Discovery of Benzocycloalkane Derivatives Efficiently Blocking

Subscriber access provided by UNIV OF PITTSBURGH

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

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

Youxin Wanga,†, Hongxia Dib,†, Feifei Chenb, Yong Xuc, Qiang Xiaoc, Xuehai Wangd, Hanwen Weia, Yanli Lua, Lingling Zhanga, Jin Zhua, Lefu Lanb, *, Jian Lia, * a

Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China

University of Science and Technology, Shanghai 200237, China b

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese

Academy of Sciences, Shanghai 201203, China c

Hubei Bio-pharmaceutical 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. [email protected] or [email protected]

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Anti-virulence 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. Analog 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 anti-fungal activity compared to NTF. In summary, 8 has the potential to be developed as a therapeutic drug, especially against intractable MRSA issues.


Page 2 of 67

Page 3 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 resistance.1 However, we seem to perpetually lose this arms race, as indicated by the widespread outbreaks of hospital-associated methicillin-resistant S. aureus (HA -MRSA),2 community-associated MRSA (CA-MRSA),3 livestock-associated MRSA (LA-MRSA)4-6 and even vancomycin-resistant S. aureus (VRSA)7 infectious diseases. It is estimated that by the year 2050, humans will suffer 10 million losses annually due to antimicrobial resistance (AMR).8 In the US alone, MRSA causes over 23 thousand deaths and over 2 million illnesses per year.9 Unfortunately, the discovery of new antibiotics has lagged far behind the growing emergence of antibiotic resistance, as for nearly 20 years, no new classes have been launched.10 Although we are capable of combining existing antibiotics to tackle immediate clinical dilemmas, as exemplified by the triple β-lactam combinations,11 the emergence of resistance is inevitable.12 Consequently, there is an urgent need for anti-infective agents with novel modes of action in the post-antibiotic era.13 Anti-virulence strategies aiming at ‘disarming’ the pathogen rather than inhibiting its growth, with weak selective pressure for the development of antibiotic resistance, are now attracting interest.14-16 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 biosynthesis is an underlying magnetic therapeutic target.17-19 Staphyloxanthin biosynthesis begins with the condensation of farnesyl diphosphate, followed by a series of important enzyme (CrtM, CrtN, CrtQ, CrtP, CrtO)-catalyzed reactions.20 A cholesterol



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

biosynthesis inhibitor, BPH-652 (Figure 1), targeting dehydrosqualene synthase (CrtM) to block staphyloxanthin biosynthesis, has provided a good lead for anti-virulence strategies along with the consequence of the susceptibility of S. aureus to the innate immune eradication.21 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 FDA-approved antifungal drug, to be an efficiency CrtN inhibitor with an established safety and efficacy profile both in vitro and in vivo.22 CHEMISTRY In this study, to develop a more potent anti-virulence 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 di-tert-butyl 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 1-benzosuberone 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 1-position/2-position = 1:4 (as determined by NMR spectra). We speculate that the reaction site was restricted as a result of the p-methylene directing effect and m-methylene space effect. Due to their highly


Page 4 of 67

Page 5 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 47 was followed by reduction with lithium aluminum hydride to afford amines 48 and 49. Treatment with di-tert-butyl 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

of

the

carboxy

group

of

commercially

attainable

2,3-dihydro-1H-indene-5-carboxylic acid and 5,6,7,8-tetrahydronaphthalene-2-carboxylic 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 4-substituted 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 Wittig-Horner olefination, 59 was yielded from phenylacetaldehyde, followed by the reduction of the ethyl ester and a bromination reaction to generate 61a-b;



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

61a-b, the commercially available (E)-1-bromobut-2-ene and (3-bromopropyl) 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 trans-cinnamaldehyde 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. 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 (3-bromopropyl) 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 skeleton compounds 1, 223 and 3. During the synthesis of compound 3, a by-product 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 2-position 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 2-position substituted products exhibited more promising potencies than their isomerides, with compound 6 revealing the strongest effect. Having established the unexpected superiority


Page 6 of 67

Page 7 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 (Table 1). The incorporation of a substituent at the 4-position of the phenyl was beneﬁcial for improving the potency, and both electron-donating and electron-withdrawing groups were tolerable (7-13 vs 6). Strikingly, compounds 8, 10 and 11 boosted the activity to a single-digit nanomolar level. Nonetheless, introducing the substituent to the 2-position or 3-position 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 N-methyl to hydrogen (26), ethyl (27) and isopropyl (28) to give insight into the effect of the N-substituent. As shown in Figure 3, the N-methyl 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.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The detailed structure-activity relationship (SAR) analysis (NTF, 1-31) is provided as follows: (1) Under the condition of the replacement of the naphthalenyl moiety of NTF with different-sized benzocycloalkane rings, the substituted positon is crucial for the potency. Generally, the 2-position is preferred to the 1-position (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 17). 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) N-substituent 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 (32-36, 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 > 300-fold greater effectiveness than the parent compound 5. Activity Target Determination of the NTF-derived Benzocycloalkane Analogs. As NTF is an established CrtN inhibitor,22 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


Page 8 of 67

Page 9 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


appeared after the expression of crtM in E. coli (Figure 6B), which was similar to that of wild-type 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 (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 8-treated wild-type S. aureus Newman (Figure 6G) with NTF-treated Newman (Figure 6F), 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 protocol,22 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 concentration;22 (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.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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. In Vitro Pigment Inhibitory Activities of Analog 8 against MRSA. 8 had been demonstrated to be a high-efficient 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 for the epidemic of CA-MRSA infectious diseases in the United States,24-25 while VISA Mu50 is a hospital-acquired MRSA strain isolated in Japan.26 As shown in Figure 7, 8 possesses a comparative ability in the pigment inhibition of USA400 MW2 and USA300 LAC (IC50 = 7.1 nM, 7.7 nM, respectively) to Mu50 at a picomole quantity (IC50 = 0.49 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 anti-virulence 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 (H2O2) or human whole blood. To verify this argument experimentally, we first compared the


Page 10 of 67

Page 11 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


susceptibility to H2O2 killing of mock-treated S. aureus Newman with that of 8-treated (1 µM) S. aureus Newman. As shown in Figure 9, the 8-treated non-pigmented S. aureus cells were more susceptible to killing by 1.5% H2O2 by a factor of ~37 (survival, 0.83% vs 30.43%). On the other hand, the survival of the known antioxidant N-acetylcysteine (NAC)-treated S. aureus cells was at a considerable level similar to that of the mock-treated 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 H2O2 (0.96% vs 14.17%, 0.98% vs 15.83%, 4.44% vs 30.67%, respectively). The addition of NAC clearly improved the survival rates of all three MRSA strains, demonstrating the addition of H2O2 exerted impact on the strain survival by the oxidation capacity and the pigment definitely acted as the protective antioxidant. Subsequently, we sought to ascertain whether 8 could decrease the whole-blood survival of S. aureus. Fresh human whole blood was added to each of 8-treated (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 8-treated 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.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 retro-orbital injection and measured the bacterial survival in the host organs. As shown in Figure 11A, the 200 mg/kg dosage 8-treatment reduced bacterial survival in the kidneys and heart by 1.45 and 1.66 log10 CFU, corresponding to 96.4% 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 log10 CFU), only a slight decrease compared to the 200 mg/kg dosage. To further evaluate the efficiency of 8 on affecting the outcome of S. aureus sepsis, we challenged animals with 2 × 107 CFU Newman bacteria (Figure 11B). The untreated mice died out within 4 days post-challenge, 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 NTF-treated 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 log10 CFU (more than a 99.9% decrease in surviving bacteria), while 37, the positive control drug, led to a reduction by only 1.59 log10 CFU at the same dosage, making it less efficacious than the 8 treatment. Upon cutting the dosage down by three


Page 12 of 67

Page 13 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 dose-dependent result of the 37 treatment was not so obvious, and in both cases, it was less efficacious than the 8 treatment. 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 log10 CFU (equivalent to a 99.99% bacteria 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 log10 CFU. Upon decreasing the dosage to 50 mg/kg, the 8 treatment still exhibited a significant effect (~2.44 log10 CFU/organ reduction), although worse than that of the high dose groups, which suggested an explicitly dose-dependent 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 log10 CFU with the prescription of 200 mg/kg 8, while 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 log10 CFU reduction), but the bacterial survival rates of the low-dosage 37 treatment were considerable compared to that of 200 mg/kg dosage group. In brief, these facts clearly reveal that 8, acting as an anti-virulence agent, could decrease both the S. aureus Newman and clinically relevant multi-drug resistant S. aureus survival with superior results to 37.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 anti-fungal drug, the question of whether 8 inherited the anti-fungal activity aroused our interest. Eventually, three naftifine sensitive strains27 were chosen to proceed with the in vitro assay. Encouragingly, 8 exhibited weak activities against all three dermatophytes compared to NTF, at a 64-fold 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 single-digit 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 N-substituents, 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 activity, being far more effective than NTF.22 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 anti-virulence, 8 (1 µM) sensitized S. aureus strains to killing by H2O2 and human whole blood. In an in vivo assay, 8 was proven to be


Page 14 of 67

Page 15 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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, 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 8-treated Mu50 model), performing better than 37. Furthermore, 8 exhibited minimal anti-fungal activity (MIC > 8 µg/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, Adamas-beta, Energy Chemical, J&K, Shanghai Chemical Reagent Co. and TCI at the highest commercial quality and were used without further purification. Analytical thin-layer chromatography (TLC) was performed on HSGF 254 (150−200 µm 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 X-4 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 AMX-400 NMR using deuterated chloroform (CDCl3), deuterated methanol (CD3OD), or deuterated dimethyl sulfoxide (DMSO-d6) as the solvent. Chemical shifts were reported



