Structural optimization of natural product nordihydroguaretic acid to discover novel analogues as AcrB inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.111910

Drug efflux pumps confer multidrug resistance to dangerous bacterial pathogens which makes these proteins promising drug targets. Herein, we present initial chemical optimization and structure-activity relationship (SAR) data around a previously described efflux pump inhibitor, nordihydroguaretic acid (NDGA). Four series of novel NDGA analogues that target Escherichia coli AcrB were designed, synthesized and evaluated for their ability to potentiate the activity of antibiotics, to inhibit AcrB-mediated substrate efflux and reduce off-target activity. Nine novel structures were identified that increased the efficacy of a panel of antibiotics, inhibited drug efflux and reduced permeabilization of the bacterial outer and inner membranes. Among them, WA7, WB11 and WD6 possessing broad-spectrum antimicrobial sensitization activity were identified as NDGA analogues with favorable properties as potential AcrB inhibitors, demonstrating moderate improvement in potency as compared to NDGA. In particular, WD6 was the most broadly active analogue improving the activity of all four classes of antibacterials tested.

Full text of DOI:10.1016/j.ejmech.2019.111910

Journal Pre-proof
Structural optimization of natural product nordihydroguaretic acid to discover novel
analogues as AcrB inhibitors
Yinhu Wang, Rawaf Alenzy, Di Song, Xingbang Liu, Yuetai Teng, Rumana Mowla,
Yingang Ma, Steven W. Polyak, Henrietta Venter, Shutao Ma
PII:
S0223-5234(19)31062-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.111910
EJMECH 111910
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 August 2019
Revised Date: 12 November 2019
Accepted Date: 24 November 2019
Please cite this article as: Y. Wang, R. Alenzy, D. Song, X. Liu, Y. Teng, R. Mowla, Y. Ma, S.W.
Polyak, H. Venter, S. Ma, Structural optimization of natural product nordihydroguaretic acid to discover
novel analogues as AcrB inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.111910.
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Graphical Abstract：
Structural optimization of natural product nordihydroguaretic acid to discover novel analogues
as AcrB inhibitors
a
a
a
c
Yinhu Wang a, b, 1, Rawaf Alenzy c, d 1, Di Song , Xingbang Liu , Yuetai Teng , Rumana Mowla ,
Yingang Ma a, Steven W. Polyak c, Henrietta Venter c, *, Shutao Ma a, *
Novel nordihydroguaretic acid analogues were designed, synthesized and evaluated as AcrB inhibitors.

Structural optimization of natural product nordihydroguaretic acid to discover
novel analogues as AcrB inhibitors
a
a
a
c
a
Yinhu Wang a, b, 1, Rawaf Alenzy c, d, 1, Di Song , Xingbang Liu , Yuetai Teng , Rumana Mowla , Yingang Ma ,
Steven W. Polyak c, Henrietta Venter c, *, Shutao Ma a, *
aDepartment of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of
Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan 250012, China
bSchool of Pharmacy, Liaocheng University, Liaocheng, China
cSchool of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia
dDepartment of Medical Laboratory, College of Applied Medical Sciences–Shaqra, Shaqra University, 11961, Saudi
Arabia
Running title: Nordihydroguaretic acid analogues
*Corresponding authors.
E-mail addresses: rietie.venter@unisa.edu.au (H. Venter), mashutao@sdu.edu.cn (S. Ma).
1These authors contributed equally
1

Abstract: Drug efflux pumps confer multidrug resistance to dangerous bacterial pathogens which makes these
proteins promising drug targets. Herein, we present initial chemical optimization and structure-activity relationship
(SAR) data around a previously described efflux pump inhibitor, nordihydroguaretic acid (NDGA). Four series of
novel NDGA analogues that target Escherichia coli AcrB were designed, synthesized and evaluated for their ability to
potentiate the activity of antibiotics, to inhibit AcrB-mediated substrate efflux and reduce off-target activity. Nine
novel structures were identified that increased the efficacy of a panel of antibiotics, inhibited drug efflux and reduced
permeabilization of the bacterial outer and inner membranes. Among them, WA7, WB11 and WD6 possessing
broad-spectrum antimicrobial sensitization activity were identified as NDGA analogues with favorable properties as
potential AcrB inhibitors, demonstrating moderate improvement in potency as compared to NDGA. In particular,
WD6 was the most broadly active analogue improving the activity of all four classes of antibacterials tested.
Keywords: Nordihydroguaretic acid; Multidrug resistance; Efflux pump; AcrB inhibitors; Structural optimization.
2

1. Introduction
The widespread prevalence and dissemination of multidrug-resistant (MDR) bacteria throughout the world poses a
serious threat to human health. In particular, infections caused by antibiotic-resistant Gram-negative bacteria, such as
Enterobacteriaceae, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii, have led to
reduced or abolished efficacy of many antibacterial agents in the clinic [1-5]. Critically, only a few new classes of
antibacterial drugs necessary to combat antibiotic resistance have been listed this century. In view of this situation,
the World Health Organization (WHO) warns that in the near future there will be no drugs available for the treatment
of serious infections caused by MDR Gram-negative bacteria unless the issue is addressed now [6-8]. Therefore,
bacterial MDR has become a serious global public health concern [6, 7, 9]. Obviously, there is an urgent need to
develop new solutions for the treatment of infections caused by MDR Gram-negative pathogens.
The over-expression of tripartite multidrug efflux complexes spanning the inner and outer membranes is one of
major mechanisms of antibiotic resistance in Gram-negative bacteria [10-15] . Efflux pumps recognize and expel a
variety of structurally diverse compounds from bacteria before they can reach their potential site of action, thereby
preventing their intracellular accumulation to threshold concentrations necessary for inhibitory activity [16-21].
In MDR Escherichia coli and other Enterobacteriaceae the major drug efflux pump contributing to antibiotic
resistance is AcrAB-TolC [22, 23]. This efflux pump spans both the inner and outer cell membranes as a tripartite
assembly consisting of an inner membrane spanning protein (AcrB), a periplasmic adaptor protein (AcrA) and
outer-membrane channel protein (TolC) [24-27]. These three separate proteins work co-operatively in a rotatory
mechanism where individual subunits become alternately protonated and then engage and subsequently disengage
substrate molecules [28, 29]. AcrB catalyses drug/H+ antiport and is the protein subunit primarily responsible for
substrate recognition [30-34]. AcrB can recognize an extremely broad range of substrates that are not limited to
antibiotics but also includes antiseptic compounds, disinfectants, dyes, solvents, fatty acids, metal cations and
virulence factors [12, 23, 35].
The inability to discover novel antibiotics to combat MDR Gram-negative bacteria prompted us to look for new
strategies capable of restoring the effectiveness of existing antibiotics. Small molecule inhibitors of the AcrAB-TolC
efflux pump provide an attractive route towards reversing MDR [36, 37]. Efflux pump inhibitors (EPIs) that appear in
the literature have been reported to restore the activities of existing antibacterial agents by binding to AcrB,
perturbing the outer membrane channel or disrupting assembly of the tripartite protein complex [19, 24, 38-42].
Despite their crucial role in MDR, there are currently no EPIs approved as antibiotic adjuvants for clinical use [43] .
The recently solved crystal and cryo-electron microscopy structures of AcrB in complex with substrates or inhibitors
have provided a solid basis for understanding the fundamental mechanism of drug export and rational design of high
efficacy, target-specific inhibitors [28, 39, 44-46]. Crystal structures of AcrB in complex with substrates minocycline
and doxorubicin [44, 47], have revealed a common substrate binding pocket that is large, flexible and rich in
phenylalanine residues that allow multidrug binding via hydrophobic and π-π stacking interactions. In addition,
certain polar residues also provide further opportunities to form hydrogen bonds, such as Asn274 and Gln176 [28].
Structures of inhibitor-bound AcrB have indicated that the inhibitor binding site partially overlaps with the
minocycline binding site in a distal binding pocket, while another part of the inhibitor is engaged in multiple
hydrophobic and π-π stacking interactions with various side chains lining an adjacent pocket known as the
hydrophobic trap including Phe178, Phe610, Phe615 and Phe628 [39, 45]. EPIs binding into this site sterically hinder
the functional rotation mechanism of the AcrB trimer, thereby disabling the activity of the entire efflux machinery [39,
45, 48, 49].
We recently identified nordihydroguaretic acid (NDGA, Fig. 1a) as a moderate EPI via virtual screening from a
library of 50 phytochemicals [50] . In our in silico binding studies, it was predicted that NDGA bound into the same
distal pocket described above that partially overlaps with the minocycline site (Fig. 1b). This interaction was
stabilized by π-π stacking interactions between the two phenyl moieties of NDGA with aromatic side chains of
Phe628, Phe178 and Phe615, and an additional hydrogen bond between a single hydroxyl group appended onto one
3

of the phenyl groups and the carbonyl of Pro326 and (Fig. 1c). Subsequent biological characterization revealed that
NDGA acted synergistically with a broad range of antibacterials such as chloramphenicol (CAM), erythromycin
(ERY) and tetraphenylphosphonium (TPP) against a MDR strain of E. coli. The mechanism of action of NDGA was
demonstrated to be through the inhibition of AcrB using whole cell assays that measured efflux of the fluorescent
Nile Red substrate. In the current study we aimed to obtain new chemotypes of NDGA with better inhibitory activity
against AcrB and to gain SAR data to direct further chemical optimization. Based upon our molecular docking studies
above, and our detailed knowledge of the structure of AcrB inhibitor binding site, we designed four series of novel
NDGA analogues for further evaluation. In each case, the hydrogen bonding donors of the two hydroxyl groups
appended onto the benzene ring on one end of the molecule were retained whilst the another end of the molecule was
modified by: (1) introduction of structurally diverse groups on the benzene ring such as hydroxyl, halogen, amino,
methyl and isopropyl groups to explore their interactions with the surrounding amino acid residues; (2) replacement
of the benzene ring with alternative larger aromatic or heterocyclic rings such as biphenyl, naphthalene and
anthracene groups, which was expected to potentiate the binding affinity for AcrB through π-π stacking or
hydrophobic interactions; (3) alteration of the length of the alkyl linker to explore its accommodation within the
hydrophobic binding site; (4) substitution of an amide-containing linker for the hydrophobic alkyl linker to probe
additional hydrogen bonding interactions with polar amino acids lining the inhibitor binding site.
<Insert Fig. 1>
2. Results and discussion
2.1 Synthetic routes
The synthetic route of the NDGA analogues (WA1-WA12 and WB1-WB11) is as outlined in Scheme 1. The starting
material benzaldehyde was subjected to the Knoevenagel condensation with malonic acid to afford
(E)-3-(3,4-dimethoxyphenyl)acrylic acid (2), which was treated with lithium aluminium tetrahydride (LiAlH4) in
tetrahydrofuran (THF) to give the corresponding alcohol (3). Then treatment of 3 with carbon tetrabromide (CBr4)
and triphenylphosphine (PPh3) produced the brominated intermediate 4. Subsequently, 4 was reacted with PPh3 in
refluxing toluene to give the phosphonium salts 5. Wittig olefination of 5 with a library of appropriate aldehydes
permitted the addition of a variety of phenyl and heterocyclic groups and gave the alkene intermediate 6 as a mixture
of geometrical isomers. Catalytic hydrogenation of these mixtures over palladium afforded the reduction intermediate
7, followed by subsequent demethylation with BBr3 to generate the NDGA analogues WA1-WA12 and WB1-WB11.
<Insert Scheme 1>
The synthetic method for 4,4'-(propane-1,3-diyl)bis(benzene-1,2-diol) (WC1) is shown in Scheme 2. Similarly,
3,4-dimethoxybenzaldehyde was used as starting material, which was subjected to aldol condensation with
3,4-dimethoxyacetophenone (8) to obtain the aldol intermediate 9. Catalytic hydrogenation of 9 gave the reduction
intermediate 10, which was treated with sodium borohydride (NaBH4) in the presence of methanol (MeOH) to
provide the corresponding alcohol 11. Then 11 was subjected to elimination to afford the alkene intermediate 12 and
catalytic hydrogenation of 12 provided the intermediate 13, which was subsequently demethylated with BBr3 to give
compound WC1.
<Insert Scheme 2>
The synthetic route for the NDGA analogue WC2 is shown in Scheme 3. Aldol condensation of 1 with acetone
afforded the ketene intermediate 14. Catalytic hydrogenation of 14 over palladium gave the ketone intermediate 15,
which was then subjected to Wolff-Kischner reduction with hydrazine hydrate in the presence of potassium hydroxide
4

