Discovery of novel small molecule TLR4 inhibitors as potent anti-inflammatory agents

Accepted Manuscript Discovery of novel small molecule TLR4 inhibitors as potent anti-inflammatory agents Yao Xu, Shujun Chen, Ying Cao, Pingzheng Zhou, Zhipeng Chen, Kui Cheng PII:

S0223-5234(18)30446-X

DOI:

10.1016/j.ejmech.2018.05.033

Reference:

EJMECH 10444

To appear in:

European Journal of Medicinal Chemistry

Received Date: 14 February 2018 Revised Date:

13 May 2018

Accepted Date: 20 May 2018

Please cite this article as: Y. Xu, S. Chen, Y. Cao, P. Zhou, Z. Chen, K. Cheng, Discovery of novel small molecule TLR4 inhibitors as potent anti-inflammatory agents, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.05.033. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Discovery of novel small molecule TLR4 inhibitors as potent anti-inflammatory agents

Yao Xu†, Shujun Chen†, Ying Cao, Pingzheng Zhou, Zhipeng Chen, Kui

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Cheng*

Guangdong Provincial Key Laboratory of New Drug Screening and Guangzhou Key

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Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515,

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China.

Phone: +86 20 6164 7192, Fax: +86 20 6164 8533 Email address: [email protected]

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†These authors contributed equally to this work.

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ACCEPTED MANUSCRIPT Abstract: Toll-like receptor 4 (TLR4) initiates innate immune response to release inflammatory cytokines and has been pathologically linked to variety of inflammatory diseases.

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Recently, we found that Carvedilol, as the classic anti-heart failure and anti-inflammatory clinic drug, could inhibit the TLR4 signaling in the TLR4 overexpressed cells. Herein, we have designed and synthesized a small library of

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novel Carvedilol derivatives and investigated their potential inhibitory activity. The

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results indicate that the most potent compound 8a (SMU-XY3) could effectively inhibited TLR4 protein and the LPS triggered alkaline phosphatase signaling in HEK-Blue hTLR4 cells. It down regulated the nitric oxide (NO) in both RAW264.7 cells and BV-2 microglial cells, in addition to blocking the TNF-α signaling in ex-vivo

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human peripheral blood mononuclear cells (PBMC). More interestingly, 8a shows higher affinity to hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) over HCN2, which probably indicates the new application of TLR4 inhibitor 8a in heart

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failure, coronary heart disease, and other inflammatory diseases.

Keywords: Toll-like receptor 4; anti-inflammatory; Carvedilol derivative; Small molecule antagonist.

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ACCEPTED MANUSCRIPT 1. Introduction Toll-like receptors (TLRs) are a class of proteins which play a key role in the innate immune system. They regulate the properties, the intensity, and the duration of a range

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of anti-pathogenic microbial factors such as type I interferons, proinflammatory cytokines, and chemokines [1]. Meanwhile, both over and low expression of TLR can lead to physical dysfunction and diseases, in order to balance a proper level of

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activation; our body adopts a series of positive or negative molecular-level regulation

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strategies to regulate TLRs-mediated signal transduction pathways [2]. Currently, thirteen TLRs have been found in mammals among which ten subtypes are expressed in humans. TLR4, discovered in 1997, was the first TLR member that simultaneously existed with several co-receptors, such as MD-2, CD14, LBP and RP105 [3].

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According to their different positions, they can be divided into two subfamilies. The first sub-family contains TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10, which are distributed on the cell membrane surface, and principally identify lipids and proteins;

membrane,

and

mainly

recognize

nucleic

acids

as

well

as

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organelle

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the other sub-family contains TLR3, TLR7, TLR8 and TLR9, which are distributed on

pathogen-associated molecular patterns (PAMPs) [4]. The key-linker molecules Mal/MyD88 and TRAM/TRIF in TLR4 signal transduction pathway can be used as targets to selectively regulate their activity [5]. TLR4 combines with its ligand protein to identify endotoxin components of gram-negative bacteria, such as the natural agonist lipopolysaccharides (LPS) and so on, inducing the release of a series of proinflammatory cytokines and IFN-β as well as the infiltration of inflammatory cells 3

ACCEPTED MANUSCRIPT infiltrating into the site of infection, mediating the collective natural immune defenses [6]. TLR4-mediated signaling can activate two downstream pathways, MyD88 dependent access and independent access. Both of them can directly activate NF-κB

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pathway leading to the activation of downstream cytokines, such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) [7, 8]. Many cardiovascular-related diseases have a close relationship with TLR4, as high blood

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pressure can increase the activity of TLR4 expression [9]; upregulation of TLR4

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expression in cardiomyocytes can promote the inflammatory response and aggravate heart failure [10]. So the development of TLR4 inhibitors may have important implications for cardiovascular-related diseases. In recent years, with the unremitting efforts of scientists, some superior TLR4 small molecule modulators are gradually

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developed. For example, the cyclohexene derivative TAK-242 can block the TLR4 signaling pathway through its interaction with the TIR region of TLR4 [11]. We previously reported that T5342126 was also a small molecule TLR4 inhibitor,

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presumably functioning by blocking MD-2, TLR4 interaction mechanism [12]. AV411,

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as a TLR4 inhibitor, was initially developed for the treatment of bronchial asthma, cerebrovascular and ocular indications and was in phase II clinic trail with good drug property [13].

TLRs are widely distributed in many organs, however, the proportion of expression

is different. TLR4 is significantly more expressed in the heart when compared to other TLRs [14]. It is well reported that the expression of TLR4 increases during the emergency injury of heart. Ligand-binding capacity and pro-inflammatory function of 4

ACCEPTED MANUSCRIPT cardiomyocyte TLR4 are upregulated, which promote inflammation and acceleration of heart failure, as well as promote inflammatory cytokine production in both infarct and remote myocardium [15]. Inhibition of TLR4 signaling pathway can effectively

the recovery of the heart from acute injury.

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reduce the expression of TLR4-related inflammatory cytokines, and it is benefical in

Carvedilol, a listed classic drug, has good anti-heart failure activity and it is a

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non-selective third-generation beta-blocker without intrinsic sympathomimetic

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activity. Meanwhile, it has excellent effect on calcium channel blocking, proliferation of smooth muscle cells inhibition, anti-inflammatory action, anti-oxidation action and among others [16]. Until now, 4 subtypes HCN channels (HCN1-4) have been discovered. HCN1 and HCN3 are mainly expressed in nerve tissue, while HCN2 and

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HCN4 are widely distributed in tissues of the brain and heart, and HCN4 is the most abundant isoform in the sinoatrial node, and its relevance to mild or severe forms of arrhythmia [17, 18]. Therefore, the use of higher affinity HCN4 inhibitors for the

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treatment of heart related diseases is particularly important and direct. Previously, it

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was reported that Carvedilol analogue VK-II-86 could prevent stress-induced ventricular tachyarrhythmias (VTs) in mutant mice and improve the survival rate of patients, indicating that extending the length of Carvedilol carbon chain was benefical for the bioactivity [19]. In our research, we found that Carvedilol could block the HCN2 and 4 in the single cell assay (data not shown). Although this inhibitory effect exists, the effect is not ideal and the selectivity is not significant. Importantly, we found that the classic anti-heart failure drug, Carvedilol, was closely linked with 5

ACCEPTED MANUSCRIPT TLR4 and it could inhibit LPS triggered TLR4 signaling in this report. In view of above points, we hypothesized that inhibition of TLR4 may play an important role in influence the HCN channel subtype. To explore this hypothesis, also

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with the carbon chain extension resulting in mind (VK-II-86) and directed by the key fragment of Carvedilol and T5342126, we have designed and synthesized a small library of novel derivatives to explore the structure-activity relationship and we

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expected to find a better TLR4 inhibitory activity compound with a higher affinity for

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HCN channel subtypes. After exploring the dominant groups of Carvedilol, we have introduced previously reported β-amino alcohol-based compounds as the backbone [20]. A series of compounds with TLR4 and HCN channel inhibitory activity were investigated. The best compound 8a (SMU-XY3), with the most potent TLR4

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inhibition activity, possesses inhibitory activity to the LPS triggered downstream inflammatory cytokines, such as TNF-α and NO both in murine and human cell lines, as well as downregulated the TLR4 protein in a dose-dependent manner. In addition,

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we also explore the selectivity of their HCN channel subtypes by measuring the

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affinity of HCN channels, and 8a has a better affinity for HCN4 than HCN2. Taken together, these results suggest that the newly developed novel TLR4 inhibitor possesses selectively HCN4 channel inhibition over HCN2, and it has great potential for the treatment of heart failure and inflammatory heart disease.

2. Results and discussion 2.1 Chemistry 6

ACCEPTED MANUSCRIPT The synthesis of compounds and basic synthetic steps are depicted in Scheme 1. Epoxide 1a and 1b were synthesized by reaction of mCPBA and the corresponding alkene. The epoxide reacts with the phenol derivative to generate the benzene epoxide

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2a-g under alkaline condition. Arylalkoxyoxiranes were synthesized via the reaction of the substituted phenols and epoxides as above (3a-d) (Scheme 1). Mannish

reaction

was

utilized

to

further

functionalize

the

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3,5-dimethyl-1H-pyrazole intermediates where paraformaldehyde provided the

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methylene source and methylamine provided the active hydrogen (4a-d). 4-hydroxy carbazole reaction with the corresponding epoxide gave carbazole epoxides of varying carbon chain lengths (5a-b). Both the electronic and steric hindrance effects often have a large effect on the activity of the compound. For this reason, we have selected

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some groups that have strong electron effects (such as fluorine), and some groups that have large steric hindrance, such as benzene rings, carbazole groups, and tert-butyl groups. Coupling 2a-d with the 2-methoxyphenoxy ethanamine of Carvedilol in

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ethanol yield compounds 6a-b, while there wasn’t any TLR4 inhibitory activity been

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observed (Table 1). Same method was used in the synthesis of derivatives of T5342126 (7a-j). Carbazole structure introduced into Carvedilol was coupled with β-aminoalcohol of different substituents (8a-f); 8a showed good TLR4 SEAP signaling inhibitory activity (Table 1). Carvedilol (9a) and the reported Carvedilol derivative 9b (VK-II-86) were synthesized to site by site evaluation the TLR4 activity. Finally, carbazole structure of carvedilol was then used as the basic skeleton and some bulky groups were introduced to investigate the hindering effect (10a-g) (Scheme 2). 7

ACCEPTED MANUSCRIPT 2.2. Biological activity. 2.2.1 In vitro TLR4 inhibition assay. In vitro assay was used to screen all the newly synthesized compounds for their

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effects on HEK-Blue hTLR4 cell lines. This cell line was obtained by co-transfection of the human TLR4, MD-2 and CD14 co-receptor genes, and an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene into HEK293 cells [21]. TLR4

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specific ligand LPS can activate the NF-κB signaling pathway to induce the SEAP

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signaling in HEK-Blue hTLR4 cells [22]. The compounds were prepared in 10 mM stock solutions by sterile DMSO and then diluted it in culture medium to the desired concentration during the cell culture.

Initially, this project was inspired by the inhibitory effect of carvedilol (9a) on

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TLR4 with IC50 of 33.22 µM. Following study observed its linker carbon extension analog 9b (VK-II-86), which also indicated similar bioactivity against TLR4. According to our previous reports, T5342126 (7a) was a potent TLR4 inhibitor. While

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extending the carbon chain of 7a the inhibitory activity was completely abolished (7a

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vs 7b, Table 1). The suppression of TLR4 production was recovered when ethoxyl group was substituted by fluoride or alkyl group (7c-g), suggesting that the electronic effect played a dominant effect in comparison to the carbon chain extension. Switching the chloride to methyl or relocating the chlorine to the para-position also can increase the bioactivity, which can further verify the substitution, compared to the carbon chain length, is critical for the TLR4 inhibition (7a vs 7i, 7j). Incorporating 2-phenoxyethanamine fragment of Carvedilol and aryl alkoxy oxiranes of T5342126, 8

ACCEPTED MANUSCRIPT we obtained compounds 6a-b, and the biological evaluation results revealed that the combination of these two fragments was not beneficial for the TLR4 activity (Table 1). Then, we turned our attention to the other part fragment of Carvedilol and T5342126,

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and we got compounds 8a-f for the SAR study. Generally, increasing the carbon chain length had no big influence for the activity (8a vs 8b). Replacing the fluoride by chloride was helpful to the TLR4 inhibition (8a vs 8e). Notably, removing one methyl

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group of the pyrazole ring significantly reduced the activity (8d vs 8f, Table 1), which

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reveals that the methyl group was essential for the in vitro activity. Last, the carbazole group of carvedilol is linked to other bulky groups to study the steric hindrance (10a-g). The results suggested that small substituents did not contribute to activity (10a, 10g), and large substituents retained varying effects (10b, 10c, 10d, 10f) except

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10e. Based on such results, the best compound 8a (IC50 =12.45±0.29 µM, Fig. 1) was selected out for further evaluation and compound 10a was chosen as a negative

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control in the following assay.

