Structure-activity relationships and optimization of acyclic acylphloroglucinol analogues as novel antimicrobial agents

Article abstract of DOI:10.1016/j.ejmech.2016.09.054

Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious threat to global public health, because it exhibits resistance to existing antibiotics and therefore high rates of morbidity and mortality. In this study, twenty-one natural product-based acylphloroglucinol congeners were synthesized, which possessed different side chains. Antibacterial screening against MRSA strains revealed that acyl moiety tailoring is a prerequisite for the antibacterial activity. Moreover, the lipophilicity, rather than the magnitude of the hydrophobic acyl tail dominates variability in activity potency. Compound 11j was identified as a promising lead for the generation of new anti-MRSA drug development. It was discovered by optimization of the side chain length in light of the potency, the breadth of the antibacterial spectrum, the rate of bactericidal action, as well as the membrane selectivity. Compound 11j exerted profound in?vitro antibacterial activity against the MRSA strain (JCSC 2172), and its MIC was 3-4 orders of magnitude lower than that of vancomycin. A preliminary mode of action study of compound 11j at the biophysical and morphology levels disclosed that the mechanism underlying its anti-MRSA activity included membrane depolarization and, to a lesser extent, membrane disruption and cell lysis.

Full text of DOI:10.1016/j.ejmech.2016.09.054

Accepted Manuscript
Structure-activity relationships and optimization of acyclic acylphloroglucinol
analogues as novel antimicrobial agents
Haibo Tan, Hongxin Liu, Liyun Zhao, Yao Yuan, Bailin Li, Yueming Jiang, Liang
Gong, Shengxiang Qiu
PII:
S0223-5234(16)30790-5
10.1016/j.ejmech.2016.09.054
EJMECH 8920
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 April 2016
Revised Date: 17 September 2016
Accepted Date: 18 September 2016
Please cite this article as: H. Tan, H. Liu, L. Zhao, Y. Yuan, B. Li, Y. Jiang, L. Gong, S. Qiu, Structure-
activity relationships and optimization of acyclic acylphloroglucinol analogues as novel antimicrobial
agents, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.054.
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ACCEPTED MANUSCRIPT
Structure-activity relationships and optimization of acyclic
acylphloroglucinol analogues as novel antimicrobial agents
Haibo Tana,1, Hongxin Liua,b,1, Liyun Zhaoa,c Yao Yuana,c, Bailin Lia,c, Yueming Jianga,
Liang Gonga,*, Shengxiang Qiua,*
*
Corresponding authors. E-mail address: lianggong@scbg.ac.cn (L. Gong),
sxqiu@scbg.ac.cn (S. Qiu). 1 These authors contributed equally to this work.

ACCEPTED MANUSCRIPT
Structure-activity relationships and optimization of acyclic
acylphloroglucinol analogues as novel antimicrobial agents
Haibo Tana,1, Hongxin Liua,b,1, Liyun Zhaoa,c Yao Yuana,c, Bailin Lia,c, Yueming
Jianga, Liang Gonga,*, Shengxiang Qiua,*
a
Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China
Botanical Garden, Chinese Academy of Sciences, Guangzhou, 510650, People's Republic of
China.
b
State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key
Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of
Applied Microbiology, Guangdong Institute of Microbiology, Guangzhou 510070, People's
Republic of China
c University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China.
* Corresponding authors. E-mail address: lianggong@scbg.ac.cn (L. Gong), sxqiu@scbg.ac.cn (S. Qiu).
1 These authors contributed equally to this work.
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Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious threat to global
public health, because it exhibits resistance to existing antibiotics and therefore high
rates of morbidity and mortality. In this study, twenty-one natural product-based
acylphloroglucinol congeners were synthesized, which possessed different side chains.
Antibacterial screening against MRSA strains revealed that acyl moiety tailoring is a
prerequisite for the antibacterial activity. Moreover, the lipophilicity, rather than the
magnitude of the hydrophobic acyl tail dominates variability in activity potency.
Compound 11j was identified as a promising lead for the generation of new
anti-MRSA drug development. It was discovered by optimization of the side chain
length in light of the potency, the breadth of the antibacterial spectrum, the rate of
bactericidal action, as well as the membrane selectivity. Compound 11j exerted
profound in vitro antibacterial activity against the MRSA strain (JCSC 2172), and its
MIC was 3-4 orders of magnitude lower than that of vancomycin. A preliminary
mode of action study of compound 11j at the biophysical and morphology levels
disclosed that the mechanism underlying its anti-MRSA activity included membrane
depolarization and, to a lesser extent, membrane disruption and cell lysis.
Keywords: anti-MRSA; acylphloroglucinol; mechanism; structure-activity
relationship.
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1. Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium
responsible for serious infections due to its high rates of morbidity and mortality [1,2].
Infection by MRSA is considered to be one of the most intractable infectious diseases
because of its emerging resistance to existing antibiotics. It is resistant to both the
β-lactam classes of antibiotics and the macrolide, fluoroquinolone, aminoglycoside,
tetracycline, and lincosamide classes of antibiotics with numerous resistance
mechanisms [3,4]. As a result, the nosocomial MRSA is considered as a predominant
pathogenic bacterium, and it created a crisis in public health systems especially in
developed countries [4]. Thus, the discovery of new therapeutic agents that are
effective against the bacteria evolving multidrug-resistant pathogens is crucial for the
future maintenance of public health.