(KOH) to provide the intermediate 16. Subsequently, BBr3 demethylation of 16 gave compound WC2.
<Insert Scheme 3>
The synthetic route for the NDGA analogues WC3 and WC4 is shown in Scheme 4. Dicarboxylic acids (17) as
the starting materials was subjected to chlorination with thionyl chloride (SOCl2), which was followed by Friedel
Crafts reaction to produce the diketone intermediate 18. Catalytic hydrogenation of 18 over palladium gave
intermediate 19, which subsequently was demethylated with BBr3 to yield compounds WC3 and WC4.
<Insert Scheme 4>
The synthetic method for the NDGA analogues WD1-WD10 is shown in Scheme 5. 3,4-Dimethoxybenzylamine
reacted with benzoic acid with different substituents to afford the amide intermediate 21 and then demethylation of 21
produced compounds WD1-WD10. When 21 possessed nitro groups on the benzene ring, a catalytic hydrogenation
reaction for 21 was first implemented, and subsequent demethylation with BBr3 to give the corresponding NDGA
analogues.
<Insert Scheme 5>
2.2. Antibacterial activity
The intrinsic antibacterial activity of all the NDGA analogues were initially assessed by determining the minimum
inhibitory concentration (MIC) against the MDR wild type E. coli BW25113 expressing AcrB that is indicated as
(+)AcrB and an isogenic strain with the acrb gene deleted that is indicated as (-)AcrB. This determined the
concentration of the NDGA analogues that could be tested without producing a direct antibacterial effect in further
antimicrobial synergy assays. The results showed that none of the compounds possessed intrinsic antibacterial
activity against E. coli BW25113 at 512 µg/mL, except WD9 with an MIC of 64 µg/mL. Likewise, none of the
compounds were active against (-)AcrB cells at 64 µg/mL except WC2 and WD9 both of which gave an MIC value
at this high concentration (data not shown). For all remaining compounds, the absence of any intrinsic antibacterial
activity at 512 µg/mL allowed us to further investigate inhibition of AcrB-mediated efflux.
2.3. Ability to reverse drug resistance
The ability of the NDGA analogues to reverse antibiotic resistance conferred by the AcrAB-TolC efflux pump was
next determined using a checkerboard assay [18, 43, 50]. Here, the MIC values of a panel of antibiotics were
measured with E. coli BW25113 in the presence of varying concentrations of the tested compounds. The spectrum of
EPI activity was explored using structurally unrelated antibacterials CAM, ERY, TTP and levofloxacin (LEV). Table
1 shows the data for the WA, WB and WC series that reduced the MIC values for at least one antibacterial agent by
2-fold or greater, whereas Table 2 shows the comparable data for the WD series. Ten compounds with antibacterial
sensitizing activity in Table 1 were all inhibitors of TPP efflux, with WC2 being the most potent compound that
reduced the MIC value by 32-fold at 256 µg/mL. WA5-WA7 and WB11 were broadly active with CAM, ERY and
TPP, with WA7 and WB11 both reducing the MIC value of TPP by 4-fold at 128 µg/mL. WA7 also had a large effect
upon CAM where it reduced the MIC value by 8-fold at the highest concentration tested. However, none of the
compounds in Table 1 improved the activity of LEV.
<Insert Table 1>
In contrast, WD2, WD6 and WD10 in Table 2 were active when tested in combination with LEV, with WD2 and
WD6 both reducing the MIC by 2-fold at the low concentration of 32 µg/mL. It is noteworthy that WD6 displayed
5

broad activity against the four antibacterials and lowered the MIC values by 4-fold for CAM, ERY and TPP at 128
µg/mL, and the MIC value by 2-fold for LEV at 32 µg/mL. Similarly, WD2, WD5, WD7 and WD8 also possessed
broad spectrum activity reducing the MIC values of three out of the four antibacterials in the screening panel. WD5
and WD9 reduced the MIC value of ERY by 4-fold at 32 µg/mL whereas WD7 decreased the MIC values of both
CAM and ERY by 4-fold at 128 µg/mL. Rifampicin (RIF) that is not a substrate of AcrB was used as a negative
control. None of the compounds altered the MIC value of RIF (16 µg/mL) consistent with these compounds likely
function through the AcrB efflux pump and not through alternative mechanism (s) of action [43, 51]. The findings
above suggest that the compounds exert their synergistic activity through the inhibition of AcrB.
<Insert Table 2>
2.4. The effect of the compounds on substrate transport
The NDGA analogues that showed bioactivity in the checkerboard assays described above were characterized further
to determine if they inhibited AcrB using whole cell efflux assays [43, 51]. The AcrAB-TolC substrate Nile Red was
employed as this probe, which is weakly fluorescent in aqueous solutions but undergoes a significant increase in
fluorescence quantum yield in non-polar environments, such as the cell membrane [52]. E. coli BW25113 were
de-energized by treatment with the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which was
followed by being preloaded with Nile Red in the absence or presence of the NDGA analogues. Following the
addition of glucose to re-energize the cells, the efflux of Nile Red was measured by fluorometry. The E. coli strain
with (-)AcrB was used as a positive control, indicating the maximum levels of Nile Red accumulation possible in the
absence of efflux. The parent compound NDGA was potent in the assay, with inhibitory activity observed at the
concentration of 50-100 µM (Fig. 2). In remarkable contrast, WA5, WA7, WC1 and WD6-WD8 were weakly active,
with inhibitory activity only observed at the highest concentration tested (500 µM). The moderate activity was
observed for WC2, WC4, WD4 and WD5 at the concentration of 100-200 µM. The most potent inhibitory activity
was measured for WA9, WA10, WB11, WC3 and WD9 at the concentration of 50-100 µM (Fig. 2), which could
completely inhibit efflux to the level of the pump-deleted strain, similar to NDGA.
<Insert Fig. 2>
2.5. The effect of the compounds on the bacterial outer membrane
Compounds that permeabilize either the inner or outer bacterial membranes may show synergistic activity with
antibiotics in a mechanism independent of AcrAB-TolC [53]. To determine if the potentiation of the antibiotic activity
observed above could be due to permeabilization of the outer membrane, the rate of hydrolysis of a chromogenic
β-lactam, nitrocefin was assayed using intact E. coli BW25513 [54] . Hydrolysis of nitrocefin by the bacterial
β-lactamase produces a colored product that can be measured spectrometrically at 490 nm. The rate of nitrocefin
hydrolysis is limited by the rate of diffusion across the outer membrane, but this may be accelerated due to outer
membrane permeabilization [51]. Polymyxin B (PMB) was used as a positive control as this agent is known to
damage the bacterial outer membrane (red line, Fig. 3). Disruption of the outer membrane was observed when cells
were treated with 256 µg/ml NDGA with increased absorbance measured over the time course that was above the
untreated control (blue lines, Fig. 3). Similar off-target activity was observed for WA9 and WA10. WC3 was
particularly toxic as the yield of product formed approached that measured for polymyxin B, so this compound was
discarded from further evaluation. The NDGA analogues with desirable low permeabilization activity included WA5,
WA7, WB11, WC1-WC4, WD2 and WD4-WD9. For these agents, we could conclude that their synergism with
known antibacterials was not due to the secondary mechanism of membrane permeabilization.
<Insert Fig. 3>
6

2.6. The effect of the compounds on the bacterial inner membrane
Finally, off-target activity caused by chemical damage to the inner membrane was addressed. As AcrAB-TolC is
powered by the proton motive force across the bacterial inner membrane, compounds that perturb the proton gradient
could also indirectly inhibit efflux [4, 11, 51]. The ability of NDGA analogues to cause depolarization of bacterial
transmembrane potential (∆ψ) was evaluated by using the fluorescent membrane potential-sensitive probe
3,3-diethyloxacarbocyanine iodide (DiOC2(3)). Bacteria were first energized by the addition of glucose to establish
the proton motive force (negative and basic inside the cell), then treated with DiOC2(3) and increased fluorescence
measured due to the aggregation of the positively charged probe as it entered cells through passive diffusion. Upon
addition of the ionophore CCCP, the ∆ψ was dissipated and the fluorescence intensity reduced to the level before
glucose stimulation (See Fig. 4, blue curves representing bacteria not challenged with compound) [18, 43, 55]. Those
compounds that disrupted the inner membrane consequently disturbed the proton gradient resulting in no
accumulation of fluorescence signal. Severe membrane disruption activity was observed with the parent compound
NDGA at concentrations of 64 µg/mL or greater (Fig. 4). This unwanted off-target activity was not investigated in the
initial report. Similar low toxicity was observed at 128 µg/mL for WA5 and WC2. However, the off-target activity
was reduced for WA7 (128 g/mL), WB11 (16 µg/mL) and WC4 (64 µg/mL). Likewise, WD4-WD8 also displayed
favorable properties with neither WD4-WD6 showing toxicity at 64 µg/mL. It was noteworthy that WD7 and WD8
was still inactive in the assay at 128 µg/mL. Unexpectedly, poor accumulation of DiOC2(3) was observed when
bacterial were treated with WD9, consistent with damage to the inner membrane. Consequently, WA7, WB11, WC2,
WC4 and WD4-WD8 with a mechanism of action consistent with the inhibition of AcrB were confirmed.
<Insert Fig. 4>
2.7. Molecular docking
To aid in the interpretation of the biological findings and the establish SAR data to guide future chemical
optimization, molecular modelling studies were performed. Crystal structures of AcrB in complex with substrates and
inhibitors have revealed that the binding pocket of AcrB is rich in phenylalanine residues Phe136, Phe178, Phe610,
Phe615, Phe628 that stabilize ligand binding through hydrophobic and π-π stacking interactions. In addition, certain
polar amino acid residues provide hydrogen bonding potential such as Asn274, Gln176 and Pro326 [4, 28, 47].
Building upon our earlier in silico binding studies with NDGA (Fig. 1), we designed the NDGA analogues that
retained the hydrogen bond donor nature of phenolic hydroxyl groups at the one end of the molecule.
Pharmacomodulations then involved the introduction of the structurally diverse groups into the benzene ring at the
other end of the molecule (WA1-WA12) to explore their interactions with the surrounding polar and non-polar amino
acid residues in the binding pocket of AcrB. Further, we replaced the benzene ring with aromatic or heterocyclic rings
owning a larger spatial structure (WB1-WB11) to explore their binding affinity for AcrB via π-π stacking or
hydrophobic interactions. In addition, we changed the length of the alkyl linker between the two benzene rings
(WC1-WC4) to explore their adaptability in the hydrophobic cavity of the binding pocket. Finally, we substituted the
alkyl linker in NDGA using an amide-containing linker (WD1-WD10) to probe additional hydrogen bonding
interaction with the amino acid residues.
After the exhaustive biological characterization of the test compounds, eight NDGA analogues specifically acting
on AcrB were identified as AcrB inhibitors in this study, namely WA7, WB11, WC2, WC4 and WD4-WD8. In order
to reasonably explain the above biological data and visually discuss the binding mode of the bioactive compounds
with AcrB, we selected WA7 and WD6 as representative compounds for molecular docking. Molecular docking
software SYBYL-X2.0 was used to predict the mode of binding in AcrB (PDB: 5ENO), and the docking results
visualized by PYMOL, as shown in Fig. 5. The docking results indicated that the binding modes of WA7 and WD6
were similar to that of the parent compound NDGA. The phenolic hydroxyl groups at the one end of the molecule
7

bond to oxygen of Pro326 by hydrogen bond interaction, while two benzene rings at both ends of the molecule were
stabilized by interactions with the amino acids Phe628 and Phe178 via π-π stacking interactions. Moreover, the
oxygen atom of the amide group on the linker of WD6 formed an additional hydrogen bond with the Val139, which
may be the reason why the bioactivity of WD series was generally better than that of the other series. The additional
binding interactions likely improve on-target recognition of AcrB that help minimize the off-target activities observed
with NDGA.
<Insert Fig. 5>
On the whole, four series of novel NDGA analogues were designed, synthesized and characterized for their ability
to inhibit the AcrAB-TolC efflux pump in E. coli. The preliminary SARs derived from this study indicate that: (1) the
sensitizing activity of the NDGA analogues is closely related to the substituents on the benzene ring in their
molecules, and hydrophilic groups are preferred; (2) the replacement of the benzene ring with aromatic or
heterocyclic rings possessing a large steric effect is unfavorable for retaining potency; (3) the intermediate linker is
best with a four-atom chain, and the introduction of the amide-containing linker can improve the efficacy.
Furthermore, NDGA was observed to possess significant and undesirable activity such as off-target effect in the
assays of measuring the permeabilization of the bacterial inner and outer membranes. Through the structural
optimization of NDGA, new chemical structural types specifically targeting AcrB were identified, which displayed
superior or comparable efficacy to NDGA, indicating a successful chemical modification in this program. More
importantly, the above SARs provide some valuable information in guiding the further structural optimization of
NDGA as well.
Noteworthy are the WD series possessing the amide-containing linker that generally exhibit better efficacy against
the AcrB-expressing strain than that of the WA, WB and WC series, which is probably due to an additional hydrogen
bond of the oxygen atom of the amide group with the Val139 in the pocket, thereby improving binding affinity for
AcrB. Among the best compounds, WD6 showed the best efficacy and decreased the MIC values by 4-fold for CAM,
ERY and TPP, and the MIC value by 2-fold for LEV at a lower concentration than that of its parent NDGA. WD5 and
WD9 lowered the MIC value of ERY by 4-fold at 32 µg/mL, whereas WD7 and WD8 at 128 µg/mL reduced the MIC
values of both CAM and ERY by 4-fold and by 2-fold, respectively. In contrast, the WA, WB and WC series all
possess a hydrophobic alkyl linker in their structure. In the WA series, WA5-WA7 were broadly active with CAM,
ERY and TPP, and particularly WA7 reduced not only the MIC value of CAM by 8-fold at 256 µg/mL, but also the
MIC value of TPP by 4-fold at 128 µg/mL. In the WB series, only WB11 containing pyrene group showed broadly
synergism with CAM, ERY and TPP, reducing the MIC value of TPP by 4-fold at 128 µg/mL. This could be because
the pyrene moiety could block the freedom of mutual rotation of the ring in the structure and might facilitate a good
fit within the AcrB binding pocket. However, none of the compounds in the WA, WB and WC series improved the
activity of LEV. Moreover, the optimal length of the alkyl linker that connects two aromatic rings was four atoms,
which could ensure that the two aromatic rings simultaneously interacted with the corresponding amino acid residues
(Phe178 and Phe628) via π-π stacking or hydrophobic interactions, thereby potentiating the binding affinity for AcrB.
Subsequently, in order to further confirm the efflux inhibition activity and specificity of AcrB, the active compounds
were selected to determine their efflux inhibition ability of Nile Red, and the effect on outer and inner membrane
permeabilization. Consequently, WA7, WB11, WC2, WC4 and WD4-WD8 were found to exhibit the following
characteristics of an ideal AcrB inhibitor, as described by Lomovskaya et al. [11, 51]: (1) it potentiated the activity of
various antibacterial agents that are substrates of AcrAB-TolC; (2) it did not reduce the MIC values of the agents that
were not its substrates (e.g., RIF); (3) it had no effect on susceptible strains ((-)AcrB); (4) it decreased the level of
extrusion of AcrB substrates (e.g., Nile Red); (4) it did not permeabilize the outer membrane; (5) it did not disturb the
proton gradient of AcrB across the inner membrane.
8

