Novel Terminal Bipheny-Based Diapophytoene Desaturases (CrtN) Inhibitors as Anti-MRSA/VISR/LRSA Agents with Reduced hERG Activity

Article abstract of DOI:10.1021/acs.jmedchem.7b01300

CrtN has been identified as an attractive and druggable target for treating pigmented Staphylococcus aureus infections. More than 100 new compounds were synthesized, which target the overwhelming the defects of the CrtN inhibitor 1. Analogues 23a and 23b demonstrated a significant activity against pigmented S. aureus Newman and 13 MRSA strains (IC₅₀ = 0.02-10.5 nM), along with lower hERG inhibition (IC₅₀ > 30 μM, ～10-fold decrease in comparison with 1). Furthermore, 23a and 23b were confirmed to reduce the staphylococcal load in the kidney and heart in a mouse model with normal treatment deeper than pretreatment ones, comparable even with vancomycin and linezolid. Remarkably, 23a could strongly block the pigment biosynthesis of these nine multidrug-resistant MRSA strains, including excellent activity against LRSA strains and VISA strains in vivo, and all of which demonstrated that 23a has a huge potential against intractable MRSA, VISA, and LRSA issues as a therapeutic drug.

Full text of DOI:10.1021/acs.jmedchem.7b01300

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Article
Novel Terminal Bipheny-Based Diapophytoene Desaturases (CrtN)
Inhibitors as Anti-MRSA/VISR/LRSA Agents with Reduced hERG Activity
Baoli Li, Shuaishuai Ni, Fei Mao, Feifei Chen, Yifu Liu,
Hanwen Wei, Wenhua Chen, Jin Zhu, Lefu Lan, and Jian Li
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01300 • Publication Date (Web): 15 Dec 2017
Downloaded from http://pubs.acs.org on December 15, 2017
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Novel Terminal Bipheny-Based Diapophytoene Desaturases
(CrtN) Inhibitors as Anti-MRSA/VISR/LRSAAgents with
Reduced hERG Activity
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a, †
a, †
a, †
b
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Baoli Li , Shuaishuai Ni , Fei Mao , Feifei Chen , Yifu Liu , Hanwen Wei ,
a
a
b,
a,
Wenhua Chen , Jin Zhu , Lefu Lan *, and Jian Li *
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
†
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
CrtN has been identified as an attractive and druggable target for treating
pigmented S. aureus infections. More than 100 new compounds were synthesized that
target the overwhelming the defects of the CrtN inhibitor 1. Analogs 23a-b
demonstrated significant activity against pigmented S. aureus Newman and thirteen
MRSA strains (IC50 = 0.02~10.5 nM), along with lower hERG inhibition (IC50 > 30
ꢀM, ~10ꢁfold decrease in comparison with 1). Furthermore, 23a-b were confirmed to
reduce the staphylococcal load in the kidney and heart in a mice model with normal
treatment deeper than pretreatment ones, comparable even with vancomycin and
linezolid. Remarkably, 23a could strongly block the pigment biosynthesis of these
nine multidrugꢁresistant MRSA strains, including excellent activity against LRSA
strains and VISA strains in vivo, all which demonstrated that 23a has huge potential
against intractable MRSA, VISA and LRSA issues as a therapeutic drug.
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Introduction
Staphylococcus aureus (S. aureus), a major human pathogen, causes serious skin
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and soft tissues infections, respiratory disease and more serious illness like
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pneumonia, endocarditis and sepsis. Infections caused by S. aureus has gained
growing concern worldwide due to its capability for the rapid development of drug
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resistance induced by antibiotic misuse. Resistance to antibiotics represents one of
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the most pressing challenges to the infectious disease community. Increasing rates
of infectious diseases caused by different methicillinꢁresistant Staphylococcus aureus
(MRSA) strains have resulted in high infiltration of hospital and community
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settings.
Antibiotics that are approved for treatment of MRSA infections
includevancomycin (VAN, glycopeptide antibiotic), linezolid (LZD, oxazolidinone
antibiotic), daptomycin (lipopeptide antibiotic), dalbavancin (lipoglycopeptide
antibiotic), oritavancin (glycopeptide antibiotic), ceftaroline (βꢁlactam antibiotic) and
tedizolid (anoxazolidinone antibiotic). The appearance of VANꢁintermediate
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resistance MRSA (VISA/MRSA) and LZDꢁresistant MRSA (LRSA/MRSA) has
especially left patients vulnerable. Recently, a report by the U.S. Centers for Disease
Control and Prevention (CDC) stated that more than 2 million people have suffered
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MRSA infections, and 23,000 people have died directly due to these infections.
Great importance has been attached by governments and organizations worldwide,
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including the ND4BB project initiated by the European Union and National Efforts
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by the United States. On 27 February 2017, the World Health Organization (WHO)
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released its first list of the world's deadliest superbugs, a catalog of 12 families of
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bacteria. The treatment of MRSA (VISA or LRSA) is urgent at high priority tiers.
Antiꢁvirulence strategies aiming at “disarming” the pathogen rather than
inhibiting growth with weak selective pressure for the development of antibiotic
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resistance are now gaining interest. Staphyloxanthin (STX) is an important virulence
factor for pigmented S. aureus. Nonpigmented S. aureus is susceptible to be killed by
reactive oxygen species. Hence, blocking STX biosynthesis is an underlying
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magnetically therapeutic target against all pigmented MRSA. STX biosynthesis
begins with the condensation of farnesyl diphosphate followed by a series of catalytic
reactions of important enzymes (CrtM, CrtN, etc.). In 2008, Eric Oldfield and
coworkers reported a CrtM inhibitor (BPHꢁ652), which was introduced in our
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previous work, and based on this work, several types of CrtM inhibitors have been
identified by the same group, and BPHꢁ652 was selected for these experiments
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because it had a good IC50 in pigment inhibition (110 nM). The same group
continued to develop a series of CrtM inhibitors (published in 2009) and the most
active compounds are halogenꢁsubstituted phosphonosulfonates, with Ki values as low
as 5 nM against the enzyme and IC50 values for STX inhibition in S. aureus as low as
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11nM.
