Structure-Activity Relationships of 6- and 8-Gingerol Analogs as Anti-Biofilm Agents

Article abstract of DOI:10.1021/acs.jmedchem.7b01426

Pseudomonas aeruginosa is a causative agent of chronic infections in immunocompromised patients. Disruption of quorum sensing circuits is an attractive strategy for treating diseases associated with P. aeruginosa infection. In this study, we designed and synthesized a series of gingerol analogs targeting LasR, a master regulator of quorum sensing networks in P. aeruginosa. Structure-activity relationship studies showed that a hydrogen-bonding interaction in the head section, stereochemistry and rotational rigidity in the middle section, and optimal alkyl chain length in the tail section are important factors for the enhancement of LasR-binding affinity and for the inhibition of biofilm formation. The most potent compound 41, an analog of (R)-8-gingerol with restricted rotation, showed stronger LasR-binding affinity and inhibition of biofilm formation than the known LasR antagonist (S)-6-gingerol. This new LasR antagonist can be used as an early lead compound for the development of anti-biofilm agents to treat P. aeruginosa infections.

Full text of DOI:10.1021/acs.jmedchem.7b01426

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Article
Structure-Activity Relationships of 6- and 8-
Gingerol Analogs as Anti-biofilm Agents
Hyunsuk Choi, So-Young Ham, Eunji Cha, Yujin Shin, Han-Shin Kim,
Jeong Kyu Bang, Sang-Hyun Son, Hee-Deung Park, and Youngjoo Byun
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01426 • Publication Date (Web): 14 Nov 2017
Downloaded from http://pubs.acs.org on November 15, 2017
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Structure-Activity Relationships of 6- and 8-Gingerol Analogs as Anti-
biofilm Agents
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†
,#
‡,#
‡
†
Hyunsuk Choi, So-Young Ham, Eunji Cha, Yujin Shin, Han-Shin Kim, Jeong Kyu
§
†
‡,⊥,*
Youngjoo Byun†,◊,*
Bang, Sang-Hyun Son, Hee-Deung Park,
†
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
§
Division of Magnetic Resonance, Korea Basic Science Institute, 161 Yeongudanji-ro,
Ochang-eup, Cheongwon-gu, Cheongju-si, Chungcheongbuk-do 28119, 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: Biofilm, Gingerol, LasR, Stereochemistry, Pseudomonas aeruginosa
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ABSTRACT
Pseudomonas aeruginosa is a causative agent of chronic infections in
immunocompromised patients. Disruption of quorum sensing circuits is an attractive
strategy for treating diseases associated with P. aeruginosa infection. In this study, we
designed and synthesized a series of gingerol analogs targeting LasR, a master regulator of
quorum sensing networks in P. aeruginosa. Structure-activity relationship studies showed
that a hydrogen-bonding interaction in the head section, stereochemistry and rotational
rigidity in the middle section, and optimal alkyl chain length in the tail section are
important factors for the enhancement of LasR-binding affinity and for the inhibition of
biofilm formation. The most potent compound 41, an analog of (R)-8-gingerol with
restricted rotation, showed stronger LasR-binding affinity and inhibition of biofilm
formation than the known LasR antagonist (S)-6-gingerol. This new LasR antagonist can
be used as an early lead compound for the development of anti-biofilm agents to treat P.
aeruginosa infections.
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INTRODUCTION
Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen that causes
infections in people with a weakened immune system, e.g., patients with cystic fibrosis,
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chronic wounds, pneumonia, AIDS, sepsis, or cancer. It is one of the six most dangerous
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bacterial species according to the Infectious Diseases Society of America. In particular, P.
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aeruginosa infection is the main cause of mortality in patients with cystic fibrosis. P.
aeruginosa can form a surface-associated community, a so-called biofilm. Biofilm cells are
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embedded in a self-produced exopolysaccharide matrix which confers antibiotic resistance.
Biofilms are involved in most of microbial infections of humans (~80% of such bacterial
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infections). Biofilms retard penetration of antibiotics and reduces the antibiotic activity,
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thus reducing treatment efficacy.
A biofilm is an aggregation of microbial cells encapsulated by self-produced
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extracellular polymeric substances on the surface. During biofilm formation, bacterial cells
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communicate with one another by means of quorum sensing (QS) network. QS is a cell-to-
cell communication system in which bacteria release and recognize chemical signals (auto-
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inducers), and QS enables bacteria to behave as a group to adapt to environmental changes.
In general, gram-negative bacteria including P. aeruginosa produce and release N-acyl-
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homoserine lactone (AHL) as a QS signal molecule. The QS mechanism of P. aeruginosa is
tightly regulated by the three main signal production and recognition systems: LasI–LasR,
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RhlI–RhlR, and PQR–MvfR. LasI in P. aeruginosa produces an extracellular diffusible N-
3-oxododecanoyl)- -homoserine lactone (OdDHL, 1a in Figure 1), which activates
(
L
expression of genes responsible for group behaviors including biofilm formation and
production of virulence factors. When OdDHL reaches a threshold concentration, the
OdDHL–LasR complex binds to the promoter regions of multiple genes affecting RhlI–RhlR
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and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS)–MvfR systems. Similarly, RhlI produces N-
butyryl-L-homoserine lactone (BHL), which is recognized by the transcriptional regulator
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RhlR. In the PQS-MvfR system, PQS and its precursors bind to the transcriptional regulator
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MvfR, resulting in transcription of target genes. Among the three systems, LasI-LasR is
considered to be a master regulator of QS networks and a key system in the biofilm formation
by P. aeruginosa.
P. aeruginosa forms biofilms and produces virulence factors through QS pathways.
Therefore, disruption of these signal production and recognition systems is an attractive
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strategy for attenuating the virulence of P. aeruginosa. One of the anti-virulence approaches
is to interrupt the interaction between chemical signals (e.g., OdDHL, BHL, and PQS) and
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their cognate receptors (e.g., LasR, RhlR, and MvfR). For instance, halogenated furanones
from the marine alga Delisea pulchra have a structure similar to AHL and can bind to LasR
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by competing with OdDHL. In addition, (Z)-4-bromo-5-(bromomethylene)furan-2(5H)-one
(Furanone C-30, 1d in Figure 1), a synthetic molecule, inhibits the expression of virulence
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factors by interfering with P. aeruginosa QS systems.
We previously demonstrated that (S)-6-gingerol (1b, Figure 1), the main component
of Ginger, reduces biofilm formation and production of virulence factors by competing with
OdDHL for LasR of P aeruginosa. RT-PCR analyses revealed that 1b reduces the expression
of genes (e.g., las, rhl, pqs, and phz genes) in the QS system and suppresses the production of
virulence factors (e.g., exoprotease, pyocyanin, and rhamnolipid), indicating that it interferes
with the interaction between OdDHL and LasR, at the top of the hierarchical QS network tree
of P. aeruginosa. Molecular modeling studies of the interaction of 1b with LasR (PDB ID:
2UVO) indicates that the 3′-hydroxyl-4′-methoxyphenyl moiety engages in a hydrogen-
bonding interaction with hydrophilic amino acids, while the alkyl side chain forms a
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hydrophobic bond. In this study, we aimed to investigate the effect of each functional group
of 1b on LasR-binding affinity and on biofilm formation by P. aeruginosa. We herein
describe the structure–activity relationship (SAR) studies of 6- and 8-gingerol analogs and
the development of novel LasR antagonists based on the chemical structure of 1b. Novel
potent biofilm inhibitors targeting LasR of P. aeruginosa have been identified from the
comprehensive SAR studies.
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<
Figure 1 >
RESULTS AND DISCUSSION
Structural modification of 1b was attempted based on key interactions between 1b
and LasR of P. aeruginosa. Chemical structure of 1b was subdivided into three parts (head,
middle, and tail sections) as shown in Figure 2. We aimed to investigate the effect of each
section on LasR-binding affinity and on biofilm formation.
<
Figure 2 >
In the head section, we tried to determine whether the hydrogen-bonding interaction
is necessary or not by replacing the methoxy group at the 3′-position and/or the hydroxyl
group at 4′-position of the phenyl moiety with other functional groups. Regarding the
modification of the middle section, we introduced a double bond between the phenyl moiety
and the carbonyl group to assess the influence of rotational flexibility. We also evaluated the
necessity of the hydroxyl group and the effect of stereochemistry of the chiral center on the
affinity for LasR and on biofilm formation. In the tail section, we tried to find the optimal
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alkyl chain length for a maximized the van der Waals interaction with the LasR hydrophobic
subpocket, which is formed by lipophilic amino acid residues (Leu36, Leu40, Ala50, Ile52,
Ala70, Val76, and Leu125).
