15.6 Hz), 7.90 (d, 1H), 7.79 (d, 1H, J = 15.6 Hz), 7.58 (s, 1H),
7.50 (t, 1H), 7.31 (dd, 1H), 7.02 (d, 1H), 6.94 (t, 1H), 6.86 (d, 1H),
4.12 (q, 2H, CH2), 1.53 (m, 3H, CH3). 13C NMR (CDCl3): δ 194.0,
163.6, 156.9, 139.6, 136.4, 131.5, 129.6, 129.1, 125.7, 125.0, 121.8,
120.1, 118.9, 118.6, 113.4, 64.5, 14.8. 8i Anal. C17H15O3Cl: C, H.
HRMS for C17H15O3Cl (M + H): theory 303.0782; found 303.0744.
(E)-1-(2-Hydroxyphenyl)-3-(2-methoxy-5-methylphenyl)-
prop-2-en-1-one (8j). 2-Hydroxyacetophenone (544 mg, 4 mmol),
2-methoxy-5-methylbenzaldehyde (601 mg, 4 mmol), and MeOH (20
mL) were combined, and KOH in methanol (40% weight/volume, 20
mL) was added at room temperature. The reaction mixture was stirred
at rt for 5 h. Workup involved evaporation of the solvent under
reduced pressure, addition of 1N HCl until the solution was at pH 1,
and extraction with ethyl acetate (twice). Each organic layer was
separated, pooled together, dried over anhydrous Na2SO4, filtered, and
concentrated to give an orange solid (1.14 g). The crude solid was
heated and sonicated in hexane, then cooled down to room
temperature and filtered to obtain a yellow solid 8j (492 mg, 46%).
The hexane filtrate was concentrated under reduced pressure to obtain
product 8j (367 mg) that was used in further reaction step without
1
issue; mp 89−94 °C; Rf 0.31 (20% ethyl acetate:hexane). H NMR
(CDCl3): δ 8.06 (d, 1H, J = 15.6 Hz), 7.86 (d, 1H, J = 15.6 Hz), 7.71
(s, 1H), 7.46 (dd, 1H), 7.38 (t, 1H), 6.83 (d, 1H), 6.63 (d, 1H), 6.45
(d, 1H), 3.97 (s, 3H, OCH3), 3.91 (s, 3H, CH3). 13C NMR (CDCl3):
δ 194.5, 164.9, 161.0, 157.7, 136.5, 136.0, 133.8, 131.0, 129.0, 126.4,
113.0 (2 × C), 112.0, 110.9, 101.6, 56.0, 55.8. 8f Anal. C17H15O4Br: C,
H. HRMS for C17H16O4Br (M + H): theory 363.0226; found
363.0204.
(E)-1-(2-Hydroxyphenyl)-3-(5-iodo-2-methoxyphenyl)prop-
2-en-1-one (8g). 2-Hydroxyacetophenone (300 mg, 2.2 mmol), 5-
iodo-2-methoxybenzaldehyde (578 mg, 2.2 mmol), and MeOH (11
mL) were combined, and KOH in methanol (40% weight/volume, 11
mL) was added at room temperature. A water condenser was attached
to the flask and the yellow solution quickly turned orange upon
heating and was stirred at reflux in an oil bath at 85 °C for 2 h.
Workup involved evaporation of the solvent under reduced pressure,
addition of 1N HCl until the solution was at pH 1, and extraction with
ethyl acetate (twice). Each organic layer was separated, pooled
together, dried over anhydrous Na2SO4, filtered, and concentrated to
give a yellow solid (782 mg). The crude solid was heated and
sonicated in hexane, then cooled down to room temperature and
filtered to obtain the majority of the pure product (415 mg). The
hexane filtrate was concentrated under reduced pressure, and column
chromatography (15% ethyl acetate:hexane) was performed to obtain
more of the desired product. The total yield of final product 8g was
542 mg (65%); mp 107−109 °C; Rf 0.44 (15% ethyl acetate:hexane).
1H NMR (CDCl3): δ 12.82 (s, 1H, OH), 8.10 (d, 1H, J = 15.6 Hz),
7.92 (m, 2H), 7.70 (d, 1H, J = 15.6 Hz), 7.66 (dd, 1H), 7.50 (dt, 1H),
7.03 (dd, 1H), 6.96 (dt, 1H), 6.73 (d, 1H), 3.92 (s, 3H, OMe). 13C
NMR (CDCl3): δ 193.9, 163.6, 158.6, 140.4, 139.1, 137.4, 136.4,
129.8, 126.2, 121.6, 120.1, 118.9, 118.6, 113.6, 82.9, 55.8. 8g Anal.
C16H15O3I: C. HRMS for C16H15O3I (M + H): theory 380.9982;
found 381.0025.
(E)-3-(5-Chloro-2-methoxyphenyl)-1-(2-hydroxyphenyl)-
prop-2-en-1-one (8h). 2-Hydroxyacetophenone (544 mg, 4 mmol),
5-chloro-2-methoxybenzaldehyde (682 mg, 4 mmol), and MeOH (20
mL) were combined, and KOH in methanol (40% weight/volume, 20
mL) was added at room temperature. A water condenser was attached
to the flask, and the yellow solution quickly turned orange upon
heating and was stirred at reflux in an oil bath at 85 °C for 3 h.
Workup involved evaporation of the solvent under reduced pressure,
addition of 1N HCl until the solution was at pH 1, and extraction with
ethyl acetate (twice). Each organic layer was separated, pooled
together, dried over anhydrous Na2SO4, filtered, and concentrated to
give a yellow solid (1.15 g). Column chromatography (10% ethyl
acetate:hexane) was performed to obtain a byproduct 13h (228 mg)
and the desired product 8h (372 mg, 32%); mp 81−82 °C; Rf 0.38
1
issue; mp 69−72 °C; Rf 0.46 (20% ethyl acetate:hexane). H NMR
(CDCl3): δ 13.00 (s, 1H, OH), 8.23 (d, 1H, J = 15.6 Hz), 7.96 (d,
1H), 7.79 (d, 1H, J = 15.6 Hz), 7.51 (t, 1H), 7.47 (s, 1H), 7.23 (d,
1H), 7.05 (d, 1H), 6.97 (t, 1H), 6.88 (d, 1H), 3.94 (s, 3H, OCH3),
2.36 (s, 3H, CH3). 13C NMR (CDCl3): δ 194.3, 163.6, 157.1, 141.3,
136.1, 132.9, 130.0, 129.9, 129.7, 123.3, 120.4, 120.2, 118.8, 118.5,
111.3, 55.7, 20.4. 8j Anal. C17H16O3·0.14H2O: C, H. HRMS for
C17H16O3 (M + H): theory 269.1172; found 269.1201.
