Synthesis, structure-activity relationship studies, and antibacterial evaluation of 4-chromanones and chalcones, as well as olympicin A and derivatives

Article abstract of DOI:10.1021/jm500853v

On the basis of recently reported abyssinone II and olympicin A, a series of chemically modified flavonoid phytochemicals were synthesized and evaluated against Mycobacterium tuberculosis and a panel of Gram-positive and -negative bacterial pathogens. Some of the synthesized compounds exhibited good antibacterial activities against Gram-positive pathogens including methicillin resistant Staphylococcus aureus with minimum inhibitory concentration as low as 0.39 μg/mL. SAR analysis revealed that the 2-hydrophobic substituent and the 4-hydrogen bond donor/acceptor of the 4-chromanone scaffold together with the hydroxy groups at 5- and 7-positions enhanced antibacterial activities; the 2′,4′-dihydroxylated A ring and the lipophilic substituted B ring of chalcone derivatives were pharmacophoric elements for antibacterial activities. Mode of action studies performed on selected compounds revealed that they dissipated the bacterial membrane potential, resulting in the inhibition of macromolecular biosynthesis; further studies showed that selected compounds inhibited DNA topoisomerase IV, suggesting complex mechanisms of actions for compounds in this series.

Full text of DOI:10.1021/jm500853v

Article
pubs.acs.org/jmc
Synthesis, Structure−Activity Relationship Studies, and Antibacterial
Evaluation of 4‑Chromanones and Chalcones, as Well as Olympicin A
and Derivatives
Li Feng,† Marcus M. Maddox,‡,# Md. Zahidul Alam,§,# Lissa S. Tsutsumi,† Gagandeep Narula,∥
David F. Bruhn,‡ Xiaoqian Wu,§ Shayna Sandhaus,∥ Robin B. Lee,‡ Charles J. Simmons,⊥
Yuk-Ching Tse-Dinh,∥ Julian G. Hurdle,§ Richard E. Lee,‡ and Dianqing Sun*,†
†Department of Pharmaceutical Sciences, The Daniel K. Inouye College of Pharmacy, University of Hawai’i at Hilo, 34 Rainbow
Drive, Hilo, Hawaii 96720, United States
‡Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105,
United States
§Department of Biology, University of Texas at Arlington, Arlington, Texas 76019, United States
∥Department of Chemistry & Biochemistry, Biomolecular Sciences Institute, Florida International University, Miami, Florida 33199,
United States
⊥Department of Chemistry, University of Hawai’i at Hilo, Hilo, Hawaii 96720, United States
S
* Supporting Information
ABSTRACT: On the basis of recently reported abyssinone II and olympicin A, a series of chemically modified flavonoid
phytochemicals were synthesized and evaluated against Mycobacterium tuberculosis and a panel of Gram-positive and -negative
bacterial pathogens. Some of the synthesized compounds exhibited good antibacterial activities against Gram-positive pathogens
including methicillin resistant Staphylococcus aureus with minimum inhibitory concentration as low as 0.39 μg/mL. SAR analysis
revealed that the 2-hydrophobic substituent and the 4-hydrogen bond donor/acceptor of the 4-chromanone scaffold together
with the hydroxy groups at 5- and 7-positions enhanced antibacterial activities; the 2′,4′-dihydroxylated A ring and the lipophilic
substituted B ring of chalcone derivatives were pharmacophoric elements for antibacterial activities. Mode of action studies
performed on selected compounds revealed that they dissipated the bacterial membrane potential, resulting in the inhibition
of macromolecular biosynthesis; further studies showed that selected compounds inhibited DNA topoisomerase IV, suggesting
complex mechanisms of actions for compounds in this series.
have been the focus of numerous basic biomedical research as
well as clinical investigation.7,8 As examples, high dietary intake
of flavonoids may offer potential to reduce the risk of various
cancers according to a number of epidemiological studies.9−13
In addition, flavonoids have been reported to display a broad
spectrum of pharmacological activities, such as antimicro-
bial,14−16 anti-inflammatory,17,18 cancer preventive19,20 and anti-
cancer,21,22 and antioxidant activities.23,24 It is also noteworthy
that some widely investigated flavonoids, such as flavone acetic
acid (FAA),25 flavopiridol,26−28 silibinin (silybin),29,30 and
quercetin31 and its derivatives32 (Figure 1), have progressed
INTRODUCTION
■
Because of the emergence and spread of multidrug resistant
microorganisms and pathogenic bacterial infections, novel
chemotype antibacterial agents demonstrating distinct modes
of action from existing antibiotics are urgently needed. Natural
products are known as rich sources of bioactive molecules and
chemical diversity and have thus provided invaluable chemical
scaffolds as well as served as an inspiration toward antibacterial
drug discovery and development.1−4 In this context, synthesis
and evaluation of natural-product-inspired compound libraries
represent an attractive approach for discovering novel anti-
bacterial agents.5
Flavonoids are a large family of polyphenolic phytochemicals,
Received: June 4, 2014
which widely exist in the plant kingdom.6 As such, flavonoids
© XXXX American Chemical Society
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Figure 1. Skeleton structures of chalcones, 4-chromanones, and representative structures of naturally occurring flavonoids including abyssinone II
and olympicin A.
to various stages of clinical trials.33 In this regard, plant-derived
phytochemicals including chemically modified flavonoids and
derivatives continue to attract great interest in the development
of novel antibiotics.34
bacterial pathogens and a detailed structure−activity relation-
ship (SAR) has been obtained. Furthermore, the antibacterial
basis of promising lead compounds and their ability to inhibit
bacterial topoisomerases such as DNA gyrase or topo IV have
also been examined.
Furthermore, chalcones (1,3-diaryl-2-propen-1-ones), one
subclass of structural analogues of flavonoids, have been
reported to exhibit diverse biological activities,35−38 in which
the enone functional group and the 2′-hydroxy group constitute
important structural motifs for antibiotic activity. From a chem-
istry point of view, chalcones and 4-chromanones are struc-
turally related, and 2′-hydroxychalcones serve as important
synthetic precursors for the synthesis of 4-chromanones
following an intramolecular conjugate addition of the phenol
on the α,β-unsaturated system.39 Notably, the 4-chromanone
derivatives containing an aromatic substituent at the 2-position,
so-called flavanones, have been identified as an important class
of bioactive heterocycles.40−42
As a result of our longstanding interest in developing natural-
product-inspired new antibacterial agents, we recently reported
the identification of abyssinone II as a promising antibacterial
lead by screening a focused flavonoid and resveratrol library.43
In addition, olympicin A, a member of the natural acyl-
phloroglucinol chemical class, was recently isolated from the
plant Hypericum olympicum and reported to exhibit potent
antibacterial activity against a panel of multidrug-resistant
(MDR) strains of clinically relevant Staphylococcus aureus,
with minimum inhibitory concentration (MIC) values ranging
from 0.5 to 1 μg/mL.44,45 Very recently, we have shown that
synthetic olympicin A also exhibited good activity against
Clostridium difficile (MIC = 1−2 μg/mL).46 Inspired by the
antibacterial activity of the natural products abyssinone II and
olympicin A, in this work we employed the 4-chromanone and
chalcone structural scaffolds as chemical starting points to
design and synthesize chemically modified flavonoid analogues.
Subsequently, several series of structurally related flavonoids
were synthesized and evaluated in vitro against a broad set of
RESULTS AND DISCUSSION
■
Synthesis of Olympicin A and Derivatives. The iso-
lation and chemical synthesis of olympicin A (2a) was originally
reported by Shiu et al., and its synthesis involved a four-step
reaction sequence. However, the overall yield was only 3.3%
from 1a.44 To improve the reaction efficiency and develop
a modular synthesis toward olympicin A and derivatives, we
evaluated diverse protecting schemes including the tert-
butyldimethylsilyl (TBDMS), the base-stable methoxymethyl
(MOM), and the p-toluenesulfonyl (Ts) groups and developed
an improved synthesis of 2a by using the Ts protecting strategy,
improving the overall yield to 40% from 1a (Scheme 1). In
particular, we found that the reported low yield may be largely
due to the instability of the TBDMS protecting group under
basic reaction conditions (K2CO3, 80 °C) when introducing
the geranyl group. To address this, the base-stable MOM
group was next applied instead of TBDMS. Following the
O-geranylation reaction, it was found that 2a decomposed
during the deprotection of the MOM groups because of the
instability of O-geranyl group under the acidic condition, and
the deprotected 1a was recovered from the reaction. Sub-
sequently the Ts group was used to protect the hydroxy
group,47 and only the tris(tosylate) 1b was obtained as the
major product because of the lack of selectivity of the Ts group
under the reaction condition. Nevertheless, we found that the
tosylate group at the 2-position of 1b was very labile, and the
geranyl group could be selectively introduced with sodium
hydride as the base. Final removal of the Ts group was per-
formed with excess sodium methoxide in methanol under
reflux48 to afford the chiral olympicin A (2a). The spectroscopic
B
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a
Scheme 1. Improved Synthesis of Olympicin A (2a)
Figure 2. Modifications based on the 4-chromanone scaffold.
As shown in Scheme 3, a series of 2-alkyl-4-chromanone
derivatives 3a−f were synthesized by reacting 2,4-dihydroxy-
acetophenone (1d) with an appropriate aliphatic aldehyde in
the presence of pyrrolidine in ethanol. This reaction was
performed in a sealed pressure tube at 150 °C for 1 h, obtaining
yields over 30%. Furthermore, the reaction time was shortened
significantly. In contrast, a lower yield (12%) was afforded
despite a longer reaction time (72 h) for the synthesis of 3b
under the conditions (ethanol, 60 °C, pyrrolidine). To optimize
the reaction conditions, a panel of different amine bases was
screened. We noted that under the same conditions, no product
was detected using DIPEA and only traces of product were
formed when using morpholine as a base monitored by HPLC,
respectively. To investigate the potential effects of the 4-carbonyl
group as a hydrogen bond acceptor, the 4-chromanones 3a−d
were further reduced by NaBH4 in methanol to provide
4-chromanol derivatives 4a−d in moderate to good yields
(64−81%).54
Accordingly, when the bicyclic (−)-myrtenal (1f) was used as
the substrate under these reaction conditions (Scheme 3,
step i), no product was obtained. Subsequently, a lower tem-
perature (75 °C) was applied (Scheme 4) to optimize the
reaction conditions. The corresponding chalcone intermediate
3g was obtained in low yield (9.5%). To improve the reaction
yield, 1d was first regioselectively protected by reacting with
MOMCl in the presence of DIPEA to provide the MOM-
protected acetophenone 1e in 86% yield. Again, when the
reaction was performed at 150 °C, only traces of 3h were
detected by HPLC. In contrast, a much higher yield (48%)
was obtained when the reaction temperature was reduced
to 75 °C. The MOM-protected 3h can be further cyclized
in ethanol in the presence of sodium acetate to yield
4-chromanone 3i as a mixture of two diastereomers.55 Final
deprotection of the O-MOM group was performed in
methanol using concentrated HCl at room temperature to
give 3j in 90% yield.
a
Reagents and conditions: (i) SOCl2, 80 °C, 2 h; (ii) AlCl3, CS2,
PhNO2, 0.5 h; (iii) TsCl, K2CO3, acetone, 1 h, 73%; (iv) geranyl
bromide, NaH, DMF, 1 h; (v) CH3ONa, MeOH, reflux, 8 h, 55%
(two-step overall yield); 52% for 2b (two-step overall yield).
data of our synthetic olympicin A were in good agreement with
those of the natural product.44 For comparison, the racemic
olympicin A (2b) was also synthesized under the same
conditions to study the potential effect of stereochemistry on
antibacterial property.
Next, to further expand the chemical diversity and investigate
the influence of 2-substitution on antibacterial activity in the
scaffold, an array of racemic olympicin derivatives (2c−e) and
enantiomeric form 2f were designed and synthesized (Scheme 2).
Compounds 1a′ and 1a were first protected by reacting with
MOMCl in the presence of diisopropylethylamine (DIPEA) to
provide 1c and 1c′ in moderate yields. The O-alkylation reaction
was subsequently carried out with appropriate alkyl bromide
using sodium hydride as a base, after which the MOM groups
were removed with hydrochloric acid to give 2c−f in moderate to
high yields (66−93%).
Synthesis of 2-Substituted 4-Chromanone and Deriv-
atives. 4-Chromanone derivatives bearing an aryl substituent in
the 2-position are normally synthesized by reacting an
acetophenone with an arylaldehyde under strong basic or
acidic conditions.49,50 However, because of the side reaction of
self-condensation of the aliphatic aldehyde,51 these conditions
are not ideal for the synthesis of 2-alkyl substituted 4-chromanones.
Several different synthetic methods to prepare such 4-chromanone
derivatives have been reported,51−53 either involving microwave
irradiation or requiring very long reaction times. Here, a sealed
pressure tube was introduced as a reaction vessel for the preparation
of 2-alkyl substituted 4-chromanones. On the basis of the scaffold,
several modification strategies (Figure 2) were applied to develop
chemical diversity and further evaluate the SAR.
Next, on the basis of the promising antibacterial activity of
3f, a focused set of 4-chromanone analogues (Scheme 5) were
subsequently designed and synthesized to investigate the
antibacterial effect of the phenol free hydroxy group at dif-
ferent positions. Compound 3f was resynthesized from the
MOM-protected 1e by using diethylamine (DEA) as a base in
a
Scheme 2. Synthesis of Olympicin A Analogues
a
Reagents and conditions: (i) MOMCl, DIPEA, DCM, 1 h; (ii) R-Br, NaH, DMF; (iii) HCl, MeOH, overnight.
C
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a
Scheme 3. Synthesis of 2-Alkylated 4-Chromanones and Derivatives
a
Reagents and conditions: (i) pyrrolidine, EtOH, 150 °C, pressure tube, 1 h; (ii) NaBH4, MeOH, rt, 24 h.
a
Scheme 4. Reactions of Acetophenone and (−)-Myrtenal
a
Reagents and conditions: (i) DIPEA, MOMCl, 0 °C, 1 h; (ii) pyrrolidine, EtOH, 75 °C, pressure tube, 1 h; (iii) NaOAc, EtOH, reflux, 24 h;
(iv) concentrated HCl, MeOH, rt, overnight.
a
using appropriate MOM-protected 1g,h in moderate yields
(58−75%).
Scheme 5. Synthesis of 4-Chromanone Analogues 3f, 3k, and 3l
To further expand the SAR and evaluate the influence of
the 5-hydroxy group, an array of 5,7-dihydroxy-4-chromanones
were synthesized. Accordingly, 2,4,6-trihydroxyacetophenone
(1j) was used to prepare 4-chromanone derivatives 5 (Scheme 6).
Unfortunately, no product was obtained under the reaction
conditions of pyrrolidine in ethanol at 150 °C in a pressure
tube. Therefore, the bis-MOM-protected acetophenone 1k
was next prepared in 79% yield.56 The corresponding MOM-
protected 4-chromanones 5 were obtained by reacting 1k with
an appropriate aldehyde in the presence of DEA in ethanol
in a pressure tube at 150 °C in moderate yields (55−70%).
a
Reagents and conditions: (i) DEA, EtOH, 150 °C, pressure tube, 1 h;
(ii) concentrated HCl, MeOH, rt, overnight.
an overall 58% yield following O-MOM deprotection. Accord-
ingly, 3k,l with the 5- or 6-hydroxy group were also synthesized
a
Scheme 6. Synthesis of 2-Substituted 4-Chromanones and Oxime Derivatives
a
Reagents and conditions: (i) DIPEA, MOMCl, 0 °C, 1 h, 79%; (ii) DEA, EtOH, 150 °C, pressure tube, 1 h; (iii) concentrated HCl, MeOH, rt,
b
24 h; (iv) pyridine, EtOH, rt, 26−72 h. Without isolating the MOM-protected intermediate.
D
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a
Scheme 7. Synthesis of Compounds 5m and 5n
a
Reagents and conditions: (i) DEA, EtOH, 150 °C, pressure tube, 4 h; (ii) HCl, MeOH, rt, 16 h; (iii) HCl, EtOH, microwave irradiation, 150 °C, 0.5 h.
a
Scheme 8. Synthesis of 2-Spiro-4-chromanones 7a−c
Next, 2-spiro-4-chromanones (7a−c) were synthesized by
reacting 1e with an appropriate cycloketone in a pressure tube
at 150 °C for 2−16 h in the presence of pyrrolidine (Scheme 8).
The following O-MOM deprotection was performed in one pot
with excess concentrated HCl. The spiro compounds 7a−c were
obtained in moderate to high yields, with 7c giving the lowest
yield (53%) probably due to the steric hindrance of the seven-
membered ring.
Synthesis of 2-Substituted Aromatic Chalcone and
Flavanone Derivatives. To systematically investigate the
SAR of the chalcone and 4-chromanone scaffolds on anti-
bacterial activities, diverse aromatic aldehydes were introduced
to synthesize a series of 2-aryl chalcone and flavanone deriv-
atives (Scheme 9). Accordingly, the Claisen−Schmidt aldol
condensations were performed at room temperature with the
addition of 60% KOH aqueous solution to the mixture of
appropriate MOM-protected acetophenone and aldehyde in
methanol.55 Subsequent removal of the MOM-protecting group
with concentrated HCl afforded the chalcone derivatives 8a−h.
Chalcone 8f was synthesized as a comparison under the same
conditions to verify the potential effect of 2′-hydroxy group on
antibacterial activity. Additionally, in comparison to 8a with the
ortho-substituted allyloxy group, regioisomers 8g and 8h (with
the allyloxy group at the meta- and para-position, respectively)
were next synthesized to investigate the influence of the
substitution at the R2 position in the scaffold. Subsequent
intramolecular conjugate additions of the phenol on the
α,β-unsaturated system of 8a−e were carried out under
microwave irradiation in the presence of catalytic amounts of
concentrated HCl to yield corresponding flavanones 8i−m in
50−75% yields.
a
Reagents and conditions: (i) pyrrolidine, EtOH, 150 °C, pressure
tube, 2−16 h; (ii) HCl, rt, overnight.
Subsequent removal of the MOM groups using concentrated
HCl in methanol at room temperature afforded the corresponding
4-chromanones in 75−94% yields. Next, to evaluate the role of
the carbonyl group at the 4-position and further explore the
chemical diversity of the 4-chromanone scaffold, a panel of
4-oximinochromanes 6a−f were produced by reacting the
corresponding 5 with an appropriate hydroxylamine in ethanol
in the presence of pyridine in high yields (80−90%). Notably,
the reaction time in this series differed from 26 to 72 h, and the
electron donating group such as MeO in 6b greatly facilitated
the reaction and reduced the reaction time (26 h).
As illustrated in Scheme 7, to synthesize the bicyclic com-
pound 5n bearing the myrtenal motif, the reaction time needed
to be extended to 4 h under the reaction conditions, and the
chalcone 5m was obtained in overall two-step 34% yield after
O-MOM deprotection. Subsequent intramolecular conjugate
addition of the phenol on the α,β-unsaturated system was
performed under microwave irradiation in the presence of
catalytic amounts of concentrated HCl to give 5n in 72% yield
as a mixture of two diastereomers.49 We also noted that the re-
action was completed in 0.5 h, which was a great improvement
in comparison to the condition of refluxing with sodium acetate
in ethanol in Scheme 4.
All the synthesized compounds were characterized by 1H and
13C NMR spectroscopy and high-resolution mass spectrometry
(HRMS), and purity was analyzed by reverse phase HPLC. The
structures of 3g and 8a were confirmed by X-ray crystallography,
a
Scheme 9. Synthesis of 2-Substituted Aromatic Chalcones and Flavanones
a
Reagents and conditions: (i) 60% KOH aq, MeOH, rt, 60 h; (ii) concentrated HCl, rt, overnight; (iii) microwave, HCl, EtOH, 150 °C, 1.5 h.
Without 2′-OH function for 8f.
b
E
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a
Table 1. Antibacterial Activities (MIC, μg/mL) of Olympicin A and Derivatives
a
No test compounds were active (>200 μg/mL) against other Gram-negative bacteria including K. pneumonia (ATCC 33495) and P. aeruginosa
b
c
(PAO1). Positive controls isoniazid and rifampin inhibited M. tuberculosis at 0.03 and 0.05 μg/mL, respectively. nd = not determined.
and their ORTEP drawings are shown in Figure S1 (Supporting
Information).
(Tables 2 and 3), the majority of compounds exhibited weak
antituberculosis activity. The 2-propyl-4-chromanol 4a showed
the most potent activity in the entire series with a MIC of
12.5 μg/mL. The reduced 4-chromanol variants 4a and 4c
(4-OH, 12.5 and 25 μg/mL, respectively) showed more potent
antituberculosis activity than their corresponding 4-chroma-
nones 3a (200 μg/mL) and 3c (50 μg/mL). In addition, the
4-oximinochromane 6a (NOH, 100 μg/mL) displayed
greater potency than 6b (NOMe, 200 μg/mL) and 6c
(NOBn, >200 μg/mL). Taken together, these results indi-
cate that the small, polar, and hydrophilic groups (e.g., −OH
and NOH) are more favorable at the 4-position of the
flavonoid scaffold for antituberculosis activity, suggesting these
polar groups may function as hydrogen bond donors when
interacting with potential biological cellular target.