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<
Scheme 1 >
First, we synthesized gingerol analogues with various alkyl chain length from 4-
gingerol to 10-gingerol to find the optimal carbon length in the tail section. As shown in
Scheme 1, compound 2 was synthesized from commercial vanillin by treatment with 10%
NaOH in acetone at 25 ºC for 16 hr in 71% yield. Compound 3 was obtained by reacting 2
with hydrogen in methanol in the presence of 10% Pd/C at 25 ºC for 2 hr in 97% yield.
Treatment of 3 with lithium diisopropylamide (LDA) at –78 ºC, followed by the addition of
appropriate aldehydes (butanal for 4, pentanal for 5, hexanal for 6, heptanal for 7, octanal for
8
, nonanal for 9, and decanal for 10), afforded the final gingerol compounds in 30–47%
21-23
yield.
By-products such as condensated or dimerized compounds were produced when
excess LDA was used and the reaction time was longer than 2 hr.
<
Figure 3 >
LasR-binding affinity of the synthesized gingerols with various alkyl chain lengths
was determined by measuring luminescence of an E. coli reporter strain. The reporter strain
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carried two plasmids, pJN105L (LasR expression plasmid) and pSC11 (lasI::lacZ fusion
25
plasmid), which enabled us to assay competitive binding of OdDHL with each gingerol
derivative 4–10. Antagonistic activities of the synthesized compounds at 1 ꢀM or 10 ꢀM
were determined by measuring luminescence in the presence of 1 ꢀM OdDHL (1a) and
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presented as relative luminescence unit (RLU) ratio. Three compounds (1b, 1c, and 1d)
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served as positive controls.
As shown in Figure 3, LasR-antagonistic activities
increased as the alkyl chain lengthened, indicating that the longer alkyl group contributed to
the affinity for LasR via the van der Waals interaction in the hydrophobic subpocket of LasR.
Just as the LasR inhibition, inhibition of biofilm formation also strengthened as the carbon
chain was extended at 10 ꢀM (Figure 3B). However, inhibition of biofilm formation
decreased with 9-gingerol (9) and 10-gingerol (10) at 100 ꢀM (Figure 3C) because of
increased bacterial growth inhibition. We previously reported that 2H-pyran-2-one analogs
show increased inhibition of LasR as the carbon chain lengthens, with the highest potency
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corresponding to the dodecyloxy group. Park et al. reported that 10- and 12-gingerol have
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anti-bacterial activity against periodontal bacteria. As shown in Figure 3D, compounds 9
and 10 with a longer alkyl chain inhibited bacterial growth significantly at 100 ꢀM as
compared with the other compounds. This effect may be due to the fact that 9 and 10 act as a
surfactant, which inhibits bacterial growth. We next examined agonistic activities of the
synthesized gingerols (4–10) in an E. coli reporter assay system in the absence of OdDHL.
None of them showed agonistic activity to LasR at 10 ꢀM (see supporting information).
<
Scheme 2 >
Based on results of LasR antagonism and biofilm formation of compounds 4–10, 6-
and 8-gingerol were selected for the structural modification in the head section. A similar
synthetic strategy was applied to prepare 6- and 8-gingerol derivatives with various
functional groups in the head section. 6-Gingerol derivatives (14a–20a) were prepared from
commercial vanillin in three steps. Briefly, treatment of various benzaldehydes with acetone
afforded analogues of compound 12 in 35–75% yield. Catalytic hydrogenation of the
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unsaturated alkene group produced analogues of 13 in 80–97% yield. 6-Gingerol analogs
(14a–20a) with various substituents in the head section were obtained by reacting 13 with n-
hexanal as shown in Scheme 2, whereas 8-gingerol derivatives (14b–20b) were done with n-
octanal instead of n-hexanal at the final step.
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<
Figure 4 >
LasR-antagonistic activities of 6- and 8-gingerol derivatives modified in the head
section were evaluated. Hydrogen-bonding effects of the methoxy group at the 3′-position
and the hydroxyl group at the 4′-position of the phenyl moiety in 6-gingerol analogs were
evaluated by introducing other functional groups. As shown in Figure 4A, removal of the
methoxy and hydroxyl group together (compound 15a) significantly decreased the LasR-
antagonistic activity, implying that at least one hydrogen-bonding interaction is required for
binding to LasR. After that, we removed only the methoxy group at the 3′-position
(compound 18a); this change increased the LasR-antagonistic activity. In contrast, removal of
the hydroxyl group at the 4′-position (Compound 20a) decreased the LasR-antagonistic
activity. These results suggested that substituents capable of hydrogen bonding at the 4′-
position were more favorable for binding to LasR. Replacement of OH at the 4′-position with
F preserved the LasR-inhibitory activity (16a vs. 1b and 17a vs. 18a), suggesting that the
functional group at 4′-position may act as a hydrogen-bonding acceptor rather than a
hydrogen-bonding donor.
LasR inhibition patterns of 8-gingerol derivatives were similar to those of 6-gingerol.
As shown in Figure 4C, compounds (17b and 18b) with a substitution by a hydrogen-bond
acceptor at the 4′-position were the most potent among the synthesized compounds. Activities
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of compounds (15b and 20b) without any hydrogen-bonding acceptor at the 4′-position were
relatively weaker than those of the other compounds. In general, LasR inhibition by 8-
gingerol derivatives was stronger than the corresponding 6-gingerol ones. The static biofilm
formation assay of 6- and 8-gingerol derivatives with variation in the head section showed a
tendency similar to that in the LasR inhibition assay (Figures 4B and 4D). Compounds with a
hydrogen-bonding acceptor at the 4′-position (16a–16b, 17a–17b, and 18a–18b) were the
most potent in the series. This result was consistent with the hypothesis that derivatives with
stronger affinity for LasR can inhibit biofilm formation more effectively. Two novel
compounds (16b and 17b) and one known compound (18b) exerted stronger inhibition of
biofilm formation than the known anti-biofilm agent 1b.
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<
Scheme 3 >
To assess the effect of rotational flexibility between the head section and the carbonyl
group, several compounds (21a–21b and 22a–22b) were prepared in 30–35% yield via
crossed aldol condensation (Scheme 3). In addition, compounds 24a and 24b were prepared
to determine the necessity of the β-hydroxy group for the LasR-binding affinity and for the
inhibition of biofilm formation. Reaction of vanillin with 2-nonanone or 2-dodecanone in the
presence of
L-proline gave compounds 23a and 23b in 45% and 60% yield, respectively.
Compounds 24a and 24b were prepared in 80% and 97% yield by subjecting 23a and 23b to
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hydrogenation conditions (H , Pd/C) for 2 hr.
<
Figure 5 >
As shown in Figure 5, compounds 21a and 21b (restricted rotation) showed slightly
stronger LasR affinity than did the corresponding compounds (6 and 8) with flexible rotation.
The derivatives (23a–23b and 24a–24b) without the β-hydroxyl group showed significantly
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weaker LasR-binding affinity and less inhibition of biofilm formation than the ones with the
β-OH group. These data suggested that the OH group at the β-position of the carbonyl group
may play a pivotal role in the binding to the LasR protein as well as in the inhibition of P.
aeruginosa biofilm formation.
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Results on in vitro LasR-binding and inhibition of biofilm formation indicated that 8-
gingerol analogs were more potent than 6-gingerol analogs. Furthermore, a racemic mixture
of 6- and 8-gingerol (6 and 8) was slightly more potent than the pure (S)-enantiomer of 6-
gingerol (1b) or 8-gingerol (1c), which are natural forms and commercially available.
Therefore, we hypothesized that the pure (R)-enantiomer of gingerol would possess stronger
LasR-binding affinity and inhibition of biofilm formation than the corresponding (S)-
enantiomer. Enantiomerically enriched (R)- and (S)-enantiomers of 8-gingerol were
synthesized by means of chiral catalysts such as D-proline and Salen’s catalyst.
<
Scheme 4 >
Scheme 4 shows the synthetic approach to enantiomerically enriched (R)-8-gingerol
29) using -proline as a chiral catalyst. Compound 25 was synthesized from 1-octanal by
treatment with -proline in acetone at room temperature for 48 hr. Acetone served as a
(
D
D
reagent and solvent in the reaction. The β-hydroxyl group in compound 25 was protected by
treatment with tert-butyldimethylsilyl chloride (TBDMSCl) and imidazole in
dichloromethane to give compound 26 (87% yield). Silyl enol ether compound 27 was
obtained by treating 26 with trimethylsilyl trifluoromethanesulfonate (TMSOTf) and N,N-
diisopropylethylamine (DIPEA). The Mukaiyama aldol reaction between silyl enol ether 27
3
and vanillin, with simultaneous removal of the TBDMS group using boron trifluoride (BF ),
3
0
afforded compound 28 (65% yield) in two steps. Catalytic hydrogenation of 28 produced
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the final compound 29 in 97% yield. The enantiomeric excess (ee) % value of compound 29
3
0
was determined by chiral HPLC analysis.
