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Discovery of Potent Benzofuran Derived Diapophytoene Desaturase (CrtN) Inhibitors with Enhanced Oral Bioavailability for the Treatment of Methicillin-Resistant S. aureus (MRSA) Infections youxin wang, Feifei Chen, Hongxia Di, Yong Xu, Qiang Xiao, Xuehai Wang, Hanwen Wei, Yanli Lu, Lingling Zhang, Jin Zhu, Chunquan Sheng, Lefu Lan, and Jian Li J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01984 • Publication Date (Web): 21 Mar 2016 Downloaded from http://pubs.acs.org on March 21, 2016

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Journal of Medicinal Chemistry

Discovery of Potent Benzofuran Derived Diapophytoene Desaturase (CrtN) Inhibitors with Enhanced Oral Bioavailability for the Treatment of Methicillin-Resistant S. aureus (MRSA) Infections Youxin Wanga,†, Feifei Chenb,†, Hongxia Dib, Yong Xuc, Qiang Xiaoc, Xuehai Wangd, Hanwen Weia, Yanli Lua, Lingling Zhanga, Jin Zhua, Chunquan Shenge, Lefu Lanb,*, Jian Lia,*

a

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

University of Science and Technology, Shanghai 200237, China b

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

Chinese Academy of Sciences, Shanghai 201203, China c

Hubei Bio-pharmaceutical Industrial Technological Institute Inc., Wuhan 430075,

China d

Humanwell Healthcare (Group) Co. Ltd, 666 Gaoxin Road, Wuhan 430075, China

e

Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical

University, Shanghai 200433, China

†

These authors contributed equally to this work.

* To whom correspondence should be addressed: [email protected] or [email protected]

ACS Paragon Plus Environment


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ABSTRACT

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


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INTRODUCTION Staphylococcus aureus (S. aureus) is a major human pathogen that causes substantial disease, and the abuse of antimicrobial chemotherapies increases concern for the rapid evolution of antimicrobial resistance.1 MRSA is a nosocomial and communal menace that is resistant against many antibacterial drugs and antiseptics. It is estimated that by 2050, worldwide drug-resistant infections will cause an additional 10 million deaths annually.2 A US Centers for Disease Control and Prevention (CDC) report3 recently issued a report to various governments and organizations outlining the concern of MRSA, including the New Drugs for Bad Bugs (ND4BB) project initiated by the European Union,4 the United States military5 and the World Health Organization (WHO).6 The Antimicrobial Resistance Global Report on Surveillance 2014 released by WHO has proven to be a global initiative to tackle the MRSA superbug and has rekindled a campaign for new, effective and safe drugs. Because of the suppression of bacterial survival or growth, conventional antibiotics are confined to a limited lifespan. It is urgent to establish a new path forward (e.g., anti-adhesion7 or anti-biofilm8 strategies) for medicinal chemists to successfully address this problem. Anti-virulence strategies are now under exploited with less selective pressure for the development of bacterial resistance. Staphyloxanthin (STX),9 a golden carotenoid pigment produced by S. aureus in human clinical isolates, enables S. aureus to flee from innate immune clearance with its numerous alternating single and double bonds,10 which can interact with reactive oxygen species (ROS) from host neutrophils.11-12 Evidence13 suggests the essential nature of STX is to act as a virulence factor with antioxidant properties; indeed, un-pigmented microbes are more susceptible to host killing. Therefore, the intervention of STX biosynthesis may offer a new avenue to treat S. aureus or even MRSA infections.14 A range of enzymes mediate the biosynthesis of STX, including dehydrosqualene synthase (CrtM) and diapophytoene desaturases (CrtN). Thus, the inhibition of these enzymes likely produces non-pigmented bacteria. In 2008, Eric Oldfield and co-workers reported a first-generation CrtM inhibitor, that is, 22 (BPH-652,15 Figure 1), which targets the biosynthesis of STX. Based on this



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pioneering work, several types of CrtM inhibitors have been subsequently identified by the same group.16-18 It is worth emphasizing that 22 is the most promising CrtM inhibitor based on the virulence factor-based concept and is now completing IND-enabling studies by AuricX Pharmaceuticals Inc., whose development is ongoing since October 2014 according to a report from Cortellis of Thomson Reuters. As the starting point for our efforts to discover new anti-virulence compounds, an in-house library of ~400 old drugs was screened using an STX inhibition assay. Finally, naftifine hydrochloride (NTF, Figure 1), an existing FDA-approved antifungal drug, was capable of prohibiting not only STX production of S. aureus Newman at nanomolar concentrations (Pigment inhibition: IC50=296.0±12.2 nM, Figure 1) but also three MRSA strains (USA300 LAC, USA400 MW2, and Mu50) at low micromolar concentrations. The in vivo performance of NTF was also encouraging in both S. aureus Newman and MRSA strains in mouse infection models. As a proof-of-concept study, we have uncovered that the inhibitory mode of action of NTF is mediated by inhibiting CrtN.19 Prior to this research, diphenylamines were reported to be the only confirmed CrtN inhibitor,20-21 but were much less effective than NTF in both inhibiting S. aureus CrtN and STX biosynthesis; in addition, their in vivo inhibitory effects on S. aureus virulence remains to be elucidated. To the best of our knowledge, none of the CrtN homologues have been identified in mammals, which differs from CrtM. Hence, the inhibition of CrtN might render fewer side effects than those observed with CrtM, making CrtN an attractive and druggable target against infections caused by pigmented S. aureus. The preferred forms of NTF dosage are cream or gel owing to its poor pharmacokinetics (PK) data, especially very low oral bioavailability (F value, Table 5). These data suggest an opportunity to modify NTF to obtain a novel drug-like candidate targeted against S. aureus infections or even MRSA infectious diseases. Our vision is to deliver an orally efficacious CrtN inhibitor by the optimization of NTF and to obtain novel NTF analogues possessing independent intellectual property rights. To this end, we designed two types of novel benzofuran skeletons (1–5, Figure 2) derived from NTF under the premise of employing the smallest possible structural


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changes. Finally, a potent benzofuran derived CrtN inhibitor with enhanced oral bioavailability, compound 5m, was identified, which could be a preclinical candidate for the treatment of MRSA infections by attenuating bacterial virulence. CHEMISTRY In this study, to further improve pharmacologic activity and oral bioavailability of the lead compound NTF and obtain novel structural scaffolds, chemical modifications were performed in four cycles. First, two novel scaffold analogs 1-2 (Figure 2) were designed to determine if the naphthalenyl moiety of NTF was necessary for pigment inhibitory activity. Second, we replaced the N-methyl group in region A (Figure 2) with various steric alkyl groups (including hydrogen atom) and 3 analogues (3a-c) were designed (Table 1). Third, six analogs (4a-f, Table 2) were prepared to estimate if the type of linkers (allyl in region B) would affect pigment inhibitory activity. Finally, we incorporated different substituted phenyls, furanyl, naphthalenyls, (cyclo)alkyls in region C, and twenty-one analogs (5a-u) were designed (Table 3). On the basis of the above design, analogs 1, 3a-c, 4a-b, and 5a-u were synthesized through the route outlined in Scheme 1. 2-Iodophenol was coupled with 1-bromo-2,2-diethoxyethane to afford 6 via nucleophilic substitution. Then, 6 was cyclized in the presence of polyphosphoric acids to afford 7-iodobenzofuran 7. After replacing the iodine atom of 7 into a cyano substituent, we obtained benzofuran-7-carbonitrile 8. Next, 9 was prepared by lithium aluminum hydride reduction from 8. On one hand, t-butyloxycarboryl group was added to compound 9 to form 10, which was then converted to the key intermediate 11 by reduction of the t-butyloxycarboryl group. On the other hand, compound 9 was coupled with trans-cinnamaldehyde via reductive amination to afford the target analog 3a. Subsequently, substitution of 3a with an ethyl group or isopropyl group afforded the target analogs 3b and 3c, respectively. Various substituted acraldehydes were reduced by sodium borohydride, followed by bromination, giving compounds 13a-x. Finally, nucleophilic substitution of compounds 13a-x with 8 provided the target analogs 1, 4a-b, and 5a-u.



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Scheme 2 depicts the synthetic route for the preparation of the analog 2. Coupling 3-bromophenol with 1-bromo-2,2-diethoxyethane generated compound 14, which was cyclized using polyphosphoric acids to generate two isomer products (i.e., 4-bromobenzofuran and 6-bromobenzofuran) with similar polarity. These products were difficult to separate, so the mixture was used for the next step without further purification. Then, a bromine atom was replaced by a cyano group, which permitted the separation of benzofuran-4-carbonitrile 16 from benzofuran-6-carbonitrile by column chromatography. Pure 16 underwent reduction, addition of t-BOC, reduction and final substitution to afford the analog 2. In parallel, analogs 5a and 4c-f were prepared in a similar process (Scheme 3). 5a and 4f were synthesized directly from 11 and commercially available corresponding

alkyl

bromides

via

nucleophilic

substitution.

While

(3-bromoprop-1-yn-1-yl)benzene 21c was not directly available, it was obtained by bromination of 3-phenylprop-2-yn-1-ol, followed by nucleophilic substitution with 11 to provide the analog 4e. The R4-substituted acid underwent esterification, reduction, bromination and final substitution to provide the analogs 4c-d. RESULTS AND DISCUSSION NTF-Derived New Scaffolds Design and Synthesis In total, 32 novel analogs (1-2, 3a-c, 4a-f, and 5a-u) of NTF were designed and synthesized. Their chemical structures are shown in Tables 1–3. These analogs were synthesized through the routes outlined in Schemes 1–3, and the details of the synthetic procedures and structural characterization are described in the Experimental Section. All analogs were confirmed to be ≥95% pure (Table S1, Supporting Information). In vitro Pigment Inhibitory Activities of Analogs 1-2, 3a-c, 4a-f, and 5a-u For the primary assay, the new NTF-derived analogs were assayed for pigment inhibitory activities against S. aureus Newman. The results are summarized in Tables 1–3. Encouragingly, the benzofuran-7-yl analog 1 displayed promising pigment inhibitory activity (pigment inhibition: IC50=247.3±18.8 nM), inheriting the activity


