On the basis of recently reported abyssinone II and olympicin A, a series of chemically modified flavonoid phytochemicals were synthesized and evaluated against Mycobacterium tuberculosis and a panel of Gram-positive and -negative bacterial pathogens. Some of the synthesized compounds exhibited good antibacterial activities against Gram-positive pathogens including methicillin resistant Staphylococcus aureus with minimum inhibitory concentration as low as 0.39 μg/mL. SAR analysis revealed that the 2-hydrophobic substituent and the 4-hydrogen bond donor/acceptor of the 4-chromanone scaffold together with the hydroxy groups at 5- and 7-positions enhanced antibacterial activities; the 2',4'-dihydroxylated A ring and the lipophilic substituted B ring of chalcone derivatives were pharmacophoric elements for antibacterial activities. Mode of action studies performed on selected compounds revealed that they dissipated the bacterial membrane potential, resulting in the inhibition of macromolecular biosynthesis; further studies showed that selected compounds inhibited DNA topoisomerase IV, suggesting complex mechanisms of actions for compounds in this series.
On the basis of recently reported abyssinone II and olympicin A, a series of chemically modified flavonoid phytochemicals were synthesized and evaluated against Mycobacterium tuberculosis and a panel of Gram-positive and -negative bacterial pathogens. Some of the synthesized compounds exhibited good antibacterial activities against Gram-positive pathogens including methicillin resistant Staphylococcus aureus with minimum inhibitory concentration as low as 0.39 μg/mL. SAR analysis revealed that the 2-hydrophobic substituent and the 4-hydrogen bond donor/acceptor of the 4-chromanone scaffold together with the hydroxy groups at 5- and 7-positions enhanced antibacterial activities; the 2',4'-dihydroxylated A ring and the lipophilic substituted B ring of chalcone derivatives were pharmacophoric elements for antibacterial activities. Mode of action studies performed on selected compounds revealed that they dissipated the bacterial membrane potential, resulting in the inhibition of macromolecular biosynthesis; further studies showed that selected compounds inhibited DNA topoisomerase IV, suggesting complex mechanisms of actions for compounds in this series.
Because of the emergence and spread of
multidrug resistant microorganisms and pathogenic bacterial infections,
novel chemotype antibacterial agents demonstrating distinct modes
of action from existing antibiotics are urgently needed. Natural products
are known as rich sources of bioactive molecules and chemical diversity
and have thus provided invaluable chemical scaffolds as well as served
as an inspiration toward antibacterial drug discovery and development.[1−4] In this context, synthesis and evaluation of natural-product-inspired
compound libraries represent an attractive approach for discovering
novel antibacterial agents.[5]Flavonoids
are a large family of polyphenolic phytochemicals, which widely exist
in the plant kingdom.[6] As such, flavonoids
have been the focus of numerous basic biomedical research as well
as clinical investigation.[7,8] As examples, high dietary
intake of flavonoids may offer potential to reduce the risk of various
cancers according to a number of epidemiological studies.[9−13] In addition, flavonoids have been reported to display a broad spectrum
of pharmacological activities, such as antimicrobial,[14−16] anti-inflammatory,[17,18] cancer preventive[19,20] and anticancer,[21,22] and antioxidant activities.[23,24] It is also noteworthy that some widely investigated flavonoids,
such as flavone acetic acid (FAA),[25] flavopiridol,[26−28] silibinin (silybin),[29,30] and quercetin[31] and its derivatives[32] (Figure 1), have progressed to various stages of clinical
trials.[33] In this regard, plant-derived
phytochemicals including chemically modified flavonoids and derivatives
continue to attract great interest in the development of novel antibiotics.[34]
Figure 1
Skeleton structures of chalcones, 4-chromanones, and representative
structures of naturally occurring flavonoids including abyssinone
II and olympicin A.
Skeleton structures of chalcones, 4-chromanones, and representative
structures of naturally occurring flavonoids including abyssinone
II and olympicin A.Furthermore, chalcones
(1,3-diaryl-2-propen-1-ones), one subclass of structural analogues
of flavonoids, have been reported to exhibit diverse biological activities,[35−38] in which the enone functional group and the 2′-hydroxy group
constitute important structural motifs for antibiotic activity. From
a chemistry point of view, chalcones and 4-chromanones are structurally
related, and 2′-hydroxychalcones serve as important synthetic
precursors for the synthesis of 4-chromanones following an intramolecular
conjugate addition of the phenol on the α,β-unsaturated
system.[39] Notably, the 4-chromanone derivatives
containing an aromatic substituent at the 2-position, so-called flavanones,
have been identified as an important class of bioactive heterocycles.[40−42]As a result of our longstanding interest in developing natural-product-inspired
new antibacterial agents, we recently reported the identification
of abyssinone II as a promising antibacterial lead by screening a
focused flavonoid and resveratrol library.[43] In addition, olympicin A, a member of the natural acylphloroglucinol
chemical class, was recently isolated from the plant Hypericum
olympicum and reported to exhibit potent antibacterial activity
against a panel of multidrug-resistant (MDR) strains of clinically
relevant Staphylococcus aureus, with minimum inhibitory
concentration (MIC) values ranging from 0.5 to 1 μg/mL.[44,45] Very recently, we have shown that synthetic olympicin A also exhibited
good activity against Clostridium difficile (MIC
= 1–2 μg/mL).[46] Inspired by
the antibacterial activity of the natural products abyssinone II and
olympicin A, in this work we employed the 4-chromanone and chalcone
structural scaffolds as chemical starting points to design and synthesize
chemically modified flavonoid analogues. Subsequently, several series
of structurally related flavonoids were synthesized and evaluated
in vitro against a broad set of bacterial pathogens and a detailed
structure–activity relationship (SAR) has been obtained. Furthermore,
the antibacterial basis of promising lead compounds and their ability
to inhibit bacterial topoisomerases such as DNA gyrase or topo IV
have also been examined.
Results and Discussion
Synthesis of Olympicin
A and Derivatives
The isolation and chemical synthesis of
olympicin A (2a) was originally reported by Shiu et al.,
and its synthesis involved a four-step reaction sequence. However,
the overall yield was only 3.3% from 1a.[44] To improve the reaction efficiency and develop a modular
synthesis toward olympicin A and derivatives, we evaluated diverse
protecting schemes including the tert-butyldimethylsilyl
(TBDMS), the base-stable methoxymethyl (MOM), and the p-toluenesulfonyl (Ts) groups and developed an improved synthesis
of 2a by using the Ts protecting strategy, improving
the overall yield to 40% from 1a (Scheme 1). In particular, we found that the reported low yield may
be largely due to the instability of the TBDMS protecting group under
basic reaction conditions (K2CO3, 80 °C)
when introducing the geranyl group. To address this, the base-stable
MOM group was next applied instead of TBDMS. Following the O-geranylation
reaction, it was found that 2a decomposed during the
deprotection of the MOM groups because of the instability of O-geranyl group under the acidic condition, and the deprotected 1a was recovered from the reaction. Subsequently the Ts group
was used to protect the hydroxy group,[47] and only the tris(tosylate) 1b was obtained as the
major product because of the lack of selectivity of the Ts group under
the reaction condition. Nevertheless, we found that the tosylate group
at the 2-position of 1b was very labile, and the geranyl
group could be selectively introduced with sodium hydride as the base.
Final removal of the Ts group was performed with excess sodium methoxide
in methanol under reflux[48] to afford the
chiral olympicin A (2a). The spectroscopic data of our
synthetic olympicin A were in good agreement with those of the natural
product.[44] For comparison, the racemic
olympicin A (2b) was also synthesized under the same
conditions to study the potential effect of stereochemistry on antibacterial
property.
Reagents and conditions: (i) SOCl2, 80 °C, 2 h; (ii) AlCl3, CS2,
PhNO2, 0.5 h; (iii) TsCl, K2CO3,
acetone, 1 h, 73%; (iv) geranyl bromide, NaH, DMF, 1 h; (v) CH3ONa, MeOH, reflux, 8 h, 55% (two-step overall yield); 52%
for 2b (two-step overall yield).Next, to further expand the chemical diversity and investigate the
influence of 2-substitution on antibacterial activity in the scaffold,
an array of racemic olympicin derivatives (2c–e) and enantiomeric form 2f were designed and
synthesized (Scheme 2). Compounds 1a′ and 1a were first protected by reacting with MOMCl
in the presence of diisopropylethylamine (DIPEA) to provide 1c and 1c′ in moderate yields. The O-alkylation
reaction was subsequently carried out with appropriate alkyl bromide
using sodium hydride as a base, after which the MOM groups were removed
with hydrochloric acid to give 2c–f in moderate to high yields (66–93%).
Scheme 2
Synthesis of Olympicin
A Analogues
Reagents and conditions: (i) MOMCl,
DIPEA, DCM, 1 h; (ii) R-Br, NaH, DMF; (iii) HCl, MeOH, overnight.
Synthesis of Olympicin
A Analogues
Reagents and conditions: (i) MOMCl,
DIPEA, DCM, 1 h; (ii) R-Br, NaH, DMF; (iii) HCl, MeOH, overnight.
Synthesis of 2-Substituted 4-Chromanone and
Derivatives
4-Chromanone derivatives bearing an aryl substituent
in the 2-position are normally synthesized by reacting an acetophenone
with an arylaldehyde under strong basic or acidic conditions.[49,50] However, because of the side reaction of self-condensation of the
aliphatic aldehyde,[51] these conditions
are not ideal for the synthesis of 2-alkyl substituted 4-chromanones.
