Discovery of Potent Benzofuran-Derived Diapophytoene Desaturase (CrtN) Inhibitors with Enhanced Oral Bioavailability for the Treatment of Methicillin-Resistant Staphylococcus aureus (MRSA) Infections

Article abstract of DOI:10.1021/acs.jmedchem.5b01984

Blocking the staphyloxanthin biosynthesis process has emerged as a new promising antivirulence strategy. Previously, we first revealed that CrtN is a druggable target against infections caused by pigmented Staphylococcus aureus (S. aureus) and that naftifine was an effective CrtN inhibitor. Here, we identify a new type of benzofuran-derived CrtN inhibitor with submicromolar IC₅₀ values that is based on the naftifine scaffold. The most potent analog, 5m, inhibits the pigment production of S. aureus Newman and three MRSA strains, with IC₅₀ values of 0.38-5.45 nM, without any impact on the survival of four strains (up to 200 μM). Notably, compound 5m (1 μM) could significantly sensitize four strains to immune clearance and could effectively attenuate the virulence of three strains in vivo. Moreover, 5m was determined to be a weak antifungal reagent (MIC > 16 μg/mL). Combined with good oral bioavailability (F = 42.2%) and excellent safety profiles, these data demonstrate that 5m may be a good candidate for the treatment of MRSA infections.

Full text of DOI:10.1021/acs.jmedchem.5b01984

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Article
Discovery of Potent Benzofuran Derived Diapophytoene Desaturase
(CrtN) Inhibitors with Enhanced Oral Bioavailability for the
Treatment of Methicillin-Resistant S. aureus (MRSA) Infections
youxin wang, Feifei Chen, Hongxia Di, Yong Xu, Qiang Xiao, Xuehai Wang, Hanwen
Wei, Yanli Lu, Lingling Zhang, Jin Zhu, Chunquan Sheng, Lefu Lan, and Jian Li
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01984 • Publication Date (Web): 21 Mar 2016
Downloaded from http://pubs.acs.org on March 21, 2016
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Discovery of Potent Benzofuran Derived Diapophytoene
Desaturase (CrtN) Inhibitors with Enhanced Oral
Bioavailability for the Treatment of Methicillin-Resistant S.
aureus (MRSA) Infections
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Youxin Wanga,†, Feifei Chenb,†, Hongxia Dib, Yong Xuc, Qiang Xiaoc, Xuehai Wangd,
Hanwen Weia, Yanli Lua, Lingling Zhanga, Jin Zhua, Chunquan Shenge, Lefu Lanb,*,
Jian Lia,*
a Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China
University of Science and Technology, Shanghai 200237, China
b
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, China
c Hubei Bioꢀpharmaceutical Industrial Technological Institute Inc., Wuhan 430075,
China
d Humanwell Healthcare (Group) Co. Ltd, 666 Gaoxin Road, Wuhan 430075, China
e Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical
University, Shanghai 200433, China
† These authors contributed equally to this work.
* To whom correspondence should be addressed: jianli@ecust.edu.cn or
llan@simm.ac.cn
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ABSTRACT
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Blocking the staphyloxanthin biosynthesis process has emerged as a new promising
antiꢀvirulence strategy. Previously, we first revealed that CrtN is a druggable target
against infections caused by pigmented S. aureus and that naftifine was an effective
CrtN inhibitor. Here, we identify a new type of benzofuranꢀderived CrtN inhibitor
with submicromolar IC50 values that is based on the naftifine scaffold. The most
potent analog, 5m, inhibits the pigment production of S. aureus Newman and three
MRSA strains, with IC50 values of 0.38 to 5.45 nM, without any impact on the
survival of four strains (up to 200 ꢁM). Notably, compound 5m (1 ꢁM) could
significantly sensitize four strains to immune clearance and could effectively attenuate
the virulence of three strains in vivo. Moreover, 5m was determined to be a weak
antiꢀfungal reagent (MIC >16 ꢁg/mL). Combined with good oral bioavailability (F =
42.2%) and excellent safety profiles, these data demonstrate that 5m may be a good
candidate for the treatment of MRSA infections.
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INTRODUCTION
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Staphylococcus aureus (S. aureus) is a major human pathogen that causes
substantial disease, and the abuse of antimicrobial chemotherapies increases concern
for the rapid evolution of antimicrobial resistance.1 MRSA is a nosocomial and
communal menace that is resistant against many antibacterial drugs and antiseptics. It
is estimated that by 2050, worldwide drugꢀresistant infections will cause an additional
10 million deaths annually.2 A US Centers for Disease Control and Prevention (CDC)
report3 recently issued a report to various governments and organizations outlining the
concern of MRSA, including the New Drugs for Bad Bugs (ND4BB) project initiated
by the European Union,4 the United States military5 and the World Health
Organization (WHO).6 The Antimicrobial Resistance Global Report on Surveillance
2014 released by WHO has proven to be a global initiative to tackle the MRSA
superbug and has rekindled a campaign for new, effective and safe drugs.
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Because of the suppression of bacterial survival or growth, conventional
antibiotics are confined to a limited lifespan. It is urgent to establish a new path
forward (e.g., antiꢀadhesion7 or antiꢀbiofilm8 strategies) for medicinal chemists to
successfully address this problem. Antiꢀvirulence strategies are now under exploited
with less selective pressure for the development of bacterial resistance.
Staphyloxanthin (STX),9 a golden carotenoid pigment produced by S. aureus in
human clinical isolates, enables S. aureus to flee from innate immune clearance with
its numerous alternating single and double bonds,10 which can interact with reactive
oxygen species (ROS) from host neutrophils.11ꢀ12 Evidence13 suggests the essential
nature of STX is to act as a virulence factor with antioxidant properties; indeed,
unꢀpigmented microbes are more susceptible to host killing. Therefore, the
intervention of STX biosynthesis may offer a new avenue to treat S. aureus or even
MRSA infections.14 A range of enzymes mediate the biosynthesis of STX, including
dehydrosqualene synthase (CrtM) and diapophytoene desaturases (CrtN). Thus, the
inhibition of these enzymes likely produces nonꢀpigmented bacteria. In 2008, Eric
Oldfield and coꢀworkers reported a firstꢀgeneration CrtM inhibitor, that is, 22
(BPHꢀ652,15 Figure 1), which targets the biosynthesis of STX. Based on this
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pioneering work, several types of CrtM inhibitors have been subsequently identified
by the same group.16ꢀ18 It is worth emphasizing that 22 is the most promising CrtM
inhibitor based on the virulence factorꢀbased concept and is now completing
INDꢀenabling studies by AuricX Pharmaceuticals Inc., whose development is ongoing
since October 2014 according to a report from Cortellis of Thomson Reuters.
As the starting point for our efforts to discover new antiꢀvirulence compounds,
an inꢀhouse library of ~400 old drugs was screened using an STX inhibition assay.
Finally, naftifine hydrochloride (NTF, Figure 1), an existing FDAꢀapproved
antifungal drug, was capable of prohibiting not only STX production of S. aureus
Newman at nanomolar concentrations (Pigment inhibition: IC50=296.0±12.2 nM,
Figure 1) but also three MRSA strains (USA300 LAC, USA400 MW2, and Mu50) at
low micromolar concentrations. The in vivo performance of NTF was also
encouraging in both S. aureus Newman and MRSA strains in mouse infection models.
As a proofꢀofꢀconcept study, we have uncovered that the inhibitory mode of action of
NTF is mediated by inhibiting CrtN.19 Prior to this research, diphenylamines were
reported to be the only confirmed CrtN inhibitor,20ꢀ21 but were much less effective
than NTF in both inhibiting S. aureus CrtN and STX biosynthesis; in addition, their in
vivo inhibitory effects on S. aureus virulence remains to be elucidated. To the best of
our knowledge, none of the CrtN homologues have been identified in mammals,
which differs from CrtM. Hence, the inhibition of CrtN might render fewer side
effects than those observed with CrtM, making CrtN an attractive and druggable
target against infections caused by pigmented S. aureus.
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The preferred forms of NTF dosage are cream or gel owing to its poor
pharmacokinetics (PK) data, especially very low oral bioavailability (F value, Table
5). These data suggest an opportunity to modify NTF to obtain a novel drugꢀlike
candidate targeted against S. aureus infections or even MRSA infectious diseases.
Our vision is to deliver an orally efficacious CrtN inhibitor by the optimization of
NTF and to obtain novel NTF analogues possessing independent intellectual property
rights. To this end, we designed two types of novel benzofuran skeletons (1–5, Figure
2) derived from NTF under the premise of employing the smallest possible structural
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changes. Finally, a potent benzofuran derived CrtN inhibitor with enhanced oral
bioavailability, compound 5m, was identified, which could be a preclinical candidate
for the treatment of MRSA infections by attenuating bacterial virulence.
CHEMISTRY
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In this study, to further improve pharmacologic activity and oral bioavailability
of the lead compound NTF and obtain novel structural scaffolds, chemical
modifications were performed in four cycles. First, two novel scaffold analogs 1ꢀ2
(Figure 2) were designed to determine if the naphthalenyl moiety of NTF was
necessary for pigment inhibitory activity. Second, we replaced the Nꢀmethyl group in
region A (Figure 2) with various steric alkyl groups (including hydrogen atom) and 3
analogues (3a-c) were designed (Table 1). Third, six analogs (4a-f, Table 2) were
prepared to estimate if the type of linkers (allyl in region B) would affect pigment
inhibitory activity. Finally, we incorporated different substituted phenyls, furanyl,
naphthalenyls, (cyclo)alkyls in region C, and twentyꢀone analogs (5a-u) were
designed (Table 3).
On the basis of the above design, analogs 1, 3a-c, 4a-b, and 5a-u were
synthesized through the route outlined in Scheme 1. 2ꢀIodophenol was coupled with
1ꢀbromoꢀ2,2ꢀdiethoxyethane to afford 6 via nucleophilic substitution. Then, 6 was
cyclized in the presence of polyphosphoric acids to afford 7ꢀiodobenzofuran 7. After
replacing the iodine atom of 7 into a cyano substituent, we obtained
benzofuranꢀ7ꢀcarbonitrile 8. Next, 9 was prepared by lithium aluminum hydride
reduction from 8. On one hand, tꢀbutyloxycarboryl group was added to compound 9
to form 10, which was then converted to the key intermediate 11 by reduction of the
tꢀbutyloxycarboryl group. On the other hand, compound 9 was coupled with
transꢀcinnamaldehyde via reductive amination to afford the target analog 3a.