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of NTF (IC50=296.0±12.2 nM), whereas the benzofuran-4-yl analog 2 possessed less than satisfactory potency (IC50=758.7±24.3 nM). Subsequently, we varied the groups on the nitrogen atom to afford the analogs 3a-c to determine whether activity may be enhanced. Evidently, once the N-methyl group was removed (3a) or converted into an ethyl or isopropyl group (3b-c), the pigment inhibition activities abated (Table 1). Analysis of the data in Table 2 revealed that when the allyl linker was replaced into propargyl (4e, IC50=164.3±15.4 nM) or vinylogue (4b, IC50=4.2±0.1 nM), the pigment inhibitory activities were greatly improved, especially for 4b (by a factor of ~61). Elimination of double bonds (4d and 4f) led to a loss of pigment inhibitory activities (IC50>1000 nM). Moreover, the introduction of branched methyl (4a) or an additional methylene moiety (4c) resulted in a decrease of pigment inhibitory activities (IC50>1000 nM). Although analog 4b showed a potent pigment inhibitory activity at the single-digit nanomolar level, in view of its oil salt form, which is unsuitable for oral dosage prescriptions, we did not select 4b for further analysis. As shown in Table 3, replacement of phenyl with various types of groups can remarkably affect the pigment inhibitory activities. Generally, (cyclo)alkyl (5a-c) or heteroaromatic (5d) groups were not beneficial; however, the introduction of large aromatic naphthalenyl groups (5e-5f), especially the naphthalene-2-yl group, was favorable. To our surprise, electron-withdrawing groups and electron-donating groups at the phenyl ring indistinguishably affected the activity. However, the substituted positions at the phenyl ring substantially affected the activity, and substitution at the para-position of the phenyl ring showed improved activity (5i vs 5p vs 5s and 5l vs 5q vs 5t). Notably, the inhibitory activity of the most potent analog 5m (IC50=4.0±0.2 nM, Table 3) increased approximately 76 times than that of the lead compound NFT (IC50=296.0±12.2 nM, Figure 2), demonstrating that the chemical modification strategy employed in this study is successful. SAR of NTF-Derived Benzofuran Analogs The SAR analysis of a set of 32 NTF-derived analogs provided important insights into the essential structural requirements for effective pigment inhibition. An analysis of the data shown in Tables 1–3 reveals some noteworthy observations of the



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SAR for compounds 1-2, 3a-c, 4a-f, and 5a-u: (1) the naphthalenyl moiety of NTF is not indispensable for pigment inhibitory activity; indeed, replacement of the naphthalenyl ring with other similar bulky aryl rings (e.g., benzofuran) can be tolerated (NTF vs 1 or 2); (2) in region A of the compounds (Figure 2), the N-methyl group is critical for high potency (see analogs 3a-c, Table 1), and functional groups that are too small (3a) or too large (3b-c) are not beneficial, leading to a loss of pigment inhibitory activity; (3) in region B of the compounds (Figure 2), among the different linkages, the potency increases in the order 1,3-pentadienyl > propargyl > allyl > 2-methyl allyl = propyl = methenecyclopropanyl (4b > 4e > NTF > 4a = 4f = 4d). Generally, the unsubstituted allyl linker is preferred and vinylogue significantly improves the activities; (4) in region C of the compounds (Figure 2), in the studied set of the R1 group (Table 3), the potency increases in the order phenyl > naphthalenyl > furanyl = (cyclo)alkyl. The para-position is the best substituted position at the phenyl ring (5i vs 5p or 5s and 5l vs 5q or 5t), and the electronic effect of the substituent is limited. Taken together, the SARs are unambiguous. A subtle interplay between linkages and the 4-substituted phenyl moiety appear to be critical for high potency. Analog 5m appears to represent the best combination of these factors, leading to ∼76-fold potency improvement compared to the lead compound NTF. Target Enzyme Determination of the NTF-Derived Benzofuran Analogs In light of our previous research that naftifine was a CrtN inhibitor,19 we reasoned that the benzofuran analogs derived from naftifine may have the same target. To validate this hypothesis, we conducted a similar HPLC experiment as before at 286 nm for 4,4’-diapophytoene (the product of CrtM and the substrate of CrtN). Expression of crtM in E. coli gave rise to a HPLC peak similar to that of wild-type S. aureus Newman with respect to both retention time and UV absorption spectra (Figures 3B–C), suggesting that this peak is 4,4’-diapophytoene. Moreover, this peak disappeared in the cells of crtM (Figure 3D) and was reinforced in the crtN mutants (Figure 3E). This comparison further proved that this peak belonged to 4,4’-diapophytoene. The peak profile of the 5m-treated wild-type S. aureus Newman (Figure 3G) was very similar to that of the profile for the naftifine-treated Newman


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(Figure 3F). In addition, the peaks of both of them were quite similar to the crtN mutants. Taken together, these data suggest that CrtN is the target of the benzofuran analogs of NTF. In vitro CrtN Enzymatic Inhibitory Activities Pigment inhibitory assays revealed that a number of analogs showed highly potent activities against the biosynthesis of STX. To further explore their potential therapeutic targets, five representative analogs (5g, 5j-k, 5m, and 5u) were assayed for CrtN enzymatic inhibitory activity using our previous protocol.19 All five analogs showed submicromolar inhibition against CrtN (Table 4), which increased up to ~40 times than that of the lead compound NTF. It was noted that the enzymatic activities were weaker than the pigment inhibition activities in vitro; the precise reason remains to be elucidated. The speculated reasons are given as follows: (i) as we proposed in our previous paper19, it is possible that 5m accumulated in the cytoplasm of S. aureus, leading to a higher intracellular concentration, (ii) enzyme CrtN catalyzed three sequential reactions in the biosynthesis of staphyloxanthin, which differed from the enzyme CrtM. The staphyloxanthin inhibition potency of the CrtN inhibitor could be enhanced through synergistic effects. Water Solubility of the Representative Analogs Attention was given to the low water solubility of NTF (6.1 mg/mL, Table 4). The overwhelming hydrophobic structure (only one nitrogen atom) in NTF was deemed likely to cause its low solubility. Replacement of the naphthalenyl ring with the benzofuran ring was favorable, leading to an increase of water solubility. In particular, the water solubility of analogs 5g (19.7 mg/mL) and 5m (10.0 mg/mL) increased 2~3 times than that of the lead compound NTF (Table 4). Because of the excellent pigment inhibitory activity and good water solubility, analog 5m was subjected to additional in vitro and in vivo investigations. In vitro Pigment Inhibitory Activities of Analog 5m against MRSA USA400 MW2 and USA300 LAC, two clones responsible for the epidemic of community-acquired MRSA (CA-MRSA) infections in the United States, along with



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Mu50, a hospital-acquired MRSA (HA-MRSA) strain with vancomycin-intermediate resistance (MRSA/VISA), were taken to investigate the effectiveness of the analog 5m. Pigment inhibition results of three MRSA strains are shown in Figure 4. We were delighted to find that the color of all strains (USA400 MW2, USA300 LAC and Mu50) faded by the inhibition of 5m to a large extent (IC50=5.45±0.36 nM, 3.39±0.03 nM and 0.38±0.02 nM, respectively). The pigment inhibitory activities of 5m against MRSA were comparable with that against S. aureus Newman. Additionally, neither the growth characteristics nor the survival of the Newman or MRSA strains was suppressed when incubated with 5m (0.2 mM or 0.05 mM, Figure 5). Thus, we conclude that 5m inhibited the production of pigment rather than inhibited the survival of the bacteria; that is to say, 5m is a potent anti-virulence candidate. Effects of 5m on Sensitizing S. aureus to Immune Clearance We next wished to determine whether the inhibition of pigment of all strains translated to susceptibility of the four colonies to innate immune clearance using two assay systems: hydrogen peroxide killing and human whole blood killing. As shown in Figure 6, after incubation with 5m (1 µM), the resulting white S. aureus Newman were more susceptible to killing by 1.5% H2O2 compared to the untreated S. aureus (mock) (survival, 1.2% vs 36.2%), which represents a decline in survival percentage by a factor of ~30. The survival percentage of USA400 WM2, USA300 LAC, and Mu50 reduced by a factor of ~20 (0.6% vs 11.7%), ~20 (0.7% vs 14.2%), and ~6 (4.3% vs 25.3%), respectively. As for human whole blood (Figure 7), which was more appealing to us, the resulting non-pigmented S. aureus were more susceptible to killing in freshly isolated human whole blood when subjected to the same concentration of 5m, causing the Newman strain survival percentage to decline by a factor of ~33 (0.8% vs 26.7%). The survival percentage of USA400 WM2, USA300 LAC, and Mu50 fell by a factor of ~8 (1.3% vs 10.2%), ~10 (1.2% vs 12.2%), and ~5 (3.0% vs 16.1%), respectively. The above results suggest that 5m indeed makes S. aureus more susceptible to immune clearance in human blood. In vivo Effects of 5m on Attenuating the Virulence of S. aureus Newman


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Next, we extended our assessment on the contribution of STX to abscess formation in a systematic S. aureus Newman infection model. After infection with 1 × 107 colony-forming units (CFU) S. aureus Newman bacteria via retro-orbital injection, the mice were sacrificed at 108 hours. We then measured bacterial survival in the host organs. As shown in Figure 8, the 5m-treated group reduced bacterial survival mainly in the kidneys and hearts by 0.85 and 1.01 log10 CFU/organ, respectively. These results clearly demonstrate that in vivo 5m-treatment attenuated the pathogenicity of S. aureus Newman. In vivo Effects of 5m on Attenuating the Virulence of MRSA We next sought to examine whether the inhibitory effect of 5m on S. aureus virulence was only restricted to the Newman strain. Mock or 5m-treated mice were infected with two distinct MRSA strains (USA400 MW2 and Mu50). As shown in Figures 9–10, in the USA400 MW2 model, we initially treated the mice with a 200 mg/kg dose to find that the staphylococcal loads of the 5m-treated group were decreased, as measured in liver by 2.35 log10 CFU. This corresponds to a 99.6% decrease in surviving bacteria and is superior than the positive group treated with 200 mg/kg 22 (1.58 log10 CFU). When we reduced the administration dose to 50 mg/kg, we observed that in both cases the bacterial survival rates were slightly elevated relative to the high dose groups, and still lower than the mock group by 0.25 log10 CFU (22-treated group) and 0.71 log10 CFU (5m-treated group). As for the kidney organs, the effect was less remarkable. In the high dosage case, the staphylococcal loads of the 5m-treated group were decreased, as measured by 1.47 log10 CFU (a 96.6% decrease in surviving bacteria). Similarly, this value is superior than the positive group treated with 200 mg/kg 22 (1.14 log10 CFU). When we reduced the administration dose to 50 mg/kg, we observed that in both cases the bacterial survival rates were slightly elevated relative to the high dose groups, and demonstrated a comparative clearance. In the Mu50 model, identical results were obtained. In the liver organs with a dose of 200 mg/kg, both inhibitors possessed excellent performance. With the treatment of 5m, the bacterial survival rates fell by 3.58 log10 CFU (a 99.97% decrease in surviving bacteria), whereas the survival rates of the 22



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treated group decreased by 2.84 log10 CFU. Although the dose of the inhibitors was reduced to 50 mg/kg, the outcomes remained encouraging. The strains of the 5m-treated group were shrunk by 2.94 log10 CFU, better than the 22 treated group, whose rates decreased by 1.87 log10 CFU, which is one order of magnitude weaker than 5m. Regarding the kidneys, the results were inconclusive. With 200 mg/kg of 5m, the bacterial colonies were inhibited by 1.11 log10 CFU. Prescribed with the same dosage of 22, only a 0.30 log10 CFU decrement was observed. Furthermore, a low dose of 50 mg/kg produced less benefit. Bacterial survival rates of the 5m and 22 treated groups decreased by only 0.68 log10 CFU and 0.25 log10 CFU, respectively. To sum up, both of the two inhibitors against STX biosynthesis, 22 and 5m, were capable of decreasing the bacterial survival in the S. aureus Newman and two MRSA strains. However, compound 5m was more successful than 22 in all experiments in this regard. In vitro Anti-fungal Activities of 5m Next, an anti-fungal experiment was conducted to examine whether 5m inherited the original anti-fungal activity of NTF. As shown in Table 5, the anti-fungal activity of the benzofuran scaffold was no longer effective. Compound 5m demonstrated weak activities in all cases, and its best MIC against Trichophyton rubrum was 128-fold worse than that of NTF, suggesting, to a certain extent, that 5m may be a selective anti-virulence active compound. In vivo Rat Pharmacokinetics Profile of 5m Because of its potency and attractive selectivity profile, compound 5m was evaluated in an in vivo rat PK model. The pharmacokinetic parameters were determined following iv injection of a single 5 mg/kg dose or po administration of a single 10 mg/kg dose in rat. Further details are shown in Table 6. Compared with the poor bioavailability of NTF (F = 0.85 %), presumably resulting in its external application, compound 5m possessed acceptable bioavailability (F = 42.2%), which is consistent with the drug-like criteria of 30% concluded by most pharmaceutical researchers.22 Moreover, the approximate 8 h half-life (t1/2) of 5m is also suitable for its oral delivery, which could be administered twice daily.


