Discovery of Benzocycloalkane Derivatives Efficiently Blocking Bacterial Virulence for the Treatment of Methicillin-Resistant S. aureus (MRSA) Infections by Targeting Diapophytoene Desaturase (CrtN)

Article abstract of DOI:10.1021/acs.jmedchem.6b00122

Antivirulence strategies are now attracting interest for the inherent mechanism of action advantages. In our previous work, diapophytoene desaturase (CrtN) was identified to be an attractive and drugable target for fighting pigmented S. aureus infections. In this research, we developed a series of effective benzocycloalkane-derived CrtN inhibitors with submicromolar IC₅₀. Analogue 8 blocked the pigment biosynthesis of three MRSA strains with a nanomolar IC₅₀ value. Corresponding to its mode of action, 8 did not function as a bactericidal agent. 8 could sensitize S. aureus to immune clearance. In vivo, 8 was proven to be efficacious in an S. aureus Newman sepsis model and abscess formation model. For two typical MRSAs, USA400 MW2 and Mu50, 8 significantly decreased the staphylococcal loads in the liver and kidneys. Moreover, 8 showed minimal antifungal activity compared to that of NTF. In summary, 8 has the potential to be developed as a therapeutic drug, especially against intractable MRSA issues.

Full text of DOI:10.1021/acs.jmedchem.6b00122

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Article
Discovery of Benzocycloalkane Derivatives Efficiently Blocking
Bacterial Virulence for the Treatment of Methicillin-Resistant S. aureus
(MRSA) Infections by Targeting Diapophytoene Desaturase (CrtN)
youxin wang, Hongxia Di, Feifei Chen, Yong Xu, Qiang Xiao, Xuehai Wang,
Hanwen Wei, Yanli Lu, Lingling Zhang, Jin Zhu, Lefu Lan, and Jian Li
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00122 • Publication Date (Web): 03 May 2016
Downloaded from http://pubs.acs.org on May 5, 2016
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Discovery of Benzocycloalkane Derivatives Efficiently Blocking
Bacterial Virulence for the Treatment of Methicillin-Resistant S.
aureus (MRSA) Infections by Targeting Diapophytoene Desaturase
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(
CrtN)
a,†
b,†
b
c
c
d
Youxin Wang , Hongxia Di , Feifei Chen , Yong Xu , Qiang Xiao , Xuehai Wang ,
a
a
a
a
b, *
a, *
Hanwen Wei , Yanli Lu , Lingling Zhang , Jin Zhu , Lefu Lan , 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
c
Hubei Bioꢀpharmaceutical Industrial Technological Institute, Inc., Wuhan 430075,
China
d
Humanwell Healthcare (Group) Co., Ltd., Wuhan 430075, 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
Antiꢀvirulence strategies are now attracting interest for the inherent mechanism of
action advantages. In our previous work, diapophytoene desaturase (CrtN) was identified
to be an attractive and drugable target for fighting pigmented S. aureus infections. In this
research, we developed a series of effective benzocycloalkaneꢀderived CrtN inhibitors
with submicromolar IC . Analog 8 blocked the pigment biosynthesis of three MRSA
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strains with a nanomolar IC value. Corresponding to its mode of action, 8 did not
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function as a bactericidal agent. 8 could sensitize S. aureus to immune clearance. In vivo,
was proven to be efficacious in an S. aureus Newman sepsis model and abscess
8
formation model. For two typical MRSAs, USA400 MW2 and Mu50, 8 significantly
decreased the staphylococcal loads in the liver and kidneys. Moreover, 8 showed minimal
antiꢀfungal activity compared to NTF. In summary, 8 has the potential to be developed as
a therapeutic drug, especially against intractable MRSA issues.
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INTRODUCTION
The pathogen Staphylococcus aureus (S. aureus) is well adapted to its human host,
compelling us in a constant evolutionary situation to match its continuously renewed
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resistance. However, we seem to perpetually lose this arms race, as indicated by the
widespread outbreaks of hospitalꢀassociated methicillinꢀresistant S. aureus (HA
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ꢀ
MRSA), communityꢀassociated MRSA (CAꢀMRSA), livestockꢀassociated MRSA
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ꢀ6
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(
LAꢀMRSA) and even vancomycinꢀresistant S. aureus (VRSA) infectious diseases. It
is estimated that by the year 2050, humans will suffer 10 million losses annually due to
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antimicrobial resistance (AMR). In the US alone, MRSA causes over 23 thousand deaths
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and over 2 million illnesses per year. Unfortunately, the discovery of new antibiotics has
lagged far behind the growing emergence of antibiotic resistance, as for nearly 20 years,
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no new classes have been launched. Although we are capable of combining existing
antibiotics to tackle immediate clinical dilemmas, as exemplified by the triple βꢀlactam
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combinations, the emergence of resistance is inevitable. Consequently, there is an
urgent need for antiꢀinfective agents with novel modes of action in the postꢀantibiotic
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era. Antiꢀvirulence strategies aiming at ‘disarming’ the pathogen rather than inhibiting
its growth, with weak selective pressure for the development of antibiotic resistance, are
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4ꢀ16
now attracting interest.
The golden carotenoid pigment (staphyloxanthin) is an
important virulence factor for pigmented S. aureus. Nonpigmented S. aureus is
susceptible to being killed by reactive oxygen species. Hence, blocking staphyloxanthin
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biosynthesis is an underlying magnetic therapeutic target.
Staphyloxanthin
biosynthesis begins with the condensation of farnesyl diphosphate, followed by a series
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of important enzyme (CrtM, CrtN, CrtQ, CrtP, CrtO)ꢀcatalyzed reactions. A cholesterol
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biosynthesis inhibitor, BPHꢀ652 (Figure 1), targeting dehydrosqualene synthase (CrtM)
to block staphyloxanthin biosynthesis, has provided a good lead for antiꢀvirulence
strategies along with the consequence of the susceptibility of S. aureus to the innate
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immune eradication. In our previous work, we demonstrated the enzyme diapophytoene
desaturase (CrtN) to be an attractive and drugable target for fighting pigmented S. aureus
infections. Simultaneously, we proved naftifine hydrochloride (NTF, Figure 1), an
FDAꢀapproved antifungal drug, to be an efficiency CrtN inhibitor with an established
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safety and efficacy profile both in vitro and in vivo.
CHEMISTRY
In this study, to develop a more potent antiꢀvirulence agent against S. aureus, NTF
was set as a lead compound, and scaffold hopping was employed to modify its structural
motifs.
Scheme 1 depicts the synthetic route for the preparation of analog 1; 38 was
achieved by the Sandmeyer reaction, followed by cyanation to afford 39. The reduction
of 39 with lithium aluminum hydride yielded 40. 41 was easily obtained by the addition
of diꢀtertꢀbutyl dicarbonate, which was further reduced to the benzyl amine 42. Then,
target compound 1 was achieved by coupling benzyl amine 42 with cinnamyl bromide.
The synthetic route for the preparation of analogs 3 and 6 is depicted in Scheme 2.
The reduction of 1ꢀbenzosuberone by triethylsilane in the presence of trifluoroacetic acid
produced 43, and then the iodination of the resulting product with NIS unexpectedly
afforded the aryl iodides 44 and 45, with a selectivity of 1ꢀposition/2ꢀposition = 1:4 (as
determined by NMR spectra). We speculate that the reaction site was restricted as a result
of the pꢀmethylene directing effect and mꢀmethylene space effect. Due to their highly
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similar molecular polarities, these two isomers were not separable by TLC, with the
reactions continuing until the last procedure. Cyanation to give the aryl cyanides 46 and
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7 was followed by reduction with lithium aluminum hydride to afford amines 48 and 49.
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Treatment with diꢀtertꢀbutyl dicarbonate gave intermediates 50 and 51, which were
further reduced to benzyl amines 52 and 53. Then, the required compounds were
achieved by coupling benzyl amines with cinnamyl bromide, and the two target
compounds 3 and 6 could be separated by flash chromatography.
Scheme 3 depicts the synthetic route for the preparation of analogs 4, 5 and 29-33.