Furthermore, in 2012, Fuyang Lin and coworkers reported Xꢁray
crystallographic structures of three inhibitors bond to CrtM, and their results provided
structural clues for the mechanism and inhibition of the headꢁtoꢁhead prenyl
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In our previous work, we demonstrated the enzyme CrtN to be an attractive and
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druggable target for fighting pigmented S. aureus infection and a potent CrtN
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inhibitor 1 (5m , Figure 1), which could inhibit the STX biosynthesis of S. aureus
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Newman and three MRSA strains, sensitize four strains to immune clearance and
effectively attenuate the virulence of three strains in vivo. However, prophylactic
administration (24 h before infection) with highꢁdosage treatment (200 mg/kg) of 1,
along with the high hERG inhibition (IC50 = 3.71 ꢀM), was impractical for clinical
application.
Figure 1
In this study, we synthesized a series of CrtN inhibitors with new scaffolds,
which not only demonstrated excellent activity against MRSA in vitro and in vivo at
lower dosage, but also overcame the disadvantages of hERG inhibition. Linezolid and
vancomycin, the lastꢁresort antimicrobial agents, were introduced as positive control
drugs into a murine model of S. aureus abscess formation to evaluate the effectiveness
of our new analogues.
Design and Chemistry
In previous work, the varied scaffold and series of different substituents about
benzofuranꢁderived CrtN inhibitors were not enough to investigate the relationship of
structure and activity comprehensively. In this study, to further improve the affinity of
the lead compound 1 and obtain novel structural scaffolds, chemical modifications
were performed in four cycles. First, in region A (Figure 1), we first explored different
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heteroaromatic rings and the related positions of substitutions (in region A) to
generate novel scaffold compounds 2aꢁj. In general, we observed that pigment
inhibition varied greatly by the scaffold hopping, and compound 2b revealed the
strongest efficiency among these derivatives, which was enough for further
modification study. Additionally, considering the structural diversity, compound 2j,
the secondꢁbest scaffold, was also identified as lead compound. Then, we replaced the
Nꢁmethyl group of either 2b or 2j in region B (Figure 1) with various steric alkyl
group (including hydrogen atom) and 6 analogues (3a-f) were designed (Table 1).
Subsequently, we incorporated different substituted phenyls, furanyl, naphthalenyls,
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(
cyclo)alkyls at the section C of either 2b or 2j, and seventyꢁfour analogs (4a-40a and
4b-40b) were further designed (Table 2ꢁ3). In addition, 10 analogs (41a-45a and
1b-45b, Table 4) were prepared to evaluate whether the varies of linkers (allyl in
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region D) affected pigment inhibitory activity.
The synthesis routes of derivatives 2a-d and 5a-40a were outlined in Scheme 1.
Intermediate 47 could be synthesized from commercial indole derivatives 46b via
reductive amination, and further coupled with transꢁ4ꢁfluorocinnamaldehyde by the
reductive amination to afford the target analog 3a. Replacing the methyl with ethyl
group or isopropyl group was to afford target analogs 3bꢁc. In parallel, 46a-b were
suffered with methylamine under the reaction of reductive amination to yield
intermediates 48aꢁb. Various substituted acraldehydes were reduced by sodium
borohydride, and accompanied with the bromination reaction for obtaining the
intermediates 49a-z and 50a-k. Finally, the nucleophilic reactions of compounds
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1a-z and 52a-k with 48a-b yielded target analogs 2a-d and 5a-40a. Meanwhile,
derivatives 2e-j, 3d-f and 5b-40b were prepared as described in scheme 2 and
synthesis of these derivatives corresponded closely to scheme 1
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Scheme 1-2
Scheme 3 outlines the synthesis strategy for the synthesis of derivatives 41a-b.
Coupling
tertꢁbutyldimethyl(propꢁ2ꢁynꢁ1ꢁyloxy)silane gave 56, which was reduced by TBAF to
give 57, and further brominated by PBr to give intermediate 58. The coupling of
of
commercially
obtained
4ꢁiodoꢁ1,1'ꢁbiphenyl
and
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intermediate 58 with either 48b or 55f yielded analogs 41a-b.
Scheme 3
Derivatives 42a-b were prepared as described in scheme 4. 49t was reduced by
sodium borohydride, followed by bromination to give intermediate 60. The
nucleophilic substitution of intermediate 60 with 48b or 55f yielded analogs 42a-b
Scheme 4
Scheme 5 outlines the synthesis strategy for the synthesis of derivatives 43a-b.
By the WittigꢁHorner olefination, 61 was yielded from [1,1'ꢁbiphenyl]ꢁ4ꢁcarbaldehyde,
then through reduction and bromination, intermediate 63 was generated. The
nucleophilic substitution of intermediate 63 with 48b or 55f yielded analogs 43a-b.
Scheme 5
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Scheme 6 outlines the synthesis strategy used for the synthesis of derivatives
44a-b. Through WittigꢁHorner olefination, intermediate 64 was yielded from
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1,1'ꢁbiphenyl]ꢁ4ꢁcarbaldehyde, then through reduction and bromination, intermediate
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66 were generated. The nucleophilic substitution of intermediate 66 with 48b or 55f
yielded analogs 44a-b.
Scheme 6
As shown in scheme 7, by the WittigꢁHorner olefination, intermediate 67 was
yielded from (E)ꢁ3ꢁ([1,1'ꢁbiphenyl]ꢁ4ꢁyl)acrylaldehyde, followed by reduction of the
ethyl ester and bromination reaction to generate 69. The nucleophilic substitution of
compounds 69 with 48b or 55f yielded analogs 45a-b.
Scheme 7
Results and Discussion
In total, 100 novel derivatives (2a-j, 3a-f, 4a-45a and 4b-45b) were designed
and synthesized. Their chemical structures are shown in Tables 1ꢁ4. The details of the
synthesis procedures and structural characterization are described in the Supporting
Information. All derivatives were confirmed to have ≥ 95% purity, and were identified
with nonꢁPAINS on the web at http://fafdrugs3.mti.univparisꢁdiderot.fr/ recommended
by editors from the ACS (American Chemical Society).