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2.2.2 8a Inhibition of LPS activated inflammatory cytokines, including NO signaling in RAW264.7 and BV-2 cell lines, as well as TNF-α in PBMC. Nitric oxide (NO) was a heteronuclear diatomic molecule that was of great

importance in physiological activities. The downstream targets of NO include granulate cyclase and NF-κB [23]. The NO content was determined using Greiss method [24]. LPS was employed to selectively activate TLR4 signaling, resulting in the activation of nitric oxide synthase and the production of NO in RAW 264.7 9

ACCEPTED MANUSCRIPT macrophage cells and microglial BV-2 cells. Inflammatory cytokines, especially TNF-α, are often overexpressed in heart failure patients and can cause cardiac hypertrophy by affecting myocardial contractility. In this experiment, we chose NO

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and TNF-α as an indicator of inflammatory cytokines. 8a has a significant dose-dependent inhibition to LPS (40 ng/mL) triggered NO signaling in Raw 264.7 cells (Fig 2A) and BV-2 cells (Fig 2B).

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Compound 10a was used as the negative control in the NO assay. Human

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peripheral blood mononuclear cells (PBMC) were employed to confirm that 8a inhibits the TLR4 mediated downstream inflammatory cytokine in ex vivo. In order to find the optimal dose of LPS, we tested the effect of different concentrations of LPS on the activation of TNF-α in PMBC cells, and finally found that 10 ng/mL possessed

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the best active effect in our system (Fig 2C). Next, we took 10 ng/mL LPS as a positive control, and added different doses of 8a to observe its inhibitory effect in PBMC. The test results showed that 8a inhibited about 80% TNF-α at 20 µM (Fig 2D).

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These data showed that 8a could inhibit the TLR4-related inflammatory cytokines

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such as NO, TNF-α both in murine and human cell lines.

2.2.3 Compound 8a targeting TLR4 to downregulate the protein expression in the Western blot analysis. Compound 8a was examined in HEK-Blue hTLR4 cells to determine its direct effect on the TLR4 protein by western blot analysis. As illustrated in Fig. 3A, LPS could significantly upregulate TLR4 about 4 folds at 40 ng/mL, and 8a inhibited 10

ACCEPTED MANUSCRIPT TLR4 in a concentration dependent manner. TLR4 protein was suppressed more than 90% by 8a at 10 µM (Fig. 3A). Viability assay showed that compound 8a did not have any significant toxicity at concentration up to 100 µM (Fig. 3B) in HEK-Blue hTLR4

2.2.4 Molecular docking studies of 8a (SMU-XY3).

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TNF-α above mentioned were not caused by the toxicity.

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cells, which further indicated the inhibition of TLR4 protein and SEAP, NO and

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The TLR4 agonist LPS binding to TLR4/MD-2 complex causes dimerization of the extracellular domains to form a TLR4/MD-2/LPS complex, a human complex crystal structure which has been solved by Park et al [25]. Previous experiments have demonstrated that 8a can inhibit LPS triggered downstream cytokines in vitro and ex

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vivo, as well as suppress the human TLR4 protein directly in the immune-precipitation assay. To examine possible explanations for the significantly TLR4 inhibition potency in human cells, we explored the predicted binding mode(s) of compound 8a with the

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TLR4/MD-2 complex by conducting molecular docking experiments in the human

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protein crystal complex (PDB 3FXI). We selected the most favorable energy configuration of 8a (shown in purple) and compared the binding of LPS (shown in red) bound to the complex (Fig. 4A). Of particular interest is the presence of a sub pocket between MD-2 and part of TLR4 in the LPS binding pocket. Computational docking studies showed that this critical site between MD-2 and TLR4 is occupied by the 3,5-dimethyl-pyrazole substituent (Fig. 4B). This result is consistent with the results of the 3,5-dimethyl-pyrazole ring derivative in cell experiments, and the activity 11

ACCEPTED MANUSCRIPT disappeared immediately after the deletion of a methyl group on this pyrazole ring (8a vs 8f). The benzyl ring of 8a is binding to the TLR4 pocket where the disaccharide group binds, while the carbazole group is well embedded in the binding pocket of LPS

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and MD-2 (Fig.4B) and the remainder of the compound is solvent exposed. These interactions suggest the greater potency of 8a in the TLR4 inhibitory activity.

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2.2.5 Electro physiological properties of 8a blockage on HCN channels

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In recent years, many reports have found that TLR4 was relevant to heart disease and blocking TLR4 could significantly reduce myocardial TNF-α, IL-1β, iNOS, and NF-κB activity [27]. Meanwhile, hyperpolarization-activated cyclic nucleotide gating (HCN) channels also involve a variety of heart diseases, like angina, heart failure and

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others [28]. To investigate whether TLR4 was associated with HCN channels, we investigated the effect of TLR4 inhibitor 8a on HCN channels. Two subtypes (HCN2 and HCN4) of HCN channels, which widely distribute in tissues of the brain and heart,

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were investigated in the study. Especially HCN4 is highly expressed in the

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myocardium [29].We first investigated the effects of 8a on currents conducted by HCN2 and HCN4 channels which were heterologous expressed in COS7 cells. Fig 5A and B show typical HCN channels currents traces elicited by a 2-s pulse applied to -120 mV and the effects on the amplitude of inward current (I/I0) were analyzed. After perfusion with 10 µM 8a, it caused a reduction of current amplitude both on HCN2 and HCN4 channels, though to a varying degree. Fig 5C demonstrated the I/I0 for HCN2 was 0.71 ± 0.06 (n = 6) and for HCN4 was 0.50 ± 0.04 (n = 7). Then we 12

ACCEPTED MANUSCRIPT induced HCN currents at different voltages, from -50 to -140 mV. Fig 5D and E showed representative tracings from HCN2 and HCN4 channels recorded in control condition or after treatment with 10 µM 8a. Fig 5F and 5G reported the

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voltage-dependence activation curves obtained in the absence or presence 10 µM 8a, demonstrating an obvious shift of the voltage dependence of activation to more negative direction after the treatment with 10 µM 8a on HCN2 and HCN4 channels

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(Table 2). Besides, 8a slowed the speed of current activation at -120 mV while it had

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no effect on the deactivation process measured at +50 mV after a test pulse to -130 mV (Table 2). Other compounds did not show higher affinity for HCN4 than 8a, such as 7a, 9a, 9b and 10a (Table 3).

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3. Conclusion

In this study, a series of Carvedilol and T5342126 derivatives were designed and synthesized, and their potential TLR4 inhibition activity was evaluated in HEK-Blue

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hTLR4 cells and human PBMC. We first found Carvedilol, a classic anti-heart failure

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drug, had strong inhibition to the TLR4 signaling. In these Carvedilol derivatives, the 3,5-dimethyl-pyrazole bridge ring, connecting with the benzyl ring and carbazole group together, contribute to the TLR4 inhibition. The optimized compound 8a displays the best inhibition activity to TLR4 and suppresses LPS-induced inflammatory cytokines, such as NO, SEAP and TNF-αin vitro and human whole blood ex vivo model, as well as directly downregulates the TLR4 protein in TLR4 overexpressed cells. More interestingly, 8a shows higher affinity for the HCN4 than 13

ACCEPTED MANUSCRIPT the HCN2 channel subtype which probably indicates blocking TLR4 may influence the HCN subtype. It also predicts that as improvements in potency, 8a derivatives will serve as useful chemical tools in studying TLR4-mediated inflammation and HCN

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channel subtypes which could pave the way for future treatment of inflammatory disease, heart disease and others.

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4. Experimental section

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4.1. Chemistry

Chemicals were purchased from Macklin or Aladdin Biochemical Technology and used without further purification. Hydrogen nuclear magnetic resonance (1H NMR) spectra and carbon nuclear magnetic resonance (13C NMR) spectra were

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recorded on a Bruker AM-400 spectrometer in CDCl3 or DMSO-d6, and chemical shifts were reported in ppm. The multiplicity of the signal was indicated as s, singlet; d, doublet; t, triplet; q, quartet; m, multiple, defined as all multi peak signals. All

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compounds were routinely checked by thin-layer chromatography (TLC), and the

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melting points were measured using yuhua X-5 microscope melting point analyzer.

General procedure for the synthesis of compound 1a-b. 3-bromoprop-1-ene (1000mg, 8.266mmol) was added dropwise to a solution of

mCPBA (2140 mg, 12.40mmol) in 50ml CH2Cl2. The reaction mixture was stirred for 24h. Afterwards NaOH (1M, 50 ml) was added and the phases were extracted with ethyl acetate. The organic layer was washed once with NaOH (4M, 50ml), followed 14

ACCEPTED MANUSCRIPT by H2O until the aqueous washing reached pH=7. The organic phase was dried over Na2SO4 and the solvent removed under reduced pressure. Yield 90% (1020 mg) of compound 1a as clear oil. Generation of the compound 1b was accomplished by using

General procedure for the synthesis of compound 2a-g.

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similar reaction conditions.

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4-Ethoxyphenol (500mg, 3.62 mmol), anhydrous potassium carbonate (366mg,

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2.650mmol) and1a (1984 mg, 14.48 mmol) were added to acetone and the resulting heterogeneous solution was refluxed for 20h. The mixture was cooled to room temperature and filtered through a pad of celite and the filtrate was concentrated under reduced pressure. The resulting oil was dissolved in toluene and washed sequentially

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with water, 5% aqueous NaOH, and water again before being dried with Na2SO4 and concentrated under reduced pressure. The resulting liquid was purified by flash chromatography on silica-gel with 20% ethyl acetate in petroleum. Yielding 83% of

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compound 2a (585 mg) as a clear liquid. Compounds 2b-g were synthesized following

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the procedure of preparation 2a.

General procedure for the synthesis of compound 3a-d. 3,5-dimethyl-1H-pyrazole (1000mg, 10.40mmol) and KOH (1675mg, 10.40mmol) were dissolved in DMSO and the resulting heterogeneous solution was stirred for 1.5 h at 80oC before being cooled to room temperature. Benzyl chloride (6 M) in 50 mL DMSO was then added over 15 mins, and the reaction mixture was stirred for a 15

ACCEPTED MANUSCRIPT further 1.5h. Upon completion as observed by TLC. The reaction was poured over water, and the resulting aqueous phase was extracted with two 20 ml portions of CHCl3. The combined organic layers were washed with 100ml of water, dried with

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anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by flash chromatography on silica-gel with 20% ethyl acetate in petroleum ether. Yielding 87% compound 3a (1999 mg) as a yellow oil. Compounds 3b-d were

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synthesized following the procedure of preparation 3a.

General procedure for the synthesis of compound 4a-d.

Paraformaldehyde (820mg, 27.2mmol) and methylamine hydrochloride (920mg, 13.6mmol) were dissolved in ethanol and stirred for 1h, then 3a (1000mg, 4.53mmol)

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was added and the reaction mixture was stirred at reflux for 16h. The mixture liquor then cooled to room temperature and quenched with aqueous NaHCO3 (15 mL). The aqueous layer was extracted 3 times with chloroform (3×15 mL) and the combined

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organic layers were washed with brine (30 mL). The organic layer was dried with

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Na2SO4 and concentrated under reduced pressure. The resulting yellow oil was purified by flash chromatography on silica-gel with 10% methanol in ethyl acetate. Yielding 77% compound 4a (872 mg) as a yellow oil. Compounds 4b-d were synthesized following the procedure of preparation 4a.

General procedure for the synthesis of compound 5a-b. 1a (897 mg, 6.55 mmol) and NaOH (222 mg, 5.55 mmol) solution were added to a 16

ACCEPTED MANUSCRIPT solution of 4-hydroxycarbazole (1000 mg, 5.55 mmol) in DMSO. The reaction mixture was stirred for 24h at 60 oC. It was then cooled to 20 oC, diluted with 25ml of water and extracted with ethyl acetate three times (3×20mL). The collected organic

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layers were washed with brine, dried with Na2SO4 and concentrated. The residue was purified by flash chromatography on silica-gel with 20% ethyl acetate in hexane. Yielding 60% of compound 5a (798 mg) as a white solid. Compound 5b was

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synthesized following the procedure of preparation 4a.

General procedure for the synthesis of compound 6a-b.

Compound 2a (100mg, 0.52mmol) , and 2-(2-methoxyphenoxy)ethanamine (150mg, 0.62mmol) were added to ethanol and the resulting heterogeneous solution was

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refluxed for 24h. The mixture was cooled to room temperature and filtered through a pad of celite and the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica-gel with 10% methanol in ethyl

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acetate. Yielding 68% compound 6a (128 mg) as a white solid. Compound 6b was

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synthesized following the procedure of preparation 6a.