The
reduction
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2
,3ꢀdihydroꢀ1Hꢀindeneꢀ5ꢀcarboxylic acid and 5,6,7,8ꢀtetrahydronaphthaleneꢀ2ꢀcarboxylic
acid yielded 54a-b, followed by the bromination of the benzyl hydroxyl group and then
an amination reaction to furnish 56a-b. Ultimately, target compounds 4, 5 and 29-33
were achieved by coupling benzyl amines 56a-b with various 4ꢀsubstituted cinnamyl
bromides.
Analogs 7-17, 19-20, 22 and 24 were synthesized through the route outlined in
Scheme 4. Various substituted acraldehydes were reduced by sodium borohydride,
followed by bromination to give compounds 58a-o. The nucleophilic substitution of
compounds 58a-o with benzyl amine 53 provided the target analogs 7-17, 19-20, 22 and
24.
In Scheme 5, we can see the synthetic route for the preparation of analogs 18, 21, 23
and 25. By the WittigꢀHorner olefination, 59 was yielded from phenylacetaldehyde,
followed by the reduction of the ethyl ester and a bromination reaction to generate 61a-b;
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1a-b, the commercially available (E)ꢀ1ꢀbromobutꢀ2ꢀene and (3ꢀbromopropyl) benzene,
reacted with 53, producing target analogs 18, 21, 23 and 25.
Scheme 6 depicts the synthetic route for the preparation of analogs 26-28.
Intermediate 49 was coupled with transꢀcinnamaldehyde via reductive amination to
afford the target analog 26, followed by substitution with an ethyl group or isopropyl
group to yield target analogs 27-28, respectively.
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The synthetic route of analogs 29-31 is summarized in Scheme 7. 62 was obtained
by the hydrolysis of 47, followed by the reduction of the carboxy group to afford 63. 63
reacted with 13a, 13c or (3ꢀbromopropyl) benzene to yield analogs 29-31.
RESULTS AND DISCUSSION
In our initial investigations, compounds 1, 2 and 3 were the only three target
structures of interest. As illustrated in Figure 2, we intended to switch the naphthalenyl
moiety of NTF into three different sizes of benzocycloalkane ring to generate novel
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skeleton compounds 1, 2 and 3.
During the synthesis of compound 3, a byꢀproduct of 6 was unexpectedly obtained.
To our surprise, compound 6 showed more potent activity than compound 3 (Figure 2).
This accidental result sparked our interest in deliberately translocating the substitution
patterns on the benzocycloalkane part of compounds 1 and 2 to the 2ꢀposition to achieve
compounds 4 and 5 (Figure 2). The pigment inhibition activities of these six different
scaffold compounds are shown in Figure 2. Intriguingly, we observed that the 2ꢀposition
substituted products exhibited more promising potencies than their isomerides, with
compound 6 revealing the strongest effect. Having established the unexpected superiority
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of compound 6, we further set out to identify more potent inhibitors based on the neoteric
framework.
Notably, the substituted positions on the phenyl ring substantially affect the activity
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(
Table 1). The incorporation of a substituent at the 4ꢀposition of the phenyl was beneficial
for improving the potency, and both electronꢀdonating and electronꢀwithdrawing groups
were tolerable (7-13 vs 6). Strikingly, compounds 8, 10 and 11 boosted the activity to a
singleꢀdigit nanomolar level. Nonetheless, introducing the substituent to the 2ꢀposition or
3ꢀposition markedly diminished the potency, except for 15, which basically maintained
the activity of 6. The replacement of the phenyl group with methyl, furyl or cyclohexyl
generated compounds (18, 19, 20 vs 6) resulted in a dramatic loss of the pigment
inhibition activity. Subsequently, we prepared compounds 21 to 25 to assess whether the
allyl linker had an influence on the pigment inhibition potency (Figure 3).
Turning the allyl to propanyl (21) or propargyl (25) and introducing a branched
methyl (22) or additional methylene (23) to allyl significantly abated the activity.
Extending the conjugation system was very beneficial for the potency (24). These results
revealed that the allyl linker was crucial for the biological activity. Additionally, we
converted the Nꢀmethyl to hydrogen (26), ethyl (27) and isopropyl (28) to give insight
into the effect of the Nꢀsubstituent. As shown in Figure 3, the Nꢀmethyl is essential for
the pigment inhibition activity. Finally, we replaced the nitrogen of 6 with oxygen to
synthesize analogs 29-31. As indicated in Figure 4, the nitrogen of 6 is very important for
the pigment inhibition activity, and oxygen is not tolerable.
SAR of NTF-derived Benzocycloalkane Analogs.
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The detailed structureꢀactivity relationship (SAR) analysis (NTF, 1-31) is provided
as follows: (1) Under the condition of the replacement of the naphthalenyl moiety of NTF
with differentꢀsized benzocycloalkane rings, the substituted positon is crucial for the
potency. Generally, the 2ꢀposition is preferred to the 1ꢀposition (1 vs 4, 2 vs 5, 3 vs 6),
and the potency improves with the size of the ring (4 vs 5 vs 6). (2) The electronic
characteristics of the substituent on the right side phenyl ring has a finite impact on the
potency, while the substituted position is fatal. The introduction of a substituent group
into the para position can significantly enhance the potency (7 vs 16, 9 vs 15, 12 vs 14 vs
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7). Replacing the phenyl ring with furanyl or (cyclo) alkyl is not tolerable (6 vs 18-20).
(3) The unsubstituted allyl linker is critical for achieving high potency, and inserting a
vinyl is beneficial (6 vs 21-25). (4) Nꢀsubstituent variation exerts a great influence on the
pigment inhibitory activity. Comparing 6 to 26-28, the original methyl is optimal. (5)
Changing the nitrogen to oxygen was not tolerable (6 vs 29, 24 vs 31).
A definite SAR is generated through the analysis mentioned above. In an effort to
confirm this SAR, the five most efficacious substitution patterns of compound 6
derivatives were introduced into compound 5 (32ꢀ36, Figure 5) due to its potency that
was second only to 6 in our initial investigation (Figure 2). All five analogues exhibited
robust biological activities, especially compounds 32 and 35, which exhibited > 300ꢀfold
greater effectiveness than the parent compound 5.
Activity Target Determination of the NTF-derived Benzocycloalkane Analogs.
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As NTF is an established CrtN inhibitor, we speculated that the benzocycloalkane
analogs derived from NTF may have the same target. To confirm this, we carried out an
HPLC experiment as before at 286 nm for the analysis of 4,4’ꢀdiapophytoene, which is
the product of CrtM and the substrate of CrtN. As shown in Figure 6, an HPLC peak
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appeared after the expression of crtM in E. coli (Figure 6B), which was similar to that of
wildꢀtype S. aureus Newman (Figure 6C) with respect to both the retention time and UV
absorption spectra. This peak faded away in the carotenoid extracts of the crtM mutants
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(
Figure 6D) and was amplified in the crtN mutants (Figure 6E). All these results suggest
this peak belongs to 4,4’ꢀdiapophytoene. Comparing the HPLC chromatograms of
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ꢀtreated wildꢀtype S. aureus Newman (Figure 6G) with NTFꢀtreated Newman (Figure
6
F), they have very similar peak profiles, and both are very similar to those of the crtN
mutants. Taken together, these data suggest that CrtN is the target of the
benzocycloalkane analogs of NTF.
In Vitro CrtN Enzymatic Inhibitory Activities.
Taking the pigment inhibition activities and structural diversity into consideration,
we carefully selected four of the best compounds (8, 10, 32, and 35) to evaluate their
capacities of inhibiting the enzymic activity of CrtN in vitro. Using our previous
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protocol, all four compounds were found to significantly inhibit the enzymatic activity
of CrtN at nanomolar concentrations (Table 2). However, the enzymatic activities were
much less effective than the inhibition of the virulence factor formation; the exact reason
needs to be elucidated. Here, we can give a preliminary hypothesis: (i) The possible
accumulation of 8 in the cytoplasm of S. aureus gives rise to a higher intracellular
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concentration; (ii) different from CrtM, the enzyme CrtN catalyzed three sequential
reactions in the biosynthesis of staphyloxanthin, so the staphyloxanthin inhibition
potency targeting CrtN could be enhanced through synergistic effects.
Water Solubility of the Representative Analogs.