3. Conclusions
In this study, thirty-seven compounds were designed, synthesized and evaluated for their AcrB inhibition activity.
The results revealed that nine compounds (WA7, WB11, WC2, WC4 and WD4-WD8) identified as ideal AcrB
inhibitors were able to reduce the MIC value of at least one antibiotic tested by 2- to 32-fold. They also inhibited Nile
red efflux at reasonable concentration and acted specifically on the AcrB efflux pump. Among the above compounds
with characteristics of ideal AcrB inhibitors, the best candidates WD6, WA7 and WB11 have broad-spectrum
antimicrobial sensitization activity. In particular, WD6 is the most active analogue improving the activity of all four
classes of antibacterials tested. Although this study obtained a promising group of potential AcrB inhibitors, the most
active compounds displayed only moderate sensitizing properties. Therefore, further optimization of NDGA
analogues with potent AcrB-targeted antibacterial activity should continue to be investigated.
4. Experimental
4.1. Chemistry
All reagents and chemicals that are of analytical grade in the present study were commercially available and used
without further purification. Analytical thin-layer chromatography (TLC) was performed using silica gel GF254
plates to monitor the reaction progress. Flash column chromatography were carried out using silica gel 60 (particle
1
size 0.040-0.063 mm) for separation and purification of crude products. H NMR and 13C NMR spectra were
recorded on Bruker instrument at 400 or 600 MHz, and chemical shifts (δ) are reported in parts per million (ppm).
Electron-spray ionization mass spectra in positive mode (ESI-MS) data were registered on API 4000 instrument
(Applied Biosystems, Connecticut, USA). Analytical high-performance liquid chromatography (HPLC) was carried
out on an Agilent 1200 Series equipment coupled to ultraviolet-visible detector (UVD) at 230 nm with a Diamonil
C18 reversed phase column (150 × 4.6 mm, 5 µm), eluted by water and acetonitrile. The effective chromatogram was
obtained using the mobile phase of acetonitrile: water (60:40, vol./vol.) with a flow rate of 1.0 mL/min. The purity of
the tested compounds as determined by analytical HPLC was more than 95%.
4.1.1. (E)-3-(3,4-Dimethoxyphenyl)acrylic acid (2)
A mixture of 3,4-dimethoxybenzaldehyde (1) (9.50 g, 57.20 mmol), malonic acid (23.80 g, 228.80 mmol) and
piperidine (1 mL) in pyridine (60 mL) was heated at 70 °C for about 6 h. Upon completion, pyridine was removed in
vacuo. The residue obtained was dissolved in 15 % solution of NaOH (100 mL), stirred at room temperature for 0.5 h,
and extracted with ethyl acetate (EtOAc) (60 mL). The water phase was acidified with concentrated HCl to a pH
value of less than 2. The resulting precipitate was filtered off, washed with water, and dried in vacuo to give
(E)-3-(3,4-dimethoxyphenyl)acrylic acid (2) (10.70 g, 90%). mp:181–183 °C.
4.1.2. 3-(3,4-Dimethoxyphenyl)propan-1-ol (3)
To a stirred suspension of LiAlH4 (2.73 g, 71.94 mmol) in anhydrous THF (50 mL) was added 2 (10.00 g, 48.03
mmol) as a solid in portions over 30 min. After addition, the resulting reaction mixture was refluxed for 5 h, cooled to
room temperature, and quenched with MeOH (20 mL). The resulting suspension was filtered under vacuum, washed
in turn with EtOAc (100 mL) and brine, and dried over anhydrous Na2SO4. The organic extracts were concentrated in
vacuo to yield a pale yellow oil (3, 7.80 g, 83%). 1H NMR (400 MHz, CDCl3) δ 6.79 (d, J = 8.1 Hz, 1H, H-5), 6.74 (d,
J = 6.1 Hz, 2H, H-2 and H-6), 3.86 (s, J = 5.7 Hz, 6H, 2×OCH3), 3.67 (t, J = 6.2 Hz, 2H, CH2OH), 2.66 (t, J = 7.6 Hz,
2H, Ph-CH2), 1.88 (q, J = 7.3 Hz, 2H, CH2CH2OH), 1.74 (s, 1H, CH2CH2OH).
4.1.3. 4-(3-Bromopropyl)-1,2-dimethoxybenzene (4)
After 3 (7.60 g, 38.73 mmol) was dissolved in dichloromethane (DCM) (50 mL), along with CBr4 (13.50 g, 40.67
mmol), the reaction mixture was cooled to 0 °C, and PPh3 (10.68 g, 40.67 mmol) was added slowly. The resultant
reaction solution was stirred for 2 h, and the reaction solvents were removed under reduced pressure. The residue was
purified by flash column chromatography (petroleum ether/EtOAc) to produce a colorless oil (4, 8.72 g, 87%).
4.1.4. (3-(3,4-Dimethoxyphenyl)propyl)triphenylphosphonium bromide (5)
9

To a solution of 4 (7.00 g, 27.01 mmol) in toluene (80 mL) were added PPh3 (21.25 g, 81.03 mmol) under an
atmosphere of nitrogen. The resulting reaction mixture was heated to reflux for 36 h. After cooling to room
temperature, the deposits were filtered, washed with toluene, and dried in vacuo to give the phosphonium salts 5
(8.70 g, 62%). mp: 169–171°C; 1H NMR (400 MHz, CDCl3) δ 8.04–7.41 (m, 15H, Ph-H), 6.90–6.65 (m, 3H, H-2、
H-5 and H-6), 3.85 (d, J = 2.8 Hz, 6H, 2×OCH3), 2.99 (t, J = 7.0 Hz, 2H, Ph-CH2 ), 1.93 (t, J = 12.3 Hz, 2H,
CH2PPh3), 1.71 (q, 2H, CH2CH2CH2).
4.1.5. General procedure for the preparation of compounds (6)
The phosphonium salts 5 (1.53 mmol) was dissolved in in dry THF (12 mL) at 0 °C, and to this solution were added
slowly n-butyllithium (1.84 mmol) and corresponding aromatic aldehyde (1.84 mmol). The reaction mixture was then
stirred for 5 h at room temperature. Upon completion of the reaction, the mixture was quenched with water (30 mL),
extracted with EtOAc (3 × 25 mL), washed with brine, and over anhydrous Na2SO4. The organic extracts were
concentrated in vacuo to give a residue, which was purified by flash column chromatography (petroleum ether/EtOAc)
to afford an alkene products 6 in the yields of 45-82%.
4.1.6. General procedure for the preparation of compounds (7)
Palladium on charcoal (10%, 50 mg) was added to a stirring solution of 6 (0.83 mmol) in MeOH–EtOAc (4:1) (10
mL) and the reaction mixture was stirred under a hydrogen balloon at room temperature for 24 h. The reaction
mixture was filtered and the residue washed with EtOAc (2 × 15 mL). The combined organic layers were
concentrated under reduced pressure to give a crude product (7), which was used directly for the next step without
further purification.
4.1.7. General procedure for the preparation of WA1-WA12 and WB1-WB11
To a solution of 7 (0.38 mmol) in DCM (8 mL) at -78 °C was added BBr3 (1.52 mmol) slowly. The resulting solution
was allowed to warm to room temperature and stirred for 1 h, which was then poured into iced water (40 mL). The
resultant mixture was extracted with DCM (3 × 20 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was
concentrated in vacuo and the residue was purified by flash column chromatography (DCM/MeOH) to produce the
crude products, which were crystallized from ether-hexane to afford the target compounds WA1-WA12 and
WB1-WB11.
4.1.7.1. 4-(4-Phenylbutyl)benzene-1,2-diol (WA1). White solid, yield 56%, mp 71–73 °C, Rf = 0.65 (DCM/MeOH =
10:1); 1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H, OH), 8.58 (s, 1H, OH), 7.29–7.22 (m, 2H, H-2ʹ and H-6ʹ), 7.19–
7.12 (m, 3H, H-3ʹ, H-4' and H-5ʹ), 6.60 (d, J = 8.0 Hz, 1H, H-5), 6.53 (d, J = 2.0 Hz, 1H, H-2 ), 6.39 (dd, J = 8.0, 2.1
Hz, 1H, H-6), 2.57 (t, J = 7.2 Hz, 2H, Phʹ-CH2), 2.41 (t, J = 7.1 Hz, 2H, Ph-CH2), 1.58–1.46 (m, 4H,
CH2CH2CH2CH2); 13C NMR (150 MHz, CDCl3) δ 143.28, 142.60, 141.21, 135.94, 128.44 (2C), 128.27 (2C), 125.66,
120.89, 115.52, 115.27, 35.82, 35.07, 31.14, 30.99; ESI-MS m/z calcd for C18H18O4F3 [M + CF3COO] – 355.1, found
355.4.
4.1.7.2. 4-(4-(p-Tolyl)butyl)benzene-1,2-diol (WA2). White solid, yield 37%, mp 70–72 °C, Rf = 0.68 (DCM/MeOH =
10:1); 1H NMR (600 MHz, DMSO-d6) δ 8.68 (s, 1H, OH), 8.57 (s, 1H, OH), 7.09–7.01 (m, 4H, H-2ʹ, H-3ʹ, H-5ʹ and
H-6ʹ), 6.59 (d, J = 7.9 Hz, 1H, H-5), 6.52 (d, J = 2.1 Hz, 1H, H-2), 6.39 (dd, J = 8.0, 2.1 Hz, 1H, H-6), 2.53 (d, J =
7.2 Hz, 2H, Ph-CH2), 2.40 (t, J = 7.3 Hz, 2H, Phʹ-CH2), 2.25 (s, 3H, CH3), 1.50 (dddd, J = 23.5, 12.1, 9.0, 5.0 Hz, 4H,
CH2CH2CH2CH2); 13C NMR (150 MHz, CDCl3) δ 143.29, 141.22, 139.51, 135.95, 135.06, 128.95 (2C), 128.30 (2C),
120.85, 115.50, 115.23, 35.36, 35.09, 31.16, 31.13, 21.00; ESI-MS m/z calcd for C19H20O4F3 [M + CF3COO]– 369.1,
found 369.2.
4.1.7.3. 4-(4-(4-Isopropylphenyl)butyl)benzene-1,2-diol (WA3). White solid, yield 48%, mp 72–74 °C, Rf = 0.24
(DCM/MeOH = 30:1); 1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H, OH), 8.59 (s, 1H, OH), 7.12 (d, J = 8.1 Hz, 2H,
H-3ʹ and H-5ʹ), 7.07 (d, J = 8.0 Hz, 2H, H-2ʹand H-6ʹ), 6.60 (d, J = 7.9 Hz, 1H, H-5), 6.54 (d, J = 2.1 Hz, 1H, H-2),
6.39 (dd, J = 8.0, 2.1 Hz, 1H, H-6), 2.82 (p, J = 6.9 Hz, 1H, Phʹ-CH), 2.53 (d, J = 6.7 Hz, 2H, Ph-CH2), 2.41 (t, J =
6.9 Hz, 2H, Phʹ-CH2), 1.57–1.44 (m, 4H, CH2CH2CH2CH2), 1.17 (d, J = 6.9 Hz, 6H, 2×CH3); 13C NMR (150 MHz,
10