General procedure for the synthesis of compound 7a-j. 2a (100mg, 0.52mol) and 4a (163 mg, 0.62 mmol) were dissolved in ethanol and warmed to 75oC and stirred until the oxirane was totally consumed as observed by TLC (20-24h). The solution was then cooled to room temperature and diluted with chloroform (15mL). The organic phase was washed with saturated sodium 17

ACCEPTED MANUSCRIPT bicarbonate, and the organic layer was dried with Na2SO4 and the solvent was removed under reduced pressure. The resulting oil was purified using flash column chromatography with 2% methanol in dichloromentane as an eluting solvent. Yielding

procedure of preparation 7a.

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General procedure for the synthesis of compound 8a-f.

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84% of 7a (200 mg) as a white solid. Compounds 7b-g were synthesized following the

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5a (500mg, 2.09mmol), and 4a (662mg, 2.51mmol) were added to ethanol and the resulting heterogeneous solution was refluxed for 24h. The mixture was cooled to room temperature and filtered through a pad of celite and the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on

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silica-gel with 10% methanol in ethyl acetate. Yielding 42% compound 8a (442 mg) as a white solid. Compounds 8b-f were synthesized following the procedure of

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preparation 8a.

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General procedure for the synthesis of compound 9a-b. Compound 5a (100mg, 0.42mmol), and 2-(2-methoxyphenoxy)ethanamine (84mg, 0.50mmol) were added to ethanol and the resulting heterogeneous solution was refluxed for 24h. The mixture was cooled to room temperature and filtered through a pad of celite and the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica-gel with 10% methanol in ethyl acetate. Yielding 83% compound 9a (176 mg) as a white solid. Compound 9b was 18

ACCEPTED MANUSCRIPT synthesized following the procedure of preparation 9a.

General procedure for the synthesis of compound 10a-g.

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5a (239mg, 1mmol), and 4-fluoroaniline (134mg, 1.2mmol) were added to ethanol and the resulting heterogeneous solution was refluxed for 24h. The mixture was cooled to room temperature and filtered through a pad of celite and the filtrate was

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concentrated under reduced pressure. The residue was purified by flash

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chromatography on silica-gel with 20% ethyl acetate in hexane. Yielding 80% compound 10a (348 mg) as a white solid. Compounds 10b-g were synthesized following the procedure of preparation 10a.

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2-(bromomethyl)oxirane. (1a)

Light yellow liquid, yield: 92%. 1H NMR (CDCl3, 400 MHz) δ 2.86 – 2.77 (m, 1H), 2.75 – 2.67 (m, 2H), 2.30 – 2.24 (m, 1H), 2.03 – 1.95 (m, 1H). 13C NMR (CDCl3, 101

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MHz) δ 51.10, 48.30, 33.20. ESI-MS: m/z calcd for C3H5BrO (M + H+) 137.0, found

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137.0. Anal calcd for C3H5O: C, 26.31; H, 3.68; O, 11.68. Found C, 26.29; H, 3.70; O, 11.71.

2-(2-bromoethyl)oxirane. (1b) Light yellow liquid, yield: 90%. 1H NMR (CDCl3, 400 MHz) δ 3.69 – 3.39 (m, 2H), 3.13 – 3.07 (m, 1H), 2.89 – 2.77 (m, 1H), 2.66 – 2.54 (m, 1H), 2.20 – 2.13 (m, 1H), 2.10 – 2.02 (m, 1H). 13C NMR (CDCl3, 101 MHz) δ 50.58, 46.90 , 35.60 , 28.92. 19

ACCEPTED MANUSCRIPT ESI-MS: m/z calcd for C4H7BrO (M + H+) 150.0, found 150.2. Anal calcd for C4H7O: C, 31.82; H, 4.67; O, 10.60. Found C, 31.87; H, 4.64; O, 10.70.

RI PT

2-(2-(4-ethoxyphenoxy)ethyl)oxirane. (2b) Clear liquid, yield: 83%. 1H NMR (CDCl3, 400 MHz) δ 6.85 (s, 4H), 4.10 – 3.96 (m, 4H), 3.19 – 3.15 (m, 1H), 2.86 – 2.83 (m, 1H), 2.62 – 2.58 (m, 1H), 2.14 – 2.05 (m, 13

C NMR (CDCl3, 101 MHz) δ

SC

1H), 1.98 – 1.90 (m, 1H), 1.44 – 1.38 (m, 3H).

M AN U

153.19 , 152.76 , 115.38 , 115.38 , 115.34 , 115.34 , 65.19 , 63.89 , 49.69 , 47.01 , 32.50 , 14.87 . ESI-MS: m/z calcd for C12H16O3 (M + H+) 208.1, found 208.2. Anal calcd for C12H16O3: C, 69.21; H, 7.74; O, 23.05. Found C, 69.18; H, 7.62; O, 23.08.

TE D

1-(2-chlorobenzyl)-3,5-dimethyl-1H-pyrazole. (3a)

Clear liquid, yield: 87%. 1H NMR (CDCl3, 400 MHz) δ 7.37 (d, J = 7.6 Hz, 1H), 7.22 – 7.14 (m, 2H), 6.56 (d, J = 8.4 Hz, 1H), 5.91 (s, 1H), 5.32 (s, 2H), 2.28 (s, 3H), 2.16

EP

(s, 3H). 13C NMR (CDCl3, 101 MHz) δ 156.58 , 129.98 , 129.86 , 114.32 , 64.62 ,

AC C

49.70 , 47.03 , 46.93 , 35.65 , 32.48 , 28.90 , 20.41 . ESI-MS: m/z calcd for C12H13ClN2 (M + H+) 220.1, found 220.4. Anal calcd for C12H13N2: C, 65.31; H, 5.94; N, 12.69 Found C, 65.41; H, 5.92; N, 12.67.

1-(1-(2-chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)-N-methylmethanamine. (4a) Yellow oil, yield: 77%. 1H NMR (CDCl3, 400 MHz) δ 7.40 – 7.36 (m, 1H), 7.24 – 7.14 (m, 2H), 6.66 (d, J = 7.6Hz, 1H), 5.33 (s, 2H), 3.87 (s, 2H), 2.52 (s, 3H), 2.36 (s, 20

ACCEPTED MANUSCRIPT 3H), 2.28 (s, 3H). 13C NMR (CDCl3, 101 MHz) δ 156.40 , 143.49 , 126.18 , 113.91 , 64.52 , 50.65 , 49.74 , 47.06 , 46.96 , 35.64 , 34.01 , 32.47 , 31.47 , 28.87 . ESI-MS: m/z calcd for C14H18ClN3 (M + H+) 263.1, found 263.3. Anal calcd for C14H18N3: C,

4-(oxiran-2-ylmethoxy)-9H-carbazole. (5a)

RI PT

63.75; H, 6.88; N, 15.93. Found C, 63.81; H, 6.84; N, 15.98.

SC

White solid, yield: 60%, m.p. 135–138oC. 1H NMR (DMSO-d6, 400 MHz) δ 11.33 (s,

M AN U

1H), 8.25 (d, J = 7.6 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H), 6.69 (d, J = 7.6 Hz, 1H), 4.57 – 4.51 (m, 1H), 4.11 – 4.05 (m, 1H), 3.56 – 3.51 (m, 1H), 2.96 – 2.92 (m, 1H), 2.87 – 2.83 (m, 1H).

13

C NMR (DMSO-d6, 101 MHz) δ 154.93 , 141.59 ,

TE D

139.42 , 126.87 , 125.10 , 122.75 , 122.04 , 119.11 , 111.98 , 110.91 , 104.69 , 101.12 , 69.16 , 50.36 , 44.20 . ESI-MS: m/z calcd for C15H13NO2 (M + H+) 239.1, found 239.3.

AC C

5.88.

EP

Anal calcd for C15H13NO2: C, 75.30; H, 5.48; N, 5.85. Found C, 75.31; H, 5.42; N,

1-(4-ethoxyphenoxy)-3-(((2-methoxyphenoxy)methyl)amino)propan-2-ol. (6a) Yellow solid, yield: 66%, m.p. 78–81oC. 1H NMR (CDCl3, 400 MHz) δ 6.98 – 6.93 (m, 2H), 6.89 (d, J = 5.2 Hz, 2H), 6.81 (d, J = 5.2 Hz, 4H), 4.16 – 4.08 (m, 3H), 4.01 – 3.95 (m, 5H), 3.85 – 3.81 (m, 2H), 3.20 – 2.80 (m, 4H), 1.44 – 1.37 (m, 3H).

13

C

NMR (CDCl3, 101 MHz) δ 153.20 , 152.77 , 149.30 , 147.81 , 121.56 , 120.85 , 115.40 , 115.31 , 113.60 , 113.42 , 111.50 , 70.58 , 68.28 , 67.38 , 63.91 , 59.56 , 21

ACCEPTED MANUSCRIPT 55.56 , 54.92 , 14.88 . ESI-MS: m/z calcd for C19H25NO5 (M + H+) 346.2, found 346.2. Anal calcd for C19H25NO5: C, 65.69; H, 7.25; N, 4.03. Found C, 65.70; H, 7.33; N,

RI PT

4.05.

1-(4-ethoxyphenoxy)-3-((2-(2-methoxyphenoxy)ethyl)amino)propan-2-ol.(6b)

Yellow solid, yield: 60%, m.p. 55–58oC. 1H NMR (CDCl3, 400 MHz) δ 6.98 – 6.89

SC

(m, 4H), 6.86 – 6.81 (m, 4H), 4.15 – 4.06 (m, 4H), 4.01 – 3.92 (m, 3H), 3.86 (s, 3H),

13

1.42 – 1.38 (m, 3H).

M AN U

3.10 – 3.05 (m, 2H), 2.88 – 2.84 (m, 1H), 2.68 – 2.62 (m, 1H), 1.98 – 1.81 (m, 2H), C NMR (CDCl3, 101 MHz) δ 153.01 , 152.97 , 152.93 ,

149.38 , 147.86 , 121.57 , 120.79 , 115.42 , 115.33 , 113.75 , 113.58 , 111.49 , 67.40 , 65.54 , 65.04 , 63.93 , 61.58 , 55.55 , 34.09 , 14.89 . ESI-MS: m/z calcd for

TE D

C20H27NO5 (M + H+) 361.2, found 361.4. Anal calcd for C20H27NO5: C, 66.46; H, 7.53; N, 3.88. Found C, 66.44; H, 7.54; N, 3.85.

EP

1-(((1-(2-chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)methyl)(methyl)amino)-3-(

AC C

4-ethoxyphenoxy)propan-2-ol. (7a T5342126) White solid, yield: 85%.m.p. 61–63 oC.1H NMR (CDCl3, 400 MHz) δ7.38 (d, J = 7.7Hz, 1H), 7.22 – 7.13 (m, 2H), 6.85 (s, 4H), 6.51 (dd, J = 7.7, 1.7Hz, 1H), 5.33 (s, 2H), 4.12 – 4.08 (m, 1H), 3.99 (q, J = 7.0 Hz, 2H), 3.93 (d, J = 4.9 Hz, 2H), 3.50 (d, J = 13.2 Hz, 2H), 3.35 (d, J = 13.2 Hz, 1H), 2.66 – 2.61 (m, 1H), 2.53 – 2.49 (m, 1H), 2.29 (s, 6H), 2.15 (s, 3H), 1.41 (t, J = 7.0 Hz, 3H).

13

C NMR (CDCl3, 101 MHz)δ

153.25 , 152.83 , 147.79 , 138.39 , 135.09 , 131.69 , 129.18 , 128.53 , 127.37 , 127.25 , 22

ACCEPTED MANUSCRIPT 115.42 , 115.42 , 115.31 , 115.31 , 113.48 , 71.06 , 66.18 , 63.91 , 59.30 , 51.49 , 50.05 , 41.88 , 14.89 , 12.12 , 9.58 . ESI-MS: m/z calcd for C25H32ClN3O3Na+ (M + Na+) 480.2, found 480.2. Anal calcd for C25H32N3O3: C, 65.56; H, 7.04; N, 9.17.

RI PT

Found C, 65.62; H, 7.11; N, 9.21.

1-(((1-(2-chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)methyl)(methyl)amino)-4-(

SC

4-ethoxyphenoxy)butan-2-ol. (7b)

M AN U

Yellow oil, yield: 84%. 1H NMR (CDCl3, 400 MHz) δ 7.37 (d, J = 8.0 Hz, 1H), 7.23 – 7.11 (m, 2H), 6.84 (d, J = 1.6 Hz, 4H), 6.52 – 6.48 (m, 1H), 5.32 (s, 2H), 4.13 – 3.91 (m, 5H), 3.53 – 3.25 (m, 2H), 2.48 – 2.31 (m, 2H), 2.27 (s, 3H), 2.23 (s, 3H), 2.12 (s, 3H), 1.93 – 1.78 (m, 2H), 1.42 – 1.37 (m, 3H).