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CONCLUSIONS In summary, we have discovered a new class of CrtN inhibitors with enhanced oral bioavailability using a scaffold hopping approach. On the basis of the structure of the lead compound NTF, 32 completely new benzofuran analogs have been synthesized and tested with pigment inhibitory assays. Five analogs (5g, 5j-k, 5m and 5u) were found to show potent pigment inhibitory activities and good physicochemical properties. Notably, the most potent analog, 5m, demonstrated pigment inhibitory capability approximately 76 times higher than that of the prototype NTF. Preliminary SARs were obtained, showing that variations in the allyl linkage and substituents at the para-position of the phenyl moiety have a very important influence on the pigment inhibitory activity, and appropriate structural optimizations of the above regions can substantially improve potency. CrtN enzymatic inhibition assays and HPLC analyses further confirmed that five analogs (5g, 5j-k, 5m, and 5u) were all CrtN inhibitors with submicromolar IC50 values, being more effective (up to 91-fold) than diphenylamines previously reported.21 Among the five analogs, compound 5m was found to have high water solubility and could effectively inhibit the STX biosynthesis of three MRSA strains (USA300 LAC, USA400 MW2, and Mu50) with IC50 values of 3.39±0.03 nM, 5.45±0.36 nM and 0.38±0.02 nM, respectively, without any impact on the survival of the Newman strain or three MRSA strains (up to 200 µM). The anti-virulence effects of 5m (1 µM) were also observed by sensitizing Newman (up to 30 times) and three MRSA strains (up to 33 times) to H2O2 and human whole blood killing, which were in good agreement with the in vivo therapeutic potential of 5m on attenuating the virulence of three S. aureus strains. The in vivo pharmacological results showed that 5m (200 mg/kg and 50 mg/kg) had both more beneficial effects on lowering abscess formation in liver and kidney than 22 (200 mg/kg and 50 mg/kg). For liver, in particular, treatment with 5m (200 mg/kg) resulted in a 99% decrease in surviving bacteria (USA400 MW2 and Mu50). To our delight, 5m possessed very weak anti-fungal activities (MIC >16 µg/mL) and good oral bioavailability (F = 42.2%).



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Moreover, the safety evaluation experiments (detailed assays in Supporting Information) showed that 5m had very weak effects on the growth of two normal human cell lines (HMEC: IC50= 67.1±2.4 µM and WI38: IC50=47.9±4.5 µM) and possessed low hERG inhibition activity (IC50=3.71 µM). Altogether, these data demonstrate that 5m may be a good candidate for discovering potential therapeutic drugs with specificity to combat pigmented S. aureus (especially MRSA). Considering only a few CrtN inhibitors reported thus far, 5m is an effective chemical biology tool for the study of STX-regulating virulence in infectious diseases. Further structural optimization of 5m is currently in progress in our laboratory. EXPERIMENTAL SECTION General Chemistry. Synthetic starting materials, reagents and solvents were purchased from Alfa Aesar, Acros, Adamas-beta, Energy Chemical, J&K, Shanghai Chemical Reagent Co. and TCI at the highest commercial quality and used without further purification. Analytical thin-layer chromatography (TLC) was performed on HSGF 254 (150−200 µm thickness; Yantai Huiyou Co., China), and the components were visualized by observation under UV light (254 nm and 365 nm). Melting points were determined on a SGW 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 spectrometer using deuterated chloroform (CDCl3), deuterated methanol (CD3OD), or deuterated dimethyl sulfoxide (DMSO-d6) as the solvent. Chemical shifts were reported in parts per million (ppm, δ) downfield from tetramethylsilane. Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). Low- and high-resolution mass spectra (LRMS and HRMS) were given with electron spray ionization (ESI) produced by a Finnigan MAT-95 and a LCQ-DECA spectrometer. HPLC data analysis of compounds 1-2, 3a-c, 4a-f and 5a-u were performed on an Agilent 1100 with a quaternary pump and diode-array detector (DAD). The peak purity was verified with UV spectra. All analogs were confirmed to be ≥95% pure.


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Preparation of salts. Taking compound 1 as an example, oily compound 1 (100.0 mg) was suspended in EtOAc (15 mL) with stirring at room temperature and then bubbled into hydrogen chloride gas for 1 min. The reaction solution was concentrated and then stirred for an additional 1 h in EtOAc/petroleum ether (1:100, v/v, 20 mL). The precipitate was filtered, washed with EtOAc to give the final compounds in the form of hydrochloride. All other end-products had experienced such salification process. Spectroscopic data given below are in their hydrochloride form. (E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-phenylprop-2-en-1-amine hydrochloride (1). A solution of intermediate 11 (120.0 mg, 0.7 mmol), 13a (146.0 mg, 0.7 mmol) and K2CO3 (112.0 mg, 0.8 mmol) in DMF (10 mL) was stirred at room temperature overnight. The mixture was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue was then purified via flash chromatography on silica gel, eluting with EtOAc/petroleum ether (1/5, v/v) to give the free base of 1 as a colorless oil. Yield: 44%. 1 was prepared by the general procedure of salification given above as a white solid, m.p. 114–118 ºC. 1H NMR (400 MHz, MeOD) δ 7.94 (s, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.58–7.47 (m, 3H), 7.38 (dt, J = 14.2, 7.4 Hz, 4H), 7.00 (d, J = 10.8 Hz, 1H), 6.95 (s, 1H), 6.48–6.32 (m, 1H), 4.82 (s, 1H), 4.65 (s, 1H), 4.07 (d, J = 38.8 Hz, 2H), 2.90 (s, 3H); HRMS (ESI) m/z calcd for C19H20NO [M+H]+ 278.1545, found 278.1539. (E)-N-(Benzofuran-4-ylmethyl)-N-methyl-3-phenylprop-2-en-1-amine hydrochloride (2). Yield: 44%. 2 was synthesized by the general procedure of 1 given above as a white solid, m.p. 173–176 ºC. 1H NMR (400 MHz, MeOD) δ 7.95 (d, J = 2.3 Hz, 1H), 7.70 (p, J = 3.8 Hz, 1H), 7.57–7.51 (m, 2H), 7.48 (d, J = 5.1 Hz, 2H), 7.44–7.33 (m, 3H), 7.17 (dd, J = 2.3, 0.9 Hz, 1H), 6.95 (d, J = 15.8 Hz, 1H), 6.44–6.33 (m, 1H), 4.68 (d, J = 64.3 Hz, 2H), 4.05 (d, J = 35.5 Hz, 2H), 2.87 (s, 3H); HRMS (ESI) m/z calcd for C19H20NO [M+H]+ for 278.1545, found 278.1539. (E)-N-(Benzofuran-7-ylmethyl)-3-phenylprop-2-en-1-amine hydrochloride (3a). A mixture of 9 (294.0 mg, 2 mmol), trans-cinnamaldehyde (264.0 mg, 2 mmol) and molecular sieve (1.0 g) in dichloromethane (25 mL) was heated at reflux for 17 h.



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Thereafter, methanol was added dropwise into the mixture and then sodium borohydride (76.0 mg, 2 mmol) was added in batches at 0 ºC. The reaction mixture was stirred for 30 min and concentrated under reduced pressure. The residue was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue was then purified via flash chromatography on silica gel, eluting with EtOAc/petroleum ether (1/1, v/v) to give the free base of 3a as a colorless oil. Yield: 65%. 3a was prepared using the general procedure of salification as a white solid, m.p. 129–130 ºC. 1H-NMR (400 MHz, MeOD) δ 7.92 (s, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.49 (t, J = 9.3 Hz, 3H), 7.43–7.29 (m, 4H), 6.99 (d, J = 1.8 Hz, 1H), 6.92 (d, J = 15.8 Hz, 1H), 6.43–6.30 (m, 1H), 4.60 (s, 2H), 3.94 (d, J = 7.2 Hz, 2H). HRMS (ESI) m/z calcd for C18H18NO [M+H]+ 264.1388, found 264.1383. (E)-N-(Benzofuran-7-ylmethyl)-N-ethyl-3-phenylprop-2-en-1-amine hydrochloride (3b). To a solution of the free base of 3a (360.0 mg, 1.4 mmol) in DMF (10 mL) was added sodium hydride (52 mg, 1.4 mmol) in batches at 0 ºC under a N2 atmosphere. The reaction mixture was stirred for 15 min and iodoethane (219.0 µL, 2.7 mmol) was added into the solution. The mixture was stirred at room temperature overnight, poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue was then purified via flash chromatography on silica gel, eluting with EtOAc/petroleum ether (1/5, v/v) to give the free base of 3b as a colorless oil. Yield: 50%. 3b was prepared using the general procedure of salification as a yellow oil. 1

H-NMR (400 MHz, MeOD) δ 7.93 (d, J = 1.8 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.53

(d, J = 7.4 Hz, 3H), 7.44–7.32 (m, 4H), 7.01 (d, J = 1.9 Hz, 1H), 6.95 (d, J = 15.8 Hz, 1H), 6.46–6.32 (m, 1H), 4.75 (s, 2H), 4.03 (dd, J = 17.7, 10.4 Hz, 2H), 3.39–3.34 (m, 2H), 1.49 (t, J = 7.2 Hz, 3H). HRMS (ESI) m/z calcd for C20H22NO [M+H]+ 292.1701, found 292.1700. (E)-N-(Benzofuran-7-ylmethyl)-N-isopropyl-3-phenylprop-2-en-1-amine hydrochloride (3c). Yield: 50%. 3c was synthesized by the general procedure of 3b given above as a yellow oil. 1H-NMR (400 MHz, MeOD) δ 7.92 (d, J = 1.8 Hz, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.52 (t, J = 7.2 Hz, 2H), 7.44 (d, J = 6.9 Hz, 2H), 7.37 (d, J = 6.2 Hz, 3H),