In vitro Pigment Inhibitory Activities against S. aureus Newman
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These synthesized compounds were evaluated for pigment inhibitory activities
against S. aureus Newman and the results are shown in Table 1ꢁ4. We replaced the
Nꢁmethyl group with various steric alkyl groups (including hydrogen), and 6 analogs
(3aꢁf) were designed (Table 1). As shown in Table 1, in the respect of the substituents
with various alkyl groups at N atom (including hydrogen atom, 2b, 2j, 3aꢁf),
compounds with Nꢁmethyl group exhibited the best pigment inhibitory activity (2b vs
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3aꢁc; 2j vs 3dꢁf), indicating that the Nꢁmethyl group is the optimal substituent for
pigment inhibition.
Table 1
3
As shown in Table 2ꢁ3, substitution of phenyl with various types of groups (R )
could significantly affect pigment inhibitory activity. Generally, (cyclo)alkyl or
heteroaromatic or 1ꢁnaphthyl groups were detrimental to pigment inhibitory activity
(
4a-9a vs 2b, 4b-9b vs 2j), while the introduction of 2ꢁnaphthyl group was favorable
10a vs 2b), but inferior to analogue 2j. Our previous studies found that the
(
electronꢁwithdrawing groups and electronꢁdonating groups at the phenyl ring may
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have minor effects on activity. To investigate the effect of substituted groups, more
electronꢁwithdrawing (fluoro, chloro, bromo, trifluoromethyl, difluoromethyl, nitro,
phenyl, formate, cyan) and electronꢁdonating groups (methyl, methoxyl, ethoxyl,
tꢁbutyl) were introduced at the phenyl ring. The results in Table 2ꢁ3 demonstrated that
there was no relationship between activity and electronic effect. However, the
substituted positions at the phenyl ring significantly affected the activity, and
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substitution at the para position of the phenyl ring showed the best activity (12a vs 24a
vs 30a, 12b vs 24b vs 30b). Moreover, the substitution at the paraꢁposition with chloro,
bromo, methyl, nitro, difluoromethyl, formate and phenyl (12a, 14a, 15a, 18a, 19a,
22a, 23a and 23b) resulted in significant improvements in activity. Especially, analog
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23a (IC50 = 2.0 ± 0.1 nM) and 23b (IC50 = 3.3 ± 0.4 nM) displayed better potency than
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other derivatives. Notably, the R group of both 23a and 23b was 4ꢁphenylphenyl
(biphenyl).
Table 2-3
As shown in Table 4, compounds 41a-45a and 41b-45b were assessed to
determine whether the allyl linker had influence on the pigment inhibition potency.
The results indicated that the change of the allyl linker moiety was detrimental to
improve pigment inhibitory activity. When the allyl linker was replaced by propargyl
(41b, IC50 = 101.8 ± 5.2 nM) or vinyl (45b, IC50=19.4 ± 0.2 nM), the pigment
inhibitory activity decreased. Additionally, other linkers listed in Table 4 were
detrimental to the pigment inhibitory activity.
Table 4
Structure-Activity Relationship
Based on the structural features and pigment inhibitory activities data, the SARs
are summarized in detail. (1) Nꢁsubstituent variation exerts great influence on pigment
inhibitory activity (2b vs 3a-c, 2j vs 3d-f), and the Nꢁmethyl is optimal; (2) The impact
of the electronic characteristics of substituent in the phenyl ring on the potency is
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weak, while the impact of the substituted position is critical. The substituent group on
the para position can enhance the potency significantly(12a vs 24a vs 30a, 12b vs 24b
vs 30b); (3) Replacement the phenyl ring with furanyl or thiophene or (cyclo)alkyl is
not tolerable to the potency (2b vs 7a/8a, 2j vs 7b/8b); (4) the unsubstituted allyl
linker is critical for high potency and the other linkers are on the contrary (2b vs
1
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41a-45a, 2j vs 41-45b).
Cytotoxicity of human embryonic kidney cell (HEK-293) and human
hepatocellular carcinoma cell (HepG2)
Based on the in vitro pigment inhibitory activities, four compounds (12a, 14a and
23a-b), whose IC50 values of pigment inhibitory activity of which are lower than 5nM,
were selected to for cytotoxicity evaluation in HEKꢁ293 and HepG2. As shown in
Table 5, in comparison with amphotericin B, all compounds displayed far less
cytotoxicity, especially, 23a (CC50 = 48.1 µM by HEKꢁ293, and CC50 = 105.8 µM by
HepG2, respectively) were superior than amphotericin B (CC50 = 4.2 µM by
HEKꢁ293, and CC50 = 3.1 µM by HepG2, respectively).
Table 5
In vitro Pigment Inhibitory Activities against four MRSA strains
29ꢁ30
USA400 MW2 and USA300 LAC,
two clones liable for the epidemic of
communityꢁacquired MRSA (CAꢁMRSA) infectious diseases in the United States;
Mu50, a hospitalꢁacquired MRSA (HAꢁMRSA) strain with VAN intermediate
resistance MRSA (MRSA/VISA); NRS271, a linezolid resistance MRSA
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1ꢁ32
(
MRSA/LRSA);
these four MRSA strains (Table S4, supporting information)
were used to investigate pigment inhibition activity of 23a-b. The results are shown in
Table 6. We were delighted to find that the color of all strains (USA400 MW2,
USA300 LAC, Mu50 and NRS271) faded by the incubation with our compounds
0
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3a-b, and the IC50 value against MRSA were comparable with that against S. aureus
Newman. Moreover, according to the results of the bacterial growth assay (Figure 2),
different from conditional antibiotics, 23a-b did not affect the growth of S. aureus
strains and MRSAs, even given a high concentration (0.2 mM).