13

C NMR (CDCl3, 101 MHz) δ

TE D

153.08 , 152.88 , 147.83 , 134.98 , 131.72 , 129.21 , 128.57 , 127.38 , 127.24 , 115.42 , 115.40 , 115.35 , 77.27 , 66.62 , 65.28 , 64.19 , 63.95 , 62.81 , 51.35 , 50.08 , 41.49 , 34.61 , 32.62 , 14.88 , 12.13 , 9.65 . ESI-MS: m/z calcd for C26H34ClN3O3 (M + H+)

EP

471.2, found 471.3. Anal calcd for C26H34ClN3O3: C, 66.16; H, 7.26; N, 8.90. Found

AC C

C, 66.22; H, 7.20; N, 8.98.

1-(((1-(2-chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)methyl)(methyl)amino)-4-( 4-fluorophenoxy)butan-2-ol. (7c) Yellow oil, yield: 80%. 1H NMR (CDCl3, 400 MHz) δ 7.37 (d, J = 7.6 Hz, 1H), 7.22 – 7.12 (m, 2H), 7.04 – 6.92 (m, 2H), 6.90 – 6.82 (m, 2H), 6.53 – 6.50 (m, 1H), 5.32 (s, 2H), 4.21 – 4.06 (m, 2H), 4.01 – 3.91 (m, 1H), 3.54 – 3.48 (m, 1H), 3.37 – 3.26 (m, 23

ACCEPTED MANUSCRIPT 1H), 2.51 – 2.45 (m, 1H), 2.39 – 2.31 (m, 1H), 2.28 – 2.24 (m, 6H), 2.13 (s, 3H), 1.96 – 1.87 (m, 1H), 1.85 – 1.75 (m, 1H). 13C NMR (CDCl3, 101 MHz) δ 154.94 , 147.75 , 138.40 , 135.05 , 131.71 , 129.20 , 128.55 , 127.36 , 127.21 , 115.79 , 115.56 , 115.45 ,

RI PT

115.37 , 113.41 , 65.30 , 64.05 , 62.84 , 62.83 , 51.40 , 50.04 , 41.56 , 34.52 , 12.10 , 9.56 . ESI-MS: m/z calcd for C24H29ClFN3O2 (M + H+) 445.2, found 445.3. Anal

SC

calcd for C24H29N3O2: C, 64.64; H, 6.55; N, 9.42. Found C, 64.73; H, 6.50; N, 9.33.

p-tolyloxy)butan-2-ol. (7d)

M AN U

1-(((1-(2-chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)methyl)(methyl)amino)-4-(

Yellow oil, yield: 75%. 1H NMR(CDCl3, 400 MHz) δ 7.38 (d, J = 8.0 Hz, 1H), 7.25 – 7.13 (m, 2H), 7.09 (d, J = 7.6 Hz, 2H), 6.83 (d, J = 8.4 Hz, 2H), 6.52 (d, J = 7.6 Hz,

TE D

1H), 5.33 (s, 2H), 4.15 – 4.11 (m, 2H), 3.99 (s, 1H), 3.56 – 3.50 (m, 1H), 3.36 – 3.31 (m, 1H), 2.53 – 2.48 (m, 1H), 2.42 – 2.37 (m, 1H), 2.32 – 2.24 (m, 9H), 2.14 (s, 3H), 1.98 – 1.79 (m, 2H). 13C NMR (CDCl3, 101 MHz) δ 156.64 , 147.82 , 139.60 , 138.59 ,

EP

138.32 , 137.91 , 129.80 , 129.80 , 129.20 , 128.57 , 127.36 , 127.23 , 114.33 , 77.16 ,

AC C

64.70 , 64.27 , 62.82 , 51.35 , 50.71 , 50.04 , 41.52 , 34.53 , 20.38 , 12.06 , 9.59 . ESI-MS: m/z calcd for C25H32ClN3O2 (M + H+) 441.2, found 441.4. Anal calcd for C25H32N3O2: C, 67.93; H, 7.30; N, 9.51. Found C, 67.88; H, 7.39; N, 9.50.

1-(((1-(2-chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)methyl)(methyl)amino)-4-( 4-ethylphenoxy)butan-2-ol.(7e)

24

ACCEPTED MANUSCRIPT Light yellow oil, yield: 79%. 1H NMR (CDCl3, 400 MHz) δ 7.38 (d, J = 7.6 Hz,1H), 7.22 – 7.16 (m, 2H), 7.10 (d, J = 8.0 Hz, 2H), 6.85 (d, J = 8.4 Hz, 2H), 6.53 (d, J = 7.6 Hz, 1H), 5.33 (s, 2H), 4.14 (t, J = 7.6 Hz, 2H), 4.03 (s, 1H), 3.58 (s, 1H), 3.39 (s, 1H),

RI PT

2.72 – 2.54 (m, 3H), 2.45 – 2.41 (m, 1H), 2.28 (s, 6H), 2.16 (s, 3H), 1.97 – 1.83 (m, 2H), 1.23 (t, J = 7.6 Hz, 3H). 13C NMR (CDCl3, 101 MHz) δ 156.79 , 147.83 , 141.44 , 136.41 , 134.96 , 131.72 , 129.21 , 128.63 , 128.63 , 127.38 , 127.23 , 114.34 , 114.34 ,

SC

98.22 , 64.67 , 64.25 , 62.82 , 51.35 , 50.76 , 50.06 , 41.50 , 34.55 , 27.90 , 15.81 ,

M AN U

12.10 , 9.63 . ESI-MS: m/z calcd for C26H34ClN3O2 (M + H+) 455.3, found 455.3. Anal calcd for C26H34N3O2: C, 68.48; H, 7.52; N, 9.21. Found C, 68.36; H, 7.56; N, 9.19.

TE D

1-(((1-(2-chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)methyl)(methyl)amino)-4-( 4-propylphenoxy)butan-2-ol. (7f)

Clear oil, yield: 82%. 1H NMR (CDCl3, 400 MHz) δ 7.38 (d, J = 7.6 Hz, 1H), 7.23 –

EP

7.13 (m, 2H), 7.10 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H), 6.52 (d, J = 7.6 Hz,

AC C

1H), 5.33 (s, 2H), 4.16 – 4.11 (m, 2H), 4.02 – 3.94 (m, 1H), 3.56 – 3.50 (m, 1H), 3.36 – 3.30 (m, 1H), 2.54 (t, J = 7.6 Hz, 2H), 2.49 – 2.45 (m, 1H), 2.41 – 2.35 (m, 1H), 2.28 – 2.25 (m, 5H), 2.14 (s, 3H), 1.97 – 1.77 (m, 2H), 1.66 – 1.59 (m, 2H), 1.29 – 1.24 (m, 1H), 0.95 (t, J = 7.3 Hz, 3H).

13

C NMR (CDCl3, 101 MHz) δ 156.84 ,

147.81 , 138.51 , 135.04 , 134.79 , 131.71 , 129.23 , 129.23 , 129.23 , 129.20 , 128.55 , 127.37 , 127.23 , 114.24 , 114.17 , 64.67 , 64.25 , 62.85 , 51.37 , 50.06 , 41.52 , 37.09 , 34.58 , 24.71 , 13.72 , 12.12 , 9.60 . ESI-MS: m/z calcd for C27H36ClN3O2 (M + H+) 25

ACCEPTED MANUSCRIPT 469.3, found 469.5. Anal calcd for C27H36N3O2: C, 68.99; H, 7.72; N, 8.94. Found C, 69.13; H, 7.63; N, 8.90.

RI PT

4-(4-(tert-butyl)phenoxy)-1-(((1-(2-chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl) methyl)(methyl)amino)butan-2-ol. (7g)

Clear oil, yield: 77%. 1H NMR (CDCl3, 400 MHz) δ 7.39 (d, J = 6.8 Hz, 1H), 7.31 (d,

SC

J = 8.4 Hz, 2H), 7.22 – 7.15 (m, 2H), 6.86 (d, J = 8.8 Hz, 2H), 6.52 (d, J = 7.6 Hz,

M AN U

1H), 5.33 (s, 2H), 4.15 (t, J = 5.2 Hz, 2H), 4.01 (s, 1H), 3.58 – 3.52 (m, 1H), 3.40 – 3.34 (m, 1H), 2.54 (s, 1H), 2.42 (s, 1H), 2.28 (s, 6H), 2.15 (s, 3H), 1.95 – 1.83 (m, 2H), 1.32 (s, 9H).

13

C NMR (CDCl3, 101 MHz) δ 156.53 , 147.80 , 143.28 , 138.46 ,

135.07 , 131.70 , 129.19 , 128.54 , 127.37 , 127.23 , 126.13 , 126.13 , 113.91 , 113.91 ,

TE D

64.63 , 64.26 , 62.88 , 51.39 , 50.06 , 41.54 , 34.58 , 33.99 , 31.47 , 31.47 , 31.47 , 31.47 , 12.12 , 9.59 . ESI-MS: m/z calcd for C28H38ClN3O2 (M + H+) 483.2, found

AC C

7.88; N, 8.66.

EP

483.2. Anal calcd for C28H38N3O2: C, 69.47; H, 7.91; N, 8.68. Found C, 69.45; H,

4-(4-ethoxyphenoxy)-1-(((1-(2-fluorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)methy l)(methyl)amino)butan-2-ol. (7h) Clear oil, yield: 82%. 1H NMR (CDCl3, 400 MHz) δ 7.27 – 7.22 (m, 1H), 7.11 – 7.01 (m, 2H), 6.85 (s, 5H), 5.29 (s, 2H), 4.10 (t, J = 6.4 Hz, 2H), 4.00 (q, J = 7.2 Hz, 3H), 3.56 – 3.50 (m, 1H), 3.36 – 3.30 (m, 1H), 2.52 (s, 1H), 2.42 – 2.36 (m, 1H), 2.26 (s, 6H), 2.18 (s, 3H), 1.91 – 1.80 (m, 2H), 1.41 (t, J = 7.0 Hz, 3H). 26

13

C NMR (CDCl3,

ACCEPTED MANUSCRIPT 101 MHz) δ 153.04 , 152.93 , 147.57 , 138.10 , 129.07 , 128.49 , 124.48 , 124.34 , 115.39 , 115.33 , 115.33 , 115.19 , 114.98 , 113.36 , 65.33 , 64.25 , 63.92 , 62.93 , 51.40 , 46.11 , 46.06 , 41.53 , 34.61 , 14.89 , 12.09 , 9.51 . ESI-MS: m/z calcd for

RI PT

C26H34FN3O3 (M + H+) 455.3, found 455.5. Anal calcd for C26H34N3O3: C, 68.55; H, 7.52; N, 9.22; O, 10.54. Found C, 68.55; H, 7.44; N, 9.23; O, 10.57.

SC

1-(((3,5-dimethyl-1-(2-methylbenzyl)-1H-pyrazol-4-yl)methyl)(methyl)amino)-4-(

M AN U

4-ethoxyphenoxy)butan-2-ol.(7i)

Light yellow oil, yield: 69%. 1H NMR (CDCl3, 400 MHz) δ 7.17 (d, J = 8.4 Hz, 2H), 7.12 – 7.08 (m, 1H), 6.85 (s, 4H), 6.45 (d, J = 7.2 Hz, 1H), 5.21 (s, 2H), 4.14 – 4.08 (m, 2H), 4.00 (q, J = 7.2 Hz, 3H), 3.56 – 3.50 (m, 1H), 3.36 – 3.30 (m, 1H), 2.55 –

TE D

2.47 (m, 1H), 2.40 (s, 1H), 2.35 (s, 3H), 2.27 (s, 6H), 2.12 (s, 3H), 1.97 – 1.80 (m, 2H), 1.41 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3, 101 MHz) δ 153.05 , 152.97 , 147.34 , 138.35 , 135.41 , 134.61 , 130.08 , 127.19 , 126.34 , 125.67 , 115.41 , 115.41 , 115.35 ,

EP

115.35 , 65.34 , 64.23 , 63.95 , 62.87 , 51.41 , 50.71 , 50.68 , 41.50 , 34.62 , 19.00 ,

AC C

14.89 , 12.09 , 9.70 . ESI-MS: m/z calcd for C27H37N3O3 (M + H+) 451.3, found 451.6. Anal calcd for C27H37N3O3: C, 71.81; H, 8.26; N, 9.30; O, 10.63 Found C, 71.70; H, 8.25; N, 9.37; O, 10.55.

1-(((1-(4-chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)methyl)(methyl)amino)-4-( 4-ethoxyphenoxy)butan-2-ol. (7j)

27

ACCEPTED MANUSCRIPT Clear oil, yield: 68%. 1H NMR (CDCl3, 400 MHz) δ 7.30 (s, 1H), 7.28 (s, 1H), 7.00 (d, J = 8.0 Hz, 2H), 6.85 (s, 4H), 5.20 (s, 2H), 4.12 – 4.08 (m, 2H), 4.00 (q, J = 6.8 Hz, 3H), 3.56 – 3.50 (m, 1H), 3.36 – 3.30 (m, 1H), 2.55 – 2.46 (m, 1H), 2.41 – 2.35 (m, 13

C

RI PT

1H), 2.26 (s, 6H), 2.14 (s, 3H), 1.97 – 1.75 (m, 2H), 1.41 (t, J = 6.8 Hz, 3H).