Different lengths of the 2-alkyl side chains in the scaffold
displayed an important relationship with antituberculosis
activity as well. Compared to 3a (three-carbon, 200 μg/mL),
3b (six-carbon, 50 μg/mL), and 3c (seven-carbon, 50 μg/mL),
compound 3d with a nine-carbon linear chain was inactive,
suggesting the nine-carbon alkyl group is too long and bulky.
To investigate the impact of the substituent at the 5-position in
the scaffold, a hydrogen bond donor and/or acceptor (hydroxy
group) was introduced. These data showed that the compounds
bearing 5,7-dihydroxy functionalities (5b, 5d, and 5f) exhibited
similar MIC values to the sole 7-hydroxychromanones 3a−c.
However, 5h (5,7-di-OH, nine-carbon chain, 100 μg/mL) was
more potent than the corresponding 3d (7-OH, nine-carbon
chain, >200 μg/mL). In addition, the position of the sole
hydroxy group in the scaffold also played a notable role in their
antituberculosis activity by comparing 3f (7-OH, 100 μg/mL)
to 3k (6-OH, 200 μg/mL) and 3l (5-OH, >200 μg/mL), with
3f bearing the 7-hydroxy function being the most potent.
In general, 2-aryl substituted compounds together with
2-spiro derivatives possessing a cyclic ring system were less
All the synthesized compounds were evaluated against
Mycobacterium tuberculosis (H37Rv) and a wide set of clinically
relevant Gram-positive and -negative bacterial pathogens
including Enterococcus faecalis (ATCC 33186), Staphylococcus
aureus (ATCC 29213 and NRS 70), Escherichia coli (K12 and
ΔtolC), Klebsiella pneumoniae (ATCC 33495), and Pseudomo-
nas aeruginosa (PAO1). Their antitubercular and antibacterial
activities are summarized in Tables 1−3.
Olympicin A Series. In the olympicin A series, olympicin A
(2a) and analogues (2b−f) showed weak antitubercular activity
with MICs of 100−200 μg/mL (Table 1). The observed weak
antituberculosis activity may be attributed to the general polar
nature of this chemical series and decreased membrane pene-
tration. In contrast, the olympicin derivatives with geranyloxy
(2a and 2b), n-hexyloxy (2d), and n-octyloxy (2e) groups
showed good to potent anti-Gram-positive activity against
E. faecalis and S. aureus strains (MIC = 0.78−3.13 μg/mL).
However, the less lipophilic olympicin derivatives 2c and 2f
with a shorter allyloxy chain exhibited about 8- to 16-fold
decrease of antibacterial activity (MIC = 6.25−12.5 μg/mL). In
terms of stereochemistry effect, the racemic olympicin A (2b)
and allyloxy derivative (2c) showed largely the same anti-
tuberculosis and anti-Gram-positive activity compared to their
corresponding chiral S-isomers 2a and 2f, respectively. Nota-
bly, the anti S. aureus activity (1.56 μg/mL) of our synthetic
sample (2a) of olympicin A is consistent with the previously
reported anti S. aureus activity (0.5−1 μg/mL) of natural
olympicin A.44 In addition, none of these olympicin analogues
were active against Gram-negative microorganisms except for
the reengineered E. coli strain (ΔtolC) with deficient efflux
activity.
4-Chromanone and Chalcone Series. Antituberculosis
Activity. In the 4-chromanone and chalcone chemical series
F
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Table 2. Antibacterial Activities (MIC, μg/mL) of 4-Chromanone Derivatives
G
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Table 2. continued
a
No test compounds were active (>200 μg/mL) against other Gram-negative bacteria including K. pneumonia (ATCC 33495) and P. aeruginosa
(PAO1). The MIC values of control antibiotics used in this study are shown in Table 1.
active against M. tuberculosis than 2-alkylated derivatives in the
entire series. Moreover, the MOM protected derivatives exhibited
comparable or more potent antitubercular activity than their
corresponding free phenol parent molecules. The SAR analysis
H
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a
Table 3. Antibacterial Activities (MIC, μg/mL) of Chalcones and Derivatives
a
No test compounds were active (>200 μg/mL) against other Gram-negative bacteria including K. pneumonia (ATCC 33495) and P. aeruginosa
(PAO1). The MIC values of control antibiotics used in this study are shown in Table 1.
above demonstrates that the 2-alkyl hydrophobic substituents
as well as the small and polar functionalities at the 4-position
(e.g., hydrogen bond donor groups OH and NOH) play impor-
tant roles in antituberculosis activities in the 4-chromanone
scaffold.
General Antibacterial Spectra. In addition to antituber-
culosis evaluation, antimicrobial assessment against representative
clinical pathogens revealed that the majority of compounds
from these series exhibited notable anti-Gram-positive bacteria
activities including against E. faecalis and S. aureus (MSSA and
MRSA) and poor activity against Gram-negative bacteria
including E. coli, K. pneumoniae, and P. aeruginosa.
Compared with the 4-chromanone flavonoid series, the
chalcone series generally exhibited more potent anti-Gram-positive
activity than their corresponding cyclized derivatives. The 2′,4′-di-
OH chalcone compound 8a having an appended 2-allyloxy
group exhibited the best activity (MIC of 0.39−6.25 μg/mL)
against MSSA and MRSA, and the o-hydroxy group appeared to
have a beneficial effect on anti-Gram-positive bacterial activity
by comparing 8a (2′,4′-di-OH, 2-allyloxy, 0.39−12.5 μg/mL) to
8f (4′-OH, 2-allyloxy, 6.25−100 μg/mL) (Table 3). Notably, this
observation regarding the importance of the o-hydroxy group in
the chalcone scaffold is also in agreement with that found in the
4-chromanone flavonoid series, whereas the corresponding hydroxy
group at the 5-position of the 4-chromanone scaffold enhanced the
antibacterial activity. However, the 2′,4′,6′-tri-OH chalcone
compounds possessing an additional 6′-hydroxy group 5m (25 to
>200 μg/mL) and 8e (25 to >200 μg/mL) showed significantly
decreased activities against Gram-positive microorganisms com-
pared to the 2′,4′-di-OH chalcones 3g (1.56−3.13 μg/mL) and 8a
(0.39−12.5 μg/mL), respectively. The notable decreased activity
for trihydroxy compounds is likely due to increased hydrophilicity
and polarity properties and thus decreased bacterial membrane
penetration. Taken together, these data suggest that two free phenol
hydroxy groups on the 4-chromanone and chalcone scaffolds are
optimal for Gram-positive antibacterial activity.
Furthermore, in the chalcone series (Table 3), the lipophilic
O-alkyl substituent at the 2-position of aromatic chalcones also
I
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had a great impact on antibacterial activities, since the chalcones
bearing diverse side chains such as the allyloxy (8a), n-hexyloxy
(8b), and n-octyloxy (8c) exhibited notable differences in their
activities; compound 8b bearing the n-hexyloxy group showed
optimal antibacterial activity against E. faecalis, MSSA, and
MRSA (1.56, 3.13, and 0.78 μg/mL, respectively). Compound
8a bearing the 2-allyloxy function demonstrated 2-fold more
potent activity against MRSA and an 8-fold reduction in activity
against E. faecalis than 8b.
corresponding 4-chromanones 3a−d, which were not active against
Gram-positive bacteria tested. In contrast, the 4-oximinochromanes
(6a and 6d-f: NOH at the 4-position) showed more potent
activity against E. faecalis and MSSA and comparable activity
against MRSA than their corresponding 4-chromanones (5h,
5b, 5d, and 5f), suggesting the free oxime NOH functionality
is preferred compared to the carbonyl group. Furthermore, by
comparison of 6a with the free 4-oxime functionality (NOH,
12.5 μg/mL), compounds 6b (NOMe, 25−100 μg/mL) and
6c (NOBn, >200 μg/mL) showed decreased antibacterial
activity against S. aureus. This observation is also consistent
with the trend toward the antituberculosis activity found in
this series, indicating that the small, polar, and hydrogen
bond donor functionalities (e.g., −OH and NOH groups)
at the 4-position may be more favorable for antibacterial prop-
erties, together with the results based upon 4-chromanols
(4a−d).
To further evaluate the importance of the 5-, 6-, and 7-OH
functionality, a set of 4-chromanone derivatives 3f (7-OH),
3k (6-OH), and 3l (5-OH) were subsequently synthesized.
Biological evaluation revealed that the 6- or 7-hydroxy
group also proved to be a determining factor for antibacterial
activities, since 3f (7-OH, 3.13−12.5 μg/mL) and 3k (6-OH,
6.25−12.5 μg/mL) demonstrated almost equally potent
antibacterial activities against Gram-positive bacteria tested.
However, their regioisomer 3l with the 5-OH substitution was
not active. The lack of antibacterial activity of 3l may be due to
the presence of intramolecular hydrogen bonding between the
carbonyl group at the 4-position and the hydroxy group at the
5-position. It should be noted that all the MOM protected
derivatives (3h, 3i, 5a, 5c, 5e, 5g, and 5i) completely lost
anti-Gram-positive bacterial activities against E. faecalis and
S. aureus, demonstrating the importance of the free phenol
hydroxy functionality and its weakly acidic nature in the
scaffold. This observation is consistent with our previous
report.43 Additionally, among the chemical series bearing a
cyclic/bicyclic ring system at the 2-position (5k, 2-cyclopentyl;
5l, 2-cyclohexyl; 5n, 2-myrtenyl; and the 2-spiro compounds
7a−c), compound 5n bearing the myrtenyl motif showed
In the flavonoid series, the length of 2-alkyl substitutions in
the 4-chromanone scaffold plays an important role in deter-
mining antibacterial activities. The 5,7-dihydroxy-4-chromanones
with long aliphatic alkyl chains 5d, 5f, 5h, and 5j (six to nine-
carbon chain, 3.13−6.25 μg/mL) showed better activity against
MRSA than the shorter chain derivative 5b (three-carbon
chain, 100 μg/mL), and notably, the 2-(2,6-dimethyl-5-
heptenyl) substituted 5j with a branched and unsaturated
alkyl chain displayed the best potency among these five
2-alkylated derivatives against three Gram-positive bacteria
tested (E. faecalis, MSSA, and MRSA; 6.25, 3.13, and 3.13 μg/mL,
respectively). These results suggest that the branched unsaturated
substitution in 5j may play an important role in enhancing
interactions and binding affinity with cellular biological target
because of its favorable lipophilicity and high conformational
flexibility, as previously noted for the prenylated derivatives.57,58
We also observed that the introduction of an additional 5-OH
group to the 7-hydroxy-4-chromanones 5b (100 μg/mL), 5d
(6.25 μg/mL), 5f (3.13 μg/mL), 5h (3.13 μg/mL), and 5n
(6.25 μg/mL) significantly improved antibacterial activity
against MRSA, compared to their corresponding 7-OH-4-
chromanones 3a−d (>200 μg/mL) and 3j (200 μg/mL) except
that 5j maintained anti-MRSA activity (3.13 μg/mL) compared
to 3f. These data demonstrate the importance of the 5-OH
group in the 7-OH-4-chromanone scaffold for antibacterial
activity.
In addition, the reduced variants 4-chromanols 4a−d (in
particular 2-n-heptyl-7-OH-4-chromanol 4c, 12.5−25 μg/mL;
and 2-n-nonyl-7-OH-4-chromanol 4d, 25−50 μg/mL) also
displayed improved antibacterial activities compared to their
Figure 3. General SAR of 4-chromanone and chalcone derivatives.
J
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a
Table 4. Cytotoxicity and Solubility Profiles of Selected Lead Compounds
molecular weight
(g/mol)
lipophilicity
b
solubility
(μg/mL)
cytotoxicity IC50
MIC against MRSA
selectivity index
c
d
compd
(cLogP)
(μg/mL)
(μg/mL)
(SI)
3f
3g
5f
288.2
284.1
278.2
306.2
304.2
321.2
293.2
296.1
340.2
323.2
340.2
368.2
3.77
3.19
3.20
4.03
3.38
4.42
3.59
3.37
4.76
3.82
4.36
5.19
nd
>200
28.8 1.8
6.9 0.3
3.13
1.56
3.13
3.13
3.13
12.5
3.13
0.39
0.78
3.13
1.56
3.13
nd
9.2
4.4
10.5
2.8
8.6
0.8
3.1
54.4
9.2
5.3
15.7
5.4
nd
>200
150.0
>200
>200
37.5 17.7
>200
>200
>200
100.0
>200
>200
nd
33.0 2.1
8.6 2.7
5h
5j
27.0 2.8
10.1 1.9
9.6 0.1
6a
6f
8a
21.2 5.4
7.2 0.4
8b
8d
16.7 0.1
24.5 2.1
17.0 0.0
5.3 1.3
8j
8k
e
thioridazine
verapamil
nd
nd
nd
nd
nd
57.3 3.2
177.5 10.6
nd
nd
f
DMSO
nd
nd
nd
nd
a
b
DMEM/FBS: Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The cLogP values of compounds
c
were calculated using ChemBioOffice Ultra, version 12.0, from CambridgeSoft Corporation. Cytotoxicity IC50, concentration that reduces viability
of Vero kidney cells by 50%. Selectivity index = (cytotoxic IC50)/(MIC against MRSA). nd = not determined. Carrier effect.
d
e
f
relatively good antibacterial activity (6.25−12.5 μg/mL) against
Gram-positive bacteria tested. In contrast, the 7-OH and 2-myrtenyl
substituted flavonoid 3j was largely inactive. Interestingly, its
corresponding ring-opened chalcone derivative 3g demonstra-
ted very good anti-Gram-positive activity (1.56−3.13 μg/mL).
A detailed SAR of the 4-chromanone and chalcone series is
summarized in Figure 3.
Solubility and Cytotoxicity Determination. Cytotoxicity
against mammalian (Vero epithelial) cells and solubility in
Dulbecco’s modified Eagle medium (DMEM) supplemented
with 10% fetal bovine serum (FBS) were evaluated for a
selected panel of 4-chromanone and chalcone lead compounds,
and the results are shown in Table 4. Overall, all the tested
compounds had good solubility (≥100 μg/mL) in the DMEM/
FBS medium used in the cytotoxicity assay except for the
4-oxime derivative 6a (37.5
17.7 μg/mL). The selectivity
indices (SI) for the compounds were calculated as the ratio of
the IC50 value of cytotoxicity against Vero monkey kidney
cell line and the MIC value against tested MRSA. Notably,
4-chromanone derivatives 5f (SI = 10.5) and 8j (SI = 15.7) and
the chalcone derivative 8a (SI = 54.4) possessed a more
favorable selectivity index (SI > 10). On the basis of these
promising antibacterial lead structures, advanced medicinal
chemistry will be applied to produce compounds with im-
proved potency and decreased cytotoxicity.
Time Kill Experiments and Mutation Selection. The
bactericidal activities of compounds 3g, 5j, 8a, and 8d were
examined against S. aureus Newman (Figure 4). The most
effective compound was the chalcone derivative 3g, which killed
more than 6 log of cells in just 2 h at 4× its MIC, but at its MIC
(1.56 μg/mL against S. aureus Newman) up to 24 h was
required to achieve a 3 log reduction in cells. Compound 3g
thus exhibited concentration-dependent killing. Compound 8a
(MIC = 6.25 μg/mL) was also rapidly bactericidal at 4× its
MIC and achieved a 6 log reduction in 6 h. Interestingly, compound
8d (6.25 μg/mL) was entirely bacteriostatic and failed to kill more
than 3 log of culture at 4 × its MIC, even up to 24 h of exposure. At
concentrations between 1× and 4× their MICs, spontaneous
mutants of S. aureus Newman could not be selected with 8a, 3g,
and 5j, but mutants arose to 8d at a frequency of 10−10. The
Figure 4. Time kill studies against S. aureus Newman exposed to 4×
MIC of compounds. Each point represents the average of two
biological replicates.
controls mupirocin and vancomycin were either bacteriostatic
or slowly bactericidal over a 24 h period, and mutants could
only be selected with mupirocin at frequencies of 10−7−10−9.
Effects on Macromolecular Synthesis and Membrane
Potential. Several key biosynthetic processes were simulta-
neously inhibited in S. aureus Newman exposed to the com-
pounds 2a, 5j, 8a, 3g, and 8d (Figure 5). These effects are
consistent with the bacterial membrane being the primary
target site of action, resulting in multiple nonspecific cellular
effects. Surprisingly, the bacteriostatic compound 8d displayed
the same time-dependent effects on macromolecular synthesis
as the bactericidal compounds. To determine if the compounds
dissipated the membrane potential of S. aureus, the fluorescent
probe DiOC2(3) was used. Compounds 2a, 5j, 3g, and 8d all
dissipated the membrane potential of S. aureus Newman in a
concentration-dependent manner. Maximum dissipation oc-
curred at 4× their MICs and was similar to the control CCCP
(carbonyl cyanide m-chlorophenylhydrazone) (Figure 6); as
expected, vancomycin (at 4× MIC) failed to affect the
membrane potential in S. aureus.
K
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Figure 5. Effects of 2a, 5j, 3g, 8a, and 8d and indicated positive controls at 4× their MICs on macromolecular synthesis in S. aureus Newman. The
standard error of the mean (SEM) is shown for three biological replicates. ERY = erythromycin (MIC = 0.78 μg/mL); RMP = rifampicin (0.12 μg/mL);
CIP = ciprofloxacin (0.25 μg/mL).
Inhibition of Bacterial Topoisomerase IV and DNA
Gyrase. Clinically, DNA gyrase and topo IV are validated and
attractive antibacterial targets for fluoroquinolone and
novobiocin antibiotics. However, because of the emergence of
target-based bacterial resistance with fluoroquinolone class and
safety concerns of novobiocin, novel DNA gyrase and
topoisomerase inhibitors are urgently needed for the treatment
of pathogenic and resistant bacterial infections.59 Thus, development
of new chemotype bacterial topo inhibitors has attracted great
interest in the scientific community, and recent examples
include bisbenzimidazoles,60 anziaic acid, and its analogues61,62
as bacterial topo IA inhibitors, and pyridylureas63 and pyr-
rolamides59 as topo II inhibitors. Previously, flavonoids have
been identified as bacterial topoisomerase inhibitors,64−66 and
we next tested to see if these promising antibacterial com-
pounds from olympicin A, 4-chromanone, and chalcone series
inhibited E. coli topo I, topo IV, and DNA gyrase. The results
are shown in Table 5 and Figure S2 (Supporting Information).
The E. coli topo I assay (relaxation of negatively supercoiled
Table 5. E. coli Topoisomerases and DNA Gyrase Inhibition
of Selected Analogues
topo I
IC50 (μM)
DNA gyrase
IC50 (μM)
topo IV
IC50 (μM)
compd
2a
2e
3g
5f
>500
>500
>500
>500
>500
>500
>500
>500
>500
500−1000
>1000
30−60
30−60
250−500
>1000
60−120
120−250
250−500
60−120
500−1000
>1000
8a
8b
8d
8j
500−1000
>1000
>1000
>1000
8k
>1000
>1000
a
b
c
b
c
known inhibitors
14−19
0.2, 100
2.1, 806
a
b
Figure 6. Dissipation of the staphylococcal membrane potential by 2a,
5j, 3g, and 8d. A representative of three biological replicates is shown.
Vancomycin (MIC = 0.8 μg/mL) and CCCP (MIC = 6.25 μg/mL)
were used as negative and positive controls.
IC50 of known inhibitor for assay: anziaic acid61 for Topo I. IC50 of
known inhibitor for assay: ciprofloxacin67 for DNA gyrase and topo IV.
c
IC50 of known inhibitor for assay: nalidixic acid68 for DNA gyrase and
topo IV.
L
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plasmid DNA) was performed at both 0.5 and 5 mM mag-
nesium chloride concentrations, and no significant inhibition
was observed against topo I at 0.5 mM compound con-
centration. The assay against E. coli gyrase (supercoiling of
relaxed plasmid DNA) and E. coli topo IV (decatenation of
catenated kinetoplast DNA) was subsequently performed.
Again for DNA gyrase, inhibition was not observed or was
weak with 8a, 3g, and 2a (MIC values of 0.39−6.25 μg/mL
against S. aureus) exhibiting IC50 values of >0.25 mM. However,
the E. coli topo IV showed more significant sensitivity toward
2a and 2e (MIC values of 0.78−1.56 μg/mL against E. faecalis
and S. aureus) with IC50 values of 30−60 μM (Table 5 and
Figure S2, Supporting Information). These results showed that
the olympicin A (2a) and its analogue 2e may serve as a
promising and novel scaffold for topo IV inhibitors.