( E ) - 1 - ( 2 - ( B e n z y l o x y ) - 6 - h y d r o x y p h e n y l ) - 3 - ( 2 -
methoxyphenyl)prop-2-en-1-one (8k). The O-benzyl derivative
10b (0.7 g, 2.89 mmol), 2-methoxybenzaldehyde (0.40 g, 2.94 mmol),
and methanol (15 mL) were combined in a KOH in methanol solution
(40% weight/volume) at room temperature. The yellow solution was
stirred at reflux at 85 °C for 19 h.20 The dark-orange solution was
concentrated to remove most of the methanol and diluted with water
(150 mL). The aqueous phase was extracted three times with ethyl
acetate (150 mL total). The organic layer was then washed once with 1
M HCl (40 mL). The organic layer was again separated, dried over
anhydrous Na2SO4, filtered, and concentrated to give 8k as a yellow−
orange solid (1.07 g crude). The crude solid was recrystallized using
25% ethyl acetate:hexane (22 mL). Chalcone 8k was obtained as an
orange solid (0.63 g; 60%); Rf 0.56 (25% ethyl acetate:hexane). In
CDCl3, two conformers were observed (3.4:1 ratio), which generated a
1
complex spectrum. H NMR (CDCl3): δ 13.49 (s, 0.77H), 8.20 (d,
1H, J = 15.75 Hz), 7.90 (d, 0.8H, J = 15.8 Hz), 7.88 (d, 0.2H, J = 15.7
Hz), 7.48 (d, 2H), 7.42−7.24 (m, 5H), 6.91 (d, 1H), 6.85 (d, 1H),
6.71 (m, 1H), 6.65 (d, 1H), 6.53 (d, 1H), 5.14 (s, 2H), 3.80 (s, 3H,
OMe). 8k Anal. C23H20O4: C, H.
(E)-1-(2-(Benzyloxy)-6-hydroxyphenyl)-3-(5-bromo-2-
methoxyphenyl)prop-2-en-1-one (8l). The O-benzyl derivative
I
dx.doi.org/10.1021/jm4003457 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
10b (566 mg, 2.3 mmol), 5-bromo-2-methoxybenzaldehyde (495 mg,
2.3 mmol), and methanol (10 mL) were combined in 9.2 mL total of
KOH in MeOH (40% weight/volume) at room temperature. The
orange solution was stirred at reflux for 3 h at 85 °C.20 The dark-
orange solution was concentrated to remove most of the methanol and
then diluted with water (150 mL). The aqueous phase was extracted
three times with ethyl acetate. The combined ethyl acetate layers were
then washed once with 1 M HCl (40 mL). The organic layer was again
separated, dried over anhydrous Na2SO4, filtered, and concentrated to
give 8l as a crude orange solid (0.1 g). The product was collected by
precipitating in 100% hexane followed by filtration and provide the
pure product 8l (830 mg, 82%); mp 121−123 °C; Rf 0.49 (25% ethyl
acetate:hexane). In CDCl3, two conformers were noticed (2:1 ratio)
with 1N HCl at 0 °C.29 A precipitate formed as the acid was added.
The flask was stored at 4 °C overnight to obtain maximal
precipitations. A light-yellow solid 9b (0.22 g, 80%) was collected
by vacuum filtration; mp 159−161 °C; Rf 0.22 (25% ethyl acetate:
1
hexane). H NMR (CDCl3): δ 8.28 (d, 1H), 7.70 (m, 2H), 7.59 (d,
1H), 7.52 (m, 1H), 7.43 (m, 1H), 6.95 (d, 1H), 6.41 (s, 1H, OH),
3.87 (s, 3H, OMe). 13C NMR (CDCl3): δ 173.6, 156.7, 156.0, 144.1,
139.5, 134.4, 133.5, 133.4, 125.5, 124.4, 121.82, 121.3, 118.4, 113.7,
112.6, 56.2. 9b Anal. C16H11O4Br·1H2O: C, H.
2-(5-Bromo-2-ethoxyphenyl)-3-hydroxy-4H-chromen-4-one
(9c). (E)-3-(5-Bromo-2-ethoxyphenyl)-1-(2-hydroxyphenyl)prop-2-
en-1-one 8c (170 mg, 0.49 mmol) was dissolved in a 3 M KOH
solution in 96% ethanol (3.5 mL). Then 35% H2O2 (1.1 mL) was
added dropwise to the mixture cooled in an ice bath (the mixture
turned viscous) and stirred for 30 min. The ice bath was removed and
the reaction stirred at room temperature for 3 h. TLC (10% ethyl
acetate:hexane) showed the completion of the reaction via the
consumption of 8c (Rf 0.42). The mixture was then acidified to pH 1
with 1N HCl at 0 °C. A precipitate formed as the acid was added. The
flask was stored at 4 °C overnight to obtain maximal precipitations. An
impure light-brown solid (96 mg) was collected by vacuum filtration
and further purified by column chromatography (100% chloroform) to
obtain 9c as a tan solid (83 mg, 47%); mp 149−155 °C; Rf 0.40 (100%
chloroform). 1H NMR (CDCl3): δ 8.28 (d, 1H), 7.72 (s, 1H), 7.70 (t,
1H), 7.55 (dd, 1H), 7.51 (d, 1H), 7.43 (t, 1H), 6.93 (d, 1H), 6.50 (s,
1H, OH), 4.11 (q, 2H, CH2), 1.35 (t, 3H, CH3). 13C NMR (CDCl3):
δ 173.3, 156.0 (2 × C), 144.2, 138.9, 134.5, 133.6, 133.4, 125.6, 124.5,
121.8, 121.3, 118.4, 114.6, 112.4, 64.8, 14.7. 9c Anal.
C17H13O4Br·0.1H2O: C, H. HRMS for C17H13O4Br (M + H): theory
361.0070; found 361.0139.
2-(5-Bromo-2,3-dimethoxyphenyl)-3-hydroxy-4H-chromen-
4-one (9d). (E)-3-(5-Bromo-2,3-dimethoxyphenyl)-1-(2-
hydroxyphenyl)prop-2-en-1-one 8d (161 mg, 0.44 mmol) was
dissolved in a 3 M KOH solution in 96% ethanol (3.2 mL). Then
35% H2O2 (1.0 mL) was added dropwise to the mixture cooled in an
ice bath (the mixture turned viscous) and stirred for 30 min. The ice
bath was removed and the reaction stirred at room temperature for 3
h. TLC (20% hexane:chloroform) showed the completion of the
reaction via the consumption of 8d (Rf 0.54). The mixture was then
acidified to pH 1 with 1N HCl at 0 °C. A precipitate formed as the
acid was added. The flask was stored at 4 °C overnight to obtain
maximal precipitations. An impure light-brown solid (98 mg) was
collected by vacuum filtration and further purified by column
chromatography (0.5% methanol:chloroform) to obtain 9d as a tan
solid (65 mg, 39%); mp 153−158 °C; Rf 0.35 (0.5% methanol:chloro-
form). 1H NMR (CDCl3): δ 8.28 (d, 1H), 7.70 (t, 1H), 7.51 (d, 1H),
7.43 (t, 1H), 7.35 (s, 1H), 7.17 (s, 1H), 6.61 (s, 1H, OH), 3.92 (s, 3H,
OMe), 3.91 (s, 3H, OMe). 13C NMR (CDCl3): δ 173.3, 156.0, 153.9,
146.9, 143.9, 138.9, 133.7, 126.1, 125.6, 124.7, 124.6, 121.3, 118.4,
117.8, 116.2, 61.4, 56.3. 9d Anal. C17H13O5Br·0.05H2O: C, H. HRMS
for C17H13O5Br (M + H): theory 377.0019; found 377.0103.
1
which generated a complex spectrum. H NMR (CDCl3): δ 13.09 (s,
0.65H), 8.04 (d, 1H, J = 15.9 Hz), 7.82 (d, 0.65H), 7.81 (d, 0.35H, J =
15.7 Hz), 7.46−7.28 (m, 8H), 6.75 (d, 1H), 6.65 (d, 1H), 6.53 (d,
1H), 5.15 (s, 2H), 3.78 (s, 3H, OMe). 13C NMR (CDCl3): δ 194.7,
164.9, 160.1, 157.5, 136.3, 135.9, 135.6, 133.3, 135.9, 135.6, 133.9,
130.5, 129.0, 128.8, 128.6, 127.6, 126.1, 112.9, 112.8, 112.3, 111.2,
102.7, 71.3, 55.7. 8l Anal. C22H19O4Br·0.05H2O: C, H.