<
Scheme 5 >
10
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26
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41
42
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55
56
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59
60
The synthetic strategy for enantiomerically enriched (R)-8-gingerol (42) using
Salen’s catalyst is described in Scheme 5. Briefly, compound 31 was synthesized from
commercial 3-buten-1-ol via treatment with sodium hydride and benzyl bromide in THF at 0
22,31
ºC for 16 hr.
3 2 2
Reaction of 31 with m-CPBA and NaHCO in CH Cl at 0 ºC for 16 hr, gave
a racemic mixture of epoxide 32 in 72% yield. The (S)-epoxide 33 was obtained by reacting
32 with (S,S)-(+)-N,Nʹ-bis(3,5-di-tert-butylsalicyclidene)-1,2-cyclohexanediaminocobalt (II)
(Salen’s catalyst). The (R)-epoxide of 32 was transformed into (R)-1,2-diol whereas the (S)-
1
epoxide remained intact, as determined by H NMR analysis during the reaction (see
supporting information). Lithiation of the terminal alkyne of 1-hexyne with n-BuLi, followed
by the addition of 33, afforded compound 34 in 57% yield via an epoxide ring–opening
reaction. The hydroxyl group of 34 was protected with TBDMSCl to obtain compound 35.
2
Debenzylation of 35 by means of H and Pd/C provided primary alcohol 36 in 89% yield.
Treatment of 36 with NaIO and RuCl oxidized the primary alcohol to the carboxylic acid,
4
3
3
2
thus producing compound 37 in 67% yield. The carboxylic acid was transformed into
Weinreb amide 38 by using N,O-dimethylhydroxylamine under peptide-coupling conditions
(HOBt and EDC). Reaction of 38 with methylmagnesium bromide in THF afforded
32
compound 39 in 90% yield. Silylenol ether 40 was generated by reacting 39 with TMSOTf
and DIPEA in dichloromethane. The Mukaiyama aldol reaction between 40 and vanillin, with
3
simultaneous deprotection of the TBDMS group using boron trifluoride (BF ), afforded
compound 41 (65% yield) in two steps. The final compound 42 was obtained in 97% yield by
reducing the double bond of α,β-unsaturated ketone 41 under catalytic hydrogenation
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conditions. The (S)-isomer of 8-gingerol (42S) was prepared in a similar way, where (R,R)-
(+)-N,Nʹ-bis(3,5-di-tert-butylsalicyclidene)-1,2-cyclohexanediaminocobalt (II) was used at
the third step (see supporting information).
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As shown Figure 6, Scheme 1 without a chiral catalyst produced a racemic mixture of 8-
gingerol (8) at an almost 1:1 ratio in a chiral HPLC experiment (Figure 6A). Scheme 4, in
which
D
-proline served as a chiral catalyst, generated 29 with a 70% ee value (Figure 6B)
29
similar to that in a previously reported reaction. As expected, Scheme 5 by means of Salen’s
catalyst afforded (S)-8-gingerol (42S) and (R)-8-gingerol (42) with ee values >95% (Figure
6C and 6D).
< Figure 6 >
< Figure 7 >
LasR-binding affinity of the synthesized (R)- and (S)-8-gingerol compounds was
evaluated in a luminescent reporter assay. The activity of (S)-8-gingerol (42S) synthesized
using Salen’s catalyst was almost the same as that of commercial (S)-8-gingerol (1c). As the
proportion of the (R)-enantiomer of 8-gingerol increased, LasR-binding affinity was
strengthened accordingly. The enantiomerically enriched (R)-8-gingerol 42 showed much
stronger LasR-binding affinity than 1c, as was the case for a racemic mixture of 8-gingerol
(8). Compound 29 with an ee value of 70% had the intermediate LasR-affinity between 8 and
42 (Figure 7A). As shown in Figure 7B, the results of the static biofilm formation assay
indicated a trend similar to that of the affinity for LasR. Compound 42 yielded 72% biofilm
formation when the effect of 1c was set to 100%. Effects of absolute configuration on the
interaction between QS chemical signals and their cognate receptors have been investigated
33-35
elsewhere.
Recently, Blackwell and co-workers studied N-(3-hydroxydodecanoyl)-L-
homoserine lactone (OH-DHL) analogs and their putative cognate receptor, AbaR in
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Acinetobacter baumannii. The (R)-isomer of OH-DHL was found to be a more active AbaR
3
5
receptor agonist than (S)-isomer of OH-DHL. In our study, the (R)-enantiomer of 8-gingerol
2 manifested stronger LasR-binding affinity than the synthesized (S)-enantiomer 42S and
4
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the commercial 1c.
Because compound 21b (a racemic mixture) with restricted rotation between the carbonyl
group and phenyl moiety was more potent than 1c (Figure 5), compound 41 was assumed to
be more potent than compound 42. As expected, compound 41 showed stronger LasR-binding
affinity and greater inhibition of biofilm formation than compound 42, as shown in Figure 7.
However, bacterial growth inhibition was not observed even at 100 ꢀM concentration of 41-
4
2 (see supporting information). In order to evaluate the binding reversibility of compound
1 to LasR, LasR binding activities of 41 (1 ꢀM) were measured for different concentrations
4
of 1a (0, 0.1, 1, 10 and 100 ꢀM). By increasing concentration of 1a, the differences in LasR
binding activities between the treatment group and the control (no treatment of 41) decreased
(See supporting information). At 100 ꢀM of 1a, LasR-agonistic activity in the treatment
group was completely recovered, and almost same as the control one. This result suggests
that 41 binds reversibly to LasR by competing with 1a.
< Figure 8 >
<
Table 1 >
A dynamic biofilm formation assay of the two most potent compounds (41 and 42)
was performed in a drip-flow reactor. Compound 1c served as a positive control, and DMSO
as a negative control. After 48-hr drip-flow reactor operation, the biofilm was stained with
Ruby and concanavalin A (ConA). Ruby (red) is a reagent for staining protein, and ConA
(green) for carbohydrate of biofilm. As shown in Figure 8A, the biofilm in the presence of
DMSO formed with typical mushroom-like morphology. By contrast, the biofilms treated
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with 1c (Figure 8B), 42 (Figure 8C), or 41 (Figure 8D) were relatively thin and sparse as
compared with the negative control. Biofilm volume and thickness with 41 were the lowest
among the three groups (Table 1). Furthermore, the biofilm treated with 41 showed a
relatively smaller volume of carbohydrates (47–74%) and proteins (23–56%) as compared to
the other groups. Comprehensive analysis of confocal laser scanning microscopy (CLSM)
images of biofilms indicated that the (R)-8-gingerol analogs 41–42 inhibited biofilm
formation more effectively than 1c did.
0
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<
Figure 9 >
To explain why compounds 41–42 showed strong LasR-binding affinity and potent
inhibition of biofilm formation, we conducted molecular docking analyses of compounds 41–
42 and their (S)-enantiomers (41S–42S) by using the crystal structure of LasR (PDB ID:
36
2
UV0). The ligands were docked to the LasR active site by means of the CDOCKER
module of Discovery Studio (Accelrys Inc., San Diego, USA). The best-docked pose of each
ligand in the active site coincided well with the crystal ligand OdDHL. Moreover, compound
41 engaged in a much greater number of hydrogen-bonding interactions with LasR than the
other three ligands did. As shown in Figure 9A, compound 41 participated in hydrogen-
bonding interactions with Tyr47, Arg61, Asp65, Asp73, and Tyr93. In particular, the OH
group at the 4′-position of the phenyl moiety was deeply projected toward Tyr93 and formed
polar interactions, which were not observed in the other ligands. Furthermore, it was
noteworthy that the β-hydroxyl group of 41 participated in strong hydrogen-bonding
interactions with the guanidinium group of Arg61. In addition, the lipophilic alkyl group
made hydrophobic contacts with lipophilic amino acid residues including Leu39, Leu40, and
Leu125. Tight packing between 41 and the surrounding amino acid residues contributed
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substantially to the stability of the protein–ligand complex; this phenomenon may explain the
strongest potency of compound 41 among the synthesized compounds. In contrast, compound
42 (Figure 9B), 41S, and 42S (see supporting information) had a relatively small number of
10
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hydrogen-bonding interactions with LasR.