Table 6
Figure 2
In vitro Water Solubility, hERG Inhibition and CrtN Enzymatic Inhibitory
Activities
Compounds with high affinity for the hERG ion channel could induce QT
interval prolongation, which is frequently related to potentially lethal arrhythmias
(
Table S2, Supporting Information). As shown in Table 7, we tested hERG inhibition
activity of 23a-b, and surprisingly found their IC50 values exceeded 30 µM, which
achieved our top priority to decrease the heart cardiotoxicity and gave us much
confidence for further development. We next evaluated their capacities of inhibiting
28
the enzymatic activity of CrtN in vitro. Using our previous protocol, both
compounds were found to significantly inhibitenzymatic activity of CrtN at
nanomolar concentrations (Table 7), However, although 23a-b represented excellent
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pigment inhibitory activity and cytotoxicity and hERG inhibition, water solubility of
23a-b decreased a lot than 1 (from 10.0 mg/mL to about 0.2 mg/mL), due to the
lipophilic biphenyl.
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1
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Table 7
In vitro Metabolic Stability of 23a-b in Human Liver Microsome
Human liver microsome assay was utilized to preliminarily evaluate the effect of
the stability of compounds in liver in vitro. As shown in Table 8, we took midazolam
as controlꢁcompound as for its fast clearance rate in human liver microsome, and
23a-b showed significant stability (t = 50.7 mins of 23a and t = 93.7 mins of 23b).
1/2
1/2
Table 8
In vitro Anti-fungal Activities of 23a-b.
2
6
Because leading compound 1 originated from the antiꢁfungal agent Naftifine,
we would like to investigate whether our compound reserved the capacity of
antifungal. As shown in Table 9, three fungus strains and three firstꢁline drugs were
chosen to proceed with the in vitro assay. Compared with the three positive control
groups, 23aꢁb exhibited weak activities against all three dermatophytes, which
verified our compounds losing theirs antifungal activities.
Tables 9
Effects of 23a-b on Sensitizing S. aureus to Immune Clearance.
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Since pigment could serve as a protective antioxidant to confer the resistance to
immune clearance, we speculated that nonꢁpigment S. aureus could be vulnerable to
be killed by the block of STX with the treatment of our CrtN inhibition. Herein, two
assays were introduced to investigate the effect of 23a-b on sensitizing S. aureus to
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2 2
immune clearance in vitro, including hydrogen peroxide (H O ) killing assay and
human whole blood killing assay. To verify this argument experimentally, we first
compared the susceptibility of mockꢁtreatment with 23aꢁtreated (1 µM) S. aureus to
H
2
O
2
killing. As shown in Figure 3, nonꢁpigmented S. aureus cells were more
vulnerable to be killed by 1.5% H O compared to the untreated S. aureus (mock)
2
2
(survival, 3.0% vs 33.3%). In parallel, the survival of the known antioxidant
nacetylcysteine (NAC)ꢁtreated S. aureus cells were far worse than the mock treatment
as expected (51.3% vs 33.3%). Similarly, the survival percentage of the three MRSA
strains greatly decreased (2.3% vs 28.7% in USA400 MW2; 2.3% vs 29.0% in
USA300 LAC; 2.7% vs 24.3% in Mu50); correspondingly, the survival rates of the
three NACꢁtreated MRSA strains worsened (54.3% vs 28.7% in USA400 MW2,
52.0% vs 29.0% in USA300 LAC, 53.0% vs 24.3% in Mu50). All results proved that
the addition of H (with strong oxidation) exerted an impact on the MRSA strain
2
O
2
survival, and the pigment definitely acted as the protective antioxidant. Subsequently,
the other experiment was to compare the effect of analogꢁtreated S. aureus with those
of nonꢁtreated ones by human whole blood killing. The fresh human whole blood was
added to 23aꢁtreated (1 µM) and untreated S. aureus, and the bacterial survival was
measured. As shown in Figure 4, untreated S. aureus survived significantly better than
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the 23aꢁtreated S. aureus (survival, 13.3% vs 1.3% in Newman, 20.0% vs 1.0% in
USA400 MW2, 12.3% vs 0.7% in USA300 LAC, 9.6% vs 0.4% in Mu50. We then
repeated the same experiment for 23b, and the analysis was identical to that of 23a.
All collected results suggested that 23aꢁb could render S. aureus more susceptible to
immune clearance.
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Figure 3-4
In vivo Effects of 23a-b on Attenuating the Virulence of S. aureus Newman
and Two MRSA Strains.
Since 23aꢁb had promising activities in vitro, we next investigated in vivo
efficacy in a systematic infection model. We subjected S. aureus Newman to a murine
model of abscess formation via retroꢁorbital injection and measured the bacterial
survival in the host organs. It was worth stressing that we first conducted the normal
treatment procedure (giving the compounds 6 h after infection). As shown in Figure
7
5
AꢁB, the mice were injected with 2.3 × 10 colonyꢁforming units (CFU) of S. aureus
Newman bacteria via retroꢁorbital injection. We provided the compounds with four
different drug regiments—i.e., both 0.4 mg b.i.d. (giving the compounds twice a
day)/4.5 d (180 mg/kg in total), and 0.1 mg b.i.d./4.5 d (45 mg/kg) in the pretreatment
case (pretreatment with drugs or compounds 24 h in advance of the infection), along
with both 0.4 mg b.i.d./3.5 d (140 mg/kg) and 0.1 mg b.i.d./3.5 d (35 mg/kg) in the
normal treatment case (administered 6 h after the infection of strains), while LZD and
VAN were set up as two positive control groups at the dosage of 0.4 mg b.i.d./4.5 d
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(
180 mg/kg) with pretreatment. The mice were sacrificed after 4.5 d, and we measured
bacterial survival in kidneys and hearts. First, we analyzed 23aꢁb in terms of
attenuating the virulence of Newman (Figure 5AꢁB). Compared to the untreated group,
the staphylococcal loads of all 23a treated groups were significantly lower in kidneys
and hearts (p < 0.01), while 23bꢁtreated groups were not as obvious. Surprisingly,
both 23aꢁtreatment and 23bꢁtreatment with low dosage in the normal treatment group
reduced bacterial load more than in the other three groups. For further investigation,
we performed the same experiment on infection with Mu50 (VISA/MRSA) and
NRS271 (LRSA/MRSA). The analysis of the data in Figure 5CꢁF revealed that
compoundꢁtreatment groups had significantly decreased Mu50 and NRS271
staphylococcal loads in the kidneys and hearts, all of which corresponded to a greater
than 95.0% decrease in surviving bacteria (significance P < 0.001), while all
lowꢁdosage compoundꢁtreatment groups were better than the highꢁdosage ones.