(E)-3-(5-Bromo-2-methoxyphenyl)-1-(1-hydroxynaphthalen-
2-yl)prop-2-en-1-one (8m). 1-(1-Hydroxynaphthalen-2-yl)ethanone
(559 mg, 3.0 mmol), 5-bromo-2-methoxybenzaldehyde (645 mg, 3.0
mmol), and MeOH (15 mL) were combined, and KOH in methanol
(40% weight/volume, 15 mL) was added at room temperature. A
water condenser was attached to the flask, and the yellow solution
quickly turned red upon heating and was stirred at reflux in an oil bath
at 85 °C for 2 h. Workup involved evaporation of the solvent under
reduced pressure, addition of 1N HCl until the solution was at pH 1,
and extraction with ethyl acetate (twice). Each organic layer was
separated, pooled together, dried over anhydrous Na2SO4, filtered, and
concentrated to give a yellow solid (1.146 g). The crude solid was
heated and sonicated in hexane, then cooled down to room
temperature and filtered to obtain 92% pure product (619 mg).
Column chromatography (10% ethyl acetate:hexane) was performed
to obtain pure 8m (565 mg, 49%); mp 117−127 °C; Rf 0.43 (10%
1
ethyl acetate:hexane). H NMR (CDCl3): δ 14.87 (s, 1H, OH), 8.50
(d, 1H), 8.20 (d, 1H, J = 15.6 Hz), 7.84 (d, 1H), 7.80 (d, 1H, J = 15.6
Hz), 7.79 (s, 1H), 7.78 (d, 1H), 7.65 (dt, 1H), 7.55 (dt, 1H), 7.49 (dd,
1H), 7.32 (d, 1H), 6.85 (d, 1H), 3.94 (s, 3H, OMe). 13C NMR
(CDCl3): δ 193.3, 164.5, 157.9, 138.7, 137.4, 134.3, 131.4, 130.2,
127.4, 125.9, 125.8, 125.5, 124.5, 124.0, 122.0, 118.2, 113.5, 113.1,
113.0, 55.9. 8m C20H15BrO3: C, H. HRMS for C20H16O3Br (M + H):
theory 385.0259; found 385.0253.
3-Hydroxy-2-(2-methoxyphenyl)-4H-chromen-4-one (9a).
Chalcone 8a (200 mg, 0.79 mmol) was dissolved in a 3 M KOH
solution in 96% ethanol (5.7 mL). Then 35% H2O2 (1.7 mL) was
added dropwise to the mixture cooled in an ice bath (the mixture
turned viscous) and stirred for 30 min. The ice bath was removed and
the reaction stirred at room temperature overnight. TLC (25% ethyl
acetate:hexane) showed the completion of the reaction via the
consumption of 8a, then the mixture was acidified to pH 1 with 1N
HCl at 0 °C. A precipitate formed as acid was added.29 The flask was
left in the refrigerator overnight to obtain maximal precipitations. A
pure white solid 9a (109 mg, 51%) was collected by vacuum filtration;
mp 210−212 °C; Rf 0.24 (25% ethyl acetate:hexane). 1H NMR
(CDCl3): δ 8.29 (d, 1H), 7.68 (m, 1H), 7.59 (d, 1H), 7.53 (d, 1H),
7.51 (m, 1H), 7.42 (m, 1H), 7.12 (m, 1H), 7.08 (d, 1H), 6.39 (s, 1H,
OH), 3.89 (s, 3H, OMe). 13C NMR (CDCl3): δ 173.3, 157.5, 156.1,
145.9, 138.8, 133.3, 132.1, 130.9, 125.5, 124.3, 121.4, 120.6, 119.7,
118.5, 111.9, 56.0. 9a Anal. C16H12O4·0.03H2O: C, H.
2-(5-Bromo-2,4-dimethoxyphenyl)-3-hydroxy-4H-chromen-
4-one (9e). (E)-3-(5-Bromo-2,4-dimethoxyphenyl)-1-(2-
hydroxyphenyl)prop-2-en-1-one 8e (208 mg, 0.57 mmol) was
dissolved in a 3 M KOH solution in 96% ethanol (4.2 mL). Then
35% H2O2 (1.2 mL) was added dropwise to the mixture cooled in an
ice bath (the mixture turned viscous) and stirred for 30 min. The ice
bath was removed and the reaction stirred at room temperature for 3
h. TLC (40% ethyl acetate:hexane) showed the completion of the
reaction via the consumption of 8e (Rf 0.33). The mixture was then
acidified to pH 1 with 1N HCl at 0 °C. A precipitate formed as the
acid was added. The flask was stored at 4 °C overnight to obtain
maximal precipitation. The product 9e (174 mg, 81%) was collected
by vacuum filtration as a tan solid; mp 196−201 °C; Rf 0.32 (50%
2-(5-Bromo-2-methoxyphenyl)-3-hydroxy-4H-chromen-4-
one (9b). (E)-3-(5-Bromo-2-methoxyphenyl)-1-(2-hydroxyphenyl)-
prop-2-en-1-one 8b (333 mg, 1 mmol) was dissolved in a 3 M KOH
solution in 96% ethanol (7.3 mL). Then 35% H2O2 (2.2 mL) was
added dropwise to the mixture cooled in an ice bath (the mixture
turned viscous) and stirred for 30 min. The ice bath was removed and
the reaction stirred at room temperature for 3 h. TLC (20% ethyl
acetate:hexane) showed the completion of the reaction via the
consumption of 8b (Rf 0.41). The mixture was then acidified to pH 1
1
ethyl acetate:hexane). H NMR (CDCl3): δ 8.26 (d, 1H), 7.75 (s,
1H), 7.68 (t, 1H), 7.51 (d, 1H), 7.41 (t, 1H), 6.60 (s, 1H), 6.51 (s, 1H,
OH), 3.98 (s, 3H, OMe), 3.90 (s, 3H, OMe). 13C NMR (CDCl3): δ
173.2, 158.6, 158.3, 156.0, 144.4, 138.8, 134.5, 133.4, 125.5, 124.4,
121.3, 118.4, 113.4, 102.2, 96.9, 56.4, 56.3. 9e Anal. C17H13O5Br: C, H.
HRMS for C17H13O5Br (M + H): theory 377.0019; found 377.0069.
J
dx.doi.org/10.1021/jm4003457 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2-(5-Bromo-2-methoxyphenyl)-3-hydroxy-5-methoxy-4H-
chromen-4-one (9f). (E)-3-(5-Bromo-2-methoxyphenyl)-1-(2-hy-
droxy-6-methoxyphenyl)prop-2-en-1-one 8f (221 mg, 75% pure, 0.52
mmol) was dissolved in a 3 M KOH solution in 96% ethanol (3.8
mL). Then 35% H2O2 (1.1 mL) was added dropwise to the mixture
cooled in an ice bath (the mixture turned viscous) and stirred for 30
min. The ice bath was removed and the reaction stirred at room
temperature for 3 h. TLC (20% ethyl acetate:hexane) showed the
completion of the reaction via the consumption of 8f (Rf 0.31). The
mixture was then acidified to pH 1 with 1N HCl at 0 °C. A precipitate
formed as the acid was added. The flask was stored at 4 °C overnight
to obtain maximal precipitations. A yellow solid (163 mg) was
collected by vacuum filtration. The crude solid was heated and
sonicated in chloroform, then cooled down to room temperature and
filtered to obtain 9f as a yellow solid (55 mg, 28% yield, 90% pure);
with 1N HCl at 0 °C. A precipitate formed as the acid was added. The
flask was stored at 4 °C overnight to obtain maximal precipitation. A
crude solid (147 mg) was collected by vacuum filtration and purified
by column chromatography (100% CHCl3) to obtain 9i as a light-
yellow solid (112 mg, 60%); mp 156−159 °C; Rf 0.34 (100% CHCl3).