Interestingly, olympicin A was also recently reported as a
moderate ATP-dependent Mycobacterium tuberculosis MurE
ligase inhibitor with an IC50 value of 75 μM.45 Studies to
determine if the olympicin A analogues 2c−f inhibit the MurE
pathway in M. tuberculosis remain to be performed. Finally, the
antibacterial mechanism of the 4-chromanone flavanone
compounds 8k and 8j (MIC values of 1.56−12.5 μg/mL
against S. aureus) may not be likely to involve topoisomerase
inhibition, as they were inactive in these topo enzyme
inhibition assays (Table 5). Collectively, no correlations
between whole-cell-based activity and topoisomerase inhibition
were observed for selected compounds, suggesting that the
membrane is likely the primary biological target responsible for
antibacterial activity and topo IV is a secondary or alternative
target. Further mechanistic studies are warranted to define the
exact mechanism of antibacterial action of these chemically
modified flavonoid and polyphenol compounds.
optimization in an effort to identify advanced experimental
candidates with antimicrobial therapeutic potential.
EXPERIMENTAL SECTION
■
Chemical Synthesis. General Procedures. Solvents and re-
agents were supplied from Aldrich, Acros, or Fisher and used without
further purification. NMR spectra were recorded on a Bruker AM-400
(400 MHz) spectrometer. High-resolution mass spectra were obtained
on an Agilent 6530 Accurate Mass Q-TOF LC/MS instrument. Reac-
tions in pressure tube were carried out using a Q-Tube reactor from Q
Labtech. Microwave reactions were conducted using a Biotage Initiator
reactor. All reactions were monitored by either TLC or HPLC.
Compounds were purified by flash chromatography on silica gel on a
Biotage Isolera One system. The purity of compounds was determined
by analytical HPLC (Shimadzu LC-20A series) using a Gemini, 3 μm,
C18, 110 Å column (50 mm × 4.6 mm, Phenomenex) and flow rate of
1 mL/min. Gradient conditions were the following: solvent A (0.1%
trifluoroacetic acid in water) and solvent B (acetonitrile), 0−2.0 min
100% A, 2.0−7.0 min 0−100% B (linear gradient), 7.0−8.0 min 100%
B, UV detection at 254 and 220 nm. All the tested compounds were
obtained with ≥95% purity by HPLC. NMR standards used are as
1
follows. H NMR: CDCl3, 7.26 ppm; CD3OD, 3.31 ppm; DMSO-d6,
2.50 ppm. 13C NMR: CDCl3, 77.16 ppm; CD3OD, 49.00 ppm;
DMSO-d6, 39.52 ppm. MOM-protected acetophenones were synthe-
sized as described in the literature.56,69,70
(S)-2-(2-Methylbutanoyl)benzene-1,3,5-triyl Tris(4-methylbenzene-
sulfonate) (1b). To a solution of 1a (150 mg, 0.715 mmol) in acetone
(11 mL) were added p-toluenesulfonic chloride (408 mg, 2.14 mmol)
and K2CO3 (845 mg, 6.15 mmol) successively. The resulting mixture
was stirred under reflux for 1 h, and then acetone was removed under
reduced pressure. The residue was diluted with water and DCM, and
the organic layer was washed with 1 M HCl aq, water, brine and dried
over Na2SO4. The solvent was removed under reduced pressure and
the crude material was adsorbed onto silica gel and subjected to silica
gel chromatography with hexane/EtOAc (first 85/15, then 75/25) to
give the product (350 mg, 0.52 mmol, 73%) as clear oil. 1H NMR (400
MHz, CDCl3): δ (ppm) 7.69 (d, J = 8.3 Hz, 2H), 7.62 (d, J = 8.4 Hz,
4H), 7.36 (d, J = 8.2 Hz, 2H), 7.30 (d, J = 8.3 Hz, 4H), 7.0 (s, 2H),
2.64−2.59 (m, 1H), 2.44 (s, 9H), 1.56−1.50 (m, 1H), 1.24−1.14 (m,
1H), 0.90 (d, J = 7.0 Hz, 3H), 0.76 (t, J = 7.4 Hz, 3H). 13C NMR (100
MHz, CDCl3): δ (ppm) 201.2, 149.8, 146.8, 146.5, 146.4, 131.7, 131.3,
130.2, 130.0, 128.5, 127.1, 114.3, 48.6, 24.5, 21.80, 21.79, 14.2, 11.2.
HRMS calculated for C32H32O10S3 (M + H)+ 673.1230, found (M + H)+
673.1229. HPLC purity: 98.6% (254 nm), tR = 8.17 min; 99.5% (220 nm),
tR = 8.17 min.
CONCLUSIONS
■
In summary, 58 olympicin A, 4-chromanone, and chalcone
derivatives containing various functionalities were synthesized
and evaluated against Mycobacterium tuberculosis and a panel
of clinically relevant Gram-positive and -negative bacterial
pathogens. Bacterial evaluation showed that this class of com-
pounds generally exhibited good activities against Gram-
positive bacteria tested. Systematic SAR study revealed that
the phenol hydroxy groups at the 5- and 7-position of the
4-chromanone scaffold were essential for antibacterial activities.
Additionally, the hydrogen bond donor/acceptor functionality
at the 4-position together with the lipophilic 2-alkyl moiety
in the scaffold also played important roles in antibacterial
activities. The flavanone derivatives bearing the lipophilic sub-
stituent on the 2-phenyl ring showed good antibacterial proper-
ties as well. In the chalcone chemical series, both hydroxy
groups at 2′- and 4′-position, as well as the bicyclic myrtenyl
motif and the 2-alkyloxy substitution on the aromatic ring,
favored anti-Gram-positive bacterial activities. The selected
compounds generally possessed favorable solubility, and 5f, 8a,
and 8j had more desirable selectivity indices ranging from 10.5
to 54.4. In addition, compounds 2a, 5j, 3g, and 8d were found
to disrupt bacterial membrane potential and have secondary
inhibitory effect on macromolecular biosynthesis of DNA,
RNA, and protein. Further evaluation of selected compounds
against bacterial topoisomerases and DNA gyrase revealed that
2a and 2e inhibited topoisomerase IV (IC50 = 30−60 μM).
Taken together, the antibacterial agents identified from this
study provide chemically modified flavonoid phytochemicals as
promising antibacterial leads for further medicinal chemistry
1-(2-Hydroxy-4,6-bis(methoxymethoxy)phenyl)-2-methyl-
butan-1-one (1c). To a suspension of 1a′ (1.12 g, 5.33 mmol) in
DCM (11 mL) at 0 °C was added DIPEA (2.78 mL, 15.9 mmol)
carefully. After stirring for 10 min, MOMCl (1.21 mL, 15.9 mmol) was
added to the solution dropwise. The resulting mixture was stirred for
1 h. Afterward, the solution was poured into sat. NH4Cl aq, and then
water and DCM were added into the mixture. The organic layer was
washed with water, brine and dried over Na2SO4. The solvent was
removed under reduced pressure and the crude material was adsorbed
onto silica gel and subjected to silica gel chromatography with hexane/
EtOAc (90/10) to give the product (1.02 g, 3.42 mmol, 64%) as pale
1
yellow solid. H NMR (400 MHz, CDCl3): δ (ppm) 13.74 (s, 1H),
6.27 (s, 2H), 5.24 (s, 2H), 5.16 (br s, 2H), 3.65−3.60 (m, 1H), 3.51
(s, 3H), 3.46 (s, 3H), 1.87−1.80 (m, 1H), 1.43−1.36 (m, 1H), 1.16 (d,
J = 6.8 Hz, 3H), 0.91 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz,
CDCl3): δ (ppm) 210.3, 167.2, 163.2, 160.1, 106.7, 97.6, 94.9, 94.4,
94.1, 56.9, 56.6, 46.6, 27.1, 16.7, 12.2. HRMS calculated for C15H22O6
(M + H)+ 299.1489, found (M + H)+ 299.1483. HPLC purity: 99.8%
(254 nm), tR = 7.42 min; 99.8% (220 nm), tR = 7.42 min.
(S)-1-(2-Hydroxy-4,6-bis(methoxymethoxy)phenyl)-2-meth-
ylbutan-1-one (1c′). Synthesis and purification were performed as
described in compound 1c. Product: 1.03 g, 3.46 mmol, 65%. 1H
NMR (400 MHz, CDCl3): δ (ppm) 13.74 (s, 1H), 6.27 (s, 2H), 5.24
(s, 2H), 5.16 (br s, 2H), 3.65−3.60 (m, 1H), 3.52 (s, 3H), 3.46 (s,
3H), 1.87−1.80 (m, 1H), 1.43−1.36 (m, 1H), 1.16 (d, J = 6.8 Hz,
M
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3H), 0.91 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm)
210.3, 167.2, 163.2, 160.1, 106.7, 97.6, 94.9, 94.4, 94.1, 56.8, 56.6, 46.6,
27.1, 16.7, 12.1. HRMS calculated for C15H22O6 (M + H)+ 299.1489,
found (M + H)+ 299.1475. HPLC purity: 99.9% (254 nm), tR = 7.38 min;
100% (220 nm), tR = 7.38 min.
163.1, 162.3, 132.1, 119.2, 105.7, 96.9, 92.3, 70.0, 46.1, 27.0, 16.7, 11.9.
HRMS calculated for C14H18O4 (M − H)− 249.1132, found (M − H)−
249.1102. HPLC purity: 99.7% (254 nm), tR = 7.16 min; 99.9% (220 nm),
tR = 7.16 min.
1-(2-(Hexyloxy)-4,6-dihydroxyphenyl)-2-methylbutan-1-one (2d).
Purified with reverse phase C18 silica gel chromatography with H2O/
MeCN (45/55) to give the product (130 mg, 0.44 mmol, 66% over
General Procedure for the Synthesis of 2a,b. To a solution
of 1b (410 mg, 0.61 mmol) in DMF (4 mL) at 0 °C was added NaH
(49 mg, 1.21 mmol) carefully. After stirring for 30 min, geranyl
bromide (198 mg, 0.91 mmol) in DMF (1 mL) was added dropwise.
The resulting mixture was stirred at room temperature for 1 h.
Afterward, the reaction was quenched with water and extracted with
EtOAc twice. The organic layer was washed with water, brine and
dried over Na2SO4. The solvent was removed under reduced pressure,
and the residue was dissolved in MeOH (10 mL), followed by the
careful addition of sodium methoxide (659 mg, 12.2 mmol). The
resulting mixture was stirred under reflux for 8 h, and then MeOH was
removed. The residue was diluted with 1 M HCl aq and extracted with
EtOAc twice. The organic layer was washed with water, brine and
dried over Na2SO4. The solvent was removed under reduced pressure
and the crude material was adsorbed onto silica gel and subjected to
silica gel chromatography with hexane/EtOAc to give the product.
(S,E)-1-(2-((3,7-Dimethylocta-2,6-dien-1-yl)oxy)-4,6-dihydroxy-
phenyl)-2-methylbutan-1-one (Olympicin A, 2a). Purified with
hexane/EtOAc (90/10) to give the product (116 mg, 0.34 mmol,
1
two steps) as pale solid. H NMR (400 MHz, CDCl3): δ (ppm) 6.01
(s, 1H), 5.92 (s, 1H), 3.96 (t, J = 6.5 Hz, 2H), 3.74−3.69 (m, 1H),
1.85−1.75 (m, 3H), 1.46−1.33 (m, 7H), 1.14 (d, J = 6.8 Hz, 3H),
0.92−0.87 (m, 6H). 13C NMR (100 MHz, CDCl3): δ (ppm) 210.8,
167.3, 163.2, 163.0, 105.6, 96.6, 91.9, 69.2, 46.0, 31.6, 29.1, 26.8, 26.1,
22.7, 17.0, 14.1, 11.9. HRMS calculated for C17H26O4 (M − H)−
293.1758, found (M − H)− 293.1713. HPLC purity: 99.2% (254 nm),
tR = 7.93 min; 99.9% (220 nm), tR = 7.92 min.
1-(2,4-Dihydroxy-6-(octyloxy)phenyl)-2-methylbutan-1-one (2e).
Purified with reverse phase C18 silica gel chromatography with H2O/
MeCN (40/60) to give the product (151 mg, 0.47 mmol, 70% over
two steps) as pale yellow solid. 1H NMR (400 MHz, CDCl3): δ (ppm)
7.33 (br, 1H), 6.01 (d, J = 1.5 Hz, 1H), 5.91 (d, J = 1.5 Hz, 1H), 3.97
(t, J = 6.5 Hz, 2H), 3.74−3.69 (m, 1H), 1.85−1.77 (m, 3H), 1.46−
1.28 (m, 11H), 1.14 (d, J = 6.8 Hz, 3H), 0.90−0.87 (m, 6H). 13C
NMR (100 MHz, CDCl3): δ (ppm) 210.7, 167.3, 163.2, 163.0, 105.6,
96.6, 91.9, 69.2, 46.1, 31.9, 29.4, 29.3, 29.1, 26.8, 26.4, 22.8, 17.0, 14.2,
11.9. HRMS calculated for C19H30O4 (M + H)+ 323.2217, found (M +
H)+ 323.2213. HPLC purity: 98.8% (254 nm), tR = 8.31 min; 99.9%
(220 nm), tR = 8.31 min.
1
55%, two-step overall yield) as pale yellow oil. H NMR (400 MHz,
CDCl3): δ (ppm) 5.98 (d, J = 2.2 Hz, 1H), 5.91 (d, J = 2.2 Hz, 1H),
5.54 (br, 1H), 5.52−5.49 (m, 1H), 5.11−5.09 (m, 1H), 4.56 (d, J = 6.6
Hz, 2H), 3.67−3.63 (m, 1H), 2.14−2.08 (m, 4H), 1.81−1.78 (m, 1H),
1.74 (s, 3H), 1.68 (s, 3H), 1.61 (s, 3H), 1.40−1.33 (m, 1H), 1.12 (d,
J = 6.7 Hz, 3H), 0.89 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz,
CDCl3): δ (ppm) 210.8, 167.3, 163.1, 162.8, 142.6, 132.1, 123.7,
118.3, 105.8, 96.6, 92.1, 65.8, 46.1, 39.6, 27.0, 26.4, 25.8, 17.8, 16.73,
16.67, 12.0. HRMS calculated for C21H30O4 (M + H)+ 347.2217,
found (M + H)+ 347.2210. HPLC purity: 99.7% (254 nm), tR = 8.18
min; 99.6% (220 nm), tR = 8.18 min.
(S)-1-(2-(Allyloxy)-4,6-dihydroxyphenyl)-2-methylbutan-1-one
(2f). Purified with hexane/EtOAc (85/15) to give the product (152 mg,
1
0.61 mmol, 90% over two steps) as white solid. H NMR (400 MHz,
CDCl3): δ (ppm) 6.11−6.02 (m, 1H), 6.00 (d, J = 2.1 Hz, 1H), 5.91 (d,
J = 2.1 Hz, 1H), 5.86 (br, 1H), 5.42 (d, J = 17.2 Hz, 1H), 5.35 (d, J =
10.4 Hz, 1H), 4.57 (d, J = 5.6 Hz, 2H), 3.69−3.64 (m, 1H), 1.84−1.79
(m, 1H), 1.42−1.35 (m, 1H), 1.13 (d, J = 6.7 Hz, 3H), 0.89 (t, J = 7.4
Hz, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm) 210.8, 167.3, 163.0,
162.3, 132.1, 119.3, 105.7, 96.9, 92.3, 70.1, 46.2, 27.0, 16.7, 11.9. HRMS
calculated for C14H18O4 (M − H)− 249.1132, found (M − H)−
249.1104. HPLC purity: 99.1% (254 nm), tR = 7.17 min; 99.6%
(220 nm), tR = 7.17 min.
(E)-1-(2-((3,7-Dimethylocta-2,6-dien-1-yl)oxy)-4,6-dihydroxy-
phenyl)-2-methylbutan-1-one (2b). Purified with hexane/EtOAc
(90/10) to give the product (110 mg, 0.32 mmol, 52% over two
steps) as pale yellow oil. 1H NMR (400 MHz, CDCl3): δ (ppm) 5.98
(d, J = 2.2 Hz, 1H), 5.91 (d, J = 2.2 Hz, 1H), 5.54 (br, 1H), 5.52−5.49
(m, 1H), 5.11−5.09 (m, 1H), 4.56 (d, J = 6.6 Hz, 2H), 3.67−3.63 (m,
1H), 2.14−2.08 (m, 4H), 1.81−1.78 (m, 1H), 1.74 (s, 3H), 1.68 (s,
3H), 1.61 (s, 3H), 1.40−1.33 (m, 1H), 1.12 (d, J = 6.7 Hz, 3H), 0.89
(t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm) 210.8,
167.3, 163.1, 162.8, 142.6, 132.1, 123.7, 118.3, 105.8, 96.6, 92.1, 65.8,
46.1, 39.6, 27.0, 26.4, 25.8, 17.8, 16.74, 16.68, 12.0. HRMS calculated
for C21H30O4 (M + H)+ 347.2217, found (M + H)+ 347.2214. HPLC
purity: 99.7% (254 nm), tR = 8.17 min; 99.6% (220 nm), tR = 8.17 min.
General Procedure for the Synthesis of 2c−f. To a solution
of 1c (200 mg, 0.67 mmol) in DMF (4 mL) at 0 °C was added NaH
(54 mg, 1.34 mmol) carefully. After stirring for 30 min, appropriate
alkyl bromide (1 mmol) in DMF (1 mL) was added dropwise. The
resulting mixture was stirred at room temperature until the complete
consumption of 1c (1−7 h). Afterward, the reaction was quenched
with water and extracted with EtOAc twice. The organic layer was
washed with water, brine and dried over Na2SO4. The solvent was
removed under reduced pressure, and the residue was dissolved in
MeOH (10 mL), followed by the addition of concentrated HCl (510 μL,
6 mmol) carefully. The resulting mixture was stirred at room temperature
overnight, and the solvent was removed under reduced pressure. The
crude material was adsorbed onto silica gel and subjected to silica gel
chromatography with hexane/EtOAc to give the product.
General Procedure for the Synthesis of 3a−e. To a solution of
1d (152 mg, 1 mmol) in ethanol (2.5 mL) were added pyrrolidine
(220 mg, 3.1 mmol) and corresponding aldehyde (6 mmol)
successively. The resulting mixture was stirred at 150 °C in a pressure
tube for 1 h. Afterward, the solution was diluted with ethyl acetate and
the organic layer was washed with 10% HCl, water, brine and dried
over Na2SO4. The solvent was removed under reduced pressure and
the crude material was adsorbed onto silica gel and subjected to silica
gel chromatography with hexane/EtOAc to give the product.
7-Hydroxy-2-propylchroman-4-one (3a). Purified with hexane/
EtOAc (87/13) to give the product (85 mg, 0.41 mmol, 41%) as light
1
brown solid. H NMR (400 MHz, CDCl3): δ (ppm) 8.13 (br, 1H),
7.79 (d, J = 8.6 Hz, 1H), 6.55 (d, J = 8.4 Hz, 1H), 6.43 (s, 1H), 4.42
(br, 1H), 2.67−2.63 (m, 2H), 1.85−1.80 (m, 1H), 1.68−1.44 (m, 3H),
0.99−0.95 (m, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm) 193.0,
164.32, 164.29, 129.5, 114.6, 110.8, 103.4, 78.0, 42.6, 37.1, 18.3, 14.0.
HRMS calculated for C12H14O3 (M − H)− 205.0870, found (M − H)−
205.0863. HPLC purity: 97.3% (254 nm), tR = 6.31 min; 98.6%
(220 nm), tR = 6.31 min.
2-Hexyl-7-hydroxychroman-4-one (3b). Purified with hexane/
EtOAc (90/10) to give the product (92 mg, 0.37 mmol, 37%) as
light brown solid. 1H NMR (400 MHz, CDCl3): δ (ppm) 7.78 (d, J =
8.7 Hz, 1H), 6.55 (dd, J = 8.6, 1.7 Hz, 1H), 6.44 (d, J = 1.7 Hz, 1H),
4.43−4.39 (m, 1H), 2.66−2.63 (m, 2H), 1.87−1.82 (m, 1H), 1.67−
1.64 (m, 1H), 1.53−1.30 (m, 8H), 0.90−0.87 (m, 3H). 13C NMR
(100 MHz, CDCl3): δ (ppm) 193.1, 164.34, 164.30, 129.4, 114.6,
110.8, 103.4, 78.3, 42.6, 35.0, 31.8, 29.2, 25.0, 22.7, 14.2. HRMS
calculated for C15H20O3 (M − H)− 247.1340, found (M − H)−
247.1331. HPLC purity: 98.4% (254 nm), tR = 7.06 min; 99.1%
(220 nm), tR = 7.06 min.
1-(2-(Allyloxy)-4,6-dihydroxyphenyl)-2-methylbutan-1-one (2c).
