Discovery and Characterization of Pure RhlR Antagonists against Pseudomonas aeruginosa Infections

Article abstract of DOI:10.1021/acs.jmedchem.0c00630

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic human pathogen that forms biofilms and produces virulence factors via quorum sensing (QS). Blocking the QS system in P. aeruginosa is an excellent strategy to reduce biofilm formation and the production of virulence factors. RhlR plays an essential role in the QS system of P. aeruginosa. We synthesized 55 analogues based on the chemical structure of 4-gingerol and evaluated their RhlR inhibitory activities using the cell-based reporter strain assay. Comprehensive structure-activity relationship studies identified the alkynyl ketone 30 as the most potent RhlR antagonist. This compound displayed selective RhlR antagonism over LasR and PqsR, strong inhibition of biofilm formation, and reduced production of virulence factors in P. aeruginosa. Furthermore, the survival rate of Tenebrio molitor larvae treated with 30 in vivo greatly improved. Therefore, compound 30, a pure RhlR antagonist, can be utilized for developing QS-modulating molecules in the control of P. aeruginosa infections.

Full text of DOI:10.1021/acs.jmedchem.0c00630

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Article
Discovery and characterization of pure RhlR antagonists
against Pseudomonas aeruginosa infections
SangJin Nam, So-Young Ham, Hongmok Kwon, Han-Shin Kim, Soohyun Moon, Jeong-
Hoon Lee, Taehyeong Lim, Sang-Hyun Son, Hee-Deung Park, and Youngjoo Byun
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00630 • Publication Date (Web): 22 Jul 2020
Downloaded from pubs.acs.org on July 24, 2020
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Discovery and characterization of pure RhlR antagonists
against Pseudomonas aeruginosa infections
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SangJin Nam,†,# So-Young Ham,‡,# Hongmok Kwon, Han-Shin Kim, Soohyun Moon,
†
§
†
Jeong-Hoon Lee, Taehyeong Lim, Sang-Hyun Son, Hee-Deung Park,‡,,*, and Youngjoo
‡
†
†
Byun†
,,*
†College of Pharmacy, Korea University, 2511 Sejong-ro, Jochiwon-eup, Sejong 30019,
Republic of Korea
‡School of Civil, Environmental and Architectural Engineering, Korea University, 145
Anam-ro, Seongbuk-Gu, Seoul 02841, Republic of Korea
§Korean Peninsula Infrastructure Cooperation Team, Korea Institute of Civil Engineering and
Building Technology (KICT), Goyang-si, Gyeonggi-do 10223, Republic of Korea
KU-KIST Graduate School of Converging Science and Technology, Korea University, 145
Anam-ro, Seongbuk-Gu, Seoul 02841, Republic of Korea
Biomedical Research Center, Korea University Guro Hospital, 148 Gurodong-ro, Guro-gu,
Seoul 08308, Republic of Korea
#
Equal contribution
*
Corresponding authors
Youngjoo Byun, Ph.D.
College of Pharmacy, Korea University, 2511 Sejong-ro, Jochiwon-eup, Sejong 30019,
Republic of Korea
Tel.: +82-44-860-1619; Fax: +82-44-860-1607; E-mail: yjbyun1@korea.ac.kr
Hee-Deung Park, Ph.D.
School of Civil, Environmental and Architectural Engineering, Korea University, 145 Anam-
ro, Seongbuk-Gu, Seoul 02841, Republic of Korea
Tel.: +82-2-3290-4861; Fax: +82-2-928-7656; E-mail: heedeung@korea.ac.kr
Key words: Pseudomonas aeruginosa, RhlR, Antagonist, Biofilm, Virulence factor
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ABSTRACT
Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic human pathogen that
forms biofilms and produces virulence factors via quorum sensing (QS). Blocking the QS
system in P. aeruginosa is an excellent strategy to reduce biofilm formation and the
production of virulence factors. RhlR plays an essential role in the QS system of P.
aeruginosa. We synthesized 55 analogs based on the chemical structure of 4-gingerol and
evaluated their RhlR inhibitory activities using cell-based reporter strain assay.
Comprehensive structure-activity relationship studies identified the alkynyl ketone 30 as the
most potent RhlR antagonist. This compound displayed selective RhlR antagonism over
LasR and PqsR, strong inhibition of biofilm formation, and reduced production of virulence
factors in P. aeruginosa. Furthermore, the survival rate of Tenebrio molitor larvae treated
with 30 in vivo greatly improved. Therefore, compound 30, a pure RhlR antagonist, can be
utilized for developing QS-modulating molecules in the control of P. aeruginosa infections.
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INTRODUCTION
Pseudomonas aeruginosa (P. aeruginosa) is a causative human pathogen that
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induces chronic disease in immune-compromised patients. It induces infections in burn
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wound, cystic fibrosis, acute leukemia, organ transplantation, and intravenous-drug
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addiction patients. The World Health Organization (WHO) categorizes it as a critical
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priority pathogen that requires considerable attention. P. aeruginosa is resistant to
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conventional antibiotic treatments mostly due to the formation of thick biofilm. It can
colonize on various surfaces by forming biofilms in which bacterial cells embedded within
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self-produced exopolysaccharides. Biofilms are widely found in medical, dental,
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agricultural, industrial, and environmental settings. In particular, biofilms in medical
settings are associated with about 80% of bacterial infections in humans and increased
antibiotic resistance.7
Quorum sensing (QS) is a bacterial cell-to-cell communication process that occurs
via chemical signal molecules and allows bacteria to share information in response to
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environmental changes. Once bacterial density reaches a certain threshold, signaling
molecules (autoinducers) bind to their cognate receptor proteins and alter gene expression
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to regulate collective behaviors. P. aeruginosa possesses three major QS systems (las, rhl,
and pqs) that are tightly interconnected with each other. Gram-negative bacteria including
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P. aeruginosa use N-acyl-L-homoserine lactones (AHLs) as autoinducers of QS. Typically,
AHLs are produced by LuxI-type synthases (e.g., LasI and RhlI) and recognized by
cytoplasmic LuxR-type receptors (e.g., LasR and RhlR). LasI and RhlI produce N-(3-oxo-
dodecanoyl)-L-homoserine lactone (OdDHL, 1a) and N-butyryl-L-homoserine lactone (BHL,
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b) as autoinducers, respectively (Figure 1). In addition to these systems, pqs circuit is the
third system regulated via Pseudomonas quinolone signal (PQS) recognized by PqsR. These
three systems are hierarchically controlled. The las system activates rhl and pqs systems,
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and the rhl system represses the pqs system. This interactive signal-network regulation of P.