CONCLUSIONS
On the basis of the chemical structure of (S)-6-gingerol, which is a potent anti-
biofilm agent demonstrated by us, a variety of 6- and 8-gingerol analogs were designed and
synthesized. These compounds were designed to evaluate the effects of the head, middle, and
tail section of 6-gingerol on LasR-binding affinity and on biofilm formation. Regarding
modification of the tail section, affinity for LasR and inhibition of biofilm formation
increased as the alkyl chain lengthened up to 8-gingerol. As for modification of the head
section, compounds with a substitution by a hydrogen-bonding acceptor group (e.g., F or OH)
at the 4′-position were the most potent, indicating that the hydrogen-bonding interaction was
essential for binding to LasR. In the variants of the middle section, β-OH of the carbonyl
group was necessary, whereas rotational rigidity between the head section and carbonyl group
was favorable for LasR-binding affinity and for inhibition of biofilm formation. To evaluate
the effects of stereochemistry, enantiomerically enriched (R)-8-gingerol was synthesized by
means of chiral catalysts such as
D-proline and Salen’s catalyst. D-Proline yielded (R)-8-
gingerol (29) with an ee value of 70% in 5 synthetic steps, whereas Salen’s catalyst afforded
enantiomerically enriched (R)-8-gingerol (42) with ee values of >95% in 12 steps.
Comprehensive SAR studies identified compounds 41–42 as strong candidates for anti-
biofilm agents, suggesting that the stereochemistry of 8-gingerol is one of the important
factors for the enhancement of LasR-binding affinity and inhibition of biofilm formation.
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6
According to the results of SAR studies and molecular modeling, compound 41 holds
promise as an early lead compound for further structural optimization in the development of
anti-biofilm agents to combat P. aeruginosa infections.
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EXPERIMENTAL SECTION
General
All the chemicals and solvents used in the reaction were purchased from Sigma-
Aldrich, TCI, or Alfa Aesar, and were used without further purification. Reactions were
monitored by TLC on 0.25 mm Merck precoated silica gel plates (60 F254). Reaction
4
progress was monitored by TLC analysis using a UV lamp and/or KMnO staining for
detection purposes. Column chromatography was performed on silica gel (230–400 mesh,
Merck, Darmstadt, Germany). NMR spectra were recorded at room temperature on either
Bruker BioSpin Avance 300 MHz NMR or Bruker Ultrashield 600 MHz Plus spectrometer.
Chemical shifts are reported in parts per million (ppm, δ) with TMS as an internal standard.
13
Coupling constant are given in Hertz. C NMR spectra were obtained by using the same
NMR spectrometers and were calibrated with CDCl
3
(δ = 77.16 ppm). Splitting patterns
1
are indicated as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad for H
NMR data. Mass spectra were obtained on a Shimadzu (MALDI-TOF) mass spectrometer
or an Agilent 6530 Accurate mass Q-TOF LC/MS spectrometer or an electrospray
ionization PE Biosystems Sciex Api 150 EX mass spectrometer single quadruple equipped
with a turbo ion spray interface. 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 × 4.6 mm, 3 ꢀm, 110 Å). RP-HPLC was performed using the following
isocratic conditions: for method A, mobile phase was acetonitrile and water (50:50, v/v);
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for method B, mobile phase was acetonitrile and water (55:45, v/v); for method C, mobile
phase was acetonitrile and water (70:30, v/v). All compounds were eluted with a flow rate
of 0.7 mL/min and monitored at UV detector: 254 nm. Purity of the tested compounds was
10
11
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>
95%.
Synthesis
(E)-4-(4-Hydroxy-3-methoxyphenyl)but-3-en-2-one (2)
To a solution of 4-hydroxy-3-methoxybenzaldehyde (1.25 g, 8.2 mmol) in acetone (50 mL)
was added 10% NaOH (3.28 mL, 8.2 mmol) dropwise. The reaction mixture was stirred at
o
25 C for 48 hr and then was quenched with water and extracted with EtOAc. The organic
layer was dried over MgSO
4
, filtered, and concentrated under reduced pressure. The crude
residue was purified by column chromatography on silica gel (eluting with a mixture of
37,38
hexane-EtOAc, 10:1 to 3:1) to furnish compound 2 (1.20 g, 71%) as yellow oil.
hexane:EtOAc = 4:1, v/v).
R = 0.25
f
(
4
-(4-Hydroxy-3-methoxyphenyl)butan-2-one (3)
To a solution of compound 2 (1.2 g, 6.2 mmol) in MeOH (20 mL) was added 10% Pd/C
200 mg, 0.187 mmol). The solution was then stirred in an atmosphere of H gas for 4 hr.
(
2
The reaction mixture was filtered through a Celite pad and concentrated under reduced
pressure. The crude residue was purified by column chromatography on silica gel
37,38
(
hexane:EtOAc = 5:1, v/v) to furnish compound 3 (1.17 g, 97%) as colorless oil.
-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)octan-3-one (4)
To a solution of compound 3 (200 mg, 1.0 mmol) in THF (5 mL) was added LDA (2.30 mL,
.2 mmol) dropwise at –78 ºC. The solution was stirred for 1 hr at the same temperature.
5
2
Butanal (0.74 mL, 8.3 mmol) was then added dropwise. The reaction mixture was stirred for
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2
2
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2
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3
3
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4
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6
4
3 hr at the same temperature, quenched with aqueous NH Cl (10 mL), and extracted with
EtOAc. The organic layer was washed with brine, dried over MgSO , filtered, and
4
concentrated under reduced pressure. The crude residue was purified by column
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chromatography on silica gel (toluene:EtOAc = 10:1 to 5:1, v/v) to furnish compound 4 (15
1
mg, 13%) as colorless oil. H NMR (300 MHz, CDCl
3
) δ 6.85 (d, J = 7.8 Hz, 1H), 6.69 (s,
1H), 6.66 (d, J = 8.7 Hz, 1H), 5.51 (s, 1H), 4.06 (brs, 1H), 3.89 (s, 3H), 2.96 (brs, 1H), 2.86 (t,
J = 6.9 Hz, 2H), 2.75 (t, J = 6.9 Hz, 2H), 2.54 (t, J = 6.6 Hz, 2H), 1.51‒1.27 (m, 4H), 0.93 (t,
13
3
J = 6.6 Hz, 3H); C NMR (75 MHz, CDCl ) δ 211.5, 146.4, 143.9, 132.6, 120.7, 114.4,
110.9, 67.4, 55.9, 49.4, 45.5, 38.6, 29.3, 24.0, 18.7, 14.0. MS (MALDI-TOF) m/z calculated
+
+
22 4
for C15H O [M] : 266.2; found: 266.1. >98% purity (as determined by RP-HPLC, method
R
B, t = 3.49 min).
Compounds 5–10 were prepared by a method similar to the one described for compound 4.
5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)nonan-3-one (5)
Compound 5 was prepared in 32% yield as colorless oil, following the same procedure as
f
described for the synthesis of 4 but with pentanal instead of butanal. R = 0.15
1
(
toluene:EtOAc = 5:1, v/v). H NMR (300 MHz, CDCl
3
) δ 6.84 (d, J = 8.1 Hz, 1H), 6.69 (s,
1H), 6.68 (d, J = 9.0 Hz, 1H), 5.50 (s, 1H), 4.04 (brs, 1H), 3.89 (s, 3H), 2.80 (s, 1H), 2.83 (t,
J = 6.6 Hz, 2H), 2.75 (t, J = 6.6 Hz, 2H), 2.72‒2.42 (m, 2H), 1.55‒1.23 (m, 6H), 0.91‒0.89
13
(
m, 3H); C NMR (75 MHz, CDCl
3
) δ 211.5, 146.4, 143.9, 132.6, 120.7, 114.3, 110.9, 67.6,
24 4
55.8, 49.3, 45.4, 36.1, 29.3, 27.6, 22.6, 14.1. MS (MALDI-TOF) m/z calculated for C16H O
+
+
R
[M] : 280.2; found: 280.1. >98% purity (as determined by RP-HPLC, method B, t = 4.20
min).