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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). Low- and high-resolution mass spectra (LRMS and HRMS) were given with 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 diode-array 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 K2CO3 (0.33 g, 2.40 mmol) in DMF (10 mL) was stirred at room temperature overnight. The mixture was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue was then purified via flash chromatography on silica gel, eluting with EtOAc/petroleum ether (1/5, v/v) to give the free base of 1 (0.32 g, 58% yield) as a


Page 16 of 67

Page 17 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


colorless oil. 1 was prepared using a general procedure of salification as a white solid. m.p. 155-156 ºC. 1H-NMR (400 MHz, MeOD) δ 7.53 (d, J = 7.5 Hz, 2H), 7.36 (dt, J = 19.5, 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, 1H), 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), 2.87 (s, 3H), 2.15 (dd, J = 14.8, 7.4 Hz, 2H);

13

C NMR (101 MHz, MeOD) δ 147.09,

146.32, 142.13, 136.69, 130.14, 129.86, 129.72, 128.37, 128.12, 127.25, 126.95, 117.78, 59.41, 58.10, 39.93, 33.94, 32.40, 26.09. HRMS (ESI) m/z calcd for C20H24N [M+H]+ 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. m.p. 165-166 ºC. 1H-NMR (400 MHz, MeOD) δ 7.54 (d, J = 7.5 Hz, 2H), 7.38 (dq, J = 13.9, 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, 1H), 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 = 13.7, 7.7 Hz, 2H), 2.94–2.72 (m, 7H), 1.97–1.71 (m, 4H); 13C 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, 127.15, 117.73, 59.64, 57.27, 40.18, 30.97, 27.40, 24.20, 23.58. HRMS (ESI) m/z calcd for C21H26N [M+H]+ 292.2065, found 292.2068. (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).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A solution of mixed 52 and 53 (0.38 g, 2.00 mmol), cinnamyl bromide (0.39 g, 2.00 mmol) and K2CO3 (0.33 g, 2.40 mmol) in DMF (10 mL) was stirred at room temperature overnight. The mixture was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue was then purified via flash chromatography on silica gel, eluting with EtOAc/petroleum ether (1/5, v/v) to 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. 186-188 ºC. 1

H-NMR (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, 4H);

13

C NMR (101 MHz, MeOD) δ 146.73, 145.08, 142.26, 136.69, 132.45, 131.30,

130.16, 129.87, 128.28, 128.12, 127.64, 117.82, 59.41, 57.85, 39.81, 37.40, 33.05, 31.02, 29.10, 28.67. HRMS (ESI) m/z calcd for C22H28N [M+H]+ 306.2222, found 306.2220. 6: m.p. 172-174 ºC. 1H-NMR (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, 1H), 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 (m, 4H), 2.81 (s, 3H), 2.01–1.78 (m, 2H), 1.66 (d, J = 4.0 Hz, 4H); 13C NMR (101 MHz, MeOD) δ 147.06, 146.01, 142.00, 136.71, 132.50, 130.96, 130.11, 129.85, 129.75, 128.44, 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 C22H28N [M+H]+ 306.2222, found 306.2216. (E)-N-((2,3-Dihydro-1H-inden-5-yl)methyl)-N-methyl-3-phenylprop-2-en-1-amine hydrochloride (4).


Page 18 of 67

Page 19 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A solution of 56a (0.32 g, 2.00 mmol), cinnamyl bromide (0.39 g, 2.00 mmol) and K2CO3 (0.33 g, 2.40 mmol) in DMF (10 mL) was stirred at room temperature overnight. The mixture was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue was then purified via flash chromatography on silica gel, eluting with EtOAc/petroleum ether (1/5, v/v) to give the free base of 4 (0.40 g, 72% yield) as a colorless oil. 4 was prepared using the general procedure of salification as a white solid. m.p. 160-161 ºC. 1H-NMR (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 Hz, 1H), 2.97 (dd, J = 13.2, 7.0 Hz, 4H), 2.81 (s, 3H), 2.21–2.08 (m, 2H); 13C NMR (101 MHz, MeOD) δ 147.78, 146.81, 141.93, 136.72, 130.15, 130.07, 129.83, 128.70, 128.11, 128.00, 126.12, 117.76, 60.56, 58.87, 39.48, 33.58, 26.49. HRMS (ESI) m/z calcd for C20H24N [M+H]+ 278.1909, found 278.1909. (E)-N-Methyl-3-phenyl-N-((5,6,7,8-tetrahydronaphthalen-2-yl)methyl)prop-2-en-1-a mine hydrochloride (5). 5 was synthesized by the general procedure of 4, given above, as a white solid. m.p. 182-184 ºC. 1H-NMR (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, 2H), 3.96 (d, J = 38.4 Hz, 2H), 2.82 (d, J = 7.7 Hz, 7H), 1.94–1.71 (m, 4H);

13

C NMR

(101 MHz, MeOD) δ 141.95, 140.61, 139.47, 136.72, 132.78, 131.09, 130.09, 129.84, 129.05, 128.11, 127.94, 117.72, 60.41, 58.89, 39.55, 30.25, 30.18, 24.10. HRMS (ESI) m/z calcd for C21H26N [M+H]+ 292.2065, found 292.2067.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(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. 188-189 ºC.

13

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, 37.24, 33.66, 29.41, 29.32, 21.30. 1H-NMR (400 MHz, MeOD) δ 7.39 (d, J = 7.8 Hz, 2H), 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 (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, 3H), 1.88 (s, 2H), 1.64 (s, 4H); 13C 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, 37.24, 33.66, 29.41, 29.32, 21.30. HRMS (ESI) m/z calcd for C23H30N [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. 164-167 ºC. 1H-NMR (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, 1H), 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), 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); 13C NMR (101 MHz, MeOD) δ 161.95, 147.02, 145.99, 141.74, 132.48, 130.95, 129.73, 129.54, 129.34, 128.49, 115.20, 114.96, 60.13, 59.16, 55.79, 39.42, 37.38, 37.24, 33.67, 29.41, 29.33. HRMS (ESI) m/z calcd for C23H30NO [M+H]+ 336.2327, found 336.2322.


Page 20 of 67

Page 21 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(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. 189-190 ºC. 1H-NMR (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), 1.67 (s, 4H);

13

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, 29.41, 29.32. HRMS (ESI) m/z calcd for C22H27FN [M+H]+ 324.2128, found 324.2122. (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). 10 was synthesized by the general procedure of 6, given above, as a white solid. m.p. 192-194 ºC. 1H-NMR (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, 1H), 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 (s, 3H), 1.88 (s, 2H), 1.64 (s, 4H);

13

C NMR (101 MHz, MeOD) δ 147.08, 146.02,

140.56, 135.88, 132.99, 132.50, 130.96, 129.85, 129.75, 128.41, 123.87, 118.83, 60.34, 58.78, 39.62, 37.38, 37.24, 33.66, 29.41, 29.31. HRMS (ESI) m/z calcd for C22H27BrN [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). 11 was synthesized by the general procedure of 6, given above, as a white solid. m.p. 200-201 ºC. 1H-NMR (400 MHz, MeOD) δ 7.53 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 (m, 1H), 2.88 (d, J = 6.8 Hz, 4H), 2.81 (s, 3H), 1.90 (s, 2H), 1.66 (s, 4H); 13C NMR (101 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 C22H27ClN [M+H]+ 340.1832, found 340.1827. (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. m.p. 182-185 ºC. 1H-NMR (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, 4H);

13

C NMR (101 MHz, MeOD) δ 149.17, 147.14, 146.04, 143.07, 139.31, 132.52,

130.98, 129.79, 129.06, 128.33, 124.98, 122.86, 60.51, 58.50, 39.80, 37.38, 37.24, 33.65, 29.40, 29.30. HRMS (ESI) m/z calcd for C22H27N2O2 [M+H]+ 351.2073, found 351.2067. (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. m.p. 203-204 ºC. 1H-NMR (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 (s, 1H), 3.94 (s, 1H), 2.98–2.68 (m, 6H), 1.90 (s, 2H), 1.66 (s, 4H); 13C NMR (101 MHz, MeOD) δ 147.10, 146.02, 140.54, 140.08, 132.52, 130.97, 129.78, 128.65, 128.38,


Page 22 of 67

Page 23 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


126.73, 121.05, 60.45, 58.63, 39.74, 37.38, 37.24, 33.65, 29.40, 29.30. HRMS (ESI) m/z calcd for C23H27F3N [M+H]+ 374.2096, found 374.2090. (E)-N-Methyl-3-(2-nitrophenyl)-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)me thyl)prop-2-en-1-amine hydrochloride (14). 14 was synthesized by the general procedure of 6, given above, as a white solid. m.p. 162-165 ºC. 1H-NMR (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 = 9.2 Hz, 7H), 1.90 (d, J = 5.1 Hz, 2H), 1.67 (s, 4H);

13

C NMR (101 MHz, MeOD) δ

149.38, 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 C22H27N2O2 [M+H]+ 351.2073, found 351.2067. (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. m.p. 152-155 ºC. 1H-NMR (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, 1H), 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,

129.35, 129.32, 128.39, 125.74, 124.41, 120.86, 116.94, 60.39, 59.03, 39.68, 37.38, 37.24, 33.67, 29.40, 29.31. HRMS (ESI) m/z calcd for C22H27FN [M+H]+ 324.2128, found 324.2122.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(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. m.p. 167-168 ºC. 1H 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), 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); 13C NMR (101 MHz, MeOD) δ 147.02, 145.98, 142.13, 139.62, 136.65, 132.52, 130.94, 130.81, 129.76, 128.69, 128.46, 125.30, 117.44, 60.24, 58.98, 39.55, 37.38, 37.24, 33.67, 29.41, 29.32, 21.36. HRMS (ESI) m/z calcd for C23H30N [M+H]+ 320.2378, found 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. m.p. 167-169 ºC. 1H 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, 1H), 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, 7.9 Hz, 7H), 1.89 (d, J = 5.4 Hz, 2H), 1.66 (s, 4H);

13


148.67, 145.70, 144.62, 137.88, 137.24, 132.67, 131.17, 129.67, 128.43, 127.01, 122.97, 121.20, 120.04, 59.07, 57.16, 38.42, 35.93, 32.28, 27.97. HRMS (ESI) m/z calcd for C22H27N2O2 [M+H]+ 351.2073, found 351.2069.