aeruginosa leads to biofilm formation, the production of virulence factors, and the
modulation of host immune response.12 Therefore, small molecules that modulate the
recognition of autoinducers toward their cognate receptors have the potential to control
virulence factors and biofilm formation of P. aeruginosa.13
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Most QS inhibitors of P. aeruginosa focus on targeting LasR because it is located
at the top of the P. aeruginosa QS network hierarchy.14-16 However, even though RhlR also
plays an important role in the QS process of P. aeruginosa, RhlR modulators have rarely
been reported.17-20 Up to date, few modulators for the interaction between BHL and RhlR as
agonists or antagonists have been reported.21-24 Blackwell and co-workers reported that BHL
analogs with RhlR agonism activity reduced the production of P. aeruginosa virulence
factors such as pyocyanin.25, 26 They also reported mixed LasR/RhlR antagonists which
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increased pyocyanin production. Bassler and co-workers recently conducted an experiment
with BHL-independent and active rhlR mutants, which showed that RhlR drives biofilm
formation and production of virulence factors in P. aeruginosa.27
Previously, we reported that (S)-6-gingerol (1c, Figure 1) from ginger extracts is a
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moderate LasR antagonist of P. aeruginosa. It reduced biofilm formation, production of
virulence factors, and expression of QS-related genes in P. aeruginosa. We performed
comprehensive structure-activity relationship (SAR) studies of gingerol analogs and
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identified the LasR antagonist that was more potent than (S)-6-gingerol. Based on our
previous studies, we hypothesized that gingerols with alkyl chain shorter than 1c might bind
to RhlR because of their structural similarity to BHL. We describe the design, synthesis, and
biochemical characterization of gingerol analogs as pure RhlR antagonists. We have
established SAR of gingerol analogs by extensively modifying their chemical structures. The
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pure and potent RhlR antagonist which we discovered in this study can be utilized in the
discovery of novel agents that inhibit biofilm formation and production of virulence factors,
as well as in the elucidation of rhl-RhlR mechanism in QS network of P. aeruginosa.
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Figure 1. Structures of endogenous and synthetic molecules interacting with the QS receptor
of Pseudomonas aeruginosa.
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RESULTS AND DISCUSSION
As an attempt to discover pure RhlR antagonists, we first screened the relative RhlR
activities of in-house gingerol analogs with various alkyl chain lengths, from 4-gingerol to 10-
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gingerol (See Supporting Scheme S1), which were previously reported. We used DMSO as
a negative control and (S)-6-gingerol (1c) and N-cyclopentylbutyramide (1d) (Figure 1) as
positive controls in in vitro biological evaluation studies. As shown in Table 1, the more potent
RhlR antagonism activities increased with shorter alkyl chain length of gingerols, suggesting
that gingerols with the shorter alkyl chain have a higher affinity for RhlR. Interestingly, all
tested gingerols showed very low RhlR agonistic activity (<4%). Compound 2 (4-gingerol)
with the shortest alkyl chain in the series was the most potent with a relative RhlR activity of
54%. As the chemical structure of 2 is more similar to that of BHL than the others, it was
assumed that it competed against BHL for binding to RhlR. Based on preliminary results, we
used 4-gingerol as an initial hit compound for further structural modification.
Table 1. Relative RhlR activity (%) of the synthesized compounds.
Relative RhlR activity
Relative RhlR activity
Relative RhlR activity
Compounda
Compound
Compound
Antagonsimb
Agonismc
Antagonsim
Agonism
Antagonsim
Agonism
1
c
75**
3**
11p
11q
11r
12a
12b
12c
12d
12e
12f
72**
2**
14b
15a
15b
16a
21
49**
2**
54**
4**
43**
1**
55**
5**
1
d
2
54**
3**
74**
3**
41**
2**
62**
2**
69**
4**
61**
5**
3
4
76**
4**
88*
4**
29**
3**
83**
3**
90*
2**
32**
3**
5
23a
23b
23c
25
6
90*
2**
64**
3**
27**
3**
41**
2**
81**
2**
34**
3**
7
8
36**
2**
71**
2**
79**
3**
65**
4**
51**
2**
45**
2**
1
1a
12g
12h
12i
27
1
1b
1c
93*
94
4**
2**
69**
76**
2**
1**
30
12**
16**
4**
4**
1
31
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1d
11e
1f
11g
51**
78**
55**
33**
1**
2**
1**
1**
12j
12k
12l
74**
47**
94
1**
2**
1**
1**
32
17**
32**
25**
38**
3**
3**
3**
3**
33a
33b
33c
34a
34b
34c
35a
35b
35c
1
12m
12n
12o
12p
12q
12r
13a
13b
14a
73**
1
1
1
1
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1
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1
1
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0
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0
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1
1h
64**
1**
98
1**
71**
4**
65**
2**
49**
1**
67**
3**
1
1i
1
1j
53**
2**
82**
3**
64**
2**
43**
3**
79**
1**
74**
3**
1
1k
1l
1m
1n
1o
1
69**
1**
85**
3**
72**
3**
60**
2**
51**
4**
67**
4**
1
1
91
1**
41**
2**
20**
2**
55**
4**
1
a
b
DMSO (negative control) and 1c and 1d (positive controls) were used. RhlR antagonism
c
activity of the compound (100 µM) in the presence of 1b (10 µM). RhlR agonism activity of
the compound (100 µM). (**) P<0.005 and (*) P<0.05 as compared with the control.
To establish SAR of 4-gingerol for RhlR antagonism, we performed structural
modification as follows: 1) variation of the substituents in the phenyl ring, 2) introduction of
double or triple bond between the phenyl ring and the carbonyl group to increase rotational
rigidity, 3) removal of β-hydroxyl group, and 4) change of absolute configuration. First, we
synthesized 34 derivatives to determine the effect of the substituents in the phenyl ring on RhlR
antagonism. 3'-OMe and 4'-OH groups of the phenyl ring in 4-gingerol were replaced with
diverse functional groups (-F, -Cl, -OH, -OMe, -OEt, -Me, and -N(CH ) ). Scheme 1 describes
3
2
the synthesis of 4-gingerol derivatives with various substituents of the phenyl ring. Compounds
0a–10r were synthesized from commercial benzaldehydes by treating 10% NaOH in acetone
1
and ethanol (or water) at 25 ℃. Reaction of 10a–10r with diisopropylamide (LDA) at –78 ℃,
followed by addition of n-butanal afforded compounds 11a–11r in 24–60% yield. Low-to-
moderate yield in this step might be because of side reactions such as elimination or dimer
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formation. Compounds 12a–12r were obtained in 40–93% yield by reducing 11a–11r with
hydrogen gas in the presence of 10% Pd/C.
a: X = OMe, Y = OH, Z = H
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O
O
OH
O
b: X = H, Y = H, Z = H
c: X = OMe, Y = H, Z = H
d: X = H, Y = OH, Z = H
e: X = OH, Y = H, Z = H
f: X = H, Y = OMe, Z = H
g: X = H, Y = F, Z = H
h: X = H, Y = Cl, Z = H
i: X = H, Y = Me, Z = H
X
Y
X
Y
X
Y
i
ii
H
Z
Z
Z
9
10a10r
11a11r
iii
j: X = H, Y = NMe , Z = H
2
k: X = OH, Y = OH , Z = H
l: X = OMe, Y = OMe, Z = H
m: X = F, Y = OH, Z = H
n: X = OEt, Y = OH, Z = H
o: X = F, Y = F, Z = H
p: X = Me, Y = Me, Z = H
q: X = OMe, Y = F, Z = H
r: X = F, Y = H, Z = OMe
O
OH
X
Y
Z
1
2a12r
Scheme 1. Synthesis of 4-gingerol derivatives with the variation of phenyl ring. Reagents and
conditions: (i) Acetone, EtOH (or water), 10% NaOH, rt, 2 to 24 h, 32%–quantitative yield;
(
ii) LDA 1.0 M in THF/hexanes, n-butanal, THF, –78 ℃, 2 to 12 h, 24–60% yield; (iii) H gas,
2
10% Pd/C, MeOH, rt, 2 h, 43–93% yield.