NMR (CDCl3, 101 MHz) δ 153.07 , 152.90 , 147.51 , 135.73 , 133.29 , 128.84 , 128.84 , 127.84 , 127.84 , 115.39 , 115.39 , 115.35 , 115.35 , 77.15 , 65.28 , 64.20 ,

SC

63.95 , 62.89 , 52.06 , 51.34 , 41.49 , 34.60 , 14.89 , 12.07 , 9.77 . ESI-MS: m/z calcd

M AN U

for C26H34ClN3O3 (M + H+) 471.2, found 471.3. Anal calcd for C26H34N3O3: C, 66.17; H, 7.25; N, 8.91; O, 10.16 Found C, 66.16; H, 7.26; N, 8.90; O, 10.17.

1-((9H-carbazol-4-yl)oxy)-3-(((1-(2-chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)

TE D

methyl)(methyl)amino)propan-2-ol.(8a)

White solid, yield: 42%, m.p. 90–92oC. 1H NMR (CDCl3, 400 MHz) δ 8.28 (d, J = 7.6 Hz, 1H), 8.22 (s, 1H), 7.42 (s, 2H), 7.35 (q, J = 7.6 Hz, 2H), 7.19 – 7.10 (m,2H), 7.07

EP

(d, J = 8.0 Hz, 1H), 6.68 (d, J = 8.0 Hz, 1H), 6.52 (d, J = 7.2 Hz, 1H), 5.27 (s, 2H),

AC C

4.38 – 4.29 (m, 2H), 4.25 – 4.20 (m, 1H), 3.58 – 3.55 (m, 1H), 3.43 – 3.39 (m, 1H), 2.83 – 2.70 (m, 2H), 2.36 (s, 3H), 2.30 (s, 3H), 2.14 (s, 3H), 1.30 – 1.26 (m, 1H). 13C NMR (CDCl3, 101 MHz) δ 155.34 , 147.81 , 140.90 , 138.69 , 138.53 , 135.04 , 131.72 , 129.19 , 128.55 , 127.39 , 127.23 , 126.67 , 124.79 , 122.84 , 122.66 , 119.41 , 112.61 , 109.92 , 103.42 , 101.19 , 64.65 , 64.34 , 62.88 , 51.33 , 50.03 , 41.51 , 34.74 , 12.08 , 9.54 . ESI-MS: m/z calcd for C29H31ClN4O2 (M + H+) 503.0, found 503.1.

28

ACCEPTED MANUSCRIPT Anal calcd for C29H31N4O2: C, 69.24; H, 6.21; N, 11.14; O, 6.36. Found C, 69.44; H, 6.27; N, 11.22; O, 6.41.

RI PT

4-((9H-carbazol-4-yl)oxy)-1-(((1-(2-chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl) methyl)(methyl)amino)butan-2-ol. (8b)

Light yellow solid, yield: 39%, m.p. 65–67oC. 1H NMR (CDCl3, 400 MHz) δ 8.31 (d,

SC

J = 8.0 Hz, 1H), 8.21 (s, 1H), 7.43 – 7.30 (m, 4H), 7.26 – 7.14 (m, 3H), 7.05 (d, J =

M AN U

8.4 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 6.52 (d, J = 6.8 Hz, 1H), 5.30 (s, 2H), 4.46 – 4.40 (m, 2H), 4.18 (s, 1H), 3.55 – 3.30 (m, 2H), 2.62 – 2.56 (m, 1H), 2.50 – 2.46 (m, 1H), 2.27 (s, 6H), 2.11 (s, 3H), 1.29 (s, 2H). 13C NMR (CDCl3, 101 MHz) δ 155.14 , 147.81 , 140.92 , 138.72 , 138.51 , 135.05 , 131.71 , 129.19 , 128.55 , 127.40 , 127.23 ,

TE D

126.60 , 124.92 , 122.86 , 122.51 , 119.56 , 113.30 , 112.66 , 109.99 , 103.77 , 101.08 , 70.24 , 66.27 , 59.73 , 51.66 , 49.98 , 41.92 , 31.53 , 12.10 , 9.56 . ESI-MS: m/z calcd for C30H33ClN4O2 (M + H+) 516.2, found 516.4. Anal calcd for C30H33N4O2: C, 69.69;

AC C

EP

H, 6.43; N, 10.84; O, 6.19. Found C, 69.77; H, 6.40; N, 10.91; O, 6.22.

1-((9H-carbazol-4-yl)oxy)-3-(((1-benzyl-3,5-dimethyl-1H-pyrazol-4-yl)methyl)(m ethyl)amino)propan-2-ol.(8c) Yellow solid, yield: 40%, m.p. 75–78oC. 1H NMR (CDCl3, 400 MHz) δ 8.36 – 8.27 (m, 2H), 7.42 – 7.30 (m, 5H), 7.26 (d, J = 7.2 Hz, 1H), 7.06 (d, J = 7.7 Hz, 3H), 6.67 (d, J = 7.2 Hz, 1H), 5.20 (s, 2H), 4.44 – 4.26 (m, 2H), 4.21 – 4.14 (m, 1H), 3.56 – 3.38 (m, 2H), 2.85 – 2.79 (m, 1H), 2.75 – 2.70 (m, 1H), 2.34 (s, 2H), 2.29 (s, 3H), 29

ACCEPTED MANUSCRIPT 2.15 (s, 3H), 2.10 (s, 3H), 1.37 – 1.31 (m, 1H).

13

C NMR (CDCl3, 101 MHz) δ

155.13 , 147.29 , 140.94 , 138.73 , 138.12 , 137.19 , 128.65 , 127.41 , 126.58 , 126.48 , 126.44 , 124.89 , 122.86 , 122.66 , 122.50 , 119.53 , 112.96 , 109.99 , 103.77 , 101.08 ,

RI PT

77.18 , 70.28 , 66.17 , 59.84 , 52.71 , 51.62 , 41.76 , 12.08 , 9.81 . ESI-MS: m/z calcd for C29H32N4O2 (M + H+) 468.3, found 468.5. Anal calcd for C29H32N4O2: C, 74.37; H,

SC

6.83; N, 11.87; O, 6.82. Found C, 74.33; H, 6.88; N, 11.96; O, 6.81.

M AN U

1-((9H-carbazol-4-yl)oxy)-3-(((1-(2-fluorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl) methyl)(methyl)amino)propan-2-ol. (8d)

Yellow solid, yield: 40%, m.p. 92–95oC. 1H NMR (CDCl3, 400 MHz) δ 8.27 (d, J = 7.6 Hz, 1H), 8.18 (s, 1H), 7.46 – 7.38 (m, 2H), 7.35 – 7.31 (m, 1H), 7.27 – 7.20 (m,

TE D

2H), 7.06 – 6.95 (m, 3H), 6.87 – 6.78 (m, 1H), 6.68 (d, J = 8.0 Hz, 1H), 5.24 (s, 2H), 4.40 – 4.28 (m, 2H), 4.26 – 4.17 (m, 1H), 3.57 – 3.42 (m, 2H), 2.91 – 2.71 (m, 2H), 2.36 (s, 3H), 2.28 (s, 3H), 2.18 (s, 3H), 1.32 – 1.29 (m, 1H).

13

C NMR (CDCl3, 101

EP

MHz) δ 155.10 , 147.66 , 140.88 , 138.68 , 129.18 , 128.54 , 126.62 , 126.62 , 124.93 ,

AC C

124.93 , 124.48 , 122.85 , 122.50 , 119.59 , 115.21 , 115.21, 115.01 , 112.67 , 109.97 , 103.76 , 101.13 , 77.16 , 70.21 , 66.13 , 59.85 , 51.65 , 41.77 , 12.11 , 9.59 . ESI-MS: m/z calcd for C29H31ClN4O2 (M + H+) 502.2, found 502.2. Anal calcd for C29H31N4O2: C, 71.58; H, 6.42; N, 11.51; O, 6.58. Found C, 71.55; H, 6.38; N, 11.44; O, 6.63.

1-((9H-carbazol-4-yl)oxy)-3-(((1-(4-chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl) methyl)(methyl)amino)propan-2-ol. (8e) 30

ACCEPTED MANUSCRIPT Yellow solid, yield: 45%, m.p. 86–88oC. 1H NMR (CDCl3, 400 MHz) δ 8.38 (s, 1H), 8.28 (d, J = 7.6 Hz, 1H), 7.39 (d, J = 4.0 Hz, 2H), 7.36 – 7.21 (m, 3H), 7.04 (d, J = 8.0 Hz, 1H), 6.97 (d, J = 8.0 Hz, 2H), 6.71 – 6.64 (m, 1H), 5.13 (s, 2H), 4.32 (s, 2H), 4.25

RI PT

– 4.17 (m, 1H), 3.56 – 3.48 (m, 1H), 3.44 – 3.38 (m, 1H), 3.23 (s ,1H), 2.89 – 2.67 (m, 2H), 2.33 (s, 3H), 2.29 (s, 3H), 2.13 (s, 3H), 1.32 – 1.26 (m, 1H). 13C NMR (CDCl3, 101 MHz) δ 155.11 , 147.55 , 140.95 , 138.07 , 135.69 , 133.27 , 128.82 , 128.82 ,

SC

127.86 , 127.86 , 126.61 , 126.61 , 124.90 , 122.85 , 122.49 , 119.53 , 113.17 , 112.64 ,

M AN U

110.02 , 103.81 , 101.07 , 70.23 , 66.20 , 59.80 , 51.98 , 51.59 , 41.81 , 12.07 , 9.76 . ESI-MS: m/z calcd for C29H31FN4O2 (M + H+) 486.2, found 486.5. Anal calcd for C29H31N4O2: C, 69.24; H, 6.21; N, 11.14; O, 6.36. Found C, 69.18; H, 6.15; N, 11.12;

TE D

O, 6.46.

1-((9H-carbazol-4-yl)oxy)-3-(((1-(2-chlorobenzyl)-3-methyl-1H-pyrazol-4-yl)meth yl)(methyl)amino)propan-2-ol.(8f)

EP

White solid, yield: 66%, m.p. 61–63oC. 1H NMR (CDCl3, 400 MHz) δ 8.28 – 8.24 (m,

AC C

1H), 8.17 (s, 1H), 7.47 – 7.30 (m, 5H), 7.28 – 7.13 (m, 3H), 7.07 (d, J = 8.0 Hz, 1H), 6.94 – 6.90 (m, 1H), 6.69 (d, J = 7.6 Hz, 1H), 5.31 (s, 2H), 4.40 – 4.28 (m, 2H), 4.26 – 4.20 (m, 1H), 3.66 – 3.58 (m, 1H), 3.54 – 3.46 (m, 1H), 2.86 – 2.79 (m, 1H), 2.77 – 2.71 (m, 1H), 2.38 (s, 3H), 2.30 (s, 2H), 2.18 (s, 1H), 1.27 – 1.33 (m, 1H). 13C NMR (CDCl3, 101 MHz) δ 155.10 , 148.24 , 140.88 , 138.68 , 134.64 , 130.61 , 129.41 , 129.17 , 129.10 , 127.18 , 126.61 , 124.94 , 122.84 , 122.49 , 119.61 , 115.35 , 112.68 , 109.97 , 103.76 , 101.15 , 77.16 , 70.20 , 66.22 , 59.65 , 52.97 , 51.69 , 42.00 , 11.97 . 31

ACCEPTED MANUSCRIPT ESI-MS: m/z calcd for C28H29ClN4O2 (M + H+) 488.3, found 488.4. Anal calcd for C28H29N4O2: C, 68.77; H, 5.98; N, 11.46; O, 6.54. Found C, 68.55; H, 5.94; N, 11.47;

RI PT

O, 6.44.

1-((9H-carbazol-4-yl)oxy)-3-((2-(2-methoxyphenoxy)ethyl)amino)propan-2-ol. (9a Carvedilol)

SC

White solid, yield: 80%, m.p. 114–116oC. 1H NMR (CDCl3, 400 MHz) δ 8.31 (s, 1H),

M AN U

8.29 (s, 1H), 7.44 – 7.22 (m, 4H), 7.07 (d, J = 8.1 Hz, 1H), 6.99 – 6.89 (m, 4H), 6.68 (d, J = 7.8 Hz, 1H), 4.32 – 4.14 (m, 5H), 3.86 (s, 3H), 3.17 – 2.95 (m, 4H), 2.61 (broad s, 2H).

13

C NMR (CDCl3, 101 MHz) δ 155.10 , 149.51 , 148.15 , 140.96 ,

138.75 , 126.62 , 124.94 , 122.92 , 122.43 , 121.54 , 120.93 , 119.57 , 113.88 , 112.58 ,

TE D

111.79 , 110.05 , 103.83 , 101.11 , 70.27 , 68.67 , 68.43 , 55.71 , 51.93 , 48.64 . m.p. 113~115 oC ESI-MS: m/z calcd for C24H26N2O4 (M + H+) 406.2, found 406.4. Anal calcd for C24H26N2O4: C, 70.92; H, 6.45; N, 6.89; O, 15.74. Found C, 70.88; H, 6.49;

AC C

EP

N, 6.78; O, 15.77.