Page 24 of 67

Page 25 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(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. m.p. 143-145 ºC. 1H-NMR (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), 1.97–1.73 (m, 5H), 1.67 (s, 4H); 13C 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, 29.34, 18.25. HRMS (ESI) m/z calcd for C17H26N [M+H]+ 244.2065, found 244.2060. (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). 19 was synthesized by the general procedure of 6, given above, as a pale yellow solid. m.p. 132-136 ºC. 1H-NMR (400 MHz, MeOD) δ 7.56 (s, 1H), 7.25 (d, J = 9.3 Hz, 3H), 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), 4.41 (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, 1H), 2.88 (d, J = 5.1 Hz, 4H), 2.78 (s, 3H), 1.90 (s, 2H), 1.67 (s, 4H);

13

C NMR (101

MHz, MeOD) δ 152.37, 147.08, 146.02, 144.84, 132.48, 130.96, 129.73, 129.60, 128.39, 115.47, 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 C20H26NO [M+H]+ 296.2014, found 296.2009. (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). 20 was synthesized by the general procedure of 6, given above, as a white solid. m.p 161-163 ºC. 1H-NMR (400 MHz, MeOD) δ 7.22 (d, J = 7.1 Hz, 3H), 6.02 (dd, J = 15.3,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 67

6.9 Hz, 1H), 5.84 (t, J = 10.6 Hz, 1H), 4.34 (d, J = 12.5Hz, 1H), 4.16 (d, J = 12.7 Hz, 1H), 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 (d, J = 10.8 Hz, 1H), 1.90 (s, 2H), 1.84–1.58 (m, 8H), 1.38–1.10 (m, 6H); 13C NMR (101 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 C22H34N [M+H]+ 312.2691, found 312.2686. N-Methyl-3-phenyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl)propan1-amine hydrochloride (21). 21 was synthesized by the general procedure of 6, given above, as a white solid. m.p. 138-140 ºC. 1H-NMR (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 Hz, 2H), 2.18–1.98 (m, 2H), 1.89 (d, J = 4.9 Hz, 2H), 1.65 (s, 4H); 13C NMR (101 MHz, MeOD) δ 146.96, 145.89, 141.36, 132.49, 130.87, 129.79, 129.65, 129.45, 128.17, 127.45, 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 C22H30N [M+H]+ 308.2378, found 308.2373. (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. m.p. 164-166 ºC. 1H-NMR (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 Hz, 1H), 2.93–2.73 (m, 7H), 1.98 (s, 3H), 1.87 (d, J = 5.3 Hz, 2H), 1.64 (s, 4H);

13

C

NMR (101 MHz, MeOD) δ 145.69, 144.55, 136.76, 136.23, 135.95, 131.43,, 129.550, 128.75, 128.05, 127.49, 127.12, 126.86, 126.28, 64.30, 59.47 , 39.19, 35.93, 32.28, 31.66,


Page 27 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


29.58, 27.99, 27.28, 15.87. HRMS (ESI) m/z calcd for C23H30N [M+H]+ 320.2378, found 320.2373. (E)-N-Methyl-4-phenyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl)but2-en-1-amine hydrochloride (23). 23 was synthesized by the general procedure of 6, given above, as a yellow oil. 1

H-NMR (400 MHz, MeOD) δ 7.36–7.13 (m, 8H), 6.30–6.18 (m, 1H), 5.73–5.63 (m,

1H), 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 (s, 2H), 1.60 (d, J = 42.4 Hz, 4H);

13

C NMR (101 MHz, MeOD) δ 147.03, 145.98,

143.88, 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 C23H30N [M+H]+ 320.2378, 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. m.p. 158-159 ºC. 1H-NMR (400 MHz, MeOD) δ 7.50 (d, J = 7.5 Hz, 2H), 7.40–7.32 (m, 2H), 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, 13.4 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 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), 2.78 (s, 3H), 1.90 (s, 2H), 1.67 (s, 4H); 13C NMR (101 MHz, MeOD) δ 147.06, 146.00, 142.49, 137.91, 137.42, 132.47, 130.95, 129.78, 129.72, 129.41, 128.41, 127.93, 127.82, 120.78, 60.08, 58.65, 39.40, 37.38, 37.24, 33.66, 29.41, 29.33. HRMS (ESI) m/z calcd for C24H30N [M+H]+ 332.2378, found 332.2373.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

N-Methyl-3-phenyl-N-((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl)prop-2yn-1-amine hydrochloride (25). 25 was synthesized by the general procedure of 6, given above, as a white solid. m.p. 171-174 ºC. 1H-NMR (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 (s, 2H), 1.67 (s, 4H);

13

C NMR (101 MHz, MeOD) δ 147.27, 146.06, 133.08, 132.60,

131.05, 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 C22H26N [M+H]+ 304.2065, found 304.2060. (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), trans-cinnamaldehyde (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 Na2SO4, filtered and condensed. The residue was then purified via flash chromatography on silica gel, eluting with EtOAc/petroleum ether (1/1, v/v) to give the free base of 26 (0.56 g, 96% yield) as a colorless oil. 26 was prepared using the general procedure of salification as a white solid. m.p. 102-105 ºC. 1H-NMR (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, 1H), 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


Page 28 of 67

Page 29 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


= 10.6, 5.3 Hz, 4H), 1.89 (d, J = 5.5 Hz, 2H), 1.66 (s, 4H); 13C 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, 119.29, 51.48, 50.18, 37.41, 37.22, 33.68, 29.45. HRMS (ESI) m/z calcd for C21H26N [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)prop2-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 N2 atmosphere. The reaction mixture was 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 Na2SO4, filtered and condensed. The residue was then purified via flash chromatography on silica gel, eluting with EtOAc/petroleum ether (1/5, v/v) to give the free base of 27 (0.33 g, 74% yield) as a colorless oil. 27 was prepared using the general procedure of salification as a yellow oil. 1H-NMR (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, 4H), 1.89 (d, J = 5.2 Hz, 2H), 1.75–1.53 (m, 4H), 1.42 (t, J = 7.2 Hz, 3H); 13C NMR (101 MHz, MeOD) δ 147.05, 146.08, 141.77, 136.71, 132.45, 131.02, 130.11, 129.86, 129.69, 128.42, 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 C23H30N [M+H]+ 320.2378, found 320.2373. (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).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

28 was synthesized by the general procedure of 27, given above, as a white solid. m.p. 170-173 ºC. 1H-NMR (400 MHz, MeOD) δ 7.44 (d, J = 6.7 Hz, 2H), 7.36 (dd, J = 15.4, 7.6 Hz, 3H), 7.24 (t, J = 7.2 Hz, 3H), 6.85 (d, J = 15.7 Hz, 1H), 6.21–6.07 (m, 1H), 4.44 (d, J = 13.3 Hz, 1H), 4.20 (d, J = 13.3 Hz, 1H), 4.02 (dd, J = 13.5, 7.5 Hz, 1H), 3.92 (dd, J = 13.6, 7.2 Hz, 1H), 3.76 (dt, J = 13.3, 6.6 Hz, 1H), 2.95–2.74 (m, 4H), 1.88 (d, J = 5.1 Hz, 2H), 1.73–1.54 (m, 4H), 1.52 (d, J = 6.6 Hz, 3H), 1.46 (d, J = 6.7 Hz, 3H); 13C NMR (101 MHz, MeOD) δ 147.00, 146.10, 140.94, 136.72, 132.49, 131.04, 130.06, 129.85, 129.69, 128.82, 127.97, 118.54, 56.43, 54.53, 53.76, 37.40, 37.23, 33.65, 29.39, 29.31, 17.17, 16.22. HRMS (ESI) m/z calcd for C24H32N [M+H]+ 334.2535, found 334.2529. (E)-2-((Cinnamyloxy)methyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene (29). A stirred suspension of sodium hydride (34 mg, 1.4 mmol) and tetrabutylammonium iodide (10 mg) was added a solution of 13a (207 mg, 1.05 mmol) in tetrahydrofuran (5 mL) at 0 °C under N2 atmosphere. After the reaction was stirred at room temperature for 30 min, a solution of 63 (176 mg, 1 mmol) in tetrahydrofuran (5 mL) was added and stirred at room temperature for 24 h. The reaction was stopped. Thereafter the aqueous layer was partitioned between EtOAc and water. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed to give 29 (228 mg, 78% yield) as yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.39 (d, J = 7.4 Hz, 2H), 7.29 (dd, J = 10.3, 4.7 Hz, 2H), 7.22 (d, J = 7.3 Hz, 1H), 7.10 – 7.01 (m, 3H), 6.61 (d, J = 15.9 Hz, 1H), 6.33 (dd, J = 14.0, 8.0 Hz, 1H), 4.48 (s, 2H), 4.15 (dd, J = 6.1, 1.3 Hz, 2H), 2.82 – 2.72 (m, 4H), 1.88 – 1.77 (m, 2H), 1.61 (d, J = 2.7 Hz, 4H). 13C NMR (101 MHz, CDCl3) δ 144.66, 144.21, 138.16, 137.00, 133.78, 130.01, 129.91, 129.59, 128.71, 127.51,