Purified with hexane/EtOAc (85/15) to give the product (157 mg,
0.63 mmol, 93% over two steps) as pale yellow solid. 1H NMR (400 MHz,
CDCl3): δ (ppm) 6.11−6.02 (m, 1H), 6.00 (d, J = 2.1 Hz, 1H), 5.91
(d, J = 2.1 Hz, 1H), 5.86 (br, 1H), 5.42 (d, J = 17.2 Hz, 1H), 5.35 (d,
J = 10.4 Hz, 1H), 4.57 (d, J = 5.6 Hz, 2H), 3.69−3.64 (m, 1H), 1.84−
1.79 (m, 1H), 1.42−1.35 (m, 1H), 1.13 (d, J = 6.7 Hz, 3H), 0.89 (t, J =
7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm) 210.9, 167.2,
N
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Journal of Medicinal Chemistry
Article
2-Heptyl-7-hydroxychroman-4-one (3c). Purified with hexane/
119.4, 119.3, 111.1, 76.6, 76.1, 43.7, 43.4, 42.30, 42.26, 37.5, 36.9, 28.9,
28.6, 25.8, 25.5, 25.4, 20.0, 19.4, 17.8. HRMS calculated for C18H24O3
(M − H)− 287.1653, found (M − H)− 287.1613. HPLC purity: 99.8%
(254 nm), tR = 7.40 min; 99.7% (220 nm), tR = 7.38 min.
EtOAc (90/10) to give the product (91 mg, 0.35 mmol, 35%) as light
1
brown solid. H NMR (400 MHz, CDCl3): δ (ppm) 7.78 (d, J = 8.7
Hz, 1H), 6.55 (dd, J = 8.7, 2.3 Hz, 1H), 6.43 (d, J = 2.2 Hz, 1H),
4.43−4.39 (m, 1H), 2.69−2.60 (m, 2H), 1.88−1.80 (m, 1H), 1.70−1.63
(m, 1H), 1.53−1.25 (m, 10H), 0.89−0.86 (m, 3H). 13C NMR (100
MHz, CDCl3): δ (ppm) 193.0, 164.4, 164.3, 129.4, 114.6, 110.9, 103.4,
78.3, 42.6, 35.0, 31.9, 29.5, 29.3, 25.0, 22.8, 14.2. HRMS calculated for
C16H22O3 (M − H)− 261.1496, found (M − H)− 261.1479. HPLC
purity: 95.0% (254 nm), tR = 7.28 min; 96.1% (220 nm), tR = 7.28 min.
7-Hydroxy-2-nonylchroman-4-one (3d). Purified with hexane/
EtOAc (90/10) to give the product (98 mg, 0.33 mmol, 33%) as light
2-(2,6-Dimethylhept-5-en-1-yl)-5-hydroxychroman-4-one (3l).
Purified by reverse phase C18 silica gel chromatography with
H2O/MeCN (30/70) to obtain a mixture of two diastereomers
(170 mg, 0.59 mmol, 59% over two steps) as pale yellow oil. 1H NMR
(400 MHz, CDCl3): δ (ppm) 11.69 (s, 2H), 7.33 (t, J = 8.3 Hz, 2H),
6.48 (dd, J = 8.3, 0.8 Hz, 2H), 6.41 (ddd, J = 8.3, 2.2, 0.8 Hz, 2H),
5.11−5.07 (m, 2H), 4.52−4.48 (m, 2H), 2.77−2.62 (m, 4H), 2.07−
1.63 (m, 8H), 1.68 (s, 6H), 1.61 (s, 6H), 1.47−1.18 (m, 6H), 0.98−
0.95 (m, 6H). 13C NMR (100 MHz, CDCl3): δ (ppm) 198.8, 198.7,
162.2, 161.84, 161.79, 138.24, 138.23, 131.7, 131.6, 124.54, 124.48,
109.2, 108.4, 107.48, 107.47, 76.2, 75.6, 43.0, 42.7, 42.21, 42.15, 37.5,
36.8, 28.9, 28.6, 25.9, 25.5, 25.4, 20.0, 19.4, 17.8. HRMS calculated for
C18H24O3 (M − H)− 287.1653, found (M − H)− 287.1604. HPLC
purity: 99.9% (254 nm), tR = 8.00 min; 100% (220 nm), tR = 7.99 min.
(E)-3-(6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)-1-(2-hydroxy-
4-(methoxymethoxy)phenyl)prop-2-en-1-one (3h). To a solution
of 1e (196 mg, 1 mmol) in ethanol (2.5 mL) were added pyrrolidine
(149 mg, 2.1 mmol) and (−)-myrtenal (300 mg, 2 mmol) successively.
The resulting mixture was stirred at 75 °C in a pressure tube for 1 h.
Afterward, the solution was diluted with ethyl acetate, and the organic
layer was washed with 10% HCl, water, brine and dried over Na2SO4.
The solvent was removed under reduced pressure and the crude
material was adsorbed onto silica gel and subjected to silica gel
chromatography with hexane/EtOAc (97/3) to give compound 3h
(160 mg, 0.48 mmol, 48%) as yellow oil. 1H NMR (400 MHz,
CDCl3): δ (ppm) 13.37 (s, 1H), 7.75 (d, J = 9.0 Hz, 1H), 7.52 (d, J =
15.1 Hz, 1H), 6.88 (d, J = 15.1 Hz, 1H), 6.61 (d, J = 2.4 Hz, 1H), 6.54
(dd, J = 8.9, 2.4 Hz, 1H), 6.21 (m, 1H), 5.20 (s, 2H), 3.47 (s, 3H),
2.68−2.67 (m, 1H), 2.55−2.41 (m, 3H), 2.19−2.18 (m, 1H), 1.39 (s,
3H), 1.18 (d, J = 9.0 Hz, 1H), 0.80 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ (ppm) 192.9, 166.2, 163.5, 146.5, 145.6, 137.3, 131.3,
116.7, 115.2, 108.1, 104.0, 94.2, 56.5, 41.6, 40.8, 38.1, 33.1, 31.3, 26.3,
21.0. HRMS calculated for C20H24O4 (M + H)+ 329.1747, found
(M + H)+ 329.1735. HPLC purity: 98.8% (254 nm), tR = 7.98 min;
98.5% (220 nm), tR = 7.98 min.
1
brown solid. H NMR (400 MHz, CDCl3): δ (ppm) 8.23 (br, 1H),
7.79 (d, J = 8.7 Hz, 1H), 6.56 (dd, J = 8.7, 2.1 Hz, 1H), 6.43 (d, J = 2.1
Hz, 1H), 4.45−4.38 (m, 1H), 2.70−2.61 (m, 2H), 1.88−1.80 (m, 1H),
1.70−1.63 (m, 1H), 1.55−1.25 (m, 14H), 0.89−0.86 (m, 3H). 13C
NMR (100 MHz, CDCl3): δ (ppm) 193.1, 164.4, 129.5, 114.6, 110.9,
103.4, 78.3, 42.5, 35.0, 32.0, 29.7, 29.6, 29.5, 29.4, 25.0, 22.8, 14.2.
HRMS calculated for C18H26O3 (M − H)− 289.1809, found (M − H)−
289.1787. HPLC purity: 99.1% (254 nm), tR = 7.71 min; 99.3% (220 nm),
tR = 7.71 min.
7-Hydroxy-2-phenethylchroman-4-one (3e). Purified with reverse
phase C18 silica gel chromatography with H2O/MeCN (45/55)
to give the product (54 mg, 0.20 mmol, 20%) as white solid. 1H NMR
(400 MHz, CDCl3): δ (ppm) 7.79 (d, J = 8.6 Hz, 1H), 7.27−7.25 (m,
2H), 7.20−7.16 (m, 3H), 6.53 (dd, J = 8.7, 1.8 Hz, 1H), 6.45 (d, J =
1.7 Hz, 1H), 4.43−4.37 (m, 1H), 2.91−2.76 (m, 2H), 2.71−2.59 (m,
2H), 2.22−2.14 (m, 1H), 2.01−1.94 (m, 1H). 13C NMR (100 MHz,
CDCl3): δ (ppm) 192.7, 164.3, 164.2, 141.0, 129.5, 128.7, 128.6,
126.3, 114.6, 111.0, 103.4, 77.1, 42.6, 36.6, 31.2. HRMS calculated
for C17H16O3 (M − H)− 267.1027, found (M − H)− 267.1012.
HPLC purity: 100% (254 nm), tR = 6.69 min; 99.5% (220 nm), tR
=
6.69 min.
General Procedure for the Synthesis of 3f, 3k, and 3l. To a
solution of corresponding MOM-protected acetophenone (196 mg,
1 mmol) in ethanol (2.5 mL) was added DEA (154 mg, 2.1 mmol)
and ( )-citronellal (309 mg, 2 mmol) successively. The resulting
mixture was stirred at 150 °C in a pressure tube for 1 h. The resulting
solution was cooled to room temperature and diluted with EtOAc.
The organic layer was washed with 10% HCl aq, water, brine and dried
over Na2SO4. The solvent was removed under reduced pressure and
the residue was dissolved in methanol (14 mL), followed by the careful
addition of concentrated HCl (340 μL, 4 mmol), and the resulting
mixture was stirred at room temperature overnight. Afterward, the
solution was concentrated and the crude material was adsorbed onto
silica gel and subjected to silica gel chromatography with hexane/
EtOAc to give the product.
(E)-1-(2,4-Dihydroxyphenyl)-3-(6,6-dimethylbicyclo[3.1.1]-
hept-2-en-2-yl)prop-2-en-1-one (3g). To a solution of 3h (130 mg,
0.396 mmol) in methanol (6 mL) was added concentrated HCl
(154 μL, 1.78 mmol) dropwise. The resulting mixture was stirred at
room temperature for 24 h, after which the solvent was removed. The
crude material was adsorbed onto silica gel and subjected to silica gel
chromatography with hexane/EtOAc (90/10) to obtain compound 3g
1
(100 mg, 0.35 mmol, 89%) as yellow solid. H NMR (400 MHz,
2-(2,6-Dimethylhept-5-en-1-yl)-7-hydroxychroman-4-one (3f).
Purified with hexane/EtOAc (90/10) to obtain a mixture of two
diastereomers (167 mg, 0.58 mmol, 58% over two steps) as white
solid. 1H NMR (400 MHz, CDCl3): δ (ppm) 7.79 (d, J = 8.6 Hz, 2H),
6.55 (d, J = 8.7 Hz, 2H), 6.43 (s, 2H), 5.98 (br, 2H), 5.11−5.08 (m,
2H), 4.55−4.50 (m, 2H), 2.69−2.56 (m, 4H), 2.04−1.90 (m, 6H),
1.82−1.69 (m, 2H), 1.68 (s, 6H), 1.60 (s, 6H), 1.44−1.32 (m, 4H),
1.26−1.19 (m, 2H), 0.97−0.94 (m, 6H). 13C NMR (100 MHz,
CDCl3): δ (ppm) 192.95, 192.86, 164.4, 164.32, 164.28, 131.7, 131.6,
129.4, 124.6, 124.5, 114.6, 110.9, 103.4, 76.2, 43.2, 42.9, 42.3, 42.2,
37.5, 36.9, 28.9, 28.5, 25.9, 25.5, 25.4, 20.0, 19.4, 17.8. HRMS
calculated for C18H24O3 (M − H)− 287.1653, found (M − H)−
287.1632. HPLC purity: 97.8% (254 nm), tR = 7.32 min; 98.0% (220
nm), tR = 7.32 min.
2-(2,6-Dimethylhept-5-en-1-yl)-6-hydroxychroman-4-one (3k).
Purified with hexane/EtOAc (92/8) to obtain a mixture of two
diastereomers (216 mg, 0.75 mmol, 75% over two steps) as yellow oil.
1H NMR (400 MHz, CDCl3): δ (ppm) 7.40 (d, J = 3.1 Hz, 2H), 7.07
(dd, J = 8.9, 3.1 Hz, 2H), 6.91 (br, 2H), 6.87 (dd, J = 8.9, 2.6 Hz, 2H),
5.11−5.08 (m, 2H), 4.51−4.45 (m, 2H), 2.71−2.58 (m, 4H), 2.04−
1.72 (m, 8H), 1.68 (s, 6H), 1.60 (s, 6H), 1.45−1.17 (m, 6H), 0.97−
0.94 (m, 6H). 13C NMR (100 MHz, CDCl3): δ (ppm) 194.2, 194.1,
156.44, 156.38, 150.5, 131.61, 131.56, 125.3, 124.61, 124.56, 121.0,
CDCl3): δ (ppm) 13.56 (s, 1H), 7.72 (d, J = 9.5 Hz, 1H), 7.51 (d, J =
15.1 Hz, 1H), 6.87 (d, J = 15.2 Hz, 1H), 6.43−6.41 (m, 2H), 6.20 (m,
1H), 2.67 (t, J = 5.4 Hz, 1H), 2.54−2.41 (m, 3H), 2.18 (br, 1H), 1.38
(s, 3H), 1.18 (d, J = 9.0 Hz, 1H), 0.79 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ (ppm) 193.0, 166.2, 163.1, 146.5, 145.8, 137.5, 132.1,
116.6, 114.5, 108.0, 103.8, 41.5, 40.8, 38.1, 33.1, 31.3, 26.3, 21.0.
HRMS calculated for C18H20O3 (M − H)− 283.1340, found (M − H)−
283.1313. HPLC purity: 99.3% (254 nm), tR = 7.48 min; 99.2%
(220 nm), tR = 7.48 min.
2-(6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)-7-(methoxymethoxy)-
chroman-4-one (3i). To a solution of 3h (60 mg, 0.183 mmol) in
ethanol (3 mL) was added sodium acetate (300 mg, 3.6 mmol). The
resulting mixture was refluxed for 24 h, after which the solvent was
removed. The residue was dissolved in ethyl acetate, and the organic
layer was washed with water, brine and dried over Na2SO4. The
solvent was removed under reduced pressure and the crude material
was adsorbed onto silica gel and subjected to silica gel chromatography
with hexane/EtOAc (95/5) to obtain a mixture of two diastereomers
3i (28 mg, 0.085 mmol, 47%) as clear oil. 1H NMR (400 MHz,
CDCl3): δ (ppm) 7.82−7.79 (m, 2H), 6.66−6.62 (m, 2H), 6.61 (s,
2H), 5.67−5.64 (m, 2H), 5.19 (s, 2H), 5.18 (s, 2H), 4.88−4.77 (m,
2H), 3.47 (s, 3H), 3.46 (s, 3H), 2.82−2.72 (m, 2H), 2.66−2.53 (m,
2H), 2.51−2.38 (m, 3H), 2.34−2.23 (m, 5H), 2.12 (br, 2H), 1.32−1.30
O
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(m, 6H), 1.28−1.14 (m, 2H), 0.87 (s, 3H), 0.74 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ (ppm) 191.6, 191.5, 163.59, 163.58, 163.50,
163.48, 145.4, 145.3, 128.72, 128.67, 122.1, 120.7, 115.9, 115.8, 111.0,
103.63, 103.58, 94.15, 94.14, 80.3, 79.8, 77.4, 56.5, 56.4, 42.6, 42.2, 41.0,
40.9, 40.8, 38.2, 38.0, 31.8, 31.7, 31.38, 31.36, 26.24. 26.20, 21.40, 21.35.
HRMS calculated for C20H24O4 (M + H)+ 329.1747, found (M + H)+
329.1736. HPLC purity: 99.6% (49.0% + 50.6%) (254 nm), tR = 7.66 and
7.68 min; 98.8% (220 nm), tR = 7.66 min. The split HPLC product
peaks further supported the product is a mixture of two diastereomers
(see Supporting Information for the details of HPLC trace).
9H), 0.90−0.87 (m, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm)
156.6, 155.9, 128.3, 118.5, 108.5, 103.1, 75.5, 65.6, 38.2, 35.6, 32.0,
29.7, 29.4, 25.2, 22.8, 14.2. HRMS calculated for C16H24O3 (M − H)−
263.1653, found (M − H)− 263.1632. HPLC purity: 95.7% (220 nm),
tR = 6.97 min.
2-Nonylchroman-4,7-diol (4d). Purified with hexane/EtOAc
(85/15) to give the product (52 mg, 0.18 mmol, 81%) as light
brown oil. 1H NMR (400 MHz, CDCl3): δ (ppm) 7.19 (d, J = 8.4 Hz,
1H), 6.54 (br, 1H), 6.34 (dd, J = 8.4, 2.4 Hz, 1H), 6.25 (d, J = 2.4 Hz,
1H), 4.83−4.81 (m, 1H), 4.02−3.99 (m, 1H), 2.61−2.59 (m, 1H),
2.32 (br, 1H), 2.25−2.20 (m, 1H), 1.75−1.53 (m, 2H), 1.49−1.27 (m,
14H), 0.90−0.87 (m, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm)
156.5, 155.8, 128.3, 118.3, 108.6, 103.1, 75.5, 65.6, 38.1, 35.5, 32.0,
29.72, 29.70, 29.5, 25.2, 22.8, 14.2. HRMS calculated for C18H28O3
(M − H)− 291.1966, found (M − H)− 291.1926. HPLC purity: 97.7%
(220 nm), tR = 7.41 min.
General Procedure for the Synthesis of 5a−l. To a solution of
1h (256 mg, 1 mmol) in ethanol (2.5 mL) were added DEA (154 mg,
2.1 mmol) and corresponding aldehyde (2 mmol) successively. The
resulting mixture was stirred at 150 °C in a pressure tube for 1 h.
Afterward, the solution was diluted with ethyl acetate and the organic
layer was washed with 10% HCl, water, brine and dried over Na2SO4.
The solvent was removed under reduced pressure and the crude
material was adsorbed onto silica gel and subjected to silica gel
chromatography with hexane/EtOAc to give the MOM-protected
product.
To a solution of appropriate MOM-protected compound (0.22 mmol)
in methanol (4 mL) was added concentrated HCl (172 μL, 1.98 mmol)
at room temperature, and the resulting mixture was stirred overnight. The
solvent was removed under reduced pressure and the crude material was
adsorbed onto silica gel and subjected to silica gel chromatography with
hexane/EtOAc to give the corresponding deprotected product.
5,7-Bis(methoxymethoxy)-2-propylchroman-4-one (5a). Purified
with hexane/EtOAc (87/13) to give the product (195 mg, 0.63 mmol,
63%) as clear oil. 1H NMR (400 MHz, CDCl3): δ (ppm) 6.37 (s, 1H),
6.29 (s, 1H), 5.24 (s, 2H), 5.16 (s, 2H), 4.39−4.35 (m, 1H), 3.51 (s,
3H), 3.46 (s, 3H), 2.64−2.53 (m, 2H), 1.83−1.79 (m, 1H), 1.66−1.43
(m, 3H), 0.96 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ
(ppm) 190.1, 164.8, 163.2, 159.5, 107.5, 97.8, 97.2, 95.1, 94.1, 77.3,
56.64, 56.57, 44.3, 37.0, 18.3, 14.0. HRMS calculated for C16H22O6
(M + H)+ 311.1489, found (M + H)+ 311.1481. HPLC purity: 98.0%
(254 nm), tR = 6.78 min; 96.3% (220 nm), tR = 6.78 min.
2-(6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)-7-hydroxy-
chroman-4-one (3j). To a solution of 3i (38 mg, 0.116 mmol) in
methanol (2 mL) was added concentrated HCl (45 μL, 0.52 mmol)
carefully. The resulting mixture was stirred at room temperature for
24 h, after which the solvent was removed. The crude material was
adsorbed onto silica gel and subjected to silica gel chromatography
with hexane/EtOAc (85/15) to obtain a mixture of two diastereomers
1
3j (30 mg, 0.106 mmol, 90%) as white solid. H NMR (400 MHz,
CDCl3): δ (ppm) 7.79−7.76 (m, 2H), 7.67 (br, 2H), 6.54−6.51 (m,
2H), 6.43−6.42 (m, 2H), 5.65−5.64 (m, 2H), 4.87−4.77 (m, 2H),
2.84−2.75 (m, 2H), 2.67−2.54 (m, 2H), 2.49−2.22 (m, 8H), 2.11 (br,
2H), 2.00 (br, 2H), 1.31−1.29 (m, 6H), 1.26−1.12 (m, 2H), 0.85 (s,
3H), 0.73 (s, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm) 192.39,
192.36, 164.0, 163.7, 145.3, 145.2, 129.4, 129.3, 122.2, 120.9, 114.89,
114.86, 110.62, 110.61, 103.5, 80.1, 79.6, 77.4, 77.1, 42.6, 42.2, 40.9,
40.83, 40.79, 40.7, 38.2, 38.0, 31.8, 31.7, 31.39, 31.36, 26.24, 26.20,
21.38, 21.36. HRMS calculated for C18H20O3 (M − H)− 283.1340,
found (M − H)− 283.1310. HPLC purity: 99.4% (49.7% + 49.7%)
(254 nm), tR = 7.09 and 7.11 min; 99.6% (50.6% + 49.0%) (220 nm),
tR = 7.09 and 7.11 min. The split HPLC product peaks further supported
the product is a mixture of two diastereomers (see Supporting
Information for the details of HPLC trace).