Subsequently, substitution of 3a with an ethyl group or isopropyl group afforded the
target analogs 3b and 3c, respectively. Various substituted acraldehydes were reduced
by sodium borohydride, followed by bromination, giving compounds 13a-x. Finally,
nucleophilic substitution of compounds 13a-x with 8 provided the target analogs 1,
4a-b, and 5a-u.
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Scheme 2 depicts the synthetic route for the preparation of the analog 2.
Coupling 3ꢀbromophenol with 1ꢀbromoꢀ2,2ꢀdiethoxyethane generated compound 14,
which was cyclized using polyphosphoric acids to generate two isomer products (i.e.,
4ꢀbromobenzofuran and 6ꢀbromobenzofuran) with similar polarity. These products
were difficult to separate, so the mixture was used for the next step without further
purification. Then, a bromine atom was replaced by a cyano group, which permitted
the separation of benzofuranꢀ4ꢀcarbonitrile 16 from benzofuranꢀ6ꢀcarbonitrile by
column chromatography. Pure 16 underwent reduction, addition of tꢀBOC, reduction
and final substitution to afford the analog 2.
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In parallel, analogs 5a and 4c-f were prepared in a similar process (Scheme 3).
5a and 4f were synthesized directly from 11 and commercially available
corresponding
alkyl
bromides
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While
(3ꢀbromopropꢀ1ꢀynꢀ1ꢀyl)benzene 21c was not directly available, it was obtained by
bromination of 3ꢀphenylpropꢀ2ꢀynꢀ1ꢀol, followed by nucleophilic substitution with 11
to provide the analog 4e. The R4ꢀsubstituted acid underwent esterification, reduction,
bromination and final substitution to provide the analogs 4c-d.
RESULTS AND DISCUSSION
NTF-Derived New Scaffolds Design and Synthesis
In total, 32 novel analogs (1-2, 3a-c, 4a-f, and 5a-u) of NTF were designed and
synthesized. Their chemical structures are shown in Tables 1–3. These analogs were
synthesized through the routes outlined in Schemes 1–3, and the details of the
synthetic procedures and structural characterization are described in the Experimental
Section. All analogs were confirmed to be ≥95% pure (Table S1, Supporting
Information).
In vitro Pigment Inhibitory Activities of Analogs 1-2, 3a-c, 4a-f, and 5a-u
For the primary assay, the new NTFꢀderived analogs were assayed for pigment
inhibitory activities against S. aureus Newman. The results are summarized in Tables
1–3. Encouragingly, the benzofuranꢀ7ꢀyl analog 1 displayed promising pigment
inhibitory activity (pigment inhibition: IC50=247.3±18.8 nM), inheriting the activity
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of NTF (IC50=296.0±12.2 nM), whereas the benzofuranꢀ4ꢀyl analog 2 possessed less
than satisfactory potency (IC50=758.7±24.3 nM). Subsequently, we varied the groups
on the nitrogen atom to afford the analogs 3a-c to determine whether activity may be
enhanced. Evidently, once the Nꢀmethyl group was removed (3a) or converted into an
ethyl or isopropyl group (3b-c), the pigment inhibition activities abated (Table 1).
Analysis of the data in Table 2 revealed that when the allyl linker was replaced
into propargyl (4e, IC50=164.3±15.4 nM) or vinylogue (4b, IC50=4.2±0.1 nM), the
pigment inhibitory activities were greatly improved, especially for 4b (by a factor of
~61). Elimination of double bonds (4d and 4f) led to a loss of pigment inhibitory
activities (IC50>1000 nM). Moreover, the introduction of branched methyl (4a) or an
additional methylene moiety (4c) resulted in a decrease of pigment inhibitory
activities (IC50>1000 nM). Although analog 4b showed a potent pigment inhibitory
activity at the singleꢀdigit nanomolar level, in view of its oil salt form, which is
unsuitable for oral dosage prescriptions, we did not select 4b for further analysis. As
shown in Table 3, replacement of phenyl with various types of groups can remarkably
affect the pigment inhibitory activities. Generally, (cyclo)alkyl (5a-c) or
heteroaromatic (5d) groups were not beneficial; however, the introduction of large
aromatic naphthalenyl groups (5e-5f), especially the naphthaleneꢀ2ꢀyl group, was
favorable. To our surprise, electronꢀwithdrawing groups and electronꢀdonating groups
at the phenyl ring indistinguishably affected the activity. However, the substituted
positions at the phenyl ring substantially affected the activity, and substitution at the
paraꢀposition of the phenyl ring showed improved activity (5i vs 5p vs 5s and 5l vs 5q
vs 5t). Notably, the inhibitory activity of the most potent analog 5m (IC50=4.0±0.2
nM, Table 3) increased approximately 76 times than that of the lead compound NFT
(IC50=296.0±12.2 nM, Figure 2), demonstrating that the chemical modification
strategy employed in this study is successful.
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SAR of NTF-Derived Benzofuran Analogs
The SAR analysis of a set of 32 NTFꢀderived analogs provided important
insights into the essential structural requirements for effective pigment inhibition. An
analysis of the data shown in Tables 1–3 reveals some noteworthy observations of the
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SAR for compounds 1-2, 3a-c, 4a-f, and 5a-u: (1) the naphthalenyl moiety of NTF is
not indispensable for pigment inhibitory activity; indeed, replacement of the
naphthalenyl ring with other similar bulky aryl rings (e.g., benzofuran) can be
tolerated (NTF vs 1 or 2); (2) in region A of the compounds (Figure 2), the Nꢀmethyl
group is critical for high potency (see analogs 3a-c, Table 1), and functional groups
that are too small (3a) or too large (3b-c) are not beneficial, leading to a loss of
pigment inhibitory activity; (3) in region B of the compounds (Figure 2), among the
different linkages, the potency increases in the order 1,3ꢀpentadienyl > propargyl >
allyl > 2ꢀmethyl allyl = propyl = methenecyclopropanyl (4b > 4e > NTF > 4a = 4f =
4d). Generally, the unsubstituted allyl linker is preferred and vinylogue significantly
improves the activities; (4) in region C of the compounds (Figure 2), in the studied set
of the R1 group (Table 3), the potency increases in the order phenyl > naphthalenyl >
furanyl = (cyclo)alkyl. The paraꢀposition is the best substituted position at the phenyl
ring (5i vs 5p or 5s and 5l vs 5q or 5t), and the electronic effect of the substituent is
limited. Taken together, the SARs are unambiguous. A subtle interplay between
linkages and the 4ꢀsubstituted phenyl moiety appear to be critical for high potency.
Analog 5m appears to represent the best combination of these factors, leading to
∼76ꢀfold potency improvement compared to the lead compound NTF.
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Target Enzyme Determination of the NTF-Derived Benzofuran Analogs
In light of our previous research that naftifine was a CrtN inhibitor,19 we
reasoned that the benzofuran analogs derived from naftifine may have the same target.
To validate this hypothesis, we conducted a similar HPLC experiment as before at 286
nm for 4,4’ꢀdiapophytoene (the product of CrtM and the substrate of CrtN).
Expression of crtM in E. coli gave rise to a HPLC peak similar to that of wildꢀtype S.
aureus Newman with respect to both retention time and UV absorption spectra
(Figures 3B–C), suggesting that this peak is 4,4’ꢀdiapophytoene. Moreover, this peak
disappeared in the cells of crtM (Figure 3D) and was reinforced in the crtN mutants
(Figure 3E). This comparison further proved that this peak belonged to
4,4’ꢀdiapophytoene. The peak profile of the 5mꢀtreated wildꢀtype S. aureus Newman
(Figure 3G) was very similar to that of the profile for the naftifineꢀtreated Newman
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(Figure 3F). In addition, the peaks of both of them were quite similar to the crtN
mutants. Taken together, these data suggest that CrtN is the target of the benzofuran
analogs of NTF.
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In vitro CrtN Enzymatic Inhibitory Activities
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Pigment inhibitory assays revealed that a number of analogs showed highly potent
activities against the biosynthesis of STX. To further explore their potential
therapeutic targets, five representative analogs (5g, 5jꢀk, 5m, and 5u) were assayed
for CrtN enzymatic inhibitory activity using our previous protocol.19 All five analogs
showed submicromolar inhibition against CrtN (Table 4), which increased up to ~40
times than that of the lead compound NTF. It was noted that the enzymatic activities
were weaker than the pigment inhibition activities in vitro; the precise reason remains
to be elucidated. The speculated reasons are given as follows: (i) as we proposed in
our previous paper19, it is possible that 5m accumulated in the cytoplasm of S. aureus,
leading to a higher intracellular concentration, (ii) enzyme CrtN catalyzed three
sequential reactions in the biosynthesis of staphyloxanthin, which differed from the
enzyme CrtM. The staphyloxanthin inhibition potency of the CrtN inhibitor could be
enhanced through synergistic effects.
Water Solubility of the Representative Analogs
Attention was given to the low water solubility of NTF (6.1 mg/mL, Table 4).
The overwhelming hydrophobic structure (only one nitrogen atom) in NTF was
deemed likely to cause its low solubility. Replacement of the naphthalenyl ring with
the benzofuran ring was favorable, leading to an increase of water solubility. In
particular, the water solubility of analogs 5g (19.7 mg/mL) and 5m (10.0 mg/mL)
increased 2~3 times than that of the lead compound NTF (Table 4). Because of the
excellent pigment inhibitory activity and good water solubility, analog 5m was
subjected to additional in vitro and in vivo investigations.
In vitro Pigment Inhibitory Activities of Analog 5m against MRSA
USA400 MW2 and USA300 LAC, two clones responsible for the epidemic of
communityꢀacquired MRSA (CAꢀMRSA) infections in the United States, along with
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Mu50, a hospitalꢀacquired MRSA (HAꢀMRSA) strain with vancomycinꢀintermediate
resistance (MRSA/VISA), were taken to investigate the effectiveness of the analog
5m. Pigment inhibition results of three MRSA strains are shown in Figure 4. We were
delighted to find that the color of all strains (USA400 MW2, USA300 LAC and Mu50)
faded by the inhibition of 5m to a large extent (IC50=5.45±0.36 nM, 3.39±0.03 nM
and 0.38±0.02 nM, respectively). The pigment inhibitory activities of 5m against
MRSA were comparable with that against S. aureus Newman. Additionally, neither
the growth characteristics nor the survival of the Newman or MRSA strains was
suppressed when incubated with 5m (0.2 mM or 0.05 mM, Figure 5). Thus, we
conclude that 5m inhibited the production of pigment rather than inhibited the
survival of the bacteria; that is to say, 5m is a potent antiꢀvirulence candidate.