4-((9H-carbazol-4-yl)oxy)-1-((2-(2-methoxyphenoxy)ethyl)amino)butan-2-ol. (9b VK-II-86)

Yellow solid, yield: 73%, m.p. 61–63oC. 1H NMR (CDCl3, 400 MHz) δ 8.30 (d, J = 7.6 Hz, 1H), 8.11 (s, 1H), 7.42 – 7.30 (m, 3H), 7.24 (t, J = 6.7 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 6.95 – 6.87 (m, 4H), 6.71 (d, J = 8.0 Hz, 1H), 4.43 (t, J = 6.0 Hz, 2H), 4.13 (t, J = 5.0 Hz, 2H), 4.10 – 4.02 (m, 1H), 3.84 (s, 3H), 3.10 – 2.99 (m, 3H), 2.74 (t, 32

ACCEPTED MANUSCRIPT J = 6.0 Hz, 1H), 2.20 – 2.07 (m, 2H), 1.65 (s, 1H).

13

C NMR (CDCl3, 101 MHz) δ

155.48 , 149.54 , 148.12 , 141.06 , 138.82 , 126.73 , 124.91 , 122.98 , 122.62 , 121.79 , 121.01 , 119.56 , 114.03 , 112.62 , 111.90 , 110.14 , 103.66 , 101.17 , 68.53 , 66.91 ,

RI PT

64.94 , 55.80 , 55.11 , 48.42 , 34.63 . ESI-MS: m/z calcd for C25H28N2O4 (M + H+) 420.2, found 420.2. Anal calcd for C25H28N2O4: C, 71.41; H, 6.71; N, 6.66; O, 15.22

SC

Found C, 71.37; H, 6.77; N, 6.42; O, 15.33.

M AN U

1-((9H-carbazol-4-yl)oxy)-3-((4-fluorophenyl)amino)propan-2-ol. (10a) White solid, yield: 80%, m.p. 93–97oC. 1H NMR (CDCl3, 400 MHz) δ 8.27 (d, J = 7.6 Hz, 1H), 8.15 (s, 1H), 7.48 – 7.40 (m, 2H), 7.36 (t, J = 8.4 Hz, 1H), 7.28 – 7.24 (m, 1H), 7.11 (d, J = 8.0 Hz, 1H), 6.93 (t, J = 8.4 Hz, 2H), 6.73 – 6.67 (m, 3H), 4.36 (d, J

TE D

= 4.8 Hz, 2H), 3.61 – 3.56 (m, 1H), 3.47 – 3.41 (m, 1H), 2.07 (s, 1H), 1.35 – 1.24 (m, 1H), 0.94 – 0.89 (m, 1H).

13

C NMR (CDCl3, 101 MHz) δ 157.51 , 154.75 , 143.96 ,

140.92 , 138.69 , 126.65 , 125.15 , 122.65 , 122.29 , 119.77 , 115.86 , 114.57 , 114.50 ,

EP

110.15 , 104.16 , 101.24 , 77.15 , 68.87 , 47.92 , 22.59 , 14.06 . ESI-MS: m/z calcd for

AC C

C21H19FN2O2 (M + H+) 350.1, found 350.1. Anal calcd for C21H19N2O2: C, 71.98; H, 5.47; N, 8.00; O, 9.13. Found C, 71.07; H, 5.64; N, 8.08; O, 9.16.

1-((9H-carbazol-4-yl)oxy)-3-([1,1'-biphenyl]-4-ylamino)propan-2-ol. (10b) Light yellow solid, yield: 81%, m.p. 183–186oC. 1H NMR (DMSO-d6, 400 MHz) δ 11.26 (s, 1H), 8.29 (d, J = 7.6 Hz, 1H), 7.56 – 7.52 (m, 2H), 7.47 (d, J = 8.0 Hz, 1H), 7.41 – 7.35 (m, 5H), 7.30 (t, J = 8.0 Hz, 1H), 7.24 – 7.19 (m, 1H), 7.18 – 7.14 (m, 1H), 33

ACCEPTED MANUSCRIPT 7.09 (d, J = 8.0 Hz, 1H), 6.75 (d, J = 8.8Hz, 2H), 6.71 (d, J = 8.0Hz, 1H), 5.92 – 5.88 (m, 1H), 5.42 – 5.30 (m, 1H), 4.25 (s, 3H), 3.55 – 3.45 (m, 1H), 3.31 – 3.25 (m, 1H). 13

C NMR (DMSO-d6, 101 MHz) δ 155.33 , 148.89 , 141.25 , 139.33 , 129.11 , 129.11 ,

RI PT

127.82 , 127.56 , 127.56 , 126.88 , 126.07 , 125.75 , 125.75 , 124.96 , 122.90 , 122.14 , 118.98 , 112.89 , 112.89 , 112.89 , 112.00 , 110.78 , 104.31 , 100.87 , 70.58 , 68.13 , 46.86 . ESI-MS: m/z calcd for C27H24N2O2 (M + H+) 408.2, found 408.3. Anal calcd

SC

for C27H24N2O2: C, 79.39; H, 5.92; N, 6.86; O, 7.83. Found C, 79.33; H, 5.81; N, 6.75;

M AN U

O, 7.88.

1-((9H-carbazol-4-yl)oxy)-3-((9H-fluoren-3-yl)amino)propan-2-ol. (10c) Red solid, yield: 77%, m.p. 199–202oC. 1H NMR (DMSO-d6, 400 MHz) δ 11.26 (s,

TE D

1H), 10.01 (t, J = 5.6 Hz, 1H), 8.27 (d, J = 8.0 Hz, 1H), 8.20 (d, J = 7.6 Hz, 1H), 8.12 (d, J = 6.8 Hz, 1H), 7.95 – 7.79 (m, 2H), 7.59 (t, J = 8.0 Hz, 1H), 7.44 (t, J = 8.0 Hz, 2H), 7.36 – 7.26 (m, 3H), 7.19 – 7.07 (m, 2H), 6.73 (d, J = 8.0 Hz, 1H), 5.72 (d, J =

13

C NMR (DMSO-d6, 101 MHz) δ 184.33 , 183.28 , 155.11 , 151.92 , 141.53 ,

AC C

1H).

EP

4.8 Hz, 1H), 4.36 (s, 1H), 4.30 – 4.26 (m, 2H), 3.83 – 3.76 (m, 1H), 3.69 – 3.57 (m,

139.32 , 135.93 , 134.88 , 134.76 , 133.81 , 132.72 , 126.88 , 126.81 , 126.63 , 124.98 , 122.91 , 122.04 , 119.06 , 119.03 , 115.46 , 112.66 , 111.97 , 110.78 , 104.47 , 100.91 , 70.35 , 67.96 , 46.08 . ESI-MS: m/z calcd for C28H24N2O2 (M + H+) 420.2, found 420.3. Anal calcd for C28H24N2O2: C, 79.98; H, 5.75; N, 6.66; O, 7.61. Found C, 79.81; H, 5.65; N, 6.56; O, 7.58.

34

ACCEPTED MANUSCRIPT (2-((3-((9H-carbazol-4-yl)oxy)-2-hydroxypropyl)amino)-5-chlorophenyl)(phenyl) methanone. (10d) Bright yellow solid, yield: 82%, m.p. 103–105oC. 1H NMR (DMSO-d6, 400 MHz)

RI PT

δ1H NMR (400 MHz, DMSO-d6) δ 11.28 (s, 1H), 8.30 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.48 – 7.40 (m, 2H), 7.35 (t, J = 7.6 Hz, 1H), 7.31 – 7.22 (m, 2H), 7.20 – 7.06 (m, 3H), 6.86 (s, 1H), 6.70 (d, J = 8.0 Hz, 2H), 5.90

SC

(s, 1H), 5.37 (s, 1H), 4.25 (s, 3H), 3.64 (s, 2H), 3.56 – 3.50 (m, 1H).

13

C NMR

M AN U

(DMSO-d6, 101 MHz) δ 150.43 , 141.53 , 139.67 , 139.33 , 135.04 , 133.51 , 131.71 , 128.95 , 128.95 , 128.80 , 126.87 , 124.98 , 122.89 , 122.05 , 119.02 , 118.04 , 117.35 , 114.57 , 111.97 , 110.77 , 104.45 , 100.90 , 70.40 , 67.82 , 60.15 , 28.40 , 21.16 , 14.48 . ESI-MS: m/z calcd for C28H23ClN2O3 (M + H+) 470.1, found 470.1. Anal

N, 5.86; O, 10.20.

TE D

calcd for C28H23N2O3: C, 71.41; H, 4.92; N, 5.95; O, 10.19. Found C, 71.33; H, 4.99;

AC C

(10e)

EP

2-((3-((9H-carbazol-4-yl)oxy)-2-hydroxypropyl)amino)anthracene-9,10-dione.

White solid, yield: 79%, m.p. 172–175oC. 1H NMR (DMSO-d6, 400 MHz) δ 11.27 (s, 1H), 8.31 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.36 (t, J = 7.2 Hz, 1H), 7.31 – 7.23 (m, 2H), 7.16 (t, J = 7.6 Hz, 1H), 7.13 – 7.07 (m, 2H), 6.87 (s, 1H), 6.71 (d, J = 8.0 Hz, 2H), 5.88 (s, 1H), 5.36 (s, 1H), 4.26 (s, 3H), 3.65 (s, 2H).

13

C NMR (DMSO-d6, 101

MHz) δ 155.31 , 149.01 , 145.07 , 142.56 , 141.96 , 141.53 , 139.36 , 130.02 , 126.88 , 35

ACCEPTED MANUSCRIPT 126.88 , 125.02 , 124.97 , 124.67 , 122.89 , 122.16 , 120.91 , 118.98 , 118.39 , 111.97 , 110.79 , 108.55 , 104.30 , 100.86 , 70.51 , 68.11 , 57.43 , 47.10 , 40.56 , 36.63 .ESI-MS: m/z calcd for C29H22N2O4 (M + H+) 462.2, found 462.4. Anal calcd

RI PT

for C29H22N2O4: C, 75.31; H, 4.79; N, 6.06; O, 13.84 Found C, 75.44; H, 4.59; N, 6.11; O, 13.97.

SC

1-((9H-carbazol-4-yl)oxy)-3-(naphthalen-2-ylamino)propan-2-ol. (10f)

M AN U

White solid, yield: 75%, m.p. 128–131oC. 1H NMR (DMSO-d6, 400 MHz) δ 11.26 (s, 1H), 8.31 (d, J = 7.6 Hz, 1H), 8.19 (d, J = 7.6 Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.48 – 7.40 (m, 3H), 7.38 – 7.32 (m, 1H), 7.30 – 7.24 (m, 2H), 7.16 - 7.06 (m, 3H), 6.73 – 6.66 (m, 2H), 4.46 – 4.40 (m, 1H), 4.34 – 4.29 (m, 2H), 3.68 – 3.63 (m, 1H), 3.48 – 13

C NMR (DMSO-d6, 101 MHz) δ 155.35 ,

TE D

3.44 (m, 1H), 1.26 – 1.14 (m, 1H).

144.27 , 141.53 , 139.33 , 134.47 , 128.40 , 127.13 , 126.87 , 126.05 , 124.95 , 124.47 , 123.50 , 122.92 , 122.15 , 121.88 , 118.95 , 116.18 , 112.01 , 110.77 , 104.31 , 103.71 ,

EP

100.87 , 70.90 , 67.67 , 47.55 . ESI-MS: m/z calcd for C25H22N2O2 (M + H+) 382.2,

AC C

found 382.2. Anal calcd for C25H22N2O2: C, 78.51; H, 5.80; N, 7.32; O, 8.37. Found C, 78.55; H, 5.89; N, 7.26; O, 8.44.

2-((9H-carbazol-4-yl)oxy)-1-(4-((methylimino)methyl)-1H-imidazol-1-yl)ethanol. (10g) Light yellow solid, yield: 50%, m.p. 285–287oC. 1H NMR (CDCl3, 400 MHz) δ 8.29 (d, J = 3.9 Hz, 2H), 7.43 – 7.40 (m, 2H), 7.34 (t, J = 5.4 Hz, 1H), 7.28 – 7.23 (m, 1H), 36

ACCEPTED MANUSCRIPT 7.06 (d, J = 8.0 Hz, 1H), 6.71 (d, J = 4.2 Hz, 1H), 4.47 – 4.38 (m, 1H), 4.36 – 4.29 (m, 2H), 3.89 – 3.72 (m, 2H), 3.66 – 3.60 (m, 2H), 1.30 – 1.24 (m, 3H). 13C NMR (CDCl3, 101 MHz) δ 155.00 , 140.94 , 138.73 , 129.83，127.42 , 126.59 , 124.95 , 122.82 ,

RI PT

122.43 , 119.56 , 112.63 , 110.04 , 103.88 , 101.16 , 71.57 , 69.26 , 68.99 , 66.97 , 15.07 . ESI-MS: m/z calcd for C19H18N4O2 (M + H+) 334.1, found 334.1. Anal calcd for C19H18N4O2: C, 68.25; H, 5.43; N, 16.76; O, 9.57. Found C, 68.25; H, 5.47; N,

M AN U

SC

16.83; O, 9.59.