5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-3-one (6)
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Compound 6 was prepared in 47% yield as colorless oil, following the same procedure as
described for the synthesis of 4 but with hexanal instead of butanal. R = 0.14 (toluene:EtOAc
f
1
=
5:1, v/v). H NMR (300 MHz, CDCl
3
) δ 6.84 (d, J = 7.8 Hz, 1H), 6.69 (s, 1H), 6.68 (d, J =
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
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36
37
38
39
40
41
42
43
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8
.7 Hz, 1H), 5.52 (s, 1H), 4.04 (brs, 1H), 3.89 (s, 3H), 2.96 (s, 1H), 2.85 (t, J = 6.6 Hz, 2H),
1
3
2.75 (t, J = 6.6 Hz, 2H), 2.68‒2.43 (m, 2H), 2.68‒2.43 (m, 8H), 0.98‒0.81 (m, 3H); C NMR
(75 MHz, CDCl ) δ 211.5, 146.4, 144.0, 132.6, 120.7, 114.4, 110.1, 67.7, 55.9, 49.4, 45.5,
3
+
+
3
6.4, 31.7, 29.3, 25.1, 22.6, 14.0. MS (MALDI-TOF) m/z calculated for C H O [M] :
17 26 4
294.2; found: 294.1. >98% purity (as determined by RP-HPLC, method B, t = 5.30 min).
R
5
-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)undecan-3-one (7)
Compound 7 was prepared in 28% yield as colorless oil, following the same procedure as
f
described for the synthesis of 4 but with heptanal instead of butanal. R = 0.15
1
(toluene:EtOAc = 5:1, v/v). H NMR (300 MHz, CDCl ) δ 6.85 (d, J = 7.8 Hz, 1H), 6.69 (s,
3
1H), 6.68 (d, J = 8.7 Hz, 1H), 5.50 (brs, 1H), 3.89 (s, 3H), 2.96 (brs, 1H), 2.86 (t, J = 6.6 Hz,
13
2H), 2.75 (t, J = 6.6 Hz, 2H), 2.72‒2.41 (m, 2H), 1.71‒1.21 (m, 10H), 0.98‒0.89 (m, 3H); C
NMR (75 MHz, CDCl
3
) δ 211.5, 146.4, 144.0, 132.7, 120.7, 114.4, 110.9, 67.7, 55.9, 49.4,
+
28 4
45.5, 36.5, 31.8, 29.3, 29.2, 25.4, 22.6, 14.1. MS (MALDI-TOF) m/z calculated for C18H O
+
[
M] : 308.2; found: 308.2. >98% purity (as determined by RP-HPLC, method B, t = 6.97
R
min).
5
-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-3-one (8)
Compound 8 was prepared in 32% yield as colorless oil, following the same procedure as
f
described for the synthesis of 4 but with octanal instead of butanal. R = 0.15 (toluene:EtOAc
1
= 5:1, v/v). H NMR (300 MHz, CDCl ) δ 6.83 (d, J = 8.1 Hz, 1H), 6.68 (s, 1H), 6.66 (d, J =
3
9.0 Hz, 1H), 5.52 (s, 1H), 4.02 (brs, 1H), 3.87 (s, 3H), 2.95 (s, 1H), 2.84 (t, J = 6.9 Hz, 2H),
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2
2
2
2
2
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3
3
3
3
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4
4
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5
5
5
5
5
5
6
2.73 (t, J = 6.9 Hz, 2H), 2.52 (t, J = 7.5Hz, 2H), 1.61‒1.12 (m, 12H), 0.88 (t, J = 6.6 Hz, 3H);
1
3
C NMR (75 MHz, CDCl ) δ 211.5, 144.0, 132.6, 120.7, 114.4, 111.0, 67.7, 55.9, 49.3, 45.4,
3
+
30 4
36.5, 31.8, 29.5, 29.2, 29.2, 25.5, 22.7, 14.1. MS (MALDI-TOF) m/z calculated for C19H O
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
+
[
M] : 322.2; found: 322.2. >98% purity (as determined by RP-HPLC, method B, t
R
= 9.56
min).
5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)tridecan-3-one (9)
Compound 9 was prepared in 32% yield as colorless oil, following the same procedure as
f
described for the synthesis of 4 but with nonanal instead of butanal. R = 0.15 (toluene:EtOAc
1
=
5:1, v/v). H NMR (300 MHz, CDCl
3
) δ 6.85 (d, J = 7.8 Hz, 1H), 6.69 (s, 1H), 6.68 (d, J =
8.7 Hz, 1H), 5.50 (brs, 1H), 4.04 (brs, 1H), 3.89 (s, 3H), 2.91 (brs, 1H), 2.85 (t, J = 6.6 Hz,
1
3
2H), 2.75 (t, J = 6.6 Hz, 2H), 2.71‒2.39 (m, 2H), 1.71‒1.15 (m, 14H), 0.89 (s, 3H); C NMR
(
75 MHz, CDCl ) δ 211.5, 144.0, 132.6, 120.7, 114.4, 111.0, 67.7, 55.9, 49.3, 45.4, 36.5, 31.8,
3
+
4
+
29.5, 29.2, 29.2, 25.5, 22.7, 14.1. MS (MALDI-TOF) m/z calculated for C20
H
32
O
[M] :
3
36.2; found: 336.2. >98% purity (as determined by RP-HPLC, method B, t
-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)tetradecan-3-one (10)
R
= 13.56 min).
5
Compound 10 was prepared in 32% yield as colorless oil, following the same procedure as
described for the synthesis of 4 but with decanal instead of butanal. R = 0.16 (toluene:EtOAc
f
1
= 5:1, v/v). H NMR (300 MHz, CDCl
3
) δ 6.85 (d, J = 7.8 Hz, 1H), 6.69 (s, 1H), 6.68 (d, J =
8.7 Hz, 2H), 5.50 (brs, 1H), 4.04 (brs, 1H), 3.89 (s, 3H), 2.90 (brs, 1H), 2.85 (t, J = 6.6 Hz,
13
2
H), 2.75 (t, J = 6.6 Hz, 2H), 2.56‒2.34 (m, 2H), 1.49‒1.18 (m, 16H), 0.98‒0.79 (m, 3H); C
NMR (75 MHz, CDCl
3
) δ 211.6, 178.4, 146.5, 144.2, 132.6, 121.5, 114.57, 111.5, 67.7, 59.3,
+
45.5, 36.4, 29.7, 26.1, 25.4, 24.7, 22.7, 14.1. MS (MALDI-TOF) m/z calculated for C H O
4
2
1
34
+
[
M] : 350.2; found: 350.2. >98% purity (as determined by RP-HPLC, method B, t
R
= 19.78
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min).
1-(3,4-Dimethoxyphenyl)-5-hydroxydecan-3-one (14a)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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59
60
To a solution of 4-(3,4-dimethoxyphenyl)butan-2-one (200 mg, 1.0 mmol) in THF (5 mL)
was added LDA (2.3 mL, 2.2 mmol) at –78 ºC. The solution was stirred for 1 hr at the same
temperature. Hexanal (0.72 mL, 8.2 mmol) was added dropwise. The reaction mixture was
stirred for 3 hr at the same temperature, quenched with aqueous NH
extracted with EtOAc. The organic layer was washed with water and brine, dried over
MgSO , filtered, and concentrated under reduced pressure. The crude residue was purified by
4
Cl (10 mL), and
4
column chromatography on silica gel (toluene:EtOAc = 10:1 to 5:1) to furnish compound 14a
1
(
97 mg, 32 %) as colorless oil. R
CDCl
.90‒2.74 (m, 4H), 2.63‒2.36 (m, 2H), 1.65‒1.28 (m, 8H), 0.90 (s, 3H); C NMR (75 MHz,
CDCl ) δ 211.5, 148.9, 147.4, 133.3, 120.0, 112.0, 67.7, 55.9, 49.3, 45.3, 36.4, 31.7, 29.2,
f
= 0.26 (hexane:EtOAc = 7:1, v/v). H NMR (300 MHz,
3
) δ 6.81 (t, J = 8.7 Hz, 1H), 6.69 (d, J = 12.3 Hz, 2H), 4.04 (brs, 1H), 3.86 (s, 6H),
1
3
2
3
+
+
25.2, 22.6, 14.0. MS (MALDI-TOF) m/z calculated for C18
H
28
O
4
[M + H] : 309.2; found:
3
09.2. >98% purity (as determined by RP-HPLC, method A, t
-(3,4-Dimethoxyphenyl)-5-hydroxydodecan-3-one (14b)
R
= 9.86 min).