CDCl3) δ 146.17, 143.29, 141.22, 139.92, 135.99, 128.32 (2C), 126.30 (2C), 120.89, 115.53, 115.26, 35.40, 35.10,
33.70, 31.23, 31.06, 24.10 (2C); ESI-MS m/z calcd for C19H23O2 [M - H] – 283.2, found 283.3.
4.1.7.4. 4-(4-(4-Hydroxyphenyl)butyl)benzene-1,2-diol (WA4). White solid, yield 51%, mp 121–123 °C, Rf = 0.56
(DCM/MeOH = 10:1); 1H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H, OH), 8.67 (s, 1H, OH), 8.57 (s, 1H, OH), 6.95
(d, J = 1.9 Hz, 1H, H-2ʹ), 6.93 (d, J = 2.2 Hz, 1H, H-6ʹ), 6.65 (d, J = 1.9 Hz, 1H, H-3ʹ), 6.63 (d, J = 2.1 Hz, 1H, H-5ʹ),
6.59 (d, J = 7.9 Hz, 1H, H-5), 6.52 (d, J = 2.1 Hz, 1H, H-2), 6.38 (dd, J = 8.0, 2.1 Hz, 1H, H-6), 2.48–2.42 (m, 2H,
Ph-CH2), 2.42–2.36 (m, 2H, Phʹ-CH2), 1.48 (m, J = 3.4 Hz, 4H, CH2CH2CH2CH2); 13C NMR (150 MHz, DMSO-d6) δ
155.64, 145.37, 143.50, 133.50, 132.74, 129.50 (2C), 119.27, 116.09, 115.83, 115.42 (2C), 34.82, 34.61, 31.39, 31.28;
ESI-MS m/z calcd for C16H17O3 [M - H] – 257.1, found 257.2.
4.1.7.5. 4-(4-(2-Hydroxyphenyl)butyl)benzene-1,2-diol (WA5). White solid, yield 51%, mp 134–136 °C, Rf = 0.59
(DCM/MeOH = 10:1); 1H NMR (400 MHz, DMSO-d6) δ 9.16 (s, 1H, OH), 8.67 (s, 1H, OH), 8.56 (s, 1H, OH), 7.03–
6.93 (m, 2H, H-6ʹ and H-4ʹ), 6.74 (d, J = 7.9 Hz, 1H, H-3ʹ), 6.68 (dd, J = 7.9, 6.7 Hz, 1H, H-5ʹ), 6.59 (d, 1H, H-5),
6.53 (d, J = 2.0 Hz, 1H, H-2), 6.39 (dd, J = 7.9, 2.0 Hz, 1H, H-6), 2.52 (d, J = 6.6 Hz, 2H, Ph-CH2), 2.39 (t, J = 6.7
Hz, 2H, Phʹ-CH2), 1.49 (m, J = 3.6 Hz, 4H, CH2CH2CH2CH2); 13C NMR (100 MHz, DMSO-d6) δ 155.47, 145.37,
143.49, 133.63, 130.19, 128.83, 127.03, 119.30, 119.23, 116.14, 115.84, 115.31, 34.96, 31.66, 29.91, 29.52; HRMS
m/z calcd for C16H17O3 [M - H] – 257.1256, found 257.1195.
4.1.7.6. 4,4'-(Butane-1,4-diyl)bis(benzene-1,2-diol) (WA6). Light brown solid, yield 56 %, mp 141–142 °C, Rf = 0.52
(DCM/MeOH = 10:1); 1H NMR (400 MHz, DMSO-d6) δ 8.56 (s, 4H, 4×OH), 6.59 (d, J = 8.0 Hz, 2H, H-5 and H-5ʹ),
6.53 (d, J = 2.1 Hz, 2H, H-2 and H-2ʹ), 6.39 (dd, J = 8.0, 2.1 Hz, 2H, H-6 and H-6ʹ), 2.45–2.33 (m, 4H, Ph-CH2 and
Phʹ-CH2), 1.47 (h, J = 3.4 Hz, 4H, CH2CH2CH2CH2); 13C NMR (100 MHz, DMSO-d6) δ 145.37 (2C), 143.49 (2C),
133.52 (2C), 119.28 (2C), 116.09 (2C), 115.83 (2C), 34.85 (2C), 31.31 (2C); ESI-MS m/z calcd for C16H17O4 [M - H]
– 273.1, found 273.3.
4.1.7.7. 5-(4-(3,4-Dihydroxyphenyl)butyl)benzene-1,2,3-triol (WA7). White solid, yield 65%, mp 192–193 °C, Rf =
1
0.14 (DCM/MeOH = 10:1); H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H, OH), 8.60 (s, 2H, 2×OH), 8.57 (s, 1H,
OH), 7.78 (s, 1H, OH), 6.60 (d, J = 7.9 Hz, 1H, H-5), 6.53 (d, J = 2.1 Hz, 1H, H-2), 6.39 (dd, J = 8.0, 2.1 Hz, 1H,
H-6), 6.05 (s, 2H, H-2ʹ and H-6ʹ), 2.38 (d, J = 7.0 Hz, 2H, Phʹ-CH2), 2.32 (d, J = 7.1 Hz, 2H, Ph-CH2), 1.45 (m, J =
3.9 Hz, 4H, CH2CH2CH2CH2); 13C NMR (100 MHz, DMSO-d6) δ 146.34 (2C), 145.38, 143.49, 133.56, 132.85,
131.17, 119.30, 116.12, 115.86, 107.45 (2C), 35.14, 34.88, 31.29, 31.15; HRMS m/z calcd for C16H17O5 [M - H] –
289.1154, found 289.1087.
4.1.7.8. 4-(4-(4-Fluorophenyl)butyl)benzene-1,2-diol (WA8). White solid, yield 64%, mp 70–72 °C, Rf = 0.53
(DCM/MeOH = 10:1); 1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H, OH), 8.58 (s, 1H, OH), 7.24–7.15 (m, 2H, H-2ʹ
and H-6ʹ), 7.11–7.03 (m, 2H, H-3ʹ and H-5ʹ), 6.60 (d, J = 7.9 Hz, 1H, H-5), 6.53 (d, J = 2.1 Hz, 1H, H-2), 6.39 (dd, J
= 7.9, 2.1 Hz, 1H, H-6), 2.56 (t, J = 7.1 Hz, 2H, Ph-CH2), 2.40 (t, J = 7.1 Hz, 2H, Phʹ-CH2), 1.56–1.43 (m, 4H,
CH2CH2CH2CH2); 13C NMR (100 MHz, CDCl3) δ 162.37, 159.95, 143.36, 141.28, 138.13, 135.81, 129.73(2C),
120.82, 115.30, 115.07 (2C), 35.05, 34.97, 31.09, 31.05; ESI-MS m/z calcd for C18H17O4F4 [M + CF3COO]– 373.1,
found 373.2.
4.1.7.9. 4-(4-(4-Chlorophenyl)butyl)benzene-1,2-diol (WA9). White solid, yield 63%, mp 93–95 °C, Rf = 0.62
(DCM/MeOH = 10:1); 1H NMR (600 MHz, DMSO-d6) δ 8.68 (s, 1H, OH), 8.58 (s, 1H, OH), 7.73 (d, J = 8.3 Hz, 2H,
H-2ʹ and H-6ʹ), 7.43 – 7.37 (m, 2H, H-3ʹ and H-5ʹ), 6.60 (d, J = 8.0 Hz, 1H, H-5), 6.52 (d, J = 2.1 Hz, 1H, H-2), 6.39
(dd, J = 8.0, 2.1 Hz, 1H, H-6), 2.67 (t, J = 7.6 Hz, 2H, Ph-CH2), 2.41 (t, J = 7.5 Hz, 2H, Phʹ-CH2), 1.59–1.52 (m, 2H,
CH2CH2CH2CH2), 1.51–1.45 (m, 2H, CH2CH2CH2CH2); 13C NMR (150 MHz, DMSO-d6) δ 145.39, 143.52, 142.72,
133.43, 128.72 (2C), 128.67 (2C), 126.05, 119.27, 116.10, 115.84, 35.46, 34.79, 31.31, 31.09; ESI-MS m/z calcd for
C16H16ClO2 [M – H] – 275.1, found 275.5.
4.1.7.10. 4-(4-(4-Bromophenyl)butyl)benzene-1,2-diol (WA10). White solid, yield 54%, mp 78–80 °C, Rf = 0.60
(DCM/MeOH = 10:1). 1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H, OH), 8.58 (s, 1H, OH), 7.23–7.16 (m, 2H, H-2ʹ
and H-6ʹ), 7.11–7.03 (m, 2H, H-3ʹ and H-5ʹ), 6.60 (d, J = 7.9 Hz, 1H, H-5), 6.53 (d, J = 2.1 Hz, 1H, H-2), 6.39 (dd, J
11