Several different synthetic methods to prepare such 4-chromanone derivatives
have been reported,[51−53] either involving microwave irradiation or requiring
very long reaction times. Here, a sealed pressure tube was introduced
as a reaction vessel for the preparation of 2-alkyl substituted 4-chromanones.
On the basis of the scaffold, several modification strategies (Figure 2) were applied to develop chemical diversity and
further evaluate the SAR.
Figure 2
Modifications based on the 4-chromanone scaffold.
Modifications based on the 4-chromanone scaffold.As shown in Scheme 3, a series of 2-alkyl-4-chromanone derivatives 3a–f were synthesized by reacting 2,4-dihydroxyacetophenone
(1d) with an appropriate aliphatic aldehyde in the presence
of pyrrolidine in ethanol. This reaction was performed in a sealed
pressure tube at 150 °C for 1 h, obtaining yields over 30%. Furthermore,
the reaction time was shortened significantly. In contrast, a lower
yield (12%) was afforded despite a longer reaction time (72 h) for
the synthesis of 3b under the conditions (ethanol, 60
°C, pyrrolidine). To optimize the reaction conditions, a panel
of different amine bases was screened. We noted that under the same
conditions, no product was detected using DIPEA and only traces of
product were formed when using morpholine as a base monitored by HPLC,
respectively. To investigate the potential effects of the 4-carbonyl
group as a hydrogen bond acceptor, the 4-chromanones 3a–d were further reduced by NaBH4 in
methanol to provide 4-chromanol derivatives 4a–d in moderate to good yields (64–81%).[54]
Scheme 3
Synthesis of 2-Alkylated 4-Chromanones and Derivatives
Reagents and conditions: (i) pyrrolidine,
EtOH, 150 °C, pressure tube, 1 h; (ii) NaBH4, MeOH,
rt, 24 h.
Synthesis of 2-Alkylated 4-Chromanones and Derivatives
Reagents and conditions: (i) pyrrolidine,
EtOH, 150 °C, pressure tube, 1 h; (ii) NaBH4, MeOH,
rt, 24 h.Accordingly, when the bicyclic (−)-myrtenal
(1f) was used as the substrate under these reaction conditions
(Scheme 3, step i), no product was obtained.
Subsequently, a lower temperature (75 °C) was applied (Scheme 4) to optimize the reaction conditions. The corresponding
chalcone intermediate 3g was obtained in low yield (9.5%).
To improve the reaction yield, 1d was first regioselectively
protected by reacting with MOMCl in the presence of DIPEA to provide
the MOM-protected acetophenone 1e in 86% yield. Again,
when the reaction was performed at 150 °C, only traces of 3h were detected by HPLC. In contrast, a much higher yield
(48%) was obtained when the reaction temperature was reduced to 75
°C. The MOM-protected 3h can be further cyclized
in ethanol in the presence of sodium acetate to yield 4-chromanone 3i as a mixture of two diastereomers.[55] Final deprotection of the O-MOM group was performed
in methanol using concentrated HCl at room temperature to give 3j in 90% yield.
Scheme 4
Reactions of Acetophenone and (−)-Myrtenal
Reagents and conditions: (i) DIPEA,
MOMCl, 0 °C, 1 h; (ii) pyrrolidine, EtOH, 75 °C, pressure
tube, 1 h; (iii) NaOAc, EtOH, reflux, 24 h; (iv) concentrated HCl,
MeOH, rt, overnight.
Reactions of Acetophenone and (−)-Myrtenal
Reagents and conditions: (i) DIPEA,
MOMCl, 0 °C, 1 h; (ii) pyrrolidine, EtOH, 75 °C, pressure
tube, 1 h; (iii) NaOAc, EtOH, reflux, 24 h; (iv) concentrated HCl,
MeOH, rt, overnight.Next, on the basis of
the promising antibacterial activity of 3f, a focused
set of 4-chromanone analogues (Scheme 5) were subsequently designed
and synthesized to investigate the antibacterial effect of the phenol
free hydroxy group at different positions. Compound 3f was resynthesized from the MOM-protected 1e by using
diethylamine (DEA) as a base in an overall 58% yield following O-MOM deprotection. Accordingly, 3k,l with the 5- or 6-hydroxy group were also synthesized using appropriate
MOM-protected 1g,h in moderate yields (58–75%).
Scheme 5
Synthesis of 4-Chromanone Analogues 3f, 3k, and 3l
Reagents and conditions:
(i) DEA, EtOH, 150 °C, pressure tube, 1 h; (ii) concentrated
HCl, MeOH, rt, overnight.
Synthesis of 4-Chromanone Analogues 3f, 3k, and 3l
Reagents and conditions:
(i) DEA, EtOH, 150 °C, pressure tube, 1 h; (ii) concentrated
HCl, MeOH, rt, overnight.To further expand
the SAR and evaluate the influence of the 5-hydroxy group, an array
of 5,7-dihydroxy-4-chromanones were synthesized. Accordingly, 2,4,6-trihydroxyacetophenone
(1j) was used to prepare 4-chromanone derivatives 5 (Scheme 6). Unfortunately, no product
was obtained under the reaction conditions of pyrrolidine in ethanol
at 150 °C in a pressure tube. Therefore, the bis-MOM-protected
acetophenone 1k was next prepared in 79% yield.[56] The corresponding MOM-protected 4-chromanones 5 were obtained by reacting 1k with an appropriate
aldehyde in the presence of DEA in ethanol in a pressure tube at 150
°C in moderate yields (55–70%). Subsequent removal of
the MOM groups using concentrated HCl in methanol at room temperature
afforded the corresponding 4-chromanones in 75–94% yields.
Next, to evaluate the role of the carbonyl group at the 4-position
and further explore the chemical diversity of the 4-chromanone scaffold,
a panel of 4-oximinochromanes 6a–f were produced by reacting the corresponding 5 with
an appropriate hydroxylamine in ethanol in the presence of pyridine
in high yields (80–90%). Notably, the reaction time in this
series differed from 26 to 72 h, and the electron donating group such
as MeO in 6b greatly facilitated the reaction and reduced
the reaction time (26 h).
Scheme 6
Synthesis of 2-Substituted 4-Chromanones
and Oxime Derivatives
Reagents and conditions:
(i) DIPEA, MOMCl, 0 °C, 1 h, 79%; (ii) DEA, EtOH, 150 °C,
pressure tube, 1 h; (iii) concentrated HCl, MeOH, rt, 24 h; (iv) pyridine,
EtOH, rt, 26–72 h.
Without isolating the MOM-protected intermediate.
Synthesis of 2-Substituted 4-Chromanones
and Oxime Derivatives
Reagents and conditions:
(i) DIPEA, MOMCl, 0 °C, 1 h, 79%; (ii) DEA, EtOH, 150 °C,
pressure tube, 1 h; (iii) concentrated HCl, MeOH, rt, 24 h; (iv) pyridine,
EtOH, rt, 26–72 h.Without isolating the MOM-protected intermediate.As illustrated in Scheme 7, to synthesize
the bicyclic compound 5n bearing the myrtenal motif,
the reaction time needed to be extended to 4 h under the reaction
conditions, and the chalcone 5m was obtained in overall
two-step 34% yield after O-MOM deprotection. Subsequent
intramolecular conjugate addition of the phenol on the α,β-unsaturated
system was performed under microwave irradiation in the presence of
catalytic amounts of concentrated HCl to give 5n in 72%
yield as a mixture of two diastereomers.[49] We also noted that the reaction was completed in 0.5 h, which was
a great improvement in comparison to the condition of refluxing with
sodium acetate in ethanol in Scheme 4.
Scheme 7
Synthesis of Compounds 5m and 5n
Reagents and conditions: (i) DEA,
EtOH, 150 °C, pressure tube, 4 h; (ii) HCl, MeOH, rt, 16 h; (iii)
HCl, EtOH, microwave irradiation, 150 °C, 0.5 h.
Synthesis of Compounds 5m and 5n
Reagents and conditions: (i) DEA,
EtOH, 150 °C, pressure tube, 4 h; (ii) HCl, MeOH, rt, 16 h; (iii)
HCl, EtOH, microwave irradiation, 150 °C, 0.5 h.Next, 2-spiro-4-chromanones (7a–c) were synthesized by reacting 1e with an appropriate
cycloketone in a pressure tube at 150 °C for 2–16 h in
the presence of pyrrolidine (Scheme 8). The
following O-MOM deprotection was performed in one
pot with excess concentrated HCl. The spiro compounds 7a–c were obtained in moderate to high yields,
with 7c giving the lowest yield (53%) probably due to
the steric hindrance of the seven-membered ring.
Scheme 8
Synthesis of 2-Spiro-4-chromanones 7a–c
Reagents
and conditions: (i) pyrrolidine, EtOH, 150 °C, pressure tube,
2–16 h; (ii) HCl, rt, overnight.
Synthesis of 2-Spiro-4-chromanones 7a–c
Reagents
and conditions: (i) pyrrolidine, EtOH, 150 °C, pressure tube,
2–16 h; (ii) HCl, rt, overnight.