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6.99 ( s, 1H), 6.90 (d, J = 16.3 Hz, 1H), 6.30–6.18 (m, 1H), 4.85 (d, J = 13.7 Hz, 1H), 4.57 (d, J = 13.7 Hz, 1H), 4.14 (d, J = 12.9 Hz, 1H), 4.04 (dd, J = 13.6, 7.2 Hz, 1H), 3.85 (dt, J = 13.3, 6.6 Hz, 1H), 1.60 (d, J = 6.6 Hz, 3H), 1.52 (d, J = 6.6 Hz, 3H); HRMS (ESI) m/z calcd for C21H24O [M+H]+ 306.1858, found 306.1862. (E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-phenylprop-2-en-1-amine hydrochloride (4a). Yield: 57%. 4a was synthesized by the general procedure of 1 given above as a white solid, m.p. 160–162 ºC. 1H-NMR (400 MHz, MeOD) δ 7.92 (s, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.52 (d, J = 7.4 Hz, 1H), 7.40–7.22 (m, 6H), 6.97 (d, J = 18.2 Hz, 1H), 6.86 (s, 1H), 4.85 (d, J = 13.3 Hz, 1H), 4.56 (d, J = 13.3 Hz, 1H), 4.11–4.05 (m, 1H), 3.98 (d, J = 12.7 Hz, 1H), 2.91 (s, 3H), 2.04 (s, 3H); HRMS (ESI) m/z calcd for C20H22NO [M+H]+ 292.1701, found 292.1707. (2E,4E)-N-(Benzofuran-7-ylmethyl)-N-methyl-5-phenylpenta-2,4-dien-1-amine hydrochloride (4b). Yield: 42%. 4b was synthesized by the general procedure of 1 given above as a yellow oil. 1H-NMR (400 MHz, MeOD) δ 7.92 (s, 1H), 7.78 (d, J = 10.8 Hz, 1H), 7.48 (d, J = 6.7 Hz, 3H), 7.43–7.31 (m, 3H), 7.30–7.20 (m, 1H), 7.04–6.90 (m, 2H), 6.83–6.65 (m, 2H), 5.96 (dt, J = 14.9, 7.4 Hz, 1H), 4.78 (d, J = 13.3 Hz, 1H), 4.57 (d, J = 13.3 Hz, 1H), 4.10–3.98 (m, 1H), 3.95–3.85 (m, 1H), 2.81 (s, 3H); HRMS (ESI) m/z calcd for C21H22NO [M+H]+ 304.1701, found 304.1695. (E)-N-(Benzofuran-7-ylmethyl)-N-methyl-4-phenylbut-3-en-1-amine hydrochloride (4c). Yield: 42%. 4c was synthesized by the general procedure of 1 given above as a yellow oil. 1H-NMR (400 MHz, MeOD) δ 7.90 (s, 1H), 7.83 (d, J = 7.5 Hz, 1H), 7.50 (d, J = 7.2 Hz, 1H), 7.42 (s, 3H), 7.33 (t, J = 7.1 Hz, 2H), 7.26 (d, J = 5.8 Hz, 1H), 7.00 (s, 1H), 6.66 (d, J = 16.3 Hz, 1H), 6.28–6.12 (m, 1H), 4.85 (d, J = 13.7 Hz, 1H), 4.63 (d, J = 13.7 Hz, 1H), 3.68–3.59 (m, 1H), 3.51-3.41 (m, 1H), 2.93 (s, 3H), 2.85– 2.77 (m, 2H); HRMS (ESI) m/z calcd for C20H22NO [M+H]+ 292.1701, found 292.1695. 1-(Benzofuran-7-yl)-N-methyl-N-(((1R,2R)-2-phenylcyclopropyl)methyl)methana mine hydrochloride (4d). Yield: 52%. 4d was synthesized by the general procedure of 1 given above as a yellow oil. 1H-NMR (400 MHz, MeOD) δ 7.86 (t, J = 4.1 Hz, 1H), 7.83–7.75 (m, 1H),



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7.53–7.43 (m, 1H), 7.42–7.31 (m, 1H), 7.28 (td, J = 7.5, 1.7 Hz, 2H), 7.22–7.11 (m, 3H), 6.97 (td, J = 5.3, 2.2 Hz, 1H), 4.85 (dd, J = 16.5, 6.5 Hz, 1H), 4.58 (dd, J = 13.3, 5.0 Hz, 1H), 3.52–3.45 (m, 1H), 3.29–3.15 (m, 1H), 2.90 (d, J = 7.3 Hz, 3H), 2.13– 2.03 (m, 1H), 1.62–1.49 (m, 1H), 1.18–1.06 (m, 1H), 0.94–0.81 (m, 1H). HRMS (ESI) m/z calcd for C20H22NO [M+H]+ 292.1701, found 292.1704. N-(Benzofuran-7-ylmethyl)-N-methyl-3-phenylpropan-1-amine

hydrochloride

(4e). Yield: 60%. 4e was synthesized by the general procedure of 1 given above as a yellow oil. 1H-NMR (400 MHz, MeOD) δ 7.93 (s, 1H), 7.84 (d, J = 7.7 Hz, 1H), 7.56 (dd, J = 14.6, 7.2 Hz, 3H), 7.52–7.37 (m, 4H), 7.02 (s, 1H), 4.89(d, J = 13.3 Hz, 1H), 4.78 (d, J = 13.3 Hz, 1H), 4.41 (d, J = 11.6 Hz, 2H), 3.05 (s, 3H). HRMS (ESI) m/z calcd for C19H18NO [M+H]+ 276.1388, found 276.1388. N-(Benzofuran-7-ylmethyl)-N-methyl-3-phenylprop-2-yn-1-amine hydrochloride (4f). Yield: 59%. 4f was synthesized by the general procedure of 1 given above as a yellow oil. 1H-NMR (400 MHz, MeOD) δ 7.91 (s, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.44 (t, J = 8.0 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H), 7.28 (d, J = 7.1 Hz, 2H), 7.22 (s, 4H), 7.0 (s, 1H), 4.76 (d, J = 13.3 Hz, 1H), 4.59 (d, J = 13.3 Hz, 1H), 3.31–3.23 (m, 1H), 3.21– 3.10 (m, 1H), 2.88 (s, 3H), 2.72 (t, J = 7.1 Hz, 2H), 2.17 (d, J = 5.6 Hz, 2H). HRMS (ESI) m/z calcd for C19H22NO [M+H]+ 280.1701, found 280.1704. (E)-N-(Benzofuran-7-ylmethyl)-N-methylbut-2-en-1-amine hydrochloride (5a). Yield: 50%. 5a was synthesized by the general procedure of 1 given above as a yellow oil. 1H-NMR (400 MHz, MeOD) δ 7.94 (s, 1H), 7.77 (dd, J = 34.7, 14.0 Hz, 1H), 7.49 (d, J = 7.4 Hz, 1H), 7.38 (dd, J = 18.9, 11.4 Hz, 1H), 7.05 –6.91 (m, 1H), 6.14 (dd, J = 14.8, 6.7 Hz, 1H), 5.81–5.63 (m, 1H), 4.73 (d, J = 17.1 Hz, 1H), 4.52 (d, J = 17.1 Hz, 1H), 3.94–3.86 (m, 1H), 3.80–3.71 (m, 1H), 2.81 (s, 3H), 1.84 (t, J = 16.1 Hz, 3H); HRMS (ESI) m/z calcd for C14H18NO [M+H]+ 216.1388, found 216.1383. (E)-N-(Benzofuran-7-ylmethyl)-3-cyclopentyl-N-methylprop-2-en-1-amine hydrochloride (5b). Yield: 39%. 5b was synthesized by the general procedure of 1 given above as a yellow oil. 1H-NMR (400 MHz, MeOD) δ 7.93 (d, J = 2.2 Hz, 1H), 7.83–7.80 (m, 1H), 7.48 (d, J = 6.9 Hz, 1H), 7.44–7.34 (m, 1H), 7.01 (d, J = 2.2 Hz, 1H), 6.10 (dd, J = 15.3, 7.8 Hz, 1H), 5.66 (dt, J = 14.9, 7.3 Hz, 1H), 4.75 (d, J = 13.3 Hz, 1H), 4.53 (d,


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J = 13.2 Hz, 1H), 3.90 (m, 1H), 3.76 (m, 1H), 2.80 (s, 3H), 2.61 (m, 1H), 1.99–1.83 (m, 2H), 1.84–1.53 (m, 4H), 1.49–1.30 (m, 2H); HRMS (ESI) m/z calcd for C18H24NO [M+H]+ 270.1858, found 270.1862. (E)-N-(Benzofuran-7-ylmethyl)-3-cyclohexyl-N-methylprop-2-en-1-amine hydrochloride (5c). Yield: 42%. 5c was synthesized by the general procedure of 1 given above as a yellow oil. 1H-NMR (400 MHz, MeOD) δ 7.94 (d, J = 2.2 Hz, 1H), 7.87–7.79 (m, 1H), 7.48 (d, J = 6.9 Hz, 1H), 7.44–7.34 (m, 1H), 7.01 (d, J = 2.2 Hz, 1H), 6.06 (dd, J = 15.3, 7.8 Hz, 1H), 5.63 (dt, J = 14.9, 7.3 Hz, 1H), 4.76 (d, J = 13.3 Hz, 1H), 4.56 (d, J = 13.4 Hz, 1H), 3.99 (m, 1H), 3.76 (m, 1H), 2.87 (s, 3H), 2.3 (m, 1H), 1.88–1.58 (m, 6H), 1.41–1.28 (m, 4H); HRMS (ESI) m/z calcd for C19H26NO [M+H]+ 284.2014, found 284.2008. (E)-N-(Benzofuran-7-ylmethyl)-3-(furan-2-yl)-N-methylprop-2-en-1-amine hydrochloride (5d). Yield: 58%. 5d was synthesized by the general procedure of 1 given above as a yellow oil. 1H-NMR (400 MHz, MeOD) δ 7.90 (d, J = 2.4 Hz, 1H), 7.79 (dd, J = 7.8, 1.1 Hz, 1H), 7.53 (d, J = 1.6 Hz, 1H), 7.48 (t, J = 5.6 Hz, 1H), 7.37 (td, J = 7.4, 3.0 Hz, 1H), 6.97 (t, J = 2.7 Hz, 1H), 6.78 (d, J = 15.6 Hz, 1H), 6.52 (d, J = 3.3 Hz, 1H), 6.51–6.44 (m, 1H), 6.28–6.13 (m, 1H), 4.78 (d, J = 13.3 Hz, 1H), 4.58 (d, J = 13.3 Hz, 1H), 4.15–4.01 (m, 1H), 3.89–3.97 (m, 1H), 2.84 (s, 3H); HRMS (ESI) m/z calcd for C17H18NO2 [M+H]+ 268.1338, found 268.1334. (E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-(naphthalen-1-yl)prop-2-en-1-amine hydrochloride (5e). Yield: 59%. 5e was synthesized by the general procedure of 1 given above as a white solid, m.p. 170–171 ºC. 1H-NMR (400 MHz, MeOD) δ 8.20 (d, J = 8.1 Hz, 1H), 7.93 (s, 3H), 7.89–7.71 (m, 3H), 7.66–7.48 (m, 4H), 7.42 (t, J = 7.5 Hz, 1H), 7.02 (s, 1H), 6.55–6.39 (m, 1H), 4.92 (d, J = 13.2 Hz, 2H), 4.69 (d, J = 13.2 Hz, 1H), 4.35– 4.22 (m, 1H), 4.20–4.06 (m, 1H), 2.94 (s, 3H); HRMS (ESI) m/z calcd for C23H22NO [M+H]+ 328.1701, found 328.1698. (E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-(naphthalen-2-yl)prop-2-en-1-amine hydrochloride (5f). Yield: 47%. 5f was synthesized by the general procedure of 1 given above as a white solid, m.p. 198–199 ºC. 1H-NMR (400 MHz, MeOD) δ 8.00–7.85 (m, 5H), 7.83 (d, J = 7.6 Hz, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.53 (s, 3H), 7.41 (t, J = 7.4 Hz, 1H),