Page 30 of 67

Page 31 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


127.01, 126.82, 73.23, 71.73, 37.58, 37.29, 33.84, 29.65; HRMS (ESI) m/z calcd for C21H25NaO [M+Na]+ 315.1725, found 315.1727. 2-((3-Phenylpropoxy)methyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene (30). 30 was synthesized by the general procedure of 29, given above, as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.29 – 7.23 (m, 2H), 7.21 – 7.14 (m, 3H), 7.11 – 7.04 (m, 3H), 4.44 (s, 2H), 3.48 (t, J = 6.4 Hz, 2H), 2.83 – 2.76 (m, 4H), 2.75 – 2.67 (m, 2H), 1.93 (ddt, J = 12.8, 9.1, 6.4 Hz, 2H), 1.87 – 1.79 (m, 2H), 1.68 – 1.59 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 143.64, 143.01, 142.20, 136.14, 129.18, 128.78, 128.64, 128.43, 125.86, 125.52, 73.02, 69.60, 36.85, 36.55, 32.89, 32.53, 31.51, 28.49, 28.41; HRMS (ESI) m/z calcd for C21H26NaO[M+Na]+ 317.1881, found 317.1878. 2-((((2E,4E)-5-Phenylpenta-2,4-dien-1-yl)oxy)methyl)-6,7,8,9-tetrahydro-5H-benzo[ 7]annulene (31). 31 was synthesized by the general procedure of 29, given above, as a yellow solid. m.p. 48-50 ºC. 1H NMR (400 MHz, CDCl3) δ 7.39 (t, J = 6.7 Hz, 2H), 7.31 (t, J = 7.6 Hz, 2H), 7.22 (dd, J = 12.9, 5.6 Hz, 1H), 7.12 – 7.05 (m, 3H), 6.79 (dd, J = 15.6, 10.5 Hz, 1H), 6.56 (dd, J = 15.5, 9.9 Hz, 1H), 6.43 (dd, J = 15.2, 10.5 Hz, 1H), 5.93 (dt, J = 15.2, 6.1 Hz, 1H), 4.49 (d, J = 8.6 Hz, 2H), 4.11 (t, J = 8.9 Hz, 2H), 2.83 – 2.73 (m, 4H), 1.86 – 1.78 (m, 2H), 1.63 (dd, J = 10.0, 4.9 Hz, 4H). 13C NMR (101 MHz, CDCl3) δ 143.70, 143.13, 137.33, 135.82, 133.04, 132.75, 130.46, 129.20, 128.89, 128.73, 128.47, 127.70, 126.52, 125.64, 72.29, 70.55, 36.83, 36.55, 32.88, 28.48; HRMS (ESI) m/z calcd for C23H26NaO [M+Na]+ 341.1881, found 341.1882.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(E)-3-(4-Bromophenyl)-N-methyl-N-((5,6,7,8-tetrahydronaphthalen-2-yl)methyl)pro p-2-en-1-amine hydrochloride (32). 32 was synthesized by the general procedure of 4, given above, as a white solid. m.p. 209-211 ºC. 1H-NMR (400 MHz, MeOD) δ 7.61–7.51 (m, 2H), 7.49–7.41 (m, 2H), 7.27–7.15 (m, 3H), 6.88 (d, J = 15.8 Hz, 1H), 6.46–6.28 (m, 1H), 4.41 (d, J = 13.0 Hz, 1H), 4.21 (d, J = 13.0 Hz, 1H), 4.03 (dd, J = 13.4, 6.7 Hz, 1H), 3.88 (dd, J = 13.3, 7.8 Hz, 1H), 2.79 (d, J = 14.7 Hz, 7H), 1.92–1.75 (m, 4H);

13


140.67, 140.57, 139.51, 135.87, 133.00, 132.77, 131.12, 129.85, 129.03, 127.89, 123.88, 118.79, 60.48, 58.74, 39.62, 30.26, 30.18, 24.09. HRMS (ESI) m/z calcd for C21H25BrN [M+H]+ 370.1170, found 370.1161. (E)-3-(4-Chlorophenyl)-N-methyl-N-((5,6,7,8-tetrahydronaphthalen-2-yl)methyl)pro p-2-en-1-amine hydrochloride (33). 33 was synthesized by the general procedure of 4, given above, as a white solid. m.p.213-215 ºC. 1H-NMR (400 MHz, MeOD) δ 7.52 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 7.29–7.16 (m, 3H), 6.90 (d, J = 15.8 Hz, 1H), 6.44–6.30 (m, 1H), 4.41 (d, J = 12.6 Hz, 1H), 4.21 (d, J = 12.6 Hz, 1H), 4.02 (d, J = 5.9 Hz, 1H), 3.90 (d, J = 7.6 Hz, 1H), 2.82 (d, J = 7.6 Hz, 7H), 1.84 (d, J = 2.7 Hz, 4H); 13C NMR (101 MHz, MeOD) δ 140.64, 140.46, 139.48, 135.75, 135.48, 132.77, 131.10, 129.96, 129.60, 129.04, 127.90, 118.72, 60.45, 58.73, 39.59, 30.25, 30.18, 24.08. HRMS (ESI) m/z calcd for C21H25ClN [M+H]+ 326.1676, found 326.1671. (E)-3-(4-Methoxyphenyl)-N-methyl-N-((5,6,7,8-tetrahydronaphthalen-2-yl)methyl)p rop-2-en-1-amine hydrochloride (34).


Page 32 of 67

Page 33 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


34 was synthesized by the general procedure of 4, given above, as a white solid. m.p. 173-174 ºC. 1H-NMR (400 MHz, MeOD) δ 7.47 (d, J = 8.4 Hz, 2H), 7.28–7.16 (m, 3H), 6.95 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 15.7 Hz, 1H), 6.19 (dt, J = 15.4, 7.7 Hz, 1H), 4.40 (d, J = 13.0 Hz, 1H), 4.18 (d, J = 12.9 Hz, 1H), 4.00 (dd, J = 13.0, 7.2 Hz, 1H), 3.90–3.76 (m, 4H), 2.94–2.69 (m, 7H), 1.84 (s, 4H);

13


161.96, 141.75, 140.61, 139.48, 132.75, 131.10, 129.53, 129.33, 129.01, 127.98, 115.20, 114.93, 60.27, 59.13, 55.79, 39.42, 30.26, 30.18, 24.10. HRMS (ESI) m/z calcd for C22H28NO [M+H]+ 322.2171, found 322.2170. (E)-N-Methyl-N-((5,6,7,8-tetrahydronaphthalen-2-yl)methyl)-3-(4-(trifluoromethyl) phenyl)prop-2-en-1-amine hydrochloride (35). 35 was synthesized by the general procedure of 4, given above, as a white solid. m.p. 210 ºC. 1H-NMR (400 MHz, MeOD) δ 7.80–7.64 (m, 4H), 7.36–7.09 (m, 3H), 6.99 (d, J = 15.8 Hz, 1H), 6.53 (d, J = 3.2 Hz, 1H), 4.40 (t, J = 19.1 Hz, 1H), 4.23 (d, J = 12.1 Hz, 1H), 4.07 (s, 1H), 3.94 (s, 1H), 2.83 (s, 7H), 1.84 (s, 4H);

13

C NMR (101 MHz,

MeOD) δ 140.69, 140.54, 140.07, 139.51, 132.80, 131.13, 129.06, 128.65, 127.88, 126.71, 121.03, 60.58, 58.58, 39.73, 30.26, 30.18, 24.09. HRMS (ESI) m/z calcd for C22H25F3N [M+H]+ 360.1939, found 360.1938. (E)-N-Methyl-3-(4-nitrophenyl)-N-((5,6,7,8-tetrahydronaphthalen-2-yl)methyl)prop2-en-1-amine hydrochloride (36). 36 was synthesized by the general procedure of 4, given above, as a white solid. m.p. 199-200 ºC. 1H-NMR (400 MHz, MeOD) δ 8.28 (d, J = 8.6 Hz, 2H), 7.77 (d, J = 8.5 Hz, 2H), 7.34–7.11 (m, 3H), 7.04 (d, J = 15.9 Hz, 1H), 6.67–6.50 (m, 1H), 4.44 (d, J = 13.0 Hz, 1H), 4.25 (d, J = 12.7 Hz, 1H), 4.10 (dd, J = 13.4, 6.9 Hz, 1H), 4.03–3.87 (m,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1H), 2.83 (d, J = 10.2 Hz, 7H), 1.84 (s, 4H);

13

C NMR (101 MHz, MeOD) δ 149.16,

143.07, 140.70, 139.51, 139.26, 132.81, 131.12, 129.08, 127.83, 124.97, 122.88, 60.63, 58.45, 39.79, 30.25, 30.18, 24.08. HRMS (ESI) m/z calcd for C21H25N2O2 [M+H]+ 337.1916, found 337.1917. 4-Iodo-2,3-dihydro-1H-indene (38). A solution of 2,3-dihydro-1H-inden-4-amine (2.00 g, 15.00 mmol) in conc. hydrochloric acid (15 mL) was treated with dropwise sodium nitrite (1.55 g, 22.50 mmol) in aqueous solution (15 mL) at 0 ºC for 15 min, and then potassium iodide (24.90 g, 150.00 mmol) in aqueous solution (90 mL) was added slowly at room temperature overnight. The mixture was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue was then purified via flash chromatography on silica gel, eluting with petroleum ether to give 38 (2.56 g, 70% yield) as a colorless oil. 1H-NMR (400 MHz, CDCl3) δ 7.53 (d, J = 7.7 Hz, 1H), 7.17 (t, J = 7.2 Hz, 1H), 6.85 (t, J = 7.5 Hz, 1H), 3.06 (dd, J = 17.6, 10.2 Hz, 2H), 2.93 (dd, J = 15.6, 8.4 Hz, 2H), 2.22–1.94 (m, 2H). 2,3-Dihydro-1H-indene-4-carbonitrile (39). To a solution of 38 (1.00 g, 4.10 mmol) in DMF (50 mL) was added copper (I) cyanide (0.73 g, 8.20 mmol) at reflux overnight. After cooling to room temperature, the mixture was poured into water with plenty of concentrated ammonium hydroxide until the mixture was clear. Thereafter, the mixture was partitioned between EtOAc and water. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue was then purified via flash chromatography on silica gel, eluting EtOAc/petroleum ether (1/25, v/v) to give 39 (0.42 g, 71% yield) as a pale