As summarized in Table 1, compound 11b without any substituent in the phenyl region,
completely lost its RhlR antagonistic property with a relative RhlR activity of 93%, indicating
that a polar functional group in the phenyl ring is required for binding to RhlR. To assess the
necessity of 4'-OH and 3'-OMe groups in the phenyl ring for RhlR antagonism, we removed
the -OH group at 4'-position (11c) and the -OMe group at 3'-position (11d). As shown in Table
1
, compound 11c displayed a reduced ability to inhibit RhlR (relative 94% RhlR activity).
However, compound 11d showed slightly stronger RhlR antagonism than 11a (51% vs. 65%).
However, compound 11f with an -OMe group at 4'-position was less potent than 11d, implying
that the presence of the polar group at 4'-position is favorable for RhlR-binding affinity. In the
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case of the 3'-position, compound 11e with an -OH group only showed stronger RhlR
antagonism than compound 11c with an -OMe group only (78% vs. 94%).
To expand the initial SAR of 4-gingerol, we synthesized a variety of mono-substituted
(11g–11j) and di-substituted (11k–11r) analogs. Among the mono-substituted compounds, 11g
with 4'-F was the most potent with a relative RhlR activity of 33%. Interestingly, compound
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1j (53%) with a bulky 4'-N(CH ) was more potent than 11a, while 11h (64%) with 4'-Cl and
3 2
11i (65%) with 4'-Me were comparable to 11a. A similar trend was observed with the di-
substituted compounds. Compound 11o with 3',4'-di-F was the most potent in this series
(relative RhlR activity, 20%). Notably, these trends indicated that the F-substitution in the
phenyl ring is preferred for the structural modification of the phenyl ring. Compound 11q (43%)
with 3'-OMe and 4'-F was more active than 11a with 3'-OMe and 4'-OH, suggesting that the F
group at 4'-position can replace the OH group of 4-gingerol. However, compound 11r (74%)
with 3'-F and 5'-OMe was less potent than 11a, confirming the necessity of the small and polar
group (i.e., -F and -OH) at 4'-position. As expected, the hydrophobic -Me group (11p, 72%) at
both 3'- and 4'-position showed decreased RhlR inhibitory activity. Compound 11n with 3'-
OEt and 4'-OH dramatically decreased the antagonistic activity of RhlR (91%), implying that
the region of RhlR which interacts with the 3'-position of 4-gingerol was highly sensitive to
the substituent size. SAR results indicated that the 4'-position is related to hydrogen-bonding
interaction with RhlR, while the 3'-position is sensitive to the bulkiness of the substituent.
The effect of rotational rigidity on RhlR antagonism was evaluated by comparing the
compounds (11a–11r) with a double bond between the phenyl ring and the carbonyl group
with the corresponding compounds (12a–12r) with a single bond. As shown in Table 1, the
compounds with a double bond (11a–11r) displayed stronger RhlR antagonism than those with
a single bond (12a–12r), irrespective of the substituents in the phenyl ring. This result implied
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5
5
5
5
5
5
5
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that the restricted rotational flexibility between the phenyl ring and the carbonyl group
significantly increased binding affinity for RhlR. Overall, the double-bond compound 11o,
substituted with the F group at both 3'- and 4'-position, showed the most potent RhlR
antagonism with low RhlR agonist activity (2%) among the compounds with variations in the
phenyl ring.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
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1
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0
1
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6
7
8
9
0
The synthesis of 4-gingerol derivatives with variations in the middle section is outlined
in Scheme S1 (See supporting information). Compounds (13a–13b) without β-hydroxyl group
were synthesized by reacting 2-heptanone with vanillin or 3,4-difluorobenzaldehyde under
basic conditions at 25 ℃ for 72 h in 30–50% yield. The α,β-unsaturated carbonyl group of 13a–
1
3b was reduced by two different reagents: 1) H and Pd/C, and 2) NaBH . The double bond
2 4
was reduced to the single bond via catalytic hydrogenation, providing compounds 14a–14b in
76% yield. The carbonyl group was reduced to the secondary alcohol by treatment with
~
NaBH to afford compounds 15a and 15b in 94% and 90% yield, respectively. Compound 15a
4
was reduced to the single-bond compound 16a in 60% yield. As shown in Table 1, compound
1
3b (41%) without the β-hydroxyl group was more potent than 11a (65%), indicating that the
β-hydroxyl group was not essential for binding to RhlR. The α,β-unsaturated carbonyl analog
(13b) was more potent than the corresponding α,β-saturated one (14b). The reduction of the
α,β-unsaturated carbonyl group to alcohol (15b, 41%) maintained the RhlR-binding affinity.
However, the RhlR antagonistic activities of 3,4-difluorophenyl ring analogs (13a–15a) were
almost similar.
Gingerol derivatives with a triple bond in the middle section was prepared by applying
synthetic route described in Scheme 2. The phenolic OH of vanillin was protected with the tert-
butyldimethylsilyl (TBDMS) group, affording compound 17. Reaction of 17 with CBr and
4
PPh in dichloromethane provided the dibromo alkene 18 in 98% yield. Elimination and
3
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lithiation of 18 in the presence of 3 equivalents of n-BuLi afforded the terminal alkyne 19 in
3
0
9
6% yield. Compound 20 was obtained by reacting 19 with Weinreb amide. The alkynyl
ketone 21 was prepared from 20 by using tetrabutylammoniumfluoride (TBAF) in 94% yield.