In a continuation to our efforts to search for active antibacterial lead compounds
from Chinese medicinal plants and their chemical derivatives, we recently reported
that rhodomyrtone (1), a natural acylphloroglucinol isolated from the traditional
Chinese medicinal plant Rhodomyrtus tomentosa (Aiton) Hassk. (Myrtaceae) shows
significant activity against a wide range of Gram-positive pathogenic bacteria,
including MRSA, with low MIC values [5,6]. The previous pharmacological
evaluations revealed that 1 might be an excellent antibiotic candidate. It exhibited
desirable antibacterial properties, including a wide spectrum of activity, rapid
bactericidal action, avoidance of antibiotic resistance, and novel mechanisms of action
[7-11]. However, the development of rhodomyrtone suffered from bottlenecks,
including a paucity of natural supply and the difficulty for total synthesis [6,12].
Recently, several natural acyclic acylphloroglucinol compounds structurally similar to
rhodomyrtone, such as callistenone C (2) [13] and semimyrtucommulone (3) [14]
(Scheme 1), were reported and showed potent antibacterial activities. With the
inspiration of this discovery, efforts towards optimization through a total synthetic
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approach accompanied by a full exploration of the structure-activity relationships
(SAR) of acyclic acylphloroglucinols were conducted and reported herein.
It is well-accepted that biophysical processes play a critical role in the
antimicrobial activity of amphipathic molecules [15]. Therefore, tuning the
amphiphilic properties by changing the molecular dimensions, as well as the
lipophilicity to modulate the biophysical balance should have a critical impact on the
biological activities of the modified molecules. Both callistenone C (2) and
semimyrtucommulone (3) are apparently amphipathic metabolites with a ‘head and
tail’ connection. Therefore, structure modification, in anticipation of profound and
diverse changes in biological activity, can be readily accessed by targeting either the
head (phloroglucinol and β-triketone) or/and tail (isobutyl and isobutyryl) moieties
through synthesis. Accordingly, a few acyclic acylphloroglucinol analogues were
prepared by replacing their isobutyryl tails with other substituent groups of different
lengths and spatial characteristics, in order to manipulate amphiphilic properties and
further modulate structure-activity relationships.
In the present study, twenty-one acyclic acylphloroglucinol congeners were
synthesized, and their antibacterial potencies were evaluated against standard
Staphylococcus aureus (SA, ATCC 6538) and methicillin-resistant S. aureus (MRSA,
JCSC 2172) strains. Significant antibacterial activities were observed for most of the
acylphloroglucinol analogues. In particular, the compounds 11i-11n exhibited
antibacterial potencies higher than that of the positive control substance vancomycin.
Furthermore, the bactericidal spectrum of compounds 11i-11p versus a panel of
Gram-positive pathogens, as well as their selectivity over normal cells was also
elaborated. Herein, the details of the total synthesis of the acyclic acylphloroglucinols
and their antibacterial evaluations and mechanistal studies were presented. Lead
optimization was also completed, based on an extensive structure-activity relationship
(SAR) study.
2. Results and discussion
2.1. Chemistry
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The synthetic route for the series of acyclic acylphloroglucinol analogues 11
employed previously reported procedures with improved reaction conditions, as
outlined in Scheme 2 [12,15]. Syncarpic acid (8) was synthesized using commercially
available phloroglucinol 4 and acyl chloride 5. Briefly, the acylphloroglucinol 6b
could be readily obtained from phloroglucinol 4 using standard aluminum
trichloride-catalyzed Friedel-Crafts acylation, compound 6b was then subjected to
selective C-tetramethylation with MeI, which afforded the desired β-triketone 7 in
excellent yield. Acid-catalyzed retro-Claisen condensation provided 8 smoothly in
80% yield. Afterwards, the key intermediate 10 was successfully accessed through a
Knoevenagel condensation. The crucial coupling reaction of 10 and the
acylphloroglucinols 6 was accomplished through base-catalyzed Michael addition,
which resulted in the acyclic acylphloroglucinols 11 in good yields, ranging from
30% to 80%. The structures of all new compounds were unambiguously confirmed by
HRMS, 1H NMR, and 13C NMR spectral measurements.
2.2. Biological evaluation
The minimum inhibitory concentrations (MIC) against MRSA cells of the
acyclic acylphloroglucinol analogues 11, using vancomycin as the positive control,
were measured using the microdilution susceptibility assay in Mueller-Hinton broth
(MHB) following CLSI guidelines. The highest concentration used for testing the
acylphloroglucinol analogues 11 was 32 µg/mL due to their potent antibacterial
activity. Results from the anti-MRSA screening demonstrated
a
clear
structure-activity relationship wherein the lipophilic ability of the acyl group tails had
a significant influence on the potency of bacterial growth inhibition (Table 1). As a
comparison, the synthesized compounds 11 were also evaluated for their antibacterial
activity against a standard, drug-susceptible S. aureus (ATCC 6538).