Page 34 of 67

Page 35 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


yellow solid. 1H-NMR (400 MHz, CDCl3) δ 7.35 (t, J = 6.6 Hz, 2H), 7.14 (t, J = 7.6 Hz, 1H), 3.04 (dd, J = 14.6, 7.1 Hz, 2H), 2.95–2.86 (m, 2H), 2.15–2.01 (m, 2H). (2,3-Dihydro-1H-inden-4-yl)methanamine (40). A solution of 39 (0.50 g, 3.49 mmol) in anhydrous tetrahydrofuran (20 mL) at -78 °C under a N2 atmosphere was treated dropwise with a suspension of lithium aluminum hydride (0.26 g, 6.98 mmol) in tetrahydrofuran (30 mL). The mixture was stirred at room temperature overnight and then carefully treated in succession with water (0.3 mL), 15% sodium hydroxide (0.3 mL) and water again (0.3 mL). The mixture was dried over anhydrous Na2SO4 and filtered through Celite, and the filter cake was washed with tetrahydrofuran. Ultimately, the filtrate was concentrated under reduced pressure to give 40 (0.46 g, 90% yield) as a pale yellow oil. The crude product was used for the next step without further purification. tert-Butyl ((2,3-dihydro-1H-inden-4-yl)methyl)carbamate (41). To a solution of 40 (1.47 g, 10.00 mmol) in tetrahydrofuran (30 mL) was added sodium hydroxide (0.48 g, 12.00 mmol), and the mixture was stirred for 5 min. After the addition of di-tert-butyl dicarbonate (2.72 mL, 12.00 mmol) in tetrahydrofuran (20 mL), the reaction mixture was stirred for another 1 h and filtered directly, and the filter cake was washed with tetrahydrofuran. The residue was then purified via flash chromatography on silica gel, eluting with EtOAc/petroleum ether (1/25, v/v) to give 41 (2.15 g, 87% yield) as a pale yellow solid. 1H-NMR (400 MHz, CDCl3) δ 7.14 (dt, J = 14.6, 7.2 Hz, 2H), 7.05 (d, J = 7.0 Hz, 1H), 4.29 (s, 2H), 2.90 (dt, J = 12.7, 7.5 Hz, 4H), 2.18–1.95 (m, 2H), 1.46 (s, 9H). 1-(2,3-Dihydro-1H-inden-4-yl)-N-methylmethanamine (42).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 67

A solution of 41 (0.25 g, 1.00 mmol) in tetrahydrofuran (10 mL) at 0 °C under a N2 atmosphere was treated dropwise with a suspension of lithium aluminum hydride (0.15 g, 4.00 mmol) in tetrahydrofuran (10 mL). The reaction mixture was stirred at reflux overnight and then carefully treated in succession with water (0.2 mL), 15% sodium hydroxide (0.2 mL) and water again (0.2 mL). The mixture was dried over anhydrous Na2SO4 and filtered through Celite, and the filter cake was washed with tetrahydrofuran. Ultimately, the filtrate was concentrated under reduced pressure to give 42 (0.14 g, 90% yield) as a yellow oil. The crude product was used for the next step without further purification. 6,7,8,9-Tetrahydro-5H-benzo[7]annulene (43). A solution of 6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-one (1.00 g, 6.16 mmol) in trifluoroacetic acid (10 mL) was treated dropwise with triethylsilane (1.48 mL, 9.24 mmol), and the reaction was stirred at 60 ºC overnight. The mixture was concentrated under reduced pressure to remove most of the trifluoroacetic acid and then poured into ice water. Next, the reaction mixture was partitioned between EtOAc and water. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue was then purified via flash chromatography on silica gel, eluting with petroleum ether to give 43 (0.81 g, 90% yield) as a clear colorless oil. 1

H-NMR (400 MHz, CDCl3) δ 7.17–6.96 (m, 4H), 2.79 (dd, J = 7.0, 4.1 Hz, 4H), 1.95–

1.73 (m, 2H), 1.65 (dd, J = 10.0, 4.8 Hz, 4H). 1-Iodo-6,7,8,9-tetrahydro-5H-benzo[7]annulene 2-iodo-6,7,8,9-tetrahydro-5H-benzo[7]annulene (45).


(44)

and

Page 37 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


To a solution of 43 (0.68 g, 4.60 mmol) in acetic acid (20 mL) was added N-iodosuccinimide (1.14 g, 5.06 mmol) under a N2 atmosphere at 70 ºC for 7 h. The reaction was stopped by adding some sodium thiosulfate aqueous solution at 0 ºC, and then some saturated sodium bicarbonate solution was added. Next, the mixture was partitioned between EtOAc and water. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue was then purified via flash chromatography on silica gel, eluting with petroleum ether to give mixed 44 and 45 (0.86 g, 69% yield) as a clear colorless oil. 1H NMR (400 MHz, acetone) δ 7.66 (dd, J = 8.0, 1.2 Hz, 1H), 7.46 (d, J = 1.8 Hz, 4H), 7.40 (dd, J = 7.9, 1.9 Hz, 4H), 7.09 (t, J = 5.4 Hz, 1H), 7.08–7.00 (m, 10H), 6.88 (d, J = 7.9 Hz, 4H), 6.77 (t, J = 7.7 Hz, 1H), 2.82–2.63 (m, 30H), 1.93–1.73 (m, 15H), 1.71–1.45 (m, 30H). 6,7,8,9-Tetrahydro-5H-benzo[7]annulene-1-carbonitrile

(46)

and

6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-carbonitrile (47). To a solution of mixed 44 and 45 (0.54 g, 2.00 mmol) in DMF (10 mL) was added copper (I) cyanide (0.36 g, 4.00 mmol) at reflux overnight. After cooling to room temperature, the mixture was poured into water with plenty of concentrated ammonium hydroxide until the mixture was clear. Thereafter, the mixture was partitioned between EtOAc and water. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue was then purified via flash chromatography on silica gel, eluting EtOAc/petroleum ether (1/25, v/v) to give mixed 46 and 47 (0.24 g, 71% yield) as a pale yellow solid. 1H NMR (500 MHz, CDCl3) δ 7.39– 7.35 (m, 2H), 7.19–7.15 (m, 1H), 2.90–2.73 (m, 4H), 1.94–1.79 (m, 2H), 1.61 (d, J = 21.6 Hz, 4H).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(6,7,8,9-Tetrahydro-5H-benzo[7]annulen-1-yl)methanamine

Page 38 of 67

(48)

and

(6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methanamine (49). A solution of mixed 46 and 47 (6.28 g, 36.70 mmol) in anhydrous tetrahydrofuran (100 mL) at -78 °C under a N2 atmosphere was treated dropwise with a suspension of lithium aluminum hydride (2.79 g, 73.40 mmol) in tetrahydrofuran (50 mL). The mixture was stirred at room temperature overnight and then carefully treated in succession with water (3 mL), 15% sodium hydroxide (3 mL) and water again (3 mL). The mixture was dried over anhydrous Na2SO4 and filtered through Celite, and the filter cake was washed with tetrahydrofuran. Ultimately, the filtrate was concentrated under reduced pressure to give mixed 48 and 49 (5.85 g, 91% yield) as a pale yellow oil. The crude product was used for the next step without further purification. tert-Butyl ((6,7,8,9-tetrahydro-5H-benzo[7]annulen-1-yl)methyl)carbamate (50) and tert-butyl ((6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methyl)carbamate (51). To a solution of mixed 48 and 49 (5.00 g, 28.50 mmol) in tetrahydrofuran (50 mL) was added sodium hydroxide (1.37 g, 34.20 mmol), and the mixture was stirred for 5 min. After the addition of di-tert-butyl dicarbonate (7.86 mL, 34.20 mmol) in tetrahydrofuran (20 mL), the reaction mixture was stirred for another 1 h and filtered directly, and the filter cake was washed with tetrahydrofuran. The residue was then purified via flash chromatography on silica gel, eluting with EtOAc/petroleum ether (1/25, v/v) to give mixed 50 and 51 (7.22 g, 92% yield) as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.15–6.79 (m, 15H), 4.33 (d, J = 4.3 Hz, 2H), 4.25 (s, 8H), 2.85– 2.79 (m, 4H), 2.79–2.69 (m, 16H), 1.88–1.76 (m, 10H), 1.62 (d, J = 2.9 Hz, 20H), 1.46 (d, J = 3.7 Hz, 45H).