Synthesis of
2-Substituted Aromatic Chalcone and Flavanone Derivatives
To systematically investigate the SAR of the chalcone and 4-chromanone
scaffolds on antibacterial activities, diverse aromatic aldehydes
were introduced to synthesize a series of 2-aryl chalcone and flavanone
derivatives (Scheme 9). Accordingly, the Claisen–Schmidt
aldol condensations were performed at room temperature with the addition
of 60% KOH aqueous solution to the mixture of appropriate MOM-protected
acetophenone and aldehyde in methanol.[55] Subsequent removal of the MOM-protecting group with concentrated
HCl afforded the chalcone derivatives 8a–h. Chalcone 8f was synthesized as a comparison
under the same conditions to verify the potential effect of 2′-hydroxy
group on antibacterial activity. Additionally, in comparison to 8a with the ortho-substituted allyloxy group, regioisomers 8g and 8h (with the allyloxy group at the meta-
and para-position, respectively) were next synthesized to investigate
the influence of the substitution at the R2 position in
the scaffold. Subsequent intramolecular conjugate additions of the
phenol on the α,β-unsaturated system of 8a–e were carried out under microwave irradiation
in the presence of catalytic amounts of concentrated HCl to yield
corresponding flavanones 8i–m in
50–75% yields.
Scheme 9
Synthesis of 2-Substituted Aromatic Chalcones
and Flavanones
Reagents and conditions: (i) 60%
KOH aq, MeOH, rt, 60 h; (ii) concentrated HCl, rt, overnight; (iii)
microwave, HCl, EtOH, 150 °C, 1.5 h.
Without 2′-OH function for 8f.
Synthesis of 2-Substituted Aromatic Chalcones
and Flavanones
Reagents and conditions: (i) 60%
KOHaq, MeOH, rt, 60 h; (ii) concentrated HCl, rt, overnight; (iii)
microwave, HCl, EtOH, 150 °C, 1.5 h.Without 2′-OH function for 8f.All the synthesized compounds were characterized
by 1H and 13C NMR spectroscopy and high-resolution
mass spectrometry (HRMS), and purity was analyzed by reverse phase
HPLC. The structures of 3g and 8a were confirmed
by X-ray crystallography, and their ORTEP drawings are shown in Figure
S1 (Supporting Information).All
the synthesized compounds were evaluated against Mycobacterium
tuberculosis (H37Rv) and a wide set of clinically relevant
Gram-positive and -negative bacterial pathogens including Enterococcus faecalis (ATCC 33186), Staphylococcus
aureus (ATCC 29213 and NRS 70), Escherichia coli (K12 and ΔtolC), Klebsiella pneumoniae (ATCC 33495), and Pseudomonas aeruginosa (PAO1).
Their antitubercular and antibacterial activities are summarized in
Tables 1–3.
Table 1
Antibacterial Activities (MIC, μg/mL) of Olympicin A and Derivativesa
No test compounds
were active (>200 μg/mL) against other Gram-negative bacteria
including K. pneumonia (ATCC 33495) and P.
aeruginosa (PAO1).
Positive controls isoniazid and rifampin inhibited M. tuberculosis at 0.03 and 0.05 μg/mL, respectively.
nd = not determined.
Table 3
Antibacterial
Activities (MIC, μg/mL) of Chalcones and Derivativesa
No test
compounds were active (>200 μg/mL) against other Gram-negative
bacteria including K. pneumonia (ATCC 33495) and P. aeruginosa (PAO1). The MIC values of control antibiotics
used in this study are shown in Table 1.
Olympicin A Series
In the olympicin A series, olympicin
A (2a) and analogues (2b–f) showed weak antitubercular activity with MICs of 100–200
μg/mL (Table 1). The observed weak antituberculosis activity may be attributed
to the general polar nature of this chemical series and decreased
membrane penetration. In contrast, the olympicin derivatives with
geranyloxy (2a and 2b), n-hexyloxy (2d), and n-octyloxy (2e) groups showed good to potent anti-Gram-positive activity
against E. faecalis and S. aureus strains (MIC = 0.78–3.13 μg/mL). However, the less
lipophilic olympicin derivatives 2c and 2f with a shorter allyloxy chain exhibited about 8- to 16-fold decrease
of antibacterial activity (MIC = 6.25–12.5 μg/mL). In
terms of stereochemistry effect, the racemic olympicin A (2b) and allyloxy derivative (2c) showed largely the same
antituberculosis and anti-Gram-positive activity compared to their
corresponding chiral S-isomers 2a and 2f, respectively. Notably, the anti S. aureus activity (1.56 μg/mL) of our synthetic sample (2a) of olympicin A is consistent with the previously reported anti S. aureus activity (0.5–1 μg/mL) of natural
olympicin A.[44] In addition, none of these
olympicin analogues were active against Gram-negative microorganisms
except for the reengineered E. coli strain (ΔtolC) with deficient efflux activity.No test compounds
were active (>200 μg/mL) against other Gram-negative bacteria
including K. pneumonia (ATCC 33495) and P.
aeruginosa (PAO1).Positive controls isoniazid and rifampin inhibited M. tuberculosis at 0.03 and 0.05 μg/mL, respectively.nd = not determined.
4-Chromanone and Chalcone Series. Antituberculosis
Activity
In the 4-chromanone and chalcone chemical series
(Tables 2 and 3), the
majority of compounds exhibited weak antituberculosis activity. The
2-propyl-4-chromanol 4a showed the most potent activity
in the entire series with a MIC of 12.5 μg/mL. The reduced
4-chromanol variants 4a and 4c (4-OH, 12.5
and 25 μg/mL, respectively) showed more potent antituberculosis
activity than their corresponding 4-chromanones 3a (200
μg/mL) and 3c (50 μg/mL). In addition, the
4-oximinochromane 6a (=NOH, 100 μg/mL) displayed
greater potency than 6b (=NOMe, 200 μg/mL)
and 6c (=NOBn, >200 μg/mL). Taken together,
these results indicate that the small, polar, and hydrophilic groups
(e.g., −OH and =NOH) are more favorable at the 4-position
of the flavonoid scaffold for antituberculosis activity, suggesting
these polar groups may function as hydrogen bond donors when interacting
with potential biological cellular target.
Table 2
Antibacterial Activities (MIC, μg/mL) of 4-Chromanone
Derivativesa
No test compounds were active (>200 μg/mL)
against other Gram-negative bacteria including K. pneumonia (ATCC 33495) and P. aeruginosa (PAO1). The MIC
values of control antibiotics used in this study are shown in Table 1.
Different lengths
of the 2-alkyl side chains in the scaffold displayed an important
relationship with antituberculosis activity as well. Compared to 3a (three-carbon, 200 μg/mL), 3b (six-carbon,
50 μg/mL), and 3c (seven-carbon, 50 μg/mL),
compound 3d with a nine-carbon linear chain was inactive,
suggesting the nine-carbon alkyl group is too long and bulky. To investigate
the impact of the substituent at the 5-position in the scaffold, a
hydrogen bond donor and/or acceptor (hydroxy group) was introduced.
These data showed that the compounds bearing 5,7-dihydroxy functionalities
(5b, 5d, and 5f) exhibited
similar MIC values to the sole 7-hydroxychromanones 3a–c. However, 5h (5,7-di-OH, nine-carbon
chain, 100 μg/mL) was more potent than the corresponding 3d (7-OH, nine-carbon chain, >200 μg/mL). In addition,
the position of the sole hydroxy group in the scaffold also played
a notable role in their antituberculosis activity by comparing 3f (7-OH, 100 μg/mL) to 3k (6-OH, 200 μg/mL)
and 3l (5-OH, >200 μg/mL), with 3f bearing the 7-hydroxy function being the most potent.In general,
2-aryl substituted compounds together with 2-spiro derivatives possessing
a cyclic ring system were less active against M. tuberculosis than 2-alkylated derivatives in the entire series. Moreover, the
MOM protected derivatives exhibited comparable or more potent antitubercular
activity than their corresponding free phenol parent molecules. The
SAR analysis above demonstrates that the 2-alkyl hydrophobic substituents
as well as the small and polar functionalities at the 4-position (e.g.,
hydrogen bond donor groups OH and =NOH) play important roles
in antituberculosis activities in the 4-chromanone scaffold.No test compounds were active (>200 μg/mL)
against other Gram-negative bacteria including K. pneumonia (ATCC 33495) and P. aeruginosa (PAO1). The MIC
values of control antibiotics used in this study are shown in Table 1.
General Antibacterial
Spectra
In addition to antituberculosis evaluation, antimicrobial
assessment against representative clinical pathogens revealed that
the majority of compounds from these series exhibited notable anti-Gram-positive
bacteria activities including against E. faecalis and S. aureus (MSSA and MRSA) and poor activity
against Gram-negative bacteria including E. coli, K. pneumoniae, and P. aeruginosa.Compared with the 4-chromanone flavonoid series, the chalcone series
generally exhibited more potent anti-Gram-positive activity than their
corresponding cyclized derivatives. The 2′,4′-di-OH
chalcone compound 8a having an appended 2-allyloxy group
exhibited the best activity (MIC of 0.39–6.25 μg/mL)
against MSSA and MRSA, and the o-hydroxy group appeared
to have a beneficial effect on anti-Gram-positive bacterial activity
by comparing 8a (2′,4′-di-OH, 2-allyloxy,
0.39–12.5 μg/mL) to 8f (4′-OH, 2-allyloxy,
6.25–100 μg/mL) (Table 3). Notably,
this observation regarding the importance of the o-hydroxy group in the chalcone scaffold is also in agreement with
that found in the 4-chromanone flavonoid series, whereas the corresponding
hydroxy group at the 5-position of the 4-chromanone scaffold enhanced
the antibacterial activity. However, the 2′,4′,6′-tri-OH
chalcone compounds possessing an additional 6′-hydroxy group 5m (25 to >200 μg/mL) and 8e (25 to
>200 μg/mL) showed significantly decreased activities against
Gram-positive microorganisms compared to the 2′,4′-di-OH
chalcones 3g (1.56–3.13 μg/mL) and 8a (0.39–12.5 μg/mL), respectively. The notable
decreased activity for trihydroxy compounds is likely due to increased
hydrophilicity and polarity properties and thus decreased bacterial
membrane penetration. Taken together, these data suggest that two
free phenol hydroxy groups on the 4-chromanone and chalcone scaffolds
are optimal for Gram-positive antibacterial activity.Furthermore,
in the chalcone series (Table 3), the lipophilic O-alkyl substituent
at the 2-position of aromatic chalcones also had a great impact on
antibacterial activities, since the chalcones bearing diverse side
chains such as the allyloxy (8a), n-hexyloxy
(8b), and n-octyloxy (8c) exhibited notable differences in their activities; compound 8b bearing the n-hexyloxy group showed optimal
antibacterial activity against E. faecalis, MSSA,
and MRSA (1.56, 3.13, and 0.78 μg/mL, respectively). Compound 8a bearing the 2-allyloxy function demonstrated 2-fold more
potent activity against MRSA and an 8-fold reduction in activity against E. faecalis than 8b.In the flavonoid
series, the length of 2-alkyl substitutions in the 4-chromanone scaffold
plays an important role in determining antibacterial activities. The
5,7-dihydroxy-4-chromanones with long aliphatic alkyl chains 5d, 5f, 5h, and 5j (six
to nine-carbon chain, 3.13–6.25 μg/mL) showed better
activity against MRSA than the shorter chain derivative 5b (three-carbon chain, 100 μg/mL), and notably, the 2-(2,6-dimethyl-5-heptenyl)
substituted 5j with a branched and unsaturated alkyl
chain displayed the best potency among these five 2-alkylated derivatives
against three Gram-positive bacteria tested (E. faecalis, MSSA, and MRSA; 6.25, 3.13, and 3.13 μg/mL, respectively).