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Drug solubility is one of the most important properties in drug discovery. Good
solubility characteristics contribute to drug absorption and bioavailability. Taking this
into consideration, we measured the aqueous solubility of the above four compounds. As
shown in Table 3, 8 exhibited the highest solubility, 4.24 mg/mL, due to the
incorporation of a hydrophilic methoxyl group, making it an attractive candidate for
further development.
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In Vitro Pigment Inhibitory Activities of Analog 8 against MRSA.
8 had been demonstrated to be a highꢀefficient pigment inhibitor of the Newman
strain. Additionally, we took three MRSA strains to evaluate the capacity of 8 to block
staphyloxanthin biosynthesis. At present, USA400 MW2 and USA300 LAC are blamed
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4ꢀ25
for the epidemic of CAꢀMRSA infectious diseases in the United States,
while VISA
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Mu50 is a hospitalꢀacquired MRSA strain isolated in Japan. As shown in Figure 7, 8
possesses a comparative ability in the pigment inhibition of USA400 MW2 and USA300
LAC (IC = 7.1 nM, 7.7 nM, respectively) to Mu50 at a picomole quantity (IC = 0.49
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nM). Importantly, incubation with 8 did not affect the growth of S. aureus strains
(Newman strain and three MRSA strains) at 0.2 mM or 0.05 mM (Figure 8), suggesting
that 8 differs from an antibiotic, and we can safely come to the conclusion that 8 is a
promising antiꢀvirulence candidate.
Effects of 8 on Sensitizing S. aureus to Immune Clearance.
S. aureus pigment could serve as a protective antioxidant to confer resistance to
immune clearance. As 8 is a powerful pigment inhibitor of S. aureus strains, we
speculated that it could sensitize S. aureus to killing by either hydrogen peroxide (H O )
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or human whole blood. To verify this argument experimentally, we first compared the
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susceptibility to H O killing of mockꢀtreated S. aureus Newman with that of 8ꢀtreated (1
2
2
µM) S. aureus Newman. As shown in Figure 9, the 8ꢀtreated nonꢀpigmented S. aureus
cells were more susceptible to killing by 1.5% H O by a factor of ~37 (survival, 0.83%
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vs 30.43%). On the other hand, the survival of the known antioxidant Nꢀacetylcysteine
(NAC)ꢀtreated S. aureus cells was at a considerable level similar to that of the
mockꢀtreated S. aureus Newman. Similarly, 8-incubated (1 µM) versions of the other
three MRSA strains (USA400 MW2, USA300 LAC, and Mu50) also induced significant
susceptibility to H O (0.96% vs 14.17%, 0.98% vs 15.83%, 4.44% vs 30.67%,
2
2
respectively). The addition of NAC clearly improved the survival rates of all three MRSA
strains, demonstrating the addition of H O exerted impact on the strain survival by the
2
2
oxidation capacity and the pigment definitely acted as the protective antioxidant.
Subsequently, we sought to ascertain whether 8 could decrease the wholeꢀblood survival
of S. aureus. Fresh human whole blood was added to each of 8ꢀtreated (1 µM) and
untreated S. aureus Newman cells, and the bacterial survival was measured. As shown in
Figure 10, the untreated S. aureus Newman survived significantly better than the
8ꢀtreated white S. aureus Newman by a factor of ~15 (survival, 26.67% vs 1.83%).
Likewise, we demonstrated that 8 could significantly impair the resistance of the other
three MRSA strains (USA400 MW2, USA300 LAC, and Mu50) to whole human blood
by factors of ~51 (10.8% vs 0.20%), ~13 (12.17% vs 0.95%) and ~40 (16.11% vs 0.40%).
All these results suggest that 8 could render S. aureus more susceptible to immune
clearance.
In Vivo Effects of 8 on Attenuating the Virulence of S. aureus Newman.
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Because 8 had inspiring activities in vitro, we next investigated the in vivo efficacy
in a systematic infection model. We subjected S. aureus Newman to a murine model of
abscess formation via retroꢀorbital injection and measured the bacterial survival in the
host organs. As shown in Figure 11A, the 200 mg/kg dosage 8ꢀtreatment reduced
bacterial survival in the kidneys and heart by 1.45 and 1.66 log CFU, corresponding to
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6.4% and 97.8% decreases, respectively. When the dosage of 8 was decreased to 50
mg/kg, the treatment also resulted in a significant reduction in the kidneys and heart (by
1.17 and 1.62 log CFU), only a slight decrease compared to the 200 mg/kg dosage. To
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0
further evaluate the efficiency of 8 on affecting the outcome of S. aureus sepsis, we
7
challenged animals with 2 × 10 CFU Newman bacteria (Figure 11B). The untreated mice
died out within 4 days postꢀchallenge, while 8 exhibited the same protective effect as
NTF, resulting in 83% animal survival. As time went on to the 8th day, over 60% of the
8
ꢀtreated mice were alive, possessing little advantage over the NTFꢀtreated group. These
initial investigations clearly prove that the in vivo 8 treatment weakened the virulence of
S. aureus Newman.
In Vivo Effects of 8 on Attenuating the Virulence of MRSA.
Along with the above encouraging in vivo outcome, the more appealing in vivo
inhibitory effects of 8 against MRSA were evaluated by two representative strain
(USA400 MW2 and Mu50) infection models. As shown in Figure 12, a 200 mg/kg
dosage 8 treatment significantly decreased the USA400 MW2 staphylococcal loads in the
liver by 4.37 log CFU (more than a 99.9% decrease in surviving bacteria), while 37, the
10
positive control drug, led to a reduction by only 1.59 log CFU at the same dosage,
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making it less efficacious than the 8 treatment. Upon cutting the dosage down by three
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quarters, the 8 treatment still resulted in a dramatic decrease in the liver by 3.87 log10
CFU; however, the 37 treatment demonstrated almost no activity. As shown in Figure 12,
the therapeutic efficiencies in the kidneys of two different dosages of 8 treatment were at
comparative levels, with a remarkable ~99.5% decrease in surviving bacteria. The
doseꢀdependent result of the 37 treatment was not so obvious, and in both cases, it was
less efficacious than the 8 treatment.
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In the Mu50 model, as shown in Figure 13, the treatment of mice with 8 lowered the
bacterial survival in the liver tissues by 4.27 log CFU (equivalent to a 99.99% bacteria
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clearance rate) with a dosage of 200 mg/kg, which was superior to 37 treatment in the
same dosage case, although 37 exhibited excellent performance by decreasing the
staphylococcal loads by 2.84 log CFU. Upon decreasing the dosage to 50 mg/kg, the 8
1
0
treatment still exhibited a significant effect (~2.44 log CFU/organ reduction), although
1
0
worse than that of the high dose groups, which suggested an explicitly doseꢀdependent
effect. The 50 mg/kg dosage 37 treatment achieved a satisfactory reduction, but slightly
worse than that of the 8 treatment. Accordingly, in the kidneys, the bacterial survival
rates shrunk drastically by 2.27 log CFU with the prescription of 200 mg/kg 8, while
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the result of the 37 treatment was not as impressive, with merely a 0.30 log10 CFU
reduction. The weakened activity of 8 came along with easing the dosage of the
administration to 50 mg/kg (0.99 log CFU reduction), but the bacterial survival rates of
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the lowꢀdosage 37 treatment were considerable compared to that of 200 mg/kg dosage
group. In brief, these facts clearly reveal that 8, acting as an antiꢀvirulence agent, could
decrease both the S. aureus Newman and clinically relevant multiꢀdrug resistant S. aureus
survival with superior results to 37.
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In Vitro Anti-fungal Activities of 8.
A drug with a specific effect is more appealing to us. As 8 was derived from the
parent compound NTF, an established antiꢀfungal drug, the question of whether 8
inherited the antiꢀfungal activity aroused our interest. Eventually, three naftifine sensitive
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strains were chosen to proceed with the in vitro assay. Encouragingly, 8 exhibited weak
activities against all three dermatophytes compared to NTF, at a 64ꢀfold worse level. This
meaningful result proved 8 to be a promising selective antibacterial candidate.