It is well-known that if the hydrophobic portion of a molecule enters into the
membrane lipid bilayer, it will create disorder in the fluid bilayer [16,17]. However, it
was still unclear whether increasing the hydrophobicity of acylphloroglucinol through
substituent modification could result in an enhancement of antibacterial activity. As
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shown in Table 1, most of the synthetic compounds exhibited significant activity
against both MRSA (JCSC 2172) cells and standard SA (ATCC 6538) cells, except
for 11a (the acyl tail was replaced by an aldehyde group), which was devoid of
noticeable activity (MIC > 32 µg/mL). This suggests that the hydrophobic acyl tail in
the phloroglucinol core is a vital element in maintaining antibacterial properties
(Table 1).
The acylphloroglucinol with an acyl group (11b) showed moderate anti-MRSA
activity with an MIC of 4 µg/mL. However, the potency tended to decrease with an
increase in the lipophilicity of their acyl tails (11b-11d). This implies a reverse
relationship between the potency of the bactericidal activity and the lipophilicity of
their acyl tails. However, with more lipophilic acyl tails introduced onto the
acylphloroglucinol core, as exemplified by analogues 11e-11l, anti-MRSA activities
dramatically increased, wherein the lipophilic ability of the acyl substituent parallels
the potency of bacterial growth inhibition. Among these analogues, compound 11j,
possessing an octanoyl substituent, exhibited the most potent anti-MRSA activity with
a MIC value of 0.25 µg/mL, which was four-times more active than vancomycin
(MIC = 1.0 µg/mL). When compounds 11k-11n, possessing even longer acyl tails,
were evaluated, they showed similar activities as that of 11j with MIC values ranging
from 0.25 to 0.5 µg/mL. When the acyl tails were longer than 14 carbons, as in
compounds 11o and 11p, these analogues were barely soluble in DMSO and showed
weaker antibacterial activities. Therefore, both the hydrophobicity of the acyl tails and,
to an even greater extent, the optimized length of the acyl tails significantly modulate
the potency.
It has been postulated that the introduction of hydrophobic branching or cyclic
rings into some antibacterial molecules could improve their biological activity [16,18].
Therefore, it was of interest to explore how the introduction of hydrophobic branching
or cyclic rings to the acylphloroglucinol acyl tails would influence the potency. To
this end, the anti-MRSA activities of 11e and 11g with added branched acyl
substituents, as well as 11q-11s with added cyclic substituents, were evaluated. All of
the congeners showed noticeable antibacterial activities, compared with their
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counterparts 11d, 11f, 11h, and 11i, respectively. On the other hand, derivatives with
aliphatic phenyl chains (11t and 11u) exhibited only marginal antibacterial effects
against MRSA and SA cells, with 11u showing slightly higher potency than 11t,
presumably due to the difference in their lipophilicity. These results indicated that the
activity of acylphloroglucinols with acyl tails against MRSA might be mainly affected
by the lipophilicity of their acyl tails rather than their spatial parameters.
All the aforementioned evidence supported the conclusion that the anti-MRSA
activity of acylphloroglucinols with acyl tails was not linearly correlated with their
lipophilicity, despite lipophilicity apparently being the principal factor influencing
antibacterial activity. Although the mechanisms behind these results were not
unequivocally determined, the properties of the compounds relative to their ability to
diffuse through and then disturb the biological membranes of microbial systems
should play a key role. Typically, small molecules easily diffuse across membranes,
but lack the ability to disturb it. However, the acylphloroglucinols with
excessively-long acyl tails also showed reduced antibacterial activity due to their poor
compatibility with the membrane lipid bilayer. These results strongly suggested that a
desirable acylphloroglucinol lead compound should have a suitable lipophilicity with
an acyl tail of 8-12 carbons.
The synthetic acylphloroglucinol analogues 11i-11p, which each exhibited
potent anti-MRSA activity, were selected for further evaluation using a broad panel of
Gram-positive pathogens. Most of these compounds showed significant antibacterial
activities against Bacillus cereus strain (ATCC 10876), B. subtilis strain (ATCC
11774), and B. thuringiensis strain (ATCC 10792) with MICs ranging from 0.13 to
0.5 µg/mL, demonstrating their superior potency to that of the control substance
vancomycin (Table 2). On the other hand, none of them demonstrated noticeable
biological effects (MIC > 32 µg/mL) against Gram-negative bacteria, such as
Escherichia coli (ATCC 8739).
Besides their profound antibacterial activity against Gram-positive pathogens,
11i-11p also demonstrated good selectivity indexes (ranging from 13.1 to 22.2), as
assessed with Raw 264.7 cells after 24 h exposure (Table 3). Among them, compound
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11j emerged as a promising candidate; with its superior selectivity, compound 11j
merits further consideration for development as a potential class of anti-MRSA
therapeutic agents.