These results suggest that the branched unsaturated substitution in 5j may play an important role in enhancing interactions and
binding affinity with cellular biological target because of its favorable
lipophilicity and high conformational flexibility, as previously noted
for the prenylated derivatives.[57,58] We also observed that
the introduction of an additional 5-OH group to the 7-hydroxy-4-chromanones 5b (100 μg/mL), 5d (6.25 μg/mL), 5f (3.13 μg/mL), 5h (3.13 μg/mL),
and 5n (6.25 μg/mL) significantly improved antibacterial
activity against MRSA, compared to their corresponding 7-OH-4-chromanones 3a–d (>200 μg/mL) and 3j (200 μg/mL) except that 5j maintained anti-MRSA
activity (3.13 μg/mL) compared to 3f. These data
demonstrate the importance of the 5-OH group in the 7-OH-4-chromanone
scaffold for antibacterial activity.In addition, the reduced
variants 4-chromanols 4a–d (in particular
2-n-heptyl-7-OH-4-chromanol 4c, 12.5–25
μg/mL; and 2-n-nonyl-7-OH-4-chromanol 4d, 25–50 μg/mL) also displayed improved antibacterial
activities compared to their corresponding 4-chromanones 3a–d, which were not active against Gram-positive
bacteria tested. In contrast, the 4-oximinochromanes (6a and 6d-f: =NOH at the 4-position)
showed more potent activity against E. faecalis and
MSSA and comparable activity against MRSA than their corresponding
4-chromanones (5h, 5b, 5d,
and 5f), suggesting the free oxime =NOH functionality
is preferred compared to the carbonyl group. Furthermore, by comparison
of 6a with the free 4-oxime functionality (=NOH,
12.5 μg/mL), compounds 6b (=NOMe, 25–100
μg/mL) and 6c (=NOBn, >200 μg/mL)
showed decreased antibacterial activity against S. aureus. This observation is also consistent with the trend toward the antituberculosis
activity found in this series, indicating that the small, polar, and
hydrogen bond donor functionalities (e.g., −OH and =NOH
groups) at the 4-position may be more favorable for antibacterial
properties, together with the results based upon 4-chromanols (4a–d).To further evaluate the importance
of the 5-, 6-, and 7-OH functionality, a set of 4-chromanone derivatives 3f (7-OH), 3k (6-OH), and 3l (5-OH)
were subsequently synthesized. Biological evaluation revealed
that the 6- or 7-hydroxy group also proved to be a determining factor
for antibacterial activities, since 3f (7-OH, 3.13–12.5
μg/mL) and 3k (6-OH, 6.25–12.5 μg/mL)
demonstrated almost equally potent antibacterial activities against
Gram-positive bacteria tested. However, their regioisomer 3l with the 5-OH substitution was not active. The lack of antibacterial
activity of 3l may be due to the presence of intramolecular
hydrogen bonding between the carbonyl group at the 4-position and
the hydroxy group at the 5-position. It should be noted that all the
MOM protected derivatives (3h, 3i, 5a, 5c, 5e, 5g, and 5i) completely lost anti-Gram-positive bacterial activities
against E. faecalis and S. aureus, demonstrating the importance of the free phenol hydroxy functionality
and its weakly acidic nature in the scaffold. This observation is
consistent with our previous report.[43] Additionally,
among the chemical series bearing a cyclic/bicyclic ring system at
the 2-position (5k, 2-cyclopentyl; 5l, 2-cyclohexyl; 5n, 2-myrtenyl; and the 2-spiro compounds 7a–c), compound 5n bearing the myrtenyl motif showed
relatively good antibacterial activity (6.25–12.5 μg/mL) against Gram-positive bacteria tested.
In contrast, the 7-OH and 2-myrtenyl substituted flavonoid 3j was largely inactive. Interestingly, its corresponding ring-opened
chalcone derivative 3g demonstrated very good anti-Gram-positive
activity (1.56–3.13 μg/mL).No test
compounds were active (>200 μg/mL) against other Gram-negative
bacteria including K. pneumonia (ATCC 33495) and P. aeruginosa (PAO1). The MIC values of control antibiotics
used in this study are shown in Table 1.A detailed SAR of the 4-chromanone
and chalcone series is summarized in Figure 3.
Figure 3
General SAR of 4-chromanone and chalcone derivatives.
General SAR of 4-chromanone and chalcone derivatives.
Solubility and Cytotoxicity Determination
Cytotoxicity against mammalian (Vero epithelial) cells and solubility
in Dulbecco’s modified Eagle medium (DMEM) supplemented with
10% fetal bovine serum (FBS) were evaluated for a selected panel of
4-chromanone and chalcone lead compounds, and the results are shown
in Table 4. Overall, all the tested compounds
had good solubility (≥100 μg/mL) in the DMEM/FBS medium
used in the cytotoxicity assay except for the 4-oxime derivative 6a (37.5 ± 17.7 μg/mL). The selectivity indices
(SI) for the compounds were calculated as the ratio of the IC50 value of cytotoxicity against Vero monkey kidney cell line
and the MIC value against tested MRSA. Notably, 4-chromanone derivatives 5f (SI = 10.5) and 8j (SI = 15.7) and the chalcone
derivative 8a (SI = 54.4) possessed a more favorable
selectivity index (SI > 10). On the basis of these promising antibacterial
lead structures, advanced medicinal chemistry will be applied to produce
compounds with improved potency and decreased cytotoxicity.
Table 4
Cytotoxicity and Solubility Profiles of Selected Lead
Compoundsa
compd
molecular weight (g/mol)
lipophilicity (cLogP)b
solubility (μg/mL)
cytotoxicity IC50 (μg/mL)c
MIC against MRSA (μg/mL)
selectivity index (SI)d
3f
288.2
3.77
>200
28.8 ± 1.8
3.13
9.2
3g
284.1
3.19
>200
6.9 ± 0.3
1.56
4.4
5f
278.2
3.20
150.0
33.0 ± 2.1
3.13
10.5
5h
306.2
4.03
>200
8.6 ± 2.7
3.13
2.8
5j
304.2
3.38
>200
27.0 ± 2.8
3.13
8.6
6a
321.2
4.42
37.5 ± 17.7
10.1 ± 1.9
12.5
0.8
6f
293.2
3.59
>200
9.6 ± 0.1
3.13
3.1
8a
296.1
3.37
>200
21.2 ± 5.4
0.39
54.4
8b
340.2
4.76
>200
7.2 ± 0.4
0.78
9.2
8d
323.2
3.82
100.0
16.7 ± 0.1
3.13
5.3
8j
340.2
4.36
>200
24.5 ± 2.1
1.56
15.7
8k
368.2
5.19
>200
17.0 ± 0.0
3.13
5.4
thioridazine
nde
nd
nd
5.3 ± 1.3
nd
nd
verapamil
nd
nd
nd
57.3 ± 3.2
nd
nd
DMSOf
nd
nd
nd
177.5 ± 10.6
nd
nd
DMEM/FBS:
Dulbecco’s modified Eagle medium (DMEM) supplemented with 10%
fetal bovine serum (FBS).
The cLogP values of compounds were calculated using ChemBioOffice
Ultra, version 12.0, from CambridgeSoft Corporation.
Cytotoxicity IC50, concentration
that reduces viability of Vero kidney cells by 50%.
Selectivity index = (cytotoxic IC50)/(MIC against MRSA).
nd = not determined.
Carrier effect.
DMEM/FBS:
Dulbecco’s modified Eagle medium (DMEM) supplemented with 10%
fetal bovine serum (FBS).The cLogP values of compounds were calculated using ChemBioOffice
Ultra, version 12.0, from CambridgeSoft Corporation.Cytotoxicity IC50, concentration
that reduces viability of Vero kidney cells by 50%.Selectivity index = (cytotoxic IC50)/(MIC against MRSA).nd = not determined.Carrier effect.