Page 39 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


N-Methyl-1-(6,7,8,9-tetrahydro-5H-benzo[7]annulen-1-yl)methanamine

(52)

and

N-methyl-1-(6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)methanamine (53). A solution of mixed 50 and 51 (7.99 g, 29.00 mmol) in tetrahydrofuran (20 mL) at 0 °C under a N2 atmosphere was treated dropwise with a suspension of lithium aluminum hydride (4.40 g, 116.00 mmol) in tetrahydrofuran (50 mL). The reaction mixture was stirred at reflux overnight and then carefully treated in succession with water (5 mL), 15% sodium hydroxide (5 mL) and water again (5 mL). The mixture was dried over anhydrous Na2SO4 and filtered through Celite, and the filter cake was washed with tetrahydrofuran. Ultimately, the filtrate was concentrated under reduced pressure to give mixed 52 and 53 (4.94 g, 90% yield) as a pale yellow oil. The crude product was used for the next step without further purification. (2,3-Dihydro-1H-inden-5-yl)methanol (54a). A solution of 2,3-dihydro-1H-indene-5-carboxylic acid (1.56 g, 9.60 mmol) in anhydrous tetrahydrofuran (20 mL) at -78 °C under a N2 atmosphere was treated dropwise with a suspension of lithium aluminum hydride (0.73 g, 19.20 mmol) in tetrahydrofuran (50 mL). The mixture was stirred at room temperature overnight and then carefully treated in succession with water (0.8 mL), 15% sodium hydroxide (0.8 mL) and water again (0.8 mL). The mixture was dried over anhydrous Na2SO4 and filtered through Celite, and the filter cake was washed with tetrahydrofuran. Ultimately, the filtrate was concentrated under reduced pressure to give 54a (1.27 g, 89% yield) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.24 (dd, J = 14.3, 6.5 Hz, 2H), 7.13 (d, J = 7.6 Hz, 1H), 4.65 (s, 2H), 2.91 (td, J = 7.5, 2.4 Hz, 4H), 2.17–1.98 (m, 2H). 5-(Bromomethyl)-2,3-dihydro-1H-indene (55a).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A solution of 54a (1.48 g, 10.00 mmol) in anhydrous ether (30 mL) was treated with phosphorus tribromide (0.32 mL, 3.34 mmol) at 0 °C under a N2 atmosphere. The reaction mixture was stirred at room temperature overnight and poured into ice water containing sodium bicarbonate. The mixture was partitioned between EtOAc and water. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed at 30 ºC. The crude produced was used for the next step without further purification, affording 55a (1.71 g, 81% yield) as a colorless oil. 1-(2,3-Dihydro-1H-inden-5-yl)-N-methylmethanamine (56a). To a solution of 55a (1.00 g, 4.74 mmol) in ethanol (20 mL) was slowly added methylamine alcohol solution (20 mL, 33 wt. % in ethanol) at room temperature overnight. The mixture was concentrated to afford crude 56a (0.69 g, 90% yield) as a yellow oil. 1H NMR (400 MHz, MeOD) δ 7.26–7.16 (m, 2H), 7.12 (d, J = 7.7 Hz, 1H), 4.03 (s, 2H), 2.90–2.77 (m, 4H), 2.59 (s, 3H), 2.00 (p, J = 7.4 Hz, 2H). (E)-3-(p-Tolyl)prop-2-en-1-ol (57a). A solution of (E)-3-(p-tolyl)acrylaldehyde (0.58 g, 4 mmol) in methanol (10 mL) was treated with sodium borohydride (0.15 g, 4 mmol) in batches at 0 °C. The reaction mixture was stirred at room temperature for 15 min and concentrated. The residue was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The crude was used for the next step without further separation, giving the title 57a (0.59 g, 99% yield) as a colorless oil. (E)-1-(3-Bromoprop-1-en-1-yl)-4-methylbenzene (58a).


Page 40 of 67

Page 41 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A solution of 57a (0.44 g, 3 mmol) in anhydrous ether (30 mL) was treated with phosphorus tribromide (94 µL, 1 mmol) at 0 °C under a N2 atmosphere. The reaction mixture was stirred at room temperature overnight and poured into ice water containing sodium bicarbonate. The mixture was partitioned between EtOAc and water. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed at 30 ºC. The crude produced was used for the next step without further purification, affording 58a (0.54 g, 85% yield) as a white solid. 1H-NMR (400 MHz, CDCl3) δ 7.27 (t, J = 7.4 Hz, 2H), 7.13 (d, J = 7.4 Hz, 2H), 6.61 (d, J = 15.6 Hz, 1H), 6.34 (dt, J = 15.6, 7.8 Hz, 1H), 4.16 (d, J = 7.7 Hz, 2H), 2.34 (s, 3H). (E)-Ethyl 4-phenylbut-2-enoate (59). To a solution of NaH (0.17 g, 4.2 mmol, 60% dispersion in mineral oil) in THF was added dropwise triethyl phosphonoacetate (794 µL, 4 mmol) for 10 min at 0 ºC. The solution was allowed to stir for another 90 min, during which time the evolution of H2 gas completely ceased. Phenylacetaldehyde (0.58 mL, 5.2 mmol) was then added dropwise. The solution was allowed to stir for 48 h at room temperature and then quenched with 10 mL saturated aqueous NH4Cl solution. The mixture was partitioned between EtOAc and water. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue was then purified via flash chromatography on silica gel, eluting with EtOAc/petroleum ether (1/5, v/v) to give 59 (0.52 g, 69% yield) as a clear colorless oil. 1H NMR (DMSO-d6, 400 MHz) δ 7.30 − 7.32 (m, 2H), 7.21 − 7.24 (m, 3H), 6.95 (dt, J = 15.5, 7.0 Hz, 1H), 5.82 (dt, J = 15.5, 1.3Hz, 1H), 4.07 (q, J = 7.0 Hz, 2H), 3.53 (dd, J = 7.1, 1.4 Hz, 2H), 1.16 (t, J = 7.0 Hz, 3H). (E)-4-Phenylbut-2-en-1-ol (60).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A solution of 59 (0.57 g, 3 mmol) in anhydrous tetrahydrofuran (25 mL) at 0 °C under a N2 atmosphere was treated dropwise with a solution of diisobutylaluminum hydride (3.0 mL, 4.5 mmol, 1.5 M in toluene). The reaction was stirred at room temperature overnight and then carefully quenched with methanol. The mixture was then filtered through Celite and condensed. The residue was then purified via flash chromatography on silica gel, eluting with EtOAc/petroleum ether (1/5, v/v) to give 60 (0.39 g, 88% yield) as a clear colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.36–7.30 (m, 3H), 7.25–7.21 (m, 2H), 5.95–5.83 (m, 1H), 5.73 (dt, J = 15.2, 5.8 Hz, 1H), 4.20–4.09 (m, 2H), 3.42 (d, J = 6.6 Hz, 2H). (E)-(4-Bromobut-2-en-1-yl)benzene (61a). a solution of 60 (445 mg, 3 mmol) in anhydrous ether (30 mL) was treated with phosphorus tribromide (0.09 mL, 1 mmol) at 0 °C under a N2 atmosphere. The reaction mixture was stirred at room temperature overnight and poured into ice water containing sodium bicarbonate. The mixture was partitioned between EtOAc and water. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed at 30 ºC. The crude product was used for the next step without further purification, affording 61a (0.54 g, 85% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.34-7.30 (m, 2H), 7.25-7.18 (m, 3H), 5.98-5.90 (m, 1H), 5.78 (dtt, J = 15.0, 7.5, 1.5 Hz, 1H), 3.97 (dd, J = 7.5, 1.0 Hz, 2H), 3.42 (d, J = 7.0 Hz, 2H). 6,7,8,9-Tetrahydro-5H-benzo[7]annulene-2-carboxylic acid (62). A solution of 47 (1.71 g, 10 mmol ) in a 1:1 2-propanol: water mixture (60 mL) was treated with potassium hydroxide (2 g, 50 mmol). The reaction was heated at reflux for 14 h and then partitioned between water and EtOAc and acidified to pH 5 with 6 N HCl.


Page 42 of 67

Page 43 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Thereafter the aqueous layer was partitioned between EtOAc and water. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed to give 62 (1.35 g, 71% yield) as white solid. 1H NMR (400 MHz, CDCl3) δ 7.84 (s, 1H), 7.26 (s, 1H), 7.18 (s, 1H), 2.85 (s, 4H), 1.85 (s, 2H), 1.66 (s, 4H). (6,7,8,9-Tetrahydro-5H-benzo[7]annulen-2-yl)methanol (63). A solution of 62 (0.95 g, 5 mmol) in anhydrous tetrahydrofuran (40 mL) at 0 °C under N2 atmosphere was treated dropwise with a suspension of lithium aluminum hydride (0.38 g, 10 mmol) in tetrahydrofuran (40 mL). The mixture was stirred at room temperature overnight and then carefully treated in succession with water (0.4 ml), 15% sodium hydroxide (0.4 mL) and then water (0.4 mL). The mixture was dried over anhydrous Na2SO4, filtered through celite, and the filter cake was washed with tetrahydrofuran. Ultimately the filtrate concentrated under reduced pressure to give 63 (0.73 g, 83% yield) as colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.10 (m, 3H), 4.63 (s, 2H), 2.79 (s, 4H), 1.83 (s, 2H), 1.64 (s, 4H). Aqueous solubility method. The aqueous solubilities of 8, 10, 32 and 35 were determined by the HPLC method. Stock solutions (800 µg/mL) of samples were dissolved completely in methanol, and then 10 µL of dilute solutions with concentrations of 50, 100, 200, 400 and 800 µg/mL were injected into the HPLC system to assess the linearity. Calibration curves were plotted as peak area versus concentration of sample. 10 mg sample was added into a 5 mL centrifuge tube, and 1 mL pure water was pipetted into the tube, and if the solution was unsaturated, remaining clear and transparent, more tested compound was added. After stirring for 24 h, the solution was filtered with a syringe filter, and the same 10 µL was



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

injected into the HPLC system. The eater solubilities were calculated by comparing the peak area of the tested compound to the calibration curves. Biological methods Pigment inhibition assay. The tested compounds were dissolved in DMSO to 20 mM as a stock concentration. Serial 2-fold dilutions were prepared from the stock solution with DMSO. S. aureus Newman bacteria were cultured in TSB (4 mL) in the presence of 40 µL inhibitors at 37 ºC for 48 h, in triplicate. 3 mL bacteria cultures then were centrifuged and washed twice with PBS, and the pigment was extracted with methanol. The OD was determined at 450 nm using a NanoDrop 2000c (Thermo scientific) spectrophotometer. IC50 values were obtained by fitting the OD data to a normal dose-response curve using Graphpad Prism 5.0 software. The IC50 values of the MRSA strains USA300 LAC, USA400 MW and Mu 50 were determined in the same way. CrtN enzyme inhibition assay. The expression of CrtN as well as the inhibition assays were carried out using our previous methods. In short, diapophytoene was purified from diapophytoene-producing E. coli BL21 (DE3)/pET28a::crtM by extraction with acetone. The emulsion of diapophytoene was prepared by dissolving 24 mg of phosphatidylcholine (Sigma-Aldrich) in 200 µL CHCl3 and mixing with 8 mg diapophytoene. The mixture was spun-dried and incubated with 2 mL 0.02 M HEPES buffer (pH 7.5) and then sonicated in ice water to obtain a homogeneous emulsion and stored at -80 °C until use. E. coli BL21 (DE3)/pET28a::crtN was sub-cultured into 1000 mL of LB broth supplemented with 50 µg/mL kanamycin to obtain an optical density at 600 nm (OD600) of approximately 0.1