CONCLUSIONS
In summary, we have designed and synthesized 36 new, efficacious CrtN inhibitors
employing a scaffold hopping approach based on the structure of NTF. Five of these
benzocycloalkane analogs showed potent pigment inhibitory activities at singleꢀdigit
nanomolar levels, and the most potent analog 32 improved the pigment inhibitory
capability by > 200 fold compared to NTF. Based on the pigment inhibition of S. aureus
Newman, unambiguous SARs were obtained. The variation of the Nꢀsubstituents, allyl
linkers and the substituted position of the phenyl moiety exerted very impressing impacts
on the pigment inhibitory activity. The four most valid pigment inhibitory compounds
were carefully selected for a CrtN enzymatic inhibition assay and showed submicromolar
2
2
activity, being far more effective than NTF. Superior aqueous solubility made 8 more
attractive than the other three analogs. We found that 8 had the capacity to block the
pigment biosynthesis of USA400 MW2, USA300 LAC, and Mu50 at comparable levels
to the Newman strain, without any bactericidal impact on these four S. aureus bacteria
(up to 200 µM). According to the concept of antiꢀvirulence, 8 (1 µM) sensitized S. aureus
strains to killing by H O and human whole blood. In an in vivo assay, 8 was proven to be
2
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efficacious in an S. aureus Newman sepsis model (more than 60% survival after 8 days)
and abscess formation model (more than 96% bacterial clearance in both kidneys and
heart) at a dosage of 200 mg/kg. Against two typical MRSAs, USA400 MW2 and Mu50,
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8
(200 mg/kg and 50 mg/kg) significantly decreased the staphylococcal loads in the liver
and kidneys (> 99% decrease, except in the kidney of the 50 mg/kg 8ꢀtreated Mu50
model), performing better than 37. Furthermore, 8 exhibited minimal antiꢀfungal activity
(MIC > 8 ꢁg/mL) compared to NTF. In total, 8 has the potential to be developed as
therapeutic drugs, especially against intractable MRSA issues, by blocking virulence.
Further research on the structural transformation of the most advanced compound 8 is
still ongoing.
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 were used without further purification. Analytical
thinꢀlayer chromatography (TLC) was performed on HSGF 254 (150−200 ꢁm thickness;
Yantai Huiyou Co., China), and components were visualized by observation under UV
light (254 nm and 365 nm). Melting points were determined on an 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 using deuterated chloroform (CDCl ), deuterated methanol (CD OD), or
3
3
deuterated dimethyl sulfoxide (DMSOꢀd ) as the solvent. Chemical shifts were reported
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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
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electron spray ionization (ESI) produced by an LCT. HPLC data analysis of compounds
1-36 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.
Preparation of salts.
To take 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
another 1 h in EtOAc/petroleum ether (1:100, v/v, 20 mL). The precipitate was filtered
and washed with EtOAc to give the final compound in the form of a hydrochloride. All
other end products were also subjected to such a salification process. The spectroscopic
data given below are for the compounds in the forms of their hydrochlorides.
(E)-N-((2,3-Dihydro-1H-inden-4-yl)methyl)-N-methyl-3-phenylprop-2-en-1-amine
hydrochloride (1).
A solution of 42 (0.32 g, 2.00 mmol), cinnamyl bromide (0.39 g, 2.00 mmol) and
K CO (0.33 g, 2.40 mmol) in DMF (10 mL) was stirred at room temperature overnight.
2
3
The mixture was poured into water and extracted with EtOAc. The combined organic
layers were washed with brine, dried over anhydrous Na SO , filtered and condensed.
2
4
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 (0.32 g, 58% yield) as a
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colorless oil. 1 was prepared using a general procedure of salification as a white solid.
1
m.p. 155ꢀ156 ºC. HꢀNMR (400 MHz, MeOD) δ 7.53 (d, J = 7.5 Hz, 2H), 7.36 (dt, J =
1
9.5, 6.4 Hz, 6H), 6.96 (d, J = 16.0 Hz, 1H), 6.46–6.29 (m, 1H), 4.52 (d, J = 11.1 Hz,
H), 4.26 (d, J = 11.8 Hz, 1H), 4.03 (d, J = 32.8 Hz, 2H), 3.00 (dd, J = 14.1, 6.7 Hz, 4H),
10
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53
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60
1
1
3
2.87 (s, 3H), 2.15 (dd, J = 14.8, 7.4 Hz, 2H); C NMR (101 MHz, MeOD) δ 147.09,
1
46.32, 142.13, 136.69, 130.14, 129.86, 129.72, 128.37, 128.12, 127.25, 126.95, 117.78,
+
5
9.41, 58.10, 39.93, 33.94, 32.40, 26.09. HRMS (ESI) m/z calcd for C H N [M+H]
2
0
24
278.1909, found 278.1903.
(
E)-N-Methyl-3-phenyl-N-((5,6,7,8-tetrahydronaphthalen-1-yl)methyl)prop-2-en-1-a
mine hydrochloride (2).
The detailed synthesis of the free base of 2 is described in the literature, as noted in
the article. 2 was prepared using the general procedure of salification as a white solid.
1
m.p. 165ꢀ166 ºC. HꢀNMR (400 MHz, MeOD) δ 7.54 (d, J = 7.5 Hz, 2H), 7.38 (dq, J =
1
3.9, 6.9 Hz, 3H), 7.33–7.27 (m, 1H), 7.24 (dd, J = 8.0, 5.4 Hz, 2H), 6.97 (d, J = 15.8 Hz,
H), 6.45–6.32 (m, 1H), 4.57 (d, J = 12.9 Hz, 1H), 4.24 (d, J = 13.5 Hz, 1H), 4.05 (dd, J
1
1
3
= 13.7, 7.7 Hz, 2H), 2.94–2.72 (m, 7H), 1.97–1.71 (m, 4H); C NMR (101 MHz, MeOD)
δ 142.26, 140.22, 138.25, 136.68, 132.55, 130.43, 130.18, 129.88, 129.52, 128.13,
1
27.15, 117.73, 59.64, 57.27, 40.18, 30.97, 27.40, 24.20, 23.58. HRMS (ESI) m/z calcd
+
for C H N [M+H] 292.2065, found 292.2068.
2
1
26
(
E)-N-Methyl-3-phenyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-1-yl)methyl)pro
p-2-en-1-amine hydrochloride (3) and
E)-N-methyl-3-phenyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl)pro
p-2-en-1-amine hydrochloride (6).
(
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A solution of mixed 52 and 53 (0.38 g, 2.00 mmol), cinnamyl bromide (0.39 g, 2.00
mmol) and K CO (0.33 g, 2.40 mmol) in DMF (10 mL) was stirred at room temperature
2
3
overnight. The mixture was poured into water and extracted with EtOAc. The combined
organic layers were washed with brine, dried over anhydrous Na SO , filtered and
0
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8
9
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condensed. The residue was then purified via flash chromatography on silica gel, eluting
with EtOAc/petroleum ether (1/5, v/v) to separately give the free bases of 3 (0.06 g, 51%
yield, based on 52) and 6 (0.30 g, 66% yield, based on 53) as colorless oils. 3 and 6 were
prepared using the general procedure of salification as white solids. 3: m.p. 186ꢀ188 ºC.
1
HꢀNMR (400 MHz, MeOD) δ 7.54 (d, J = 7.2 Hz, 2H), 7.44–7.34 (m, 3H), 7.35–7.26
(
m, 2H), 7.22 (t, J = 7.5 Hz, 1H), 6.98 (d, J = 15.8 Hz, 1H), 6.45–6.33 (m, 1H), 4.60 (s,
1H), 4.35 (s, 1H), 4.03 (s, 2H), 3.03–2.88 (m, 4H), 2.84 (s, 3H), 1.88 (s, 2H), 1.65 (s,
13
4
1
2
H); C NMR (101 MHz, MeOD) δ 146.73, 145.08, 142.26, 136.69, 132.45, 131.30,
30.16, 129.87, 128.28, 128.12, 127.64, 117.82, 59.41, 57.85, 39.81, 37.40, 33.05, 31.02,
+
9.10, 28.67. HRMS (ESI) m/z calcd for C H N [M+H] 306.2222, found 306.2220.