Effects of 5m on Sensitizing S. aureus to Immune Clearance
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We next wished to determine whether the inhibition of pigment of all strains
translated to susceptibility of the four colonies to innate immune clearance using two
assay systems: hydrogen peroxide killing and human whole blood killing. As shown
in Figure 6, after incubation with 5m (1 ꢁM), the resulting white S. aureus Newman
were more susceptible to killing by 1.5% H2O2 compared to the untreated S. aureus
(mock) (survival, 1.2% vs 36.2%), which represents a decline in survival percentage
by a factor of ~30. The survival percentage of USA400 WM2, USA300 LAC, and
Mu50 reduced by a factor of ~20 (0.6% vs 11.7%), ~20 (0.7% vs 14.2%), and ~6 (4.3%
vs 25.3%), respectively. As for human whole blood (Figure 7), which was more
appealing to us, the resulting nonꢀpigmented S. aureus were more susceptible to
killing in freshly isolated human whole blood when subjected to the same
concentration of 5m, causing the Newman strain survival percentage to decline by a
factor of ~33 (0.8% vs 26.7%). The survival percentage of USA400 WM2, USA300
LAC, and Mu50 fell by a factor of ~8 (1.3% vs 10.2%), ~10 (1.2% vs 12.2%), and ~5
(3.0% vs 16.1%), respectively. The above results suggest that 5m indeed makes S.
aureus more susceptible to immune clearance in human blood.
In vivo Effects of 5m on Attenuating the Virulence of S. aureus Newman
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Next, we extended our assessment on the contribution of STX to abscess
formation in a systematic S. aureus Newman infection model. After infection with 1 ×
107 colonyꢀforming units (CFU) S. aureus Newman bacteria via retroꢀorbital
injection, the mice were sacrificed at 108 hours. We then measured bacterial survival
in the host organs. As shown in Figure 8, the 5mꢀtreated group reduced bacterial
survival mainly in the kidneys and hearts by 0.85 and 1.01 log10 CFU/organ,
respectively. These results clearly demonstrate that in vivo 5mꢀtreatment attenuated
the pathogenicity of S. aureus Newman.
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In vivo Effects of 5m on Attenuating the Virulence of MRSA
We next sought to examine whether the inhibitory effect of 5m on S. aureus
virulence was only restricted to the Newman strain. Mock or 5mꢀtreated mice were
infected with two distinct MRSA strains (USA400 MW2 and Mu50). As shown in
Figures 9–10, in the USA400 MW2 model, we initially treated the mice with a 200
mg/kg dose to find that the staphylococcal loads of the 5mꢀtreated group were
decreased, as measured in liver by 2.35 log10 CFU. This corresponds to a 99.6%
decrease in surviving bacteria and is superior than the positive group treated with 200
mg/kg 22 (1.58 log10 CFU). When we reduced the administration dose to 50 mg/kg,
we observed that in both cases the bacterial survival rates were slightly elevated
relative to the high dose groups, and still lower than the mock group by 0.25 log10
CFU (22ꢀtreated group) and 0.71 log10 CFU (5mꢀtreated group). As for the kidney
organs, the effect was less remarkable. In the high dosage case, the staphylococcal
loads of the 5mꢀtreated group were decreased, as measured by 1.47 log10 CFU (a 96.6%
decrease in surviving bacteria). Similarly, this value is superior than the positive
group treated with 200 mg/kg 22 (1.14 log10 CFU). When we reduced the
administration dose to 50 mg/kg, we observed that in both cases the bacterial survival
rates were slightly elevated relative to the high dose groups, and demonstrated a
comparative clearance. In the Mu50 model, identical results were obtained. In the
liver organs with a dose of 200 mg/kg, both inhibitors possessed excellent
performance. With the treatment of 5m, the bacterial survival rates fell by 3.58 log10
CFU (a 99.97% decrease in surviving bacteria), whereas the survival rates of the 22
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treated group decreased by 2.84 log10 CFU. Although the dose of the inhibitors was
reduced to 50 mg/kg, the outcomes remained encouraging. The strains of the
5mꢀtreated group were shrunk by 2.94 log10 CFU, better than the 22 treated group,
whose rates decreased by 1.87 log10 CFU, which is one order of magnitude weaker
than 5m. Regarding the kidneys, the results were inconclusive. With 200 mg/kg of 5m,
the bacterial colonies were inhibited by 1.11 log10 CFU. Prescribed with the same
dosage of 22, only a 0.30 log10 CFU decrement was observed. Furthermore, a low
dose of 50 mg/kg produced less benefit. Bacterial survival rates of the 5m and 22
treated groups decreased by only 0.68 log10 CFU and 0.25 log10 CFU, respectively.
To sum up, both of the two inhibitors against STX biosynthesis, 22 and 5m, were
capable of decreasing the bacterial survival in the S. aureus Newman and two MRSA
strains. However, compound 5m was more successful than 22 in all experiments in
this regard.
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In vitro Anti-fungal Activities of 5m
Next, an antiꢀfungal experiment was conducted to examine whether 5m inherited
the original antiꢀfungal activity of NTF. As shown in Table 5, the antiꢀfungal activity
of the benzofuran scaffold was no longer effective. Compound 5m demonstrated
weak activities in all cases, and its best MIC against Trichophyton rubrum was
128ꢀfold worse than that of NTF, suggesting, to a certain extent, that 5m may be a
selective antiꢀvirulence active compound.
In vivo Rat Pharmacokinetics Profile of 5m
Because of its potency and attractive selectivity profile, compound 5m was
evaluated in an in vivo rat PK model. The pharmacokinetic parameters were
determined following iv injection of a single 5 mg/kg dose or po administration of a
single 10 mg/kg dose in rat. Further details are shown in Table 6. Compared with the
poor bioavailability of NTF (F = 0.85 %), presumably resulting in its external
application, compound 5m possessed acceptable bioavailability (F = 42.2%), which is
consistent with the drugꢀlike criteria of 30% concluded by most pharmaceutical
researchers.22 Moreover, the approximate 8 h halfꢀlife (t1/2) of 5m is also suitable for
its oral delivery, which could be administered twice daily.
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CONCLUSIONS
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In summary, we have discovered a new class of CrtN inhibitors with enhanced
oral bioavailability using a scaffold hopping approach. On the basis of the structure of
the lead compound NTF, 32 completely new benzofuran analogs have been
synthesized and tested with pigment inhibitory assays. Five analogs (5g, 5jꢀk, 5m and
5u) were found to show potent pigment inhibitory activities and good
physicochemical properties. Notably, the most potent analog, 5m, demonstrated
pigment inhibitory capability approximately 76 times higher than that of the prototype
NTF. Preliminary SARs were obtained, showing that variations in the allyl linkage
and substituents at the paraꢀposition of the phenyl moiety have a very important
influence on the pigment inhibitory activity, and appropriate structural optimizations
of the above regions can substantially improve potency.
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CrtN enzymatic inhibition assays and HPLC analyses further confirmed that five
analogs (5g, 5jꢀk, 5m, and 5u) were all CrtN inhibitors with submicromolar IC50
values, being more effective (up to 91ꢀfold) than diphenylamines previously
reported.21 Among the five analogs, compound 5m was found to have high water
solubility and could effectively inhibit the STX biosynthesis of three MRSA strains
(USA300 LAC, USA400 MW2, and Mu50) with IC50 values of 3.39±0.03 nM,
5.45±0.36 nM and 0.38±0.02 nM, respectively, without any impact on the survival of
the Newman strain or three MRSA strains (up to 200 ꢁM). The antiꢀvirulence effects
of 5m (1 ꢁM) were also observed by sensitizing Newman (up to 30 times) and three
MRSA strains (up to 33 times) to H2O2 and human whole blood killing, which were in
good agreement with the in vivo therapeutic potential of 5m on attenuating the
virulence of three S. aureus strains. The in vivo pharmacological results showed that
5m (200 mg/kg and 50 mg/kg) had both more beneficial effects on lowering abscess
formation in liver and kidney than 22 (200 mg/kg and 50 mg/kg). For liver, in
particular, treatment with 5m (200 mg/kg) resulted in a 99% decrease in surviving
bacteria (USA400 MW2 and Mu50). To our delight, 5m possessed very weak
antiꢀfungal activities (MIC >16 ꢁg/mL) and good oral bioavailability (F = 42.2%).
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Moreover, the safety evaluation experiments (detailed assays in Supporting
Information) showed that 5m had very weak effects on the growth of two normal
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human cell lines (HMEC: IC50= 67.1±2.4
ꢁ
M and WI38: IC50=47.9±4.5
ꢁM) and
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possessed low hERG inhibition activity (IC50=3.71
ꢁM). Altogether, these data
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demonstrate that 5m may be a good candidate for discovering potential therapeutic
drugs with specificity to combat pigmented S. aureus (especially MRSA).
Considering only a few CrtN inhibitors reported thus far, 5m is an effective chemical
biology tool for the study of STXꢀregulating virulence in infectious diseases. Further
structural optimization of 5m is currently in progress in our laboratory.
EXPERIMENTAL SECTION
General Chemistry.
Synthetic starting materials, reagents and solvents were purchased from Alfa
Aesar, Acros, Adamasꢀbeta, Energy Chemical, J&K, Shanghai Chemical Reagent Co.
and TCI at the highest commercial quality and used without further purification.
Analytical thinꢀlayer chromatography (TLC) was performed on HSGF 254 (150−200
ꢁm thickness; Yantai Huiyou Co., China), and the components were visualized by
observation under UV light (254 nm and 365 nm). Melting points were determined on
a SGW Xꢀ4 melting point apparatus without correction. The products were purified
by recrystallization or column chromatography on silica gel (200–300 mesh).
Reaction yields were not optimized. Nuclear magnetic resonance (NMR)
spectroscopy was performed on a Bruker AMXꢀ400 NMR spectrometer using
deuterated chloroform (CDCl3), deuterated methanol (CD3OD), or deuterated
dimethyl sulfoxide (DMSOꢀd6) as the solvent. Chemical shifts were reported in parts
per million (ppm, δ) downfield from tetramethylsilane. Proton coupling patterns were
described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad
(br). Lowꢀ and highꢀresolution mass spectra (LRMS and HRMS) were given with
electron spray ionization (ESI) produced by a Finnigan MATꢀ95 and a LCQꢀDECA
spectrometer. HPLC data analysis of compounds 1-2, 3a-c, 4a-f and 5a-u were
performed on an Agilent 1100 with a quaternary pump and diodeꢀarray detector
(DAD). The peak purity was verified with UV spectra. All analogs were confirmed to
be ≥95% pure.