Encouragingly, all lowꢁdosage groups were not inferior to the positiveꢁcontrolled ones;
especially, 23aꢁtreatment with low dosage (0.1 mg b.i.d./3.5 d) in the normal
treatment case displayed best activity again among the groups.
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Figure 5
Along with the encouraging outcome in vivo described above, we concluded
2
3aꢁtreatment with low dosage in the normal treatment group to be the group with the
greatest potential. To confirm the most appropriate treatment dosage, we lowered the
dosage to 0.05 mg b.i.d./3.5 d (17.5 mg/kg in total) and 0.01 mg b.i.d./3.5 d (3.5
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mg/kg). As shown in Figure 6AꢁB, 23a (17.5 mg/kg) significantly decreased the
Mu50 staphylococcal loads in kidneys by 1.09 log10 CFU (more than 91.8% decrease)
and in hearts by 2.70 log10 CFU (more than 99.8% decrease); however, it was not
comparable to the 0.1 mg b.i.d./3.5 d (35 mg/kg) group. Generally, we confirmed
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23aꢁtreatment with 0.1 mg b.i.d./3.5 d (35 mg/kg) dosage in normal treatment still as
the best treatment. To further evaluate the efficacy of 23a at affecting the outcome of
8
S. aureus sepsis, we challenged animals with 2 × 10 CFU Newman bacteria. The
untreated mice nearly died out (90% animal) within 4 days, with 23a resulting in 80%
animal survival. By the eighth day, more than 70% of the 23aꢁtreated mice were alive,
demonstrating a slight advantage over the 1ꢁtreated group (Figure 7). This
investigation clearly proved that the in vivo 23aꢁtreatment weakened the virulence of
S. aureus Newman.
Figure 6-7
In vitro Pigment Inhibitory Activities against additional 9 MRSA Strains.
In addition to four S. aureus strains above, 9 additional multidrugꢁresistant
33
MRSA strains—NRS70, NRS100, NRS108, LRSA56, LRSA202, LRSA205,
3
4
35
HS663, NF65Y and XN108 (Table S4, supporting information), were selected to
further explore the effect of the lead compound 23a against multiple resistant bacteria.
First, the pigment inhibition of 23a were conducted, and the results were shown in
Table 10 indicated that 23a could strongly block the pigment biosynthesis of these
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nine MRSA strains (IC50=0.02ꢁ10.5 nM), which meant the adaptability of our
compound against MRSAs in vitro.
Table 10
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In vivo Effects of 23a on Attenuating the Virulence of S. aureus LRSA102,
LRSA56, NF65Y and XN108.
Following, based on in vitro pigment inhibitory activities (Table 10) and MIC
values of the nine MRSA strains (Table S4, Supporting Information), we chose four
representative MRSA strains, LRSA56 (LRSA), LRSA202 (LRSA), NF65Y (VISA)
and XN108 (VISA) to further investigate in vivo effects of 23a in vivo (0.1 mg
b.i.d./3.5 d, in normal treatment, Figure 8).
7
As shown in Figure 8A , the mice were injected with 5.0 × 10 CFU of S. aureus
LRSA56 bacteria via retroꢁorbital injection. Compared with untreated group,
23aꢁtreatment decreased the LRSA56 loads in kidneys by 0.77 log10 CFU (more than
83.0% decrease) and in hearts by 0.73 log10 CFU (more than 81.4% decrease).
Compared with LZDꢁtreated group, 23aꢁtreatment decreased the LRSA56 loads in
kidneys by 0.34 log10 CFU (more than 54.3% decrease) and in hearts by 0.55 log10
CFU (more than 71.8% decrease), however, it showed no significant difference
compared to the VANꢁtreatment group. As shown in Figure 7B, the analysis was just
like Figure 7A, 23a could be comparable to LZD, but worse than VAN.
As shown in Figure 7CꢁD, for VISAꢁresistance bacteria NF65Y and XN108, all
the 23aꢁtreatment groups had significantly reduced S. aureus loads (p < 0.001), and
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was comparable to the positive control groups, even though LAZ and VAN were
given at higher doses. Especially, in Figure 7C, 23aꢁtreatment group showed
extraordinary activity, exceeding VAN with significance p < 0.01. Consequently, we
were quite confident that 23a was the best candidate for further development.
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Figure 8
In vivo Effects of 23a on Attenuating the Virulence of S. aureus Newman,
Mu50 and NRS271 with Oral Administration.
Because all in vivo investigations above were through intraperitoneal injection,
oral administration was utilized for more investigations about curative effects of 23a
(0.4 mg b.i.d./3.5 d, 0.2 mg b.i.d./3.5 d and 0.1 mg b.i.d./3.5 d, in normal treatment,
Figure 9). We kept VAN and LZD as positive control groups (0.4 mg b.i.d./4.5 d, in
pretreatment).
7
As shown in Figure 9AꢁB, the mice were injected with 3.1× 10 CFU of S.
aureus Newman bacteria via retroꢁorbital injection. In the kidneys, compared with
untreated group, 23aꢁtreatment decreased the Newman loads by 1.00 log10 CFU
(90.0% decrease), but there was no significant difference in hearts. Compared with
VANꢁtreated group, 23aꢁtreatment decreased the Newman loads in hearts by 0.38
log10 CFU (more than 55.3% decrease with significance p < 0.1.). However, all
23aꢁtreatment groups were not better than positiveꢁcontrolled groups. As for Figure
9CꢁF, the analysis was just like that of Figure 9A, the best dosage of the three
23aꢁtreatment groups was still 0.2 mg b.i.d./ 3.5 d, and not better than VAN and
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LZDꢁtreatment groups. Consequently, the most appropriate dose of 23a was 0.2 mg
b.i.d./ 3.5 d with oral administration, which was not comparable to intraperitoneal
injection.
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Figure 9
In vitro Effects of 23a on CYP Enzymatic Inhibitory Activity.