2
2
28
1
6
: m.p. 172ꢀ174 ºC. HꢀNMR (400 MHz, MeOD) δ 7.53 (d, J = 7.0 Hz, 2H), 7.36
(ddd, J = 10.7, 10.0, 5.3 Hz, 3H), 7.25 (dd, J = 12.0, 9.5 Hz, 3H), 6.93 (d, J = 15.8 Hz,
H), 6.45–6.27 (m, 1H), 4.42 (s, 1H), 4.24 (s, 1H), 4.03 (s, 1H), 3.91 (s, 1H), 2.91–2.84
1
1
3
(
m, 4H), 2.81 (s, 3H), 2.01–1.78 (m, 2H), 1.66 (d, J = 4.0 Hz, 4H); C NMR (101 MHz,
MeOD) δ 147.06, 146.01, 142.00, 136.71, 132.50, 130.96, 130.11, 129.85, 129.75,
1
28.44, 128.11, 117.70, 60.30, 58.94, 39.57, 37.38, 37.24, 33.66, 29.41, 29.32. HRMS
+
(
ESI) m/z calcd for C H N [M+H] 306.2222, found 306.2216.
22 28
(
E)-N-((2,3-Dihydro-1H-inden-5-yl)methyl)-N-methyl-3-phenylprop-2-en-1-amine
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A solution of 56a (0.32 g, 2.00 mmol), cinnamyl bromide (0.39 g, 2.00 mmol) and
K CO (0.33 g, 2.40 mmol) in DMF (10 mL) was stirred at room temperature overnight.
2
3
The mixture was poured into water and extracted with EtOAc. The combined organic
layers were washed with brine, dried over anhydrous Na SO , filtered and condensed.
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2
4
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 4 (0.40 g, 72% yield) as a
colorless oil. 4 was prepared using the general procedure of salification as a white solid.
1
m.p. 160ꢀ161 ºC. HꢀNMR (400 MHz, MeOD) δ 7.60–7.48 (m, 2H), 7.48–7.32 (m, 5H),
7
.28 (d, J = 7.8 Hz, 1H), 6.93 (d, J = 15.8 Hz, 1H), 6.41–6.30 (m, 1H), 4.46 (d, J = 13.0
Hz, 1H), 4.24 (d, J = 12.9 Hz, 1H), 4.05 (dd, J = 13.2, 7.2 Hz, 1H), 3.89 (dd, J = 13.0, 7.8
1
3
Hz, 1H), 2.97 (dd, J = 13.2, 7.0 Hz, 4H), 2.81 (s, 3H), 2.21–2.08 (m, 2H); C NMR (101
MHz, MeOD) δ 147.78, 146.81, 141.93, 136.72, 130.15, 130.07, 129.83, 128.70, 128.11,
1
28.00, 126.12, 117.76, 60.56, 58.87, 39.48, 33.58, 26.49. HRMS (ESI) m/z calcd for
+
C H N [M+H] 278.1909, found 278.1909.
20
24
(
E)-N-Methyl-3-phenyl-N-((5,6,7,8-tetrahydronaphthalen-2-yl)methyl)prop-2-en-1-a
mine hydrochloride (5).
was synthesized by the general procedure of 4, given above, as a white solid. m.p.
5
1
1
82ꢀ184 ºC. HꢀNMR (400 MHz, MeOD) δ 7.53 (d, J = 6.9 Hz, 2H), 7.42–7.31 (m, 3H),
7.28–7.13 (m, 3H), 6.92 (d, J = 15.8 Hz, 1H), 6.42–6.28 (m, 1H), 4.31 (d, J = 64.0 Hz,
13
2
H), 3.96 (d, J = 38.4 Hz, 2H), 2.82 (d, J = 7.7 Hz, 7H), 1.94–1.71 (m, 4H); C NMR
(101 MHz, MeOD) δ 141.95, 140.61, 139.47, 136.72, 132.78, 131.09, 130.09, 129.84,
1
29.05, 128.11, 127.94, 117.72, 60.41, 58.89, 39.55, 30.25, 30.18, 24.10. HRMS (ESI)
+
m/z calcd for C H N [M+H] 292.2065, found 292.2067.
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(
E)-N-Methyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl)-3-(p-tolyl)pr
op-2-en-1-amine hydrochloride (7).
7
was synthesized by the general procedure of 6, given above, as a white solid. m.p.
0
1
2
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0
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0
13
1
88ꢀ189 ºC. C NMR (101 MHz, MeOD) δ 147.04, 146.00, 142.02, 140.38, 133.94,
132.48, 130.96, 130.46, 129.73, 128.46, 128.08, 116.48, 60.23, 59.05, 39.50, 37.38,
1
3
7.24, 33.66, 29.41, 29.32, 21.30. HꢀNMR (400 MHz, MeOD) δ 7.39 (d, J = 7.8 Hz,
H), 7.21 (dd, J = 17.3, 8.8 Hz, 5H), 6.86 (d, J = 15.6 Hz, 1H), 6.37–6.15 (m, 1H), 4.38
2
(s, 1H), 4.19 (s, 1H), 3.98 (s, 1H), 3.87 (s, 1H), 2.93–2.81 (m, 4H), 2.78 (s, 3H), 2.34 (s,
13
3
H), 1.88 (s, 2H), 1.64 (s, 4H); C NMR (101 MHz, MeOD) δ 147.04, 146.00, 142.02,
1
40.38, 133.94, 132.48, 130.96, 130.46, 129.73, 128.46, 128.08, 116.48, 60.23, 59.05,
39.50, 37.38, 37.24, 33.66, 29.41, 29.32, 21.30. HRMS (ESI) m/z calcd for C H N
2
3
30
+
[
M+H] 320.2378, found 320.2373.
(E)-3-(4-ethoxyphenyl)-N-methyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)m
ethyl)prop-2-en-1-amine hydrochloride (8).
8
was synthesized by the general procedure of 6, given above, as a white solid. m.p.
1
164ꢀ167 ºC. HꢀNMR (400 MHz, MeOD) δ 7.47 (d, J = 8.6 Hz, 2H), 7.26 (d, J = 10.0
Hz, 3H), 6.93 (t, J = 13.2 Hz, 2H), 6.86 (d, J = 15.7 Hz, 1H), 6.19 (dt, J = 15.4, 7.5 Hz,
1
H), 4.42 (d, J = 12.9 Hz, 1H), 4.19 (d, J = 12.9 Hz, 1H), 4.01 (dd, J = 13.1, 7.1 Hz, 1H),
13
3.90–3.77 (m, 4H), 2.94–2.83 (m, 4H), 2.80 (s, 3H), 1.96–1.82 (m, 2H), 1.67 (s, 4H); C
NMR (101 MHz, MeOD) δ 161.95, 147.02, 145.99, 141.74, 132.48, 130.95, 129.73,
1
2
29.54, 129.34, 128.49, 115.20, 114.96, 60.13, 59.16, 55.79, 39.42, 37.38, 37.24, 33.67,
+
9.41, 29.33. HRMS (ESI) m/z calcd for C H NO [M+H] 336.2327, found 336.2322.
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(
E)-3-(4-Fluorophenyl)-N-methyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)
methyl)prop-2-en-1-amine hydrochloride (9).
9
was synthesized by the general procedure of 6, given above, as a white solid. m.p.
10
11
12
13
14
15
16
17
18
19
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1
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89ꢀ190 ºC. HꢀNMR (400 MHz, MeOD) δ 7.66–7.47 (m, 2H), 7.25 (d, J = 8.4 Hz, 3H),
7.14 (t, J = 8.5 Hz, 2H), 6.91 (d, J = 15.8 Hz, 1H), 6.38–6.20 (m, 1H), 4.31 (d, J = 70.2
Hz, 2H), 3.95 (d, J = 41.8 Hz, 2H), 2.86 (t, J = 11.5 Hz, 4H), 2.81 (s, 3H), 1.90 (s, 2H),
1
3
1
.67 (s, 4H); C NMR (101 MHz, MeOD) δ 147.03, 145.99, 133.17, 132.52, 130.95,
130.16, 130.07, 129.77, 128.45, 117.70, 116.74, 60.26, 58.85, 39.54, 37.38, 37.24, 33.66,
+
2
9.41, 29.32. HRMS (ESI) m/z calcd for C H FN [M+H] 324.2128, found 324.2122.