Time Kill
Experiments and Mutation Selection
The bactericidal activities
of compounds 3g, 5j, 8a, and 8d were examined against S. aureus Newman
(Figure 4). The most effective compound was
the chalcone derivative 3g, which killed more than 6
log of cells in just 2 h at 4× its MIC, but at its MIC (1.56
μg/mL against S. aureus Newman) up to 24 h
was required to achieve a 3 log reduction in cells. Compound 3g thus exhibited concentration-dependent killing. Compound 8a (MIC = 6.25 μg/mL) was also rapidly bactericidal
at 4× its MIC and achieved a 6 log reduction in 6 h. Interestingly,
compound 8d (6.25 μg/mL) was entirely bacteriostatic
and failed to kill more than 3 log of culture at 4 × its MIC,
even up to 24 h of exposure. At concentrations between 1× and
4× their MICs, spontaneous mutants of S. aureus Newman could not be selected with 8a, 3g, and 5j, but mutants arose to 8d at a
frequency of 10–10. The controls mupirocin and vancomycin
were either bacteriostatic or slowly bactericidal over a 24 h period,
and mutants could only be selected with mupirocin at frequencies of
10–7–10–9.
Figure 4
Time kill studies against S. aureus Newman exposed to 4× MIC of compounds. Each
point represents the average of two biological replicates.
Time kill studies against S. aureus Newman exposed to 4× MIC of compounds. Each
point represents the average of two biological replicates.
Effects on Macromolecular Synthesis and Membrane
Potential
Several key biosynthetic processes were simultaneously
inhibited in S. aureus Newman exposed to the compounds 2a, 5j, 8a, 3g, and 8d (Figure 5). These effects are consistent
with the bacterial membrane being the primary target site of action,
resulting in multiple nonspecific cellular effects. Surprisingly,
the bacteriostatic compound 8d displayed the same time-dependent
effects on macromolecular synthesis as the bactericidal compounds.
To determine if the compounds dissipated the membrane potential of S. aureus, the fluorescent probe DiOC2(3) was used. Compounds 2a, 5j, 3g, and 8d all
dissipated the membrane potential of S. aureus Newman
in a concentration-dependent manner. Maximum dissipation occurred
at 4× their MICs and was similar to the control CCCP (carbonyl
cyanide m-chlorophenylhydrazone) (Figure 6); as expected, vancomycin (at 4× MIC) failed
to affect the membrane potential in S. aureus.
Figure 5
Effects of 2a, 5j, 3g, 8a, and 8d and indicated positive controls at 4× their MICs on
macromolecular synthesis in S. aureus Newman. The
standard error of the mean (SEM) is shown for three biological replicates.
ERY = erythromycin (MIC = 0.78 μg/mL); RMP = rifampicin (0.12
μg/mL); CIP = ciprofloxacin (0.25 μg/mL).
Figure 6
Dissipation of the staphylococcal membrane potential by 2a, 5j, 3g, and 8d.
A representative of three biological replicates is shown. Vancomycin
(MIC = 0.8 μg/mL) and CCCP (MIC = 6.25 μg/mL) were used
as negative and positive controls.
Effects of 2a, 5j, 3g, 8a, and 8d and indicated positive controls at 4× their MICs on
macromolecular synthesis in S. aureus Newman. The
standard error of the mean (SEM) is shown for three biological replicates.
ERY = erythromycin (MIC = 0.78 μg/mL); RMP = rifampicin (0.12
μg/mL); CIP = ciprofloxacin (0.25 μg/mL).Dissipation of the staphylococcal membrane potential by 2a, 5j, 3g, and 8d.
A representative of three biological replicates is shown. Vancomycin
(MIC = 0.8 μg/mL) and CCCP (MIC = 6.25 μg/mL) were used
as negative and positive controls.
Inhibition of Bacterial Topoisomerase IV and DNA Gyrase
Clinically, DNA gyrase and topo IV are validated and attractive antibacterial
targets for fluoroquinolone and novobiocin antibiotics. However, because
of the emergence of target-based bacterial resistance with fluoroquinolone
class and safety concerns of novobiocin, novel DNA gyrase and topoisomerase
inhibitors are urgently needed for the treatment of pathogenic and
resistant bacterial infections.[59] Thus,
development of new chemotype bacterial topo inhibitors has attracted
great interest in the scientific community, and recent examples include
bisbenzimidazoles,[60] anziaic acid, and
its analogues[61,62] as bacterial topo IA inhibitors,
and pyridylureas[63] and pyrrolamides[59] as topo II inhibitors. Previously, flavonoids
have been identified as bacterial topoisomerase inhibitors,[64−66] and we next tested to see if these promising antibacterial compounds
from olympicin A, 4-chromanone, and chalcone series inhibited E. coli topo I, topo IV, and DNA gyrase. The results are
shown in Table 5 and Figure S2 (Supporting Information). The E. coli topo I assay (relaxation of negatively supercoiled plasmid DNA)
was performed at both 0.5 and 5 mM magnesium chloride concentrations,
and no significant inhibition was observed against topo I at 0.5 mM
compound concentration. The assay against E. coli gyrase (supercoiling of relaxed plasmid DNA) and E. coli topo IV (decatenation of catenated kinetoplast DNA) was subsequently
performed. Again for DNA gyrase, inhibition was not observed or was
weak with 8a, 3g, and 2a (MIC
values of 0.39–6.25 μg/mL against S. aureus) exhibiting IC50 values of >0.25 mM. However, the E. coli topo IV showed more significant sensitivity toward 2a and 2e (MIC values of 0.78–1.56 μg/mL
against E. faecalis and S. aureus) with IC50 values of 30–60 μM (Table 5 and Figure S2, Supporting Information). These results showed that the olympicin A (2a) and
its analogue 2e may serve as a promising and novel scaffold
for topo IV inhibitors. Interestingly, olympicin A was also recently
reported as a moderate ATP-dependent Mycobacterium tuberculosis MurE ligase inhibitor with an IC50 value of 75 μM.[45] Studies to determine if the olympicin A analogues 2c–f inhibit the MurE pathway in M. tuberculosis remain to be performed. Finally, the antibacterial
mechanism of the 4-chromanone flavanone compounds 8k and 8j (MIC values of 1.56–12.5 μg/mL against S. aureus) may not be likely to involve topoisomerase inhibition,
as they were inactive in these topo enzyme inhibition assays (Table 5). Collectively, no correlations between whole-cell-based
activity and topoisomerase inhibition were observed for selected compounds,
suggesting that the membrane is likely the primary biological target
responsible for antibacterial activity and topo IV is a secondary
or alternative target. Further mechanistic studies are warranted to
define the exact mechanism of antibacterial action of these chemically
modified flavonoid and polyphenol compounds.
Table 5
E. coli Topoisomerases and DNA Gyrase Inhibition of Selected
Analogues
compd
topo I IC50 (μM)
DNA gyrase IC50 (μM)
topo IV IC50 (μM)
2a
>500
500–1000
30–60
2e
>500
>1000
30–60
3g
>500
250–500
60–120
5f
>500
>1000
120–250
8a
>500
500–1000
250–500
8b
>500
>1000
60–120
8d
>500
>1000
500–1000
8j
>500
>1000
>1000
8k
>500
>1000
>1000
known inhibitors
14–19a
0.2,b 100c
2.1,b 806c
IC50 of known inhibitor for assay: anziaic
acid[61] for Topo I.
IC50 of known inhibitor for assay: ciprofloxacin[67] for DNA gyrase and topo IV.
IC50 of known inhibitor for assay: nalidixic
acid[68] for DNA gyrase and topo IV.
IC50 of known inhibitor for assay: anziaic
acid[61] for Topo I.IC50 of known inhibitor for assay: ciprofloxacin[67] for DNA gyrase and topo IV.IC50 of known inhibitor for assay: nalidixic
acid[68] for DNA gyrase and topo IV.
Conclusions
In
summary, 58 olympicin A, 4-chromanone, and chalcone derivatives containing
various functionalities were synthesized and evaluated against Mycobacterium tuberculosis and a panel of clinically relevant
Gram-positive and -negative bacterial pathogens. Bacterial evaluation
showed that this class of compounds generally exhibited good activities
against Gram-positive bacteria tested. Systematic SAR study revealed
that the phenol hydroxy groups at the 5- and 7-position of the 4-chromanone
scaffold were essential for antibacterial activities. Additionally,
the hydrogen bond donor/acceptor functionality at the 4-position together
with the lipophilic 2-alkyl moiety in the scaffold also played important
roles in antibacterial activities. The flavanone derivatives bearing
the lipophilic substituent on the 2-phenyl ring showed good antibacterial
properties as well. In the chalcone chemical series, both hydroxy groups
at 2′- and 4′-position, as well as the bicyclic myrtenyl
motif and the 2-alkyloxy substitution on the aromatic ring, favored
anti-Gram-positive bacterial activities. The selected compounds generally
possessed favorable solubility, and 5f, 8a, and 8j had more desirable selectivity indices ranging
from 10.5 to 54.4. In addition, compounds 2a, 5j, 3g, and 8d were found to disrupt bacterial
membrane potential and have secondary inhibitory effect on macromolecular
biosynthesis of DNA, RNA, and protein. Further evaluation of selected
compounds against bacterial topoisomerases and DNA gyrase revealed
that 2a and 2e inhibited topoisomerase IV
(IC50 = 30–60 μM). Taken together, the antibacterial
agents identified from this study provide chemically modified flavonoid
phytochemicals as promising antibacterial leads for further medicinal
chemistry optimization in an effort to identify advanced experimental
candidates with antimicrobial therapeutic potential.