1H NMR (CDCl3): δ 8.21 (d, 1H), 7.61 (t, 1H), 7.51 (d, 1H), 7.43 (t,
1H), 7.37−7.31 (m, 2H), 6.90 (d, 1H), 6.50 (s, 1H, OH), 4.04 (q, 2H,
CH2), 1.27 (t, 3H, CH3). 13C NMR (CDCl3): δ 172.4, 155.1, 154.6,
143.4, 138.0, 132.6, 130.6, 129.7, 124.6, 124.5, 123.5, 120.4, 120.3,
117.4, 113.2, 63.9, 13.8. 9i Anal. C17H13O4Cl: C, H. HRMS for
C17H13O4Cl (M + H): theory 317.0575; found 317.0613.
3-Hydroxy-2-(2-methoxy-5-methylphenyl)-4H-chromen-4-
one (9j). (E)-1-(2-Hydroxyphenyl)-3-(2-methoxy-5-methylphenyl)-
prop-2-en-1-one 8j (346 mg, 90% pure, 1.29 mmol) was dissolved
in a 3 M KOH solution in 96% ethanol (9.4 mL). Then 35% H2O2
(2.6 mL) was added dropwise to the mixture cooled in an ice bath (the
mixture turned viscous) and stirred for 30 min. The ice bath was
removed and the reaction stirred at room temperature for 3 h. TLC
(20% ethyl acetate:hexane) showed the completion of the reaction via
the consumption of 8j (Rf 0.46). The mixture was then acidified to pH
1 with 1N HCl at 0 °C. A precipitate formed as the acid was added.
The flask was stored at 4 °C overnight to obtain maximal
precipitations. A light-brown solid 9j (259 mg, 71%) was collected
by vacuum filtration; mp 158−164 °C; Rf 0.32 (20% ethyl
1
mp 207−213 °C; Rf 0.29 (100% chloroform). H NMR (CDCl3): δ
8.35 (d, 1H), 7.58 (t, 1H), 7.43 (dd, 1H), 7.27 (s, 1H, OH), 6.92 (d,
1H), 6.80 (d, 1H), 6.63 (d, 1H), 4.01 (s, 3H, OMe), 3.89 (s, 3H,
OMe). 13C NMR (CDCl3): δ 182.2, 166.9, 158.6, 157.6, 147.3, 138.4,
134.0, 133.4, 123.5, 113.1, 112.4, 110.9, 105.3, 104.9, 104.4, 56.3, 55.9.
Mass spectrum for C17H13BrO5 (M + H): theory 377, LC-MS
indicated a facile loss of methyl during ionization (M = 363).
3-Hydroxy-2-(5-iodo-2-methoxyphenyl)-4H-chromen-4-one
(9g). (E)-1-(2-Hydroxyphenyl)-3-(5-iodo-2-methoxyphenyl)prop-2-
en-1-one 8g (251 mg, 0.66 mmol) was dissolved in a 3 M KOH
solution in 96% ethanol (4.8 mL). Then 35% H2O2 (1.5 mL) was
added dropwise to the mixture cooled in an ice bath (the mixture
turned viscous) and stirred for 30 min. The ice bath was removed and
the reaction stirred at room temperature for 3 h. TLC (15% ethyl
acetate:hexane) showed the completion of the reaction via the
consumption of 8g (Rf 0.44). The mixture was then acidified to pH 1
with 1N HCl at 0 °C. A precipitate formed as the acid was added. The
flask was stored at 4 °C overnight to obtain maximal precipitations. A
light-brown solid (173 mg) was collected by vacuum filtration and
purified by column chromatography (25% ethyl acetate:hexane) to
obtain 9g as a tan solid (115 mg, 44%); mp 153−159 °C; Rf 0.27 (25%
1
acetate:hexane). H NMR (CDCl3): δ 8.28 (dd, 1H), 7.68 (dt, 1H),
7.52 (d, 1H), 7.41 (dt, 1H), 7.39 (d, 1H), 7.30 (dd, 1H), 6.98 (d, 1H),
6.44 (s, 1H, OH), 3.86 (s, 3H, OMe), 2.37 (s, 3H, Me). 13C NMR
(CDCl3): δ 173.3, 156.0, 155.4, 146.3, 138.8, 133.3, 132.6, 131.2,
130.0, 125.5, 124.3, 121.5, 119.5, 118.5, 112.0, 56.1, 20.4. 9j Anal.
C17H14O4: C, H. HRMS for C17H15O4 (M + H): theory 283.0965;
found 283.0945.
2-Benzoxy-6-hydroxyacetophenone (10b).30 To a solution
containing 2,6-dihydroxyacetophenone 10a (5 g, 33 mmol) in acetone
(50 mL) were added benzyl bromide (6.65 g, 4.6 mL, 40.0 mmol), KI
(9 g, 53 mmol), and K2CO3 (15 g, 106 mmol). The reaction mixture
was heated to reflux overnight under a N2 atmosphere, then cooled
and filtered.31 The filtrate was concentrated under reduced pressure,
and the residue was redissolved in ethyl acetate and washed with H2O
(50 mL). The water layer was washed three times with ethyl acetate.
The organic layers were combined, dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure to give a crude solid
(12 g). The solid was purified by flash chromatography (20%
ethylacetate/hexane) on a silica gel column (700 g silica). Elution with
20% ethyl acetate and hexane gave recovered starting material 10a
(0.65 g) and the desired product 10b (4.0 g) as a light-yellow solid.
Taking into account the recovered starting material, the yield of 10b
1
ethyl acetate:hexane). H NMR (CDCl3): δ 8.27 (d, 1H), 7.85 (s,
1H), 7.76 (dd, 1H), 7.69 (t, 1H), 7.52 (d, 1H), 7.42 (t, 1H), 6.84 (d,
1H), 6.42 (s, 1H, OH), 3.86 (s, 3H, OMe). 13C NMR (CDCl3): δ
173.2, 157.4, 156.0, 143.9, 140.5, 139.1, 138.9, 133.5, 125.6, 124.5,
122.1, 121.2, 118.5, 114.1, 82.2, 56.1. 9g Anal. C16H11O4I: C, H.
HRMS for C16H11O4I (M + H): theory 394.9775; found 394.9851.