= 7.9, 2.1 Hz, 1H, H-6), 2.56 (t, J = 7.1 Hz, 2H, Ph-CH2), 2.40 (t, J = 7.1 Hz, 2H, Phʹ-CH2), 1.56–1.45 (m, 4H,
CH2CH2CH2CH2). 13C NMR (100 MHz, CDCl3) δ 143.30, 142.64, 141.25, 135.93, 128.46 (2C), 128.30 (2C), 125.68,
120.89, 115.56, 115.31, 35.84, 35.09, 31.17, 31.02. ESI-MS m/z calcd for C18H17BrO4F3 [M + CF3COO] – 433.0,
found 433.4.
4.1.7.11. 4-(4-(Tert-butyl)phenyl)butyl)benzene-1,2-diol (WA11). White solid, yield 76%, mp 88–90 °C, Rf = 0.19
(DCM/MeOH = 30:1); 1H NMR (600 MHz, DMSO-d6) δ 8.66 (s, 1H, OH), 8.57 (s, 1H, OH), 7.29–7.24 (m, 2H, H-3ʹ
and H-5ʹ), 7.09 (d, J = 8.2 Hz, 2H, H-2ʹ and H-6ʹ), 6.60 (d, J = 7.9 Hz, 1H, H-5), 6.54 (d, J = 2.1 Hz, 1H, H-2), 6.40
(dd, J = 8.0, 2.1 Hz, 1H, H-6), 2.53 (t, J = 7.2 Hz, 2H, Ph-CH2), 2.41 (t, J = 7.1 Hz, 2H, Phʹ-CH2), 1.57–1.47 (m, 4H,
CH2CH2CH2CH2), 1.26 (s, 9H, 3×CH3); 13C NMR (100 MHz, CDCl3) δ 148.45, 143.28, 141.22, 139.55, 135.99,
128.08 (2C), 125.17 (2C), 120.90, 115.55, 115.28, 35.29, 35.11, 34.37, 31.46(3C), 31.28, 31.01; ESI-MS m/z calcd
for C21H27O4 [M + HCOO] – 343.2, found 343.5.
4.1.7.12. 4-(4-(4-Aminophenyl)butyl)benzene-1,2-diol (WA12). White solid, yield 24%, mp 126–128 °C, Rf = 0.53
(DCM/MeOH = 10:1); 1H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H, OH), 8.55 (s, 1H, OH), 6.83–6.77 (m, 2H, H-2ʹ
and H-6ʹ), 6.59 (d, J = 7.9 Hz, 1H, H-5), 6.52 (d, J = 2.1 Hz, 1H, H-2), 6.48–6.43 (m, 2H, H-3ʹ and H-5ʹ), 6.38 (dd, J
= 7.9, 2.1 Hz, 1H, H-6), 4.79 (s, 2H, NH2), 2.39 (h, J = 2.9 Hz, 4H, 2×Ph-CH2), 1.46 (m, J = 3.6 Hz, 4H,
CH2CH2CH2CH2); 13C NMR (100 MHz, DMSO-d6) δ 148.15, 145.36, 143.48, 133.55, 129.69, 129.12 (2C), 119.26,
116.09, 115.82, 112.27 (2C), 34.87, 34.65, 31.55, 31.31; ESI-MS m/z calcd for C16H20NO2 [M + H]+ 258.1, found
258.2.
4.1.7.13. 4-(4-(Thiophen-2-yl)butyl)benzene-1,2-diol (WB1). White solid, yield 64%, mp 151–153 °C, Rf = 0.23
1
(DCM/MeOH = 30:1); H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H, OH), 8.57 (s, 1H, OH), 8.48 (dd, J = 5.2, 1.7
Hz, 1H, H-3ʹ), 7.74 (td, J = 7.6, 1.8 Hz, 1H, H-5ʹ), 7.28 (d, J = 7.8 Hz, 1H, H-6ʹ), 7.23 (ddd, J = 7.5, 4.9, 1.1 Hz, 1H,
H-4ʹ), 6.60 (d, J = 8.0 Hz, 1H, H-5), 6.53 (d, J = 2.0 Hz, 1H, H-2), 6.39 (dd, J = 8.0, 2.1 Hz, 1H, H-6), 2.77–2.70 (m,
2H, Py-CH2), 2.41 (t, J = 7.5 Hz, 2H, Ph-CH2), 1.65 (ddd, J = 15.4, 8.8, 6.7 Hz, 2H, CH2CH2CH2CH2), 1.51 (h, J =
7.3, 6.8 Hz, 2H, CH2CH2CH2CH2); 13C NMR (150 MHz, DMSO-d6) δ 145.52, 143.81, 134.69, 134.05, 132.49,
131.97, 130.86, 129.16, 119.36, 116.09, 115.93, 35.77, 28.59, 28.11, 24.03; ESI-MS m/z calcd for C15H18NO2 [M +
H]+ 244.1, found 244.4.
4.1.7.14. 4-(4-(Tetrahydrofuran-2-yl)butyl)benzene-1,2-diol (WB2). Yellow oil, yield 45%, Rf = 0.30 (DCM/MeOH =
1
30:1); H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 2H, OH), 6.61 (d, J = 7.9 Hz, 1H, H-5), 6.55 (d, J = 2.1 Hz, 1H,
H-2), 6.41 (dd, J = 8.0, 2.1 Hz, 1H, H-6), 4.23 (tt, J = 8.5, 4.3 Hz, 1H, CHOCH2), 3.60–3.50 (m, 2H, CHOCH2), 2.39
(t, J = 7.2 Hz, 2H, Ph-CH2), 1.95 (dd, J = 27.0, 15.3, 10.4, 7.5, 5.6 Hz, 4H, OCH2CH2CH2), 1.81 (qd, J = 8.5, 4.1 Hz,
2H, PhCH2CH2CH2CH2), 1.60–1.19 (m, 4H, PhCH2CH2CH2CH2); 13C NMR (150 MHz, CDCl3) δ 143.40, 141.34,
135.62, 120.79, 115.49, 115.31, 56.89, 39.11, 37.34, 35.02, 33.06, 30.91, 30.60, 27.12; ESI-MS m/z calcd for
C14H24NO3 [M + NH4]+ 254.1, found 254.3.
4.1.7.15. 4-(4-(Pyridin-4-yl)butyl)benzene-1,2-diol (WB3). White solid, yield 42%, mp 134–136 °C, Rf = 0.42
(DCM/MeOH = 10:1); 1H NMR (400 MHz, DMSO-d6) δ 8.69 (s, 1H, OH), 8.59 (s, 1H, OH), 8.43 (d, J = 5.5 Hz, 2H,
H-3ʹ and H-5ʹ), 7.24–7.16 (m, 2H, H-2ʹ and H-6ʹ ), 6.60 (d, J = 7.9 Hz, 1H, H-5), 6.53 (d, J = 2.1 Hz, 1H, H-2), 6.40
(dd, J = 8.0, 2.1 Hz, 1H, H-6), 2.59 (t, J = 7.4 Hz, 2H, Py-CH2), 2.41 (t, J = 7.3 Hz, 2H, Ph-CH2), 1.61–1.44 (m, 4H,
CH2CH2CH2CH2); 13C NMR (100 MHz, CDCl3) δ 153.23, 148.22 (2C), 144.57, 142.67, 134.21, 124.53 (2C), 119.99,
115.12, 114.92, 35.12, 34.91, 30.84, 29.44; ESI-MS m/z calcd for C15H18NO2 [M + H]+ 244.1, found 244.3.
4.1.7.16. 4-(4-([1,1'-Biphenyl]-4-yl)butyl)benzene-1,2-diol (WB4). White solid, yield 86%, mp 128–130 °C, Rf = 0.27
(DCM/MeOH = 30:1); 1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H, OH), 8.58 (s, 1H, OH), 7.64 (d, J = 1.5 Hz, 1H,
H-2ʺ), 7.62 (t, J = 1.2 Hz, 1H, H-6ʺ), 7.57 (d, J = 1.9 Hz, 1H, H-3ʹ), 7.55 (d, J = 1.9 Hz, 1H, H-5ʹ), 7.44 (t, J = 7.7 Hz,
2H, H-2ʺ and H-6ʺ), 7.37–7.31 (m, 1H, H-4ʺ), 7.26 (d, J = 8.0 Hz, 2H, H-2ʹ and H-6ʹ), 6.60 (d, J = 7.9 Hz, 1H, H-5),
6.55 (d, J = 2.0 Hz, 1H, H-2), 6.41 (dd, J = 8.0, 2.1 Hz, 1H, H-6), 2.62 (t, J = 7.2 Hz, 2H, Phʹ-CH2), 2.43 (t, J = 7.1
Hz, 2H, Ph-CH2), 1.56 (ddd, J = 20.2, 11.5, 5.4 Hz, 4H,CH2CH2CH2CH2); 13C NMR (150 MHz, DMSO-d6) δ 145.40,
143.54, 142.04, 140.60, 138.02, 133.43, 129.33 (4C), 127.57, 127.00 (2C), 126.93 (2C), 119.30, 116.13, 115.85,
12

35.06, 34.82, 31.34, 31.01; ESI-MS m/z calcd for C22H26O2N [M + NH4]+ 336.2, found 336.6.
1
4.1.7.17. 4-(4,4-Diphenylbutyl)benzene-1,2-diol (WB5). Yellow oil, yield 57%, Rf = 0.38 (DCM/MeOH = 30:1); H
NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H, OH), 8.56 (s, 1H, OH), 7.31–7.22 (m, 8H, Ar-H), 7.14 (dd, J = 6.0, 3.1 Hz,
2H, Ar-H), 6.58 (d, J = 7.9 Hz, 1H, H-5), 6.49 (d, J = 2.0 Hz, 1H, H-2), 6.35 (dd, J = 8.0, 2.0 Hz, 1H, H-6), 3.93 (t, J
= 7.9 Hz, 1H, Ph-CH-Ph), 2.41 (t, J = 7.6 Hz, 2H, Ph-CH2), 1.99 (q, J = 7.9 Hz, 2H, CH2CH2CH2CHPh2), 1.39 (td, J
= 7.6, 7.1, 3.7 Hz, 2H, CH2CH2CH2CHPh2); 13C NMR (100 MHz, CDCl3) δ 143.30, 141.72, 141.23, 141.13, 138.63,
135.90, 128.84 (2C), 128.71 (2C), 128.68, 127.02 (2C), 126.99 (2C), 120.87, 115.51, 115.26, 35.45, 35.08, 31.18,
30.96; ESI-MS m/z calcd for C24H22O4F3 [M + CF3COO] – 431.2, found 431.6.
4.1.7.18. 4-(4-(Naphthalen-1-yl)butyl)benzene-1,2-diol (WB6). White solid, yield 34%, mp 136–138 °C, Rf = 0.59
1
(DCM/MeOH = 10:1); H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H, OH), 8.57 (s, 1H, OH), 8.03 (dd, J = 7.8, 1.6
Hz, 1H, H-8ʹ), 7.93–7.85 (m, 1H, H-5ʹ), 7.75 (d, J = 8.1 Hz, 1H, H-4ʹ), 7.51 (pd, 2H, H-6ʹ and H-3ʹ), 7.40 (dd, J = 8.1,
7.0 Hz, 1H, H-7ʹ), 7.33 (dd, J = 7.0, 1.3 Hz, 1H, H-2ʹ), 6.61 (d, J = 7.9 Hz, 1H, H-5), 6.56 (d, J = 2.1 Hz, 1H, H-2),
6.42 (dd, J = 8.0, 2.1 Hz, 1H, H-6), 3.04 (t, J = 7.2 Hz, 2H, Arʹ-CH2), 2.45 (t, J = 7.1 Hz, 2H, Ph-CH2), 1.69–1.58 (m,
4H, CH2CH2CH2CH2); 13C NMR (150 MHz, DMSO-d6) δ 145.41, 143.53, 138.91, 133.91, 133.40, 131.81, 129.01,
126.72, 126.35, 126.31, 126.03, 125.94, 124.22, 119.31, 116.15, 115.84, 34.80, 32.56, 31.67, 30.55; ESI-MS m/z
calcd for C22H20O4F3 [M + CF3COO] – 405.1, found 405.6.
4.1.7.19. 4-(4-(6-Hydroxynaphthalen-2-yl)butyl)benzene-1,2-diol (WB7). White solid, yield 51%, mp 147–149 °C, Rf
= 0.55 (DCM/MeOH = 10:1); 1H NMR (400 MHz, DMSO-d6) δ 9.58 (s, 1H, Arʹ-OH), 8.67 (s, 1H, Ph-OH ), 8.57 (s,
1H, Ph-OH), 7.66 (d, J = 8.7 Hz, 1H, H-8ʹ), 7.58 (d, J = 8.4 Hz, 1H, H-4ʹ), 7.51 (d, J = 1.6 Hz, 1H, H-5ʹ), 7.23 (dd, J
= 8.4, 1.7 Hz, 1H, H-3ʹ), 7.06 (d, J = 2.4 Hz, 1H, H-7ʹ), 7.03 (dd, 1H, H-2ʹ), 6.59 (d, J = 7.9 Hz, 1H, H-5 ), 6.53 (d, J
= 2.1 Hz, 1H, H-2), 6.39 (dd, J = 8.0, 2.1 Hz, 1H, H-6), 2.68 (t, J = 7.4 Hz, 2H, Arʹ-CH2), 2.42 (t, J = 7.4 Hz, 2H,
Ph-CH2), 1.57 (dq, J = 31.9, 7.5 Hz, 4H, CH2CH2CH2CH2); 13C NMR (150 MHz, DMSO-d6) δ 155.11, 145.39,
143.52, 136.82, 133.46, 133.42, 129.17, 128.28, 127.99, 126.39, 126.33, 119.28, 118.94, 116.11, 115.84, 108.95,
35.39, 34.85, 31.36, 31.03; ESI-MS m/z calcd for C21H21O4 [M + HCOO] – 353.1, found 353.3.
4.1.7.20. 4-(4-(1-Methyl-1H-indol-3-yl)butyl)benzene-1,2-diol (WB8). Yellow oil, yield 44%, Rf = 0.24 (DCM/MeOH
1
= 30:1); H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H, OH), 8.57 (s, 1H, OH), 7.48 (dt, J = 7.8, 1.0 Hz, 1H, H-6ʹ),
7.35 (dd, J = 8.2, 0.9 Hz, 1H, H-7ʹ), 7.11 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H, I H-4ʹ), 7.04 (s, 1H, H-2ʹ), 6.98 (ddd, J = 7.9,
7.0, 1.0 Hz, 1H, H-5ʹ), 6.60 (d, J = 7.9 Hz, 1H, H-5), 6.54 (d, J = 2.1 Hz, 1H, H-2), 6.40 (dd, J = 8.0, 2.1 Hz, 1H,
H-6), 3.71 (s, 3H, CH3), 2.66 (d, J = 7.3 Hz, 2H, Ph-CH2 ), 2.43 (t, J = 7.2 Hz, 2H, Arʹ-CH2), 1.59 (dq, J = 21.8, 7.9
Hz, 4H, CH2CH2CH2CH2); 13C NMR (150 MHz, CDCl3) δ 143.32, 141.32, 137.04, 135.97, 127.96, 126.12, 121.38,
120.87, 120.85, 119.11, 118.45, 115.50, 115.20, 109.13, 35.09, 32.56, 31.40, 29.88, 24.94; ESI-MS m/z calcd for
C19H22NO2 [M + H]+ 296.2, found 296.3.
4.1.7.21. 4-(4-(1-Isopentyl-1H-indol-3-yl)butyl)benzene-1,2-diol (WB9). Yellow oil, yield 44%, Rf = 0.13
(DCM/MeOH = 10:1); 1H NMR (400 MHz, DMSO-d6) δ 8.65 (s, 1H, OH), 8.56 (s, 1H, OH), 7.48 (dt, J = 7.9, 1.0 Hz,
1H, H-6ʹ), 7.37 (dt, J = 8.2, 0.9 Hz, 1H, H-7ʹ), 7.13–7.06 (m, 2H, H-4ʹ and H-2ʹ), 6.96 (ddd, J = 7.9, 7.0, 1.0 Hz, 1H,
H-5ʹ), 6.59 (d, J = 7.9 Hz, 1H, H-5), 6.54 (d, J = 2.1 Hz, 1H, H-2), 6.40 (dd, J = 8.0, 2.1 Hz, 1H, H-6), 4.10 (t, J = 7.3
Hz, 2H, NCH2CH2), 2.67 (t, J = 7.2 Hz, 2H, Ph-CH2), 2.43 (t, J = 7.3 Hz, 2H, Arʹ-CH2), 1.70–1.58 (m, 4H,
CH2CH2CH2CH2), 1.58–1.38 (m, 2H, NCH2CH2), 1.09 (t, J = 7.0 Hz, 1H, CH(CH3)2), 0.90 (d, J = 6.6 Hz, 6H,
2×CH3); 13C NMR (150 MHz, CDCl3) δ 143.26, 141.25, 136.27, 136.04, 128.01, 124.90, 121.20, 120.87, 119.32,
119.18, 118.34, 115.51, 115.21, 109.26, 44.34, 39.12, 35.06, 31.39, 29.81, 25.79, 25.00, 22.49 (2C); ESI-MS m/z
calcd for C23H30NO2 [M + H]+ 352.2, found 352.9.
4.1.7.22. 4-(4-(3,4,5,6-Tetrahydroanthracen-9-yl)butyl)benzene-1,2-diol (WB10). White solid, yield 65%, mp 158–
160 °C, Rf = 0.25 (DCM/MeOH = 30:1); 1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H, OH), 8.59 (s, 1H, OH), 7.94–
7.89 (m, 1H, H-6ʹ), 7.72 (dd, J = 7.5, 2.0 Hz, 1H, H-2ʹ), 7.45 (s, 1H, H-10ʹ), 7.37 (td, J = 7.2, 1.6 Hz, 2H, H-3ʹ and
H-9ʹ), 6.62 (d, J = 7.9 Hz, 1H, H-5), 6.54 (d, J = 2.1 Hz, 1H, H-2), 6.45 (dd, J = 8.0, 2.1 Hz, 1H, H-6), 3.02 – 2.95 (m,
2H, Arʹ-CH2), 2.90 (t, J = 6.3 Hz, 2H, CH2), 2.85 (t, J = 6.5 Hz, 2H, CH2), 2.48 (d, J = 7.4 Hz, 2H, Ph-CH2), 1.81 (q,
13