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7.13 (d, J = 15.6 Hz, 1H), 7.01 (s, 1H), 6.62–6.47 (m, 1H), 4.86 (d, J = 13.4 Hz, 1H), 4.66 (d, J = 13.4 Hz, 1H), 4.28–4.14 (m, 1H), 4.14–3.98 (m, 1H), 2.91 (s, 3H); HRMS (ESI) m/z calcd for C23H22NO [M+H]+ 328.1701, found 328.1701. (E)-N-(Benzofuran-7-ylmethyl)-3-(4-methoxyphenyl)-N-methylprop-2-en-1-amin e hydrochloride (5g). Yield: 49%. 5g was synthesized by the general procedure of 1 given above as a white solid, m.p. 151–152 ºC. 1H-NMR (400 MHz, MeOD) δ 7.93 (s, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.49 (t, J = 9.2 Hz, 3H), 7.40 (t, J = 7.4 Hz, 1H), 7.04–6.93 (m, 3H), 6.90 (d, J = 15.8 Hz, 1H), 6.25 (dt, J = 15.2, 7.5 Hz, 1H), 4.83 (d, J = 13.3 Hz, 1H), 4.61 (d, J = 13.3 Hz, 1H), 4.14–4.05 (m, 1H), 4.02–3.90 (m, 1H), 3.83 (s, 3H), 2.94– 2.81 (m, 3H); HRMS (ESI) m/z calcd for C20H22NO2 [M+H]+ 308.1651, found 308.1653. (E)-Methyl4-(3-((benzofuran-7-ylmethyl)(methyl)amino)prop-1-en-1-yl)benzoate hydrochloride (5h). Yield: 85%. 5h was synthesized by the general procedure of 1 given above as a white solid, m.p. 163–165 ºC. 1H-NMR (400 MHz, MeOD) δ 8.05 (d, J = 7.8 Hz, 2H), 7.94 (s, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.65 (d, J = 7.8 Hz, 2H), 7.52 (d, J = 7.4 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.02 (d, J = 12.1 Hz, 2H), 6.62–6.49 (m, 1H), 4.85 (d, J = 13.2 Hz, 1H), 4.65 (d, J = 13.4 Hz, 1H), 4.22–4.12 (m, 1H), 4.10–3.98 (m, 1H), 3.93 (s, 3H), 2.89 (s, 3H). HRMS (ESI) m/z calcd for C21H22NO3 [M+H]+ 336.1600, found 336.1597. (E)-N-(Benzofuran-7-ylmethyl)-3-(4-fluorophenyl)-N-methylprop-2-en-1-amine hydrochloride (5i). Yield: 50%. 5i was synthesized by the general procedure of 1 given above as a white solid, m.p. 148–150 ºC. 1H-NMR (400 MHz, MeOD) δ 7.90 (s, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.55 (d, J = 5.6 Hz, 2H), 7.48 (d, J = 7.2 Hz, 1H), 7.37 (t, J = 7.5 Hz, 1H), 7.11 (t, J = 8.3 Hz, 2H), 6.98 (s, 1H), 6.91 (d, J = 15.8 Hz, 1H), 6.41–6.25 (m, 1H), 4.80 (d, J = 13.2 Hz, 1H), 4.60 (d, J = 12.9 Hz, 1H), 4.15–4.02 (m, 1H), 4.02– 3.87 (m, 1H), 2.84 (s, 3H); HRMS (ESI) m/z calcd for C19H19FNO [M+H]+ 296.1451, found 296.1449. (E)-N-(Benzofuran-7-ylmethyl)-3-(4-chlorophenyl)-N-methylprop-2-en-1-amine hydrochloride (5j).


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Yield: 61%. 5j was synthesized by the general procedure of 1 given above as a white solid, m.p. 183–184 ºC. 1H-NMR (400 MHz, MeOD) δ 7.90 (s, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.50 (d, J = 8.3 Hz, 3H), 7.38 (d, J = 7.1 Hz, 3H), 6.96 (d, J = 14.9 Hz, 1H), 6.91 (d, J = 15.7 Hz, 1H), 6.47–6.32 (m, 1H), 4.81 (d, J = 13.2 Hz, 1H), 4.61 (d, J = 13.4 Hz, 1H), 4.18–4.04 (m, 1H), 4.03–3.89 (m, 1H), 2.84 (s, 3H); HRMS (ESI) m/z calcd for C19H19ClNO [M+H]+ 312.1155, found 312.1148. (E)-N-(Benzofuran-7-ylmethyl)-3-(4-bromophenyl)-N-methylprop-2-en-1-amine hydrochloride (5k). Yield: 46%. 5k was synthesized by the general procedure of 1 given above as a white solid, m.p. 192–194 ºC. 1H-NMR (400 MHz, MeOD) δ 7.93 (s, 1H), 7.81 (t, J = 10.5 Hz, 1H), 7.66–7.24 (m, 6H), 6.98 (d, J = 20.3 Hz, 1H), 6.92 (d, J = 15.8 Hz, 1H), 6.57–6.33 (m, 1H), 4.84 (d, J = 13.4 Hz, 1H), 4.63 (d, J = 13.2 Hz, 1H), 4.21– 4.07 (m, 1H), 4.06–3.89 (m, 1H), 2.87 (s, 3H); HRMS (ESI) m/z calcd for C19H19BrNO [M+H]+ 358.0630, found 358.0634. (E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-(4-nitrophenyl)prop-2-en-1-amine hydrochloride (5l). Yield: 59%. 5l was synthesized by the general procedure of 1 given above as a white solid, m.p. 194–195 ºC. 1H-NMR (400 MHz, MeOD) δ 8.28 (d, J = 8.4 Hz, 2H), 7.94 (s, 1H), 7.80 (dd, J = 21.2, 8.0 Hz, 3H), 7.53 (d, J = 7.3 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.14–6.96 (m, 2H), 6.71–6.55 (m, 1H), 4.86 (d, J = 13.3 Hz, 1H), 4.67 (d, J = 13.3 Hz, 1H), 4.27–4.13 (m, 1H), 4.15–3.90 (m, 1H), 2.94 (s, 3H); HRMS (ESI) m/z calcd for C19H19N2O3 [M+H]+ 323.1396, found 323.1393. (E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-(4-(trifluoromethyl)phenyl)prop-2-e n-1-amine hydrochloride (5m). Yield: 43%. 5m was synthesized by the general procedure of 1 given above as a white solid, m.p. 162–164 ºC. 1H-NMR (400 MHz, MeOD) δ 7.94 (s, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.72 (s, 4H), 7.53 (d, J = 7.4 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.03 (d, J = 15.8 Hz, 2H), 6.64–6.49 (m, 1H), 4.86 (d, J = 13.7 Hz, 1H), 4.66 (d, J = 12.9 Hz, 1H), 4.24–4.12 (m, 1H), 4.09–3.97 (m, 1H), 2.93 (s, 3H); HRMS (ESI) m/z calcd for C20H19F3NO [M+H]+ 346.1419, found 346.1406. (E)-N-(Benzofuran-7-ylmethyl)-3-(4-(tert-butyl)phenyl)-N-methylprop-2-en-1-am ine hydrochloride (5n).



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Yield: 49%. 5n was synthesized by the general procedure of 1 given above as a white solid, m.p. 139–141 ºC. 1H-NMR (400 MHz, MeOD) δ 7.93 (s, 1H), 7.81 (t, J = 11.2 Hz, 1H), 7.59–7.35 (m, 6H), 7.01 (s, 1H), 6.93 (d, J = 15.9 Hz, 1H), 6.45–6.27 (m, 1H), 4.84 (d, J = 13.4 Hz, 1H), 4.62 (d, J = 13.3 Hz, 1H), 4.17–4.07 (m, 1H), 4.07–3.92 (m, 1H), 2.86 (s, 3H), 1.34 (s, 9H); HRMS (ESI) m/z calcd for C23H28NO [M+H]+ 334.2171, found 334.2172. (E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-(p-tolyl)prop-2-en-1-amine hydrochloride (5o). Yield: 67%. 5o was synthesized by the general procedure of 1 given above as a white solid, m.p. 180–182 ºC. 1H-NMR (400 MHz, MeOD) δ 7.93 (s, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.51 (d, J = 7.3 Hz, 1H), 7.41 (t, J = 10.2 Hz, 3H), 7.22 (d, J = 7.3 Hz, 2H), 7.01 (s, 1H), 6.92 (d, J = 15.6 Hz, 1H), 6.41–6.28 (m, 1H), 4.83 (d, J = 13.5 Hz, 1H), 4.62 (d, J = 13.1 Hz, 1H), 4.18–4.07 (m, 1H), 4.04–3.89 (m, 1H), 2.86 (s, 3H), 2.37 (s, 3H); HRMS (ESI) m/z calcd for C20H22NO [M+H]+ 292.1701, found 292.1707. (E)-N-(Benzofuran-7-ylmethyl)-3-(2-fluorophenyl)-N-methylprop-2-en-1-amine hydrochloride (5p). Yield: 53%. 5p was synthesized by the general procedure of 1 given above as a yellow oil. 1H-NMR (400 MHz, MeOD) δ 7.93 (s, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.64 (dd, J = 17.1, 9.5 Hz, 1H), 7.53 (d, J = 7.3 Hz, 1H), 7.39 (dd, J = 14.8, 7.3 Hz, 2H), 7.23 (t, J = 7.5 Hz, 1H), 7.12 (dd, J = 19.1, 12.9 Hz, 2H), 7.04–6.94 (m, 1H), 6.60– 6.47 (m, 1H), 4.84 (d, J = 13.4 Hz, 1H), 4.65 (d, J = 13.2 Hz, 1H), 4.22–4.10 (m, 1H), 4.08–3.98 (m, 1H), 2.91 (s, 3H); HRMS (ESI) m/z calcd for C19H19FNO [M+H]+ 296.1451, found 296.1452. (E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-(2-nitrophenyl)prop-2-en-1-amine hydrochloride (5q). Yield: 74%. 5q was synthesized by the general procedure of 1 given above as a white solid, m.p. 44–46 ºC. 1H-NMR (400 MHz, MeOD) δ 8.06 (d, J = 8.2 Hz, 1H), 7.94 (s, 1H), 7.85–7.71 (m, 3H), 7.66–7.51 (m, 2H), 7.40 (dd, J = 15.2, 7.2 Hz, 2H), 7.01 (s, 1H), 6.50–6.27 (m, 1H), 4.87 (d, J = 13.3 Hz, 1H), 4.68 (d, J = 13.3 Hz, 1H), 4.25–4.15 (m, 1H), 4.14–3.99 (m, 1H), 2.95 (s, 3H); HRMS (ESI) m/z calcd for C19H19N2O3 [M+H]+ 323.1396, found 323.1393.