General Procedure for the Synthesis of 4a−d. To a solution of
appropriate 4-chromanone 3a−d (0.22 mmol) in methanol (3 mL)
was added sodium borohydride (16.6 mg, 0.44 mmol) every 1.5 h for a
total of 9 h. The resulting mixture was allowed to stir at room tem-
perature overnight. The mixture was cooled to 0 °C, quenched with
saturated NH4Cl aq carefully, and extracted with EtOAc twice. The
combined organic layer was washed with water, brine and dried over
Na2SO4. The solvent was removed under reduced pressure and the
crude material was adsorbed onto silica gel and subjected to silica gel
chromatography with hexane/EtOAc to give the product.
2-Propylchroman-4,7-diol (4a). Purified with hexane/EtOAc
(85/15) to give the product (29 mg, 0.14 mmol, 64%) as light
brown oil. 1H NMR (400 MHz, CDCl3): δ (ppm) 7.19 (d, J = 8.4 Hz,
1H), 6.64 (br, 1H), 6.34 (dd, J = 8.4, 2.4 Hz, 1H), 6.24 (d, J = 2.4 Hz,
1H), 4.83−4.79 (m, 1H), 4.04−4.98 (m, 1H), 2.68 (br, 1H), 2.33 (br,
1H), 2.23−2.17 (m, 1H), 1.74−1.62 (m, 2H), 1.59−1.39 (m, 3H),
0.94 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm)
156.6, 155.8, 128.3, 118.3, 108.6, 103.1, 75.2, 65.5, 38.0, 37.6, 18.4,
14.1. HRMS calculated for C12H16O3 (M − H2O + H)+ 191.1067,
found (M − H2O + H)+ 191.1058. HPLC purity: 95.3% (220 nm),
tR = 5.91 min.
2-Hexylchroman-4,7-diol (4b). Purified with hexane/EtOAc
(85/15) to give the product (42 mg, 0.17 mmol, 77%) as light
brown oil. 1H NMR (400 MHz, CDCl3): δ (ppm) 7.18 (d, J = 8.4 Hz,
1H), 6.72 (br, 1H), 6.34 (dd, J = 8.4, 2.4 Hz, 1H), 6.25 (d, J = 2.4 Hz,
1H), 4.82−4.80 (m, 1H), 4.03−3.97 (m, 1H), 2.75−2.73 (m, 1H),
2.43 (br, 1H), 2.24−2.19 (m, 1H), 1.76−1.52 (m, 3H), 1.50−1.30 (m,
7H), 0.90−0.87 (m, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm)
156.5, 155.8, 128.3, 118.3, 108.6, 103.1, 75.5, 65.6, 38.0, 35.5, 31.9,
29.3, 25.2, 22.7, 14.2. HRMS calculated for C15H22O3 (M − H2O + H)+
233.1536, found (M − H2O + H)+ 233.1534. HPLC purity: 97.8%
(220 nm), tR = 6.73 min.
5,7-Dihydroxy-2-propylchroman-4-one (5b). Purified with hexane/
EtOAc (85/15) to give the product (46 mg, 0.207 mmol, 94%) as
1
white solid. H NMR (400 MHz, CDCl3): δ (ppm) 12.03 (s, 1H),
7.24 (br, 1H), 5.97 (s, 1H), 5.93 (s, 1H), 4.41−4.36 (m, 1H), 2.73−
2.57 (m, 2H), 1.86−1.79 (m, 1H), 1.69−1.42 (m, 3H), 0.97 (t, J = 7.2
Hz, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm) 197.0, 165.3, 164.2,
163.7, 103.2, 96.5, 95.5, 41.6, 36.9, 18.2, 14.0. HRMS calculated for
C12H14O4 (M − H)− 221.0819, found (M − H)− 221.0802. HPLC
purity: 96.7% (254 nm), tR = 6.54 min; 100% (220 nm), tR = 6.54 min.
2-Hexyl-5,7-bis(methoxymethoxy)chroman-4-one (5c). Purified
with hexane/EtOAc (90/10) to give the product (220 mg, 0.63 mmol,
1
63%) as clear oil. H NMR (400 MHz, CDCl3): δ (ppm) 6.37 (s, 1H),
6.30 (s, 1H), 5.24 (s, 2H), 5.16 (s, 2H), 4.39−4.32 (m, 1H), 3.51 (s, 3H),
3.47 (s, 3H), 2.65−2.53 (m, 2H), 1.84−1.77 (m, 1H), 1.67−1.60 (m,
1H), 1.55−1.30 (m, 8H), 0.90−0.87 (m, 3H). 13C NMR (100 MHz,
CDCl3): δ (ppm) 190.0, 164.8, 163.2, 159.5, 107.5, 97.8, 97.3, 95.1, 94.1,
77.6, 56.6, 56.5, 44.3, 34.9, 31.8, 29.2, 24.9, 22.7, 14.2. HRMS calculated
for C19H28O6 (M + H)+ 353.1959, found (M + H)+ 353.1954. HPLC
purity: 99.5% (254 nm), tR = 7.49 min; 99.3% (220 nm), tR = 7.49 min.
2-Hexyl-5,7-dihydroxychroman-4-one (5d). Purified with hexane/
EtOAc (85/15) to give the product (44 mg, 0.165 mmol, 75%) as
1
white solid. H NMR (400 MHz, CDCl3): δ (ppm) 12.02 (s, 1H),
2-Heptylchroman-4,7-diol (4c). Purified with hexane/EtOAc
(85/15) to give the product (39 mg, 0.15 mmol, 68%) as light
brown oil. 1H NMR (400 MHz, CDCl3): δ (ppm) 7.25 (d, J = 8.4 Hz,
1H), 6.39 (dd, J = 8.4, 2.5 Hz, 1H), 6.26 (d, J = 2.4 Hz, 1H), 5.82 (br,
1H), 4.88−4.82 (m, 1H), 4.07−4.01 (m, 1H), 2.29−2.23 (m, 1H),
2.11−2.09 (m, 1H), 1.92 (br, 1H), 1.78−1.38 (m, 5H), 1.33−1.28 (m,
6.99 (br, 1H), 5.96 (d, J = 1.9 Hz, 1H), 5.93 (d, J = 1.9 Hz, 1H), 4.40−
4.33 (m, 1H), 2.72−2.57 (m, 2H), 1.86−1.78 (m, 1H), 1.69−1.62 (m,
1H), 1.54−1.30 (m, 8H), 0.90−0.87 (m, 3H). 13C NMR (100 MHz,
CDCl3): δ (ppm) 197.0, 165.3, 164.2, 163.7, 103.2, 96.5, 95.5, 77.7,
41.6, 34.9, 31.8, 29.1, 24.9, 22.7, 14.2. HRMS calculated for C15H20O4
P
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Journal of Medicinal Chemistry
Article
(M − H)− 263.1289, found (M − H)− 263.1264. HPLC purity: 97.9%
(254 nm), tR = 7.26 min; 99.1% (220 nm), tR = 7.26 min.
2-Cyclopentyl-5,7-dihydroxychroman-4-one (5k). Purified with
hexane/EtOAc (85/15) to give the product (174 mg, 0.70 mmol, 70%
1
2-Heptyl-5,7-bis(methoxymethoxy)chroman-4-one (5e). Purified
with hexane/EtOAc (90/10) to give the product (220 mg, 0.60 mmol,
60%) as clear oil. 1H NMR (400 MHz, CDCl3): δ (ppm) 6.37 (s, 1H),
6.29 (s, 1H), 5.24 (s, 2H), 5.16 (s, 2H), 4.38−4.31 (m, 1H), 3.51 (s,
3H), 3.46 (s, 3H), 2.64−2.52 (m, 2H), 1.85−1.76 (m, 1H), 1.68−1.59
(m, 1H), 1.54−1.27 (m, 10H), 0.89−0.86 (m, 3H). 13C NMR (100
MHz, CDCl3): δ (ppm) 190.0, 164.8, 163.2, 159.6, 107.5, 97.8, 97.3,
95.1, 94.2, 77.6, 56.64, 56.57, 44.3, 34.9, 31.9, 29.5, 29.3, 25.0, 22.8,
14.2. HRMS calculated for C20H30O6 (M + H)+ 367.2115, found (M +
H)+ 367.2114. HPLC purity: 99.6% (254 nm), tR = 7.69 min; 98.8%
(220 nm), tR = 7.69 min.
over two steps) as white solid. H NMR (400 MHz, DMSO-d6): δ
(ppm) 12.12 (s, 1H), 10.73 (s, 1H), 5.83 (s, 2H), 4.26−4.20 (m, 1H),
2.80−2.73 (m, 1H), 2.61−2.56 (m, 1H), 2.18−2.12 (m, 1H), 1.84−
1.78 (m, 1H), 1.72−1.42 (m, 6H), 1.32−1.27 (m, 1H). 13C NMR
(100 MHz, DMSO-d6): δ (ppm) 196.4, 166.5, 163.4, 163.0, 101.8,
95.6, 94.8, 80.7, 43.3, 40.1, 28.3, 27.7, 25.0, 24.9. HRMS calculated for
C14H16O4 (M − H)− 247.0976, found (M − H)− 247.0954. HPLC
purity: 99.6% (254 nm), tR = 6.84 min; 99.6% (220 nm), tR = 6.84 min.
2-Cyclohexyl-5,7-dihydroxychroman-4-one (5l). Purified with
hexane/EtOAc (85/15) to give the product (168 mg, 0.64 mmol,
1
64% over two steps) as white solid. H NMR (400 MHz, DMSO-d6):
2-Heptyl-5,7-dihydroxychroman-4-one (5f). Purified with hexane/
δ (ppm) 12.12 (s, 1H), 10.74 (s, 1H), 5.84 (s, 2H), 4.24−4.18 (m,
1H), 2.85−2.77 (m, 1H), 2.55−2.51 (m, 1H), 1.91−1.88 (m, 1H),
1.75−1.63 (m, 5H), 1.28−1.01 (m, 5H). 13C NMR (100 MHz,
DMSO-d6): δ (ppm) 196.7, 166.5, 163.4, 163.0, 101.8, 95.6, 94.7, 80.9,
40.9, 38.3, 27.58, 27.56, 25.9, 25.43, 25.36. HRMS calculated for
C15H18O4 (M − H)− 261.1132, found (M − H)− 261.1106. HPLC
purity: 99.1% (254 nm), tR = 7.05 min; 99.5% (220 nm), tR = 7.05 min.
(E)-3-(6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)-1-(2,4,6-
trihydroxyphenyl)prop-2-en-1-one (5m). To a solution of 1h
(256 mg, 1 mmol) in ethanol (2.5 mL) were added DEA (154 mg,
2.1 mmol) and (−)-myrtenal (300 mg, 2 mmol) successively. The
resulting mixture was stirred at 150 °C in a pressure tube for 4 h.
Afterward, the solution was diluted with ethyl acetate and the organic
layer was washed with 10% HCl, water, brine and dried over Na2SO4.
The solvent was removed under reduced pressure, and the residue
was dissolved in methanol (15 mL), followed by the addition of
concentrated HCl (765 μL, 9 mmol) dropwise. The resulting mixture
was stirred at room temperature for 16 h, after which the solvent was
removed. The crude material was adsorbed onto silica gel and
subjected to silica gel chromatography with hexane/EtOAc (75/25) to
EtOAc (90/10) to give the product (49 mg, 0.176 mmol, 80%) as
white solid. H NMR (400 MHz, CDCl3): δ (ppm) 12.03 (s, 1H),
1
6.57 (br, 1H), 5.96 (s, 1H), 5.93 (s, 1H), 4.39−4.34 (m, 1H), 2.72−
2.57 (m, 2H), 1.86−1.79 (m, 1H), 1.69−1.62 (m, 1H), 1.54−1.26 (m,
10H), 0.90−0.87 (m, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm)
196.9, 165.0, 164.3, 163.6, 103.3, 96.5, 95.4, 77.8, 41.7, 34.9, 31.9, 29.4,
29.3, 25.0, 22.8, 14.2. HRMS calculated for C16H22O4 (M − H)−
277.1445, found (M − H)− 277.1420. HPLC purity: 98.6% (254 nm),
tR = 7.47 min; 98.8% (220 nm), tR = 7.47 min.
5,7-Bis(methoxymethoxy)-2-nonylchroman-4-one (5g). Purified
with hexane/EtOAc (90/10) to give the product (218 mg, 0.55 mmol,
55%) as clear oil. 1H NMR (400 MHz, CDCl3): δ (ppm) 6.37 (s, 1H),
6.29 (s, 1H), 5.24 (s, 2H), 5.16 (s, 2H), 4.37−4.31 (m, 1H), 3.51 (s,
3H), 3.47 (s, 3H), 2.64−2.53 (m, 2H), 1.81−1.76 (m, 1H), 1.67−1.59
(m, 1H), 1.54−1.26 (m, 14H), 0.89−0.85 (m, 3H). 13C NMR (100
MHz, CDCl3): δ (ppm) 190.1, 164.8, 163.2, 159.6, 107.5, 97.8, 97.3,
95.1, 94.2, 77.6, 56.64, 56.57, 44.3, 34.9, 32.0, 29.63, 29.63, 29.5, 29.4,
25.0, 22.8, 14.2. HRMS calculated for C22H34O6 (M + H)+ 395.2428,
found (M + H)+ 395.2407. HPLC purity: 99.9% (254 nm), tR = 8.09
min; 99.5% (220 nm), tR = 8.09 min.
1
obtain compound 5m (102 mg, 0.34 mmol, 34%) as yellow solid. H
5,7-Dihydroxy-2-nonylchroman-4-one (5h). Purified with hexane/
NMR (400 MHz, CD3OD): δ (ppm) 7.55 (d, J = 15.4 Hz, 1H), 7.38
(d, J = 15.4 Hz, 1H), 6.11 (s, 1H), 5.81 (s, 2H), 2.65−2.64 (m, 1H),
2.57−2.47 (m, 3H), 2.18−2.17 (m, 1H), 1.38 (s, 3H), 1.18 (d, J = 9.0
Hz, 1H), 0.82 (s, 3H). 13C NMR (100 MHz, CD3OD): δ (ppm)
194.6, 166.2, 165.9, 148.3, 144.2, 135.5, 125.5, 105.9, 96.0, 42.9, 42.0,
38.7, 33.7, 32.0, 26.6, 21.2. HRMS calculated for C18H20O4 (M − H)−
299.1289, found (M − H)− 299.1240. HPLC purity: 99.6% (254 nm),
tR = 7.29 min; 99.9% (220 nm), tR = 7.29 min.
EtOAc (90/10) to give the product (55 mg, 0.18 mmol, 82%) as white
1
solid. H NMR (400 MHz, CDCl3): δ (ppm) 12.05 (s, 1H), 5.95 (s,
1H), 5.92 (s, 1H), 5.44 (br, 1H), 4.40−4.35 (m, 1H), 2.72−2.58 (m,
2H), 1.86−1.80 (m, 1H), 1.69−1.61 (m, 1H), 1.54−1.26 (m, 14H),
0.90−0.88 (m, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm) 197.1,
165.4, 164.2, 163.7, 103.2, 96.5, 95.5, 77.7, 41.6, 34.8, 32.0, 29.62,
29.58, 29.5, 29.4, 24.9, 22.8, 14.2. HRMS calculated for C18H26O4
(M − H)− 305.1758, found (M − H)− 305.1719. HPLC purity: 99.7%
(254 nm), tR = 7.87 min; 99.2% (220 nm), tR = 7.87 min.
2-(2,6-Dimethylhept-5-en-1-yl)-5,7-bis(methoxymethoxy)-
chroman-4-one (5i). Purified with hexane/EtOAc (90/10) to obtain a
mixture of two diastereomers (275 mg, 0.70 mmol, 70%) as clear oil.
1H NMR (400 MHz, CDCl3): δ (ppm) 6.35 (s, 2H), 6.27 (s, 2H),
2-(6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)-5,7-dihydroxy-
chroman-4-one (5n). To a suspension of 5m (57 mg, 0.19 mmol) in
ethanol (2.5 mL) was added catalytic amount of concentrated HCl.
The resulting mixture was stirred at 150 °C under microwave irradia-
tion for 30 min. The solvent was removed under reduced pressure and
the crude material was adsorbed onto silica gel and subjected to silica
gel chromatography with hexane/EtOAc (75/25) to obtain a mixture
of two diastereomers 5n (41 mg, 0.136 mmol, 72%) as pale yellow
5.22 (s, 4H), 5.14 (s, 4H), 5.07−5.05 (m, 2H), 4.46−4.41 (m, 2H),
3.49 (s, 6H), 3.44 (s, 6H), 2.61−2.45 (m, 4H), 1.99−1.68 (m, 8H),
1.65 (s, 6H), 1.57 (s, 6H), 1.41−1.14 (m, 6H), 0.93−0.91 (m, 6H).
13C NMR (100 MHz, CDCl3): δ (ppm) 189.9, 189.8, 164.72, 164.67,
1
solid. H NMR (400 MHz, CDCl3): δ (ppm) 12.05 (br, 2H), 5.98−
5.94 (m, 4H), 5.68−5.67 (m, 2H), 4.86−4.75 (m, 2H), 2.89−2.80 (m,
2H), 2.69−2.55 (m, 2H), 2.53−2.45 (m, 2H), 2.43−2.26 (m, 6H),
2.16−2.15 (m, 2H), 1.34−1.33 (m, 6H), 1.29−1.16 (m, 2H), 0.88 (s,
3H), 0.78 (s, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm) 196.82,
196.80, 165.2, 164.2, 163.5, 145.0, 144.8, 122.4, 121.2, 103.21, 103.18,
96.55, 96.54, 95.7, 79.5, 79.0, 42.5, 42.2, 40.9, 40.8, 39.8, 39.7, 38.2,
38.0, 31.7, 31.6, 31.4, 31.3, 26.20, 26.16, 21.4, 21.3. HRMS calculated
for C18H20O4 (M − H)− 299.1289, found (M − H)− 299.1247. HPLC
purity: 99.7% (254 nm), tR = 7.53 min; 99.9% (220 nm), tR = 7.52 min.
General Procedure for the Synthesis of 6a−f. To a suspension
of corresponding hydroxyamine (0.8 mmol) in ethanol (1 mL) was
added pyridine (64 mg, 0.8 mmol). After stirring at room temperature
for 15 min, the solution of appropriate 4-chromanone (0.1 mmol)
in ethanol (2 mL) was added. The resulting mixture was stirred for
26−72 h at room temperature, after which the solvent was removed.
EtOAc was added to the residue, and the organic layer was washed
with water, brine and dried over Na2SO4. The solvent was removed
under reduced pressure and the crude material was adsorbed onto
163.1, 159.49, 159.48, 131.5, 131.4, 124.54, 124.48, 107.5, 97.8, 97.22,
97.21, 95.0, 94.1, 76.1, 75.5, 56.53, 56.46, 44.9, 44.5, 42.11, 42.06, 37.4,
36.8, 28.7, 28.4, 25.8, 25.4, 25.3, 19.9, 19.4, 17.7. HRMS calculated for
C22H32O6 (M + H)+ 393.2272, found (M + H)+ 393.2263. HPLC
purity: 98.8% (254 nm), tR = 7.71 min; 98.4% (220 nm), tR = 7.71 min.
2-(2,6-Dimethylhept-5-en-1-yl)-5,7-dihydroxychroman-4-one
(5j). Purified with hexane/EtOAc (90/10) to obtain a mixture of two
1
diastereomers (54 mg, 0.176 mmol, 80%) as white solid. H NMR
(400 MHz, CDCl3): δ (ppm) 12.02 (s, 2H), 7.25 (br, 2H), 5.97 (s,
2H), 5.94 (s, 2H), 5.09−5.08 (m, 2H), 4.51−4.46 (m, 2H), 2.73−2.56
(m, 4H), 2.04−1.70 (m, 8H), 1.68 (s, 6H), 1.60 (s, 6H), 1.53−1.32
(m, 4H), 1.25−1.18 (m, 2H), 0.98−0.94 (m, 6H). 13C NMR (100
MHz, CDCl3): δ (ppm) 197.1, 197.0, 165.4, 164.2, 163.67, 163.65,
131.72, 131.67, 124.54, 124.48, 103.2, 96.6, 95.62, 95.60, 76.3, 75.7,
42.3, 42.14, 42.08, 41.9, 37.5, 36.8, 28.9, 28.5, 25.8, 25.5, 25.4, 20.0,
19.4, 17.8. HRMS calculated for C18H24O4 (M − H)− 303.1602, found
(M − H)− 303.1564. HPLC purity: 98.4% (254 nm), tR = 7.50 min;
98.4% (220 nm), tR = 7.50 min.