The study on CYP drug metabolizing enzymatic could be used to predict the side
effect of compounds in liver in vitro. The data in Table 11 showed the CYP enzymatic
inhibition of 23a, and the results indicated that 23a exhibited no significant inhibition
of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4M and
CYP3A4T (IC50 > 3 µM), which preliminarily proved that our compound had no side
effect in liver.
Table 11
In vivo Rat and Mice Pharmacokinetics (PK) Profile of 23a.
Because of its potency and attractive adaptability, compound 23a was further
evaluated by both rat (Table 12) and mice (Table 13) PK model. Initially, 23a was
characterized by low clearance (1750 mL/h/kg in rat and 1840 mL/h/kg in mice) and a
terminal halfꢁlife after I.V. administration (2.46 h in rat and 2.05 h in mice), while the
peak serum concentrations were observed at 2 h by P.O. administration. As shown in
Table 12 and Table 13, the terminal halfꢁlife after P.O. administration was 1.29 h in rat
and 3.35 h in mice. The oral bioavailability showed obvious difference between
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species of 23a which was 16.3% in rat and 83.3% in mice, which was high enough for
a candidate drug. Furthermore, we supplemented PK study of intraperitoneal (I.P.)
injection in rat as shown in Table12. Surprisingly, all the PK parameters were
significantly higher than the two other methods of administrations, especially the
terminal halfꢁlife reached 7.61 h, which made us confident that I.P. was the most
appropriate method of administration for 23a.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 12-13
Pharmacological Safety
The favorable PK profile of 23a along with its highly desirable inhibitory
potency against CrtN warranted its use in in vitro safety studies. The maximum
tolerated dose of 23a was determined for acute toxicity in rat. 30 rat were randomly
divided into three groups and given single oral doses of 60 mg/kg, 250 mg/kg, or 1000
mg/kg of 23a on the first day. The animals treated with 23a did not exhibit any
poisoning symptoms or mortality immediately or during the postꢁtreatment period of 2
weeks. In addition, no abnormal behaviors or significant changes in the water/food
consumption and body weight were observed during the period of the experiments.
Therefore, 23a was well tolerated up to a dose of 1000 mg/kg with no acute toxicity.
Resistance Development Study of 23a
As the drug resistance development was the central issue to design and
evaluation of new antibiotics, we wondered whether these CrtN inhibitors could avoid
2 2
the development of drug resistance under sequential H O selection pressure.
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2
2
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Erythromycin, a macrolides antibiotics, was included as a positive control to compare
with these CrtN inhibitors for their drug resistance development. In the initial
susceptibility testing, we determined the MIC value of erythromycin and H
0.125 ꢀg/ml and 0.15‰ (v/v), respectively. Subsequent passaging for erythromycin
was started with MIC/2 (0.0625ꢀg/ml). And for H with CrtN inhibitors, we chose
a concentration of 0.15‰ (v/v) H 2, which imposed a selected pressure for bacteria
2 2
O were
0
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9
0
2 2
O
2
O
growth and leading to a lagged time for bacteria to reach the exponential phase, and
IC90 concentrations of 23a-b for pigment production to initiate passaging. As it was
shown in Figure.10, the MIC value of erythromycin against S.aureus Newman strain
started to increase once beginning passaging, and the value had increased by a factor
of over 16 after 12 passages. By contrast, the IC50 value of the CrtN inhibitors for
pigment production of 12th passage of S. aureus Newman (under IC90 concentrations
of 23a or 23b) was basically unchanged (1.8nM vs 2.0 nM for 23a, 2.6 nM vs 3.3 nM
for 23b, Table 12). Meanwhile, if CrtN was mutated under the sequential H O + CrtN
2
2
inhibitors selection pressure, colored single colony would be appeared on the TSA
plate containing these drugs. As shown in Fig. 11，when ~1000 CFUs of the 12th
passage bacteria were spread onto TSA plate containing IC90 concentrations of 23a or
23b, all the colonies still remained nonꢁpigmented. In contrast, pigmented bacteria
were observed from TSA plate without CrtN inhibitors. In total, 100 TSA plates
containing IC90 concentrations of 23a or 23b were included to determine the
frequency of mutations. The frequencies of mutations at IC90 concentrations of CrtN
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ꢁ
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inhibitors for pigment production were less than ~ 1×10 for both 23a-b after 12
sequential passaging.
Figure 10-11
Table 14
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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3
3
3
4
4
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4
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4
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5
5
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6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
0
1
2
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9
0
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2
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4
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7
8
9
0
Finally, the MIC values of 23a for the three other pathogens were investigated,
including enterococcus faecium, S. aureus Newman, and pseudomonas aeruginosa,
which all belong to the “ESKAPE” family. As shown in Table 15, the three values of
MIC of 23a were all above 500 µg/mL, which proved our inhibitor do not affect the
growth of these pathogens.
Table 15
Conclusion
In summary, based on the results of scaffold hopping, 3ꢁsubstituted benzofuran
and 7ꢁsubstituted indole were key skeletons. Subsequently, with the variation of the
Nꢁsubstituents, allyl linkers and phenyl moiety, 100 new analogs were synthesized. To
investigate the pigment inhibition of S. aureus Newman, the unambiguous SARs were
obtained. The most valid four pigment inhibitors 12a, 14a and 23aꢁb (pigment
inhibition IC50 < 5nM) were chosen among the analogs to test their cytotoxicity for
HEKꢁ293 and HepG2 cells, in order to keep the diversity of main structure, we
maintained 23a-b for further study. 23a-b had the capacity to block the pigment
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6
biosynthesis of USA400 MW2, USA300 LAC, Mu50 and NRS271 at a comparable
level to the Newman strain. Notably, the hERG inhibition activity of 23a (IC50 = 34.8
ꢀM) and 23b (IC50 > 40 ꢀM) were largely improved compared to 1 (IC50 = 3.71 ꢀM), ~
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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9
0
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8
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0
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2
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5
6
7
8
9
0
10 fold decrease. We found that 23aꢁb had submicromolar activity in CrtN enzymatic
inhibition assay without any bactericidal impact on S. aureus bacteria (up to 200 µM)
and proper human miarosome stability (50.7 mins of 23a and 93.7mins of 23b for
halfꢁlife time). Furthermore, 23aꢁb abandoned the antifungal activities (MIC > 8
ꢀ
g/mL). According to the concept of antivirulence, 23aꢁb (1 µM)ꢁsensitized S. aureus
strains were killed by H O and human whole blood. In the in vivo assay, 23aꢁb were
2
2
proven efficacious in the S. aureus Newman and multidrug resistant MRSA (Mu50
and NRS271) model, and 23a treatment with 0.1 mg b.i.d./3.5 d (35 mg/kg in total) in
the normal treatment group was preferred. Next, all compoundꢁtreatment groups
significantly decreased Mu50 (VISA/MRSA) loads in hearts and kidneys, which were
compared with the efficacy of the positive groups, VAN and LZD. Regarding NRS271
(LRSA/MRSA), 23aꢁb strongly decreased the NRS271 loads in kidneys and hearts (>
99% decrease), which were also comparable with the positive control groups.