In order to understand the anti-bacterial nature of acylphloroglucinols, time-kill
experiments were carried out using the most sensitive MRSA strain (JCSC 2172). The
time-kill curves of 11j at 0.5-fold, 1-fold, 2-fold, and 4-fold of the MIC over a 24 h
treatment period in comparison with the known bactericidal and bacteriostatic agent
vancomycin were measured (Fig. 1). Compound 11j suppressed more than 90% of
bacterial growth within 10 h at the 0.5-fold, 1-fold, and 2-fold of the MIC values,
indicating the presence of a considerable bacteriostatic effect. In the treatment with a
4-fold MIC value, MRSA (JCSC 2172) survival in the presence of 11j dramatically
decreased with increasing time, reaching a sharp reduction (99.99%) in CFU after 24
h exposure. In particular, MRSA (JCSC 2172) cells were completely killed (after
incubation for 24 h) and rapidly achieved 3-log reductions (99.9% reduction) within
only 4 h at the 4-fold MIC level. Meanwhile, vancomycin only achieved a 3-log
reduction at the same concentration in approximately 10 h. In this respect, compound
11j exhibited a distinctly superior advantage over vancomycin, since the rapidity of
the time-kill effect is critical to prevent microbes gaining resistance. In summary, the
experimental evidence suggested that the acyclic acylphloroglucinol analogue 11j
mainly exerted bacteriostatic activity at 0.5 to 2-fold of MIC, and mainly bactericidal
activity at a dose of 4-fold of MIC.
2.3 Bactericidal mechanism study
It was previously reported from this laboratory that the natural xanthone
α-mangostin, which is biogenetically related to the acylphloroglucinol family, exerted
potent anti-MRSA activity via a mechanism that depolarized the bacterial cytoplasm
membrane [13,19]. In the present experiments, after the MRSA (JCSC 2172) cells
were treated with serial concentrations (0.5 × MIC, 1 × MIC, 2 × MIC, 4 × MIC, 8 ×
MIC) of 11j, a sharp increase in the fluorescence intensity was observed, as
monitored in a fluorescence probe experiment (Fig. 2A). This suggested that the mode
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of bactericidal action of 11j might be attributed to its significant dissipation of
membrane potential.
A SYTOX green assay was employed to decipher whether compound 11j also
caused microbial membrane disruption. Unlike the positive control substance melittin,
which caused an immediate and unambiguous increase in the fluorescence intensity
within 60 s, treatment with 11j resulted in only a weak increase in the fluorescence
intensity, even at the 8-fold MIC value (Fig. 2B). This suggested that 11j was not a
typical membrane disruption agent, even though it did promote permeability of the
cytoplasm membrane to some extent (Fig. 2C), implying that compound 11j might
possess a unique mechanism of action to prevent microbial survival. The morphology
of the 11j-treated MRSA (JCSC 2172) cells was examined using scanning electron
microscopy which showed that 11j could induce cell lysis after treatment with an
8-fold MIC dose for 4 h (Fig. 3).
3. Conclusions
In summary, a synthetic natural product-based pool of twenty one
acylphloroglucinol congeners were specifically designed with enhanced structural
diversity by modifying the pendant acyl moiety. Antibacterial screening against
MRSA (JCSC 2172) strains revealed an unambiguous structure-activity relationship,
which indicated that the acyl tail was the prerequisite for anti-bacterial activity, and
that its lipophilicity, rather than its spatial dimension, was critical for activity. As a
result, compound 11j was identified as a very promising lead compound by tuning the
length of the acyl tail and the potency of activity, which was greater than that of
vancomycin. In addition, compound 11j possessed other advantages, including
breadth of the antibacterial spectrum, rapidity of bactericidal action, and excellent
membrane selectivity. Studies of its biophysical and morphological mechanism of
action revealed that the effects included membrane depolarization and, to lesser
extents, membrane disruption and cell lysis. Thus, a new approach for modifying
acylphloroglucinol scaffolds has effectively yielded a lead compound, 11j, which
possesses excellent antimicrobial activities, high selectivity, and other favorable
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drug-like properties. Overall, these compounds represent a new generation of
anti-MRSA agents that possess a unique anti-bacterial mechanism, and therefore
awaits further study.
4. Experimental Section
4.1. Materials and experimental apparatus
All of the reactions were carried out under a nitrogen atmosphere with dry
solvents under anhydrous conditions, unless otherwise noted. Reagents were
purchased at high commercial quality, and used without further purification.
Thin-layer chromatography (TLC) was conducted with 0.25 mm Tsingdao silica gel
plates (60 F-254) and visualized by exposure to UV light (254 nm) or stained with
potassium permanganate. Silica gel (ZCX-II, 200-300 mesh) used for flash column
chromatography was purchased from Qing Dao Hai Yang Chemical Industry Co. of
1
China. H NMR and 13C NMR spectra were recorded on a Brüker Advance 500
instrument (1H: 500 MHz, 13C: 125 MHz). Chemical shifts were reported in parts per
million relative to CDCl3 (1H NMR; 7.27 ppm, 13C NMR; 77.00 ppm). The following
abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet, br = broad. Mass spectrometric data were obtained
using an ABI-Q Star Elite high resolution mass spectrometer. Dichloroethane (DCE)
was distilled from calcium hydride. Yields referred to chromatographically purified
products unless otherwise stated.
4.2. Chemical synthesis
4.2.1 General procedure for the synthesis of compounds 6c-6k
Aluminum trichloride (1.33 g, 10 mmol) was slowly and carefully added to a
solution of phloroglucinol 4 (315 mg, 2.5 mmol) in CH2ClCH2Cl/PhNO2 (1:1, 10 mL)
o
at 0 C. After stirring at this temperature for 10 min under nitrogen atmosphere, acid
chloride 5 (3.0 mmol) was added. Then, the ice bath was removed, and the mixture
o
was stirred at 80 C for 3 h. The crude reaction mixture was cooled to room
temperature and quenched with water (25 mL). The mixture was extracted with
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EtOAc (5 × 25 mL), washed with brine, and concentrated in vacuum. The crude
product was purified by flash chromatography (silica gel, hexane:EtOAc = 2:l). Their
spectra data were referred the reported relative references.