Enantiomerically-enriched alkynyl alcohol analogs (23b–23c) were prepared from 20 by using
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
chiral
catalysts
such
as
RuCl[(R,R)-TsDPEN(mesitylene)]
or
RuCl[(S,S)-
3
1
TsDPEN(mesitylene)] , followed by desilylation with TBAF. Compound 23a, a racemic
mixture, was obtained from 20 by treatment of NaBH and subsequent desilylation. The alkynyl
4
alcohols (23a–23c) were analyzed by chiral reversed-phase (RP)-HPLC. As compound 23a
was a racemic mixture, two distinct peaks were observed at 9.91 min and 12.58 min in chiral
HPLC with a 1:1 ratio (See supporting information). Compound 23b with (R)-configuration
was eluted at 12.54 min while compound 23c with (S)-configuration was eluted at 9.88 min
with ee values >99% (Supporting Figure S1). Z-selective reduction of the alkyne (22a) to cis-
isomer (25) was accomplished by the treatment of Lindlar catalyst and 1,4-benzoquinone under
32
an atmosphere of hydrogen, followed by desilylation with TBAF. The alkanyl alcohol 27 was
obtained from 22a in 94% yield via catalytic hydrogenation and subsequent desilylation.
O
O
O
O
Br
O
O
H
H
i
ii
iii
Br
O
O
O
HO
Si
Si
Si
17
18
19
O
O
vii
v
v
O
OH
O
HO
HO
HO
25

Si
O
O
2
4
iv
vi
O
O
OH
OH
Si
Si
2
2
2a: racemic
2b: (R)-configuration
O
O
20
viii
22c: (S)-configuration
O
HO
Si
v
v
26
27
O
OH

O
O
HO
HO
21
23a: racemic
23b: (R)-configuration
23c: (S)-configuration
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2
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3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 2. Synthesis of 4-gingerol derivatives with the variation of rotational rigidity and
absolute configuration. Reagents and conditions: (i) tert-Butyldimethylsilyl chloride,
imidazole, CH Cl , rt, 18 h, 94% yield; (ii) PPh , CBr , CH Cl , rt, 3 h, 98% yield; (iii) n-
2
2
3
4
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
BuLi 1.6 M in hexanes (3.0 eq.), THF, rt, 2 h, 96% yield; (iv) n-BuLi 1.6 M in hexanes (1.1
eq.), N-methoxy-N-methylhexanamide, THF, –78 ℃ to rt, 16 h, 75% yield; (v) n-Bu NF, 0.1
4
M in THF, rt, 1 h, 94% yield (for 21), 94% yield (for 23a), 94% yield (for 23b), 93% yield
(
for 23c), 70% yield (for 25), and 94% yield (for 27); (vi) NaBH , MeOH, rt, 1 h, 77% yield
4
(for 22a), RuCl[(R,R)-TsDPEN(mesitylene)], KOH, 2-propanol, 4 h, 88% yield (for 22b),
and RuCl[(S,S)-TsDPEN(mesitylene)], KOH, 2-propanol, 4 h, 68% yield (for 22c); (vii)
Lindlar cat., H gas, 1,4-benzoquinone, MeOH, 0 ℃, 1 h (from 22a); (viii) H gas, 10% Pd/C,
2
2
MeOH, rt, 2 h, 94% yield (from 22a).
The alkynyl ketone 21 displayed strong relative RhlR activity (29%) than the
corresponding alkenyl ketone 13b (41%) and alkanyl ketone 14b (49%) (Table 1). Similarly,
the alkynyl alcohol 23a (32%) was also more potent than the alkene 15b (41%) and the alkane
27 (45%). The relative RhlR activities of the alkynyl ketone (21) or the alkyny alcohol
compounds (23a–23c) were <34%. They were more potent than the reported RhlR antagonist
(
1d, 54%). Regarding the effect of absolute configuration on RhlR affinity, the (R)-isomer 23b
27%) was stronger than the corresponding (S)-isomer 23c (34%) and the racemate 23a (32%).
(
The (Z)-alkenyl alcohol 25 with a relative RhlR activity of 79% was much weaker than the (E)-
alkenyl alcohol 15b (41%), implying that the cis-isomer may not be properly located in the
RhlR active site. Overall, SAR data indicated that the carbonyl group or the hydroxyl group at
the γ-position from the phenyl group is important for binding to RhlR.
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Based on the SAR studies of 4-gingerol analogs, we synthesized 3,4-difluorophenyl
derivatives which showed the most potent RhlR-binding activity in the variation of the phenyl
ring. As described in Scheme 3, the alkynyl ketones (30–32) and the alkynyl alcohols (33a–
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3c, 34a–34c, and 35a–35c) were prepared from commercial 3,4-difluorobenzaldehyde by
applying the same synthetic strategy used for 4-gingerol derivatives. The purity and ee ratio of
the final compounds were analyzed using chiral HPLC (See Supporting Figure S2). The
alkynyl ketones (30–32) were more potent than the corresponding alkene and alkane
compounds, with relative RhlR activities <17% at 100 μM. In addition, the (R)-stereoisomers
(33b–35b) were also more active than the corresponding (S)-stereoisomers (33c–35c) or the
racemates (33a–35a). Furthermore, the compounds with the shorter alkyl chain length (30 and
3
3
3a–33c) were stronger than the corresponding ones with the longer alkyl chain lengths (31,
2, 34a–34c, and 35a–35c). The alkynyl ketone with the n-propyl group (30) displayed the
most potent activity in this series, with a relative RhlR activity of 12%, which is a much
stronger activity profile than the reported antagonist 1d (54%).
O
O
F
F
Br
F
F
F
F
n
F
F
i
ii
iii
H
Br
30: n = 1
1: n = 2
2: n = 3
3
3
28
29
33a: n=1, racemic
OH
33b
: n=1, (R)-configuration
3
3c: n=1, (S)-configuration

n
F
F
34a: n=2, racemic
iv
3
3
3
3
4b: n=2, (R)-configuration
4c: n=2, (S)-configuration
5a: n=3, racemic
5b: n=3, (R)-configuration
35c: n=3, (S)-configuration
Scheme 3. Synthesis of 3,4-difluorophenyl derivatives. Reagents and conditions: (i) PPh3,
CBr , CH Cl , rt, 3 h, 97% yield; (ii) n-BuLi 1.6 M in hexanes (3.0 eq.), THF, rt, 2 h; (iii) n-
4
2
2
BuLi 1.6 M in hexanes (1.1 eq.), appropriate Weinreb amides, THF, –78 ℃ to rt, 16 h, 72%
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
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5
5
5
5
5
5
6
yield (for 30), 88% yield (for 31), and 72% yield (for 32); (iv) NaBH , MeOH, rt, 1 h (for
4
3
3a–35a), 88% yield (for 33a), 80% yield (for 34a), and 86% yield (for 35a), RuCl[(R,R)-
TsDPEN(mesitylene)], KOH, 2-propanol, 4 h (for 33b–35b), 86% yield (for 33b), 82% yield
for 34b), and 80% yield (for 35b), and RuCl[(S,S)-TsDPEN(mesitylene)], KOH, 2-propanol,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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6
7
8
9
0
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
4
h (for 33c-35c), 86% yield (for 33c), 82% yield (for 34c), and 82% yield (for 35c).