2-(5-Chloro-2-methoxyphenyl)-3-hydroxy-4H-chromen-4-
one (9h). (E)-3-(5-Chloro-2-methoxyphenyl)-1-(2-hydroxyphenyl)-
prop-2-en-1-one 8h (221 mg, 0.77 mmol) was dissolved in a 3 M
KOH solution in 96% ethanol (5.9 mL). Then 35% H2O2 (1.6 mL)
was added dropwise to the mixture cooled in an ice bath (the mixture
turned viscous) and stirred for 30 min. The ice bath was removed and
the reaction stirred at room temperature for 3 h. TLC (10% ethyl
acetate:hexane) showed the completion of the reaction via the
consumption of 8h (Rf 0.38). The mixture was then acidified to pH 1
with 1N HCl at 0 °C. A precipitate formed as the acid was added. The
flask was stored at 4 °C overnight to obtain maximal precipitation. A
light-pink solid 9h (114 mg, 49%) was collected by vacuum filtration;
1
was 58%; Rf 0.52 (3:1 hexanes:ethyl acetate). H NMR (CDCl3): δ
13.29 (s, 1H), 7.37 (m, 6H), 6.59 (d, 1H, J = 8.4 Hz), 6.46 (d, 1H, J =
8.4 Hz), 5.13 (s, 2H), 2.62 (s, 3H); matched lit spectrum.28
5-(Benzyloxy)-2-(2-methoxyphenyl)-4H-chromen-4-one
(11a). Ketone 8k (550 mg, 1.53 mmol) was dissolved in DMSO (100
mL), and iodine (38 mg, 0.15 mmol) was added. The mixture was
heated to 140 °C and stirred overnight with a water condenser and
drying tube attached. The light-yellow solution was cooled and 1 M
HCl added until the solution turned permanently cloudy, followed by
extraction with ethyl acetate three times.20 The organic layers were
pooled and washed once with brine. The organic layer was separated,
dried over anhydrous Na2SO4, filtered, and concentrated to give crude
11a (1.05 g). The solid residue was recrystallized from ethyl
acetate:hexane (1:3 by volume) overnight to give pure 11a (380
mg; 70%, from first crop). The filtrate was concentrated and a second
crop obtained (56 mg; using 20% ethyl acetate:hexane). Total yield of
11a: 436 mg, 80%; Rf 0.10 (20% ethyl acetate:hexane). 1H NMR
(CDCl3): δ 7.90 (d, 1H), 7.65 (d, 2H), 7.55−7.42 (m, 2H), 7.40 (m,
2H), 7.30 (m, 1H), 7.26 (s, 1H), 7.15−7.05 (m, 3H), 7.04 (d, 1H),
6.84 (d, 1H), 5.30 (s, 2H), 3.93 (s, 3H, OMe). 13C NMR (CDCl3): δ
178.6, 158.53, 158.50, 158.47, 158.01, 136.7, 133.4, 132.2, 129.1,
128.5, 127.6, 126.6, 120.7, 120.5, 115.1, 114.3, 111.7, 110.5, 108.3,
70.9, 55.6. 11a Anal. C23H18O4·0.04H2O: C, H.
1
mp 160−167 °C; Rf 0.33 (100% chloroform). H NMR (CDCl3): δ
8.28 (d, 1H), 7.69 (t, 1H), 7.57 (s, 1H), 7.52 (d, 1H), 7.44 (dd, 1H),
7.43 (t, 1H), 7.00 (d, 1H), 6.46 (s, 1H, OH), 3.87 (s, 3H, OMe). 13C
NMR (CDCl3): δ 173.3, 156.1, 156.0, 144.1, 139.0, 133.6, 131.6,
130.6, 125.6 (2 × C), 124.5, 121.3, 121.1, 118.5, 113.2, 56.3. 9h Anal.
C16H11O4Cl: C, H. HRMS for C16H11O4Cl (M + H): theory
303.0419; found 303.0488.
2-(5-Chloro-2-ethoxyphenyl)-3-hydroxy-4H-chromen-4-one
(9i). (E)-3-(5-Chloro-2-ethoxyphenyl)-1-(2-hydroxyphenyl)prop-2-
en-1-one 8i (178 mg, 0.59 mmol) was dissolved in a 3 M KOH
solution in 96% ethanol (4.3 mL). Then 35% H2O2 (1.2 mL) was
added dropwise to the mixture cooled in an ice bath (the mixture
turned viscous) and stirred for 30 min. The ice bath was removed and
the reaction stirred at room temperature for 3 h. TLC (30%
chloroform:hexane) showed the completion of the reaction via the
consumption of 8i (Rf 0.25). The mixture was the acidified to pH 1
K
dx.doi.org/10.1021/jm4003457 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
5-(Benzyloxy)-2-(5-bromo-2-methoxyphenyl)-4H-chromen-
4-one (11b). Bromo-chalcone 8l (439 mg, 1 mmol) was dissolved in
DMSO (67 mL), and iodine (25 mg) was added to the mixture at
room temperature. The mixture was refluxed at 140 °C for 3 h. The
flask was cooled to room temperature. Work up involved adding 1 M
HCl until the solution reached pH 1, followed by extraction with ethyl
acetate (three times).20 The organic layer was washed once with brine,
separated, and dried over anhydrous Na2SO4, filtered, and
concentrated to give a crude solid. Column chromatography was
performed (1:1 ethyl acetate and hexane) to give pure product 11b
(0.24 g, 55%); mp 153−155 °C; Rf 0.38 (50% ethyl acetate:hexane).
1H NMR (CDCl3): δ 8.02 (s, 1H), 7.65 (d, 2H), 7.54 (m, 2H), 7.40
(m, 2H), 7.31 (d, 1H), 7.13 (d, 1H), 7.08 (s, 1H), 6.92 (d, 1H), 6.85
(d, 1H), 5.30 (s, 2H), 3.93 (s, 3H, OMe). 13C NMR (CDCl3): δ
178.4, 158.5, 158.4, 157.1, 156.7, 136.6, 134.5, 133.6, 131.5, 128.6,
127.6, 126.6, 122.3, 114.8, 113.5, 113.1, 110.5, 108.4, 70.9, 56.0. 11b
Anal. C23H17O4Br·0.06H2O: C, H.
2-(2-Methoxyphenyl)-4H-chromen-4-one (11c). Chalcone 8a
(150 mg, 0.59 mmol) was dissolved in DMSO (39 mL), and iodine
(14.4 mg, 0.06 mmol) was added. The mixture was refluxed in DMSO
via an oil bath at 140 °C overnight.20 The orange solution was cooled
to room temperature, and 1 M HCl was added until the solution pH
was 1, followed by extraction with ethyl acetate four times. The
organic layers were pooled, washed once with brine, dried over
anhydrous Na2SO4, filtered, and concentrated under reduced pressure
to give a crude solid. The solid residue was recrystallized from ethyl
acetate/hexane (1:3) overnight to give a solid (180 mg). Column
chromatography was performed (25% ethyl acetate:hexane) to provide
pure 11c (104 mg, 70%); mp 93−94 °C; Rf 0.12 (25% ethyl
acetate:hexane). 1H NMR (CDCl3): δ 8.24 (d, 1H), 7.91 (d, 1H), 7.68
(m, 1H), 7.54 (d, 1H), 7.49 (m, 1H), 7.41 (m, 1H), 7.15 (s, 1H), 7.12
(m, 1H), 7.06 (d, 1H), 3.95 (s, 3H, OMe). 13C NMR (CDCl3): δ
178.9, 160.9, 158.0, 156.5, 133.5, 132.4, 129.3, 125.7, 124.9, 123.9,
120.9, 120.7, 118.0, 112.7, 111.8, 55.7. 11c Anal. C16H12O3·0.05H2O:
C, H.
pressure, and column chromatography was performed (20% ethyl
acetate:hexane) to obtain the pure product 12b (55 mg, 70%); mp
1
175−180 °C; Rf 0.28 (20% ethyl acetate:hexane). H NMR (CDCl3):
δ 12.57 (s, 1H, OH), 8.02 (s, 1H), 7.56 (m, 2H), 7.10 (s, 1H), 7.00
(m, 1H), 6.95 (d, 1H), 6.82 (d, 1H), 3.94 (s, 3H, OMe). 13C NMR
(CDCl3): δ 183.9, 160.7, 160.1, 157.2, 156.5, 135.4, 135.3, 131.8,
121.9, 113.6, 113.2, 111.7, 111.2, 110.8, 107.0, 56.1. 12b Anal.
C16H11BrO4: C, H.