4.2.2. General procedure for the synthesis of intermediate compounds 6l-6u
To a solution of phloroglucinol 4 (315 mg, 2.5 mmol) in acid chloride 5 (3.0
mmol) was added methanesulfonic acid (720 mg, 7.5 mmol). After stirring at room
temperature for 10 min under nitrogen atmosphere, the mixture was stirred at 60-80
oC for another 1 h. Then, the crude mixture was cooled to room temperature and
quenched with water (25 mL). The mixture was extracted with EtOAc (5 × 25 mL),
washed with brine, and concentrated in vacuum. The crude product was purified by
flash chromatography (silica gel, hexane:EtOAc = 2:l). Their spectra data were
referred the reported relative references.
4.2.3 The synthesis of 4-acyl-5-hydroxy-2,2,6,6-tetramethylcyclohex-4-ene-1,3-dione
7
Sodium methoxide (7.18 g, 133 mmol) was slowly dissolved in anhydrous
methanol (60 mL) at 0oC. To this clear solution, acylphloroglucinol 6b (2.75 g, 16.5
mmol) was added carefully and the mixture stirred for 10 min under nitrogen
atmosphere. After that, methyl iodide (14.2 mL, 228 mmol) was slowly added. The
ice bath was then removed, and the mixture stirred at room temperature for 24 h. The
crude mixture was quenched with 2 N HCl (60 mL) and extracted with CHCl3 (5 × 60
mL), washed with brine, and concentrated in vacuum. The crude product was purified
by flash chromatography (silica gel, hexane:EtOAc = 5:l) to provide 7 (3.17 g, 14.2
mmol, 86% yield) as a yellow rod-like crystal. 1H NMR (500 MHz, CDCl3): δ 1.33 (s,
6H), 1.42 (s, 6H), 2.57 (s, 3H); 13C NMR (125 MHz, CDCl3): δ 23.8, 24.3, 27.4, 52.0,
56.7, 109.4, 196.7, 199.1, 201.7, 210.0.
4.2.4 Synthesis of syncarpic acid 8
A flame-dried 50 mL flask was charged with 6 N HCl (30 mL) and 7 (3.17 g,
14.2 mmol), and the reaction mixture was stirred vigorously under reflux for 24 h.
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The mixture was cooled to room temperature and extracted with EtOAc (5 × 50 mL).
The combined organic phases were washed with brine (30 mL), dried over Na2SO4,
and filtered. Removal of solvent by rotary evaporation and purification by flash
column chromatography (silica gel, hexane:EtOAc = 1:l) afforded 8 (2.17 g, 11.4
mmol, 80% yield). 1H NMR (500 MHz, CDCl3): ketone: δ 1.31 (s, 12H), 3.61 (s, 2H);
enol: δ 1.40 (s, 12H), 5.74 (br d, J = 2.3 Hz, 1H), 8.00 (br s, 1H); 13C NMR (100 MHz,
CDCl3): ketone: δ 21.8, 50.2, 59.1, 204.3, 208.9; enol: δ 24.5, 51.2, 59.1, 101.7, 191.9,
204.3, 212.6.
4.2.5 The synthesis of 2,2,4,4-tetramethyl-6-(3-methylbutylidene)cyclohexane-1,3,5-
trione 10
Syncarpic acid 8 (91 mg, 0.5 mmol) was dissolved in CH2Cl2 (5 mL), and
isovaleraldehyde 9 (86 mg, 1.0 mmol) and proline (5.7 mg, 0.5 mmol) were added.
The resulting mixture was stirred for 30 min at room temperature, then purified by 3
cm long flash chromatography (silica gel, CH2Cl2) to afford the desired product 10
(125 mg, 100% yield) as a colorless oil. 1H NMR (500 MHz, CDCl3): δ 0.95 (d, J =
6.7 Hz, 6H), 1.30 (s, 6H), 1.31 (s, 6H), 1.89 (m, J = 6.7 Hz,1H), 2.59 (dd, J = 3.0, 3.0
Hz, 2H), 7.51 (dd, J = 3.0 Hz, 1H); 13C NMR (125 MHz, CDCl3): δ 21.9, 22.3, 22.6,
28.7, 38.9, 57.9, 58.6, 133.1, 159.1, 196.4, 199.5, 208.8.
4.2.6 General procedure for the synthesis of acylphloroglucinol analogues 11
Sodium hydride (30.0 mg, 0.75 mmol, 60% in mineral oil) was carefully added
to a solution of acylphloroglucinol 6 (0.5 mmol) in THF (8 mL), then the unsaturated
triketone 10 (62.5 mg, 0.25 mmol) in THF (2 mL) was slowly added. The resulting
mixture was stirred for 0.5 h at room temperature, and then quenched with 1 N HCl (3
mL) and extracted with EtOAc (3 × 5 mL), washed with brine, and concentrated in
vacuum. The crude product was purified by flash chromatography (silica gel,
hexane:EtOAc = 10:l to 2:l) to provide derivatives of the general structure 11 with
yields ranging from 30% to 80%. The yields and spectra data of 11 were seen in the
supporting information.