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Preparation of salts.
Taking compound 1 as an example, oily compound 1 (100.0 mg) was suspended
in EtOAc (15 mL) with stirring at room temperature and then bubbled into hydrogen
chloride gas for 1 min. The reaction solution was concentrated and then stirred for an
additional 1 h in EtOAc/petroleum ether (1:100, v/v, 20 mL). The precipitate was
filtered, washed with EtOAc to give the final compounds in the form of hydrochloride.
All other endꢀproducts had experienced such salification process. Spectroscopic data
given below are in their hydrochloride form.
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(E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-phenylprop-2-en-1-amine
hydrochloride (1).
A solution of intermediate 11 (120.0 mg, 0.7 mmol), 13a (146.0 mg, 0.7 mmol)
and K2CO3 (112.0 mg, 0.8 mmol) in DMF (10 mL) was stirred at room temperature
overnight. The mixture was poured into water and extracted with EtOAc. The
combined organic layers were washed with brine, dried over anhydrous Na2SO4,
filtered and condensed. The residue was then purified via flash chromatography on
silica gel, eluting with EtOAc/petroleum ether (1/5, v/v) to give the free base of 1 as a
colorless oil. Yield: 44%. 1 was prepared by the general procedure of salification
given above as a white solid, m.p. 114–118 ºC. 1H NMR (400 MHz, MeOD) δ 7.94 (s,
1H), 7.83 (d, J = 7.7 Hz, 1H), 7.58–7.47 (m, 3H), 7.38 (dt, J = 14.2, 7.4 Hz, 4H), 7.00
(d, J = 10.8 Hz, 1H), 6.95 (s, 1H), 6.48–6.32 (m, 1H), 4.82 (s, 1H), 4.65 (s, 1H), 4.07
(d, J = 38.8 Hz, 2H), 2.90 (s, 3H); HRMS (ESI) m/z calcd for C19H20NO [M+H]+
278.1545, found 278.1539.
(E)-N-(Benzofuran-4-ylmethyl)-N-methyl-3-phenylprop-2-en-1-amine
hydrochloride (2).
Yield: 44%. 2 was synthesized by the general procedure of 1 given above as a
1
white solid, m.p. 173–176 ºC. H NMR (400 MHz, MeOD) δ 7.95 (d, J = 2.3 Hz,
1H), 7.70 (p, J = 3.8 Hz, 1H), 7.57–7.51 (m, 2H), 7.48 (d, J = 5.1 Hz, 2H), 7.44–7.33
(m, 3H), 7.17 (dd, J = 2.3, 0.9 Hz, 1H), 6.95 (d, J = 15.8 Hz, 1H), 6.44–6.33 (m, 1H),
4.68 (d, J = 64.3 Hz, 2H), 4.05 (d, J = 35.5 Hz, 2H), 2.87 (s, 3H); HRMS (ESI) m/z
calcd for C19H20NO [M+H]+ for 278.1545, found 278.1539.
(E)-N-(Benzofuran-7-ylmethyl)-3-phenylprop-2-en-1-amine hydrochloride (3a).
A mixture of 9 (294.0 mg, 2 mmol), transꢀcinnamaldehyde (264.0 mg, 2 mmol)
and molecular sieve (1.0 g) in dichloromethane (25 mL) was heated at reflux for 17 h.
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Thereafter, methanol was added dropwise into the mixture and then sodium
borohydride (76.0 mg, 2 mmol) was added in batches at 0 ºC. The reaction mixture
was stirred for 30 min and concentrated under reduced pressure. The residue was
poured into water and extracted with EtOAc. The combined organic layers were
washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue
was then purified via flash chromatography on silica gel, eluting with
EtOAc/petroleum ether (1/1, v/v) to give the free base of 3a as a colorless oil. Yield:
65%. 3a was prepared using the general procedure of salification as a white solid, m.p.
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129–130 ºC. HꢀNMR (400 MHz, MeOD) δ 7.92 (s, 1H), 7.77 (d, J = 7.7 Hz, 1H),
7.49 (t, J = 9.3 Hz, 3H), 7.43–7.29 (m, 4H), 6.99 (d, J = 1.8 Hz, 1H), 6.92 (d, J = 15.8
Hz, 1H), 6.43–6.30 (m, 1H), 4.60 (s, 2H), 3.94 (d, J = 7.2 Hz, 2H). HRMS (ESI) m/z
calcd for C18H18NO [M+H]+ 264.1388, found 264.1383.
(E)-N-(Benzofuran-7-ylmethyl)-N-ethyl-3-phenylprop-2-en-1-amine
hydrochloride (3b).
To a solution of the free base of 3a (360.0 mg, 1.4 mmol) in DMF (10 mL) was
added sodium hydride (52 mg, 1.4 mmol) in batches at 0 ºC under a N2 atmosphere.
The reaction mixture was stirred for 15 min and iodoethane (219.0 ꢁL, 2.7 mmol) was
added into the solution. The mixture was stirred at room temperature overnight,
poured into water and extracted with EtOAc. The combined organic layers were
washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue
was then purified via flash chromatography on silica gel, eluting with
EtOAc/petroleum ether (1/5, v/v) to give the free base of 3b as a colorless oil. Yield:
50%. 3b was prepared using the general procedure of salification as a yellow oil.
1HꢀNMR (400 MHz, MeOD) δ 7.93 (d, J = 1.8 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.53
(d, J = 7.4 Hz, 3H), 7.44–7.32 (m, 4H), 7.01 (d, J = 1.9 Hz, 1H), 6.95 (d, J = 15.8 Hz,
1H), 6.46–6.32 (m, 1H), 4.75 (s, 2H), 4.03 (dd, J = 17.7, 10.4 Hz, 2H), 3.39–3.34 (m,
2H), 1.49 (t, J = 7.2 Hz, 3H). HRMS (ESI) m/z calcd for C20H22NO [M+H]+
292.1701, found 292.1700.
(E)-N-(Benzofuran-7-ylmethyl)-N-isopropyl-3-phenylprop-2-en-1-amine
hydrochloride (3c).
Yield: 50%. 3c was synthesized by the general procedure of 3b given above as a
1
yellow oil. HꢀNMR (400 MHz, MeOD) δ 7.92 (d, J = 1.8 Hz, 1H), 7.79 (d, J = 7.7
Hz, 1H), 7.52 (t, J = 7.2 Hz, 2H), 7.44 (d, J = 6.9 Hz, 2H), 7.37 (d, J = 6.2 Hz, 3H),
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6.99 ( s, 1H), 6.90 (d, J = 16.3 Hz, 1H), 6.30–6.18 (m, 1H), 4.85 (d, J = 13.7 Hz, 1H),
4.57 (d, J = 13.7 Hz, 1H), 4.14 (d, J = 12.9 Hz, 1H), 4.04 (dd, J = 13.6, 7.2 Hz, 1H),
3.85 (dt, J = 13.3, 6.6 Hz, 1H), 1.60 (d, J = 6.6 Hz, 3H), 1.52 (d, J = 6.6 Hz, 3H);
HRMS (ESI) m/z calcd for C21H24O [M+H]+ 306.1858, found 306.1862.
(E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-phenylprop-2-en-1-amine
hydrochloride (4a).
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Yield: 57%. 4a was synthesized by the general procedure of 1 given above as a
white solid, m.p. 160–162 ºC. 1HꢀNMR (400 MHz, MeOD) δ 7.92 (s, 1H), 7.81 (d, J
= 7.8 Hz, 1H), 7.52 (d, J = 7.4 Hz, 1H), 7.40–7.22 (m, 6H), 6.97 (d, J = 18.2 Hz, 1H),
6.86 (s, 1H), 4.85 (d, J = 13.3 Hz, 1H), 4.56 (d, J = 13.3 Hz, 1H), 4.11–4.05 (m, 1H),
3.98 (d, J = 12.7 Hz, 1H), 2.91 (s, 3H), 2.04 (s, 3H); HRMS (ESI) m/z calcd for
C20H22NO [M+H]+ 292.1701, found 292.1707.
(2E,4E)-N-(Benzofuran-7-ylmethyl)-N-methyl-5-phenylpenta-2,4-dien-1-amine
hydrochloride (4b).
Yield: 42%. 4b was synthesized by the general procedure of 1 given above as a
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yellow oil. HꢀNMR (400 MHz, MeOD) δ 7.92 (s, 1H), 7.78 (d, J = 10.8 Hz, 1H),
7.48 (d, J = 6.7 Hz, 3H), 7.43–7.31 (m, 3H), 7.30–7.20 (m, 1H), 7.04–6.90 (m, 2H),
6.83–6.65 (m, 2H), 5.96 (dt, J = 14.9, 7.4 Hz, 1H), 4.78 (d, J = 13.3 Hz, 1H), 4.57 (d,
J = 13.3 Hz, 1H), 4.10–3.98 (m, 1H), 3.95–3.85 (m, 1H), 2.81 (s, 3H); HRMS (ESI)
m/z calcd for C21H22NO [M+H]+ 304.1701, found 304.1695.
(E)-N-(Benzofuran-7-ylmethyl)-N-methyl-4-phenylbut-3-en-1-amine
hydrochloride (4c).
Yield: 42%. 4c was synthesized by the general procedure of 1 given above as a
yellow oil. 1HꢀNMR (400 MHz, MeOD) δ 7.90 (s, 1H), 7.83 (d, J = 7.5 Hz, 1H), 7.50
(d, J = 7.2 Hz, 1H), 7.42 (s, 3H), 7.33 (t, J = 7.1 Hz, 2H), 7.26 (d, J = 5.8 Hz, 1H),
7.00 (s, 1H), 6.66 (d, J = 16.3 Hz, 1H), 6.28–6.12 (m, 1H), 4.85 (d, J = 13.7 Hz, 1H),
4.63 (d, J = 13.7 Hz, 1H), 3.68–3.59 (m, 1H), 3.51ꢀ3.41 (m, 1H), 2.93 (s, 3H), 2.85–
2.77 (m, 2H); HRMS (ESI) m/z calcd for C20H22NO [M+H]+ 292.1701, found
292.1695.
1-(Benzofuran-7-yl)-N-methyl-N-(((1R,2R)-2-phenylcyclopropyl)methyl)methana
mine hydrochloride (4d).