Experimental Section
Chemical Synthesis. General Procedures
Solvents and reagents were supplied from Aldrich, Acros, or Fisher
and used without further purification. NMR spectra were recorded on
a Bruker AM-400 (400 MHz) spectrometer. High-resolution mass spectra
were obtained on an Agilent 6530 Accurate Mass Q-TOF LC/MS instrument.
Reactions in pressure tube were carried out using a Q-Tube reactor
from Q Labtech. Microwave reactions were conducted using a Biotage
Initiator reactor. All reactions were monitored by either TLC or HPLC.
Compounds were purified by flash chromatography on silica gel on a
Biotage Isolera One system. The purity of compounds was determined
by analytical HPLC (Shimadzu LC-20A series) using a Gemini, 3 μm,
C18, 110 Å column (50 mm × 4.6 mm, Phenomenex) and flow
rate of 1 mL/min. Gradient conditions were the following: solvent
A (0.1% trifluoroacetic acid in water) and solvent B (acetonitrile),
0–2.0 min 100% A, 2.0–7.0 min 0–100% B (linear
gradient), 7.0–8.0 min 100% B, UV detection at 254 and 220
nm. All the tested compounds were obtained with ≥95% purity
by HPLC. NMR standards used are as follows. 1H NMR: CDCl3, 7.26 ppm; CD3OD, 3.31 ppm; DMSO-d6, 2.50 ppm. 13C NMR: CDCl3, 77.16
ppm; CD3OD, 49.00 ppm; DMSO-d6, 39.52 ppm. MOM-protected acetophenones were synthesized as described
in the literature.[56,69,70]
To
a solution of 1a (150 mg, 0.715 mmol) in acetone (11
mL) were added p-toluenesulfonic chloride (408 mg,
2.14 mmol) and K2CO3 (845 mg, 6.15 mmol) successively.
The resulting mixture was stirred under reflux for 1 h, and then acetone
was removed under reduced pressure. The residue was diluted with water
and DCM, and the organic layer was washed with 1 M HCl aq, water,
brine and dried over Na2SO4. The solvent was
removed under reduced pressure and the crude material was adsorbed
onto silica gel and subjected to silica gel chromatography with hexane/EtOAc
(first 85/15, then 75/25) to give the product (350 mg, 0.52 mmol,
73%) as clear oil. 1H NMR (400 MHz, CDCl3):
δ (ppm) 7.69 (d, J = 8.3 Hz, 2H), 7.62 (d, J = 8.4 Hz, 4H), 7.36 (d, J = 8.2 Hz, 2H),
7.30 (d, J = 8.3 Hz, 4H), 7.0 (s, 2H), 2.64–2.59
(m, 1H), 2.44 (s, 9H), 1.56–1.50 (m, 1H), 1.24–1.14
(m, 1H), 0.90 (d, J = 7.0 Hz, 3H), 0.76 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm) 201.2, 149.8, 146.8, 146.5, 146.4, 131.7, 131.3,
130.2, 130.0, 128.5, 127.1, 114.3, 48.6, 24.5, 21.80, 21.79, 14.2,
11.2. HRMS calculated for C32H32O10S3 (M + H)+ 673.1230, found (M + H)+ 673.1229. HPLC purity: 98.6% (254 nm), tR = 8.17 min; 99.5% (220 nm), tR = 8.17
min.
To a suspension of 1a′ (1.12 g, 5.33 mmol) in DCM (11 mL) at 0 °C was added DIPEA
(2.78 mL, 15.9 mmol) carefully. After stirring for 10 min, MOMCl (1.21
mL, 15.9 mmol) was added to the solution dropwise. The resulting mixture
was stirred for 1 h. Afterward, the solution was poured into sat.
NH4Cl aq, and then water and DCM were added into the mixture.
The organic layer was washed with water, brine and dried over Na2SO4. The solvent was removed under reduced pressure
and the crude material was adsorbed onto silica gel and subjected
to silica gel chromatography with hexane/EtOAc (90/10) to give the
product (1.02 g, 3.42 mmol, 64%) as pale yellow solid. 1H NMR (400 MHz, CDCl3): δ (ppm) 13.74 (s, 1H), 6.27
(s, 2H), 5.24 (s, 2H), 5.16 (br s, 2H), 3.65–3.60 (m, 1H),
3.51 (s, 3H), 3.46 (s, 3H), 1.87–1.80 (m, 1H), 1.43–1.36
(m, 1H), 1.16 (d, J = 6.8 Hz, 3H), 0.91 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm) 210.3, 167.2, 163.2, 160.1, 106.7, 97.6, 94.9, 94.4,
94.1, 56.9, 56.6, 46.6, 27.1, 16.7, 12.2. HRMS calculated for C15H22O6 (M + H)+ 299.1489,
found (M + H)+ 299.1483. HPLC purity: 99.8% (254 nm), tR = 7.42 min; 99.8% (220 nm), tR = 7.42 min.
To a solution of 1b (410 mg, 0.61 mmol) in DMF (4 mL)
at 0 °C was added NaH (49 mg, 1.21 mmol) carefully. After stirring
for 30 min, geranyl bromide (198 mg, 0.91 mmol) in DMF (1 mL) was
added dropwise. The resulting mixture was stirred at room temperature
for 1 h. Afterward, the reaction was quenched with water and extracted
with EtOAc twice. The organic layer was washed with water, brine and
dried over Na2SO4. The solvent was removed under
reduced pressure, and the residue was dissolved in MeOH (10 mL), followed
by the careful addition of sodium methoxide (659 mg, 12.2 mmol). The
resulting mixture was stirred under reflux for 8 h, and then MeOH
was removed. The residue was diluted with 1 M HCl aq and extracted
with EtOAc twice. The organic layer was washed with water, brine and
dried over Na2SO4. The solvent was removed under
reduced pressure and the crude material was adsorbed onto silica gel
and subjected to silica gel chromatography with hexane/EtOAc to give
the product.
(S,E)-1-(2-((3,7-Dimethylocta-2,6-dien-1-yl)oxy)-4,6-dihydroxyphenyl)-2-methylbutan-1-one
(Olympicin A, 2a)
To a solution of 1c (200 mg, 0.67
mmol) in DMF (4 mL) at 0 °C was added NaH (54 mg, 1.34 mmol)
carefully. After stirring for 30 min, appropriate alkyl bromide (1
mmol) in DMF (1 mL) was added dropwise. The resulting mixture was
stirred at room temperature until the complete consumption of 1c (1–7 h). Afterward, the reaction was quenched with
water and extracted with EtOAc twice. The organic layer was washed
with water, brine and dried over Na2SO4. The
solvent was removed under reduced pressure, and the residue was dissolved
in MeOH (10 mL), followed by the addition of concentrated HCl (510
μL, 6 mmol) carefully. The resulting mixture was stirred at
room temperature overnight, and the solvent was removed under reduced
pressure. The crude material was adsorbed onto silica gel and subjected
to silica gel chromatography with hexane/EtOAc to give the product.
To a solution of 1d (152 mg, 1 mmol) in ethanol (2.5 mL) were added pyrrolidine (220
mg, 3.1 mmol) and corresponding aldehyde (6 mmol) successively. The
resulting mixture was stirred at 150 °C in a pressure tube for
1 h. Afterward, the solution was diluted with ethyl acetate and the
organic layer was washed with 10% HCl, water, brine and dried over
Na2SO4. The solvent was removed under reduced
pressure and the crude material was adsorbed onto silica gel and subjected
to silica gel chromatography with hexane/EtOAc to give the product.
General Procedure for the
Synthesis of 3f, 3k, and 3l
To a solution of corresponding MOM-protected acetophenone
(196 mg, 1 mmol) in ethanol (2.5 mL) was added DEA (154 mg, 2.1 mmol)
and (±)-citronellal (309 mg, 2 mmol) successively. The resulting
mixture was stirred at 150 °C in a pressure tube for 1 h. The
resulting solution was cooled to room temperature and diluted with
EtOAc. The organic layer was washed with 10% HCl aq, water, brine
and dried over Na2SO4. The solvent was removed
under reduced pressure and the residue was dissolved in methanol (14
mL), followed by the careful addition of concentrated HCl (340 μL,
4 mmol), and the resulting mixture was stirred at room temperature
overnight. Afterward, the solution was concentrated and the crude
material was adsorbed onto silica gel and subjected to silica gel
chromatography with hexane/EtOAc to give the product.
To a solution of 3h (60 mg,
0.183 mmol) in ethanol (3 mL) was added sodium acetate (300 mg, 3.6
mmol). The resulting mixture was refluxed for 24 h, after which the
solvent was removed. The residue was dissolved in ethyl acetate, and
the organic layer was washed with water, brine and dried over Na2SO4. The solvent was removed under reduced pressure
and the crude material was adsorbed onto silica gel and subjected
to silica gel chromatography with hexane/EtOAc (95/5) to obtain a
mixture of two diastereomers 3i (28 mg, 0.085 mmol, 47%)
as clear oil. 1H NMR (400 MHz, CDCl3): δ
(ppm) 7.82–7.79 (m, 2H), 6.66–6.62 (m, 2H), 6.61 (s,
2H), 5.67–5.64 (m, 2H), 5.19 (s, 2H), 5.18 (s, 2H), 4.88–4.77
(m, 2H), 3.47 (s, 3H), 3.46 (s, 3H), 2.82–2.72 (m, 2H), 2.66–2.53
(m, 2H), 2.51–2.38 (m, 3H), 2.34–2.23 (m, 5H), 2.12
(br, 2H), 1.32–1.30 (m, 6H), 1.28–1.14 (m, 2H), 0.87
(s, 3H), 0.74 (s, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm) 191.6, 191.5, 163.59, 163.58, 163.50, 163.48, 145.4,
145.3, 128.72, 128.67, 122.1, 120.7, 115.9, 115.8, 111.0, 103.63,
103.58, 94.15, 94.14, 80.3, 79.8, 77.4, 56.5, 56.4, 42.6, 42.2, 41.0,
40.9, 40.8, 38.2, 38.0, 31.8, 31.7, 31.38, 31.36, 26.24. 26.20, 21.40,
21.35. HRMS calculated for C20H24O4 (M + H)+ 329.1747, found (M + H)+ 329.1736.
HPLC purity: 99.6% (49.0% + 50.6%) (254 nm), tR = 7.66 and 7.68 min; 98.8% (220 nm), tR = 7.66 min. The split HPLC product peaks further supported
the product is a mixture of two diastereomers (see Supporting Information for the details of HPLC trace).