Q
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silica gel and subjected to silica gel chromatography with hexane/
EtOAc to give oxime compound.
General Procedure for the Synthesis of 7a−c. To a solution of
1e (196 mg, 1 mmol) in ethanol (2.5 mL) were added pyrrolidine
(149 mg, 2.1 mmol) and corresponding cycloketone (2 mmol) suc-
cessively. The resulting mixture was stirred at 150 °C in a pressure
tube for 2−16 h. The resulting solution was cooled to room tempera-
ture, and concentrated HCl (1.02 mL, 12 mmol) was added. The
resulting mixture was then stirred at room temperature overnight.
Afterward, the solution was diluted with EtOAc, and the organic layer
was washed with water, brine and dried over Na2SO4. The solvent was
removed under reduced pressure and the crude material was adsorbed
onto silica gel and subjected to silica gel chromatography with hexane/
EtOAc to give the product.
5,7-Dihydroxy-2-nonylchroman-4-one oxime (6a). Purified with
hexane/EtOAc (85/15) to give the product (29 mg, 0.09 mmol, 90%)
as white solid. 1H NMR (400 MHz, CD3OD): δ (ppm) 5.89 (d,
J = 2.3 Hz, 1H), 5.85 (d, J = 2.3 Hz, 1H), 3.99−3.93 (m, 1H), 3.24
(dd, J = 17.1, 3.5 Hz, 1H), 2.32 (dd, J = 17.1, 11.4 Hz, 1H), 1.79−1.70
(m, 1H), 1.67−1.59 (m, 1H), 1.57−1.30 (m, 14H), 0.92−0.88 (m,
3H). 13C NMR (100 MHz, CD3OD): δ (ppm) 161.5, 160.7, 159.5,
154.7, 98.8, 97.3, 96.2, 76.2, 36.1, 33.1, 30.69, 30.68, 30.62, 30.5, 29.0,
26.2, 23.7, 14.5. HRMS calculated for C18H27NO4 (M − H)− 320.1867,
found (M − H)− 320.1818. HPLC purity: 99.9% (254 nm), tR
7.62 min; 99.5% (220 nm), tR = 7.62 min.
=
7-Hydroxyspiro[chroman-2,1′-cyclopentan]-4-one (7a). Purified
with hexane/EtOAc (90/10) to give the product (170 mg, 0.78 mmol,
5,7-Dihydroxy-2-nonylchroman-4-one O-Methyloxime (6b). Puri-
fied with hexane/EtOAc (90/10) to give the product (27 mg, 0.081
1
78% over two steps) as white solid. H NMR (400 MHz, CDCl3):
1
δ (ppm) 7.78 (d, J = 8.6 Hz, 1H), 7.12 (br, 1H), 6.49 (dd, J = 8.6, 2.3
Hz, 1H), 6.36 (d, J = 2.3 Hz, 1H), 2.78 (s, 2H), 2.09−2.04 (m, 2H),
1.87−1.83 (m, 2H), 1.72−1.59 (m, 4H). 13C NMR (100 MHz,
CDCl3): δ (ppm) 192.5, 163.7, 162.8, 129.2, 114.9, 110.1, 104.0, 90.5,
46.8, 37.7, 24.0. HRMS calculated for C13H14O3 (M − H)− 217.0870,
mmol, 81%) as white solid. H NMR (400 MHz, CDCl3): δ (ppm)
10.96 (s, 1H), 6.04 (d, J = 2.3 Hz, 1H), 5.96 (d, J = 2.3 Hz, 1H), 5.60
(br, 1H), 4.03−3.98 (m, 1H), 3.94 (s, 3H), 3.18 (dd, J = 17.2, 3.0 Hz,
1H), 2.36 (dd, J = 17.2, 11.6 Hz, 1H), 1.82−1.73 (m, 1H), 1.66−1.57
(m, 1H), 1.54−1.27 (m, 14H), 0.90−0.86 (m, 3H). 13C NMR (100
MHz, CDCl3): δ (ppm) 159.7, 159.1, 158.7, 154.1, 98.3, 97.0, 95.8,
75.1, 62.5, 35.0, 32.0, 29.67, 29.66, 29.59, 29.5, 28.6, 25.2, 22.8, 14.3.
HRMS calculated for C19H29NO4 (M − H)− 334.2024, found
(M − H)− 334.1977. HPLC purity: 99.8% (254 nm), tR = 8.13 min;
99.1% (220 nm), tR = 8.13 min.
found (M − H)− 217.0854. HPLC purity: 99.1% (254 nm), tR
6.26 min; 98.7% (220 nm), tR = 6.26 min.
=
7-Hydroxyspiro[chroman-2,1′-cyclohexan]-4-one (7b). Purified
with hexane/EtOAc (90/10) to give the product (207 mg, 0.89 mmol,
1
89% over two steps) as white solid. H NMR (400 MHz, CDCl3): δ
(ppm) 7.77 (d, J = 8.6 Hz, 1H), 7.45 (br, 1H), 6.50 (dd, J = 8.6, 2.2
Hz, 1H), 6.42 (d, J = 2.2 Hz, 1H), 2.66 (s, 2H), 1.99−1.96 (m, 2H),
1.72−1.59 (m, 3H), 1.50−1.44 (m, 4H), 1.34−1.25 (m, 1H). 13C
NMR (100 MHz, CDCl3): δ (ppm) 192.6, 164.0, 162.2, 129.0, 114.6,
110.1, 103.9, 80.5, 47.9, 35.0, 25.3, 21.6. HRMS calculated for
C14H16O3 (M − H)− 231.1027, found (M − H)− 231.1007. HPLC
purity: 98.2% (254 nm), tR = 6.47 min; 98.8% (220 nm), tR = 6.47 min.
7-Hydroxyspiro[chroman-2,1′-cycloheptan]-4-one (7c). Purified
by reverse phase C18 silica gel chromatography with H2O/MeCN
(65/35) to give the product (130 mg, 0.53 mmol, 53% over two steps)
5,7-Dihydroxy-2-nonylchroman-4-one O-Benzyloxime (6c). Puri-
fied with hexane/EtOAc (92/8) to give the product (34 mg, 0.083
mmol, 83%) as white solid. H NMR (400 MHz, CDCl3): δ (ppm)
1
10.89 (s, 1H), 7.39−7.31 (m, 5H), 6.01 (d, J = 2.4 Hz, 1H), 5.94 (d,
J = 2.3 Hz, 1H), 5.45 (br, 1H), 5.12 (s, 2H), 4.02−3.96 (m, 1H), 3.22
(dd, J = 17.2, 3.0 Hz, 1H), 2.40 (dd, J = 17.2, 11.6 Hz, 1H), 1.80−1.72
(m, 1H), 1.65−1.57 (m, 1H), 1.54−1.27 (m, 14H), 0.91−0.87 (m,
3H). 13C NMR (100 MHz, CDCl3): δ (ppm) 159.7, 159.1, 158.7,
154.5, 137.0, 128.72, 128.68, 128.5, 98.4, 96.9, 95.8, 76.7, 75.0, 35.0,
32.0, 29.67, 29.65, 29.58, 29.4, 28.9, 25.1, 22.8, 14.3. HRMS calculated
for C25H33NO4 (M + H)+ 412.2482, found (M + H)+ 412.2472.
HPLC purity: 99.1% (254 nm), tR = 8.36 min; 98.7% (220 nm), tR =
8.37 min.
5,7-Dihydroxy-2-propylchroman-4-one Oxime (6d). Purified with
hexane/EtOAc (83/17) to give the product (20 mg, 0.084 mmol,
84%) as white solid. 1H NMR (400 MHz, CD3OD): δ (ppm) 5.90 (s,
1H), 5.85 (s, 1H), 3.98 (br, 1H), 3.24 (d, J = 17.0, 1H), 2.33 (dd, J =
17.0, 11.4 Hz, 1H), 1.75−1.72 (m, 1H), 1.63−1.47 (m, 3H), 1.0−0.96
(m, 3H). 13C NMR (100 MHz, CD3OD): δ (ppm) 161.5, 160.7,
159.5, 154.8, 98.8, 97.3, 96.2, 76.0, 38.2, 28.0, 19.4, 14.3. HRMS
calculated for C12H15NO4 (M − H)− 236.0928, found (M − H)−
236.0908. HPLC purity: 99.9% (254 nm), tR = 6.35 min; 100% (220 nm),
tR = 6.34 min.
1
as white solid. H NMR (400 MHz, CDCl3): δ (ppm) 8.26 (br, 1H),
7.75 (d, J = 8.6 Hz, 1H), 6.51 (dd, J = 8.6, 2.2 Hz, 1H), 6.40 (d, J = 2.2
Hz, 1H), 2.70 (s, 2H), 2.09−2.03 (m, 2H), 1.77−1.61 (m, 6H), 1.57−
1.50 (m, 2H), 1.43−1.36 (m, 2H). 13C NMR (100 MHz, CDCl3): δ
(ppm) 193.1, 164.6, 162.5, 128.9, 114.3, 110.2, 103.9, 84.7, 48.5, 38.4,
29.4, 22.1. HRMS calculated for C15H18O3 (M − H)− 245.1183, found
(M − H)− 245.1154. HPLC purity: 97.4% (254 nm), tR = 6.67 min;
97.0% (220 nm), tR = 6.67 min.
General Procedure for the Synthesis of 8a−h. To a solution of
corresponding acetophenone (1 mmol) and appropriate benzaldehyde
(1.5 mmol) in methanol (10 mL) was added 60% KOH aqueous
solution (1.5 mL) dropwise at room temperature. The resulting
solution was stirred for 60 h at room temperature, after which the
reaction mixture was neutralized with 10% HCl aq and extracted with
ethyl acetate twice. The organic layer was washed with water, brine
and dried over Na2SO4. The solvent was removed under reduced
pressure, and the residue was dissolved in methanol (14 mL), followed
by the careful addition of concentrated HCl (340 μL, 4 mmol). The
obtained mixture was stirred at room temperature until the MOM-
protected chalcone was gone, and then the mixture was concentrated.
The crude material was adsorbed onto silica gel and subjected to silica
gel chromatography with hexane/EtOAc to afford the chalcones.
(E)-3-(2-(Allyloxy)phenyl)-1-(2,4-dihydroxyphenyl)prop-2-en-1-
one (8a). Purified with hexane/EtOAc (90/10) to give the product
2-Hexyl-5,7-dihydroxychroman-4-one Oxime (6e). Purified with
hexane/EtOAc (86/14) to give the product (22 mg, 0.08 mmol, 80%)
1
as white solid. H NMR (400 MHz, CD3OD): δ (ppm) 5.90 (d, J =
2.3 Hz, 1H), 5.85 (d, J = 2.3 Hz, 1H), 3.97−3.91 (m, 1H), 3.24 (dd,
J = 17.1, 3.1 Hz, 1H), 2.32 (dd, J = 17.1, 11.4 Hz, 1H), 1.78−1.69 (m,
1H), 1.65−1.49 (m, 2H), 1.45−1.32 (m, 7H), 0.92−0.89 (m, 3H). 13C
NMR (100 MHz, CD3OD): δ (ppm) 161.4, 160.6, 159.5, 154.7, 98.8,
97.3, 96.2, 76.2, 36.0, 32.9, 30.3, 29.0, 26.2, 23.6, 14.4. HRMS
calculated for C15H21NO4 (M − H)− 278.1398, found (M − H)−
278.1354. HPLC purity: 99.8% (254 nm), tR = 7.02 min; 99.9% (220 nm),
tR = 7.01 min.
1
(175 mg, 0.59 mmol, 59% over two steps) as yellow solid. H NMR
2-Heptyl-5,7-dihydroxychroman-4-one Oxime (6f). Purified with
hexane/EtOAc (90/10) to give the product (25 mg, 0.085 mmol,
85%) as white solid. 1H NMR (400 MHz, CD3OD): δ (ppm) 5.90 (s,
1H), 5.85 (s, 1H), 3.95 (br, 1H), 3.24 (d, J = 17.0 Hz, 1H), 2.32 (dd,
J = 17.0, 11.4 Hz, 1H), 1.75−1.73 (m, 1H), 1.63−1.33 (m, 11H), 0.90
(br, 3H). 13C NMR (100 MHz, CD3OD): δ (ppm) 161.5, 160.7,
159.5, 154.7, 98.8, 97.3, 96.2, 76.2, 36.0, 33.0, 30.6, 30.3, 29.0, 26.2,
23.7, 14.4. HRMS calculated for C16H23NO4 (M − H)− 292.1554,
found (M − H)− 292.1512. HPLC purity: 99.9% (254 nm), tR = 7.22
min; 99.8% (220 nm), tR = 7.22 min.
(400 MHz, CDCl3): δ (ppm) 13.49 (s, 1H), 8.18 (d, J = 15.6 Hz, 1H),
7.81 (d, J = 9.4 Hz, 1H), 7.74 (d, J = 15.6 Hz, 1H), 7.62 (d, J = 7.6 Hz,
1H), 7.36 (t, J = 8.4 Hz, 1H), 7.0 (t, J = 7.5 Hz, 1H), 6.94 (d, J = 8.4
Hz, 1H), 6.43−6.41 (m, 2H), 6.22 (s, 1H), 6.16−6.09 (m, 1H), 5.47
(d, J = 17.2 Hz, 1H), 5.35 (d, J = 10.6 Hz, 1H), 4.65 (d, J = 5.3 Hz,
2H). 13C NMR (100 MHz, CDCl3): δ (ppm) 192.8, 166.5, 162.9,
158.1, 140.5, 133.0, 132.2, 132.0, 130.1, 124.2, 121.4, 121.2, 118.4,
114.8, 112.8, 107.9, 103.9, 69.5. HRMS calculated for C18H16O4
(M − H)− 295.0976, found (M − H)− 295.0955. HPLC purity: 100%
(254 nm), tR = 7.20 min; 99.9% (220 nm), tR = 7.20 min.
R
dx.doi.org/10.1021/jm500853v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(E)-1-(2,4-Dihydroxyphenyl)-3-(2-(hexyloxy)phenyl)prop-2-en-1-
one (8b). Purified by reverse phase C18 silica gel chromatography with
H2O/MeCN (43/57) to give the product (170 mg, 0.50 mmol, 50%
over two steps) as yellow solid. 1H NMR (400 MHz, CDCl3): δ
(ppm) 13.61 (s, 1H), 8.11 (d, J = 15.6 Hz, 1H), 7.81−7.77 (m, 2H),
7.57 (dd, J = 7.6, 1.2 Hz, 1H), 7.35 (t, J = 7.9 Hz, 1H), 6.99−6.92 (m,
3H), 6.45−6.43 (m, 2H), 4.08−4.04 (m, 2H), 1.93−1.86 (m, 2H),
1.56−1.49 (m, 2H), 1.39−1.32 (m, 4H), 0.92−0.88 (m, 3H). 13C
NMR (100 MHz, CDCl3): δ (ppm) 193.0, 166.4, 163.2, 158.8, 141.1,
132.14, 132.06, 130.7, 123.8, 121.3, 120.7, 114.6, 112.3, 108.1, 103.8,
68.7, 31.7, 29.4, 26.1, 22.7, 14.1. HRMS calculated for C21H24O4 (M − H)−
339.1602, found (M − H)− 339.1550. HPLC purity: 99.6% (254 nm), tR =
8.00 min; 98.9% (220 nm), tR = 8.01 min.
(E)-1-(2,4-Dihydroxyphenyl)-3-(2-(octyloxy)phenyl)prop-2-en-1-
one (8c). Purified by reverse phase C18 silica gel chromatography with
H2O/MeCN (45/55) to give the product (195 mg, 0.53 mmol, 53%
over two steps) as yellow solid. 1H NMR (400 MHz, CDCl3): δ
(ppm) 13.64 (s, 1H), 8.12 (d, J = 15.6 Hz, 1H), 7.80 (s, 1H), 7.77 (d,
J = 6.2 Hz, 1H), 7.57 (d, J = 7.5 Hz, 1H), 7.35 (t, J = 8.2 Hz, 1H), 7.14
(br, 1H), 6.97 (t, J = 7.5 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 6.46−6.44
(m, 2H), 4.06−4.03 (m, 2H), 1.92−1.85 (m, 2H), 1.55−1.48 (m, 2H),
1.39−1.27 (m, 8H), 0.88−0.85 (m, 3H). 13C NMR (100 MHz,
CDCl3): δ (ppm) 193.0, 166.3, 163.3, 158.8, 141.1, 132.2, 132.1,
130.6, 123.8, 121.2, 120.7, 114.6, 112.3, 108.2, 103.8, 68.7, 31.9, 29.5,
29.4, 29.3, 26.4, 22.8, 14.2. HRMS calculated for C23H28O4 (M + H)+
369.2060, found (M + H)+ 369.2057. HPLC purity: 99.5% (254 nm),
tR = 8.36 min; 99.4% (220 nm), tR = 8.36 min.
1H), 7.53 (s, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.37 (t, J = 7.7 Hz, 1H),
7.05 (dd, J = 8.0, 2.0 Hz, 1H), 6.43 (dd, J = 8.9, 2.3 Hz, 1H), 6.30 (d,
J = 2.2 Hz, 1H), 6.11−6.03 (m, 1H), 5.44 (dd, J = 17.3, 1.4 Hz, 1H),
5.29 (d, J = 10.5 Hz, 1H), 4.65 (d, J = 5.2 Hz, 2H). 13C NMR (100
MHz, DMSO-d6): δ (ppm) 191.5, 165.9, 165.3, 158.6, 143.7, 136.0,
133.6, 133.3, 130.0, 122.2, 121.5, 117.7, 117.4, 114.1, 113.0, 108.3,
102.6, 68.4. HRMS calculated for C18H16O4 (M + H)+ 297.1121,
found (M + H)+ 297.1107. HPLC purity: 99.7% (254 nm), tR = 7.22
min; 99.6% (220 nm), tR = 7.22 min.
(E)-3-(4-(Allyloxy)phenyl)-1-(2,4-dihydroxyphenyl)prop-2-en-1-
one (8h). Purified with hexane/EtOAc (90/10) to give the product
1
(130 mg, 0.44 mmol, 44% over two steps) as yellow solid. H NMR
(400 MHz, DMSO-d6): δ (ppm) 13.54 (s, 1H), 10.70 (br, 1H), 8.19
(d, J = 9.0 Hz, 1H), 7.87−7.75 (m, 4H), 7.04 (d, J = 8.7 Hz, 2H), 6.41
(dd, J = 8.9, 2.3 Hz, 1H), 6.29 (d, J = 2.3 Hz, 1H), 6.10−6.01 (m, 1H),
5.42 (dd, J = 17.3, 1.4 Hz, 1H), 5.28 (d, J = 10.5 Hz, 1H), 4.65 (d, J =
5.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 191.5, 165.8,
165.1, 160.4, 143.7, 133.4, 133.0, 131.0, 127.4, 118.6, 117.8, 115.1,
113.0, 108.2, 102.6, 68.4. HRMS calculated for C18H16O4 (M + H)+
297.1121, found (M + H)+ 297.1108. HPLC purity: 99.8% (254 nm),
tR = 7.18 min; 99.6% (220 nm), tR = 7.18 min.
General Procedure for the Synthesis of 8i−m. The obtained
appropriate chalcone (0.2 mmol) was dissolved in ethanol (2.5 mL)
with a catalytic amount of concentrated HCl and then heated at 150 °C
under microwave irradiation for 1.5 h. The solvent was removed under
reduced pressure and the crude material was adsorbed onto silica gel
and subjected to silica gel chromatography with hexane/EtOAc to afford
the corresponding flavanones.
2-(2-(Allyloxy)phenyl)-7-hydroxychroman-4-one (8i). Purified
with hexane/EtOAc (90/10) to give the product (30 mg, 0.1 mmol,
50%) as pale yellow solid. 1H NMR (400 MHz, CDCl3): δ (ppm) 7.85
(d, J = 8.7 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.54 (br, 1H), 7.30 (t, J =
8.5 Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H), 6.58
(dd, J = 8.7, 2.2 Hz, 1H), 6.51 (d, J = 2.1 Hz, 1H), 6.03−5.95 (m, 1H),
5.87 (dd, J = 11.3, 5.0 Hz, 1H), 5.34 (d, J = 17.2 Hz, 1H), 5.24 (d, J =
10.6 Hz, 1H), 4.61−4.51 (m, 2H), 2.97−2.86 (m, 2H). 13C NMR (100
MHz, CDCl3): δ (ppm) 192.6, 164.5, 163.9, 154.9, 133.0, 129.6, 129.5,
127.8, 126.7, 121.2, 117.8, 114.8, 112.0, 111.0, 103.6, 75.0, 69.0, 43.4.
HRMS calculated for C18H16O4 (M − H)− 295.0976, found (M − H)−
295.0949. HPLC purity: 98.0% (254 nm), tR = 6.76 min; 99.0% (220 nm),
tR = 6.76 min.