To elucidate the binding mode of compound 30 in RhlR, we performed in silico
molecular docking studies of 30 and 1d with a RhlR homology model. Compound 30 interacted
with RhlR via a π-π stacking interaction with Tyr72 and a hydrogen-bond with Trp 68, while
1d made hydrogen-bonding interactions with Asp81, Thr121, and Ser135 (Figure 2). Trp 68
26
was reported as one of key amino acids in a BHL-independent and active rhlR mutants. As
the alkyl chain length gets longer (30 vs. 31 and 32), the phenyl moiety made weaker π-π
stacking interactions with Tyr72 (See Supporting Figure S3), elucidating the importance of the
π-π stacking interaction in 3,4-difluorophenyl analogs.
Figure 2. The docked poses of 30 (yellow) and 1d (pink) with the RhlR homology model. Red
and black dotted lines represent - stacking and hydrogen-bond interactions, respectively.
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Table 2. In vitro biological data of the representative compounds.
O
OH
O
OH
O
OH
O
OH
O
O
O
HO
HO
F
F
F
F
HO
11a
11c
11k
11o
13a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
OH
OH
OH
F
F
F
F
F
F
F
F
F
F
F
F
30
31
32
33a
33b
33c
Relative RhlR activity (%)
Antagonismb Agonismc
Relative biological activity (%)
Biofilm formationd Bacterial growthe Rhamnolipid productionf
Compounda
1
0
100
100
1000
10
86
84
82
100
10
100
101
98
10
100
86*
75**
3**
5**
71**
96*
76*
68*
1
c
1
d
74*
54**
4**
37**
69**
95*
102
96
58**
45**
73**
65**
4**
5**
65**
73*
57*
1
1a
1c
1k
95
1
89
94
2**
4**
88
73**
95*
91
83
60**
43**
3**
5**
84*
72**
104*
56*
50*
1
99
1
1
1o
3a
60**
20**
2**
5**
72*
53**
101
103
103
100
100
100
98
105*
103
63*
50**
53**
51**
4**
3**
80*
61**
64**
62*
3
0
1
2
31**
12**
4**
4**
68*
47**
99
57**
42*
29**
16**
4**
6**
75*
55**
89**
59**
49**
3
3
39**
17**
3**
5**
71*
56**
88**
60**
58**
45**
32**
3**
5**
73**
60**
104*
73*
49**
3
3a
3b
3c
3
45**
59**
25**
38**
3**
3**
5**
5**
74*
79*
60**
64**
102
99
60*
59*
48**
52**
3
98
a
b
DMSO (negative control) and 1c and 1d (positive controls) were used. RhlR antagonism
c
activity of the compound (10 or 100 µM) in the presence of 1b (10 µM). RhlR agonism
d
activity of the compound (100 or 1000 µM). Biofilm formed by P. aeruginosa at 10 or 100
e
µM compound in static conditions. Growth of P. aeruginosa at 10 or 100 µM compound for
f
2
4 h. Rhamnolipid produced by P. aeruginosa at 10 or 100 µM compound. (**) P<0.005 and
(*) P<0.05 as compared with the control.
To validate the SAR results and compare the activities under the same experimental
conditions, we selected 11 representative compounds (11a, 11c, 11k, 11o, 13a, 30, 31, 32, 33a,
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
3b, and 33c) and determined RhlR antagonism, RhlR agonism, static biofilm formation,
bacterial growth, and rhamnolipid production (Table 2). The removal of the OH group at the
4'-position of the phenyl ring (11c) significantly reduced the RhlR antagonism activity profile.
In contrast, replacement of -OMe at the 3'-position of the phenyl ring with -OH (11k) increased
RhlR antagonism. Comparison of 11o and 13a confirmed that the β-hydroxyl group in the
middle section is not essential for RhlR activity. The alkynyl ketone compounds (30–32) with
restricted rotational flexibility exhibited increased RhlR activity. We also confirmed that the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(R)-alkynol (33b) is a better RhlR antagonist than the racemate (33a) and the (S)-alkynol (33c).
Next, we determined the RhlR agonism activity of the selected compounds. As summarized in
Table 2, none of the synthesized compounds showed RhlR agonism activity even at 1000 μM.
However, the reported RhlR antagonist 1d exhibited RhlR agonism with a relative RhlR
agonistic activity of 37% at 1000 μM. Compared with the control, compounds 11o, 13a, 30,
31, and 32 significantly inhibited biofilm formation at 100 μM. In particular, compound 30
showed approximately 47% relative biofilm formation of P. aeruginosa, which is much more
potent than (S)-6-gingerol (1c) and the known RhlR antagonist (1d). Next, we measured the
effect of bacterial growth inhibition by the compounds at 10 and 100 μM concentrations.
Compounds 31 and 32 slightly inhibited bacterial growth at 100 μM. However, compound 30
had no effect on bacterial growth at the same concentration. Furthermore, we examined the
3
3
production of rhamnolipid, a representative virulence factor in P. aeruginosa. RhlR is known
to directly regulate the expression of rhamnolipid production enzymes. As shown in Table 2,
the most potent RhlR antagonist 30 (13% of relative RhlR activity) displayed the lowest
rhamnolipid production (42%) among the selected compounds. Moreover, compound 30
showed stronger inhibition activities on biofilm formation and rhamnolipid production without
inhibiting bacterial growth than the other compounds (7, 8, 11d, 11f, 21, 23a, 23b, and 23c)
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with high RhlR activity but not included in the representative set (See Supporting Information
Table S1).
Next, we performed the comprehensive biological assays of the most potent RhlR
antagonist (30) against P. aeruginosa, using 1c, 1d, and 4-gingerol (12a) as positive controls.
First, the RhlR IC values of all four compounds were measured at different concentrations
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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(
0–100 μM). The IC of 1c, 1d, 12a, and 30 were 945, 86, 218, and 26 μM, respectively (Figure
50
3A), confirming that compound 30 was the most potent RhlR antagonist. We also determined
the agonist and antagonist activities of the compounds against LasR and PqsR as las and pqs
1
2
systems are also involved in the QS processes of P. aeruginosa. All four compounds showed
no LasR- and PqsR-agonistic activity nor PqsR-antagonism activity at 0.1 and 10 μM,
respectively (Figures 3B and 3C). However, (S)-6-gingerol (1c) displayed moderate LasR
antagonism as previously reported.28 4-Gingerol (12a) exhibited no LasR antagonism,
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9
confirming the result of our previous study. The most potent RhlR antagonist 30 showed high
selectivity for RhlR over LasR and PqsR, indicating that it is a pure RhlR antagonist.
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Figure 3. In vitro biological activities of compounds 1c, 1d, 12a, and 30. DMSO (negative
control) and 1c, 1d, and 12a (positive controls) were used. (A) Relative RhlR activity dose-
response curves. (B) Relative QS receptor (LasR and PqsR) agonism activity at 0.1 and 10 μM
of compound, respectively. (C) Relative QS receptor (LasR and PqsR) antagonism activity at
0
.1 and 10 μM of compound, respectively. (D) Static biofilm formation of P. aeruginosa at 10
or 100 μM of compound. (**) P<0.005 and (*) P<0.05 as compared with the control.