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(E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-(m-tolyl)prop-2-en-1-amine hydrochloride (5r). Yield: 50%. 5r was synthesized by the general procedure of 1 given above as a white solid, m.p. 161–162 ºC. 1H NMR (400 MHz, MeOD) δ 7.94 (s, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.51 (d, J = 7.4 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.38–7.23 (m, 3H), 7.19 (d, J = 7.4 Hz, 1H), 7.01 (s, 1H), 6.93 (d, J = 15.8 Hz, 1H), 6.39 (dt, J = 15.5, 7.6 Hz, 1H), 4.84 (d, J = 13.2 Hz, 1H), 4.63 (d, J = 12.6 Hz, 1H), 4.11 (d, J = 6.7 Hz, 1H), 4.00 (d, J = 7.7 Hz, 1H), 2.90 (s, 3H), 2.38 (s, 3H). HRMS (ESI) m/z calcd for C20H22NO [M+H]+ 292.1701, found 292.1700. (E)-N-(Benzofuran-7-ylmethyl)-3-(3-fluorophenyl)-N-methylprop-2-en-1-amine hydrochloride (5s). Yield: 49%. 5s was synthesized by the general procedure of 1 given above as a white solid, m.p. 130 ºC. 1H NMR (400 MHz, MeOD) δ 7.94 (s, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.52 (d, J = 7.4 Hz, 1H), 7.40 (dd, J = 16.9, 9.6 Hz, 2H), 7.33 (t, J = 9.3 Hz, 2H), 7.11 (t, J = 8.3 Hz, 1H), 7.01 (s, 1H), 6.95 (d, J = 15.8 Hz, 1H), 6.53–6.40 (m, 1H), 4.84 (d, J = 13.4 Hz, 1H), 4.65 (d, J = 13.0 Hz, 1H), 4.13 (d, J = 6.0 Hz, 1H), 4.02 (d, J = 7.5 Hz, 1H), 2.91 (s, 3H). HRMS (ESI) m/z calcd for C19H19FNO [M+H]+ 296.1451, found 296.1453. (E)-N-(Benzofuran-7-ylmethyl)-N-methyl-3-(3-nitrophenyl)prop-2-en-1-amine hydrochloride (5t). Yield: 55%. 5t was synthesized by the general procedure of 1 given above as a yellow oil. 1H NMR (400 MHz, MeOD) δ 8.38 (s, 1H), 8.20 (d, J = 8.2 Hz, 1H), 7.96–7.88 (m, 2H), 7.82 (dd, J = 20.2, 4.8 Hz, 1H), 7.63 (dd, J = 14.8, 6.8 Hz, 1H), 7.54 (d, J = 7.3 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.05 (d, J = 15.8 Hz, 1H), 6.97 (t, J = 9.5 Hz, 1H), 6.69–6.53 (m, 1H), 4.85 (d, J = 13.3 Hz, 1H), 4.66 (d, J = 13.3 Hz, 1H), 4.20–4.12 (m, 1H), 4.08–4.01 (m, 1H), 2.92 (s, 3H); HRMS (ESI) m/z calcd for C19H19N2O3 [M+H]+ 323.1396, found 323.1396. (E)-N-(Benzofuran-7-ylmethyl)-3-(2,4-dichlorophenyl)-N-methylprop-2-en-1-ami ne hydrochloride (5u). Yield: 57%. 5u was synthesized by the general procedure of 1 given above as a white solid, m.p. 49–51 ºC. 1H-NMR (400 MHz, MeOD) δ 7.90 (s, 1H), 7.78 (d, J = 7.5 Hz, 1H), 7.69 (d, J = 7.7 Hz, 1H), 7.50 (s, 2H), 7.36 (d, J = 7.2 Hz, 2H), 7.24 (d, J = 15.9 Hz, 1H), 6.97 (s, 1H), 6.54–6.33 (m, 1H), 4.80 (d, J = 12.8 Hz,, 1H), 4.63 (d, J



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= 12.8 Hz, 1H), 4.17–3.95 (m, 2H), 2.81 (s, 3H); HRMS (ESI) m/z calcd for C19H18Cl2NO [M+H]+ 346.0765, found 346.0750. 1-(2,2-Diethoxyethoxy)-2-iodobenzene (6). To a solution of 2-iodophenol (30.0 g, 136 mmol) in DMF (200 mL) was slowly treated with sodium hydride (6.0 g, 250 mmol), and then the reaction was stirred at room temperature for 30 min. Thereafter, 1-bromo-2,2-diethoxyethane (31.0 mL, 205 mmol) was added dropwise and the reaction was heated to 90 ºC overnight. After cooling to room temperature, the mixture was partitioned between EtOAc and water. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue was then purified via flash chromatography on silica gel, eluting with EtOAc/petroleum ether (1/25, v/v) to give 6 as a clear colorless oil. Yield: 93%. 1H-NMR (400 MHz, CDCl3) δ 7.77 (dd, J = 7.8, 1.4 Hz, 1H), 7.29 (dd, J = 11.7, 4.4 Hz, 1H), 6.86–6.78 (m, 1H), 6.72 (td, J = 7.7, 1.2 Hz, 1H), 4.90 (t, J = 5.2 Hz, 1H), 4.04 (d, J = 5.2 Hz, 2H), 3.82 (tt, J = 14.1, 7.1 Hz, 2H), 3.77–3.69 (m, 2H), 1.28–1.23 (m, 6H). 7-Iodobenzofuran (7). A stirred mixture of polyphosphoric acid (85.0 g) and 6 (40.0 g, 119 mmol) in toluene (200 mL) was heated at reflux overnight. The reaction mixture was allowed to cool to room temperature, and 1 M aq NaOH (20 mL) was added. The mixture was partitioned between EtOAc and water. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue was then purified via flash chromatography on silica gel, eluting with petroleum ether to give 7 as a clear colorless oil. Yield: 55%. 1H-NMR (400 MHz, Acetone) δ 7.95 (s, 1H), 7.70 (dd, J = 17.8, 7.5 Hz, 2H), 7.09 (d, J = 9.0 Hz, 2H). Benzofuran-7-carbonitrile (8). A solution of 7 (17.0 g, 70 mmol) and copper (I) cyanide (12.5 g, 139 mmol) in DMF (150 mL) was heated at reflux overnight. The reaction was then cooled and poured into water with plenty of concentrated ammonium hydroxide until the mixture was clear. Thereafter, the mixture was partitioned between EtOAc and water. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The residue was then purified via flash chromatography on silica gel, eluting EtOAc/petroleum ether (1/25, v/v) to give 8 as a white solid. Yield: 90%. 1H-NMR (400 MHz, CDCl3) δ 7.84 (t, J = 11.2 Hz, 1H), 7.77 (s, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.33 (t, J = 7.7 Hz, 1H), 6.88 (s, 1H).


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Benzofuran-7-ylmethanamine (9). To a solution of 8 (9.0 g, 63 mmol) in tetrahydrofuran (100 mL) at -78 °C under a N2 atmosphere was treated dropwise with a suspension of lithium aluminum hydride (4.8 g, 126 mmol) in tetrahydrofuran (100 mL). The mixture was stirred at room temperature overnight and then carefully treated in succession with water (5 ml), 15% sodium hydroxide (5 mL) and water (5 mL). The mixture was dried over anhydrous Na2SO4, filtered through celite, and the filter cake was washed with tetrahydrofuran. Ultimately, the filtrate concentrated under reduced pressure to give 9 as a yellow oil. Yield: 97%. 1H-NMR (400 MHz, MeOD) δ 7.77 (d, J = 1.9 Hz, 1H), 7.40 (t, J = 8.6 Hz, 1H), 7.29 (t, J = 7.7 Hz, 1H), 7.24 (d, J = 7.3 Hz, 1H), 7.00 (s, 1H), 4.05 (s, 2H). tert-Butyl (benzofuran-7-ylmethyl)carbamate (10). To a solution of 9 (9.0 g, 61 mmol) in tetrahydrofuran (100 mL) was added sodium hydroxide (4.6 g, 122 mmol) and stirred for 5 min. After the addition of di-tert-butyl dicarbonate (21.1 mL, 92 mmol) in tetrahydrofuran (100 mL), the reaction mixture was stirred for another 1 h and then filtered directly. The filter cake was washed with tetrahydrofuran. The residue was then purified via flash chromatography on silica gel, eluting with EtOAc/petroleum ether (1/25, v/v) to give 10 as a pale yellow solid. Yield: 83%. 1H-NMR (400 MHz, CDCl3) δ 7.64 (d, J = 1.6 Hz, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.37–7.10 (m, 2H), 6.78 (d, J = 2.1 Hz, 1H), 4.63 (s, 2H), 1.47 (s, 9H). 1-(Benzofuran-7-yl)-N-methylmethanamine (11). To a solution of 10 (12.6 g, 51 mmol) in tetrahydrofuran (100 mL) at 0 °C under a N2 atmosphere was treated dropwise with a suspension of lithium aluminum hydride (7.7 g, 204 mmol) in tetrahydrofuran (50 mL). The reaction mixture was stirred at reflux overnight and then carefully treated in succession with water (8 mL), 15% sodium hydroxide (8 mL) and water (8 mL). The mixture was dried over anhydrous Na2SO4 and filtered through celite, and the filter cake was washed with tetrahydrofuran. Ultimately, the filtrate was concentrated under reduced pressure to give 11 as a yellow oil. Yield: 95%. 1H-NMR (400 MHz, CDCl3) δ 7.63 (d, J = 2.0 Hz, 1H), 7.51 (dt, J = 10.5, 5.2 Hz, 1H), 7.32–7.14 (m, 2H), 6.78 (d, J = 2.1 Hz, 1H), 4.07 (s, 2H), 2.48 (s, 3H). (E)-3-Phenylprop-2-en-1-ol (12a). To a solution of cinnamaldehyde (264.0 mg, 2 mmol) in methanol (10 mL) was treated with sodium borohydride (76.0 mg, 2 mmol) in batches at 0 ºC. The reaction