2-(5-Bromo-2-methoxyphenyl)chroman-4-one (13b). Bromo-
chalcone 8b (600 mg, 1.8 mmol) and sodium acetate (1.48 g, 18
mmol) were added together in 1-butanol (18 mL). The mixture was
refluxed at 118 °C (with a reflux condenser and a drying tube
attached) for a week. The mixture was then cooled to room
temperature.22 The volatiles were removed under reduced pressure,
water was added, and the mixture was extracted three times with
CH2Cl2. The organic layer was separated, washed once with brine,
separated, and dried over anhydrous Na2SO4, filtered, and
concentrated to give a yellow oily solid. Column chromatography
was performed (40% hexane:chloroform) to give pure product 13b
(0.18g. 11%); mp 101−103 °C; Rf 0.73 (40% hexane:chloroform). 1H
NMR (CDCl3): δ 7.94 (d, 1H), 7.78 (s, 1H), 7.53 (m, 1H), 7.44 (d,
1H), 7.07 (m, 2H), 6.80 (d, 1H), 5.78 (d, 1H), 3.83 (s, 3H, OMe),
3.02−2.78 (m, 2H). 13C NMR (CDCl3): δ 192.2, 161.7, 154.7, 136.1,
131.9, 129.8, 129.3, 127.1, 121.7, 121.0, 118.1, 113.3, 112.3, 74.2, 55.7,
43.6. 13b Anal. C16H13O3Br: C, H.
2-(5-Chloro-2-methoxyphenyl)chroman-4-one (13h). This
byproduct was isolated (228 mg, 20%) during the synthesis of
compound 8h (see 8h for details); mp 51−60 °C; Rf 0.36 (10% ethyl
acetate:hexane). 1H NMR (CDCl3): δ 7.87 (d, 1H), 7.57 (d, 1H), 7.45
(t, 1H), 7.22 (dd, 1H), 7.01 (d, 1H), 7.00 (t, 1H), 6.77 (d, 1H), 5.71
(dd, 1H), 3.76 (s, 3H, OMe), 2.91 (dd, 1H), 2.76 (dd, 1H). 13C NMR
(CDCl3): δ 192.2, 161.7, 154.2, 136.2, 129.4, 128.9, 127.1, 126.5,
126.1, 121.7, 121.0, 118.1, 111.8, 74.3, 55.7, 43.6. 13h Anal.
C16H13O3Cl: C, H. HRMS for C16H14O3Cl (M + H): theory
289.0626; found 289.0639.
2-(5-Bromo-2-methoxyphenyl)-4H-chromen-4-one (11d).
Bromo-chalcone 8b (250 mg, 0.75 mmol) was dissolved in DMSO
(50 mL), and iodine (18.7 mg, 0.07 mmol) was added. The mixture
was refluxed in DMSO via an oil bath at 140 °C for 4 h.20 The orange
solution was cooled to room temperature, and 1 M HCl was added
until the solution pH was 1, followed by extraction with ethyl acetate
four times. The organic layers were pooled, washed once with brine,
dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure to give a crude solid. Column chromatography was
performed (25% ethyl acetate:hexane) to provide pure product 11d
(240 mg, 95% yield); mp 161−164 °C; Rf 0.12 (25% ethyl
(E)-3-(5-Bromo-2-methoxyphenyl)acrylic acid (15). The start-
ing chalcone 8m ((E)-3-(5-bromo-2-methoxyphenyl)-1-(1-hydroxy-
naphthalen-2-yl)prop-2-en-1-one, 316 mg, 0.82 mmol) was combined
with 40% KOH in EtOH (6.1 mL) and 30% H2O2 (1.8 mL) and
stirred for 30 min at 0 °C and then at rt for an additional 3 h. Aqueous
acid (1 M HCl) was added to give a pH of 1, and the resultant
precipitation was stored at 4 °C overnight and then collected by
filtration and dried to give the crude cinnamic acid derivative 15 as an
orange solid (157 mg, 74%). 1H NMR (DMSO-d6): δ 12.42 (br s, 1H,
COOH), 7.88 (d, 1H, J = 2 Hz), 7.73 (d, 1H, β-vinyl CH, J = 16 Hz),
7.55 (dd, 1H, J = 8.9 and 2 Hz), 7.06 (d, 1H, J = 8.9 Hz), 6.59 (d, 1H,
J = 16 Hz), 3.86 (s, 3H, OCH3). 13C NMR (DMSO-d6): δ 167.5,
156.8, 136.9, 133.8, 130.4, 124.7, 120.8, 114.0, 112.3, 56.0. Rf (1%
MeOH in CHCl3) = 0.4.
Biological Investigations. The efficacy of each compound on the
US28 receptor was investigated in luciferase based reporter gene assay
PathDetect trans Elk1 (Agilent, Stratagene)23 as described previ-
ously.13 Briefly, the human embryonic kidney (HEK) 293T cells were
transiently transfected with 0.1 μg Elk1, 5 μg Luc, and 5 μg of US28
vector or empty pcDNA3 vector (mock cells). Five hours after
transfection, the cells were washed with DMEM containing 1% FBS,
harvested, seeded in a white half area 96-well plate (20,000 cells/well),
and incubated with the indicated concentrations of test compounds.
The incubation buffer consisted of DMEM, 1% FBS, 2 mM L-
glutamine, 1% penicillin−streptomycin, and 1% DMSO. The cells
were incubated at 37 °C in the humid atmosphere with 5% CO2 for
additional 20−24 h. The luciferase substrate BrightGlo (Promega) was
used according to the manufacturer’s instruction. The luminescence
intensity was acquired using microplate reader Victor3V (Perkin-
Elmer). The basal luminescence of mock transfected cells was
approximately 500 RLU and the basal luminescence of US28-
transfected cells was 4−5-fold higher (2000−2500 RLU). As the
inhibitors gave differential responses in terms of their ability to reduce
US28 mediated signaling, their relative EC50 values were determined
by dosing the US28 transfected HEK cells with increasing
1
acetate:hexane). H NMR (CDCl3): δ 8.23 (d, 1H), 8.04 (s, 1H),
7.70 (m, 1H), 7.57 (m, 2H), 7.42 (m, 1H), 7.15 (s, 1H), 6.94 (d, 1H),
3.94 (s, 3H, OMe). 13C NMR (CDCl3): δ 178.7, 159.0, 157.1, 156.4,
134.8, 133.8, 131.7, 125.7, 125.1, 123.8, 122.6, 118.1, 113.6, 113.13,
113.10, 56.0. 11d Anal. C16H11O3Br·0.17H2O: C, H.
5-Hydroxy-2-(2-methoxyphenyl)-4H-chromen-4-one (12a).
Flavone 11a (321 mg, 0.89 mmol) was dissolved in a solution of
acetic acid (34 mL) and water (8.5 mL) at room temperature. The
system was then attached to a water condenser and refluxed at 110 °C
for 24 h.21 The solvent was removed under reduced pressure, and the
residue was heated and sonicated in hexane and filtered to give a crude
solid (201 mg). Column chromatography was performed (20% ethyl
acetate:hexane) to obtain pure product 12a (162 mg, 67%); mp 128−
1
130 °C; Rf 0.31 (20% ethyl acetate:hexane). H NMR (CDCl3): δ
12.69 (s, 1H, OH), 7.91 (d, 1H), 7.52 (m, 2H), 7.12 (m, 1H), 7.11 (s,
1H), 7.06 (d, 1H), 6.98 (m, 1H), 6.80 (d, 1H), 3.95 (s, 3H, OMe).