Yield: 52%. 4d was synthesized by the general procedure of 1 given above as a
yellow oil. 1HꢀNMR (400 MHz, MeOD) δ 7.86 (t, J = 4.1 Hz, 1H), 7.83–7.75 (m, 1H),
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7.53–7.43 (m, 1H), 7.42–7.31 (m, 1H), 7.28 (td, J = 7.5, 1.7 Hz, 2H), 7.22–7.11 (m,
3H), 6.97 (td, J = 5.3, 2.2 Hz, 1H), 4.85 (dd, J = 16.5, 6.5 Hz, 1H), 4.58 (dd, J = 13.3,
5.0 Hz, 1H), 3.52–3.45 (m, 1H), 3.29–3.15 (m, 1H), 2.90 (d, J = 7.3 Hz, 3H), 2.13–
2.03 (m, 1H), 1.62–1.49 (m, 1H), 1.18–1.06 (m, 1H), 0.94–0.81 (m, 1H). HRMS (ESI)
m/z calcd for C20H22NO [M+H]+ 292.1701, found 292.1704.
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hydrochloride
(4e).
Yield: 60%. 4e was synthesized by the general procedure of 1 given above as a
yellow oil. 1HꢀNMR (400 MHz, MeOD) δ 7.93 (s, 1H), 7.84 (d, J = 7.7 Hz, 1H), 7.56
(dd, J = 14.6, 7.2 Hz, 3H), 7.52–7.37 (m, 4H), 7.02 (s, 1H), 4.89(d, J = 13.3 Hz, 1H),
4.78 (d, J = 13.3 Hz, 1H), 4.41 (d, J = 11.6 Hz, 2H), 3.05 (s, 3H). HRMS (ESI) m/z
calcd for C19H18NO [M+H]+ 276.1388, found 276.1388.
N-(Benzofuran-7-ylmethyl)-N-methyl-3-phenylprop-2-yn-1-amine hydrochloride
(4f).
Yield: 59%. 4f was synthesized by the general procedure of 1 given above as a
yellow oil. 1HꢀNMR (400 MHz, MeOD) δ 7.91 (s, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.44
(t, J = 8.0 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H), 7.28 (d, J = 7.1 Hz, 2H), 7.22 (s, 4H), 7.0
(s, 1H), 4.76 (d, J = 13.3 Hz, 1H), 4.59 (d, J = 13.3 Hz, 1H), 3.31–3.23 (m, 1H), 3.21–
3.10 (m, 1H), 2.88 (s, 3H), 2.72 (t, J = 7.1 Hz, 2H), 2.17 (d, J = 5.6 Hz, 2H). HRMS
(ESI) m/z calcd for C19H22NO [M+H]+ 280.1701, found 280.1704.
(E)-N-(Benzofuran-7-ylmethyl)-N-methylbut-2-en-1-amine hydrochloride (5a).
Yield: 50%. 5a was synthesized by the general procedure of 1 given above as a
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yellow oil. HꢀNMR (400 MHz, MeOD) δ 7.94 (s, 1H), 7.77 (dd, J = 34.7, 14.0 Hz,
1H), 7.49 (d, J = 7.4 Hz, 1H), 7.38 (dd, J = 18.9, 11.4 Hz, 1H), 7.05 –6.91 (m, 1H),
6.14 (dd, J = 14.8, 6.7 Hz, 1H), 5.81–5.63 (m, 1H), 4.73 (d, J = 17.1 Hz, 1H), 4.52 (d,
J = 17.1 Hz, 1H), 3.94–3.86 (m, 1H), 3.80–3.71 (m, 1H), 2.81 (s, 3H), 1.84 (t, J =
16.1 Hz, 3H); HRMS (ESI) m/z calcd for C14H18NO [M+H]+ 216.1388, found
216.1383.
(E)-N-(Benzofuran-7-ylmethyl)-3-cyclopentyl-N-methylprop-2-en-1-amine
hydrochloride (5b).
Yield: 39%. 5b was synthesized by the general procedure of 1 given above as a
1
yellow oil. HꢀNMR (400 MHz, MeOD) δ 7.93 (d, J = 2.2 Hz, 1H), 7.83–7.80 (m,
1H), 7.48 (d, J = 6.9 Hz, 1H), 7.44–7.34 (m, 1H), 7.01 (d, J = 2.2 Hz, 1H), 6.10 (dd, J
= 15.3, 7.8 Hz, 1H), 5.66 (dt, J = 14.9, 7.3 Hz, 1H), 4.75 (d, J = 13.3 Hz, 1H), 4.53 (d,
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J = 13.2 Hz, 1H), 3.90 (m, 1H), 3.76 (m, 1H), 2.80 (s, 3H), 2.61 (m, 1H), 1.99–1.83
(m, 2H), 1.84–1.53 (m, 4H), 1.49–1.30 (m, 2H); HRMS (ESI) m/z calcd for
C18H24NO [M+H]+ 270.1858, found 270.1862.
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(E)-N-(Benzofuran-7-ylmethyl)-3-cyclohexyl-N-methylprop-2-en-1-amine
hydrochloride (5c).
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Yield: 42%. 5c was synthesized by the general procedure of 1 given above as a
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yellow oil. HꢀNMR (400 MHz, MeOD) δ 7.94 (d, J = 2.2 Hz, 1H), 7.87–7.79 (m,
1H), 7.48 (d, J = 6.9 Hz, 1H), 7.44–7.34 (m, 1H), 7.01 (d, J = 2.2 Hz, 1H), 6.06 (dd, J
= 15.3, 7.8 Hz, 1H), 5.63 (dt, J = 14.9, 7.3 Hz, 1H), 4.76 (d, J = 13.3 Hz, 1H), 4.56 (d,
J = 13.4 Hz, 1H), 3.99 (m, 1H), 3.76 (m, 1H), 2.87 (s, 3H), 2.3 (m, 1H), 1.88–1.58 (m,
6H), 1.41–1.28 (m, 4H); HRMS (ESI) m/z calcd for C19H26NO [M+H]+ 284.2014,
found 284.2008.
(E)-N-(Benzofuran-7-ylmethyl)-3-(furan-2-yl)-N-methylprop-2-en-1-amine
hydrochloride (5d).
Yield: 58%. 5d was synthesized by the general procedure of 1 given above as a
yellow oil. 1HꢀNMR (400 MHz, MeOD) δ 7.90 (d, J = 2.4 Hz, 1H), 7.79 (dd, J = 7.8,
1.1 Hz, 1H), 7.53 (d, J = 1.6 Hz, 1H), 7.48 (t, J = 5.6 Hz, 1H), 7.37 (td, J = 7.4, 3.0
Hz, 1H), 6.97 (t, J = 2.7 Hz, 1H), 6.78 (d, J = 15.6 Hz, 1H), 6.52 (d, J = 3.3 Hz, 1H),
6.51–6.44 (m, 1H), 6.28–6.13 (m, 1H), 4.78 (d, J = 13.3 Hz, 1H), 4.58 (d, J = 13.3 Hz,
1H), 4.15–4.01 (m, 1H), 3.89–3.97 (m, 1H), 2.84 (s, 3H); HRMS (ESI) m/z calcd for
C17H18NO2 [M+H]+ 268.1338, found 268.1334.
(E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-(naphthalen-1-yl)prop-2-en-1-amine
hydrochloride (5e).
Yield: 59%. 5e was synthesized by the general procedure of 1 given above as a
white solid, m.p. 170–171 ºC. 1HꢀNMR (400 MHz, MeOD) δ 8.20 (d, J = 8.1 Hz, 1H),
7.93 (s, 3H), 7.89–7.71 (m, 3H), 7.66–7.48 (m, 4H), 7.42 (t, J = 7.5 Hz, 1H), 7.02 (s,
1H), 6.55–6.39 (m, 1H), 4.92 (d, J = 13.2 Hz, 2H), 4.69 (d, J = 13.2 Hz, 1H), 4.35–
4.22 (m, 1H), 4.20–4.06 (m, 1H), 2.94 (s, 3H); HRMS (ESI) m/z calcd for C23H22NO
[M+H]+ 328.1701, found 328.1698.
(E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-(naphthalen-2-yl)prop-2-en-1-amine
hydrochloride (5f).
Yield: 47%. 5f was synthesized by the general procedure of 1 given above as a
white solid, m.p. 198–199 ºC. 1HꢀNMR (400 MHz, MeOD) δ 8.00–7.85 (m, 5H), 7.83
(d, J = 7.6 Hz, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.53 (s, 3H), 7.41 (t, J = 7.4 Hz, 1H),
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7.13 (d, J = 15.6 Hz, 1H), 7.01 (s, 1H), 6.62–6.47 (m, 1H), 4.86 (d, J = 13.4 Hz, 1H),
4.66 (d, J = 13.4 Hz, 1H), 4.28–4.14 (m, 1H), 4.14–3.98 (m, 1H), 2.91 (s, 3H); HRMS
(ESI) m/z calcd for C23H22NO [M+H]+ 328.1701, found 328.1701.
(E)-N-(Benzofuran-7-ylmethyl)-3-(4-methoxyphenyl)-N-methylprop-2-en-1-amin
e hydrochloride (5g).
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Yield: 49%. 5g was synthesized by the general procedure of 1 given above as a
white solid, m.p. 151–152 ºC. 1HꢀNMR (400 MHz, MeOD) δ 7.93 (s, 1H), 7.82 (d, J
= 7.6 Hz, 1H), 7.49 (t, J = 9.2 Hz, 3H), 7.40 (t, J = 7.4 Hz, 1H), 7.04–6.93 (m, 3H),
6.90 (d, J = 15.8 Hz, 1H), 6.25 (dt, J = 15.2, 7.5 Hz, 1H), 4.83 (d, J = 13.3 Hz, 1H),
4.61 (d, J = 13.3 Hz, 1H), 4.14–4.05 (m, 1H), 4.02–3.90 (m, 1H), 3.83 (s, 3H), 2.94–
2.81 (m, 3H); HRMS (ESI) m/z calcd for C20H22NO2 [M+H]+ 308.1651, found
308.1653.
(E)-Methyl4-(3-((benzofuran-7-ylmethyl)(methyl)amino)prop-1-en-1-yl)benzoate
hydrochloride (5h).