To a solution of 3i (38 mg,
0.116 mmol) in methanol (2 mL) was added concentrated HCl (45 μL,
0.52 mmol) carefully. The resulting mixture was stirred at room temperature
for 24 h, after which the solvent was removed. The crude material
was adsorbed onto silica gel and subjected to silica gel chromatography
with hexane/EtOAc (85/15) to obtain a mixture of two diastereomers 3j (30 mg, 0.106 mmol, 90%) as white solid. 1H
NMR (400 MHz, CDCl3): δ (ppm) 7.79–7.76 (m,
2H), 7.67 (br, 2H), 6.54–6.51 (m, 2H), 6.43–6.42 (m,
2H), 5.65–5.64 (m, 2H), 4.87–4.77 (m, 2H), 2.84–2.75
(m, 2H), 2.67–2.54 (m, 2H), 2.49–2.22 (m, 8H), 2.11
(br, 2H), 2.00 (br, 2H), 1.31–1.29 (m, 6H), 1.26–1.12
(m, 2H), 0.85 (s, 3H), 0.73 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ (ppm) 192.39, 192.36, 164.0, 163.7, 145.3,
145.2, 129.4, 129.3, 122.2, 120.9, 114.89, 114.86, 110.62, 110.61,
103.5, 80.1, 79.6, 77.4, 77.1, 42.6, 42.2, 40.9, 40.83, 40.79, 40.7,
38.2, 38.0, 31.8, 31.7, 31.39, 31.36, 26.24, 26.20, 21.38, 21.36.
HRMS calculated for C18H20O3 (M –
H)− 283.1340, found (M – H)− 283.1310. HPLC purity: 99.4% (49.7% + 49.7%) (254 nm), tR = 7.09 and 7.11 min; 99.6% (50.6% + 49.0%) (220 nm), tR = 7.09 and 7.11 min. The split HPLC product
peaks further supported the product is a mixture of two diastereomers
(see Supporting Information for the details
of HPLC trace).
General Procedure for the Synthesis of 4a–d
To a solution of appropriate
4-chromanone 3a–d (0.22 mmol) in
methanol (3 mL) was added sodium borohydride (16.6 mg, 0.44 mmol)
every 1.5 h for a total of 9 h. The resulting mixture was allowed
to stir at room temperature overnight. The mixture was cooled to 0
°C, quenched with saturated NH4Cl aq carefully, and
extracted with EtOAc twice. The combined organic layer was washed
with water, brine and dried over Na2SO4. The
solvent was removed under reduced pressure and the crude material
was adsorbed onto silica gel and subjected to silica gel chromatography
with hexane/EtOAc to give the product.
To a solution of 1h (256 mg, 1 mmol) in ethanol (2.5 mL) were added DEA (154 mg, 2.1
mmol) and corresponding aldehyde (2 mmol) successively. The resulting
mixture was stirred at 150 °C in a pressure tube for 1 h. Afterward,
the solution was diluted with ethyl acetate and the organic layer
was washed with 10% HCl, water, brine and dried over Na2SO4. The solvent was removed under reduced pressure and
the crude material was adsorbed onto silica gel and subjected to silica
gel chromatography with hexane/EtOAc to give the MOM-protected product.To a solution of appropriate MOM-protected compound (0.22 mmol)
in methanol (4 mL) was added concentrated HCl (172 μL, 1.98
mmol) at room temperature, and the resulting mixture was stirred overnight.
The solvent was removed under reduced pressure and the crude material
was adsorbed onto silica gel and subjected to silica gel chromatography
with hexane/EtOAc to give the corresponding deprotected product.
To a solution of 1h (256 mg,
1 mmol) in ethanol (2.5 mL) were added DEA (154 mg, 2.1 mmol) and
(−)-myrtenal (300 mg, 2 mmol) successively. The resulting mixture
was stirred at 150 °C in a pressure tube for 4 h. Afterward,
the solution was diluted with ethyl acetate and the organic layer
was washed with 10% HCl, water, brine and dried over Na2SO4. The solvent was removed under reduced pressure, and
the residue was dissolved in methanol (15 mL), followed by the addition
of concentrated HCl (765 μL, 9 mmol) dropwise. The resulting
mixture was stirred at room temperature for 16 h, after which the
solvent was removed. The crude material was adsorbed onto silica gel
and subjected to silica gel chromatography with hexane/EtOAc (75/25)
to obtain compound 5m (102 mg, 0.34 mmol, 34%) as yellow
solid. 1H NMR (400 MHz, CD3OD): δ (ppm)
7.55 (d, J = 15.4 Hz, 1H), 7.38 (d, J = 15.4 Hz, 1H), 6.11 (s, 1H), 5.81 (s, 2H), 2.65–2.64 (m,
1H), 2.57–2.47 (m, 3H), 2.18–2.17 (m, 1H), 1.38 (s,
3H), 1.18 (d, J = 9.0 Hz, 1H), 0.82 (s, 3H). 13C NMR (100 MHz, CD3OD): δ (ppm) 194.6, 166.2,
165.9, 148.3, 144.2, 135.5, 125.5, 105.9, 96.0, 42.9, 42.0, 38.7,
33.7, 32.0, 26.6, 21.2. HRMS calculated for C18H20O4 (M – H)− 299.1289, found (M
– H)− 299.1240. HPLC purity: 99.6% (254 nm), tR = 7.29 min; 99.9% (220 nm), tR = 7.29 min.
To a suspension of 5m (57
mg, 0.19 mmol) in ethanol (2.5 mL) was added catalytic amount of concentrated
HCl. The resulting mixture was stirred at 150 °C under microwave
irradiation for 30 min. The solvent was removed under reduced pressure
and the crude material was adsorbed onto silica gel and subjected
to silica gel chromatography with hexane/EtOAc (75/25) to obtain a
mixture of two diastereomers 5n (41 mg, 0.136 mmol, 72%)
as pale yellow solid. 1H NMR (400 MHz, CDCl3): δ (ppm) 12.05 (br, 2H), 5.98–5.94 (m, 4H), 5.68–5.67
(m, 2H), 4.86–4.75 (m, 2H), 2.89–2.80 (m, 2H), 2.69–2.55
(m, 2H), 2.53–2.45 (m, 2H), 2.43–2.26 (m, 6H), 2.16–2.15
(m, 2H), 1.34–1.33 (m, 6H), 1.29–1.16 (m, 2H), 0.88
(s, 3H), 0.78 (s, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm) 196.82, 196.80, 165.2, 164.2, 163.5, 145.0, 144.8,
122.4, 121.2, 103.21, 103.18, 96.55, 96.54, 95.7, 79.5, 79.0, 42.5,
42.2, 40.9, 40.8, 39.8, 39.7, 38.2, 38.0, 31.7, 31.6, 31.4, 31.3,
26.20, 26.16, 21.4, 21.3. HRMS calculated for C18H20O4 (M – H)− 299.1289,
found (M – H)− 299.1247. HPLC purity: 99.7%
(254 nm), tR = 7.53 min; 99.9% (220 nm), tR = 7.52 min.
General Procedure for the
Synthesis of 6a–f
To a suspension
of corresponding hydroxyamine (0.8 mmol) in ethanol (1 mL) was added
pyridine (64 mg, 0.8 mmol). After stirring at room temperature for
15 min, the solution of appropriate 4-chromanone (0.1 mmol) in ethanol
(2 mL) was added. The resulting mixture was stirred for 26–72
h at room temperature, after which the solvent was removed. EtOAc
was added to the residue, and the organic layer was washed with water,
brine and dried over Na2SO4. The solvent was
removed under reduced pressure and the crude material was adsorbed
onto silica gel and subjected to silica gel chromatography with hexane/EtOAc
to give oxime compound.
To a solution
of 1e (196 mg, 1 mmol) in ethanol (2.5 mL) were added
pyrrolidine (149 mg, 2.1 mmol) and corresponding cycloketone (2 mmol)
successively. The resulting mixture was stirred at 150 °C in
a pressure tube for 2–16 h. The resulting solution was cooled
to room temperature, and concentrated HCl (1.02 mL, 12 mmol) was added.
The resulting mixture was then stirred at room temperature overnight.
Afterward, the solution was diluted with EtOAc, and the organic layer
was washed with water, brine and dried over Na2SO4. The solvent was removed under reduced pressure and the crude material
was adsorbed onto silica gel and subjected to silica gel chromatography
with hexane/EtOAc to give the product.