(E)-1-(2,4-Dihydroxyphenyl)-3-(4-(piperidin-1-yl)phenyl)prop-2-
en-1-one (8d). Purification was performed without chromatography
because of the poor solubility of product. The crude solid was washed
with small amount of methanol three times to give the product
1
(210 mg, 0.65 mmol, 65% over two steps) as yellow solid. H NMR
(400 MHz, DMSO-d6): δ (ppm) 8.19 (d, J = 9.0 Hz, 1H), 7.98−7.92
(m, 3H), 7.77 (d, J = 15.4 Hz, 1H), 7.67 (br, 2H), 6.45 (dd, J = 8.9, 2.2
Hz, 1H), 6.33 (d, J = 2.2 Hz, 1H), 3.49−3.46 (m, 4H), 1.88 (br, 4H),
1.64 (br, 2H). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 191.7,
166.3, 165.8, 133.5, 131.0, 113.5, 108.8, 103.1, 24.0. HRMS calculated
for C20H21NO3 (M − H)− 322.1449, found (M − H)− 322.1407.
HPLC purity: 99.0% (254 nm), tR = 5.99 min; 98.8% (220 nm), tR =
5.98 min.
(E)-3-(2-(Allyloxy)phenyl)-1-(2,4,6-trihydroxyphenyl)prop-2-en-1-
one (8e). Purified with hexane/EtOAc (70/30) to give the product
2-(2-(Hexyloxy)phenyl)-7-hydroxychroman-4-one (8j). Purified by
1
(106 mg, 0.34 mmol, 34% over two steps) as yellow solid. H NMR
reverse phase C18 silica gel chromatography with H2O/MeCN (50/50)
1
to give the product (35 mg, 0.1 mmol, 52%) as pale yellow solid. H
(400 MHz, CD3OD): δ (ppm) 8.25 (d, J = 15.8 Hz, 1H), 8.10 (d, J =
15.8 Hz, 1H), 7.61 (dd, J = 7.7, 1.3 Hz, 1H), 7.34−7.30 (m, 1H), 7.0−
6.94 (m, 2H), 6.16−6.09 (m, 1H), 5.86 (s, 2H), 5.45 (dd, J = 17.3, 1.6
Hz, 1H), 5.29 (dd, J = 10.6, 1.4 Hz, 1H), 4.63 (d, J = 5.2 Hz, 2H). 13C
NMR (100 MHz, CD3OD): δ (ppm) 194.5, 166.4, 166.1, 158.9, 138.2,
134.6, 132.4, 129.5, 129.1, 126.0, 122.0, 117.8, 113.9, 106.0, 96.0, 70.3.
HRMS calculated for C18H16O5 (M − H)− 311.0925, found (M − H)−
311.0877. HPLC purity: 97.5% (254 nm), tR = 6.93 min; 97.2%
(220 nm), tR = 6.93 min.
NMR (400 MHz, CDCl3): δ (ppm) 7.86 (d, J = 8.7 Hz, 1H), 7.58 (d,
J = 7.6 Hz, 1H), 7.31 (t, J = 8.5 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 6.89
(d, J = 8.2 Hz, 1H), 6.59 (dd, J = 8.7, 2.2 Hz, 1H), 6.52 (d, J = 2.1 Hz,
1H), 5.86−5.82 (m, 1H), 4.03−3.93 (m, 2H), 2.96−2.87 (m, 2H),
1.79−1.72 (m, 2H), 1.45−1.37 (m, 2H), 1.31−1.27 (m, 4H), 0.87−0.84
(m, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm) 192.8, 164.5, 164.1,
155.5, 129.52, 129.50, 127.5, 126.5, 120.7, 114.7, 111.5, 111.0, 103.6,
75.1, 68.3, 43.3, 31.6, 29.2, 25.9, 22.6, 14.1. HRMS calculated for
C21H24O4 (M − H)− 339.1602, found (M − H)− 339.1563. HPLC
purity: 99.9% (254 nm), tR = 7.65 min; 99.6% (220 nm), tR = 7.65 min.
7-Hydroxy-2-(2-(octyloxy)phenyl)chroman-4-one (8k). Purified by
reverse phase C18 silica gel chromatography with H2O/MeCN
(42/58) to give the product (37 mg, 0.1 mmol, 50%) as pale yellow
solid. 1H NMR (400 MHz, CDCl3): δ (ppm) 7.86 (d, J = 8.7 Hz, 1H),
7.58 (dd, J = 7.5, 1.0 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.02 (t, J = 7.5
Hz, 1H), 6.89 (d, J = 8.2 Hz, 1H), 6.60 (dd, J = 8.7, 2.2 Hz, 1H), 6.53
(d, J = 2.2 Hz, 1H), 5.86−5.82 (m, 1H), 4.03−3.93 (m, 2H), 2.97−
2.87 (m, 2H), 1.79−1.72 (m, 2H), 1.44−1.37 (m, 2H), 1.32−1.24 (m,
8H), 0.87−0.83 (m, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm)
192.9, 164.6, 164.2, 155.5, 129.53, 129.49, 127.5, 126.5, 120.7, 114.6,
111.5, 111.0, 103.6, 75.1, 68.3, 43.3, 31.9, 29.4, 29.3, 29.2, 26.2, 22.7,
14.2. HRMS calculated for C23H28O4 (M + H)+ 369.2060, found (M + H)+
369.2056. HPLC purity: 99.9% (254 nm), tR = 8.05 min; 99.9% (220 nm),
tR = 8.05 min.
(E)-3-(2-(Allyloxy)phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one
(8f). Purified with hexane/EtOAc (80/20) to give the product
1
(205 mg, 0.73 mmol, 73% over two steps) as pale yellow solid. H
NMR (400 MHz, CDCl3): δ (ppm) 8.42 (br, 1H), 8.16 (d, J = 15.8
Hz, 1H), 8.01−7.99 (m, 2H), 7.70 (d, J = 15.8 Hz, 1H), 7.61 (dd, J =
7.7, 1.4 Hz, 1H), 7.36−7.31 (m, 1H), 7.03−6.96 (m, 3H), 6.91 (d, J =
8.3 Hz, 1H), 6.11−6.02 (m, 1H), 5.43 (dd, J = 17.2, 1.4 Hz, 1H), 5.29
(dd, J = 10.6, 1.3 Hz, 1H), 4.61−4.59 (m, 2H). 13C NMR (100 MHz,
CDCl3): δ (ppm) 190.8, 161.6, 158.0, 140.6, 132.9, 131.8, 131.5,
130.5, 129.6, 124.3, 122.8, 121.1, 118.1, 115.9, 112.7, 69.3. HRMS
calculated for C18H16O3 (M − H)− 279.1027, found (M − H)−
279.0988. HPLC purity: 99.6% (254 nm), tR = 6.99 min; 99.8% (220 nm),
tR = 6.99 min.
(E)-3-(3-(Allyloxy)phenyl)-1-(2,4-dihydroxyphenyl)prop-2-en-1-
one (8g). Purified with hexane/EtOAc (90/10) to give the product
1
(113 mg, 0.38 mmol, 38% over two steps) as yellow solid. H NMR
(400 MHz, DMSO-d6): δ (ppm) 13.40 (s, 1H), 10.77 (br, 1H), 8.23
(d, J = 9.0 Hz, 1H), 7.98 (d, J = 15.4 Hz, 1H), 7.76 (d, J = 15.4 Hz,
7-Hydroxy-2-(4-(piperidin-1-yl)phenyl)chroman-4-one (8l). Puri-
fied with hexane/EtOAc (84/16) to give the product (37 mg, 0.11 mmol,
S
dx.doi.org/10.1021/jm500853v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
57%) as pale yellow solid. 1H NMR (400 MHz, CD3OD): δ (ppm) 7.73
(d, J = 8.7 Hz, 1H), 7.37−7.35 (m, 2H), 7.02−6.99 (m, 2H), 6.50 (dd,
J = 8.7, 2.2 Hz, 1H), 6.36 (d, J = 2.2 Hz, 1H), 5.39 (dd, J = 12.9, 2.9
Hz, 1H), 3.19−3.16 (m, 4H), 3.06 (dd, J = 16.9, 12.9 Hz, 1H), 2.70
(dd, J = 16.9, 3.0 Hz, 1H), 1.75−1.69 (m, 4H), 1.63−1.59 (m, 2H).
13C NMR (100 MHz, CD3OD): δ (ppm) 193.6, 166.8, 165.6, 153.8,
131.2, 129.9, 128.5, 117.7, 115.1, 115.0, 111.7, 103.8, 81.0, 51.9, 44.8,
26.8, 25.4. HRMS calculated for C20H21NO3 (M − H)− 322.1449,
found (M − H)− 322.1401. HPLC purity: 97.4% (254 nm), tR = 5.22
min; 98.7% (220 nm), tR = 5.22 min.
membrane potential evaluated using the software FlowJo X 10.0.7 as
described.76 Carbonyl cyanide m-chlorophenylhydrazone (CCCP;
Sigma-Aldrich) and vancomycin were used as positive and negative
controls, respectively.
Assay of Relaxation Activity of E. coli Topoisomerase I.
Recombinant E. coli topoisomerase I (EcTopI) was purified with
published procedures.77 CsCl gradient purified plasmid DNA (160 ng)
was added to relaxation reaction buffer containing 10 mM Tris-HCl,
pH 8.0, 50 mM NaCl, 0.1 mg/mL gelatin, and 0.5 mM MgCl2. The
resulting solution was divided into 9.5 μL aliquots, to which 0.5 μL of
the 20 mM compounds dissolved in DMSO was added for final
compound concentrations of 500 μM. The compound control reaction
received only DMSO. EcTopI (20 ng) in 10 μL of the relaxation
reaction buffer was then added to each DNA/compound reaction
mixture. The reaction mixtures were then incubated at 37 °C for
30 min before the reaction was terminated, and the mixtures were
loaded onto a 1% agarose gel for electrophoretic analysis in TAE buffer
(40 mM Tris−acetate, pH 8.1, 2 mM EDTA) as described previously.78
E. coli DNA Gyrase Supercoiling Assay. The E. coli DNA gyrase
supercoiling assay was performed according to the manufacturer’s
protocol. Briefly, 1.25 U of E. coli DNA gyrase (New England Biolabs)
was added to 20 μL of DNA gyrase reaction mixture (35 mM Tris-
HCl, pH 7.5, 24 mM KCl, 4 mM MgCl2, 2 mM DTT, 1.75 mM ATP,
5 mM spermidine, 0.1 mg/mL BSA, 6.5% glycerol) containing 300 ng
of relaxed plasmid DNA substrate (New England Biolabs) in the
presence of indicated concentration of compound. After incubation at
37 °C for 30 min, the reactions were stopped and analyzed by agarose
gel electrophoresis similarly as the topoisomerase I relaxation assay.78
IC50 values (compound concentrations at which only 50% of the input
DNA was converted to supercoiled form) were calculated by spot
densitometry analysis with AlphaView, AlphaImager Mini (Protein
Simple). The IC50 values represent the average from at least two
experiments performed separately.
E. coli Topoisomerase IV Decatenation Assay. For the assay
of decatenation activity of topoisomerase IV,79 0.25 U of E. coli
topoisomerase IV (Topogen) was added to 20 μL of decatenation
reaction mixture (40 mM HEPES-KOH, pH 8.0, 100 mM potassium
glutamate, 10 mM magnesium acetate, 10 mM dithiothreitol, 50 μg of
BSA/mL) containing 290 ng of kinetoplast DNA substrate (Topogen)
and 1 mM ATP in the presence of indicated concentration of com-
pound. After incubation at 37 °C for 30 min, the reactions were
stopped by the addition of stop buffer (50 mM EDTA, 50% glycerol,
and 0.5% (v/v) bromophenol blue). The DNA samples were
electrophoresed in a 1% agarose gel containing 0.5 μg/mL ethidium
bromide in TAE buffer also containing 0.5 μg/mL ethidium bromide.
Visualization of the bands was carried out by exposure to UV light.
IC50 values (compound concentrations at which only 50% of kineto-
plast DNA was converted into monomeric products) were calculated
by spot densitometry analysis with AlphaView, AlphaImager Mini
(Protein Simple). The IC50 values represent the average from at least
two experiments performed separately.
2-(2-(Allyloxy)phenyl)-5,7-dihydroxychroman-4-one (8m). Purified
with hexane/EtOAc (85/15) to give the product (47 mg, 0.15 mmol,
75%) as white solid. 1H NMR (400 MHz, CD3OD): δ (ppm) 7.54 (d,
J = 7.6 Hz, 1H), 7.30 (td, J = 7.9, 1.6 Hz, 1H), 7.03−6.99 (m, 2H),
6.11−6.01 (m, 1H), 5.95 (d, J = 2.1 Hz, 1H), 5.90 (d, J = 2.1 Hz, 1H),
5.73 (dd, J = 12.7, 3.2 Hz, 1H), 5.38 (dd, J = 17.3, 1.6 Hz, 1H), 5.25
(dd, J = 10.6, 1.4 Hz, 1H), 4.65−4.56 (m, 2H), 2.99−2.91 (m, 1H),
2.83−2.78 (m, 1H). 13C NMR (100 MHz, CD3OD): δ (ppm) 197.7,
168.3, 165.5, 165.0, 156.4, 134.6, 130.5, 128.8, 127.5, 122.0, 117.6,
113.2, 103.3, 97.2, 96.2, 75.7, 70.0, 43.1. HRMS calculated for
C18H16O5 (M − H)− 311.0925, found (M − H)− 311.0877. HPLC
purity: 99.8% (254 nm), tR = 7.14 min; 99.6% (220 nm), tR
=
7.14 min.
MIC Determination. MIC values were determined against M.
tuberculosis (H37Rv) and other bacteria using the standard microbroth
dilution method exactly as previously described,71 which is based on
the methods by the Clinical and Laboratory Standards Institute.72,73
The maximum test concentration used was 200 μg/mL.
Cytotoxicity Assays. Cytotoxicity was assessed in vitro using Vero
cells (kidney epithelial cells, ATCC CCL-81) as described pre-
viously.74 In brief, monolayers of cells cultured in Dulbecco’s modified
Eagle medium (DMEM)/10% fetal bovine serum (FBS) were
trypsinized, seeded at approximately 10% confluence in white-walled
96-well plates, and incubated overnight to allow adherence. Medium
was replaced with DMEM/FBS containing 2-fold serial dilutions of
test compounds. Detection was performed using MTT (CellTiter96,
Promega) with overnight solubilization according to the manufac-
turer’s instructions.
Approximation of Solubility in Cell Culture Media. The 2-fold
serial dilutions in clear, round-bottom 96-well plates were prepared in
DMEM with 5% FBS. Plates were incubated at 37 °C overnight. The
highest compound concentration that did not result in visible
precipitation of compound was used to approximate the solubility
limit in cell culture medium.
Time Kill Experiments. The ability of compounds to kill S. aureus
Newman was evaluated using standard approaches.75 Overnight
cultures were diluted 1:25 in fresh Mueller−Hinton broth and
grown to OD600nm ≈ 0.3 before being exposed to compounds at
various increments of their MICs. The number of viable bacteria over
time was then determined.
Macromolecular Synthesis. The effects of compounds on key
macromolecular processes were determined as described,76 using
radiolabeled precursors from American Radiolabeled Chemicals, Inc.:
[methyl-3H]thymidine, [5,6-3H]uridine, and [4,5-3H]leucine for DNA,
RNA, and protein, respectively. Experiments were performed on three
independent mid-logarithmic cultures (OD600nm ≈ 0.3). The anti-
biotics ciprofloxacin (DNA, from Sigma-Aldrich), rifampicin (RNA,
from TCI America), and erythromycin (protein, from Calbiochem)
were used as positive controls.
ASSOCIATED CONTENT
* Supporting Information
■
S
NMR and HRMS spectra and HPLC profiles of all synthetic
compounds; ORTEP views of 3g and 8a; inhibition data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Analysis of the Membrane Potential. The effects of compounds
on the membrane potential of S. aureus were evaluated by flow
cytometry using the fluorescent probe diethyloxacarbocyanine dye
DiOC2(3). The emission of red fluorescence of DiOC2(3) from cells is
dependent on the membrane potential, while green fluorescence
emission is independent of the membrane potential. Therefore, the
ratio of red to green fluorescence provides a measure of the effects of
compounds on the membrane potential. These experiments were
performed as previously described,76 using S. aureus Newman grown
to OD600nm ≈ 0.3 in Mueller−Hinton II. Samples were analyzed using
BD LSR II flow cytometer and the effects of compounds on the
AUTHOR INFORMATION
Corresponding Author
*Phone: +1-808-933-2960. Fax: +1-808-933-2974. E-mail:
dianqing@hawaii.edu.
■
Author Contributions
#M.M.M and Md.Z.A contributed equally to the work.
Notes
The authors declare no competing financial interest.
T
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Journal of Medicinal Chemistry
Article
(14) Gradisar, H.; Pristovsek, P.; Plaper, A.; Jerala, R. Green tea
catechins inhibit bacterial DNA gyrase by interaction with its ATP
binding site. J. Med. Chem. 2006, 50, 264−271.
ACKNOWLEDGMENTS
■
We thank financial support from the National Institutes of
Health Grants P20GM103466 and R15AI092315 and Hawaii
Community Foundation’s Leahi Fund to Treat & Prevent
Pulmonary Disease Grant 12ADVC-57499 (D.S.), National
Institutes of Health Grants R01AI069313 (Y.-C.T.-D.) and
R01AT006732 (J.G.H.), and the American Lebanese Syrian
Associated Charities (ALSAC), St. Jude Children’s Research
Hospital (R.E.L.). We thank Dr. Benjamin Clark for his assis-
tance with the measurement of high-resolution mass spectra.
́ ́
(15) Favela-Hernandez, J. M. J.; García, A.; Garza-Gonzalez, E.;
Rivas-Galindo, V. M.; Camacho-Corona, M. R. Antibacterial and
antimycobacterial lignans and flavonoids from Larrea tridentata.
Phytother. Res. 2012, 26, 1957−1960.
(16) Cushnie, T. P. T.; Lamb, A. J. Recent advances in understanding
the antibacterial properties of flavonoids. Int. J. Antimicrob. Agents
2011, 38, 99−107.
(17) Ballesteros, J. F.; Sanz, M. J.; Ubeda, A.; Miranda, M. A.; Iborra,
S.; Paya, M.; Alcaraz, M. J. Synthesis and pharmacological evaluation of
2′-hydroxychalcones and flavones as inhibitors of inflammatory
mediators generation. J. Med. Chem. 1995, 38, 2794−2797.
(18) Wu, J.; Li, J.; Cai, Y.; Pan, Y.; Ye, F.; Zhang, Y.; Zhao, Y.; Yang,
S.; Li, X.; Liang, G. Evaluation and discovery of novel synthetic
chalcone derivatives as anti-inflammatory agents. J. Med. Chem. 2011,
54, 8110−8123.
(19) Takemura, H.; Sakakibara, H.; Yamazaki, S.; Shimoi, K. Breast
cancer and flavonoidsa role in prevention. Curr. Pharm. Des. 2013,
19, 6125−6132.
(20) Park, E. J.; Pezzuto, J. M. Flavonoids in cancer prevention. Anti-
Cancer Agents Med. Chem. 2012, 12, 836−851.
(21) Echeverria, C.; Santibanez, J. F.; Donoso-Tauda, O.; Escobar,
C.; Ramirez-Tagle, R. Structural antitumoral activity relationships of
synthetic chalcones. Int. J. Mol. Sci. 2009, 10, 221−231.
(22) Loa, J.; Chow, P.; Zhang, K. Studies of structure−activity
relationship on plant polyphenol-induced suppression of human liver
cancer cells. Cancer Chemother. Pharmacol. 2009, 63, 1007−1016.
(23) Stevenson, D.; Hurst, R. Polyphenolic phytochemicalsjust
antioxidants or much more? Cell. Mol. Life Sci. 2007, 64, 2900−2916.
(24) Erasto, P.; Bojase-Moleta, G.; Majinda, R. R. T. Antimicrobial
and antioxidant flavonoids from the root wood of Bolusanthus speciosus.
Phytochemistry 2004, 65, 875−880.
(25) Weiss, R. B.; Greene, R. F.; Knight, R. D.; Collins, J. M.; Pelosi,
J. J.; Sulkes, A.; Curt, G. A. Phase I and clinical pharmacology study of
intravenous flavone acetic acid (NSC 347512). Cancer Res. 1988, 48,
5878−5882.
(26) Senderowicz, A. M. Flavopiridol: the first cyclin-dependent
kinase inhibitor in human clinical trials. Invest. New Drugs 1999, 17,
313−320.
(27) Burdette-Radoux, S.; Tozer, R. G.; Lohmann, R. C.; Quirt, I.;
Ernst, D. S.; Walsh, W.; Wainman, N.; Colevas, A. D.; Eisenhauer, E.