2
2
27
(
E)-3-(4-Bromophenyl)-N-methyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)
methyl)prop-2-en-1-amine hydrochloride (10).
1
0 was synthesized by the general procedure of 6, given above, as a white solid.
1
m.p. 192ꢀ194 ºC. HꢀNMR (400 MHz, MeOD) δ 7.54 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.2
Hz, 2H), 7.23 (d, J = 8.9 Hz, 3H), 6.87 (d, J = 15.6 Hz, 1H), 6.36 (dt, J = 15.2, 7.4 Hz,
1
H), 4.30 (d, J = 71.2 Hz, 2H), 3.93 (d, J = 49.8 Hz, 2H), 2.85 (d, J = 6.4 Hz, 4H), 2.79
1
3
(s, 3H), 1.88 (s, 2H), 1.64 (s, 4H); C NMR (101 MHz, MeOD) δ 147.08, 146.02,
1
5
40.56, 135.88, 132.99, 132.50, 130.96, 129.85, 129.75, 128.41, 123.87, 118.83, 60.34,
8.78, 39.62, 37.38, 37.24, 33.66, 29.41, 29.31. HRMS (ESI) m/z calcd for C H BrN
2
2
27
+
[M+H] 384.1327, found 384.1321.
E)-3-(4-Chlorophenyl)-N-methyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)
methyl)prop-2-en-1-amine hydrochloride (11).
(
1
1 was synthesized by the general procedure of 6, given above, as a white solid.
1
m.p. 200ꢀ201 ºC. HꢀNMR (400 MHz, MeOD) δ 7.53 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4
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Hz, 2H), 7.26 (d, J = 8.3 Hz, 3H), 6.91 (d, J = 15.9 Hz, 1H), 6.43–6.29 (m, 1H), 4.43 (d,
J = 12.9 Hz, 1H), 4.21 (d, J = 13.0 Hz, 1H), 4.04 (dd, J = 12.9, 6.8 Hz, 1H), 3.97–3.80
1
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(
m, 1H), 2.88 (d, J = 6.8 Hz, 4H), 2.81 (s, 3H), 1.90 (s, 2H), 1.66 (s, 4H); C NMR (101
0
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9
0
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7
8
9
0
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0
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0
MHz, MeOD) δ 147.06, 146.01, 140.48, 135.75, 135.49, 132.51, 130.96, 129.97, 129.77,
129.61, 128.42, 118.75, 60.32, 58.78, 39.60, 37.38, 37.24, 33.66, 29.41, 29.32. HRMS
+
(
ESI) m/z calcd for C H ClN [M+H] 340.1832, found 340.1827.
2
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(E)-N-Methyl-3-(4-nitrophenyl)-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)me
thyl)prop-2-en-1-amine hydrochloride (12).
12 was synthesized by the general procedure of 6, given above, as a white solid.
1
m.p. 182ꢀ185 ºC. HꢀNMR (400 MHz, MeOD) δ 8.28 (d, J = 8.6 Hz, 2H), 7.77 (d, J = 8.6
Hz, 2H), 7.26 (t, J = 10.1 Hz, 3H), 7.04 (d, J = 15.9 Hz, 1H), 6.67–6.53 (m, 1H), 4.35 (d,
J = 65.6 Hz, 2H), 4.03 (d, J = 49.6 Hz, 2H), 2.98–2.76 (m, 7H), 1.90 (s, 2H), 1.66 (s,
1
3
4
1
2
H); C NMR (101 MHz, MeOD) δ 149.17, 147.14, 146.04, 143.07, 139.31, 132.52,
30.98, 129.79, 129.06, 128.33, 124.98, 122.86, 60.51, 58.50, 39.80, 37.38, 37.24, 33.65,
+
9.40, 29.30. HRMS (ESI) m/z calcd for C H N O [M+H] 351.2073, found 351.2067.
2
2
27
2
2
(E)-N-Methyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl)-3-(4-(trifluor
omethyl)phenyl)prop-2-en-1-amine hydrochloride (13).
13 was synthesized by the general procedure of 6, given above, as a white solid.
1
m.p. 203ꢀ204 ºC. HꢀNMR (400 MHz, MeOD) δ 7.71 (s, 4H), 7.26 (d, J = 8.6 Hz, 3H),
7
.00 (d, J = 15.7 Hz, 1H), 6.51 (dt, J = 15.3, 7.5 Hz, 1H), 4.43 (s, 1H), 4.25 (s, 1H), 4.07
13
(
s, 1H), 3.94 (s, 1H), 2.98–2.68 (m, 6H), 1.90 (s, 2H), 1.66 (s, 4H); C NMR (101 MHz,
MeOD) δ 147.10, 146.02, 140.54, 140.08, 132.52, 130.97, 129.78, 128.65, 128.38,
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26.73, 121.05, 60.45, 58.63, 39.74, 37.38, 37.24, 33.65, 29.40, 29.30. HRMS (ESI) m/z
+
calcd for C H F N [M+H] 374.2096, found 374.2090.
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thyl)prop-2-en-1-amine hydrochloride (14).
14 was synthesized by the general procedure of 6, given above, as a white solid.
1
m.p. 162ꢀ165 ºC. HꢀNMR (400 MHz, MeOD) δ 8.06 (d, J = 8.2 Hz, 1H), 7.76 (q, J = 7.6
Hz, 2H), 7.61 (t, J = 6.7 Hz, 1H), 7.34 (t, J = 12.9 Hz, 1H), 7.26 (t, J = 10.4 Hz, 3H), 6.32
(dt, J = 15.2, 7.4 Hz, 1H), 4.43 (s, 1H), 4.27 (s, 1H), 4.08 (s, 1H), 3.96 (s, 1H), 2.88 (d, J
13
=
9.2 Hz, 7H), 1.90 (d, J = 5.1 Hz, 2H), 1.67 (s, 4H); C NMR (101 MHz, MeOD) δ
1
49.38, 147.12, 146.05, 137.51, 134.78, 132.52, 132.30, 130.98, 130.82, 130.43, 129.81,
128.32, 125.62, 123.03, 60.35, 58.27, 39.78, 37.36, 37.25, 33.66, 29.40, 29.32. HRMS
+
(
ESI) m/z calcd for C H N O [M+H] 351.2073, found 351.2067.
2
2
27
2
2
(E)-3-(2-Fluorophenyl)-N-methyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)
methyl)prop-2-en-1-amine hydrochloride (15).
15 was synthesized by the general procedure of 6, given above, as a white solid.
1
m.p. 152ꢀ155 ºC. HꢀNMR (400 MHz, MeOD) δ 7.65 (t, J = 7.6 Hz, 1H), 7.39 (d, J = 6.2
Hz, 1H), 7.24 (dd, J = 19.8, 10.6 Hz, 4H), 7.16 (t, J = 9.7 Hz, 1H), 7.06 (d, J = 16.0 Hz,
1
H), 6.52–6.38 (m, 1H), 4.43 (d, J = 13.1 Hz, 1H), 4.23 (d, J = 13.0 Hz, 1H), 4.08 (dd, J
= 13.0, 7.0 Hz, 1H), 4.01–3.83 (m, 1H), 2.97–2.63 (m, 7H), 1.82 (s, 2H), 1.66 (s, 4H);
13
C NMR (101 MHz, MeOD) δ 147.07, 146.01, 134.03, 132.52, 131.85, 130.96, 129.77,
1
29.35, 129.32, 128.39, 125.74, 124.41, 120.86, 116.94, 60.39, 59.03, 39.68, 37.38,
+
3
7.24, 33.67, 29.40, 29.31. HRMS (ESI) m/z calcd for C H FN [M+H] 324.2128,
2
2
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found 324.2122.
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(
E)-N-Methyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl)-3-(m-tolyl)p
rop-2-en-1-amine hydrochloride (16).
16 was synthesized by the general procedure of 6, given above, as a white solid.