Yield: 85%. 5h was synthesized by the general procedure of 1 given above as a
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white solid, m.p. 163–165 ºC. HꢀNMR (400 MHz, MeOD) δ 8.05 (d, J = 7.8 Hz,
2H), 7.94 (s, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.65 (d, J = 7.8 Hz, 2H), 7.52 (d, J = 7.4
Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.02 (d, J = 12.1 Hz, 2H), 6.62–6.49 (m, 1H), 4.85
(d, J = 13.2 Hz, 1H), 4.65 (d, J = 13.4 Hz, 1H), 4.22–4.12 (m, 1H), 4.10–3.98 (m, 1H),
3.93 (s, 3H), 2.89 (s, 3H). HRMS (ESI) m/z calcd for C21H22NO3 [M+H]+ 336.1600,
found 336.1597.
(E)-N-(Benzofuran-7-ylmethyl)-3-(4-fluorophenyl)-N-methylprop-2-en-1-amine
hydrochloride (5i).
Yield: 50%. 5i was synthesized by the general procedure of 1 given above as a
white solid, m.p. 148–150 ºC. 1HꢀNMR (400 MHz, MeOD) δ 7.90 (s, 1H), 7.79 (d, J
= 7.6 Hz, 1H), 7.55 (d, J = 5.6 Hz, 2H), 7.48 (d, J = 7.2 Hz, 1H), 7.37 (t, J = 7.5 Hz,
1H), 7.11 (t, J = 8.3 Hz, 2H), 6.98 (s, 1H), 6.91 (d, J = 15.8 Hz, 1H), 6.41–6.25 (m,
1H), 4.80 (d, J = 13.2 Hz, 1H), 4.60 (d, J = 12.9 Hz, 1H), 4.15–4.02 (m, 1H), 4.02–
3.87 (m, 1H), 2.84 (s, 3H); HRMS (ESI) m/z calcd for C19H19FNO [M+H]+ 296.1451,
found 296.1449.
(E)-N-(Benzofuran-7-ylmethyl)-3-(4-chlorophenyl)-N-methylprop-2-en-1-amine
hydrochloride (5j).
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Yield: 61%. 5j was synthesized by the general procedure of 1 given above as a
white solid, m.p. 183–184 ºC. 1HꢀNMR (400 MHz, MeOD) δ 7.90 (s, 1H), 7.79 (d, J
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= 7.7 Hz, 1H), 7.50 (d, J = 8.3 Hz, 3H), 7.38 (d, J = 7.1 Hz, 3H), 6.96 (d, J = 14.9 Hz,
1H), 6.91 (d, J = 15.7 Hz, 1H), 6.47–6.32 (m, 1H), 4.81 (d, J = 13.2 Hz, 1H), 4.61 (d,
J = 13.4 Hz, 1H), 4.18–4.04 (m, 1H), 4.03–3.89 (m, 1H), 2.84 (s, 3H); HRMS (ESI)
m/z calcd for C19H19ClNO [M+H]+ 312.1155, found 312.1148.
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(E)-N-(Benzofuran-7-ylmethyl)-3-(4-bromophenyl)-N-methylprop-2-en-1-amine
hydrochloride (5k).
Yield: 46%. 5k was synthesized by the general procedure of 1 given above as a
white solid, m.p. 192–194 ºC. 1HꢀNMR (400 MHz, MeOD) δ 7.93 (s, 1H), 7.81 (t, J
= 10.5 Hz, 1H), 7.66–7.24 (m, 6H), 6.98 (d, J = 20.3 Hz, 1H), 6.92 (d, J = 15.8 Hz,
1H), 6.57–6.33 (m, 1H), 4.84 (d, J = 13.4 Hz, 1H), 4.63 (d, J = 13.2 Hz, 1H), 4.21–
4.07 (m, 1H), 4.06–3.89 (m, 1H), 2.87 (s, 3H); HRMS (ESI) m/z calcd for
C19H19BrNO [M+H]+ 358.0630, found 358.0634.
(E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-(4-nitrophenyl)prop-2-en-1-amine
hydrochloride (5l).
Yield: 59%. 5l was synthesized by the general procedure of 1 given above as a
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white solid, m.p. 194–195 ºC. HꢀNMR (400 MHz, MeOD) δ 8.28 (d, J = 8.4 Hz,
2H), 7.94 (s, 1H), 7.80 (dd, J = 21.2, 8.0 Hz, 3H), 7.53 (d, J = 7.3 Hz, 1H), 7.41 (t, J =
7.6 Hz, 1H), 7.14–6.96 (m, 2H), 6.71–6.55 (m, 1H), 4.86 (d, J = 13.3 Hz, 1H), 4.67 (d,
J = 13.3 Hz, 1H), 4.27–4.13 (m, 1H), 4.15–3.90 (m, 1H), 2.94 (s, 3H); HRMS (ESI)
m/z calcd for C19H19N2O3 [M+H]+ 323.1396, found 323.1393.
(E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-(4-(trifluoromethyl)phenyl)prop-2-e
n-1-amine hydrochloride (5m).
Yield: 43%. 5m was synthesized by the general procedure of 1 given above as a
white solid, m.p. 162–164 ºC. 1HꢀNMR (400 MHz, MeOD) δ 7.94 (s, 1H), 7.82 (d, J
= 7.8 Hz, 1H), 7.72 (s, 4H), 7.53 (d, J = 7.4 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.03 (d,
J = 15.8 Hz, 2H), 6.64–6.49 (m, 1H), 4.86 (d, J = 13.7 Hz, 1H), 4.66 (d, J = 12.9 Hz,
1H), 4.24–4.12 (m, 1H), 4.09–3.97 (m, 1H), 2.93 (s, 3H); HRMS (ESI) m/z calcd for
C20H19F3NO [M+H]+ 346.1419, found 346.1406.
(E)-N-(Benzofuran-7-ylmethyl)-3-(4-(tert-butyl)phenyl)-N-methylprop-2-en-1-am
ine hydrochloride (5n).
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Yield: 49%. 5n was synthesized by the general procedure of 1 given above as a
white solid, m.p. 139–141 ºC. 1HꢀNMR (400 MHz, MeOD) δ 7.93 (s, 1H), 7.81 (t, J
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= 11.2 Hz, 1H), 7.59–7.35 (m, 6H), 7.01 (s, 1H), 6.93 (d, J = 15.9 Hz, 1H), 6.45–6.27
(m, 1H), 4.84 (d, J = 13.4 Hz, 1H), 4.62 (d, J = 13.3 Hz, 1H), 4.17–4.07 (m, 1H),
4.07–3.92 (m, 1H), 2.86 (s, 3H), 1.34 (s, 9H); HRMS (ESI) m/z calcd for C23H28NO
[M+H]+ 334.2171, found 334.2172.
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(E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-(p-tolyl)prop-2-en-1-amine
hydrochloride (5o).
Yield: 67%. 5o was synthesized by the general procedure of 1 given above as a
white solid, m.p. 180–182 ºC. 1HꢀNMR (400 MHz, MeOD) δ 7.93 (s, 1H), 7.82 (d, J
= 7.7 Hz, 1H), 7.51 (d, J = 7.3 Hz, 1H), 7.41 (t, J = 10.2 Hz, 3H), 7.22 (d, J = 7.3 Hz,
2H), 7.01 (s, 1H), 6.92 (d, J = 15.6 Hz, 1H), 6.41–6.28 (m, 1H), 4.83 (d, J = 13.5 Hz,
1H), 4.62 (d, J = 13.1 Hz, 1H), 4.18–4.07 (m, 1H), 4.04–3.89 (m, 1H), 2.86 (s, 3H),
2.37 (s, 3H); HRMS (ESI) m/z calcd for C20H22NO [M+H]+ 292.1701, found
292.1707.
(E)-N-(Benzofuran-7-ylmethyl)-3-(2-fluorophenyl)-N-methylprop-2-en-1-amine
hydrochloride (5p).
Yield: 53%. 5p was synthesized by the general procedure of 1 given above as a
yellow oil. 1HꢀNMR (400 MHz, MeOD) δ 7.93 (s, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.64
(dd, J = 17.1, 9.5 Hz, 1H), 7.53 (d, J = 7.3 Hz, 1H), 7.39 (dd, J = 14.8, 7.3 Hz, 2H),
7.23 (t, J = 7.5 Hz, 1H), 7.12 (dd, J = 19.1, 12.9 Hz, 2H), 7.04–6.94 (m, 1H), 6.60–
6.47 (m, 1H), 4.84 (d, J = 13.4 Hz, 1H), 4.65 (d, J = 13.2 Hz, 1H), 4.22–4.10 (m, 1H),
4.08–3.98 (m, 1H), 2.91 (s, 3H); HRMS (ESI) m/z calcd for C19H19FNO [M+H]+
296.1451, found 296.1452.
(E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-(2-nitrophenyl)prop-2-en-1-amine
hydrochloride (5q).
Yield: 74%. 5q was synthesized by the general procedure of 1 given above as a
white solid, m.p. 44–46 ºC. 1HꢀNMR (400 MHz, MeOD) δ 8.06 (d, J = 8.2 Hz, 1H),
7.94 (s, 1H), 7.85–7.71 (m, 3H), 7.66–7.51 (m, 2H), 7.40 (dd, J = 15.2, 7.2 Hz, 2H),
7.01 (s, 1H), 6.50–6.27 (m, 1H), 4.87 (d, J = 13.3 Hz, 1H), 4.68 (d, J = 13.3 Hz, 1H),
4.25–4.15 (m, 1H), 4.14–3.99 (m, 1H), 2.95 (s, 3H); HRMS (ESI) m/z calcd for
C19H19N2O3 [M+H]+ 323.1396, found 323.1393.
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(E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-(m-tolyl)prop-2-en-1-amine
hydrochloride (5r).
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Yield: 50%. 5r was synthesized by the general procedure of 1 given above as a
white solid, m.p. 161–162 ºC. 1H NMR (400 MHz, MeOD) δ 7.94 (s, 1H), 7.83 (d, J =
7.7 Hz, 1H), 7.51 (d, J = 7.4 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.38–7.23 (m, 3H),
7.19 (d, J = 7.4 Hz, 1H), 7.01 (s, 1H), 6.93 (d, J = 15.8 Hz, 1H), 6.39 (dt, J = 15.5, 7.6
Hz, 1H), 4.84 (d, J = 13.2 Hz, 1H), 4.63 (d, J = 12.6 Hz, 1H), 4.11 (d, J = 6.7 Hz, 1H),
4.00 (d, J = 7.7 Hz, 1H), 2.90 (s, 3H), 2.38 (s, 3H). HRMS (ESI) m/z calcd for
C20H22NO [M+H]+ 292.1701, found 292.1700.