Page 44 of 67

Page 45 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and grown to an OD600 of ~0.5. The expression of 6His-CrtN was induced with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) overnight at 16 °C. The cells were harvested, and the pellets were suspended in 30 mL HEPES buffer and lysed at 4 °C by sonication. The enzyme activity was determined in triplicate, with a total of 700 µL of the following: 276.5 µL 0.02 M HEPES buffer (pH 7.5), 50 µL diapophytoene emulsion, 70 µL different concentrations of compounds or mock (ddH2O), 3.5 µL FAD stock solution (10 mM), and 300 µL CrtN lysate (~1.41 mg CrtN, as estimated by western blot using a known concentration of the purified 6His-crtN protein). The tests were performed under an anaerobic atmosphere by adding a final concentration of 20 U/mL glucose oxidase (Sigma-Aldrich, G2133), 20000 U/mL catalase (Sigma-Aldrich, C1345), and 2 mM glucose as an oxygen-trapping system. The reaction mixture was started by adding the lysate and incubating overnight at 37 ºC. The reaction was terminated by methanol. The pigments were extracted twice against 700 µL chloroform. The organic phase was combined, concentrated, and re-dissolved in 200 µL chloroform and analyzed by reading the OD value under 450 nm. The IC50 values were obtained by fitting the OD data to a normal dose-response curve using Graphpad Prism 5.0 software. Bacterial growth assays of S. aureus Newman and MRSA strains. 8 was dissolved in DMSO to 20 mM as a stock concentration and diluted with fresh TSB medium to produce a final concentration of either 0.2 mM or 0.05 mM. 100 µL of the dilution was distributed in 96-well plates, as well as growth controls (containing equal amounts of DMSO). 60 µL paraffin wax was covered onto the dilutions to avoid the evaporation of the medium. The dilutions were plated at 37 ºC for 4 h to sufficiently dissolve the compound. Four S. aureus strains, including Newman, USA300 LAC,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

USA400 WM2 and Mu50, were cultured overnight and diluted with fresh medium to obtain an optical density at 600 nm (OD600) of ~1.0. Test and growth control wells were inoculated with 5 µL of a bacterial suspension (final OD600 ≈ 0.05). The 96-well plates were incubated at 37 ºC overnight, and the OD600 was recorded every half an hour with a Synergy 2 (Biotek) plate reader following the manufacturer’s instructions. Hydrogen peroxide killing and human whole blood killing. For H2O2 killing assay, four strains, including Newman, USA300 LAC, USA400 WM2 and Mu50, were diluted 1:100 in TSB and grown at 37 °C for 24 h with or without 1 µM compound 8 or N-Acetyl-Cysteine (NAC). The bacteria were washed twice with PBS and then diluted to a concentration of 4 × 106 CFU per 250 µL reaction mixture in a 2-mL Eppendorf tube. After H2O2 was added to a final concentration of 1.5%, the tubes were sealed with Parafilm M laboratory film and incubated for 30 min at 37 ºC. The reaction was stopped by 1000 U/mL exogenous catalase (Sigma-Aldrich). Bacterial survival was assessed by serial dilutions on TSA plates for the enumeration of CFU. For the human whole blood killing assay, overnight cultured strains were centrifuged and suspended in sterile PBS to generate a suspension of 1 × 107 CFU/mL. Whole blood (360 µL) from a healthy human volunteer was collected using a BD VACUTAINER PT tube and then mixed with 40 µL bacterial sample, which resulted in a concentration of 1 × 106 CFU/mL. The tubes were incubated at 37 ºC for 6 h, and then the dilutions were plated on TSA agar plates for enumeration of the surviving CFUs. S. aureus systemic infection models.


Page 46 of 67

Page 47 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6-8 week old female BALB/c mice (SIPPR-BK Lab Animal, Ltd.) were housed under specified pathogen-free conditions. The mice received intraperitoneal injections of 50 mg/kg or 200 mg/kg 37 or 8 in 12 h intervals for 108 h (4.5 d). Twelve hours after the first injection of inhibitors, the mice were challenged with three strains. For the mouse model of abscess formation, the mice were challenged with 100 µL of a bacterial suspension of either 1 × 107 CFU of S. aureus Newman, 4 × 107 CFU of S. aureus USA400 MW2, or 1.6 × 108 CFU of S. aureus Mu50. The mice were sacrificed 5 days post-infection. The kidneys, hearts and livers were aseptically removed and homogenized in 1 mL PBS plus 0.1% Triton X-100 to obtain single-cell suspensions. Serial dilutions of each organ were plated on TSA (Difco) plates, and the CFUs were counted after overnight incubation at 37 ºC. The statistical significance was determined by the Mann-Whitney Test (two-tailed). For the lethal challenge experiments, female BALB/c mice were infected with 2 × 107 CFU of S. aureus Newman, and the log-rank test was used to analyze mortality data. Anti-fungal assays. The in vitro antifungal activity was performed by means of the minimal inhibitory concentrations (MIC) using the serial dilution method in 96-well microtest plates. Test fungal strains were obtained from the ATCC or were clinical isolates. The MIC determination was performed according to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations with RPMI 1640 (Sigma) buffered with 0.165 M MOPS (Sigma) as the test medium. The MIC value was defined as the lowest concentration of test compounds that resulted in a culture with turbidity less than or equal to 80% inhibition when compared with the growth of the control. Test



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 67

compounds were dissolved in DMSO serially diluted in growth medium. The dermatophytes were incubated at 28 °C. The growth MIC was determined at 7 days for filamentous fungi. ASSOCIATED CONTENT Supporting Information. HPLC reports for the purity check of analogs 1-36. 1H and

13

C NMR spectra of

representative compounds. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * For J.L.: phone, +86-21-64252584; fax, +86-21-64252584; Email, [email protected]; For L.L.: phone, +86-21-50803109; E-mail, [email protected]. Author Contributions †

These authors contributed equally to this work.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT Financial support of this research provided by the National Natural Science Foundation of China (Grants 21222211, 21372001 and 21472207), the “Shu Guang” project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation (Grant 14SG28), the Program for New Century Excellent


Page 49 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Talents in University (Grant NCET-12-0853), and the Fundamental Research Funds for the Central Universities is gratefully acknowledged. ABBREVIATIONS S. aureus, Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus; STX, staphyloxanthin; NTF, naftifine hydrochloride; CrtM, dehydrosqualene synthase; CrtN,

diapophytoene

desaturases;

SAR,

structure-activity

relationship;

IC50,

half-maximal inhibitory concentration; MIC, minimum inhibitory concentration; HPLC, high-performance

liquid

chromatography;

MS,

mass

chromatography;

CFU,

colony-forming unit; PBS, phosphate-buffered saline; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; EtOH, ethanol; EtOAc, ethyl acetate, MeOH, methanol; THF, tetrahydrofuran; CH2Cl2, dichloromethane; CH3CN, acetonitrile; Et3N, triethylamine.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

REFERENCES (1) Berendonk, T. U.; Manaia, C. M.; Merlin, C.; Fatta-Kassinos, D.; Cytryn, E.; Walsh, F.; Burgmann, H.; Sorum, H.; Norstrom, M.; Pons, M. N.; Kreuzinger, N.; Huovinen, P.; Stefani, S.; Schwartz, T.; Kisand, V.; Baquero, F.; Martinez, J. L. Tackling antibiotic resistance: the environmental framework. Nat. Rev. Microbiol. 2015, 13, 310-317. (2) Barber, M. Methicillin-resistant staphylococci. J. Clin. Pathol. 1961, 14, 385-393. (3) David, M. Z.; Glikman, D.; Crawford, S. E.; Peng, J.; King, K. J.; Hostetler, M. A.; Boyle-Vavra, S.; Daum, R. S. What is community-associated methicillin-resistant Staphylococcus aureus? J. Infect. Dis. 2008, 197, 1235-1243. (4) Wulf, M.; Voss, A. MRSA in livestock animals-an epidemic waiting to happen? Clin. Microbiol. Infect. 2008, 14, 519-521. (5) van Cleef, B. A.; Monnet, D. L.; Voss, A.; Krziwanek, K.; Allerberger, F.; Struelens, M.; Zemlickova, H.; Skov, R. L.; Vuopio-Varkila, J.; Cuny, C.; Friedrich, A. W.; Spiliopoulou, I.; Paszti, J.; Hardardottir, H.; Rossney, A.; Pan, A.; Pantosti, A.; Borg, M.; Grundmann, H.; Mueller-Premru, M.; Olsson-Liljequist, B.; Widmer, A.; Harbarth, S.; Schweiger, A.; Unal, S.; Kluytmans, J. A. Livestock-associated methicillin-resistant Staphylococcus aureus in humans, Europe. Emerg. Infect. Dis. 2011, 17, 502-505. (6) Nicholson, T. L.; Shore, S. M.; Smith, T. C.; Frana, T. S. Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) isolates of swine origin form robust biofilms. PloS one 2013, 8, e73376.