A. Phase II trial of flavopiridol, a cyclin dependent kinase inhibitor, in
untreated metastatic malignant melanoma. Invest. New Drugs 2004, 22,
315−322.
(28) Jones, J. A.; Rupert, A. S.; Poi, M.; Phelps, M. A.; Andritsos, L.;
Baiocchi, R.; Benson, D. M.; Blum, K. A.; Christian, B.; Flynn, J.;
Penza, S.; Porcu, P.; Grever, M. R.; Byrd, J. C. Flavopiridol can be
safely administered using a pharmacologically derived schedule and
demonstrates activity in relapsed and refractory non-Hodgkin’s
lymphoma. Am. J. Hematol. 2014, 89, 19−24.
ABBREVIATIONS USED
■
ATCC, American Type Culture Collection; CCCP, carbonyl
cyanide m-chlorophenylhydrazone; Cip, ciprofloxacin; DEA,
diethylamine; DIPEA, diisopropylethylamine; DMEM, Dulbecco’s
modified Eagle medium; DMSO, dimethylsulfoxide; FBS,
fetal bovine serum; HPLC, high-performance liquid chroma-
tography; HRMS, high-resolution mass spectrometry; MIC,
minimum inhibitory concentration; MOM, methoxymethyl;
MSSA, methicillin-sensitive Staphylococcus aureus; MRSA,
methicillin-resistant Staphylococcus aureus; NMR, nuclear
magnetic resonance; SAR, structure−activity relationship;
SEM, standard error of mean; SI, selectivity index; Topo,
topoisomerase; TBDMS, tert-butyldimethylsilyl; Ts, p-toluene-
sulfonyl
REFERENCES
■
(1) Cushnie, T. P. T.; Lamb, A. J. Antimicrobial activity of flavonoids.
Int. J. Antimicrob. Agents 2005, 26, 343−356.
(2) Gutierrez-Lugo, M.-T.; Bewley, C. A. Natural products, small
molecules, and genetics in tuberculosis drug development. J. Med.
Chem. 2008, 51, 2606−2612.
(3) Negi, A. S.; Kumar, J. K.; Luqman, S.; Saikia, D.; Khanuja, S. P. S.
Antitubercular potential of plants: a brief account of some important
molecules. Med. Res. Rev. 2010, 30, 603−645.
(4) Walsh, C. Where will new antibiotics come from? Nat. Rev.
Microbiol. 2003, 1, 65−70.
(5) Shang, S.; Tan, D. S. Advancing chemistry and biology through
diversity-oriented synthesis of natural product-like libraries. Curr. Opin.
Chem. Biol. 2005, 9, 248−258.
(6) Havsteen, B. Flavonoids, a class of natural products of high
pharmacological potency. Biochem. Pharmacol. 1983, 32, 1141−1148.
(7) Harborne, J. B.; Williams, C. A. Advances in flavonoid research
since 1992. Phytochemistry 2000, 55, 481−504.
(8) Middleton, E., Jr.; Kandaswami, C.; Theoharides, T. C. The
effects of plant flavonoids on mammalian cells: implications for
inflammation, heart disease, and cancer. Pharmacol. Rev. 2000, 52,
673−751.
(29) Loguercio, C.; Festi, D. Silybin and the liver: from basic research
to clinical practice. World J. Gastroenterol. 2011, 17, 2288−2301.
(30) Cheung, C. W. Y.; Gibbons, N.; Johnson, D. W.; Nicol, D. L.
Silibinina promising new treatment for cancer. Anti-Cancer Agents
Med. Chem. 2010, 10, 186−195.
(9) Knekt, P.; Jarvinen, R.; Seppanen, R.; Heliovaara, M.; Teppo, L.;
Pukkala, E.; Aromaa, A. Dietary flavonoids and the risk of lung cancer
and other malignant neoplasms. Am. J. Epidemiol. 1997, 146, 223−230.
(10) Knekt, P.; Kumpulainen, J.; Jarvinen, R.; Rissanen, H.;
Heliovaara, M.; Reunanen, A.; Hakulinen, T.; Aromaa, A. Flavonoid
intake and risk of chronic diseases. Am. J. Clin. Nutr. 2002, 76, 560−
568.
(11) Le Marchand, L.; Murphy, S. P.; Hankin, J. H.; Wilkens, L. R.;
Kolonel, L. N. Intake of flavonoids and lung cancer. J. Natl. Cancer Inst.
2000, 92, 154−160.
(12) Wang, Y.; Stevens, V. L.; Shah, R.; Peterson, J. J.; Dwyer, J. T.;
Gapstur, S. M.; McCullough, M. L. Dietary flavonoid and
proanthocyanidin intakes and prostate cancer risk in a prospective
cohort of US men. Am. J. Epidemiol. 2014, 179, 974−986.
(13) Andrews, L. Dietary flavonoids for the prevention of colorectal
cancer. Clin. J. Oncol. Nurs. 2013, 17, 671−672.
(31) Ferry, D. R.; Smith, A.; Malkhandi, J.; Fyfe, D. W.; deTakats, P.
G.; Anderson, D.; Baker, J.; Kerr, D. J. Phase I clinical trial of the
flavonoid quercetin: pharmacokinetics and evidence for in vivo
tyrosine kinase inhibition. Clin. Cancer Res. 1996, 2, 659−668.
(32) Mulholland, P. J.; Ferry, D. R.; Anderson, D.; Hussain, S. A.;
Young, A. M.; Cook, J. E.; Hodgkin, E.; Seymour, L. W.; Kerr, D. J.
Pre-clinical and clinical study of QC12, a water-soluble, pro-drug of
quercetin. Ann. Oncol. 2001, 12, 245−248.
(33) Bast, A.; Haenen, G.; Bruynzeel, A.; Van der Vijgh, W.
Protection by flavonoids against anthracycline cardiotoxicity: from
chemistry to clinical trials. Cardiovasc. Toxicol. 2007, 7, 154−159.
U
dx.doi.org/10.1021/jm500853v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(34) Savoia, D. Plant-derived antimicrobial compounds: alternatives
to antibiotics. Future Microbiol. 2012, 7, 979−990.
inhibitors specific for lanosterol 14α-demethylase of fungi. J. Med.
Chem. 2003, 46, 474−485.
(35) Jandial, D. D.; Blair, C. A.; Zhang, S.; Krill, L. S.; Zhang, Y. B.;
Zi, X. Molecular targeted approaches to cancer therapy and prevention
using chalcones. Curr. Cancer Drug Targets 2014, 14, 181−200.
(36) Kumar, D.; Kumar, M.; Kumar, A.; Singh, S. K. Chalcone and
curcumin derivatives: a way ahead for malarial treatment. Mini-Rev.
Med. Chem. 2013, 13, 2116−2133.
(53) Kabbe, H.-J.; Widdig, A. Synthesis and reactions of 4-
chromanones. Angew. Chem., Int. Ed. 1982, 21, 247−256.
(54) Pouget, C.; Fagnere, C.; Basly, J.-P.; Leveque, H.; Chulia, A.-J.
Synthesis and structure of flavan-4-ols and 4-methoxyflavans as new
potential anticancer drugs. Tetrahedron 2000, 56, 6047−6052.
(55) Maiti, A.; Cuendet, M.; Croy, V. L.; Endringer, D. C.; Pezzuto, J.
M.; Cushman, M. Synthesis and biological evaluation of abyssinone II
and its analogues as aromatase inhibitors for chemoprevention of
breast cancer. J. Med. Chem. 2007, 50, 2799−2806.
(37) Bukhari, S. N. A.; Franzblau, S. G.; Jantan, I.; Jasamai, M.
Current prospects of synthetic curcumin analogs and chalcone
derivatives against Mycobacterium tuberculosis. Med. Chem. 2013, 9,
897−903.
(56) Vogel, S.; Heilmann, J. Synthesis, cytotoxicity, and antioxidative
activity of minor prenylated chalcones from Humulus lupulus. J. Nat.
Prod. 2008, 71, 1237−1241.
(57) Jung, D. H.; Lee, Y. R.; Kim, S. H. New synthetic routes to
biologically interesting geranylated flavanones and geranylated
chalcones: first total synthesis of ( )-prostratol F, xanthoangelol,
and ( )-lespeol. Helv. Chim. Acta 2010, 93, 635−647.
(38) Lin, Y.-M.; Zhou, Y.; Flavin, M. T.; Zhou, L.-M.; Nie, W.; Chen,
F.-C. Chalcones and flavonoids as anti-tuberculosis agents. Bioorg.
Med. Chem. 2002, 10, 2795−2802.
(39) Cabrera, M.; Simoens, M.; Falchi, G.; Lavaggi, M. L.; Piro, O. E.;
Castellano, E. E.; Vidal, A.; Azqueta, A.; Monge, A.; de Cerai
́
n, A. L.;
Sagrera, G.; Seoane, G.; Cerecetto, H.; Gonzalez, M. Synthetic
́
chalcones, flavanones, and flavones as antitumoral agents: Biological
evaluation and structure−activity relationships. Bioorg. Med. Chem.
2007, 15, 3356−3367.
(58) Tello, M.; Kuzuyama, T.; Heide, L.; Noel, J.; Richard, S. The
ABBA family of aromatic prenyltransferases: broadening natural
product diversity. Cell. Mol. Life Sci. 2008, 65, 1459−1463.
(59) Basarab, G. S.; Hill, P. J.; Garner, C. E.; Hull, K.; Green, O.;
Sherer, B. A.; Dangel, P. B.; Manchester, J. I.; Bist, S.; Hauck, S.; Zhou,
F.; Uria-Nickelsen, M.; Illingworth, R.; Alm, R.; Rooney, M.; Eakin, A.
E. Optimization of pyrrolamide topoisomerase II inhibitors toward
identification of an antibacterial clinical candidate (AZD5099). J. Med.
Chem. 2014, 57, 6060−6082.
(60) Nimesh, H.; Sur, S.; Sinha, D.; Yadav, P.; Anand, P.; Bajaj, P.;
Virdi, J. S.; Tandon, V. Synthesis and biological evaluation of novel
bisbenzimidazoles as Escherichia coli topoisomerase IA inhibitors and
potential antibacterial agents. J. Med. Chem. 2014, 57, 5238−5257.
(61) Cheng, B.; Cao, S.; Vasquez, V.; Annamalai, T.; Tamayo-
Castillo, G.; Clardy, J.; Tse-Dinh, Y.-C. Identification of anziaic acid, a
lichen depside from Hypotrachyna sp., as a new topoisomerase poison
inhibitor. PLoS One 2013, 8, e60770.
(40) Chiari, M. E.; Vera, D. M. A.; Palacios, S. M.; Carpinella, M. C.
Tyrosinase inhibitory activity of a 6-isoprenoid-substituted flavanone
isolated from Dalea elegans. Bioorg. Med. Chem. 2011, 19, 3474−3482.
(41) Tsuchiya, H.; Sato, M.; Miyazaki, T.; Fujiwara, S.; Tanigaki, S.;
Ohyama, M.; Tanaka, T.; Iinuma, M. Comparative study on the
antibacterial activity of phytochemical flavanones against methicillin-
resistant Staphylococcus aureus. J. Ethnopharmacol. 1996, 50, 27−34.
(42) Pouget, C.; Fagnere, C.; Basly, J.-P.; Besson, A.-E.; Champavier,
Y.; Habrioux, G.; Chulia, A.-J. Synthesis and aromatase inhibitory
activity of flavanones. Pharm. Res. 2002, 19, 286−291.
(43) Sun, D.; Hurdle, J. G.; Lee, R.; Lee, R.; Cushman, M.; Pezzuto, J.
M. Evaluation of flavonoid and resveratrol chemical libraries reveals
abyssinone II as a promising antibacterial lead. ChemMedChem 2012,
7, 1541−1545.
(44) Shiu, W. K. P.; Rahman, M. M.; Curry, J.; Stapleton, P.; Zloh,
M.; Malkinson, J. P.; Gibbons, S. Antibacterial acylphloroglucinols
from Hypericum olympicum. J. Nat. Prod. 2012, 75, 336−343.
(45) Shiu, W. K. P.; Malkinson, J. P.; Rahman, M. M.; Curry, J.;
Stapleton, P.; Gunaratnam, M.; Neidle, S.; Mushtaq, S.; Warner, M.;
Livermore, D. M.; Evangelopoulos, D.; Basavannacharya, C.; Bhakta,
S.; Schindler, B. D.; Seo, S. M.; Coleman, D.; Kaatz, G. W.; Gibbons, S.
A new plant-derived antibacterial is an inhibitor of efflux pumps in
Staphylococcus aureus. Int. J. Antimicrob. Agents 2013, 42, 513−518.
(46) Wu, X.; Alam, M. Z.; Feng, L.; Tsutsumi, L. S.; Sun, D.; Hurdle,
J. G. Prospects for flavonoid and related phytochemicals as nature-
inspired treatments for Clostridium difficile infection. J. Appl. Microbiol.
2014, 116, 23−31.
(62) Lin, H.; Annamalai, T.; Bansod, P.; Tse-Dinh, Y.-C.; Sun, D.
Synthesis and antibacterial evaluation of anziaic acid and its analogues
as topoisomerase I inhibitors. MedChemComm 2013, 4, 1613−1618.
(63) Basarab, G. S.; Manchester, J. I.; Bist, S.; Boriack-Sjodin, P. A.;
Dangel, B.; Illingworth, R.; Sherer, B. A.; Sriram, S.; Uria-Nickelsen,
M.; Eakin, A. E. Fragment-to-hit-to-lead discovery of a novel
pyridylurea scaffold of ATP competitive dual targeting type II
topoisomerase inhibiting antibacterial agents. J. Med. Chem. 2013,
56, 8712−8735.
̌
(64) Plaper, A.; Golob, M.; Hafner, I.; Oblak, M.; Solmajer, T.; Jerala,
R. Characterization of quercetin binding site on DNA gyrase. Biochem.
Biophys. Res. Commun. 2003, 306, 530−536.
(65) Russo, P.; Del Bufalo, A.; Cesario, A. Flavonoids acting on DNA
topoisomerases: recent advances and future perspectives in cancer
therapy. Curr. Med. Chem. 2012, 19, 5287−5293.
(66) Constantinou, A.; Mehta, R.; Runyan, C.; Rao, K.; Vaughan, A.;
Moon, R. Flavonoids as DNA topoisomerase antagonists and poisons:
structure−activity relationships. J. Nat. Prod. 1995, 58, 217−225.
(67) Black, M. T.; Stachyra, T.; Platel, D.; Girard, A.-M.; Claudon,
M.; Bruneau, J.-M.; Miossec, C. Mechanism of action of the antibiotic
NXL101, a novel nonfluoroquinolone inhibitor of bacterial type II
topoisomerases. Antimicrob. Agents Chemother. 2008, 52, 3339−3349.
(68) Hoshino, K.; Kitamura, A.; Morrissey, I.; Sato, K.; Kato, J.;
Ikeda, H. Comparison of inhibition of Escherichia coli topoisomerase
IV by quinolones with DNA gyrase inhibition. Antimicrob. Agents
Chemother. 1994, 38, 2623−2627.
(69) Kagawa, H.; Shigematsu, A.; Ohta, S.; Harigaya, Y. Preparative
monohydroxyflavanone syntheses and a protocol for gas chromatog-
raphy-mass spectrometry analysis of monohydroxyflavanones. Chem.
Pharm. Bull. 2005, 53, 547−554.
(70) Kuo, S.-C.; Lee, H.-Z.; Juang, J.-P.; Lin, Y.-T.; Wu, T.-S.; Chang,
J.-J.; Lednicer, D.; Paull, K. D.; Lin, C. M.; Hamel, E.; Lee, K.-H.
Synthesis and cytotoxicity of 1,6,7,8-substituted 2-(4′-substituted
phenyl)-4-quinolones and related compounds: identification as
(47) Simon, O.; Reux, B.; La Clair, J. J.; Lear, M. J. Total synthesis
confirms laetirobin as a formal Diels−Alder adduct. Chem.Asian J.
2010, 5, 342−351.
(48) Sarmah, M. P.; Shashidhar, M. S.; Sureshan, K. M.; Gonnade, R.
G.; Bhadbhade, M. M. Sulfonate protecting groups. Synthesis of O-
and C-methylated inositols: D- and L-ononitol, D- and L-laminitol,
mytilitol and scyllo-inositol methyl ether. Tetrahedron 2005, 61, 4437−
4446.
(49) Wallen, E. A. A.; Dahlen, K.; Grotli, M.; Luthman, K. Synthesis
of 3-aminomethyl-2-aryl-8-bromo-6-chlorochromones. Org. Lett. 2007,
9, 389−391.
(50) Poonia, N. S.; Chhabra, K.; Kumar, C.; Sharma, T. C.; Bhagwat,
V. W. Coordinative role of alkali cations in organic synthesis. 2. The
chalcone−flavanone system. J. Org. Chem. 1977, 42, 3311−3313.
́
(51) Friden-Saxin, M.; Pemberton, N.; da Silva Andersson, K.;
Dyrager, C.; Friberg, A.; Grotli, M.; Luthman, K. Synthesis of 2-alkyl-
substituted chromone derivatives using microwave irradiation. J. Org.
Chem. 2009, 74, 2755−2759.
(52) Ji, H.; Zhang, W.; Zhang, M.; Kudo, M.; Aoyama, Y.; Yoshida,
Y.; Sheng, C.; Song, Y.; Yang, S.; Zhou, Y.; Lu, J.; Zhu, J. Structure-
based de novo design, synthesis, and biological evaluation of non-azole
V
dx.doi.org/10.1021/jm500853v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
antimitotic agents interacting with tubulin. J. Med. Chem. 1993, 36,
1146−1156.
(71) Lee, R. E.; Hurdle, J. G.; Liu, J.; Bruhn, D. F.; Matt, T.;
Scherman, M. S.; Vaddady, P. K.; Zheng, Z.; Qi, J.; Akbergenov, R.;
Das, S.; Madhura, D. B.; Rathi, C.; Trivedi, A.; Villellas, C.; Lee, R. B.;
Rakesh; Waidyarachchi, S. L.; Sun, D.; McNeil, M. R.; Ainsa, J. A.;
Boshoff, H. I.; Gonzalez-Juarrero, M.; Meibohm, B.; Bottger, E. C.;
Lenaerts, A. J. Spectinamides: a new class of semisynthetic
antituberculosis agents that overcome native drug efflux. Nat. Med.
2014, 20, 152−158.
(72) Susceptibility Testing of Mycobacteria, Nocardiae, and Other
Aerobic Actinomycetes: Approved Standard. Clinical and Laboratory
Standards Institute (CLSI), Wayne, PA, U.S., 2003; Vol. 23, M24-A.
(73) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria
That Grow Aerobically: Approved Standard, 7th ed.; Clinical and
Laboratory Standards Institute (CLSI), Wayne, PA, U.S., 2006; Vol.
26, M7-A7.
(74) Rakesh; Bruhn, D.; Madhura, D. B.; Maddox, M.; Lee, R. B.;
Trivedi, A.; Yang, L.; Scherman, M. S.; Gilliland, J. C.; Gruppo, V.;
McNeil, M. R.; Lenaerts, A. J.; Meibohm, B.; Lee, R. E. Antitubercular
nitrofuran isoxazolines with improved pharmacokinetic properties.
Bioorg. Med. Chem. 2012, 20, 6063−6072.
(75) Hurdle, J. G.; O’Neill, A. J.; Chopra, I. Anti-staphylococcal
activity of indolmycin, a potential topical agent for control of
staphylococcal infections. J. Antimicrob. Chemother. 2004, 54, 549−
552.
(76) Cherian, P. T.; Wu, X.; Maddox, M. M.; Singh, A. P.; Lee, R. E.;
Hurdle, J. G. Chemical modulation of the biological activity of
reutericyclin: a membrane-active antibiotic from Lactobacillus reuteri.
Sci. Rep. 2014, 4, 4721 DOI: 10.1038/srep04721.
(77) Sorokin, E. P.; Cheng, B.; Rathi, S.; Aedo, S. J.; Abrenica, M. V.;
Tse-Dinh, Y.-C. Inhibition of Mg2+ binding and DNA religation by
bacterial topoisomerase I via introduction of an additional positive
charge into the active site region. Nucleic Acids Res. 2008, 36, 4788−
4796.
(78) Zhu, C.-X.; Tse-Dinh, Y.-C. The acidic triad conserved in type
IA DNA topoisomerases is required for binding of Mg(II) and
subsequent conformational change. J. Biol. Chem. 2000, 275, 5318−
5322.
(79) Peng, H.; Marians, K. J. Escherichia coli topoisomerase IV.
Purification, characterization, subunit structure, and subunit inter-
actions. J. Biol. Chem. 1993, 268, 24481−24490.
W
dx.doi.org/10.1021/jm500853v | J. Med. Chem. XXXX, XXX, XXX−XXX