13C NMR (CDCl3): δ 184.0, 161.9, 160.8, 158.2, 156.7, 135.2, 132.9,
129.3, 120.8, 120.3, 111.8, 111.2, 110.8, 107.0, 55.7. 12a Anal.
C16H12O4: C, H.
2-(5-Bromo-2-methoxyphenyl)-5-hydroxy-4H-chromen-4-
one (12b). Bromo-flavone 11b (100 mg, 0.23 mmol) was dissolved in
a solution of acetic acid (11.3 mL) and water (2.82 mL) at room
temperature. The system was then attached to a water condenser and
refluxed at 110 °C for 24 h.21 The solvent was removed under reduced
L
dx.doi.org/10.1021/jm4003457 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
concentrations of each compound. In this case, the EC50 value is the
concentration of the compound needed to provoke a response halfway
between the compound’s maximal response (change in luminescence)
and the no-compound control in the same US28 transfected cell line.
A plot of relative luminescence versus log of the compound
concentration provided a dose−response curve from which the %
efficacy was derived via nonlinear regression methods using the
algorithms in PRISM 5.0 (GraphPad Software, San Diego, CA). The
data is tabulated in Table 1.
Pharmacological Significance of Chalcone Scaffold: A Review. Curr.
Med. Chem. 2012, 19, 209−225.
(2) Lin, Y. M.; Zhou, Y.; Flavin, M. T.; Zhou, L. M.; Niea, W.;
Chenb, F. C. Chalcones and Flavonoids as Anti-Tuberculosis Agents.
Bioorg. Med. Chem. 2002, 10, 2795−2802.
(3) Babu, M. A.; Shakya, N.; Prathipati, P.; Kaskhedikar, S. G.;
Saxena, A. K. Development of 3D-QSAR Models for 5-Lipoxygenase
Antagonists: Chalcones. Bioorg. Med. Chem. 2002, 10, 4035−4041.
(4) Sogawa, S.; Nihro, Y.; Ueda, H.; Izumi, A.; Miki, T.; Matsumoto,
H.; Satoh, T. 3,4-Dihydroxychalcones as Potent 5-Lipoxygenase and
Cyclooxygenase Inhibitors. J. Med. Chem. 1993, 36, 3904−3909.
(5) Zhao, F.; Zhao, Q. J.; Zhang, D. Z.; Jin, Y. S.; Zhang, W.
Synthesis and Protein Tyrosine Phosphatase 1B-Inhibitory Activity of
Chalcones. Asian J. Chem. 2011, 23, 5339−5342.
(6) Buckwold, V.; Wilson, R.; Nalca, A.; Beer, B.; Voss, T.; Turpin, J.;
Buckheit, R.; Wei, J.; Wenzelmathers, M.; Walton, E. Antiviral activity
of hop constituents against a series of DNA and RNA viruses. Antiviral
Res. 2004, 61, 57−62.
(7) Wang, Y.; Chen, Y.; Wang, J.; Chen, J.; Aggarwal, B. B.; Pang, X.;
Liu, M. Xanthohumol, a Prenylated Chalcone Derived from Hops,
Suppresses Cancer Cell Invasion through Inhibiting the Expression of
CXCR4 Chemokine Receptor. Curr. Mol. Med. 2012, 12, 153−162.
(8) Chua, A. W.; Hay, H. S.; Rajendran, P.; Shanmugam, M. K.; Li,
F.; Bist, P.; Koay, E. S.; Lim, L. H.; Kumar, A. P.; Sethi, G. Butein
downregulates chemokine receptor CXCR4 expression and function
through suppression of NF-kappaB activation in breast and pancreatic
tumor cells. Biochem. Pharmacol. 2010, 80, 1553−1562.
(9) Evers, D.; Chao, C.; Wang, X.; Zhang, Z.; Huong, S.; Huang, E.
Human cytomegalovirus-inhibitory flavonoids: studies on antiviral
activity and mechanism of action. Antiviral Res. 2005, 68, 124−134.
(10) Kim, H. J.; Woo, E. R.; Shin, C. G.; Park, H. A new flavonol
glycoside gallate ester from Acer okamotoanum and its inhibitory
activity against human immunodeficiency virus-1 (HIV-1) integrase. J.
Nat. Prod. 1998, 61, 145−148.
(11) Hachet-Haas, M.; Balabanian, K.; Rohmer, F.; Pons, F.;
Franchet, C.; Lecat, S.; Chow, K. Y.; Dagher, R.; Gizzi, P.; Didier,
B.; Lagane, B.; Kellenberger, E.; Bonnet, D.; Baleux, F.; Haiech, J.;
Parmentier, M.; Frossard, N.; Arenzana-Seisdedos, F.; Hibert, M.;
Galzi, J. L. Small neutralizing molecules to inhibit actions of the
chemokine CXCL12. J. Biol. Chem. 2008, 283, 23189−23199.
(12) Gao, J. L.; Murphy, P. M. Human Cytomegalovirus Open
Reading Frame US28 Encodes a Functional Beta Chemokine
Receptor. J. Biol. Chem. 1994, 269, 28539−28542.
(13) Kralj, A.; Wetzel, A.; Mahmoudian, S.; Stamminger, T.;
Tschammer, N.; Heinrich, M. R. Identification of novel allosteric
modulators for the G-protein coupled US28 receptor of human
cytomegalovirus. Bioorg. Med. Chem. Lett. 2011, 21, 5446−5450.
(14) Boomker, J. M.; van Luyn, M. J.; The, T. H.; de Leij, L. F.;
Harmsen, M. C. US28 actions in HCMV infection: lessons from a
versatile hijacker. Rev. Med. Virol. 2005, 15, 269−282.
CHO Assay.25a The CHO assay was conducted to assess the
general toxicity of each flavonoid drug. Briefly, cell growth was assayed
in sterile 96-well microtiter plates (Costar 3599, Corning, NY, USA) in
the presence of each drug. Experiments were conducted in triplicate.
CHO cells were plated at 10000 cells/mL. Drug solutions were
prepared in 100% DMSO and dosed so that the final DMSO
concentration was <1%. For example, 1 μL of drug solution was added
to the CHO cells plated in each well in 100 μL of media. Drug
additions occurred after an initial overnight incubation of CHO cells in
each well. After drug was added, the cells were incubated in 5% CO2
for 48 h at 37 °C. The MTS reagent (Promega Cell Titer 96 AQueous
nonradiactive cell proliferation reagent) was added (20 μL), and the
CHO cells were incubated for an additional 4 h and then absorbance
at 490 nm was measured on a BioTek Synergy MX plate reader.
Controls run using 1% DMSO in the media showed no toxicity over
the 48 h period compared to CHO cells grown in media only. IC50
values were determined from the corresponding plot of relative
absorbance at 490 nm vs drug concentration. The data is tabulated in
Table 1.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR spectra for compounds 7a−7d, 8a−8m, 9a−9j, 11a−
11d, 12a−12b, 13b, 13h, and 15, 13C NMR spectra for 7a−7d,
8a−8j, 8l, 8m, 9a−9j, 11a−11d, 12a−12b, 13b, 13h, and 15,
elemental analyses of compounds 8a−8m, 9a−9e, 9g−9j, 11a−
11d, 12a−12b, 13b, and 13h, a graphical comparison of the
assay results obtained with 8a, 11b, and 11d in US28-
transfected 293T HEK cells, and 3D plots showing the relative
performance of selected members of each structural class (8, 9,
and 11) in terms of efficacy, % effect on mock cells, and CHO
toxicity. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
* P h o n e : 4 0 7 - 7 4 5 - 2 1 3 0 . F a x : 4 0 7 - 2 6 6 - 1 1 9 9 .