Purified by reverse phase C18 silica gel
chromatography with H2O/MeCN (65/35) to give the product
(130 mg, 0.53 mmol, 53% over two steps) as white solid. 1H NMR (400 MHz, CDCl3): δ (ppm) 8.26 (br, 1H), 7.75
(d, J = 8.6 Hz, 1H), 6.51 (dd, J = 8.6, 2.2 Hz, 1H), 6.40 (d, J = 2.2 Hz, 1H), 2.70
(s, 2H), 2.09–2.03 (m, 2H), 1.77–1.61 (m, 6H), 1.57–1.50
(m, 2H), 1.43–1.36 (m, 2H). 13C NMR (100 MHz, CDCl3): δ (ppm) 193.1, 164.6, 162.5, 128.9, 114.3, 110.2,
103.9, 84.7, 48.5, 38.4, 29.4, 22.1. HRMS calculated for C15H18O3 (M – H)− 245.1183,
found (M – H)− 245.1154. HPLC purity: 97.4%
(254 nm), tR = 6.67 min; 97.0% (220 nm), tR = 6.67 min.
General Procedure for the
Synthesis of 8a–h
To a solution
of corresponding acetophenone (1 mmol) and appropriate benzaldehyde
(1.5 mmol) in methanol (10 mL) was added 60% KOH aqueous solution
(1.5 mL) dropwise at room temperature. The resulting solution was
stirred for 60 h at room temperature, after which the reaction mixture
was neutralized with 10% HCl aq and extracted with ethyl acetate twice.
The organic layer was washed with water, brine and dried over Na2SO4. The solvent was removed under reduced pressure,
and the residue was dissolved in methanol (14 mL), followed by the
careful addition of concentrated HCl (340 μL, 4 mmol). The obtained
mixture was stirred at room temperature until the MOM-protected chalcone
was gone, and then the mixture was concentrated. The crude material
was adsorbed onto silica gel and subjected to silica gel chromatography
with hexane/EtOAc to afford the chalcones.
The obtained appropriate chalcone (0.2 mmol) was dissolved in ethanol
(2.5 mL) with a catalytic amount of concentrated HCl and then heated
at 150 °C under microwave irradiation for 1.5 h. The solvent
was removed under reduced pressure and the crude material was adsorbed
onto silica gel and subjected to silica gel chromatography with hexane/EtOAc
to afford the corresponding flavanones.
MIC values were determined against M. tuberculosis (H37Rv) and other bacteria using the standard
microbroth dilution method exactly as previously described,[71] which is based on the methods by the Clinical
and Laboratory Standards Institute.[72,73] The maximum
test concentration used was 200 μg/mL.
Cytotoxicity Assays
Cytotoxicity was assessed in vitro using Vero cells (kidney epithelial
cells, ATCC CCL-81) as described previously.[74] In brief, monolayers of cells cultured in Dulbecco’s modified
Eagle medium (DMEM)/10% fetal bovine serum (FBS) were trypsinized,
seeded at approximately 10% confluence in white-walled 96-well plates,
and incubated overnight to allow adherence. Medium was replaced with
DMEM/FBS containing 2-fold serial dilutions of test compounds. Detection
was performed using MTT (CellTiter96, Promega) with overnight solubilization
according to the manufacturer’s instructions.
Approximation
of Solubility in Cell Culture Media
The 2-fold serial dilutions
in clear, round-bottom 96-well plates were prepared in DMEM with 5%
FBS. Plates were incubated at 37 °C overnight. The highest compound
concentration that did not result in visible precipitation of compound
was used to approximate the solubility limit in cell culture medium.
Time Kill Experiments
The ability of compounds to kill S. aureus Newman was evaluated using standard approaches.[75] Overnight cultures were diluted 1:25 in fresh
Mueller–Hinton broth and grown to OD600nm ≈
0.3 before being exposed to compounds at various increments of their
MICs. The number of viable bacteria over time was then determined.
Macromolecular Synthesis
The effects of compounds on key
macromolecular processes were determined as described,[76] using radiolabeled precursors from American
Radiolabeled Chemicals, Inc.: [methyl-3H]thymidine, [5,6-3H]uridine, and [4,5-3H]leucine for DNA, RNA, and
protein, respectively. Experiments were performed on three independent
mid-logarithmic cultures (OD600nm ≈ 0.3). The antibiotics
ciprofloxacin (DNA, from Sigma-Aldrich), rifampicin (RNA, from TCI
America), and erythromycin (protein, from Calbiochem) were used as
positive controls.
Analysis of the Membrane Potential
The effects of compounds on the membrane potential of S.
aureus were evaluated by flow cytometry using the fluorescent
probe diethyloxacarbocyanine dye DiOC2(3). The emission
of red fluorescence of DiOC2(3) from cells is dependent
on the membrane potential, while green fluorescence emission is independent
of the membrane potential. Therefore, the ratio of red to green fluorescence
provides a measure of the effects of compounds on the membrane potential.
These experiments were performed as previously described,[76] using S. aureus Newman grown
to OD600nm ≈ 0.3 in Mueller–Hinton II. Samples
were analyzed using BD LSR II flow cytometer and the effects of compounds
on the membrane potential evaluated using the software FlowJo X 10.0.7
as described.[76] Carbonyl cyanide m-chlorophenylhydrazone (CCCP; Sigma-Aldrich) and vancomycin
were used as positive and negative controls, respectively.
Assay
of Relaxation Activity of E. coli Topoisomerase I
Recombinant E. coli topoisomerase I (EcTopI) was
purified with published procedures.[77] CsCl
gradient purified plasmid DNA (160 ng) was added to relaxation reaction
buffer containing 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.1 mg/mL gelatin,
and 0.5 mM MgCl2. The resulting solution was divided into
9.5 μL aliquots, to which 0.5 μL of the 20 mM compounds
dissolved in DMSO was added for final compound concentrations of 500
μM. The compound control reaction received only DMSO. EcTopI
(20 ng) in 10 μL of the relaxation reaction buffer was then
added to each DNA/compound reaction mixture. The reaction mixtures
were then incubated at 37 °C for 30 min before the reaction was
terminated, and the mixtures were loaded onto a 1% agarose gel for
electrophoretic analysis in TAE buffer (40 mM Tris–acetate,
pH 8.1, 2 mM EDTA) as described previously.[78]
E. coli DNA Gyrase Supercoiling Assay
The E. coli DNA gyrase supercoiling assay was performed according
to the manufacturer’s protocol. Briefly, 1.25 U of E. coli DNA gyrase (New England Biolabs) was added to 20
μL of DNA gyrase reaction mixture (35 mM Tris-HCl, pH 7.5, 24
mM KCl, 4 mM MgCl2, 2 mM DTT, 1.75 mM ATP, 5 mM spermidine,
0.1 mg/mL BSA, 6.5% glycerol) containing 300 ng of relaxed plasmid
DNA substrate (New England Biolabs) in the presence of indicated concentration
of compound. After incubation at 37 °C for 30 min, the reactions
were stopped and analyzed by agarose gel electrophoresis similarly
as the topoisomerase I relaxation assay.[78] IC50 values (compound concentrations at which only 50%
of the input DNA was converted to supercoiled form) were calculated
by spot densitometry analysis with AlphaView, AlphaImager Mini (Protein
Simple). The IC50 values represent the average from at
least two experiments performed separately.
E. coli Topoisomerase IV Decatenation Assay
For the assay of decatenation
activity of topoisomerase IV,[79] 0.25 U
of E. coli topoisomerase IV (Topogen) was added to
20 μL of decatenation reaction mixture (40 mM HEPES-KOH, pH
8.0, 100 mM potassium glutamate, 10 mM magnesium acetate, 10 mM dithiothreitol,
50 μg of BSA/mL) containing 290 ng of kinetoplast DNA substrate
(Topogen) and 1 mM ATP in the presence of indicated concentration
of compound. After incubation at 37 °C for 30 min, the reactions
were stopped by the addition of stop buffer (50 mM EDTA, 50% glycerol,
and 0.5% (v/v) bromophenol blue). The DNA samples were electrophoresed
in a 1% agarose gel containing 0.5 μg/mL ethidium bromide in
TAE buffer also containing 0.5 μg/mL ethidium bromide. Visualization
of the bands was carried out by exposure to UV light. IC50 values (compound concentrations at which only 50% of kinetoplast
DNA was converted into monomeric products) were calculated by spot
densitometry analysis with AlphaView, AlphaImager Mini (Protein Simple).
The IC50 values represent the average from at least two
experiments performed separately.
Authors: Catherine Wing Ying Cheung; Norma Gibbons; David Wayne Johnson; David Lawrence Nicol Journal: Anticancer Agents Med Chem Date: 2010-03 Impact factor: 2.505
Authors: Danielle D Jandial; Christopher A Blair; Saiyang Zhang; Lauren S Krill; Yan-Bing Zhang; Xiaolin Zi Journal: Curr Cancer Drug Targets Date: 2014 Impact factor: 3.428
Authors: Richard E Lee; Julian G Hurdle; Jiuyu Liu; David F Bruhn; Tanja Matt; Michael S Scherman; Bernd Meibohm; Erik C Böttger; Anne J Lenaerts; Pavan K Vaddady; Zhong Zheng; Jianjun Qi; Rashid Akbergenov; Sourav Das; Dora B Madhura; Chetan Rathi; Ashit Trivedi; Cristina Villellas; Robin B Lee; Samanthi L Waidyarachchi; Dianqing Sun; Michael R McNeil; Jose A Ainsa; Helena I Boshoff; Mercedes Gonzalez-Juarrero Journal: Nat Med Date: 2014-01-26 Impact factor: 53.440
Figure 1
Scheme 1
Scheme 2
Figure 2
Scheme 3
Scheme 4
Scheme 5
Scheme 6
Scheme 7
Scheme 8
Scheme 9
Figure 3
Figure 4
Figure 5
Figure 6