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(E)-N-(Benzofuran-7-ylmethyl)-3-(3-fluorophenyl)-N-methylprop-2-en-1-amine
hydrochloride (5s).
Yield: 49%. 5s was synthesized by the general procedure of 1 given above as a
white solid, m.p. 130 ºC. 1H NMR (400 MHz, MeOD) δ 7.94 (s, 1H), 7.82 (d, J = 7.8
Hz, 1H), 7.52 (d, J = 7.4 Hz, 1H), 7.40 (dd, J = 16.9, 9.6 Hz, 2H), 7.33 (t, J = 9.3 Hz,
2H), 7.11 (t, J = 8.3 Hz, 1H), 7.01 (s, 1H), 6.95 (d, J = 15.8 Hz, 1H), 6.53–6.40 (m,
1H), 4.84 (d, J = 13.4 Hz, 1H), 4.65 (d, J = 13.0 Hz, 1H), 4.13 (d, J = 6.0 Hz, 1H),
4.02 (d, J = 7.5 Hz, 1H), 2.91 (s, 3H). HRMS (ESI) m/z calcd for C19H19FNO [M+H]+
296.1451, found 296.1453.
(E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-(3-nitrophenyl)prop-2-en-1-amine
hydrochloride (5t).
Yield: 55%. 5t was synthesized by the general procedure of 1 given above as a
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yellow oil. H NMR (400 MHz, MeOD) δ 8.38 (s, 1H), 8.20 (d, J = 8.2 Hz, 1H),
7.96–7.88 (m, 2H), 7.82 (dd, J = 20.2, 4.8 Hz, 1H), 7.63 (dd, J = 14.8, 6.8 Hz, 1H),
7.54 (d, J = 7.3 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.05 (d, J = 15.8 Hz, 1H), 6.97 (t, J
= 9.5 Hz, 1H), 6.69–6.53 (m, 1H), 4.85 (d, J = 13.3 Hz, 1H), 4.66 (d, J = 13.3 Hz, 1H),
4.20–4.12 (m, 1H), 4.08–4.01 (m, 1H), 2.92 (s, 3H); HRMS (ESI) m/z calcd for
C19H19N2O3 [M+H]+ 323.1396, found 323.1396.
(E)-N-(Benzofuran-7-ylmethyl)-3-(2,4-dichlorophenyl)-N-methylprop-2-en-1-ami
ne hydrochloride (5u).
Yield: 57%. 5u was synthesized by the general procedure of 1 given above as a
white solid, m.p. 49–51 ºC. 1HꢀNMR (400 MHz, MeOD) δ 7.90 (s, 1H), 7.78 (d, J =
7.5 Hz, 1H), 7.69 (d, J = 7.7 Hz, 1H), 7.50 (s, 2H), 7.36 (d, J = 7.2 Hz, 2H), 7.24 (d, J
= 15.9 Hz, 1H), 6.97 (s, 1H), 6.54–6.33 (m, 1H), 4.80 (d, J = 12.8 Hz,, 1H), 4.63 (d, J
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= 12.8 Hz, 1H), 4.17–3.95 (m, 2H), 2.81 (s, 3H); HRMS (ESI) m/z calcd for
C19H18Cl2NO [M+H]+ 346.0765, found 346.0750.
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1-(2,2-Diethoxyethoxy)-2-iodobenzene (6).
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To a solution of 2ꢀiodophenol (30.0 g, 136 mmol) in DMF (200 mL) was slowly
treated with sodium hydride (6.0 g, 250 mmol), and then the reaction was stirred at
room temperature for 30 min. Thereafter, 1ꢀbromoꢀ2,2ꢀdiethoxyethane (31.0 mL, 205
mmol) was added dropwise and the reaction was heated to 90 ºC overnight. After
cooling to room temperature, the mixture was partitioned between EtOAc and water.
The combined organic layers were washed with brine, dried over anhydrous Na2SO4,
filtered and condensed. The residue was then purified via flash chromatography on
silica gel, eluting with EtOAc/petroleum ether (1/25, v/v) to give 6 as a clear colorless
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oil. Yield: 93%. HꢀNMR (400 MHz, CDCl3) δ 7.77 (dd, J = 7.8, 1.4 Hz, 1H), 7.29
(dd, J = 11.7, 4.4 Hz, 1H), 6.86–6.78 (m, 1H), 6.72 (td, J = 7.7, 1.2 Hz, 1H), 4.90 (t, J
= 5.2 Hz, 1H), 4.04 (d, J = 5.2 Hz, 2H), 3.82 (tt, J = 14.1, 7.1 Hz, 2H), 3.77–3.69 (m,
2H), 1.28–1.23 (m, 6H).
7-Iodobenzofuran (7).
A stirred mixture of polyphosphoric acid (85.0 g) and 6 (40.0 g, 119 mmol) in
toluene (200 mL) was heated at reflux overnight. The reaction mixture was allowed to
cool to room temperature, and 1 M aq NaOH (20 mL) was added. The mixture was
partitioned between EtOAc and water. The combined organic layers were washed
with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue was
then purified via flash chromatography on silica gel, eluting with petroleum ether to
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give 7 as a clear colorless oil. Yield: 55%. HꢀNMR (400 MHz, Acetone) δ 7.95 (s,
1H), 7.70 (dd, J = 17.8, 7.5 Hz, 2H), 7.09 (d, J = 9.0 Hz, 2H).
Benzofuran-7-carbonitrile (8).
A solution of 7 (17.0 g, 70 mmol) and copper (I) cyanide (12.5 g, 139 mmol) in
DMF (150 mL) was heated at reflux overnight. The reaction was then cooled and
poured into water with plenty of concentrated ammonium hydroxide until the mixture
was clear. Thereafter, the mixture was partitioned between EtOAc and water. The
combined organic layers were washed with brine, dried over anhydrous Na2SO4,
filtered and condensed. The residue was then purified via flash chromatography on
silica gel, eluting EtOAc/petroleum ether (1/25, v/v) to give 8 as a white solid. Yield:
90%. 1HꢀNMR (400 MHz, CDCl3) δ 7.84 (t, J = 11.2 Hz, 1H), 7.77 (s, 1H), 7.61 (d, J
= 7.5 Hz, 1H), 7.33 (t, J = 7.7 Hz, 1H), 6.88 (s, 1H).
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Benzofuran-7-ylmethanamine (9).
To a solution of 8 (9.0 g, 63 mmol) in tetrahydrofuran (100 mL) at ꢀ78 °C under
a N2 atmosphere was treated dropwise with a suspension of lithium aluminum hydride
(4.8 g, 126 mmol) in tetrahydrofuran (100 mL). The mixture was stirred at room
temperature overnight and then carefully treated in succession with water (5 ml), 15%
sodium hydroxide (5 mL) and water (5 mL). The mixture was dried over anhydrous
Na2SO4, filtered through celite, and the filter cake was washed with tetrahydrofuran.
Ultimately, the filtrate concentrated under reduced pressure to give 9 as a yellow oil.
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Yield: 97%. HꢀNMR (400 MHz, MeOD) δ 7.77 (d, J = 1.9 Hz, 1H), 7.40 (t, J = 8.6
Hz, 1H), 7.29 (t, J = 7.7 Hz, 1H), 7.24 (d, J = 7.3 Hz, 1H), 7.00 (s, 1H), 4.05 (s, 2H).
tert-Butyl (benzofuran-7-ylmethyl)carbamate (10).
To a solution of 9 (9.0 g, 61 mmol) in tetrahydrofuran (100 mL) was added
sodium hydroxide (4.6 g, 122 mmol) and stirred for 5 min. After the addition of
diꢀtertꢀbutyl dicarbonate (21.1 mL, 92 mmol) in tetrahydrofuran (100 mL), the
reaction mixture was stirred for another 1 h and then filtered directly. The filter cake
was washed with tetrahydrofuran. The residue was then purified via flash
chromatography on silica gel, eluting with EtOAc/petroleum ether (1/25, v/v) to give
10 as a pale yellow solid. Yield: 83%. 1HꢀNMR (400 MHz, CDCl3) δ 7.64 (d, J = 1.6
Hz, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.37–7.10 (m, 2H), 6.78 (d, J = 2.1 Hz, 1H), 4.63
(s, 2H), 1.47 (s, 9H).
1-(Benzofuran-7-yl)-N-methylmethanamine (11).
To a solution of 10 (12.6 g, 51 mmol) in tetrahydrofuran (100 mL) at 0 °C under
a N2 atmosphere was treated dropwise with a suspension of lithium aluminum hydride
(7.7 g, 204 mmol) in tetrahydrofuran (50 mL). The reaction mixture was stirred at
reflux overnight and then carefully treated in succession with water (8 mL), 15%
sodium hydroxide (8 mL) and water (8 mL). The mixture was dried over anhydrous
Na2SO4 and filtered through celite, and the filter cake was washed with
tetrahydrofuran. Ultimately, the filtrate was concentrated under reduced pressure to
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give 11 as a yellow oil. Yield: 95%. HꢀNMR (400 MHz, CDCl3) δ 7.63 (d, J = 2.0
Hz, 1H), 7.51 (dt, J = 10.5, 5.2 Hz, 1H), 7.32–7.14 (m, 2H), 6.78 (d, J = 2.1 Hz, 1H),
4.07 (s, 2H), 2.48 (s, 3H).
(E)-3-Phenylprop-2-en-1-ol (12a).
To a solution of cinnamaldehyde (264.0 mg, 2 mmol) in methanol (10 mL) was
treated with sodium borohydride (76.0 mg, 2 mmol) in batches at 0 ºC. The reaction
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mixture was stirred at room temperature for 15 min and concentrated. The residue was
poured into water and extracted with EtOAc. The combined organic layers were
washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The crude
was used for the next step without further separation to give 12a as a yellow oil.
(E)-(3-Bromoprop-1-en-1-yl)benzene(13a).
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To a solution of 12a (357.0 mg, 2.7 mmol) in anhydrous ether (20 mL) was
treated with phosphorus tribromide (84.0 ꢁL, 0.9 mmol) at 0 ºC under a N2
atmosphere。 The reaction mixture was stirred at room temperature overnight and
poured into ice water containing sodium bicarbonate. The mixture was partitioned
between EtOAc and water. The combined organic layers were washed with brine,
dried over anhydrous Na2SO4, filtered and condensed at 30 ºC. The crude was used
for the next step without further purification to afford 13a as a white solid. Yield:
85%. 1HꢀNMR (400 MHz, CDCl3) δ 7.42–7.36 (m, 2H), 7.33 (t, J = 7.5 Hz, 2H), 7.27
(t, J = 5.2 Hz, 1H), 6.65 (d, J = 15.6 Hz, 1H), 6.40 (dt, J = 15.6, 7.8 Hz, 1H), 4.16 (d,
J = 7.8 Hz, 2H).