E-mail: Otto.Phanstiel@ucf.edu.
Notes
(15) Sodhi, A.; Montaner, S.; Gutkind, J. S. Viral hijacking of G-
protein-coupled-receptor signalling networks. Nature Rev. Mol. Cell
Biol. 2004, 5, 998−1012.
The authors declare no competing financial interest.
(16) Maussang, D.; Vischer, H. F.; Leurs, R.; Smit, M. J. Herpesvirus-
encoded G protein-coupled receptors as modulators of cellular
function. Mol. Pharmacol. 2009, 76, 692−701.
ACKNOWLEDGMENTS
■
M-T.N. was supported by the University of Florida College of
Pharmacy summer research internship program. N.T. was
financially supported by Collaborative Research Center 796
(SFB796) of German Research Foundation (DFG). O.P. was
financially supported by the UCF College of Medicine startup
funds. We wish to thank Dr. Meenu Madan for her assistance in
generating the Supporting Information comparative graphs,
Figures S2 and S3.
(17) Vomaske, J.; Nelson, J. A.; Streblow, D. N. Human
Cytomegalovirus US28: A Functionally Selective Chemokine Binding
Receptor. Infect. Disord .Drug Targets 2009, 9, 548−556.
(18) Maussang, D.; Verzijl, D.; van Walsum, M.; Leurs, R.; Holl, J.;
Pleskoff, O.; Michel, D.; van Dongen, G. A.; Smit, M. J. Human
cytomegalovirus-encoded chemokine receptor US28 promotes tumori-
genesis. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 13068−13073.
(19) Tschammer, N. Virally Encoded G protein-Coupled Receptors:
Overlooked Therapeutic Opportunities? Annu. Rep. Med. Chem. 2012,
47, 379−392.
REFERENCES
■
(20) Smith, J. A.; Maloney, D. J.; Hecht, S. M.; Deborah, A.
Lannigan, Structural basis for the activity of the RSK-specific inhibitor,
SL0101. Bioorg. Med. Chem. 2007, 15, 5018−5034.
(1) (a) Dimmock, J. R.; Elias, D. W.; Beazely, M. A.; Kandepu, N. M.
Bioactivities of chalcones. Curr. Med. Chem. 1999, 6, 1125−1149.
(b) Sahu, N. K.; Balbhadra, S. S.; Choudhary, J.; Kohli, D. V. Exploring
M
dx.doi.org/10.1021/jm4003457 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(21) Yamasaki, K.; Hishiki, R.; Kato, E.; Kawabata., J. Study of
Kaempferol Glycoside as an Insulin Mimic Reveals Glycon to be the
Key Active Structure. ACS Med. Chem. Lett. 2011, 2, 17−21.
(22) (a) Chimenti, J. A. A new series of flavones, thioflavones, and
flavanones as selective monoamine oxidase-B inhibitors. Bioorg. Med.
Chem. 2010, 18, 1273−1279. (b) House, H. O. Modern Synthetic
Reactions, 2nd ed.; W. A. Benjamin: Menlo Park, CA, 1972; p 325;
(c) Doering, W. E.; Speers, L. The Peracetic Acid Cleavage of
Unsymmetrical Ketones. J. Am. Chem. Soc. 1950, 72, 5515−5518.
(d) Fujishiro, K.; Mitamura, S. The Baeyer−Villiger Reaction of
Polycyclic Aromatic Aldehydes: Preparation of Polycyclic Phenols.
Bull. Chem. Soc. Jpn. 1988, 61, 4464−4466.
(23) PathDetect in Vivo Signal Transduction Pathway trans-Reporting
Systems, Instruction Manual; Agilent Technologies, Inc.: Santa Clara,
CA, 2011; 219000-12, revision B.
(24) Bright-Glo Luciferase Assay System, Technical Manual; Promega
Corporation: Madison, WI, Sept 2011; part no. TM052.
(25) (a) Phanstiel, O., IV; Kaur, N.; Delcros, J.-G. Structure−activity
investigations of polyamine−anthracene conjugates and their uptake
via the polyamine transporter. Amino Acids 2007, 33, 305−313.
(b) Parisini, E.; Metrangolo, P.; Pilati, T.; Resnati, G.; Terraneo, G.
Halogen bonding in halo-carbon−protein complexes: a structural
survey. Chem. Soc. Rev. 2011, 40, 2267−2278.
(26) Hesselgesser, J.; Ng, H. P.; Liang, M.; Zheng, W.; May, K.;
Bauman, J. G.; Monahan, S.; Islam, I.; Wei, G. P.; Ghannam, A.; Taub,
D. D.; Rosser, M.; Snider, R. M.; Morrissey, M. M.; Perez, H. D.;
Horuk, R. Identification and Characterization of Small Molecule
Functional Antagonists of the CCR1 Chemokine Receptor. J. Biol.
Chem. 1998, 273, 15687−15692.
(27) Vischer, H. F.; Hulshof, J. W.; Hulscher, S.; Fratantoni, S. A.;
Verheij, M. H.; Victorina, J.; Smit, M. J.; de Esch, I. J.; Leurs, R.
Identification of novel allosteric nonpeptidergic inhibitors of the
human cytomegalovirus-encoded chemokine receptor US28. Bioorg.
Med. Chem. 2010, 18, 675−688.
(28) Casarosa, P.; Menge, W. M.; Minisini, R.; Otto, C.; van Heteren,
J.; Jongejan, A.; Timmerman, H.; Moepps, B.; Kirchhoff, F.; Mertens,
T.; Smit, M. J.; Leurs, R. Identification of the first nonpeptidergic
inverse agonist for a constitutively active viral-encoded G protein-
coupled receptor. J. Biol. Chem. 2003, 278, 5172−5178.
(29) Chan, K. F.; Zhao, Y.; Chow, T. W. C.; Yan, C. S. W.; Ma, D. K.;
Burkett, B. A.; Wong, I. L. K.; Chow, L. M. C.; Chan, T. H. Flavonoid
Dimers as Bivalent modulators for P-Glycoprotein-Based Multidrug
Resistance: Structure−Activity Relationships. ChemMedChem 2009, 4,
594−614.
(30) (a) Murata, T.; Shimada, M.; Sakakibera, S.; Yohino, T.;
Masuda, T.; Shintani, T.; Sato, H.; Koriyama, Y.; Fukushima, K.;
Nunami, N.; Yamauchi, M. Synthesis and structure−activity relation-
ships of novel IKK-β inhibitors. Part 3: Orally active anti-inflammatory
agents. Bioorg. Med. Chem. Lett. 2004, 14, 4019−4022. (b) Pinto, D.
G.; Silva, A. M. S.; Cavaleiro, J. A. Synthesis of 3-(2-benzyloxy-6-
hydroxyphenyl)-1-methylpyrazoles by the reaction of chromones with
methylhydrazine. J. Heterocycl. Chem. 2000, 37, 1629−1634. (c) Patil,
A. D.; Deshpande, V. H. Indian J. Chem., Sect. B: Org. Chem. Incl. Med.
Chem. 1983, 22, 109−113.
(31) Jung, S.-H.; Cho, S.-H.; Dang, T. H.; Lee, J.-H.; Ju, J.-H.; Kim,
M.-K.; Lee, S.-H.; Ryu, J.-C.; Kim., Y. Structural requirement of
isoflavonones for the inhibitory activity of interleukin-5. Eur. J. Med.
Chem. 2003, 38, 537−545.
N
dx.doi.org/10.1021/jm4003457 | J. Med. Chem. XXXX, XXX, XXX−XXX