Considering 23a had a slight advantage over 23b, we preferred 23a as the candidate
drug and then proved it to be efficacious in S. aureus Newman sepsis model, with
more than 70% survival after 8 d. Considering no better inhibitory activity under the
lower dosage, the 0.1 mg b.i.d./3.5 d (35 mg/kg) case was guaranteed to be the most
appropriate dosage. The pigment inhibitory activity of 23a against another nine MRSA
strains was at the same level to the Newman strain, which demonstrated that 23a had a
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broad spectrum bactericidal effect. Then two LRSA strains (LRSA56 and LRSA202)
and two VISA strains (NF65Y and XN108) among the nine strains were tested in vivo,
and 23a showed extraordinary activity again, comparable to LZD and VAN. Moreover,
23a showed no inhibitory activity on CYP enzymatic, and is characterized by an
acceptable halfꢁlife, high volume of distribution and low clearance. 23a was a
potential antibiotic and had 16.3% orally bioavailable in rat and 83.8% orally
bioavailable in mice. When we tried I.P. as method of administration in rat, 23a
showed extreme stability (7.61 h for halfꢁlife time). Lastly, we verified that 23a was
not easy to induce the resistance development, and used 23b as contrast. We developed
1
1
1
1
1
1
1
1
1
1
2
2
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2
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0
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2 2
12 passages of S. aureus Newman under pressure of 23a-b and H O , and made sure
that the frequencies of mutations at IC90 concentrations of CrtN inhibitors for pigment
ꢁ5
production were < ~ 1×10 . In total, 23a has the potential to be developed as
therapeutic drugs, especially against intractable MRSA (VISA and LRSA) issues, by
blocking virulence, and this class of antibiotics holds great promise in treatment of
infections by difficult human pathogens.
Experimental Section
General Chemistry
Reagents and solvents were obtained from commercial suppliers at high quality
and were used without further purification. TLC was performed on a HSGF 254
2
(150ꢁ200 ꢀm thickness; Yantai Huiyou Co., China). UV light and I were used to
monitor synthetic progress. Column chromatography was performed on silica gel
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(
200ꢁ300 mesh), eluted with ethyl acetate and petroleum ether. NMR spectra data
were obtained on a Bruker AMXꢁ400 NMR using TMS as an internal standard.
1
Chemical shifts were provided in parts per million. H NMR data were reported from
0
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0
the aspect of multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet and br = broad), coupling constant (Hz) and integrated value. Lowꢁ and
highꢁresolution mass spectral (MS) data were acquired with electron spray ionization
(ESI) produced by a Finnigan MATꢁ95 and LCQꢁDECA spectrometer. The purity of
each compound (> 95%) was determined by HPLC on an Agilent 1100 with a
quaternary pump and diodeꢁarray detector (DAD). The melting points of each
compound were determined on an SGW Xꢁ4 melting point apparatus.
Preparation of Salts.
Taking 4a as an example, to a solution of oily derivative 4a (100 mg) in ethyl
ether (10 mL) stirred at room temperature bubbled with hydrogen chloride gas for 1
min. After stirring for 15 min, the solvent was removed by rotary evaporation and the
residue was suspended in ethyl acetate/petroleum ether (1:100, v/v, 10 mL) for an
additional hour of agitation. The precipitate was filtrated and washed with ethyl
acetate to obtain the final compound in the form of hydrochloride. All other final
derivatives underwent through this process to yield an amorphous, solid form or oil.
Spectroscopic data reported below are in their hydrochloride form.
(E)-N-(benzofuran-2-ylmethyl)-N-methyl-3-(4-(trifluoromethyl))phenyl)pro
p-2-en-1-amine hydrochloride (2a).
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A solution of intermediate 48a (X=O, 163.0 mg, 1 mmol), 51a (290.0 mg, 1.1
2 3
mmol) and K CO (280.0 mg, 2 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 Na SO ,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
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4
4
4
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5
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5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
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7
8
9
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2
4
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 2a as
white solid. Yield: 44%. 2a was prepared by the general procedure given above as
o
white solid, m.p. 161ꢁ162 C. 1H NMR (400 MHz, MeOD) δ 7.72 – 7.63 (m, 5H),
7
.60 – 7.54 (m, 1H), 7.45 – 7.36 (m, 1H), 7.35 – 7.27 (m, 1H), 7.20 (s, 1H), 7.02 (d, J
=
15.8 Hz, 1H), 6.56 – 6.46 (m, 1H), 4.66 (s, 2H), 4.10 (dd, J = 14.3, 7.1 Hz, 2H),
13
2.95 (s, 3H); C NMR (101 MHz, MeOD) δ 155.67, 146.49, 139.13, 138.76, 127.49,
1
27.22, 127.22, 125.67, 125.50, 125.33, 125.29, 124.59, 123.34, 121.54, 119.72,
+
111.05, 110.86, 57.44, 51.29, 39.09. HRMS (ESI) m/z calcd for C20
46.1419, found 346.1418.
19 3
H F NO (M+H)
3
(E)-N-(benzofuran-3-ylmethyl)-N-methyl-3-(4-(trifluoromethyl))phenyl)pro
p-2-en-1-amine hydrochloride (2b).