As shown in Figure 3D, all four compounds strongly inhibited P. aeruginosa biofilm
formation by 14–32% (10 μM) and 29–53% (100 μM), without affecting bacterial growth (See
Supporting Figure S4). Dynamic biofilm inhibition by using a drip-flow reactor was analyzed
at 48 h of the reactor operation. As shown in Figure 4A, control condition displayed typical
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mushroom morphology with 44.15 μm /μm volume and 53.90 μm thickness. However,
treatment with the other compounds (1c, 1d, and 30) showed thinner and sparser formation of
biofilm, as well as smaller volume and thickness, than that with the control. In particular,
compound 30 inhibited biofilm formation by 74% and decreased the amount of carbohydrate
and protein by 39 and 72%, respectively, as compared with the control (Figures 4B and 4C).
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Figure 4. Biofilm formation by P. aeruginosa with compounds 1c, 1d, and 30 at 10 μM in
flow conditions. DMSO (negative control) and 1c, 1d, and 12a (positive controls) were used.
(A) CLSM images with biofilm volume and thickness. (B) The amount of carbohydrate in EPS.
(
C) The amount of protein in EPS. (**) P<0.005 and (*) P<0.05 as compared with the control.
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In the next virulence production experiment, compound 30 reduced significantly
rhamnolipid production by P. aeruginosa at 10 and 100 M concentrations (Figure 5A),
indicating that RhlR antagonists can regulate rhamnolipid production and play an important
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role in biofilm development stages. In general, rhamnolipid increases the hydrophobicity of
cells and bacterial twitching motility, affecting attachment and maturation in biofilm formation
stages.35 P. aeruginosa rhlA mutants which are down-regulated for the synthesis of
36
rhamnolipid did not form mushroom-like biofilms, suggesting that RhlR antagonist-induced
reduction of rhamnolipid production may control biofilm formation in P. aeruginosa.
Figure 5. The production of virulence factors and expression of QS-induced genes by P.
aeruginosa with compounds 1c, 1d, 12a, and 30. DMSO (C, negative control) and 1c, 1d, and
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2a (positive controls) were used. (A) Rhamnolipid production by wild-type P. aeruginosa at
0 or 100 μM compound. (B) Relative rhlA expression of biofilm cells at 10 μM compound.
1
(C) Pyocyanin production by wild-type P. aeruginosa and rhlR mutants at 10 μM compound.
D) Relative QS-related gene expression of wild-type P. aeruginosa and rhlR mutants. (**)
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(
P<0.005 and (*) P<0.05 as compared with the control.
We hypothesized that compound 30-induced inhibition of biofilm formation and
virulence factor production are closely associated with the rhl system. To verify this hypothesis,
we measured rhamnolipid synthesis gene (rhlA) expression in compound 30-treated biofilm
cells using RT-qPCR (Figure 5B). The RhlR antagonists (1d, 12a, and 30) down-regulated
rhlA expression of biofilm cells more significantly (65–79%) than compound 1c (38%),
indicating that rhlA expression can be controlled by RhlR antagonists. These RT-qPCR results
demonstrated that compound 30 could inhibit P. aeruginosa biofilm formation and virulence
factor production by down-regulating the rhlA expression of P. aeruginosa. However, the rhl
system was reported to negatively modulate the pqs system, leading to increased pyocyanin
1
2
production. Considering the relationship between the rhl and pqs systems, Blackwell and co-
workers developed an RhlR agonist and used it as an anti-virulence strategy against
26
pathogens. The RhlR agonist strongly inhibited pyocyanin production by suppressing pqs
signaling. Based on their results, we expected that RhlR antagonists would increase pyocyanin
production, thus negatively affecting biofilm formation and virulence production. Surprisingly,
rhlR mutants produced less pyocyanin than wild-type P. aeruginosa as shown in Figure 5C. In
addition, compound 30 reduced pyocyanin production by 44% as compared with the control.
However, there was no difference of virulence factor production between compound 30 and
the control in rhlR mutants (Figure 5C), indicating that compound 30 reduced pyocyanin
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production by blocking RhlR. This difference might be related to the stage when pyocyanin is
produced in P. aeruginosa. RhlR agonists are suggested to suppress the pqs system only before
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basal pyocyanin is produced. According to the microarray experiment by Givskov and co-
workers, rhlR mutants repressed rhl system-related genes, especially rhamnolipid production-
related genes (rhlA and rhlB), but insignificantly affect las- or pqs-related system genes.37
Pyocyanin production-related genes (phzA-D) were also down-regulated in rhlR mutants.
Bassler and co-workers reported the down-regulation of pyocyanin biosynthetic genes (phzA1-
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G1) and decreased pyocyanin production in rhlR mutants. Similarly, according to our RT-
qPCR experiment results, rhlR mutants decreased rhlA and phzC1 expression (P<0.005)
without affecting the expression of other QS-related genes (lasR, lasB, and pqsR) (Figure 5D).
Figure 6. Mortality of T. molitor larvae injected P. aeruginosa with compounds 1c, 1d, 12a,
and 30 at 10 μM. DMSO (negative control) and 1c, 1d, and 12a (positive controls) were used.
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Encouraged by in vitro assay results, we determined the effect of compound 30 on
Tenebrio molitor (T. molitor) larvae mortality. Larvae injected with P. aeruginosa started to
die in initial incubation time and 70% of them died at the end of the 20-day incubation period
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(Figure 6). The survival rate of compound 30-treated larvae was greatly improved, with
approximately 80% larvae surviving at the end of the incubation period. In addition, larvae
injected with rhlR mutants of P. aeruginosa showed a 90% survival rate after 20 days.
Moreover, when treated rhlR mutants with different concentrations of rhamnolipid (0.01–10
μM) in larvae, their mortality was increased depending on the rhamnolipid concentration,
emphasizing the key role of RhlR in the production of virulence factor (Figure S5). Based on
in vitro and in vivo results, we propose that the pure RhlR antagonist 30 inhibits rhamnolipid
and pyocyanin production by inactivating of virulence factor production gene expression (e.g.,
rhl and phz genes) as described in a working model (Figure 7).
Figure 7. A working model to control QS system of P. aeruginosa by compound 30.