4.2. Biology assays

4.2.1 The IC50 of the tested Compounds in HEK Blue cells.

HEK-blue hTLR4 cells (InvivoGen) were cultured in 96-well plates (4 × 104 cells

first

day in

200

TE D

per well) (Thermo Scientific) at 37°C with condition of 5% CO2 for 24 hours on the µL DMEM,

supplemented

with

10%

FBS

and

1%

penicillin/streptomycin. After 24 hours, nonadherent cells and medium were removed

EP

and replaced with unsupplemented fresh DMEM medium. The cells were treated with

AC C

indicated concentrations of compounds and 20 ng/ml LPS (InvivoGen) in 200 µL DMEM totally. After added compounds, cells were cultured for another 24 hours. On the third day, a sample buffer (50µL) was collected and transferred from each well of the cell culture supernatants to a transparent 96-well plate. Each well was treated with 50µL of QUANTI-Blue (InvivoGen) buffer and incubated at 37°C in dark place. Then measure the purple color by using a plate reader at an absorbance of 620nm (OD620) at 15-30 minutes. Finally, analyze the data by using Origin 8.5 software. 37

ACCEPTED MANUSCRIPT

4.2.2 NO activation in Raw 264.7 cells and BV-2 cells. The free nitrite was monitored by using a modification of the colorimetric Griess

RI PT

assay [24]. Briefly, dissolving 85% strong phosphoric acid (6 mL) and sulfanilic acid (1.0 g) in 96 mL water to make 100 mL obtained substrate solution A, then dissolving naphthyl ethylenediamine dihydrochloride (0.1 g) in 100 mL water to make substrate

SC

solution B.

M AN U

Raw 264.7 cells (mouse leukemic monocyte macrophage cell line) or BV-2 microglial cells were cultured in medium of DMEM supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml), seeded in 96-well plates at 50,000 cells of density per well, and pre-incubated for 24 hours at 37°C in a 5% CO2

TE D

humidified incubator. Removed nonadherent cells away and replaced with fresh unsupplemented DMEM medium after 24 hours incubation [29]. The adherent macrophages or BV-2 were treated with 40 ng/mL LPS (InvivoGen) and different

EP

concentrations of 8a or 10a. Cells were then incubated for an additional 24 hours.

AC C

After incubation, 50 µL of medium and standard nitrite solution was collected and added to a transparent 96-well plate, then added 50 µL substrate solution A and incubated for 10 minutes in dark. After that, another 50 µL substrate solution B was added and incubated in 37 °C incubator for 10 min. Finally, absorbance was detected at 450 nm by a microplate reader (Thermo Scientific) and afforded the content of nitric oxide by Origin 8.5 software.

38

ACCEPTED MANUSCRIPT 4.2.3 ELISA for TNF-α in PBMC. Human TNF-α Elisa assay kits (BD) were used according to the manufacturer’s instructions. PBMC were cultivated in 6-well plates at density of 1 ×106 cells per well

RI PT

with indicated concentrations of compound with/without LPS (10 ng/mL) in 3 ml medium of RPMI 1640, and incubate for 24 hours at 37°C in a 5% CO2 humidified incubator. After 24 hours, collect and froze cell culture supernatants at −80°C until

SC

ready for measuring the cytokine production. Use cytokine specific capture antibodies,

M AN U

detection antibodies, and recombinant human cytokine to quantify TNF-α cytokine production. The cytokine level in each sample was conducted in triplicate assays.

4.2.4 Western Blot.

TE D

HEK-Blue hTLR4 cells were cultured 24h in 6-well plates at density of 1×106 cells per well in 3 mL DMEM medium with 10% FBS and 1% penicillin/streptomycin. Removed nonadherent cells away and replaced with fresh unsupplemented DMEM

EP

medium after 24 hours incubation. The adherent HEK-Blue hTLR4 cells were treated

AC C

with 40 ng/mL LPS (InvivoGen) and different concentrations of 8a for 24h. Then, cells were washed in 1 × PBS buffer and lysed in cell lysisbuffer (Life Sciences) on ice for 15 min. The lysates were cleared by centrifugation at 4 °C, and the protein concentrations were measured by BCA analysis (Boster, China). Lysates containing 30 mg of total proteins were fractionated on a 10% SDS polyacrylamide gel and transferred to Hybond-P PVDF Membrane (Merck Millipore). Membranes were blocked with 5% (w/v) skim milk (Becton, Dickinson and Company) in TBST and 39

ACCEPTED MANUSCRIPT incubated with TLR4 primary antibody overnight at 4 oC on the shaker. After washing five times with TBST, membranes were incubated with their respective secondary antibody for 1 h in room temperature on the shaker. Detection was performed with

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ECL Chemiluminescent Substrate Reagent Kits (Life Sciences) and the Fluor Chem R Multifunctional Imaging Analysis System (Protein Simple). During this operation, the following primary and secondary antibodies were employed: rat-GAPDH (1:2000, Sciences),

rabbit-TLR4

BOSTER

(1:1000,

Biological Life

Technology), Sciences),

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goat-anti-rabbit-TLR4-HRP

(1:1000,

SC

Life

goat-anti-rat-GAPDH/TLR4-HRP (1:2500, BOSTER).

4.2.5 Transfection and Electrophysiology.

To express HCN channels in heterologous systems, COS7 cells were cultured in

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DMEM with supplemental 10% FBS, and maintained at a 6-well plate overnight, then transiently transfected with 1.5 µg plasmid DNA of HCN2 or HCN4 into cells using

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Lipofectamine 2000TM reagent (InvitrogenTM, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s specifications while the density of growth cells were

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70~90%. 24-48h after transfection, cells were split and redistributed onto coverslips pre-coated with poly-L-lysine (Sigma-Aldrich, St Louis, MO, USA) for the following electrophysiological experiments. Electrophysiological experiments were recorded using an Axopatch-700B amplifier interface to Digidata 1550B data acquisition system using the pClamp 10.6 software (Molecular Devices, USA). Recording pipettes were pulled from micropipette glass usingP-97 horizontal micropipette puller (Sutter Instrument, USA), with resistances of 40

ACCEPTED MANUSCRIPT 2-5 MΩ when filled with the intracellular solution containing (in mM): 140 KCl, 1 MgCl2, 5 EGTA and 10 HEPES, pH adjusted to 7.2 with KOH. The extracellular solution contained (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10

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HEPES, pH adjusted to 7.4 with NaOH. Data were filtered at 2 kHz and digitized at 20 kHz.

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4.2.6. Molecular docking studies.

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The crystal structure of TLR4 and MD-2 in complex with LPS (PDB 3FXI) was downloaded from Protein Data Bank. 3D structures of the selected compounds were generated and optimized by the Glide 7.4 program (Glide, Schrödinger, LLC, New York, NY, 2018).The TLR4/MD-2 conformations were prepared using standard Glide

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protocols. This includes addition of hydrogens, restrained energy-minimizations of the protein structure with the Optimized Potentials for Liquid Simulations-All Atom (OPLS-AA) force field, and finally setting up the Glide grids using the Protein and

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Ligand Preparation Module [30].

Acknowledgments

This work was supported, in part, by the introduction of scientific research project

of start-up support in Southern Medical University of China (No. C1033269), Youth Pearl River Scholar Program of Guangdong Province (No. C1034007) and National Natural Science Foundation of China (No. K1011638).

41

ACCEPTED MANUSCRIPT Appendix A. Supplementary data Supporting Information Available: Supplementary data for the representative NMR

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spectra associated with this article can be found in the online version at http://

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[1] H. Negishi, H. Yanai, A. Nakajima, R. Koshiba, K. Atarashi, A. Matsuda, K. Matsuki, S. Miki, T. Doi, A. Aderem, J. Nishio, S.T. Smale, K. Honda, T. Taniguchi, Cross-interference of RLR and TLR signaling pathways modulates antibacterial T cell responses, Nat. Immunol. 13 (2012) 659-666, https://doi.org/ 10.1038/ni.2307. [2] L. Ahlers, A. Goodman, Nucleic acid sensing and innate immunity: signaling pathways controlling viral pathogenesis and autoimmunity, Curr. Clin. Microbiol. Rep. 3 (2016) 132–141, https://doi.org/10.1007/s40588-016-0043-5. [3]J.K. Ryu, S.J. Kim, S.H. Rah, J.I. Kang, H.E. Jung, D. Lee, H.K. Lee, J.O. Lee, B.S. Park, T.Y. Yoon, H.M. Kim, Reconstruction of LPS Transfer Cascade Reveals Structural Determinants within LBP, CD14, and TLR4-MD-2 for Efficient LPS Recognition and Transfer, Immunity. 46 (2017) 38-50, https://doi.org/10.1016/j.immuni.2016.11.007. [4] J. Kato, N.M. Agalave, Pattern recognition receptors in chronic pain: Mechanisms and therapeutic implications, Eur. J. Pharmacol. 788 (2016) 261-273, https://doi.org/10.1016/j.ejphar.2016.06.039. [5] S. De, H. Zhou, D. DeSantis, C.M. Croniger, X. Li, G.R. Stark, Erlotinib protects against LPS-induced endotoxicity because TLR4 needs EGFR to signal, Proc. Natl. Acad. Sci. USA. 112 (2015) 9680-9685, https://doi.org/10.1073/pnas.1511794112. [6] F. Peri, M. Piazza, Therapeutic targeting of innate immunity with Toll-like receptor 4 (TLR4) antagonists, Biotechnol. Adv. 30 (2012) 251-260, https://doi.org/10.1016/j.biotechadv.2011.05.014. [7] P. Xiang, T. Chen, Y. Mou, H. Wu, P. Xie, G. Lu, X. Gong, Q. Hu, Y. Zhang, H. Ji, NZ suppresses TLR4/NF-κB signalings and NLRP3 inflammasome activation in LPS-induced RAW264.7 macrophages, Inflamm. Res. 64 (2015) 799-808, https://doi.org/10.1007/s00011-015-0863-4. [8] L. Yu, M. Yin, X. Yang, M. Lu, F. Tang, H. Wang, Calpain inhibitor attenuates atherosclerosis and inflammation in atherosclerotic rats through eNOS/NO/NF-κB pathway, Can. J. Physiol. Pharmacol. 7 (2017) 1-8, https://doi.org/10.1139/cjpp-2016-0652. [9] R. Eissler, C. Schmaderer, K. Rusai, L. Kühne, D. Sollinger, T. Lahmer, O. Witzke, J. Lutz, U. Heemann, M. Baumann, Hypertension augments cardiac Toll-like receptor 4 expression and activity, Hypertens Res. 5 (2011) 551-558, https://doi.org/10.1038/hr.2010.270. [10] L. Liu, Y. Wang, Z. Cao , M. Wang, X. Liu, T. Gao, Q. Hu, W. Yuan, L. Lin, Up-regulated TLR4 in cardiomyocytes exacerbates heart failure after long-term 42