J = 6.6, 6.1 Hz, 2H, CH2CH2CH2CH2), 1.78–1.65 (m, 4H, 2×CH2), 1.55–1.43 (m, 2H, CH2CH2CH2CH2); 13C NMR
(150 MHz, DMSO-d6) δ 145.43, 143.54, 136.13, 135.90, 133.36, 133.34, 132.20, 130.64, 128.08, 125.57, 125.37,
124.99, 123.88, 119.34, 116.17, 115.84, 34.79, 32.04, 30.70, 29.69, 27.69, 26.76, 23.66, 22.81; ESI-MS m/z calcd for
C26H26O4F3 [M + CF3COO] – 459.2, found 459.6.
4.1.7.23. 4-(4-(Pyren-4-yl)butyl)benzene-1,2-diol (WB11). White solid, yield 57%, mp 66–68 °C, Rf = 0.23
(DCM/MeOH = 30:1); 1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H, OH), 8.58 (s, 1H, OH), 8.32 (d, J = 9.3 Hz, 1H,
H-8ʹ), 8.27 (dd, J = 7.6, 2.4 Hz, 2H, H-4ʹ and H-6ʹ), 8.20 (dd, J = 8.5, 5.0 Hz, 2H, H-2ʹ and H-7ʹ), 8.12 (d, J = 1.6 Hz,
2H, H-4ʹ and H-9ʹ), 8.05 (t, J = 7.6 Hz, 1H, H-10ʹ), 7.93 (d, J = 7.8 Hz, 1H, H-3ʹ), 6.60 (d, J = 7.9 Hz, 1H, H-5), 6.57
(d, J = 2.2 Hz, 1H, H-2), 6.42 (dd, J = 8.0, 2.1 Hz, 1H, H-6), 3.21 (d, J = 7.4 Hz, 2H, Arʹ-CH2), 2.47 (d, J = 7.7 Hz,
2H, Ph-CH2), 1.84-1.64 (m, 4H, CH2CH2CH2CH2); 13C NMR (100 MHz, DMSO-d6) δ 145.43, 143.55, 137.53,
133.42, 131.37, 130.89, 129.65, 128.49, 127.92, 127.61, 126.88, 126.57, 125.94, 125.36, 125.18, 124.70, 124.65,
–
123.98, 123.06, 119.34, 116.18, 115.87, 34.85, 32.96, 31.64, 31.58; HRMS m/z calcd for C26H21O2 [M – H]
365.1620, found 365.1558.
4.1.8. (E)-1,3-Bis(3,4-dimethoxyphenyl)prop-2-en-1-one (9)
To a solution of 3,4-dimethoxybenzaldehyde (1) (3.00 g, 18.05 mmol) and 3,4-dimethoxyacetophenone (8) (3.25 g,
18.05 mmol) in 45 mL of ethanol (EtOH) was added NaOH (0.80 g, 19.86 mmol). The solution was allowed to warm
to 75 °C and stirred for 6 h. After concentrating under reduced pressure, the residue was poured in water (30 mL) and
extracted with EtOAc (3 × 20 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by flash column chromatography (petroleum ether/EtOAc) to afford
5.30 g of 9 as a yellow solid in a yield of 90%. mp: 110–112 °C.
4.1.9. 1,3-Bis(3,4-dimethoxyphenyl)propan-1-one (10)
The title compound 10 was synthesized in a yield of 97% in a manner similar to the synthesis of 7.
4.1.10. 1,3-Bis(3,4-dimethoxyphenyl)propan-1-ol (11)
NaBH4 (0.29 g, 7.67 mmol) was added to a solution of 10 (1.95 g, 5.90 mmol) in MeOH (30 mL), and the resulting
mixture was stirred at 0 °C for 1 h. Water (20 mL) was added to the mixture, which was extracted with EtOAc (3 × 20
mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was
purified using flash column chromatography with elution system of petroleum ether/EtOAc to generate 1.89 g of 11
as yellow oil, in a yield of 96%.
4.1.11. 4,4'-(Prop-1-ene-1,3-diyl)bis(1,2-dimethoxybenzene)( 12)
To a solution of 11 (1.00 g, 3.01 mmol) in dioxane (25 mL) was added 7-8 drops of H2SO4, and the reaction mixture
was refluxed for 5 h before pouring into iced water (20 mL). The mixture was extracted with EtOAc (3 × 20 mL).
Then washed with saturated NaHCO3 and brine, dried with anhydrous Na2SO4, and concentrated in vacuo to afford
0.64 g yellow oil in a yield of 68%.
4.1.12. 1,3-Bis(3,4-dimethoxyphenyl)propane (13)
The compound 13 was synthesized in a manner similar to the synthesis of 7, white solid in a yield of 82%. mp:
62-63 °C.
4.1.13. 4,4'-(Propane-1,3-diyl)bis(benzene-1,2-diol) (WC1)
The target compound WC1 was synthesized in a manner similar to the synthesis of WA1-WA12, The desired product
WC1 was obtained as white solid in a yield of 71%. mp: 115–117 °C, Rf = 0.50 (DCM/MeOH = 10:1); 1H NMR (400
MHz, DMSO-d6) δ 8.69 (s, 2H, 2 × OH), 8.58 (s, 2H, 2 × OH), 6.61 (d, J = 7.9 Hz, 2H, H-5 and H-5ʹ), 6.55 (d, J =
2.0 Hz, 2H, H-2 and H-2ʹ), 6.41 (dd, J = 8.0, 2.0 Hz, 2H, H-6 and H-6ʹ), 2.38 (t, J = 7.6 Hz, 4H, 2 × Ph-CH2), 1.70 (p,
J = 7.6 Hz, 2H, CH2CH2CH2); 13C NMR (100 MHz, DMSO-d6) δ 145.43 (2C), 143.53 (2C), 133.38 (2C), 119.30
–
(2C), 116.11 (2C), 115.90 (2C), 34.48 (2C), 33.65; HRMS m/z calcd for C15H15O4 [M - H] 259.1049, found
259.1018.
4.1.14. (1E,4E)-1,5-Bis(3,4-dimethoxyphenyl)penta-1,4-dien-3-one (14)
To a solution of 3,4-dimethoxybenzaldehyde (1) (3.00 g, 18.05 mmol) and acetone (0.87 g, 9.03 mmol) in EtOH-H2O
14

mixture (4:1, 30 mL) at 0 °C was added NaOH (1.81 g, 45.18 mmol) slowly. the resulting solution was raised to room
temperature and stirred for 1 h. the precipitate was filtered off, washed with water, and recrystallized from EtOH to
give a solid (2.78 g, 87%). mp: 84–86 °C.
4.1.15. 1,5-Bis(3,4-dimethoxyphenyl)pentan-3-one (15)
The title compound 15 was synthesized in a manner similar to the synthesis of 7. White solid, yield 95%, mp: 83–
85°C.
4.1.16. 1,5-Bis(3,4-dimethoxyphenyl)pentane(16)
Hydrazine hydrate (0.56 g, 11.14 mmol) was added dropwise to a stirring mixture of 15 (2.00 g, 5.57 mmol) and
KOH (0.78 g, 13.93 mmol) in diethylene glycol (30 mL). After the addition was complete, the resulting mixture was
heated at 105°C for 5 h under a nitrogen atmosphere before pouring into water (100 mL).The resultant mixture was
extracted with EtOAc (3 × 30 mL). The combined organic extracts were washed with water and brine, dried over
anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by flash column chromatography (petroleum
ether/EtOAc) to give 0.56 g of 16 as a white solid in a yield of 96%. mp: 56–58 °C.
4.1.17. 4,4'-(Pentane-1,5-diyl)bis(benzene-1,2-diol) (WC2)
The title compound WC2 was prepared according to the procedure depicted for compounds WA1-WA12. The desired
product WC2 was obtained as a white solid in a yield of 47%. mp: 129–131 °C, Rf = 0.53 (DCM/MeOH = 10:1); 1H
NMR (400 MHz, DMSO-d6) δ 8.66 (s, 2H, 2 × OH), 8.56 (s, 2H, 2 × OH ), 6.60 (d, J = 7.9 Hz, 2H, H-5 and H-5ʹ),
6.53 (d, J = 2.1 Hz, 2H, H-2 and H-2ʹ), 6.39 (dd, J = 8.0, 2.1 Hz, 2H, H-6 and H-6ʹ ), 2.36 (t, J = 7.6 Hz, 4H, 2 ×
Ph-CH2), 1.48 (p, J = 7.6 Hz, 4H, 2 × Ph-CH2CH2), 1.28–1.22 (m, 2H, CH2CH2CH2CH2CH2); 13C NMR (150 MHz,
DMSO-d6) δ 145.37 (2C), 143.48 (2C), 133.58 (2C), 119.25 (2C), 116.09 (2C), 115.83 (2C), 34.99 (2C), 31.62 (2C),
28.77; ESI-MS m/z calcd for C17H19O4 [M - H] – 287.1, found 287.2.
4.1.18. General procedure for the preparation of compounds 1,6-bis(3,4-dimethoxyphenyl)hexane-1,6-dione (18a)
and 1,7-bis(3,4-dimethoxyphenyl)heptane-1,7-dione (18b)
To a solution of 17 (7.20 mmol) in DCM (20 mL) at room temperature was slowly added SOCl2 (5 mL). The mixture
was heated at reflux for 4.5 h and the reaction solvents were removed in vacuo. The crude product was re-dissolved
in DCM (5 mL), and was added dropwise to a stirring mixture of veratrole (2.00 g, 14.40 mmol) and aluminum
trichloride (AlCl3) (2.02 g, 15.12 mmol) in DCM (20 mL). The resulting solution was stirred at 0 °C for 5 h before
pouring into ice water (50 mL). The mixture was extracted with DCM (3 × 20 mL), washed with saturated NaHCO3
(20 mL) and brine. The combined organic layer was removed in vacuo to afford 18. 18a: white solid, yield 98%, mp:
149–151 °C; 18b: white solid, yield 95%, mp: 156–158 °C.
4.1.19. General procedure for the preparation of compounds 1,6-bis(3,4-dimethoxyphenyl)hexane (19a) and
1,7-bis(3,4-dimethoxyphenyl)heptane (19b)
The title compounds 19a and 19b were prepared according to the procedure depicted for compound 7. 19a: white
solid, yield 55%, mp: 66-69°C; 19b: white solid, yield 66%, mp: 92–94 °C.
4.1.20. General procedure for the preparation of compounds WC3 and WC4
The target compound WC3 and WC4 were synthesized in a manner similar to the synthesis of WA1-WA12.
4.1.20.1. 4,4'-(hexane-1,6-diyl)bis(benzene-1,2-diol) (WC3). White solid in a yield of 80%. mp: 131–133 °C, Rf =
0.52 (DCM/MeOH = 10:1); 1H NMR (400 MHz, DMSO-d6) δ 8.66 (s, 2H, 2 × OH), 8.56 (s, 2H, 2 × OH), 6.60 (d, J
= 7.9 Hz, 2H, H-5 and H-5ʹ ), 6.53 (d, J = 2.1 Hz, 2H, H-2 and H-2ʹ), 6.39 (dd, J = 8.0, 2.1 Hz, 2H, H-6 and H-6ʹ),
2.36 (t, J = 7.6 Hz, 4H, 2 × Ph-CH2), 1.46 (t, J = 7.4 Hz, 4H, CH2CH2CH2CH2CH2CH2), 1.29–1.22 (m, 4H,
CH2CH2CH2CH2CH2CH2); 13C NMR (150 MHz, DMSO-d6) δ 145.37 (2C), 143.47 (2C), 133.57 (2C), 119.25 (2C),
116.08 (2C), 115.83 (2C), 34.97 (2C), 31.66 (2C), 28.95 (2C); ESI-MS m/z calcd for C18H21O4 [M - H] – 301.2, found
301.4.
4.1.20.2. 4,4'-(Heptane-1,7-diyl)bis(benzene-1,2-diol) (WC4).White solid in a yield of 62%,. mp 101–103 °C, Rf =
0.20 (DCM/MeOH = 30:1); 1H NMR (600 MHz, DMSO-d6) δ 8.67 (s, 2H, 2 × OH), 8.56 (s, 2H, 2 × OH), 6.60 (d, J
= 7.9 Hz, 2H, H-5 and H-5ʹ), 6.53 (d, J = 2.1 Hz, 2H, H-2 and H-2ʹ ), 6.39 (dd, J = 8.0, 2.1 Hz, 2H, H-6 and H-6ʹ),
15