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CONCLUSIONS
RhlR is one of the key LuxR-type receptors in the QS network of P. aeruginosa and
considered as an attractive target protein for the discovery of biofilm inhibitors. We
systemically synthesized 55 gingerol analogues and evaluated their relative RhlR activities
using QS reporter strain assay. Comprehensive SAR studies identified the alkynyl ketone 30
as the most potent RhlR antagonist. Compound 30 displayed high RhlR antagonism (no
RhlR agonism at 1000 M), strong inhibition of biofilm formation in static and dynamic
settings, and reduced production of virulence factors such as rhamnolipid and pyocyanin in
P. aeruginosa. It also displayed selective affinity for RhlR over LasR and PqsR. Furthermore,
compound 30 significantly increased the in vivo survival rate of T. molitor larvae as
compared with the control. In conclusion, the pure and potent RhlR antagonist 30 which we
discovered for the first time can be utilized for investigating rhl-related QS mechanism and
developing a novel anti-virulence strategy to control P. aeruginosa infections.
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EXPERIMENTAL SECTION
General
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All chemicals and solvents used in the reaction were purchased from Sigma-Aldrich, TCI, and
Acros and were used without further purification. Reaction progress was monitored by TLC on
pre-coated silica gel plates with silica gel 60F254 (Merck; Darmstadt, Germany) and visualized
by UV254 light and/or KMnO staining for detection purposes. Column chromatography was
4
performed on silica gel (Silica gel 60; 230‒400 mesh ASTM, Merck, Darmstadt, Germany).
Nuclear magnetic resonance (NMR) spectra were recorded at room temperature on either a
1
13
Bruker BioSpin Avance 300 MHz NMR ( H, 300 MHz; C, 75 MHz) or a Bruker Ultrashield
1
13
6
00 MHz Plus ( H, 600 MHz; C, 150 MHz) spectrometer. All chemical shifts are reported in
parts per million (ppm) from tetramethylsilane (δ = 0) and were measured relative to the solvent
1
13
in which the sample was analyzed (CDCl : δ 7.26 for H NMR, δ 77.0 for C NMR; MeOH-
3
1
13
1
d : δ 3.31 for H NMR, δ 49.0 for C NMR). The H NMR shift values are reported as chemical
4
shift (δ), the corresponding integral, multiplicity (s = singlet, br = broad, d = doublet, t = triplet,
q = quartet, m = multiplet, dd = doublet of doublets, td = triplet of doublets, qd = quartet of
doublets), coupling constant (J in Hz) and assignments. High-resolution mass spectra (HRMS)
were recorded on an Agilent 6530 Accurate Mass Q-TOF LC/MS spectrometer. The purity of
all final compounds was measured by analytical reverse-phase HPLC on an Agilent 1260
Infinity (Agilent) with a C18 column (Phenomenex, 150 mm×4.6 mm, 3 μm, 110Å). RP-HPLC
was performed using the following isocratic conditions: for method A, mobile phase was
acetonitrile and water (30:70, v/v); for method B, mobile phase was acetonitrile and water with
0.1% TFA (30:70, v/v); for method C, mobile phase was acetonitrile and water (40:60, v/v);
for method D, mobile phase was acetonitrile and water (50:50, v/v); for method E, mobile phase
was acetonitrile and water (55:45, v/v); for method F, mobile phase was acetonitrile and water
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(
60:40, v/v); for method G, mobile phase was methanol and water (50:50, v/v); for method H,
mobile phase was methanol and water (60:40, v/v); for method I, mobile phase was methanol
and water (70:30, v/v). All compounds were eluted with a flow rate of 1 mL/min and monitored
at UV detector (220 nm). The purity of the tested compounds was >95%.
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Chemical Synthesis
29
Compounds 2−8 were reported by our previous study. The chemical structure and name for
compounds 2−8 can be found in the Supporting Information (Scheme S1).
General procedure A for compounds 11a–11r
To a stirred solution of benzylideneacetone compound in THF (20 mL) was added LDA (1.2–
3.6 eq.) at –78 ℃. The solution was stirred under argon for 1 h at the same temperature then n-
butanal (10.0 eq.) was added dropwise. The reaction mixture was vigorously stirred at the same
temperature until TLC analysis indicated complete conversion (typically 3–24 h), quenched
with aqueous NH Cl (10 mL), and extracted with EtOAc (3 × 25 mL). The organic layer was
4
washed with brine, dried over MgSO , filtered, and concentrated under reduced pressure. The
4
crude residue was purified by column chromatography on silica gel to furnish compounds.
(E)-5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)oct-1-en-3-one (11a)
Compound 11a was prepared in 60% yield as yellow oil, following the same procedure as
described in the general procedure A with (E)-4-(4-hydroxy-3-methoxyphenyl)but-3-en-2-one
(300 mg, 1.56 mmol), LDA (4.10 mL, 2.4 eq.) and stirring for 3 h. The crude residue was
purified by column chromatography on silica gel (Toluene/EtOAc = 6:1 to 3:1, v/v). R = 0.40
f
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(
Toluene/EtOAc = 2:1, v/v). H NMR (300 MHz, CDCl ) δ 7.53 (d, J = 16.1 Hz, 1H), 7.13 (dd,
3
J = 1.8 and 8.2 Hz, 1H), 7.08 (d, J = 1.7 Hz, 1H), 6.96 (d, J = 8.2 Hz, 1H), 6.61 (d, J = 16.1
Hz, 1H), 5.94 (s, 1H), 4.23–4.09 (m, 1H), 3.96 (s, 3H), 3.30 (d, J = 2.9 Hz, 1H), 2.90 (dd, J =
1
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.7 and 17.1 Hz, 1H), 2.75 (q, J = 9.0 Hz, 1H), 1.59–1.36 (m, 4H), 0.97 (t, J = 7.2 Hz, 3H); C
NMR (75 MHz, CDCl ) δ 201.03, 148.50, 146.89, 143.87, 126.72, 124.15, 123.76, 114.87,
3
109.51, 67.65, 55.98, 46.52, 38.68, 18.75, 14.05, 13.97. HRMS m/z calculated for C H O
1
5
20
4
–
[
M–H] : 263.1289; found: 263.1304. >95% purity (as determined by RP-HPLC, method C, t
R
=
6.34 min, method G, t = 7.33 min).
R
(
E)-5-Hydroxy-1-phenyloct-1-en-3-one (11b)
Compound 11b was prepared in 36% yield as yellow oil, following the same procedure as
described in the general procedure A with (E)-4-phenylbut-3-en-2-one (300 mg, 2.05 mmol),
LDA (2.70 mL, 1.2 eq.) and stirring for 2 h. The crude residue was purified by column
chromatography on silica gel (Toluene/EtOAc = 6:1 to 3:1, v/v). R = 0.40 (Toluene/EtOAc =
f
1
2
:1, v/v). H NMR (600 MHz, CDCl ) δ 7.58 (d, J = 16.2 Hz, 1H), 7.56–7.54 (m, 2H), 7.43–
3
7.39 (m, 3H), 6.74 (d, J = 16.2 Hz, 1H), 4.19–4.13 (m, 1H), 3.17 (d, J = 3.30 Hz, 1H), 2.88
(
dd, J = 2.6 and 17.3 Hz, 1H), 2.77 (q, J = 9.1 Hz, 1H), 1.62–1.36 (m, 4H), 0.96 (t, J = 7.1 Hz,
1
3
3
H); C NMR (150 MHz, CDCl ) δ 211.24, 140.75, 128.56, 128.29, 126.22, 67.36, 49.32,
3
–
4
5.06, 38.59, 29.53, 18.65, 13.98. HRMS m/z calculated for C H O [M–H] : 217.1234;
14 18
2
found: 217.1252. >95% purity (as determined by RP-HPLC, method C, t = 12.91 min, method
R
G, t = 15.79 min).