0
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0
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1
m.p. 167ꢀ168 ºC. H NMR (400 MHz, MeOD) δ 7.41–7.22 (m, 6H), 7.18 (d, J = 7.3 Hz,
1H), 6.89 (d, J = 15.7 Hz, 1H), 6.32 (dd, J = 15.5, 7.6 Hz, 1H), 4.43 (d, J = 12.9 Hz, 1H),
4
.21 (d, J = 12.9 Hz, 1H), 4.04 (dd, J = 13.1, 7.0 Hz, 1H), 3.88 (dd, J = 13.1, 8.0 Hz, 1H),
1
3
2
.92–2.82 (m, 4H), 2.81 (s, 3H), 2.37 (s, 3H), 1.90 (d, J = 5.3 Hz, 2H), 1.67 (s, 4H); C
NMR (101 MHz, MeOD) δ 147.02, 145.98, 142.13, 139.62, 136.65, 132.52, 130.94,
1
2
30.81, 129.76, 128.69, 128.46, 125.30, 117.44, 60.24, 58.98, 39.55, 37.38, 37.24, 33.67,
+
9.41, 29.32, 21.36. HRMS (ESI) m/z calcd for C H N [M+H] 320.2378, found
23 30
320.2378.
E)-N-Methyl-3-(3-nitrophenyl)-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)me
thyl)prop-2-en-1-amine hydrochloride (17).
(
17 was synthesized by the general procedure of 6, given above, as a white solid.
1
m.p. 167ꢀ169 ºC. H NMR (400 MHz, MeOD) δ 8.41 (s, 1H), 8.23 (d, J = 8.2 Hz, 1H),
7.94 (d, J = 7.7 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 7.26 (dd, J = 12.6, 10.0 Hz, 3H), 7.04
(d, J = 15.8 Hz, 1H), 6.64–6.47 (m, 1H), 4.46 (d, J = 12.9 Hz, 1H), 4.26 (d, J = 12.9 Hz,
1
H), 4.10 (dd, J = 13.2, 6.9 Hz, 1H), 3.96 (dd, J = 13.2, 7.8 Hz, 1H), 2.87 (dd, J = 12.5,
1
3
7.9 Hz, 7H), 1.89 (d, J = 5.4 Hz, 2H), 1.66 (s, 4H); C NMR (101 MHz, MeOD) δ
1
1
48.67, 145.70, 144.62, 137.88, 137.24, 132.67, 131.17, 129.67, 128.43, 127.01, 122.97,
21.20, 120.04, 59.07, 57.16, 38.42, 35.93, 32.28, 27.97. HRMS (ESI) m/z calcd for
+
C H N O [M+H] 351.2073, found 351.2069.
22
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E)-N-Methyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl)but-2-en-1-a
mine hydrochloride (18).
18 was synthesized by the general procedure of 6, given above, as a white solid.
10
11
12
13
14
15
16
17
18
19
20
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1
m.p. 143ꢀ145 ºC. HꢀNMR (400 MHz, MeOD) δ 7.22 (dd, J = 9.3, 7.0 Hz, 3H), 6.10 (dq,
J = 13.1, 6.5 Hz, 1H), 5.66 (dt, J = 14.0, 6.9 Hz, 1H), 4.35 (d, J = 12.9 Hz, 1H), 4.11 (d, J
=
12.8 Hz, 1H), 3.85–3.74 (m, 1H), 3.70–3.57 (m, 1H), 2.94–2.80 (m, 4H), 2.72 (s, 3H),
1
3
1
.97–1.73 (m, 5H), 1.67 (s, 4H); C NMR (101 MHz, MeOD) δ 146.96, 145.95, 139.94,
132.46, 130.91, 129.72, 128.45, 120.30, 59.90, 58.62, 39.19, 37.37, 37.24, 33.67, 29.42,
+
2
9.34, 18.25. HRMS (ESI) m/z calcd for C H N [M+H] 244.2065, found 244.2060.
1
7
26
(
E)-3-(Furan-2-yl)-N-methyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methy
l)prop-2-en-1-amine hydrochloride (19).
1
9 was synthesized by the general procedure of 6, given above, as a pale yellow
1
solid. m.p. 132ꢀ136 ºC. HꢀNMR (400 MHz, MeOD) δ 7.56 (s, 1H), 7.25 (d, J = 9.3 Hz,
3
H), 6.77 (d, J = 15.7 Hz, 1H), 6.52 (d, J = 12.2 Hz, 2H), 6.18 (dt, J = 15.4, 7.6 Hz, 1H),
.41 (d, J = 12.8 Hz, 1H), 4.18 (d, J = 12.9 Hz, 1H), 4.08–3.97 (m, 1H), 3.93–3.75 (m,
4
1
3
1H), 2.88 (d, J = 5.1 Hz, 4H), 2.78 (s, 3H), 1.90 (s, 2H), 1.67 (s, 4H); C NMR (101
MHz, MeOD) δ 152.37, 147.08, 146.02, 144.84, 132.48, 130.96, 129.73, 129.60, 128.39,
1
15.47, 112.77, 112.20, 60.23, 58.68, 39.42, 37.38, 37.24, 33.66, 29.40, 29.33. HRMS
+
(ESI) m/z calcd for C H NO [M+H] 296.2014, found 296.2009.
2
0
26
(
E)-3-Cyclohexyl-N-methyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl)
prop-2-en-1-amine hydrochloride (20).
2
0 was synthesized by the general procedure of 6, given above, as a white solid. m.p
1
1
61ꢀ163 ºC. HꢀNMR (400 MHz, MeOD) δ 7.22 (d, J = 7.1 Hz, 3H), 6.02 (dd, J = 15.3,
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.9 Hz, 1H), 5.84 (t, J = 10.6 Hz, 1H), 4.34 (d, J = 12.5Hz, 1H), 4.16 (d, J = 12.7 Hz,
H), 3.80 (dd, J = 18.1, 10.6 Hz, 1H), 3.69–3.53 (m, 1H), 2.87 (s, 4H), 2.76 (s, 3H), 2.27
1
1
3
(d, J = 10.8 Hz, 1H), 1.90 (s, 2H), 1.84–1.58 (m, 8H), 1.38–1.10 (m, 6H); C NMR (101
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
MHz, MeOD) δ 150.45, 147.40, 147.06, 146.02, 132.43, 130.97, 129.71, 128.50, 59.98,
58.80, 53.28, 42.07, 39.69, 39.29, 37.85, 37.39, 33.70, 33.44, 29.44 , 27.08, 26.90.
+
HRMS (ESI) m/z calcd for C H N [M+H] 312.2691, found 312.2686.
2
2
34
N-Methyl-3-phenyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl)propan-
1-amine hydrochloride (21).
21 was synthesized by the general procedure of 6, given above, as a white solid.
1
m.p. 138ꢀ140 ºC. HꢀNMR (400 MHz, MeOD) δ 7.35–7.25 (m, 2H), 7.26–7.10 (m, 6H),
4.19 (d, J = 50.0 Hz, 2H), 3.13 (s, 2H), 2.90–2.83 (m, 4H), 2.80 (s, 3H), 2.71 (t, J = 7.4
13
Hz, 2H), 2.18–1.98 (m, 2H), 1.89 (d, J = 4.9 Hz, 2H), 1.65 (s, 4H); C NMR (101 MHz,
MeOD) δ 146.96, 145.89, 141.36, 132.49, 130.87, 129.79, 129.65, 129.45, 128.17,
1
27.45, 60.69, 56.04, 40.21, 37.34, 37.22, 33.64, 33.42, 29.41, 29.31, 26.87. HRMS
+
(ESI) m/z calcd for C H N [M+H] 308.2378, found 308.2373.
2
2
30
(E)-N,2-Dimethyl-3-phenyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl)
prop-2-en-1-amine hydrochloride (22).
22 was synthesized by the general procedure of 6, given above, as a white solid.
1
m.p. 164ꢀ166 ºC. HꢀNMR (400 MHz, MeOD) δ 7.48–7.10 (m, 8H), 6.79 (s, 1H), 4.39 (d,
J = 13.0 Hz, 1H), 4.24 (t, J = 11.5 Hz, 1H), 3.98 (t, J = 12.4 Hz, 1H), 3.81 (d, J = 12.8
1
3
Hz, 1H), 2.93–2.73 (m, 7H), 1.98 (s, 3H), 1.87 (d, J = 5.3 Hz, 2H), 1.64 (s, 4H); C
NMR (101 MHz, MeOD) δ 145.69, 144.55, 136.76, 136.23, 135.95, 131.43,, 129.550,
1
28.75, 128.05, 127.49, 127.12, 126.86, 126.28, 64.30, 59.47 , 39.19, 35.93, 32.28, 31.66,
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Page 27 of 67
Journal of Medicinal Chemistry
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+
2
9.58, 27.99, 27.28, 15.87. HRMS (ESI) m/z calcd for C H N [M+H] 320.2378, found
23 30
3
20.2373.