2.36 (t, J = 7.6 Hz, 4H, 2 × Ph-CH2), 1.45 (q, J = 7.4 Hz, 4H, CH2CH2CH2CH2CH2CH2CH2), 1.25 (ddd, J = 21.9,
10.9, 5.8 Hz, 6H, CH2CH2CH2CH2CH2CH2CH2); 13C NMR (150 MHz, DMSO-d6) δ 145.36 (2C), 143.46 (2C),
133.59 (2C), 119.25 (2C), 116.09 (2C), 115.83 (2C), 34.97 (2C), 31.69 (2C), 29.24, 29.10 (2C); ESI-MS m/z calcd for
C21H24O6F3 [M + CF3COO] – 429.2, found 429.5.
4.1.21. General procedure for the preparation of compounds 21
To
a
solution
of
corresponding
aromatic
acid
(1.16
mmol)
and
2-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) (1.16 mmol) in CH3CN (20 mL)
was slowly added 3,4-dimethoxyphenethylamine (20) (0.20 g, 1.10 mmol) and N,N-diisopropylethylamine (DIEA)
(0.28 g, 2.20 mmol). The reaction mixture was allowed to stir for 12 h at room temperature, the solvents were
removed under reduced pressure, and then water (20 mL) was added. The aqueous suspension was extracted with
EtOAc (3 × 15 mL), and the resulting organic layer was washed with 1 M HCl, saturated NaHCO3 and brine in turn,
and was then dried over anhydrous Na2SO4. After removal of the solvent from the organic extracts in vacuo, the
residue was purified by flash column chromatography with elution system of petroleum ether/EtOAc to afford the
desired compounds 21 in yields ranging from 65-87%.
4.1.22. General procedure for the preparation of compound 22
Palladium on charcoal (10%, 40 mg) was added to a stirring solution of 21 containing nitro groups (0.77 mmol) in
MeOH–EtOAc mixture (1:1) (20 mL) and the resulting mixture was stirred under a hydrogen balloon at room
temperature for 24 h. The mixture was filtered and the residue was extracted with EtOAc (2 × 10 mL) and
concentrated in vacuo to yield a crude product 22, which was sufficiently pure to be directly used in next step.
4.1.23. General procedure for the preparation of compounds WD1-WD10
The target compounds WD1-WD10 were prepared according to the procedure depicted for compounds WA1-WA12.
4.1.23.1. 4-(Tert-butyl)-N-(3,4-dihydroxyphenethyl)benzamide (WD1). Yellow solid, yield 90%, mp 118–120 °C, Rf =
0.25 (DCM/MeOH = 10:1); 1H NMR (400 MHz, DMSO-d6) δ 8.74 (s, 1H, OH), 8.62 (s, 1H, OH), 8.41 (t, J = 5.6 Hz,
1H, NH), 7.75 (d, J = 8.2 Hz, 2H, H-2ʹ and H-6ʹ), 7.46 (d, J = 8.2 Hz, 2H, H-3ʹ and H-5ʹ), 6.66–6.58 (m, H-5 and
H-2), 6.46 (dd, J = 8.0, 2.0 Hz, 1H, H-6), 3.43–3.35 (m, 2H, NHCH2), 2.64 (t, J = 7.5 Hz, 2H, Ph-CH2), 1.29 (s, 9H);
13C NMR (100 MHz, DMSO-d6) δ 166.40, 154.19, 145.52, 143.97, 132.43, 130.79, 127.40 (2C), 125.43 (2C), 119.69,
116.47, 115.95, 41.70, 35.16, 35.04, 31.43 (3C); ESI-MS m/z calcd for C19H24NO3 [M + H]+ 314.2, found 314.4.
4.1.23.2. N-(3,4-Dihydroxyphenethyl)-2-hydroxybenzamide (WD2). White solid, yield 80%, mp 138–140 °C, Rf =
1
0.21 (DCM/MeOH = 10:1); H NMR (400 MHz, DMSO-d6) δ 12.60 (s, 1H, Phʹ-OH), 8.84 (t, J = 5.6 Hz, 1H, NH),
8.75 (s, 1H, Ph-OH), 8.64 (s, 1H, Ph-OH), 7.82 (dd, J = 8.0, 1.5 Hz, 1H, H-2ʹ), 7.42–7.33 (m, 1H, H-6ʹ), 6.88 (t, J =
7.9 Hz, 2H, H-3ʹ and H-5ʹ), 6.68–6.58 (m, 2H, H-5 and H-2), 6.47 (dd, J = 8.0, 2.0 Hz, 1H, H-6), 3.48–3.39 (m, 2H,
NHCH2), 2.67 (t, J = 7.5 Hz, 2H, Ph-CH2); 13C NMR (100 MHz, DMSO-d6) δ 169.24, 160.50, 145.57, 144.06, 134.05,
130.43, 128.11, 119.70, 118.98, 117.82, 116.45, 116.00, 115.72, 41.40, 34.80; ESI-MS m/z calcd for C15H16NO4 [M +
H]+ 274.1, found 274.4.
4.1.23.3. N-(3,4-Dihydroxyphenethyl)-4-formylbenzamide (WD3). Brown solid, yield 80%, mp 120–122 °C, Rf = 0.19
(DCM/MeOH = 15:1); 1H NMR (400 MHz, DMSO-d6) δ 8.70 (s, 2H, 2 × OH), 8.61 (t, J = 5.6 Hz, 1H, NH), 7.77 (d,
J = 8.5 Hz, 2H, H-2ʹ and H-6ʹ), 7.67 (d, J = 8.4 Hz, 2H, H-3ʹ and H-5ʹ), 6.67–6.56 (m, 2H, H-5 and H-2), 6.46 (dd, J
= 8.0, 2.1 Hz, 1H, H-6), 3.36 (m, 2H, NHCH2), 2.64 (t, J = 7.5 Hz, 2H, Ph-CH2); 13C NMR (100 MHz, DMSO-d6) δ
165.57, 145.53, 144.00, 134.22, 131.72 (2C), 130.67, 129.72 (2C), 125.21, 119.70, 116.47, 115.97, 41.81, 35.00;
ESI-MS m/z calcd for C15H15BrNO3 [M + H]+ 336.0, found 336.3.
4.1.23.4. 3,5-Diamino-N-(3,4-dihydroxyphenethyl)benzamide (WD4). White solid, yield 70%, mp: 185–187 °C, Rf =
0.29 (DCM/MeOH = 10:1); 1H NMR (400 MHz, DMSO-d6) δ 8.75 (s, 1H, OH), 8.61 (s, 1H, OH), 7.98 (t, J = 5.7 Hz,
H, NH), 6.63 (d, J = 7.9 Hz, 1H, H-2ʹ ), 6.59 (d, J = 2.1 Hz, 1H, H-6ʹ), 6.45 (dd, J = 8.0, 2.1 Hz, 1H, H-5), 6.19 (d, J
= 2.0 Hz, 2H, H-2 and h-6), 5.92 (s, 1H, H-4), 4.83 (s, 4H, 2× NH2), 3.28 (dt, J = 8.5, 6.0 Hz, 2H, NHCH2), 2.58 (dd,
J = 8.9, 6.5 Hz, 2H, Ph-CH2); 13C NMR (100 MHz, DMSO-d6) δ 168.10, 149.39 (2C), 145.50, 143.91, 137.02, 130.88,
16

119.63, 116.41, 115.97, 102.50 (2C), 102.43, 41.55, 35.23; ESI-MS m/z calcd for C15H18N3O3 [M + H]+ 288.1, found
288.3.
4.1.23.5. N-(3,4-Dihydroxyphenethyl)-3,4-dihydroxybenzamide (WD5). White solid, yield 87%, mp 166–168 °C, Rf =
0.21 (DCM/MeOH = 10:1); 1H NMR (400 MHz, DMSO-d6) δ 9.41 (s, 1H, OH), 9.07 (s, 1H, OH), 8.74 (s, 1H, OH),
8.62 (s, 1H, OH), 8.16 (t, J = 5.7 Hz, 1H, NH), 7.26 (d, J = 2.2 Hz, 1H, H-6ʹ), 7.15 (dd, J = 8.2, 2.1 Hz, 1H, H-2ʹ),
6.73 (d, J = 8.2 Hz, 1H, H-5ʹ), 6.66–6.55 (m, 2H, H-5 and H-2), 6.45 (dd, J = 8.0, 2.1 Hz, 1H, H-6), 3.30 (d, J = 7.1
Hz, 2H, NHCH2), 2.60 (t, J = 7.6 Hz, 2H, Ph-CH2); 13C NMR (100 MHz, DMSO-d6) δ 166.38, 148.62, 145.51,
145.22, 143.93, 130.89, 126.45, 119.65, 119.27, 116.43, 115.94, 115.51, 115.23, 41.67, 35.27; ESI-MS m/z calcd for
C15H16NO5 [M + H]+ 290.1, found 290.5.
4.1.23.6. N-(3,4-Dihydroxyphenethyl)-3,4,5-trihydroxybenzamide (WD6). White solid, yield 38%, mp 198–200 °C, Rf
= 0.17 (DCM/MeOH = 10:1); 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 2H, 2 × OH), 8.74 (s, 1H, OH), 8.62 (s, 2H,
OH), 8.08 (t, J = 5.6 Hz, 1H, NH), 6.80 (s, 2H, H-2ʹ and H-6ʹ), 6.63 (d, J = 7.9 Hz, 1H, H-5), 6.59 (d, J = 2.1 Hz, 1H,
H-2), 6.44 (dd, J = 8.0, 2.1 Hz, 1H, H-6), 3.29 (dd, J = 8.4, 5.9 Hz, 2H, NHCH2), 2.59 (dd, J = 8.8, 6.5 Hz, 2H,
Ph-CH2); 13C NMR (100 MHz, DMSO-d6) δ 166.64, 145.81 (2C), 145.50, 143.91, 136.54, 130.91, 125.53, 119.65,
116.42, 115.94, 107.11 (2C), 41.66, 35.24; ESI-MS m/z calcd for C15H16NO6 [M + H]+ 306.1, found 306.6.
4.1.23.7. 4-Amino-N-(3,4-dihydroxyphenethyl)benzamide (WD7). White solid, yield 72%, mp 194–195 °C, Rf = 0.23
(DCM/MeOH = 10:1); 1H NMR (400 MHz, DMSO-d6) δ 8.77 (s, 1H, OH), 8.65 (s, 1H, OH), 8.04 (t, J = 5.7 Hz, 1H,
NH), 7.54 (d, J = 8.5 Hz, 2H, H-3ʹ and H-5ʹ), 6.67 – 6.58 (m, 2H, H-5 and H-2ʹ), 6.52 (d, J = 8.4 Hz, 2H, H-2 and
H-6ʹ), 6.45 (dd, J = 8.0, 2.1 Hz, 1H, H-6), 5.59 (s, 2H, NH2), 3.31 (dd, J = 8.7, 5.9 Hz, 2H, NHCH2), 2.60 (dd, J = 8.8,
6.5 Hz, 2H, Ph-CH2); 13C NMR (100 MHz, DMSO-d6) δ 166.53, 151.87, 145.50, 143.91, 130.97, 129.06 (2C), 121.93,
119.65, 116.44, 115.94, 113.00 (2C), 41.61, 35.44; ESI-MS m/z calcd for C15H17N2O3 [M + H]+ 273.1, found 273.4.
4.1.23.8. 2-Amino-N-(3,4-dihydroxyphenethyl)benzamide (WD8). Brown solid, yield 81%, mp 168–170 °C, Rf = 0.25
(DCM/MeOH = 10:1); 1H NMR (400 MHz, DMSO-d6) δ 8.80 (s, 1H, OH), 8.68 (s, 1H, OH), 8.25 (t, J = 5.6 Hz, 1H,
NH), 7.43 (dd, J = 8.0, 1.5 Hz, 1H, H-6ʹ), 7.12 (ddd, J = 8.4, 7.1, 1.5 Hz, 1H, H-4ʹ), 6.70–6.61 (m, 3H, H-5, H-3ʹ and
H-5ʹ), 6.52–6.44 (m, 2H, H-2 and H-6), 6.38 (s, 2H, NH2), 3.38–3.28 (m, 2H, NHCH2), 2.70–2.58 (m, 2H, Ph-CH2);
13C NMR (100 MHz, DMSO-d6) δ 168.54, 160.80, 145.57, 143.99, 134.05, 130.45, 128.06, 119.70, 118.98, 117.77,
116.45, 116.00, 115.74, 41.40, 34.80; ESI-MS m/z calcd for C15H17N2O3 [M + H]+ 273.1, found 273.3.
4.1.23.9. N-(3,4-Dihydroxyphenethyl)-4-methylbenzamide (WD9). White solid, yield 61%, mp 121–123 °C, Rf = 0.21
(DCM/MeOH = 15:1); 1H NMR (400 MHz, DMSO-d6) δ 8.75 (s, 1H, OH), 8.63 (s, 1H, OH), 8.42 (t, J = 5.6 Hz, 1H,
NH), 7.77–7.69 (m, 2H, H-2ʹ and H-6ʹ), 7.25 (d, J = 7.9 Hz, 2H, H-3ʹ and H-5ʹ), 6.67–6.59 (m, 2H, H-5 and H-2),
6.47 (dd, J = 8.0, 2.0 Hz, 1H, H-6), 3.46–3.35 (m, 2H, NHCH2), 2.64 (dd, J = 8.7, 6.5 Hz, 2H, Ph-CH2), 2.34 (s, 3H,
CH3); 13C NMR (100 MHz, DMSO-d6) δ 166.46, 149.08, 147.69, 141.30, 132.52, 132.35, 129.21 (2C), 127.58 (2C),
120.93, 113.03, 112.39, 41.48, 35.16, 21.37; ESI-MS m/z calcd for C16H18NO3 [M + H]+ 272.1, found 272.5.
4.1.23.10. N-(3,4-Dihydroxyphenethyl)-4-(trifluoromethyl)benzamide (WD10). White solid, yield 54%, mp 127–
129 °C, Rf = 0.17 (DCM/MeOH = 15:1); 1H NMR (400 MHz, DMSO-d6) δ 8.80 (t, J = 5.6 Hz, 1H, NH), 8.77 (s, 1H,
OH), 8.66 (s, 1H, OH), 8.22–8.06 (m, 2H, H-2ʹ and H-6ʹ), 7.90 (d, 1H, H-3ʹ), 7.72 (t, J = 7.8 Hz, 1H, H-5ʹ), 6.62 (s,
2H, H-5 and H-2 ), 6.47 (dd, J = 8.0, 2.1 Hz, 1H, H-6), 3.41 (dt, J = 8.2, 6.0 Hz, 2H, NHCH2), 2.66 (dd, J = 8.6, 6.5
Hz, 2H, Ph-CH2); 13C NMR (150 MHz, DMSO-d6) δ 165.01, 145.56, 144.03, 135.94, 131.71, 130.59, 130.08, 128.13,
125.38, 124.20, 123.58, 119.69, 116.45, 115.95, 41.89, 34.95; ESI-MS m/z calcd for C16H15NF3O3 [M + H]+ 326.1,
found 326.4.
4.2. Antibacterial evaluation
4.2.1. Antimicrobial assays
Antimicrobial assays to measure the MIC values of tested compounds were performed applying the 96-well
microtitre broth dilution method as previously described [18].
4.2.2 Checkerboard titration assay
17