Page 50 of 67

Page 51 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(7) Melo-Cristino, J.; Resina, C.; Manuel, V.; Lito, L.; Ramirez, M. First case of infection with vancomycin-resistant Staphylococcus aureus in Europe. Lancet 2013, 382, 205. (8) Shallcross, L. J.; Howard, S. J.; Fowler, T.; Davies, S. C. Tackling the threat of antimicrobial resistance: from policy to sustainable action. Philos. T. R. Soc. B. 2015, 370, 20140082. (9) Antibiotic resistance: the global threat (U.S.). http://stacks.cdc.gov/view/cdc/31340 , National Center for Emerging and Zoonotic Infectious Diseases (U.S.), 2015. (10) Bragginton, E. C.; Piddock, L. J. V. UK and European Union public and charitable funding from 2008 to 2013 for bacteriology and antibiotic research in the UK: an observational study. Lancet Infect. Dis. 2014, 14, 857-868. (11) Gonzales, P. R.; Pesesky, M. W.; Bouley, R.; Ballard, A.; Biddy, B. A.; Suckow, M. A.; Wolter, W. R.; Schroeder, V. A.; Burnham, C.-a. D.; Mobashery, S.; Chang, M.; Dantas, G. Synergistic, collaterally sensitive β-lactam combinations suppress resistance in MRSA. Nat. Chem. Biol. 2015, 11, 855-861. (12) Vuong, C.; Yeh, A. J.; Cheung, G. Y.; Otto, M. Investigational drugs to treat methicillin—resistant Staphylococcus aureus. Expert Opin. on Investig. Drugs. 2016, 25, 73-93. (13) Arias, C. A.; Murray, B. E. Antibiotic-resistant bugs in the 21st century--a clinical super-challenge. New. Engl. J. Med. 2009, 360, 439-443. (14) Clatworthy, A. E.; Pierson, E.; Hung D. T. Targeting virulence: a new paradigm for antimicrobial therapy. Nat. Chem. Biol. 2007, 3, 541-548. (15) Wang, R.; Braughton, K. R.; Kretschmer, D.; Bach, T. H.; Queck, S. Y.; Li, M.; Kennedy, A. D.; Dorward, D. W.; Kleba-noff, S. J.; Peschel, A.; DeLeo, F. R.; Otto, M.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Identification of novel cytolytic peptides as key virulence determinants for community-associated MRSA. Nat. Med. 2007, 13, 1510-1514. (16) Escaich, S. Antivirulence as a new antibacterial approach for chemotherapy. Curr. Opin. Chem. Biol. 2008, 12, 400-408. (17) Russo, T. A.; Spellberg, B.; Johnson, J. R. Important complexities of the anti-virulence target paradigm: a novel ostensibly resistance-avoiding approach for treating infections. J. Infect. Dis. 2016, 213, 901-903. (18) Clauditz, A.; Resch, A.; Wieland, K. P.; Peschel, A.; Gotz, F. Staphyloxanthin plays a role in the fitness of Staphylococcus aureus and its ability to cope with oxidative stress. Infect. Immun. 2006, 74, 4950-4953. (19) Liu, G. Y.; Essex, A.; Buchanan, J. T.; Datta, V.; Hoffman, H. M.; Bastian, J. F.; Fierer, J.; Nizet, V. Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity. J. Exp. Med. 2005, 202, 209-215. (20) Pelz, A.; Wieland, K. P.; Putzbach, K.; Hentschel, P.; Albert, K.; Gotz, F. Structure and biosynthesis of staphyloxanthin from Staphylococcus aureus. J. Biol. Chem. 2005, 280, 32493-32498. (21) Liu, C. I.; Liu, G. Y.; Song, Y.; Yin, F.; Hensler, M. E.; Jeng, W. Y.; Nizet, V.; Wang, A. H.; Oldfield, E. A cholesterol biosynthesis inhibitor blocks Staphylococcus aureus virulence. Science. 2008, 319, 1391-1394. (22) Chen, F.; Di, H.; Wang, Y.; Cao, Q.; Xu, B.; Zhang, X.; Yang, N.; Liu, G.; Yang, C. G.; Xu, Y.; Jiang, H.; Lian, F.; Zhang, N.; Li, J.; Lan, L. Small molecule targeting of a


Page 52 of 67

Page 53 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


diapophytoene desaturase inhibits S. aureus virulence. Nat. Chem. Bio. 2016, 12, 174-179. (23) Stütz, A.; Georgopoulos, A.; Granitzer, W.; Petranyi, G.; Berney, D. Synthesis and structure-activity relationships of naftifine-related allylamine antimycotics. J. Med Chem. 1986, 29, 112-125. (24) David,

M.Z.;

Daum,

R.S.

Community-associated

methicillin-resistant

Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clin. Microbiol. Rev. 2010, 23, 616-687. (25) de Matos PD; de Oliveira TL; Cavalcante FS; Ferreira DC; Iorio NL; Pereira EM; Chamon RC; Dos Santos KR. Molecular markers of antimicrobial resistance in methicillin-resistant Staphylococcus aureus SCCmec IV presenting different genetic backgrounds. Microb. Drug. Resist. 2016, April 5, doi:10.1089/mdr.2015.0255. (26) Kuroda, M.; Ohta, T.; Uchiyama, I.; Baba, T.; Yuzawa, H.; Kobayashi, I.; Cui, L.; Oguchi, A.; Aoki, K.-i.; Nagai, Y.; Lian, J.; Ito, T.; Kanamori, M.; Matsumaru, H.; Maruyama, A.; Murakami, H.; Hosoyama, A.; Mizutani-Ui, Y.; Takahashi, N. K.; Sawano, T.; Inoue, R.-i.; Kaito, C.; Sekimizu, K.; Hirakawa, H.; Kuhara, S.; Goto, S.; Yabuzaki, J.; Kanehisa, M.; Yamashita, A.; Oshima, K.; Furuya, K.; Yoshino, C.; Shiba, T.; Hattori, M.; Ogasawara, N.; Hayashi, H.; Hiramatsu, K. Whole genome sequencing of meticillin-resistant Staphylococcus aureus. Lancet. 2001, 357, 1225-1240. (27) Monk, J.; Brogden, R. Naftifine. Drugs 1991, 42, 659-672.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

Page 54 of 67

Page 55 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FIGURES Figure 1. Chemical structures and pigment inhibition activities of 37 and naftifine hydrochloride (NTF).

Figure 2. Chemical structures and pigment inhibition activities of analogs 1-6 of NTF.

Figure 3. Structures and pigment inhibition activities of the linker-modified and N-substituted analogs of compound 6.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Structures and pigment inhibition activities of compounds 29-31.

Figure 5. Structures and pigment inhibition activities of the analogs of compound 5.

Figure 6. 8 treatment resulted in the inhibition of the in vivo function of CrtN. (A–G) HPLC chromatograms (absorption at 286 nm) of the carotenoid extracts from E. coli (A), E. coli expressing S. aureus crtM (B), wild-type S. aureus Newman (C), crtM mutant (D), crtN mutant (E), NTF-treated wild-type S. aureus Newman (F) and 8-treated wild-type S. aureus Newman (G) strains. Insets on the right show the absorbance spectra of the indicated HPLC peaks. mAu, milli-absorbance units. Absorbance (Abs) represents the amount of light absorbed by the sample.


Page 56 of 67

Page 57 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. Effect of 8 on pigment production of S. aureus USA400 MW2 (A), USA300 LAC (B), Mu50 (C). Data are presented as means and SEM, n = 3 independent experiments.

Figure 8. Effect of 8 on the bacterial growth of S. aureus Newman (A), USA400 MW2 (B), USA300 LAC (C), and Mu50 (D).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 9. Effect of 8 and NAC on the susceptibility of S. aureus to killing by H2O2; *p< 0.05, **p< 0.01, ***p < 0.001 via two-tailed t-test (n = three biological replicates, each with two technical replicates).

Figure 10. Effect of 8 on the susceptibility of S. aureus to killing by human whole blood; *p< 0.05, **p< 0.01, ***p < 0.001 via two-tailed t-test (n = three biological replicates, each with two technical replicates).


Page 58 of 67

Page 59 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 11. Effect of 8 on S. aureus survival in the kidneys and hearts of mice (n = 13) challenged with 1 × 107 CFU Newman bacteria( A) and in protecting mice (n = 15) from lethal S. aureus infection challenged with 2 × 107 CFU Newman bacteria (B).

Figure 12. Effect of 8 on S. aureus bacterial survival in the livers (A) and kidneys (B) of mice (n = 13) challenged with 4×107 CFU USA400 MW2 bacteria. Statistical significance determined by Mann-Whitney Test (two-tailed): *p< 0.05, **p< 0.01, ***p1000

11

8.8

18

>50000

12

12.4

19

>2500

13

10.0

20

>10000

The values given are the IC50 values for pigment inhibition in S. aureus Newman, in

nM. Table 2. CrtN Inhibition Activities of the Four Most Valid Compounds for Pigment Inhibition.

a

Compd.

IC50 a (µM)

Compd.

IC50 a (µM)

8

0.32

32

0.45

10

0.41

35

0.50

The values given are the IC50 values against CrtN, in µM.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 66 of 67

Table 3. Aqueous Solubilities of the Four Most Valid Compounds for Pigment Inhibition. Solubilitya Compound

Solubilitya Compound

(mg/mL)

a

(mg/mL)

8

4.24

32

2.10

10

0.57

35

0.69

The values given are the solubility in water, in mg/mL.

Table 4. Antifungal activity of 8. Antifungal Activity MIC (µg/mL) Compd. Trichophyton rubrum

Microsporum gypseum

Tinea barbae

Ketoconazole

0.5

2

0.0625

Voriconazole

0.03125

0.25

0.03125

Fluconazole

1

8

2

NTF

0.125

0.25

0.125

8

8

16

8


Page 67 of 67

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC 228x54mm (300 x 300 DPI)


Discovery of Benzocycloalkane Derivatives Efficiently Blocking

Recommend Documents