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mixture was stirred at room temperature for 15 min and concentrated. The residue was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The crude was used for the next step without further separation to give 12a as a yellow oil. (E)-(3-Bromoprop-1-en-1-yl)benzene(13a). To a solution of 12a (357.0 mg, 2.7 mmol) in anhydrous ether (20 mL) was treated with phosphorus tribromide (84.0 µL, 0.9 mmol) at 0 ºC under a N2 atmosphere。 The reaction mixture was stirred at room temperature overnight and poured into ice water containing sodium bicarbonate. The mixture was partitioned between EtOAc and water. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed at 30 ºC. The crude was used for the next step without further purification to afford 13a as a white solid. Yield: 85%. 1H-NMR (400 MHz, CDCl3) δ 7.42–7.36 (m, 2H), 7.33 (t, J = 7.5 Hz, 2H), 7.27 (t, J = 5.2 Hz, 1H), 6.65 (d, J = 15.6 Hz, 1H), 6.40 (dt, J = 15.6, 7.8 Hz, 1H), 4.16 (d, J = 7.8 Hz, 2H). 1-Bromo-3-(2,2-diethoxyethoxy)benzene (14). Compound 14 was synthesized by the general procedure of compound 6 given above as a clear colorless oil. Yield: 95%. 1H-NMR (400 MHz, CDCl3) δ 7.07–7.14 (m, 3H), 6.86–6.78 (m, 1H), 4.82 (t, J = 5.2 Hz, 1H), 3.98 (d, J = 5.2 Hz, 2H), 3.72– 3.80 (m, 2H), 3.57–3.70 (m, 2H), 1.5 (t, J = 7.8 Hz, 6H). 4-Bromobenzofuran (15). Compound 15 was synthesized by the general procedure of compound 7 given above. Additionally, another product (6-bromobenzofuran) was synthesized at the same time. The crude mixture was used directly for next step without separation. Benzofuran-4-carbonitrile (16). Compound 16 was synthesized by the general procedure of compound 8 given above. The crude was purified via flash chromatography on silica gel, eluting with ethyl acetate/petroleum ether (1/25, v/v) to give 16 as a white solid. Yield: 30% for two steps. 1H-NMR (400 MHz, CDCl3) δ 7.85 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 2.1 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.33 (t, J = 7.7 Hz, 1H), 6.88 (d, J = 2.2 Hz, 1H). Benzofuran-4-ylmethanamine (17). Compound 17 was synthesized by the general procedure given above using 9. The crude mixture was used directly for next step without separation. Yield: 94%.


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1-(Benzofuran-4-yl)-N-methylmethanamine (18). Compound 18 was synthesized by the general procedure of compound 11 given above. Yield: 70% for 2 steps.1H-NMR (400 MHz, CDCl3) δ 7.64 (d, J = 2.3 Hz, 1H), 7.44 (d, J = 8 Hz, 1H), 7.24(t, J = 2.1 Hz, 1H), 7.16–7.18 (m, 1H), 6.89–6.88 (m, 1H), 3.99 (s, 2H), 2.47(s, 3H). (E)-Ethyl 4-phenylbut-3-enoate (19a). To a solution of trans-styrylacetic acid (0.973 g, 6.2 mmol) in ethanol (10 mL) was added thionyl chloride (893.0 µL, 13.3 mmol) dropwise. The reaction mixture was heated at reflux for 2 h and concentrated under reduced pressure. The residue was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and condensed. The crude was used for next step without further separation to give 19a as a clear colorless oil. Yield: 98%. 1H-NMR (400 MHz, CDCl3) δ 7.36 (t, J = 8.3 Hz, 2H), 7.31 (t, J = 7.5 Hz, 2H), 7.24 (dd, J = 13.2, 6.1 Hz, 1H), 6.49 (d, J = 15.9 Hz, 1H), 6.38–6.25 (m, 1H), 4.17 (q, J = 7.1 Hz, 2H), 3.24 (d, J = 7.0 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H). (E)-4-Phenylbut-3-en-1-ol (20a). To a solution of 19a (551.0 mg, 2.9 mmol) in anhydrous tetrahydrofuran (25 mL) at 0 °C under a N2 atmosphere was treated dropwise with a solution of diisobutylaluminium hydride (3.0 mL, 1.5 M in toluene). The reaction was stirred at room temperature overnight and then carefully quenched with methanol. The mixture was filtered through celite and condensed. The residue was then purified via flash chromatography on silica gel, eluting with EtOAc/petroleum ether (1/5, v/v) to give 20a as a clear colorless oil. Yield: 71%. 1H-NMR (400 MHz, CDCl3) δ 7.40–7.19 (m, 5H), 6.50 (d, J = 15.9 Hz, 1H), 6.26–6.14 (m, 1H), 3.80–3.71 (m, 2H), 2.49 (q, J = 6.4 Hz, 2H). (E)-(4-Bromobut-1-en-1-yl)benzene (21a). Compound 21a was synthesized by the general procedure of compound 13a given above as a yellow oil. Yield: 57%. 1H-NMR (400 MHz, CDCl3) δ 7.41 (d, J = 7.5 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.26 (t, J = 7.1 Hz, 1H), 6.51 (d, J = 15.7 Hz, 1H), 6.20 (td, J = 7.0 Hz, J = 15.7 Hz, 1H), 3.51 (t, J = 7.2 Hz, 2H), 2.81 (q, J = 7.1 Hz, 2H) Determination of water solubility. The water solubilities of NTF, 5g, 5j, 5k, 5m and 5u were determined by an HPLC method. Stock solutions (800 µg/mL) of the samples were prepared in



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methanol. Then, 10 µL dilute solutions with concentrations of 50, 100, 200, 400 and 800 µg/mL were injected into the HPLC system to assess linearity. Calibration curves were plotted as peak area versus concentration of the sample. Next, 10 mg of the sample was added into a 5 mL centrifuge tube, and 1 mL pure water was pipetted into the tube. If the solution was unsaturated and remained clear and transparent, more testing of the compound was needed. After stirring for 24 h, the solution was filtered with a syringe, and the HPLC system was injected with the same 10 µL. Water solubility was calculated by comparing the peak area of the tested compound to the calibration curves 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. Then, 3 mL bacteria cultures were centrifuged and washed twice with PBS. The pigment was extracted with methanol. The OD was determined at 450 nm using a NanoDrop 2000c (Thermo scientific) spectrophotometer. IC50 values were obtained by fitting the OD data to a normal dose-response curve using the Graphpad Prism 5.0 software. IC50 values of the MRSA strains USA300 LAC, USA400 MW and Mu 50 were conducted in the same way. High-performance liquid chromatography (HPLC). Strains were cultured overnight in tubes and diluted in 50 mL of TSB fresh medium (100 µM naftifine hydrochloride or 1 µM of 5m was used where appropriate, as indicated). Cells were further cultured with shaking at 37 °C for 24 h and harvested by centrifugation. Briefly, wet cells harvested from 50 mL of cultures were extracted with 20 mL of acetone. To this, 10 mL of hexane and 10 mL of aqueous NaCl (10%, wt/vol) were added, and the mixture was shaken vigorously to remove the oily lipids. The upper phase containing the carotenoids was dried with anhydrous MgSO4 and concentrated in a rotary evaporator. Finally, a 300-µL acetonitrile/isopropanol mixture (85:15, v/v) was used to dissolve the extracts. The dissolved extracts were filtered, and 50 µL samples were analyzed through a Spherisorb ODS2 column (250 × 4.6 mm; particle diameter, 5 µm; Waters) and eluted with an acetonitrile-isopropanol mixture (85:15, v/v) at a flow rate of 1 mL/min using an Alliance high-pressure liquid


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chromatography (HPLC) system (Agilent 1260 Infinity) equipped with a photodiode array detector. CrtN enzyme inhibition assay. The expression of CrtN and the inhibition assay were performed using our previously reported methods.19 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 phosphatidylcholine (Sigma-Aldrich) in 200 µL CHCl3 and mixed with 8 mg diapophytoene. The mixture was spun drying and incubated with 2 mL 0.02 M HEPES buffer (pH 7.5), then sonicated in ice water to obtain a homogeneous emulsion and stored at -80 °C until use. E. coli BL21 (DE3)/pET28a::crtN was sub-cultured into 1,000 mL of LB broth supplemented with 50 µg/mL kanamycin to obtain an optical density at 600 nm (OD600) of approximately 0.1 and grown to an OD600

of

~0.5.

Expression

of

6His-CrtN

was

induced

with

0.5

mM

isopropyl-β-D-thiogalactoside (IPTG) overnight at 16 °C. Cells were harvested, and the pellets were suspended in 30 mL HEPES buffer and lysed at 4 °C by sonication. Enzyme activity was performed in triplicate, with a total of 700 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, estimated by Western blot using known concentration of the purified 6His-crtN protein). The tests were performed under 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 initiated by adding the lysate and incubated overnight at 37 ºC. The reaction was terminated by methanol. Pigments were extracted twice against 700 µL chloroform. The organic phase was combined, concentrated, re-dissolved in 200 µL chloroform, and analyzed by reading OD value under 450 nm. IC50 values were obtained by fitting the OD data to a normal dose-response curve using the Graphpad Prism 5.0 software. Bacterial growth assays of S. aureus Newman and MRSA strains. 5m was dissolved in DMSO to 20 mM as a stock concentration and diluted with fresh TSB medium enabling the final concentration of 5m of either 0.2 mM or 0.05 mM. 100 µL of the dilution was distributed into 96-well plates and growth controls (containing an equal amount of DMSO). A 60-µL paraffin wax was used to cover the



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dilutions to avoid the evaporation of the medium. The dilutions were plated at 37 ºC for 4 h to dissolve the compounds sufficiently. Four S. aureus strains, including Newman, USA300 LAC, 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 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 the H2O2 killing assay, four strains, including Newman, USA300 LAC, USA400 WM2 and Mu50, were cultured in TSB for 24 h with or without compound 5m (40 µL). 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 with 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 to determine the CFU. Regarding 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 a 40 µL bacterial sample, which resulted in 1 × 106 CFU/mL. The tubes were incubated at 37 °C for 6 h, and then dilutions were plated on a TSA agar plate to determine the surviving CFU. S. aureus systemic infection models. The 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 of 22 or 5m. For this, 76 mg of the compounds were dissolved in sterile water with a concentration of 2 mg/mL, and 8 mL of them were further diluted to 0.5 mg/mL. Aliquots of 200 µL (2 mg/mL) or 200 µL (0.5 mg/mL) of the compounds were intraperitoneal injected once and in 12 h intervals for 108 h (4.5 d) to reach a total dose of 200 mg/kg or 50 mg/kg (the average weight of the mouse used for the study was 18 g). As a negative control, the same volume of sterile water was injected. Twelve hours after the first injection of the inhibitor, the


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mice were challenged with three strains. For the mouse model of abscess formation, mice were challenged with 100 µL of 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 murine were sacrificed 5 days post infection. Kidneys, hearts or 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 onto TSA (Difco) plates, and CFUs were counted after overnight incubation at 37 °C. The statistical significance was determined by the Mann-Whitney test (two-tailed). Anti-fungal assays. In vitro antifungal activity was measured 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 compounds were dissolved in DMSO serially diluted in growth medium. The dermatophytes were incubated at 28 °C. Growth MIC was determined at 7 days for filamentous fungi. Pharmacokinetic Studies. Compounds NTF and 5m were tested in pharmacokinetic studies on Sprague−Dawley (male) rats that were obtained from Shanghai Sippr-BK Laboratory Animal Co. Ltd. The animals were housed in a room with controlled temperature and humidity and allowed free access to food and water. For studying both compounds, the rats were divided into 2 groups (3 rats/group), and each group received the compounds intravenously at a dose of 5 mg/kg or orally at a dose of 10 mg/kg, respectively. Blood samples of the orally administered rats were collected at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h postdose, and blood samples of the intravenously injected rats were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h postdose. All blood samples were centrifuged at 8000 rpm and 4 °C for 6 min. The obtained plasma was collected and stored at −80 °C. All plasma samples were analyzed within 1 week after



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Page 32 of 49

collection. Plasma concentration-time data were analyzed, and the pharmacokinetic parameters were calculated. ASSOCIATED CONTENT Supporting Information. HPLC reports to establish the purity of the analogs 1-2, 3a-c, 4a-f, and 5a-u, and the methods of the cell proliferation assay and the hERG inhibition assay. 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;

E-mail,

[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 for this research provided by the National Natural Science Foundation of China (Grants 21222211, 21372001 and 21472207), the “Shu Guang” project supported by the Shanghai Municipal Education Commission and Shanghai Education Development Foundation (Grant 14SG28), the Program for New Century Excellent 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; ND4BB, New Drugs for Bad Bugs; CrtM, dehydrosqualene synthase; CrtN, diapophytoene desaturases; ROS, reactive oxygen species; PK, pharmacokinetics; SAR, structure-activity relationship;


Page 33 of 49

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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.