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4.3. Bacterial stains and cells
S. aureus strain (ATCC 6538), B. cereus strain (ATCC 10876), B. subtilis strain
(ATCC 11774), and B. thuringiensis strain (ATCC 10792) were obtained from
Guangdong Institute of Microbiology (Guangzhou, China). We sincerely thank T. Ito
and K. Hiramatsu for providing us the SCCmec type strains of clinically isolated
MRSA (JCSC 2172). Raw 264.7 cells were obtained from Kunming Institute of
Zoology, Chinese Academy of Sciences (Kunming, China).
4.4 Minimal inhibitory concentration (MIC) assays
MIC determinations for 11a-11u were performed using the Mueller-Hinton broth
(MHB) microdilution method following CLSI guidelines according to a modification
of the Clinical and Laboratory Standards Institute. Resazurin was added as a viability
indicator. Colorimetric MIC end-points were interpreted as the lowest concentration
that remained blue (indicating no growth) or the first dilution that changed from blue
to slightly purple (equivalent to prominent growth inhibition).
To every well in a 96-well microtiter plate was added 100 µL of cation-adjusted
Mueller containing 5 × 105 CFU/mL of bacteria with resazurin as viability indicator
(0.06 mg/mL). Vancomycin and 11a-11u were first dissolved in DMSO to make 320
µg/mL stock solutions. A 20 µL sample of the solutions and another 180 µL of cation
adjusted Mueller solution were then added to the first line of wells in a 96-well
microtiter plate. After the solutions were uniformly mixed, 100 µL of this solution
was removed to the second line and uniformly mixed. The other lines were further
replicated with the same operation to prepare final concentrations ranging from 32 to
o
0.13 µg/mL. The 96-well microtiter plates were then incubated for 8-12 h at 37 C.
The MIC value was determined based on the color change of the solutions.
Experiments were repeated at least three times with three parallel simples for each
time and found to be reproducible. For clarity, data from only one experiment was
presented.
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4.5 Selectivity over cytotoxic activity
Cells were cultured in RPMI-1640 or DMEM media supplemented with 10%
heat-inactivated fetal bovine serum (FBS), 1% 100 mg/mL penicillin, and 100 mg/mL
o
streptomycin. All of the cell lines were maintained in culture at 37 C in an
atmosphere of 5% CO2. Cell proliferation or cell proliferation or viability was
measured using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide] assay. Cells were seeded in each well containing 100 µL medium in 96-well
microtiter plates under identical conditions. All of the compounds tested were
dissolved and eventually further diluted in dimethylsulfoxide (DMSO), and three
panels of wells were used for each concentration of each compound. Results were
expressed as half maximal inhibitory concentration (IC50, mg/mL). The effects of the
test compounds on cell viability were calculated using cells treated with DMSO as
control. The data were subjected to linear regression analysis and the regression lines
were plotted for the best straight-line fit. The IC50 (inhibition of cell viability)
concentrations were calculated using the respective regression equation.
Exponentially growing Raw 264.7 cells were plated in 96-well tissue plates at a
density of 4 × 104 cells/well. Adherent cell lines were previously incubated for 24 h to
ensure adhesion to the wells in an atmosphere of 5% CO2. Compounds 11i-11p were
applied at various concentrations (ranging from 1.5625 to 100 µg/mL) and control
cells were treated with DMSO at the highest concentration used in test wells (0.5%). 1
h prior to the end of the incubation period, 20 mL of MTT solution (5 mg/mL in PBS,
5% MTT) was added to each well and the plates further incubated at 37 oC for another
4 h. Supernatants were removed and 150 µL DMSO was added to each well to
dissolve the formazan crystals. The mixture was oscillated for 10 min at room
temperature and the absorbance was measured at 490 nm (Biotek Synergy 4).
Experiments were repeated at least three times with three parallel simples for each
time and found to be reproducible. Data from one experiment was presented.
4.6 Time-kill studies
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The bacterial strain MRSA (JCSC 2172) used in the time-kill studies was
selected from isolated colonies on a 24 h tryptic soy agar (TSA) plate. The inocula
was suspended and adjusted in 0.31 mM phosphate buffer to obtain a bacterial
suspension with 106 to 107 CFU/mL. The inoculum was then treated with various
concentrations (0.5 ×, 1 ×, 2 ×, 4 ×, and 8 × MIC) of 11j and vancomycin. The
mixtures were incubated at 35 oC. Culture aliquots were removed at 30 min, 2 h, 4 h,
8 h, and 24 h to measure the viable plate counts. The initial drug-free control count
was 2.5 × 106 CFU/mL, which increased to almost 1.6 × 109 CFU/mL after 24 h. The
aliquots were diluted serially 10-fold using D/E neutralization broth, and 20 µL of
each dilution was plated on TSA plates using the surface-spread plate method. The
plates were then incubated at 35 °C for 48 h. Bacterial viability was determined by
counting the colonies grown on the plates. Bactericidal activity was defined as a 3-log
reduction of viable count in cultures treated with an antimicrobial compound
compared to an untreated control. The detection limit for a reliable plate count was
100 CFU/mL for both 11j and vancomycin. The results were expressed as the log
value of the number of surviving bacteria in the anti-MRSA assay at various time
intervals. Experiments were repeated at least two times and found to be reproducible.