Yield: 57%. 2b was synthesized by the general procedure given above as white
o
1
solid, m.p. 165ꢁ166 C. HꢁNMR (400 MHz, MeOD) δ 8.13 (s, 1H), 7.82 (d, J = 8.1
Hz, 1H), 7.73–7.64 (m, 5H), 7.60 (d, J = 7.8 Hz, 1H), 7.41 (dtd, J = 17.8, 7.3, 1.2 Hz,
2
H), 7.01 (d, J = 15.9 Hz, 1H), 6.59–6.47 (m, 1H), 4.61 (d, J = 37.0 Hz, 2H),
1
3
4
.24–4.07 (m, 1H), 4.00 (s, 1H), 2.92 (s, 3H). C NMR (101 MHz, MeOD) δ 157.89,
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
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3
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3
4
4
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4
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1
55.54, 147.94, 139.07, 138.78, 130.18, 127.26, 127.26, 126.49, 125.30, 124.15,
123.48, 120.12, 119.50, 119.27, 111.52, 109.86, 57.26, 47.87, 38.56. HRMS (ESI)
+
m/z calcd for C20
H
19
F
3
NO (M+H) 346.1419, found 346.1418.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
(E)-N-(benzo[b]thiophen-2-ylmethyl)-N-methyl-3-(4-(trifluoromethyl))phen
yl)prop-2-en-1-amine hydrochloride (2c).
Yield: 42%. 2c was synthesized by the general procedure given above as white
o
1
solid. m.p. 223ꢁ224 C. HꢁNMR (400 MHz, MeOD) δ 7.99 – 7.85 (m, 2H), 7.75 –
7.63 (m, 5H), 7.49 – 7.37 (m, 2H), 7.02 (d, J = 15.8 Hz, 1H), 6.53 (dt, J = 15.2, 7.4
13
Hz, 1H), 4.73 (s, 2H), 4.02 (s, 2H), 2.94 (s, 3H); C NMR (101 MHz, MeOD) δ
1
1
41.02, 139.18, 139.11, 138.93, 130.89, 129.50, 127.30, 127.30, 125.57, 125.51,
25.33, 125.30, 124.80, 124.12, 122.81, 122.08, 119.48, 57.18, 53.43, 38.51.HRMS
+
(ESI) m/z calcd for C20
H
19
F
3
NS (M+H) 362.1190, found 360.1193.
(E)-N-(benzo[b]thiophen-3-ylmethyl)-N-methyl-3-(4-(trifluoromethyl))phen
yl)prop-2-en-1-amine hydrochloride (2d).
Yield: 50%. 2d was synthesized by the general procedure given above as white
o
solid. m.p. 198ꢁ200 C. 1H NMR (400 MHz, MeOD) δ 8.02 (s, 3H), 7.68 (s, 3H), 7.50
(d, J = 19.3 Hz, 2H), 6.99 (d, J = 16.0 Hz, 1H), 6.53 (s, 1H), 4.71 (d, J = 53.3 Hz, 2H),
13
4.09 (d, J = 40.8 Hz, 2H), 2.90 (s, 3H); C NMR (101 MHz, MeOD) δ 140.38, 139.08,
138.87, 137.86, 131.76, 127.27, 127.27, 125.50, 125.35, 125.31, 125.27, 124.98,
124.78, 124.48, 122.81, 121.27, 119.55, 57.55, 51.26, 38.81. HRMS (ESI) m/z calcd
+
for C20
H
18
F
3
NS [M+H] 361.1112, found 361.1114.
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(
E)-N-((1H-indol-2-yl)methyl)-N-methyl-3-(4-(trifluoromethyl)phenyl)prop-
2-en-1-amine hydrochloride (2e).
Yield: 25%. 2e was synthesized by the general procedure given above as white
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
oil. H NMR (400 MHz, Acetone) δ 7.98 (s, 1H), 7.71 – 7.64 (m, 4H), 7.59 (d, J = 8.0
Hz, 1H), 7.42 (d, J = 8.3 Hz, 1H), 7.20 (t, J = 7.4 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H),
7
.00 (d, J = 15.8 Hz, 1H), 6.55 – 6.45 (m, 1H), 4.62 (t, J = 11.2 Hz, 1H), 4.50 (d, J =
13.9 Hz, 1H), 4.11 (dd, J = 13.1, 7.0 Hz, 1H), 3.94 (dd, J = 13.4, 7.7 Hz, 1H), 2.89 (s,
13
3
1
1
H); C NMR (101 MHz, MeOD) δ 139.12, 138.76, 137.21, 127.90, 127.25, 127.25,
25.78, 125.50, 125.32, 125.28, 124.51, 122.84, 120.46, 119.80, 119.63, 111.21,
+
06.12, 57.05, 52.11, 38.49. HRMS (EI) m/z calcd for C20
H
19
F
3
N
2
(M) 344.1500,
found 344.1496.
(E)-N-((1H-indol-3-yl)methyl)-N-methyl-3-(4-(trifluoromethyl)phenyl)prop-
2
-en-1-amine hydrochloride (2f).
Yield: 78%. 2f was synthesized by the general procedure given above as white
o
1
solid. m.p. 165ꢁ168 C. H NMR (400 MHz, MeOD) δ 7.64 (d, J = 7.9 Hz, 1H), 7.62 –
7
7
.54 (m, 4H), 7.35 (t, J = 7.5 Hz, 1H), 7.26 (s, 1H), 7.12 (dd, J = 11.0, 4.0 Hz, 1H),
.05 (dd, J = 11.0, 3.9 Hz, 1H), 6.66 (d, J = 15.9 Hz, 1H), 6.48 (dt, J = 15.9, 6.8 Hz,
13
1H), 3.86 (s, 2H), 3.33 (d, J = 6.9 Hz, 2H); C NMR (101 MHz, MeOD) δ 139.09，
1
38.51, 136.68, 128.08, 127.19, 127.05, 127.05, 126.00, 125.37, 125.33, 124.57,
122.18, 120.09, 119.94, 117.59, 111.65, 102.64, 95.65, 56.57, 50.82, 38.09. HRMS
+
(
EI) m/z calcd for C20
H F
19 3
N
2
(M) 344.1500, found 344.1499.
29
ACS Paragon Plus Environment