1
Compound 14b was obtained in 32% yield as colorless oil, following the same procedure as
f
described for the synthesis of 14a but with octanal instead of hexanal. R = 0.26
1
(
hexane:EtOAc = 7:1, v/v). H NMR (300 MHz, CDCl
3
) δ 6.81 (t, J = 8.7 Hz, 1H), 6.70 (d, J
=
12.3 Hz, 2H), 4.02 (brs, 1H), 3.86 (s, 6H), 2.90‒2.74 (m, 4H), 2.68‒2.32 (m, 2H), 1.75‒
13
1
.28 (m, 10H), 0.80 (s, 3H); C NMR (75 MHz, CDCl
3
) δ 211.5, 148.9, 147.4, 133.3, 120.0,
111.6, 111.3, 67.7, 55.9, 49.3, 45.3, 36.4, 33.8, 29.7, 25.7, 24.5, 22.6, 14.0. MS (MALDI-
+
+
32 4
TOF) m/z calculated for C20H O [M] : 336.2; found: 336.2. >98% purity (as determined by
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3
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
R
RP-HPLC, method B, t = 14.10 min).
5-Hydroxy-1-phenyldecan-3-one (15a)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
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9
0
Compound 15a was prepared in 40% yield as colorless oil, following the same procedure as
described for the synthesis of 14a but with 4-phenylbutan-2-one instead of 4-(3,4-
1
dimethoxyphenyl)butan-2-one. R
CDCl
2.93 (t, J = 7.5 Hz, 2H), 2.77 (t, J = 7.5 Hz, 2H), 2.62‒2.46 (m, 2H), 1.49‒1.30 (m, 8H),
f
= 0.43 (hexane:EtOAc = 5:1, v/v). H NMR (300 MHz,
3
) δ 7.30 (t, J = 7.5 Hz, 2H), 7.21 (d, J = 7.2 Hz, 3H), 4.05 (brs, 1H), 2.99 (brs, 1H),
13
0
.95‒0.82 (m, 3H); C NMR (75 MHz, CDCl
3
) δ 211.3, 140.7, 128.6, 128.3, 126.2, 67.6,
+
24 2
49.3, 45.1, 36.4, 31.7, 29.5, 25.2, 22.6, 14.1. MS (MALDI-TOF) m/z calculated for C16H O
+
[
M + H] : 249.2; found: 249.9. >98% purity (as determined by RP-HPLC, method A, t
R
=
16.57 min).
5-Hydroxy-1-phenyldodecan-3-one (15b)
Compound 15b was prepared in 38% yield as colorless oil, following the same procedure as
f
described for the synthesis of 15a but with octanal instead of hexanal. R = 0.43
1
(
hexane:EtOAc = 5:1, v/v). H NMR (300 MHz, CDCl
3
) δ 7.31 (t, J = 7.5 Hz, 2H), 7.20 (d, J
=
7.2 Hz, 3H), 4.04 (brs, 1H), 2.98 (brs, 1H), 2.93 (t, J = 7.5 Hz, 2H), 2.79 (t, J = 7.5 Hz, 2H),
1
3
2.57‒2.46 (m, 2H), 1.60‒1.28 (m, 10H), 0.96‒0.83 (m, 3H); C NMR (75 MHz, CDCl ) δ
3
211.0, 152.6, 149.4, 147.3, 137.1, 120.3, 115.9, 67.7, 56.2, 49.4, 45.1, 36.5, 31.8, 29.5, 29.2,
+
+
25.5, 22.7, 14.1. MS (MALDI-TOF) m/z calculated for C18
H
28
O
2
[M + Na] : 299.2; found:
2
99.1. >98% purity (as determined by RP-HPLC, method B, t
-(4-Fluoro-3-methoxyphenyl)-5-hydroxydecan-3-one (16a)
R
= 23.28 min).
1
Compound 16a was prepared in 33% yield as colorless oil, following the same procedure as
described for the synthesis of 14a but with 4-(4-fluoro-3-methoxyphenyl)butan-2-one instead
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of 4-(3,4-dimethoxyphenyl)butan-2-one. R
f
= 0.32 (hexane:EtOAc = 5:1, v/v). H NMR (300
MHz, CDCl ) δ 6.99 (t, J = 9.8 Hz, 1H), 6.79 (d, J = 8.1 Hz, 1H), 6.71 (brs, 1H), 4.05 (s, 1H),
3
3
.89 (s, 3H), 2.94‒2.84 (m, 3H), 2.77 (t, J = 7.3 Hz, 2H), 2.57‒2.46 (m, 2H), 1.49‒1.30 (m,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3
8
3
H), 0.98‒0.81 (m, 3H); C NMR (75 MHz, CDCl ) δ 211.0, 152.6, 149.4, 147.5, 137.1,
120.3, 116.1, 113.6., 67.7, 56.2, 49.4, 45.1, 36.4, 34.5, 32.8, 31.7, 29.1, 25.1, 22.6, 14.1. MS
‒
‒
(
MALDI-TOF) m/z calculated for C17
3
H25FO [M – H] : 295.2; found: 295.0. >98% purity
(
as determined by RP-HPLC, method A, t = 15.63 min).
R
1
-(4-Fluoro-3-methoxyphenyl)-5-hydroxydodecan-3-one (16b)
Compound 16b was prepared in 38% yield as colorless oil, following the same procedure as
f
described for the synthesis of 16a but with octanal instead of hexanal. R = 0.32
1
(hexane:EtOAc = 5:1, v/v). H NMR (300 MHz, CDCl
3
) δ 6.99 (t, J = 9.8 Hz, 1H), 6.80 (d, J
=
8.1 Hz, 1H), 6.70 (brs, 1H), 4.04 (s, 1H), 3.88 (s, 3H), 3.02–2.81 (m, 3H), 2.76 (t, J = 6.9
13
Hz, 2H), 2.57–2.49 (m, 2H), 1.48–1.28 (m, 10H), 0.97–0.80 (m, 3H); C NMR (75 MHz,
CDCl ) δ 211.0, 152.6, 149.4, 147.5, 137.1, 120.9, 116.1, 114.0, 67.7, 56.2, 49.4, 45.1, 36.4,
4.5, 32.8, 31.7, 29.1, 26.1, 25.1, 22.6, 14.0. MS (MALDI-TOF) m/z calculated for
3
3
+
+
C
19
H
29FO
3
[M + H] : 325.2; found: 325.3. >98% purity (as determined by RP-HPLC,
method B, t = 21.19 min).
R
1
-(4-Fluorophenyl)-5-hydroxydecan-3-one (17a)
Compound 17a was prepared in 40% yield as colorless oil, following the same procedure as
described for the synthesis of 14a but with 4-(4-fluorophenyl)butan-2-one instead of 4-(3,4-
1
dimethoxyphenyl)butan-2-one. R
f
= 0.29 (hexane:EtOAc = 5:1, v/v). H NMR (300 MHz,
CDCl ) δ 7.22–7.13 (m, 2H), 6.97 (d, J = 8.7 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 4.03 (s, 1H),
3
2
.99 (s, 1H), 2.88 (t, J = 6.6 Hz, 2H), 2.75 (t, J = 6.6 Hz, 2H), 2.68–2.39 (m, 2H), 1.64–1.40
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4
5
5
5
5
5
5
5
5
5
5
6
1
3
3
(m, 8H), 0.90 (s, 3H); C NMR (75 MHz, CDCl ) δ 211.0, 148.6, 147.4, 132.5, 115.1, 112.9,
1
16.1, 67.7, 63.2, 49.4, 45.1, 36.4, 34.8, 32.2, 31.0, 29.3, 26.1, 25.4, 22.7, 14.0. MS (MALDI-
–
2
–
TOF) m/z calculated for C16
H23FO
[M – H] : 265.4; found: 264.9. >98% purity (as
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
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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
determined by RP-HPLC, method A, t
R
= 17.31 min).
1
-(4-Fluorophenyl)-5-hydroxydodecan-3-one (17b)
Compound 17b was prepared in 45% yield as colorless oil, following the same procedure as
described for the synthesis of 17a but with by using octanal instead of hexanal. R = 0.29
f
1
(
hexane:EtOAc = 5:1, v/v). H NMR (300 MHz, CDCl
3
) δ 7.18–7.08 (m, 2H), 6.97 (d, J =
8
.4 Hz, 1H), 6.94 (d, J = 8.7 Hz, 1H), 4.03 (brs, 1H), 3.00 (s, 1H), 2.88 (t, J = 6.6 Hz, 2H),
1
3
2
.75 (t, J = 6.6 Hz, 2H), 2.68–2.39 (m, 2H), 1.63–1.28 (m, 10H), 0.88 (s, 3H); C NMR (75
) δ 211.5, 145.7, 144.1, 132.5, 120.6, 114.4, 114.3, 111.8, 67.7, 64.4, 49.3, 45.5,
6.4, 31.8, 29.8, 29.3, 25.5, 22.7, 14.9, 14.1. MS (MALDI-TOF) m/z calculated for
MHz, CDCl
3
3
+
+
C
18 2
H27FO [M] : 294.2; found: 294.0. >98% purity (as determined by RP-HPLC, method B,
t
R
= 23.81 min).