Data from one experiment was presented.
4.7 Cytoplasmic membrane depolarization assay
The effect of 11j on the membrane potential of MRSA (JCSC 2172) was
investigated using a modified version of the method described by Wu and Hancock
[20]. Briefly, MRSA (JCSC 2172) cells were grown to exponential phase and then
harvested. Bacteria were suspended and washed with buffer solution (5 mM HEPES
at pH 7) until an optical density OD 0.1 at 600 nm (OD600) was obtained. The cell
suspension was then incubated with 0.4 µM 3,3-dipropylthiacarbocyanine (DiSC3-5),
better-known as a cytoplasmic membrane-potential sensitive dye, was used to localize
in the bacterial polarized membrane and exhibit self-quenched fluorescence, for 20
o
min at 37 C with shaking. Then, KCl at 100 mM was added to equilibrate the
cytoplasmic and external K+ concentrations. The desired concentration of 11j was
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added into a stirred cuvette, and the change in fluorescence was monitored at an
excitation wavelength of 660 nm and an emission wavelength of 675 nm. DMSO and
vancomycin were used as the negative controls with no effect on the depolarization.
Experiments were repeated at least three times and found to be reproducible. Data
from one experiment are presented.
4.8 SYTOX green assay
In order to understand the effects of test compounds on bacterial membrane
structure, a membrane-impermeable cationic cyanine dye SYTOX green was utilized,
which can lead to an enhanced fluorescence signal through binding to nucleic acids.
The protocol was based on the method of Rathinakumar et al. [21]. Briefly, MRSA
(JCSC 2172) cells were suspended in 0.85% NaCl buffer until an OD600 of 0.2 was
obtained. The bacterial suspension was incubated with serial concentrations of 11j
respectively for 4 h, was then incubated with 3 µM of SYTOX green for 5 min. The
fluorescence was measured at an excitation wavelength of 504 nm and an emission
wavelength of 523 nm. Melittin, a cell lytic factor, was used as a positive control.
Experiments were repeated at least three times and found to be reproducible. Data
from one experiment are presented.
4.9 Scanning electron microscopy
For the observations using scanning electron microscopy, exponential-phase
o
bacteria were treated with compounds at 8 × MIC for 4 h at 37 C. Then, the cells
were washed twice, suspended in PBS, and prefixed in 0.1 M phosphate buffer (pH
7.2) containing 2% glutaraldehyde and 2.5% paraformaldehyde overnight. After
washing six times with 0.1 M phosphate buffer, the samples were post-fixed in 1%
osmium tetroxide for 2 h. After washing another three times with 0.1 M phosphate
buffer, the samples were dehydrated through a graded ethanol series and subjected to
freeze-drying (JFD-310, JEOL, Tokyo, Japan). Following this, samples were mounted
on stubs and coated with gold in a sputter coater (JFC-1600, JEOL, Tokyo, Japan),
16

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and then examined and photographed under a scanning electron microscope
(JSM-6360LV, JEOL, Tokyo, Japan).
Acknowledgments
We thank the National Science and Technology Major Projects of China
(2014ZX10005002-005), National Natural Science Foundation of China (NSFC) (No.
81502949), and Natural Science Foundation of Guangdong Province
(2015A030310482) for financial support. We thank Professor Emeritus Geoffrey A.
Cordell, Natural Products Inc., IL, USA, for his review of the manuscript prior to
submission.
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Captions
Scheme 1. Structures of selected natural acylphloroglucinols.
Scheme 2. Syntheses of acylphloroglucinol analogues.
Table 1. In vitro antibacterial activity (MIC) of compounds 11a-11u against MRSA
(JCSC 2172) and standard S. aureus (ATCC 6538) cells.
Table 2. Antibacterial spectra of compounds 11i-11p compared with vancomycin
against a panel of Gram-positive pathogens.
Table 3. The selectivity of anti-MRSA versus antiproliferative (IC50) activities
against Raw 264.7 cells.
Fig. 1. Time-kill kinetics of MRSA (JCSC 2172) cells treated with vancomycin and
compound 11j at various concentrations. Experiments were repeated at least two
times and found to be reproducible. Data from one experiment was presented.
Fig. 2. Antibacterial action of compound 11j. A) 11j-induced membrane
depolarization of MRSA (JCSC 2172) cells using of the DiSC3-5 probe; B) SYTOX
green assay of MRSA (JCSC 2172) cells immediately after adding 11j at 8-fold MIC,
which showed that 11j could disrupt the cytoplasmic membrane; melittin (10 µg/mL)
was used as the positive control; C) SYTOX green assay of MRSA (JCSC 2172) cells
treated with 11j for 4 h, which demonstrated that 11j caused permeabilization of the
cytoplasm membrane; melittin was used as the positive control. MIC values: 11j =
0.25 µg/mL, melittin = 1.25 µg/mL. Experiments were repeated at least three times
and found to be reproducible. Data from one experiment was presented.
20

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Fig. 3. Scanning electron microscopy: (A) Untreated MRSA (JCSC 2172) cells
showing intact morphology, and (B) MRSA (JCSC 2172) cells treated with 11j (8 ×
MIC) for 4 h, showing that 11j induced cell lysis.