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myocardial infarction, J Cell Mol Med. 12 (2015) 2728-2740, https://doi.org/10.1111/jcmm.12659. [11] I. Gárate, B. García-Bueno, J. Madrigal, J. Caso, L. Alou, M. Gómez-Lus, J. Leza, T. Kitazaki, Toll-like 4 receptor inhibitor TAK-242 decreases neuroinflammation in rat brain frontal cortex after stress, J. Neuroinflammation. 11 (2014) 8-22, https://doi.org/10.1186/1742-2094-11-8. [12] S. Chavez, A. Martinko, C. Lau, M. Pham, K. Cheng, D. Bevan, T. Mollnes, H. Yin, Development of β-amino alcohol derivatives that inhibit Toll-like receptor 4 mediated inflammatory response as potential antiseptics, J Med Chem. 13 (2011) 4659-4669, https://doi.org/10.1021/jm2003365. [13]G. Zhu, Y. Xu, X. Cen, K.S. Nandakumar, S. Liu, K. Cheng, Targeting pattern-recognition receptors to discover new small molecule immune modulators, Eur J Med Chem. 144 (2017) 82-92, https://doi.org/10.1016/j.ejmech.2017.12.026. [14] Y. Yang, J. Lv, S. Jiang, Z. Ma, D. Wang, W. Hu, C. Deng, C. Fan, S. Di, Y. Sun, W. Yi, The emerging role of Toll-like receptor 4 in myocardial inflammation, Cell Death Dis. 7 (2016) e2234, https://doi.org/10.1038/cddis.2016.140. [15] L. Liu , Y. Wang, Z. Cao, M. Wang, X. Liu, T. Gao, Q. Hu, W. Yuan, L. Lin, Up-regulated TLR4 in cardiomyocytes exacerbates heart failure after long-term myocardial infarction, J Cell Mol Med. 12 (2015) 2728-2740, https://doi.org/10.1111/jcmm.12659. [16] K. Amirshahrokhi, A.R. Khalili, Carvedilol attenuates paraquat-induced lung injury by inhibition of proinflammatory cytokines, chemokine MCP-1, NF-κB activation and oxidative stress mediators, Cytokine. 88 (2016) 144-153, https://doi.org/10.1016/j.cyto.2016.09.004. [17] M. Baruscotti, A. Barbuti, A. Bucchi, The cardiac pacemaker current, J Mol Cell Cardiol, 1 (2010) 55-64, https://doi.org/10.1016/j.yjmcc.2009.06.019. [18] D. Difrancesco, HCN4, Sinus Bradycardia and Atrial Fibrillation, CIRC-Arrhythmia Elec. Rev. 4 (2015) 9-13, https://doi.org/ 10.15420/aer.2015.4.1.9. [19] Q. Zhou, J. Xiao, D. Jiang, R. Wang, K. Vembaiyan, A. Wang, C.D. Smith, C. Xie, W. Chen, J. Zhang, Carvedilol and its new analogs suppress arrhythmogenic store overload-induced Ca2+ release, Nat. Med. 17 (2011) 1003-1009, https://doi.org/10.1038/nm.2406. [20] S.A. Chavez, A.J. Martinko, C. Lau, M.N. Pham, K. Cheng, D.E. Bevan, T.E. Mollnes, H. Yin, Development of beta-amino alcohol derivatives that inhibit Toll-like receptor 4 mediated inflammatory response as potential antiseptics, J. Med. Chem. 54 (2011) 4659-4669, https://doi.org/ 10.1021/jm2003365. [21] M. Oda,H. Yamamoto, M. Shibutani, M. Nakano,K. Yabiku, T. Tarui, N. Kameyama, D. Shirakawa, S. Obayashi, N. Watanabe, H. Nakase, M. Suenaga, Y. Matsunaga, M. Nagahama, H. Takahashi, H. Imagawa, M. Kurosawa, Y. Terao, M. Nishizawa, J. Sakurai, Vizantin inhibits endotoxin-mediated immune responses via the TLR 4/MD-2 complex, J. Immunol. 193 (2014) 4507-4514, https://doi.org/10.4049/jimmunol.1401796. [22] K. Tombácz, D. Mwangi, D. Werling, A.J. Gibson, Comparison of cellular assays for TLR activation and development of a species-specific reporter cell line for cattle, 43

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44

ACCEPTED MANUSCRIPT O O

OH

NH2

O

H N

O

O

n

(vi) OH

6a-b

O O

(i)

Br

O

R1

Br

n

6a: n=1 6b: n=2

O

(ii)

R1

1a-b

7a: n=1; R1=-ethoxy; R2=o-Cl 7b: n=2; R1=-ethoxy; R2=o-Cl 7c: n=2; R1=-F; R2=o-Cl 7d: n=2; R1=-Me; R2=o-Cl 7e: n=2; R1=-Et; R2=o-Cl 7f: n=2; R1=-Pr; R2=o-Cl 7g: n=2; R1=-t-Bu; R2=o-Cl 7h: n=2; R1=-ethoxy; R2=o-F 7i: n=2; R1=-ethoxy; R2=o-Me 7j: n=2; R1=-ethoxy; R2=p-Cl

R2

n

n

2a-g

N

OH (vi)

O

N

N n

R2 +

R3

R3

R3 Cl

NH N

R2

(iii)

(iv)

N N

HN

3a-d

4a-d

(R3= H or Me) H N

O

7a-j

R1

N N

(v)

(vi)

H N

HN

O

N

n

OH

n

1a-b

R3

N

+

Br

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R2

N

8a-f HO

n

O

O

5a-b O NH2

HN

O

n

N H

O

SC

O

8a: n=1; R2=o-Cl; R3=-Me 8b: n=2; R2=o-Cl; R3=-Me R2 8c: n=1; R2=-H; R3=-Me 8d: n=1; R2=p-Cl; R3=-Me 8e: n=1; R2=o-F; R3=-Me 8f: n=1; R2=o-Cl; R3=-H

OH

(vi)

O

9a: n=1 9b: n=2

9a-b

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Scheme 1.Synthesis of compounds6a-b, 7a-j, 8a-f and 9a-b. Reagents and conditions: (i) mCPBA, CH2Cl2, rt, 24h. (ii) K2CO3, acetone, rf, 20h. (iii) Substituent pyrazole, KOH, DMSO, 80oC, 1.5h; then Benzyl chloride, rt, 2h (iv) CH3NH2, (CHO)n, EtOH, rf,16h. (v) DMSO, NaOH, 60oC, 24h. (vi) EtOH, rf, 24h.

Scheme 2. Synthesis of compounds 10a-g.

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ACCEPTED MANUSCRIPT Table 1. The compounds IC50 values in human HEK-Blue hTLR4 cell. Antagonist IC50 (µM)a

Compd

6a

NAb

8c

31.54±2.15

6b

NA

8d

13.64±1.03

34.17±5.46

8e

NA

8f

7a (T5342126)

7c

55.39±9.16

Antagonist IC50 (µM)a

34.64±1.21

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7b

Structure

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Structure

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Compd

9a

NA

32.22±0.01

(Carvedilol)

7d

51.54±13.67

7e

26.02±2.82

9b

32.55±1.16

NA

24.68±1.57

10b

21.71±1.45

16.68±0.23

10c

12.39±0.83

NA

10d

21.12±0.55

7i

54.60±3.12

10e

NA

7j

25.92±0.17

10f

28.53±1.55

8a

12.45±0.29

10g

NA

8b

15.11±1.94

7g

7h

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7f

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10a

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(VK-II-86)

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Data were mean ± SD. bNA means no inhibition effect.

Table 2. Biophysical characteristics of inhibition of 10 µM 8a on HCN2 and HCN4 channels V1/2 (mV)

Tact (ms), -120mV

Channel

Tdeact (ms), +50mV

∆V1/2 (mV) Control

10 µM 8a

Control

10 µM 8a

Control

10 µM 8a

-89.9 ± 0.9

-113.8 ± 0.4

-21.4 ± 2.6

242.9 ± 24.0

891.6 ± 115.7＊

66.0±4.2

53.1± 2.0

HCN4

-102.1 ± 0.9

-126.9± 3.7

-28.8 ± 2.1

780.9 ± 59.2

2150.0 ± 346.8＊

92.9±9.5

79.9± 1.9

*

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P<0.001 vs Control.

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HCN2

Test compounds

I/I0[a] on HCN2

I/I0 on HCN4

T5342126 (7a)

0.73±0.04

0.75±0.04

SMU-XY3 (8a)

0.70±0.07

0.50±0.04

Carvedilol (9a)

0.66±0.06

0.75±0.04

VK-II-86 (9b)

0.96±0.02

0.78±0.03

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0.82±0.07

10a [a]

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Table 3. Inhibition effects of 8a and a series of compounds on HCN2 and HCN4 channels.

1.00±0.07

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I0 is the amplitude of inward HCN currents at -120 mV in control condition, while I is the amplitude of inward HCN2 currents in the presence of the corresponding compound at 10 µM. Each compound was tested on at least at two different days and in 3-8 cells.

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ACCEPTED MANUSCRIPT (B) 0.9

0.7 0.6 0.5 0.4

0.8 0.7 0.6 0.5 0.4

0.3

0.3 0

5 2 6 .5 1.5 3.1 6.2 12

25

20 ng/mL LPS + [8a] 20 ng/mL LPS + [7a] 20 ng/mL LPS + [Carvedilol]

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20ng/mL LPS+ [8a]

0.8

TLR4 SEAP signals (OD620)

TLR4 SEAP signals (OD620)

(A)

10

50 100 lank b

100

[Compound] / µM

[Compound] / µM

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Fig. 1. SEAP signal analysis with different TLR4 antagonists in HEK-Blue hTLR4 cells. (A) Cells were treated with 20 ng/mL LPS and different concentrations of 8a ranging from 1.56 to 100 µM for 24 h. SEAP release in the conditioned medium was determined by Quanti-Blue, and the absorbance was detected at 620 nm. (B) 8a, 7a, and Carvedilol inhibit the LPS (20 ng/mL) stimulated SEAP signaling in a dose-dependent manner. Data represent the mean of two independent experiments carried out in triplicate.

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40 ng/mL LPS + [8a] 40 ng/mL LPS + [10a]

0.4

Nitric Oxide (OD560)

Nitric Oxide (OD560)

40 ng/mL LPS + [8a] 40 ng/mL LPS + [10a]

0.20

0.15

0.10

0.05 1

BV-2 cells

(B)

RAW 264.7 cells 0.25

10

0.3

0.2

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(A)

0.1 1

100

10

TNF-α (OD 450)

OD 450

0.6 0.5 0.4 0.3 0.2 0.1

0.01

0.1

1

10

[LPS]/(ng/mL)

100

10 ng/mL LPS + [8a] [8a]

1.4

SC

(D)

TNF−α in PBMC

0.7

1.2 1.0 0.8 0.6

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(C)

100

[Compound] / µM

[Compound] / µM

0.4 0.2 0.0

0

1

10 20 [8a] / µM

50

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Fig. 2. Compound 8a inhibit of TLR4-related inflammatory cytokines. (A) Raw 264.7 cells were incubated with LPS and indicated concentration of 8a or 10a for 24 hours, and the inhibition was measured by the absorbance of NO signals in the culture medium supernatants at OD560. Compound 10a was as a negative control. (B) 8a inhibits the NO signaling in a dose-dependent manner in BV-2 cells. (C) LPS activates TNF-α in a dose-dependent manner in PBMC. (D) Compound 8a inhibits TNF-α in PBMC. PBMC were co-cultured with concentrations of 8a with/without 10 ng/mL LPS for 24 h. The TNF-α expression in the supernatant was measured via ELISA. Results shown are averages of three independent experiments and error bars represent mean ± SD.

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Fig. 3. Western blot analysis for compound 8a's effect on the LPS activated TLR4 signaling in HEK-Blue hTLR4 cells and its viability. (A) 8a inhibits LPS triggered TLR4 in cellular environment. HEK Blue hTLR4 cells, which overexpress the human TLR4, were treated with LPS (40 ng/mL) and 8a (0 to 20 µM) for 24 hours, and cell lysates were immuno precipitated and detected by anti-TLR4 antibody in the Western blotting analysis. GAPDH served as the cell lysates input control. (B) HEK-Blue hTLR4 cells were incubated with LPS (40 ng/mL) and 8a (0 to 100 µM) for 24 hours, thenthe viability was detected by CCK8 at the absorbance of 450 nm. Data shown are mean ± SD of representative data of three independent experiments. **P < 0.05.

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Figure 4. Computational docking studies of compounds 8a to TLR4/MD-2 complex. (A) Molecular docking of compound 8a (purple) in the LPS (red line) binding pocket of human TLR4/MD-2 complex (PDB 3FXI). Overlay of binding geometry between compound 8a (purple) and LPS (red) with TLR4 (green) and MD-2 (cyan). (B) Magnified view of the interface of TLR4 and MD-2. The benzyl ring of 8a is binding to TLR4, and the carbazole group is located in the MD-2 pocket.

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Fig. 5. Inhibition effects of 8a on HCN2 and HCN4 channels. (A, B) Typical HCN2 (A) and HCN4 (B) current traces in the absence or presence of 10 µM 8a elicited by hyperpolarizing step to -120 mV from a holding potential of -40 mV. Tail currents were measured at +50 mV and the protocol was applied every 15 s until achieving stable inhibition. (C) The inhibitory effects of 10 µM 8a on HCN2 and HCN4 channels obtained at -120 mV. (D, E) Representative traces of HCN2 (D) and HCN4 (E) currents in the absence (upper) or presence (bottom) of 10 µM 8a, HCN currents were elicited by a series hyperpolarizing steps ranging from -50 mV to -140 mV in -10 mV decrements from a holding potential of -40 mV. Voltage was then returned to +50 mV to measure the tail currents. (F, G) Normalized voltage-dependent activation curves of HCN2 (F) and HCN4 (G) channels before and after treatment with 10 µM 8a. The dotted curve represented data normalized to its own peak amplitude. Data were fitted with a Boltzmann equation to estimate the V1/2 for channel activation. Error bars indicate S.E.M.

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ACCEPTED MANUSCRIPT Highlights for this paper

We first prove that Carvediol can inhibit TLR4 in vitro. A series of novel Carvedilol derivatives were generated to investigate their TLR4

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inhibition with compound 8a optimized. 8a has a higher affinity for HCN4 channel subtypes than HCN2.

Inhibitors of TLR4 may affect the HCN pathway for the treatment of

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cardiovascular disease.