(
E)-N-Methyl-4-phenyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl)but-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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35
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37
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47
48
49
50
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52
53
54
55
56
57
58
59
60
2
-en-1-amine hydrochloride (23).
23 was synthesized by the general procedure of 6, given above, as a yellow oil.
1
HꢀNMR (400 MHz, MeOD) δ 7.36–7.13 (m, 8H), 6.30–6.18 (m, 1H), 5.73–5.63 (m,
1
H), 4.33 (d, J = 13.0 Hz, 1H), 4.12 (d, J = 12.8 Hz, 1H), 3.84 (dd, J = 13.1, 6.7 Hz, 1H),
3.74–3.60 (m, 1H), 3.47 (t, J = 32.1 Hz, 2H), 2.84 (t, J = 10.0 Hz, 4H), 2.73 (s, 3H), 1.90
13
(s, 2H), 1.60 (d, J = 42.4 Hz, 4H); C NMR (101 MHz, MeOD) δ 147.03, 145.98,
1
43.88, 140.20, 132.43, 130.93, 129.72, 128.31, 127.51, 120.01, 59.98, 58.35, 39.73,
+
39.39, 37.35, 37.23, 33.65, 29.41, 29.32. HRMS (ESI) m/z calcd for C H N [M+H]
2
3
30
3
20.2378, found 320.2373.
(2E,4E)-N-Methyl-5-phenyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl
)penta-2,4-dien-1-amine hydrochloride (24).
24 was synthesized by the general procedure of 6, given above, as a white solid.
1
m.p. 158ꢀ159 ºC. HꢀNMR (400 MHz, MeOD) δ 7.50 (d, J = 7.5 Hz, 2H), 7.40–7.32 (m,
2
H), 7.27 (dd, J = 18.1, 7.7 Hz, 4H), 6.98 (dd, J = 15.3, 10.6 Hz, 1H), 6.75 (dd, J = 28.9,
3.4 Hz, 2H), 5.92 (dt, J = 14.8, 7.5 Hz, 1H), 4.39 (d, J = 12.8 Hz, 1H), 4.17 (d, J = 13.0
1
Hz, 1H), 3.96 (dd, J = 13.1, 7.4 Hz, 1H), 3.87–3.75 (m, 1H), 2.84 (d, J = 30.7 Hz, 4H),
13
2
1
1
.78 (s, 3H), 1.90 (s, 2H), 1.67 (s, 4H); C NMR (101 MHz, MeOD) δ 147.06, 146.00,
42.49, 137.91, 137.42, 132.47, 130.95, 129.78, 129.72, 129.41, 128.41, 127.93, 127.82,
20.78, 60.08, 58.65, 39.40, 37.38, 37.24, 33.66, 29.41, 29.33. HRMS (ESI) m/z calcd
+
for C H N [M+H] 332.2378, found 332.2373.
2
4
30
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Journal of Medicinal Chemistry
Page 28 of 67
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N-Methyl-3-phenyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl)prop-2-
yn-1-amine hydrochloride (25).
25 was synthesized by the general procedure of 6, given above, as a white solid.
0
1
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9
0
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9
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8
9
0
1
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7
8
9
0
1
m.p. 171ꢀ174 ºC. HꢀNMR (400 MHz, MeOD) δ 7.58 (d, J = 7.5 Hz, 2H), 7.52–7.37 (m,
3H), 7.28 (d, J = 11.3 Hz, 3H), 4.34 (d, J = 49.5 Hz, 4H), 2.99 (s, 3H), 2.88 (s, 4H), 1.90
1
3
(
s, 2H), 1.67 (s, 4H); C NMR (101 MHz, MeOD) δ 147.27, 146.06, 133.08, 132.60,
1
31.05, 130.87, 129.79, 127.97, 122.25, 91.56, 78.08, 59.88, 46.19, 40.19, 37.42, 37.27,
+
33.68, 29.40, 29.31. HRMS (ESI) m/z calcd for C H N [M+H] 304.2065, found
2
2
26
3
04.2060.
(E)-3-Phenyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl)prop-2-en-1-a
mine hydrochloride (26).
A mixture of 49 (0.35 g, 2 mmol), transꢀcinnamaldehyde (0.26 g, 2 mmol) and
molecular sieve (1 g) in dichloromethane (25 mL) was heated at reflux for 17 h.
Thereafter, methanol was added into the mixture, and then sodium borohydride (0.08 g, 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
Na SO , filtered and condensed. The residue was then purified via flash chromatography
2
4
on silica gel, eluting with EtOAc/petroleum ether (1/1, v/v) to give the free base of 26
(0.56 g, 96% yield) as a colorless oil. 26 was prepared using the general procedure of
1
salification as a white solid. m.p. 102ꢀ105 ºC. HꢀNMR (400 MHz, MeOD) δ 7.50 (d, J =
7
.1 Hz, 2H), 7.36 (dt, J = 20.1, 7.0 Hz, 3H), 7.31–7.13 (m, 3H), 6.89 (d, J = 15.7 Hz,
H), 6.37 – 6.25 (m, 1H), 4.17 (d, J = 12.5 Hz, 2H), 3.85 (d, J = 7.1 Hz, 2H), 2.86 (dd, J
1
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Page 29 of 67
Journal of Medicinal Chemistry
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=
10.6, 5.3 Hz, 4H), 1.89 (d, J = 5.5 Hz, 2H), 1.66 (s, 4H); C NMR (101 MHz, MeOD)
δ 146.43, 145.81, 139.95, 136.92, 131.44, 130.84, 130.03, 129.82, 128.57, 127.93,
19.29, 51.48, 50.18, 37.41, 37.22, 33.68, 29.45. HRMS (ESI) m/z calcd for C H N
1
2
1
26
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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49
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60
+
[
M+H] 292.2065, found 292.2060.
(E)-N-Ethyl-3-phenyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl)prop-
2
-en-1-amine hydrochloride (27).
To a solution of 26 (0.41 g, 1.4 mmol) in DMF (10 mL) was added sodium hydride
(0.05 g, 1.4 mmol) in batches at 0 ºC under a N atmosphere. The reaction mixture was
2
stirred for 15 min, and iodoethane (0.22 mL, 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
Na SO , filtered and condensed. The residue was then purified via flash chromatography
2
4
on silica gel, eluting with EtOAc/petroleum ether (1/5, v/v) to give the free base of 27
(
0.33 g, 74% yield) as a colorless oil. 27 was prepared using the general procedure of
1
salification as a yellow oil. HꢀNMR (400 MHz, MeOD) δ 7.52 (d, J = 7.0 Hz, 2H), 7.45–
7.32 (m, 3H), 7.25 (t, J = 9.0 Hz, 3H), 6.91 (d, J = 15.8 Hz, 1H), 6.39–6.23 (m, 1H), 4.34
(
s, 2H), 3.95 (dd, J = 16.0, 9.9 Hz, 2H), 3.26 (dd, J = 14.4, 7.2 Hz, 2H), 2.99–2.75 (m,
1
3
4
H), 1.89 (d, J = 5.2 Hz, 2H), 1.75–1.53 (m, 4H), 1.42 (t, J = 7.2 Hz, 3H); C NMR (101
MHz, MeOD) δ 147.05, 146.08, 141.77, 136.71, 132.45, 131.02, 130.11, 129.86, 129.69,
1
28.42, 128.07, 117.38, 57.51, 55.38, 37.39, 37.23, 33.65, 29.41, 29.31, 9.31. HRMS
+
(
ESI) m/z calcd for C H N [M+H] 320.2378, found 320.2373.
23 30
(
E)-N-Isopropyl-3-phenyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl)p
rop-2-en-1-amine hydrochloride (28).
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