Checkerboard titration assay was used to assess the possible synergism between the antibiotics and the tested
compounds as previously described [18].
4.2.3. Nile red efflux assay
The ability of tested compounds to inhibit substrate transport mediated by AcrB was evaluated by determining the
reduction in Nile Red efflux as previously described [18].
4.2.4. Nitrocefin uptake assay
The ability of tested compounds to permeabilize outer membrane of E. coli BW25513 was assessed by using
nitrocefin uptake assay as described previously [18, 51].
4.2.5. Measurement of the electrochemical gradient over the inner membrane
The influence of tested compounds on the membrane potential (∆ψ) across the inner-membrane of E. coli BW25513
was investigated by determining fluorescence intensity of the fluorescent probe DiOC2 (3) as previously described [18,
51].
Conflicts of interest
The authors declare that this study was carried out only with public funding. There is no funding or no agreement
with commercial for profit firms.
Acknowledgments
This research was supported financially by the National Natural Science Foundation of China (81973179 and
81673284), the National Health and Medical Research Council of Australia (GN1147538), Key research and
development project of Shandong Province (2017CXGC1401) and Major Project of Research and development of
Shandong Province (2019GSF108051). RA is the recipient of a PhD scholarship from the Government of Saudi
Arabia.
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fluorescent membrane probes in a combined real-time influx and efflux Assay, Antimicrob. Agents Chemother.
60 (2016) 1974-1983.
[55] H. Venter, R.A. Shilling, S. Velamakanni, L. Balakrishnan, H.W. Van Veen, An ABC transporter with a
secondary-active multidrug translocator domain, Nature 426 (2003) 866-870.
21

Fig. 1. Molecular docking of NDGA [50]. (a) Chemical structure of NDGA; (b) Overlay of NDGA (yellow) on the
crystal structure of AcrB with minocycline (red); (c) The interaction of NDGA with the amino acids of AcrB.
Fig. 2. Inhibition of AcrB-mediated Nile Red efflux. Wild-type resistant cells with active pump (solid black line),
∆AcrB drug-sensitive cells (grey line) or wild-type cells in the presence of the compounds tested (dotted lines) were
preloaded with Nile Red before the start of fluorescence measurements. Efflux was triggered at 100 sec by the
addition of 0.2% glucose (indicated by arrow). Representative fluorescent traces are shown for triplicate experiments
with different batches of cells.
Fig. 3. The effect of compounds tested on outer membrane permeability. E. coli were treated with 10 µM CCCP to
inhibit the efflux of nitrocefin. Nitrocefin was added to the cells that received no compound (blue circles), cells
treated with the outer membrane permeabilizer polymyxin B (red squares) or the test compounds (red triangles).
Nitrocefin hydrolysis by the periplasmic β-lactamase was observed as an increase in absorbance at 490 nm.
Representative traces are shown from duplicate experiments performed on different days.
Fig. 4. The effect of the selected compounds tested on pmf of inner membrane. Bacterial suspensions were either left
untreated (solid blue line) or exposed to 8-256 µg/mL test compounds (broken red line) for 10 min after which the
potentiometric probe, DiOC2(3) was added and the fluorescence monitored until it plateaued. Cells were then
re-energized with 0.5% glucose and the establishment of a membrane potential (inside negative) was measured as an
increase in fluorescence until it plateaued. The membrane potential was then disrupted by the addition of the proton
ionophore CCCP (observed as a sharp drop in fluorescence intensity).
Fig. 5. In silico docking studies. The molecular interactions of AcrB with (a) WA7 and (b) WD6.
Scheme 1 Reagents and conditions: a) CH2(COOH)2, piperidine, pyridine, reflux, 90%; b) LiAlH4, THF, 83%; c)
PPh3, CBr4, 87%; d) PPh3, toluene, reflux, 62%; e) ArCHO, n-butyllithium, THF, 45-82%; f) Pd/C, H2, rt; g) BBr3,
37-76% in two steps for WA1-WA12, 34-86% in two steps for WB1-WB11.
Scheme 2 Reagents and conditions: a) NaOH, EtOH, reflux, 90%; b) Pd/C, H2, MeOH, rt, 97%; c) NaBH4, MeOH, rt,
96%; d) H2SO4, 1,4-dioxane, 68%; e) Pd/C, H2, MeOH, rt, 82%; f) BBr3, 71%.
Scheme 3 Reagents and conditions: a) acetone, NaOH, EtOH, 87%; b) Pd/C, H2, MeOH/AcOEt, rt, 95%; c)
N2H4·H2O, KOH, glycol, 96%; d) BBr3, 47%.
Scheme 4 Reagents and conditions: a) SOCl2, 1,2-dimethoxybenzene, then AlCl3, 95-98%; b) Pd/C, H2,
MeOH/EtOAc, rt, 55-66%; c) BBr3, 62-80%.
Scheme 5 Reagents and conditions: a) TBTU, DIEA, CH3CN, 65-87%; b) Pd/C, H2, MeOH/EtOAc, rt; c) BBr3, 38-90%
for WD1-WD3, WD5, WD6, WD9 and WD10, 70-81% for WD4, WD7 and WD8.
22

Table 1. The synergistic effect of NDGA analogues (Series WA-WC) with different antibacterials against wild-type drug-resistant strain E. coli BW25113 expressing AcrB that is
indicated as (+)AcrB.
Conc.
(µg/mL)
16
Conc.
(µg/mL)
16
MIC (µg/mL)
CAM ERY LEV
MIC (µg/mL)
TPP
512
Compd.
WA5
R1
Compd.
WB11
R1
TPP
512
512
512
512
512
1024
1024
512
512
512
512
512
512
256
256
1024
1024
512
512
512
1024
1024
512
512
512
1024
1024
128
64
RIF
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
CAM
8
ERY
64
64
32
32
32
64
64
32
32
32
64
64
64
64
64
64
64
64
64
64
64
64
64
64
64
64
4
LEV
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.004
RIF
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
8
4
64
64
64
64
32
64
64
64
32
32
64
64
32
32
32
64
64
64
64
64
64
64
64
64
64
64
64
64
32
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.03
0.03
0.03
32
32
8
512
64
4
64
8
512
128
256
16
4
128
256
16
4
256
4
4
256
8
8
1024
1024
512
32
8
32
8
WA6
WA7
WA9
64
8
WC1
WC2
WC3
WC4
64
8
128
256
16
8
128
256
16
8
512
4
8
512
8
8
1024
1024
1024
512
32
8
32
8
64
8
64
8
128
256
16
8
128
256
16
8
1
8
32
16
16
16
16
16
8
8
512
32
32
8
512
64
64
8
512
128
256
16
128
256
16
8
512
8
512
8
1024
512
32
8
32
8
WA10
64
8
64
8
512
128
256
32
8
128
256
0
8
512
8
8
512
8
NONE (+)AcrB
NONE (-)AcrB
8
1024
32
64
8
0
2
NDGA
128
256
4
2
CAM = Chloramphenicol; ERY = Erythromycin; TPP = Tetraphenylphosphonium; LEV = levofloxacin; RIF = Rifampicin
23

Table 2. The synergistic effect of NDGA derivatives (Series WD) with different antibacterials against wild-type drug-resistant strain E. coli BW25113 expressing AcrB that is
indicated as (+)AcrB.
Conc.
(µg/mL)
8
Conc.
(µg/mL)
8
MIC (µg/mL)
MIC (µg/mL)
TPP
Compd.
WD2
R2
Compd.
WD7
R2
CAM ERY
TPP
1024
1024
1024
512
LEV
0.06
0.06
0.03
0.03
0.03
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.03
0.03
0.03
RIF
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
CAM
ERY
64
64
32
32
16
64
64
64
64
32
64
32
16
N/D
N/D
64
64
64
64
64
64
64
64
32
64
4
LEV
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
N/D
N/D
0.06
0.06
0.06
0.06
0.03
0.06
0.03
0.03
0.03
0.06
0.004
RIF
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
8
8
8
8
4
8
8
8
8
8
8
8
8
8
8
8
4
4
2
2
4
4
4
4
2
64
64
64
64
64
64
64
64
64
32
64
64
32
32
32
64
64
32
32
32
32
32
32
16
16
8
4
1024
1024
1024
1024
512
16
16
2-OH
32
4-NH2
32
4
64
64
4
128
8
512
128
8
2
1024
1024
1024
1024
1024
512
8
1024
1024
1024
1024
512
16
16
8
WD3
WD4
WD5
4-Br
32
WD8
WD9
2-NH2
4-CH3
4-CF3
32
8
64
64
4
128
8
128
8
4
8
1024
1024
1024
N/D
16
512
16
8
3,5-di-NH2
3,4-di-OH
32
512
32
8
64
512
64
N/D
N/D
8
128
8
512
128
8
N/D
1024
1024
512
1024
1024
1024
1024
1024
1024
1024
128
16
16
8
32
32
8
WD10
NDGA
64
512
64
8
128
8
512
128
32
8
1024
1024
512
8
16
64
8
WD6 3,4,5-tri-OH
32
128
256
0
4
64
512
2
64
128
256
NONE (+)AcrB
NONE (-)AcrB
8
1024
32
0
2
CAM = Chloramphenicol; ERY = Erythromycin; TPP = Tetraphenylphosphonium; LEV = levofloxacin; RIF = Rifampicin
24

Fig. 1
a
b
c
25

Fig. 2
Glucose
30
25
20
15
10
5
(-) AcrB
(+) AcrB+ WD4 (100 µM)
(+) AcrB+ WD4 (50 µM)
(+) AcrB
50
100 150 200 250
Time (sec)
26

27

Fig. 3
28