1-Bromo-3-(2,2-diethoxyethoxy)benzene (14).
Compound 14 was synthesized by the general procedure of compound 6 given
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above as a clear colorless oil. Yield: 95%. HꢀNMR (400 MHz, CDCl3) δ 7.07–7.14
(m, 3H), 6.86–6.78 (m, 1H), 4.82 (t, J = 5.2 Hz, 1H), 3.98 (d, J = 5.2 Hz, 2H), 3.72–
3.80 (m, 2H), 3.57–3.70 (m, 2H), 1.5 (t, J = 7.8 Hz, 6H).
4-Bromobenzofuran (15).
Compound 15 was synthesized by the general procedure of compound 7 given
above. Additionally, another product (6ꢀbromobenzofuran) was synthesized at the
same time. The crude mixture was used directly for next step without separation.
Benzofuran-4-carbonitrile (16).
Compound 16 was synthesized by the general procedure of compound 8 given
above. The crude was purified via flash chromatography on silica gel, eluting with
ethyl acetate/petroleum ether (1/25, v/v) to give 16 as a white solid. Yield: 30% for
two steps. 1HꢀNMR (400 MHz, CDCl3) δ 7.85 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 2.1 Hz,
1H), 7.61 (d, J = 7.5 Hz, 1H), 7.33 (t, J = 7.7 Hz, 1H), 6.88 (d, J = 2.2 Hz, 1H).
Benzofuran-4-ylmethanamine (17).
Compound 17 was synthesized by the general procedure given above using 9.
The crude mixture was used directly for next step without separation. Yield: 94%.
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1-(Benzofuran-4-yl)-N-methylmethanamine (18).
Compound 18 was synthesized by the general procedure of compound 11 given
above. Yield: 70% for 2 steps.1HꢀNMR (400 MHz, CDCl3) δ 7.64 (d, J = 2.3 Hz, 1H),
7.44 (d, J = 8 Hz, 1H), 7.24(t, J = 2.1 Hz, 1H), 7.16–7.18 (m, 1H), 6.89–6.88 (m, 1H),
3.99 (s, 2H), 2.47(s, 3H).
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(E)-Ethyl 4-phenylbut-3-enoate (19a).
To a solution of transꢀstyrylacetic acid (0.973 g, 6.2 mmol) in ethanol (10 mL)
was added thionyl chloride (893.0 ꢁL, 13.3 mmol) dropwise. The reaction mixture
was heated at reflux for 2 h and concentrated under reduced pressure. The residue was
poured into water and extracted with EtOAc. The combined organic layers were
washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The crude
was used for next step without further separation to give 19a as a clear colorless oil.
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Yield: 98%. HꢀNMR (400 MHz, CDCl3) δ 7.36 (t, J = 8.3 Hz, 2H), 7.31 (t, J = 7.5
Hz, 2H), 7.24 (dd, J = 13.2, 6.1 Hz, 1H), 6.49 (d, J = 15.9 Hz, 1H), 6.38–6.25 (m, 1H),
4.17 (q, J = 7.1 Hz, 2H), 3.24 (d, J = 7.0 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H).
(E)-4-Phenylbut-3-en-1-ol (20a).
To a solution of 19a (551.0 mg, 2.9 mmol) in anhydrous tetrahydrofuran (25 mL)
at 0 °C under a N2 atmosphere was treated dropwise with a solution of
diisobutylaluminium hydride (3.0 mL, 1.5 M in toluene). The reaction was stirred at
room temperature overnight and then carefully quenched with methanol. The mixture
was filtered through celite and condensed. The residue was then purified via flash
chromatography on silica gel, eluting with EtOAc/petroleum ether (1/5, v/v) to give
20a as a clear colorless oil. Yield: 71%. 1HꢀNMR (400 MHz, CDCl3) δ 7.40–7.19 (m,
5H), 6.50 (d, J = 15.9 Hz, 1H), 6.26–6.14 (m, 1H), 3.80–3.71 (m, 2H), 2.49 (q, J = 6.4
Hz, 2H).
(E)-(4-Bromobut-1-en-1-yl)benzene (21a).
Compound 21a was synthesized by the general procedure of compound 13a
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given above as a yellow oil. Yield: 57%. HꢀNMR (400 MHz, CDCl3) δ 7.41 (d, J =
7.5 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.26 (t, J = 7.1 Hz, 1H), 6.51 (d, J = 15.7 Hz,
1H), 6.20 (td, J = 7.0 Hz, J = 15.7 Hz, 1H), 3.51 (t, J = 7.2 Hz, 2H), 2.81 (q, J = 7.1
Hz, 2H)
Determination of water solubility.
The water solubilities of NTF, 5g, 5j, 5k, 5m and 5u were determined by an
HPLC method. Stock solutions (800 ꢁg/mL) of the samples were prepared in
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methanol. Then, 10 ꢁL dilute solutions with concentrations of 50, 100, 200, 400 and
800 ꢁg/mL were injected into the HPLC system to assess linearity. Calibration curves
were plotted as peak area versus concentration of the sample. Next, 10 mg of the
sample was added into a 5 mL centrifuge tube, and 1 mL pure water was pipetted into
the tube. If the solution was unsaturated and remained clear and transparent, more
testing of the compound was needed. After stirring for 24 h, the solution was filtered
with a syringe, and the HPLC system was injected with the same 10 ꢁL. Water
solubility was calculated by comparing the peak area of the tested compound to the
calibration curves
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Pigment inhibition assay.
The tested compounds were dissolved in DMSO to 20 mM as a stock
concentration. Serial 2ꢀfold dilutions were prepared from the stock solution with
DMSO. S. aureus Newman bacteria were cultured in TSB (4 mL) in the presence of
40 ꢁL inhibitors at 37 ºC for 48 h in triplicate. Then, 3 mL bacteria cultures were
centrifuged and washed twice with PBS. The pigment was extracted with methanol.
The OD was determined at 450 nm using a NanoDrop 2000c (Thermo scientific)
spectrophotometer. IC50 values were obtained by fitting the OD data to a normal
doseꢀresponse curve using the Graphpad Prism 5.0 software. IC50 values of the MRSA
strains USA300 LAC, USA400 MW and Mu 50 were conducted in the same way.
High-performance liquid chromatography (HPLC).
Strains were cultured overnight in tubes and diluted in 50 mL of TSB fresh
medium (100 ꢁM naftifine hydrochloride or 1 ꢁM of 5m was used where appropriate,
as indicated). Cells were further cultured with shaking at 37 °C for 24 h and harvested
by centrifugation. Briefly, wet cells harvested from 50 mL of cultures were extracted
with 20 mL of acetone. To this, 10 mL of hexane and 10 mL of aqueous NaCl (10%,
wt/vol) were added, and the mixture was shaken vigorously to remove the oily lipids.
The upper phase containing the carotenoids was dried with anhydrous MgSO4 and
concentrated in a rotary evaporator. Finally, a 300ꢀꢁL acetonitrile/isopropanol mixture
(85:15, v/v) was used to dissolve the extracts. The dissolved extracts were filtered,
and 50 ꢁL samples were analyzed through a Spherisorb ODS2 column (250 × 4.6 mm;
particle diameter, 5 ꢁm; Waters) and eluted with an acetonitrileꢀisopropanol mixture
(85:15, v/v) at a flow rate of 1 mL/min using an Alliance highꢀpressure liquid
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chromatography (HPLC) system (Agilent 1260 Infinity) equipped with a photodiode
array detector.
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CrtN enzyme inhibition assay.
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The expression of CrtN and the inhibition assay were performed using our
previously reported methods.19 In short, diapophytoene was purified from
diapophytoeneꢀproducing E. coli BL21 (DE3)/pET28a::crtM by extraction with
acetone. The emulsion of diapophytoene was prepared by dissolving 24 mg
phosphatidylcholine (SigmaꢀAldrich) in 200 ꢁL CHCl3 and mixed with 8 mg
diapophytoene. The mixture was spun drying and incubated with 2 mL 0.02 M
HEPES buffer (pH 7.5), then sonicated in ice water to obtain a homogeneous
emulsion and stored at ꢀ80 °C until use. E. coli BL21 (DE3)/pET28a::crtN was
subꢀcultured into 1,000 mL of LB broth supplemented with 50 ꢁg/mL kanamycin to
obtain an optical density at 600 nm (OD600) of approximately 0.1 and grown to an
OD600 of ~0.5. Expression of 6HisꢀCrtN was induced with 0.5 mM
isopropylꢀβꢀDꢀthiogalactoside (IPTG) overnight at 16 °C. Cells were harvested, and
the pellets were suspended in 30 mL HEPES buffer and lysed at 4 °C by sonication.
Enzyme activity was performed in triplicate, with a total of 700 ꢁL of the
following: 276.5 ꢁL 0.02 M HEPES buffer (pH 7.5), 50 ꢁL diapophytoene emulsion,
70 ꢁL different concentrations of compounds or mock (ddH2O), 3.5 ꢁL FAD stock
solution (10 mM), and 300 ꢁL CrtN lysate (~1.41 mg CrtN, estimated by Western blot
using known concentration of the purified 6HisꢀcrtN protein). The tests were
performed under anaerobic atmosphere by adding a final concentration of 20 U/mL
glucose oxidase (SigmaꢀAldrich, G2133), 20000 U/mL catalase (SigmaꢀAldrich,
C1345), and 2 mM glucose as an oxygenꢀtrapping system. The reaction mixture was
initiated by adding the lysate and incubated overnight at 37 ºC. The reaction was
terminated by methanol. Pigments were extracted twice against 700 ꢁL chloroform.
The organic phase was combined, concentrated, reꢀdissolved in 200 ꢁL chloroform,
and analyzed by reading OD value under 450 nm. IC50 values were obtained by fitting
the OD data to a normal doseꢀresponse curve using the Graphpad Prism 5.0 software.
Bacterial growth assays of S. aureus Newman and MRSA strains.
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5m was dissolved in DMSO to 20 mM as a stock concentration and diluted with
fresh TSB medium enabling the final concentration of 5m of either 0.2 mM or 0.05
mM. 100 µL of the dilution was distributed into 96ꢀwell plates and growth controls
(containing an equal amount of DMSO). A 60ꢀµL paraffin wax was used to cover the
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