5
-Hydroxy-1-(4-hydroxyphenyl)decan-3-one (18a)
Compound 18a was prepared in 40% yield as colorless oil, following the same procedure as
described for the synthesis of 14a but with 4-(4-hydroxyphenyl)butan-2-one instead of 4-
1
(3,4-dimethoxyphenyl)butan-2-one. R
MHz, CDCl
f
= 0.23 (hexane:EtOAc = 5:1, v/v). H NMR (300
3
) δ 7.04 (d, J = 7.2 Hz, 2H), 6.75 (d, J = 7.2 Hz, 2H), 5.77 (brs, 1H), 4.05 (s, 1H),
.19 (brs, 1H), 2.84 (t, J = 6.6 Hz, 2H), 2.74 (t, J = 6.6 Hz, 2H), 2.68–2.41 (m, 2H), 1.69–
3
1
13
3
.12 (m, 8H), 0.89 (s, 3H); C NMR (75 MHz, CDCl ) δ 211.2, 159.7, 142.4, 129.5, 120.6,
114.1, 111.5, 67.7, 55.1, 49.3, 45.0, 36.5, 31.7, 29.6, 25.1, 22.6, 22.2, 14.0. MS (MALDI-
+
+
TOF) m/z calculated for C16
H
24
O
3
[M + H] : 265.2; found: 265.9. >98% purity (as
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R
determined by RP-HPLC, method A, t = 6.60 min).
5-Hydroxy-1-(4-hydroxyphenyl)dodecan-3-one (18b)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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45
46
47
48
49
50
51
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56
57
58
59
60
Compound 18b was prepared in 25% yield as colorless oil, following the same procedure as
f
described for the synthesis of 18a but with octanal instead of hexanal. R = 0.23
1
(
hexane:EtOAc = 5:1, v/v). H NMR (300 MHz, CDCl
3
) δ 7.05 (d, J = 8.1Hz, 2H), 6.76 (d, J
=
8.1 Hz, 2H), 5.09 (brs, 1H), 4.04 (s, 1H), 3.15 (brs, 1H), 2.85 (t, J = 6.6 Hz, 2H), 2.74 (t, J
13
= 6.6 Hz, 2H), 2.68–2.45 (m, 2H), 1.58–1.15 (m, 10H), 0.89 (s, 3H); C NMR (75 MHz,
CDCl
3
) δ 211.2, 159.7, 142.4, 129.7, 129.5, 129.4, 120.6, 114.1, 111.5, 67.7, 55.2, 49.4, 45.0,
+
36.4, 31.8, 29.5, 29.2, 25.5, 22.6, 14.1. MS (MALDI-TOF) m/z calculated for C18
H
28
O
3
[M
+
+
R
H] : 293.2; found: 293.1. >98% purity (as determined by RP-HPLC, method B, t = 9.05
min).
1-(3-Ethoxy-4-hydroxyphenyl)-5-hydroxydecan-3-one (19a)
Compound 19a was prepared in 35% yield as colorless oil, following the same procedure as
described for the synthesis of 14a but with 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one instead
1
of 4-(3,4-dimethoxyphenyl)butan-2-one. R
MHz, CDCl
.10 (q, J = 7.2 Hz, 2H), 2.84 (t, J = 6.6 Hz, 2H), 2.76 (t, J = 6.6 Hz, 2H), 2.67–2.45 (m, 2H),
f
= 0.32 (hexane:EtOAc = 4:1, v/v). H NMR (300
3
) δ 6.84 (d, J = 7.8 Hz, 1H), 6.65 (s, 1H), 6.82–6.61 (m, 1H), 5.59 (brs, 1H),
4
13
1.45 (t, J = 7.2 Hz, 3H), 1.58–1.15 (m, 8H), 0.90 (s, 3H); C NMR (75 MHz, CDCl
3
) δ 200.8,
1
2
2
48.1, 146.8, 142.6, 127.1, 124.2, 123.4, 114.8, 109.3, 56.0, 45.7, 40.7, 31.9, 29.5, 29.3, 29.3,
+
+
4.6, 22.7, 14.1. MS (MALDI-TOF) m/z calculated for C16
H
24
O
3
[M + H] : 265.4; found:
65.9. >98% purity (as determined by RP-HPLC, method A, t
R
= 9.25 min).
1-(3-Ethoxy-4-hydroxyphenyl)-5-hydroxydodecan-3-one (19b)
Compound 19b was prepared in 27% yield as colorless oil, following the same procedure as
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5
6
f
described for the synthesis of 19a but with octanal instead of hexanal. R = 0.32
1
(
hexane:EtOAc = 4:1, v/v). H NMR (300 MHz, CDCl ) δ 6.85 (d, J = 7.8 Hz, 1H), 6.68 (s,
3
1H), 6.66 (d, J = 7.2 Hz, 1H), 5.56 (brs, 1H), 4.10 (q, J = 7.2 Hz, 2H), 2.84 (t, J = 6.6 Hz,
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
2H), 2.74 (t, J = 6.6 Hz, 2H), 2.91–2.45 (m, 2H), 1.46 (t, J = 6.9 Hz, 3H), 1.84–1.19 (m, 10H),
1
3
0.90 (s, 3H); C NMR (75 MHz, CDCl
3
) δ 211.1, 159.7, 142.4, 129.7, 129.5, 129.4, 120.6,
114.0, 111.5 67.7, 55.2, 49.4, 45.0, 36.4, 31.8, 29.8, 29.5, 29.2, 25.5, 22.6, 14.0; MS
+
+
(
MALDI-TOF) m/z calculated for C H O [M + H] : 293.2; found: 293.1. >98% purity (as
18 28 3
determined by RP-HPLC, method B, t = 12.91 min).
R
5-Hydroxy-1-(3-methoxyphenyl)decan-3-one (20a)
Compound 20a was prepared in 32% yield as colorless oil, following the same procedure as
described for the synthesis of 14a but with 4-(3-methoxyphenyl)butan-2-one instead of 4-
1
(
3,4-dimethoxyphenyl)butan-2-one. R = 0.34 (hexane:EtOAc = 5:1, v/v). H NMR (300
f
MHz, CDCl
3
) δ 7.22 (t, J = 7.8 Hz, 1H), 6.79–6.72 (m, 3H), 4.04 (brs, 1H), 3.81 (s, 3H), 2.96
(s, 1H), 2.90 (t, J = 7.2 Hz, 2H), 2.78 (t, J = 7.2 Hz, 2H), 2.61–2.49 (m, 2H), 1.68–1.21 (m,
13
8
H), 0.90 (s, 3H); C NMR (75 MHz, CDCl
3
) δ 211.2, 159.7, 142.4, 129.5, 120.6, 114.1,
111.5, 67.7, 55.1, 49.3, 45.0, 36.5, 31.7, 29.6, 25.1, 22.6, 14.0. MS (MALDI-TOF) m/z
+
3
+
calculated for C H O [M] : 278.2; found: 278.0. >98% purity (as determined by RP-
17
26
R
HPLC, method A, t = 15.50 min).
5-Hydroxy-1-(3-methoxyphenyl)dodecan-3-one (20b)
Compound 20b was prepared in 34% yield as colorless oil, following the same procedure as
f
described for the synthesis of 20a but with octanal instead of hexanal. R = 0.34
1
(hexane:EtOAc = 5:1, v/v). H NMR (300 MHz, CDCl ) δ 7.22 (t, J = 7.8 Hz, 1H), 6.79–6.73
3
(m, 3H), 4.04 (brs, 1H), 3.81 (s, 3H), 2.95–2.86 (m, 3H), 2.82–2.74 (m, 2H), 2.52 (t, J = 9.0
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7
8
9
1
3
Hz, 2H), 1.61–1.18 (m, 10H), 0.88 (s, 3H); C NMR (75 MHz, CDCl
3
) δ 211.2, 159.7, 142.4,
1
29.7, 120.6, 114.1, 111.5, 67.7, 55.2, 49.3, 45.0, 36.5, 31.8, 29.6, 29.2 25.5, 22.7, 14.1. MS
+
+
(MALDI-TOF) m/z calculated for C19
H
30
O
3
[M + H] : 307.2; found: 307.2. >98% purity (as
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
determined by RP-HPLC, method B, t
R
= 21.69 min).