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REFERENCES (1) O'Connell, K. M.; Hodgkinson, J. T.; Sore, H. F.; Welch, M.; Salmond, G. P.; Spring, D. R. Combating multidrug-resistant bacteria: current strategies for the discovery of novel antibacterials. Angew. Chem. Int. Edit. 2013, 52, 10706–10733. (2) 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. (3) Antibiotic

resistance:

the

global

threat

(U.S.).

http://stacks.cdc.gov/view/cdc/31340, National Center for Emerging and Zoonotic Infectious Diseases (U.S.): February 27, 2015. (4) Rex, J. H. ND4BB: addressing the antimicrobial resistance crisis. Nat. Rev. Microbiol. 2014, 12, 231–232. (5) National

action

plan

for

combating

antibotic-resistant

bacteria.

www.whitehouse.gov/sites/default/files/docs/national_action_plan_for_combating _antibotic-resistant_bacteria.pdf , White House: March. 2015. (6) Antimicrobial

resistance

global

report

on

surveillance

http://www.who.int/drugresistance/documents/surveillancereport/en/,

2014. World

Health Organzition: April, 2014. (7) Cascioferro, S.; Cusimano, M. G.; Schillaci, D. Antiadhesion agents against Gram-positive pathogens. Future Microbiol. 2014, 9, 1209–1220. (8) Sommer, R.; Joachim, I.; Wagner, S.; Titz, A. New approaches to control infections: anti-biofilm strategies against gram-negative bacteria. Chimia 2013, 67, 286–290. (9) Liu, G. Y.; Nizet, V. Color me bad: microbial pigments as virulence factors. Trends Microbiol. 2009, 17, 406–413. (10) 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.


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(11) Spaan, A. N.; Surewaard, B. G.; Nijland, R.; van Strijp, J. A. Neutrophils versus Staphylococcus aureus: a biological tug of war. Annu. Rev. Microbial. 2013, 67, 629–650. (12) Rigby, K. M.; DeLeo, F. R. Neutrophils in innate host defense against Staphylococcus aureus infections. Semin. Immunopathol. 2012, 34, 237–259. (13) Rasko,

D.

A.;

Sperandio,

V.

Anti-virulence

strategies

to

combat

bacteria-mediated disease. Nat. Rev. Drug Discov. 2010, 9, 117–128. (14) Oldfield, E.; Feng, X. Resistance-resistant antibiotics. Trends Pharmacol. Sci. 2014, 35, 664–674. (15) 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. (16) Song,Y.; Lin, F. Y.; Yin, F.; Hensler, M.; Rodrı´gues Poveda, C. A.; Mukkamala, D.; Cao, R.; Wang, H.; Morita, C. T.; Gonza´lez Pacanowska, D.; Nizet, V.; Oldfield,

E.

Phosphonosulfonates

are

potent,

selective

inhibitors

of

dehydrosqualene synthase and staphyloxanthin biosynthesis in Staphylococcus aureus. J. Med. Chem. 2009, 52, 976–988. (17) Song, Y.; Liu, C. I.; Lin, F. Y.; No, J. H.; Hensler, M.; Liu, Y. L.; Jeng, W. Y.; Low, J.; Liu, G. Y.; Nizet, V.; Wang, A. H. J.; Oldfield, E. Inhibition of staphyloxanthin virulence factor biosynthesis in Staphylococcus aureus: in vitro, in vivo, and crystallographic results. J. Med. Chem. 2009, 52, 3869–3880. (18) Zhang, Y.; Lin, F. Y.; Li, K.; Zhu, W.; Liu, Y. L.; Cao, R.; Pang, R.; Lee, E.; Axelson, J.; Hensler, M.; Wang, K.; Molohon, K. J.; Wang, Y.; Mitchell, D. A.; Nizet, V.; Oldfield, E. HIV-1 integrase inhibitor-inspired antibacterials targeting isoprenoid biosynthesis. ACS Med. Chem. Lett. 2012, 3, 402−406. (19) 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 diapophytoene desaturase inhibits S. aureus virulence. Nat. Chem. Bio. 2016, 12, 174−179.



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(20) Hammond, R. K.; White, D. C. Inhibition of vitamin K2 and carotenoid synthesis in Staphylococcus aureus by diphenylamine. J. bacteriol. 1970, 103, 611−615. (21) Raisig, A.; Sandmann, G. Functional properties of diapophytoene and related desaturases of C(30) and C(40) carotenoid biosynthetic pathways. Biochim. Biophys. Acta. 2001, 1533, 164−170. (22) Hann, M. M.; Oprea, T. I. Pursuing the leadlikeness concept in pharmaceutical research. Curr. Opin. Chem. Biol. 2004, 8, 255−263.


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FIGURES Figure 1. Structures of 22 and naftifine hydrochloride (NTF).

Figure 2. Scaffold hopping (1-2) and three chemical modification regions (3-5, A-C) of the lead compound NTF. 5a-u C

3a-c A N

N .HCl

O

O 1

N .HCl

B 4a-f

Scaffold pigment inhibition: IC50=247.3 18.8nM Hopping N .HCl

Naftifine hydrochloride (NTF) pigment inhibition: IC50=296.0 12.2 nM O

2

pigment inhibition: IC50=758.7 24.3 nM

Figure 3. 5m 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), naftifine-treated wild-type S. aureus Newman (F) and 5m-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.



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. The dose-response curves and IC50 values of 5m in the pigment formation of S. aureus USA400 MW2 (A), USA300 LAC (B), and Mu50 (C). Data are presented as the means ± SEM, n = 3 independent experiments.

Figure 5. The growth curve and effect of 5m on the bacterial growth of S. aureus Newman (A), USA400 MW2 (B), USA300 LAC (C), and Mu50 (D).

Figure 6. Effect of 5m on the susceptibility to hydrogen peroxide killing. S. aureus Newman (A), USA400 MW2 (B), USA300 LAC (C), and Mu50 (D); **p< 0.01***p < 0.001 via two-tailed t-test (n = three biological replicates, each with two technical replicates).


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Figure 7. Effect of 5m on the susceptibility to human whole blood killing. S. aureus Newman (A), USA400 MW2 (B), USA300 LAC (C), and Mu50 (D); *p< 0.05, **p< 0.01, ***p < 0.001 via two-tailed t-test (n = three biological replicates, each with two technical replicates).

Figure 8. Effect of 5m treatment on the survival of S. aureus Newman bacteria in the kidneys and hearts of mice (n = 13) challenged with 1×107 CFU S. aureus Newman bacteria. Statistical significance determined by the Mann-Whitney test (two-tailed): *p< 0.05, **p< 0.01. Each symbol represents the value for an individual mouse. Horizontal bars indicate the observation means, and dashed lines mark the limits of detection.



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Figure 9. Effect of 5m and 22 treatment on the survival of S. aureus bacteria in the livers (A) and kidneys (B) of mice (n = 13) challenged with 4×107 CFU USA400 MW2 bacteria. Statistical significance determined by the Mann-Whitney test (two-tailed): *p< 0.05, **p< 0.01, ***p1000

3c

isopropyl

>1000

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

Table 2. Chemical structures of 4a-f and their pigment inhibitory activities against S. aureus Newman. N O

Compd.

Linker (X)

X .HCl

S. aureus Newman



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Page 46 of 49

IC50 (nM)a

a

4a

>1000

4b

4.2±0.1

4c

>1000

4d

>1000

4e

164.3±15.4

4f

>1000


Table 3. Chemical structures of 5a-u and their pigment inhibitory activities against S. aureus Newman.

S. aureus Compd.

R1

Newman

S. aureus Compd.

IC50 (nM)a

R1

Newman IC50 (nM)a

5a

>1000

5l

71.7±2.5

5b

>1000

5m

4.0±0.2

5c

>1000

5n

46.7±3.2

5d

>1000

5o

35. 2±3.8


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a

5e

887.7±60.0

5p

288.3±18.2

5f

17. 1±1.5

5q

>1000

5g

11.2±1.1

5r

>1000

5h

16.9±1.5

5s

513. 0±11.1

5i

31.2±2.1

5t

>1000

5j

6.3±0.1

5u

8.3±0.3

5k

6.7±0.1


Table 4. Enzyme (CrtN IC50), pigment (S. aureus Newman, IC50) and water solubility results of NTF and its five representative analogs. CrtN

S. aureus Newman

Solubility

IC50 (nM)a

IC50 (nM)b

(mg/mL)c

NTF

8830.0±109.1

296.0±12.2

6.1

5g

683.7±68.1

11.08±1.06

19.7

5j

219.0±16.8

6.20±1.02

7.4

5k

355.1±26.1

6.44±1.02

3.9

5m

338.8±28.3

3.93±1.02

10.0

5u

740.2±55.6

8.21±1.03

7.2

Compd.



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

Page 48 of 49

The values given are the IC50 values against CrtN, in nM. bThe values given are the

IC50 values for pigment inhibition in S. aureus Newman，in nM. cThe values given are the solubility in water, in mg/mL. Table 5. Pharmacokinetic parameters for NTF and 5m after single dosing in ratsa. CLiv compd

species

Vss

AUC/dose

(L/kg)

((ng/(mL•h))/(mg/kg)

T1/2 (h) (L/hr/kg)

F (%)

NTF

rat

3.28

1.13

8.23

25.98

0.85

5m

rat

3.26

8.23

10.67

1299.47

42.2

a

IV dose=5 mg/kg, PO dose=10 mg/kg. CL: the total plasma clearance, T1/2: terminal

half-life (h), Vss: the volume of distribution at steady state, AUC/dose: the extrapolated area under the plasma concentration-time curve from zero to infinity, F (%): bioavailability after a single oral dosing relative to a single IV dosing corrected by doses and dose normalized AUC (AUC/dose). Table 6. Antifungal activity of 5m. 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

5m

16

32

>64


Page 49 of 49

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TOC 215x98mm (300 x 300 DPI)


CrtN - ACS Publications - American Chemical Society

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