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(
E)-5-Hydroxy-1-(3-methoxyphenyl)oct-1-en-3-one (11c)
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Journal of Medicinal Chemistry
Page 28 of 89
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Compound 11c was prepared in 41% yield as yellow oil, following the same procedure as
described in the general procedure A with (E)-4-(3-methoxyphenyl)but-3-en-2-one (300 mg,
1.70 mmol), LDA (2.23 mL, 1.2 eq.) and stirring for 4 h. The crude residue was purified by
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column chromatography on silica gel (Hexane/EtOAc = 10:1 to 6:1, v/v). R = 0.48
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(
Hexane/EtOAc = 2:1, v/v). H NMR (600 MHz, CDCl ) δ 7.55 (d, J = 16.1 Hz, 1H), 7.33 (t,
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J = 7.9 Hz, 1H), 7.16 (d, J = 7.6 Hz, 1H), 7.08 (s, 1H), 6.98 (d, J = 8.2 Hz, 1H), 6.73 (d, J =
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6.2 Hz, 1H), 4.17 (brs, 1H), 3.86 (s, 3H), 3.22 (brs, 1H), 2.91–2.88 (m, 1H), 2.80–2.76 (m,
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H), 1.61–1.40 (m, 4H), 0.97 (t, J = 7.1 Hz, 3H); C NMR (150 MHz, CDCl ) δ 201.04, 159.98,
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43.48, 135.60, 130.02, 126.65, 121.15, 116.66, 113.20, 67.61, 55.35, 46.86, 38.69, 18.75,
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4.04. HRMS m/z calculated for C H O [M–H] : 247.1339; found: 247.1344. >95% purity
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(
as determined by RP-HPLC, method C, t = 14.25 min, method G, t = 19.69 min).
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(E)-5-Hydroxy-1-(4-hydroxyphenyl)oct-1-en-3-one (11d)
Compound 11d was prepared in 43% yield as yellow oil, following the same procedure as
described in the general procedure A with (E)-4-(4-hydroxyphenyl)but-3-en-2-one (250 mg,
1.54 mmol), LDA (4.10 mL, 2.4 eq.) and stirring for 6 h. The crude residue was purified by
column chromatography on silica gel (Hexane/EtOAc = 10:1 to 3:1, v/v). R = 0.42
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(
Hexane/EtOAc = 2:1, v/v). H NMR (600 MHz, MeOD) δ 7.61 (d, J = 16.1 Hz, 1H), 7.53 (d,
J = 8.6 Hz, 1H), 6.84 (d, J = 8.6 Hz, 1H), 6.72 (d, J = 16.1 Hz, 1H), 4.14–4.12 (m, 1H), 2.88–
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.84 (m, 1H), 2.78–2.75 (m, 1H), 1.55–1.41 (m, 4H), 0.97 (t, J = 7.1 Hz, 3H); C NMR (150
MHz, MeOD) δ 200.62, 160.22, 144.09, 130.21, 125.86, 123.16, 115.54, 67.81, 39.25, 18.45,
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2.98. HRMS m/z calculated for C H O [M–H] : 233.1183; found: 233.1213. >95% purity
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(as determined by RP-HPLC, method C, t = 6.18 min, method G, t = 8.08 min).
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Page 29 of 89
Journal of Medicinal Chemistry
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(
E)-5-Hydroxy-1-(3-hydroxyphenyl)oct-1-en-3-one (11e)
Compound 11e was prepared in 34% yield as yellow oil, following the same procedure as
described in the general procedure A with (E)-4-(3-hydroxyphenyl)but-3-en-2-one (250 mg,
1.54 mmol), LDA (4.10 mL, 2.4 eq.) and stirring for 6 h. The crude residue was purified by
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column chromatography on silica gel (Hexane/EtOAc = 10:1 to 3:1, v/v). R = 0.30
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(Hexane/EtOAc = 2:1, v/v). H NMR (600 MHz, MeOD) δ 7.58 (d, J = 16.1 Hz, 1H), 7.24 (t,
J = 7.9 Hz, 1H), 7.11 (d, J = 7.6 Hz, 1H), 7.06 (s, 1H), 6.87 (dd, J = 8.1 and 1.8 Hz, 1H), 6.80
(d, J = 16.2 Hz, 1H), 4.16–4.11 (m, 1H), 2.90–2.86 (m, 1H), 2.79–2.77 (m, 1H), 1.54–1.40 (m,
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H), 0.97 (t, J = 7.1 Hz, 3H); C NMR (150 MHz, MeOD) δ 200.49, 157.73, 143.65, 135.85,
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29.65, 126.18, 119.78, 117.53, 114.14, 67.66, 39.25, 18.45, 12.96. HRMS m/z calculated for
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C H O [M–H] : 233.1183; found: 233.1200. >95% purity (as determined by RP-HPLC,
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method C, t = 5.68 min, method G, t = 8.17 min).
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(E)-5-Hydroxy-1-(4-methoxyphenyl)oct-1-en-3-one (11f)
Compound 11f was prepared in 48% yield as white oil, following the same procedure as
described in the general procedure A with (E)-4-(4-methoxyphenyl)but-3-en-2-one (300 mg,
1.70 mmol), LDA (2.30 mL, 1.2 eq.) and stirring for 2 h. The crude residue was purified by
column chromatography on silica gel (Toluene/EtOAc = 10:1 to 3:1, v/v). R = 0.55
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(Toluene/EtOAc = 2:1, v/v). H NMR (600 MHz, CDCl ) δ 7.55–7.50 (m, 3H), 6.93 (d, J =
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.7, 2H), 6.62 (d, J = 16.2 Hz, 1H), 4.14 (brs, 1H), 3.85 (s, 3H), 3.27 (brs, 1H), 2.88–2.85 (m,
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1H), 2.75–2.71 (m, 1H), 1.62–1.42 (m, 4H), 0.95 (t, J = 7.2 Hz, 3H); C NMR (150 MHz,
CDCl ) δ 201.01, 161.83, 143.39, 130.19, 126.87, 124.18, 114.49, 67.73, 55.41, 46.71, 38.75,
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8.75, 14.04. HRMS m/z calculated for C H O [M–H] : 247.1339; found: 247.1357. >95%
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purity (as determined by RP-HPLC, method C, t = 12.88 min, method G, t = 18.64 min).
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