21

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Scheme 1. Structures of selected natural acylphloroglucinols.
22

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HO
OH
For A:
6b-6k.
For B:
6l-6v.
OH
A: AlCl3, PhNO2 or
B: MsA, 80 oC, 3 h
O
O
+
Cl
R
25-85% yield
OH
R
HO
HO
OH
6
5
4
O
O
OH
NaOCH3
6 N HCl
CH3OH, CH3I
reflux, 12 h
O
92%
80%
O
O
OH
OH
6b
7
O
Proline
CH2Cl2, 0.5 h
NaH, THF
HO
OH
O
O
OH
O
O
O
H
9
10
6
8
OH
R
R = H (a), CH3 (b), C2H5 (c), nꢀC3H7 (d), iꢀ
C3H7 (e), nꢀC4H9 (f), iꢀC4H9 (g), nꢀC5H11 (h), nꢀ
C6H13 (i), nꢀC7H15 (j), nꢀC8H17 (k), nꢀC9H19 (l),
nꢀC10H21 (m), nꢀC11H23 (n), nꢀC13H27 (o), nꢀ
C15H31 (p), cꢀC4H7 (q), cꢀC5H9 (r), cꢀC6H11 (s),
CH2Ph (t), C2H4Ph (u).
HO
O
OH
HO
O
OH
O
R
11
Scheme 2. Syntheses of acylphloroglucinol analogues.
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Table 1. In vitro antibacterial activity (MIC) of compounds 11a-11u against MRSA
(JCSC 2172) and standard S. aureus (ATCC 6538) cells.
Compounds
R
MIC (µg/mL)
MRSA JCSC
S. aureus
2172
ATCC 6538
H
>32
>32
4.0
11a
11b
CH3
4.0
8.0
C2H5
8.0
11c
n-C3H7
i-C3H7
n-C4H9
i-C4H9
n-C5H11
n-C6H13
n-C7H15
n-C8H17
n-C9H19
n-C10H21
n-C11H23
n-C13H27
n-C15H31
c-C4H7
c-C5H9
c-C6H11
CH2Ph
C2H4Ph
-
32.0
16.0
8.0
32.0
16.0
4.0
11d
11e
11f
8.0
8.0
11g
2.0
2.0
11h
0.5
0.5
11i
0.25
0.5
0.25
0.25
0.25
0.25
0.5
11j
11k
0.25
0.5
11l
11m
11n
0.5
1.0
0.5
11o
2.0
1.0
11p
16.0
>32
4.0
16.0
32
11q
11r
4.0
11s
16.0
8.0
16.0
4.0
11t
11u
1.0
0.5
Vancomycin
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Table 2. Antibacterial spectra of compounds 11i-11p compared with vancomycin
against a panel of Gram-positive pathogens.
Compounds
R
MIC (µg/mL)
B. cereus
B. thuringiensis
B. subtilis
E. coli
n-C6H13
n-C7H15
n-C8H17
n-C9H19
n-C10H21
n-C11H23
n-C13H27
n-C15H31
-
0.5
0.25
0.5
0.5
0.25
0.5
0.25
0.13
0.25
0.25
0.25
0.25
8.0
>32
>32
>32
>32
>32
>32
>32
>32
>32
11i
11j
11k
0.5
1.0
11l
0.25
0.5
0.5
11m
1.0
11n
2.0
4.0
11o
2.0
8.0
16.0
0.13
11p
1.0
0.25
Vancomycin
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Table 3. The selectivity of anti-MRSA versus antiproliferative (IC50) activities
against Raw 264.7 cells.
Compounds
R
MICMRSA
IC50 (µg/mL)
Selectivity
ratioa
µg/mL
Raw 264.7
n-C6H13
n-C7H15
n-C8H17
n-C9H19
n-C10H21
n-C11H23
n-C13H27
n-C15H31
0.5
0.25
0.5
8.8
5.1
16.7
20.4
16.4
20.4
22.2
15.8
13.1
17.5
11i
11j
8.2
11k
11l
0.25
0.5
5.1
11.1
7.9
11m
11n
11o
11p
0.5
1.0
13.1
17.5
1.0
a Selectivity ratio = IC50/MIC against MRSA (JSJC 2172).
26

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Fig. 1. Time-kill kinetics of MRSA (JCSC 2172) cells treated with vancomycin and
compound 11j at various concentrations. Experiments were repeated at least two
times and found to be reproducible. Data from one experiment was presented.
27

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A
B
C
Fig. 2. Antibacterial action of compound 11j. A) 11j-induced membrane depolarization
of MRSA (JCSC 2172) cells using of the DiSC3-5 probe; B) SYTOX green assay of
MRSA (JCSC 2172) cells immediately after added 11j at 8-fold MIC, which showed
that 11j could disrupt the cytoplasmic membrane; melittin (10 µg/mL) was used as the
positive control; C) SYTOX green assay of MRSA (JCSC 2172) cells treated with 11j
for 4 h, which demonstrated that 11j caused permeabilization of the cytoplasmic
membrane; melittin was used as the positive control. MIC values: 11j = 0.25 µg/mL,
melittin = 1.25 µg/mL. Experiments were repeated at least three times and found to be
reproducible. Data from one experiment are presented.
28