(E)-5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)dec-1-en-3-one (21a)
To a solution of compound 12 (150 mg, 0.7 mmol) in THF (5 mL) was added LDA (1.7 mL,
1 M in THF/hexanes) at –78 ºC under Ar. The solution was stirred for 1 hr at the same
temperature. Hexanal (0.52 mL, 6.2 mmol) was slowly added. The reaction mixture was
stirred for 3 hr at the same temperature. The reaction mixture was quenched with aqueous
NH
brine, dried over MgSO
residue was purified by column chromatography on silica gel (eluting with a mixture of
4
Cl (10 mL) and extracted with EtOAc. The organic layer was washed with water and
4
, filtered, and concentrated under reduced pressure. The crude
f
hexane:EtOAc, 10:1 to 3:1, v/v) to furnish compound 21a (70 mg, 31%). R = 0.25
1
(
hexane:EtOAc = 5:1, v/v). H NMR (300 MHz, CDCl
3
) δ 7.53 (d, J = 16.2 Hz, 1H), 7.12 (d,
J = 8.1 Hz, 1H), 7.07 (s, 1H), 6.95 (d, J = 8.1 Hz, 1H), 6.61 (d, J = 16.2 Hz, 1H), 4.15 (brs,
1
3
1H), 3.95 (s, 3H), 2.93–2.71 (m, 1H), 2.39 (s, 1H), 1.82–1.25 (m, 8H), 0.92 (s, 3H);
C
NMR (75 MHz, CDCl ) δ 201.0, 148.6, 146.9, 143.9, 126.7, 124.1 123.8, 114.9, 109.5, 68.1,
3
+
56.0, 46.5, 36.5, 31.8, 25.2, 22.6, 14.1. MS (MALDI-TOF) m/z calculated for C17
H
24
O
4
[M
+
+
H] : 293.2; found :293.1. >98% purity (as determined by RP-HPLC, method A, t
R
= 6.91
min).
(E)-5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodec-1-en-3one (21b)
Compound 21b was prepared in 31% yield, following the same procedure as described for
the synthesis of 21a but with octanal instead of hexanal. R = 0.25 (hexane:EtOAc = 5:1, v/v).
f
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9
1
1
1
1
1
1
1
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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
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
H NMR (300 MHz, CDCl
3
) δ 7.53 (d, J = 16.2 Hz, 1H), 7.11 (d, J = 8.1 Hz, 1H), 7.08 (s,
1
H), 6.94 (d, J = 8.1 Hz, 1H), 6.60 (d, J = 16.2 Hz, 1H), 4.15 (brs, 1H), 3.91 (s, 3H), 2.93–
13
2
.68 (m, 1H), 2.35 (s, 1H), 2.75–1.21 (m, 10H), 0.89 (s, 3H); C NMR (75 MHz, CDCl
3
) δ
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
2
00.1, 148.5, 146.9, 143.8, 126.7, 124.1, 123.7, 114.9, 109.5, 68.0, 56.0, 46.5, 36.6, 31.8,
+
+
29.6, 29.2, 25.5, 22.6, 14.0. MS (MALDI-TOF) m/z calculated for C19
H
28
O
3
[M + Na] :
3
43.4; found: 343.2. >98% purity (as determined by RP-HPLC, method B, t = 11.97 min).
R
(E)-1-(4-Fluoro-3-methoxyphenyl)-5-hydroxydec-1-en-3-one (22a)
Compound 22a was prepared in 40% yield, following the same procedure as described for the
synthesis of 21a but with (E)-4-(4-fluoro-3-methoxyphenyl)but-3-en-2-one instead of
1
compound 12. R
f
= 0.32 (hexane:EtOAc = 5:1, v/v). H NMR (300 MHz, CDCl
3
) δ 7.53 (d, J
= 16.2 Hz, 1H), 7.15 (d, J = 8.1 Hz, 2H), 7.11 (s, 1H), 6.66 (d, J = 16.2 Hz, 1H), 4.16 (brs,
13
1
H), 3.95 (s, 3H), 3.2 (s, 1H), 2.93–2.72 (m, 2H), 1.63–1.33 (m, 8H), 0.89 (s, 3H); C NMR
(75 MHz, CDCl ) δ 200.1, 155.7, 152.3, 148.2, 148.0, 142.6, 130.9, 126.2, 122.2, 116.5,
3
1
12.3, 67.9, 58.0, 46.9, 36.6, 31.8, 25.6, 25.3, 22.7, 22.6, 14.0. MS (MALDI-TOF) m/z
+
+
calculated for C17
H25FO
3
[M] : 294.2; found: 294.0. >98% purity (as determined by RP-
HPLC, method A, t
R
= 16.32 min).
(E)-1-(4-Fluoro-3-methoxyphenyl)-5-hydroxydodec-1-en-3-one (22b)
Compound 22b was prepared in 31% yield, following the same procedure as described for
f
the synthesis of 22a but with octanal instead of hexanal. R = 0.32 (hexane:EtOAc = 5:1, v/v).
1
H NMR (300 MHz, CDCl
3
) δ 7.55 (d, J = 16.2 Hz, 1H), 7.27 (s, 1H), 7.12 (d, J = 8.1 Hz,
2
H), 6.66 (d, J = 16.2 Hz, 1H), 4.15 (brs, 1H), 3.95 (s, 3H), 3.2 (s, 1H), 2.93–2.79 (m, 2H),
1
3
1.60–1.30 (m, 10H), 0.89 (s, 3H); C NMR (75 MHz, CDCl ) δ 200.1, 155.7, 152.3, 148.2,
3
1
48.0, 142.6, 130.9, 126.2, 122.2, 116.5, 112.3, 67.9, 58.0, 46.9, 36.6, 31.8, 29.5, 29.3 25.6,
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+
+
3
25.3, 22.7, 22.6, 14.0. MS (MALDI-TOF) m/z calculated for C19H27FO [M] : 323.2; found:
3
23.2 >98% purity (as determined by RP-HPLC, method B, t = 22.13 min).
R
(E)-1-(4-Hydroxy-3-methoxyphenyl)dec-1-en-3-one (23a)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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To a solution of 4-hydroxy-3-methoxybenzaldehyde (913 mg, 6.0 mmol) in MeOH (10 mL)
was added (L)-proline (86 mg, 0.75 mmol) and nonan-2-one (0.87 mL, 5.0 mmol) at 25 ºC
under Ar. After 30 min, triethylamine (0.21 mL, 1.5 mmol) was introduced. The reaction
mixture was stirred 25 ºC for 48 hr, and then quenched with water and extracted with EtOAc.
The combined organic layer was dried over MgSO
4
, filtered, and concentrated under reduced
pressure. The crude residue was purified by column chromatography on silica gel
(hexane:EtOAc = 10:1 to 3:1, v/v) to furnish compound 23a (746 mg, 45%) as a fluffy white
1
solid. R
f
= 0.32 (hexane:EtOAc = 5:1, v/v). H NMR (300 MHz, CDCl
3
) δ 7.51 (d, J = 16.2
Hz, 1H), 7.12 (d, J = 7.8 Hz, 1H), 7.08 (s, 1H), 6.94 (d, J = 8.1 Hz, 1H), 6.63 (d, J = 16.2 Hz,
1H), 5.94 (s, 1H), 3.95 (s, 3H), 2.66 (t, J = 7.2 Hz, 2H), 1.78–1.56 (m, 2H), 1.54–1.19 (m,
13
8
H), 0.88 (s, 3H); C NMR (75 MHz, CDCl
3
) δ 201.0, 148.4, 147.0, 142.8, 126.9, 123.9,
123.3, 114.9, 109.6, 55.9, 40.6, 31.7, 29.3, 29.1, 24.6, 22.6, 14.0. MS (MALDI-TOF) m/z
+
+
24 3
calculated for C14H O [M] : 276.2; found: 276.2. >98% purity (as determined by RP-
HPLC, method A, t = 24.61 min).
R
(E)-1-(4-Hydroxy-3-methoxyphenyl)dodec-1-en-3-one (23b)
Compound 23b was prepared in 60% yield as a fluffy white solid, following the same
f
procedure as described for the synthesis of 23a but with octanal instead of hexanal. R = 0.32
1
(
hexane:EtOAc = 5:1, v/v). H NMR (300 MHz, CDCl
3
) δ 7.51 (d, J = 16.2 Hz, 1H), 7.12 (d,
J = 7.8 Hz, 1H), 7.08 (s, 1H), 6.94 (d, J = 8.1 Hz, 1H), 6.63 (d, J = 16.2 Hz, 1H), 5.92 (s, 1H),
3.95 (s, 3H), 2.66 (t, J = 7.2 Hz, 2H), 1.78–1.56 (m, 2H), 1.51–1.13 (m, 12H), 0.88 (s, 3H);
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