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

Article abstract of DOI:10.1016/j.ejmech.2018.05.033

Toll-like receptor 4 (TLR4) initiates innate immune response to release inflammatory cytokines and has been pathologically linked to variety of inflammatory diseases. 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 novel Carvedilol derivatives and investigated their potential inhibitory activity. The 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 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 failure, coronary heart disease, and other inflammatory diseases.

Full text of DOI:10.1016/j.ejmech.2018.05.033

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
10.1016/j.ejmech.2018.05.033
EJMECH 10444
DOI:
Reference:
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.
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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
*
Cheng
Guangdong Provincial Key Laboratory of New Drug Screening and Guangzhou Key
Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School
of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515,
China.
Phone: +86 20 6164 7192, Fax: +86 20 6164 8533
Email address: Chengk@smu.edu.cn
†
These authors contributed equally to this work.
1

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Abstract:
Toll-like receptor 4 (TLR4) initiates innate immune response to release inflammatory
cytokines and has been pathologically linked to variety of inflammatory diseases.
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
novel Carvedilol derivatives and investigated their potential inhibitory activity. The
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
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
failure, coronary heart disease, and other inflammatory diseases.
Keywords: Toll-like receptor 4; anti-inflammatory; Carvedilol derivative; Small
molecule antagonist.
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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
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
activation; our body adopts a series of positive or negative molecular-level regulation
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].
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;
the other sub-family contains TLR3, TLR7, TLR8 and TLR9, which are distributed on
organelle membrane, and mainly recognize nucleic acids as well as
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
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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
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
pressure can increase the activity of TLR4 expression [9]; upregulation of TLR4
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
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,
presumably functioning by blocking MD-2, TLR4 interaction mechanism [12]. AV411,
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
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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
reduce the expression of TLR4-related inflammatory cytokines, and it is benefical in
the recovery of the heart from acute injury.
Carvedilol, a listed classic drug, has good anti-heart failure activity and it is a
non-selective third-generation beta-blocker without intrinsic sympathomimetic
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
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
treatment of heart related diseases is particularly important and direct. Previously, it
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
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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
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
expected to find a better TLR4 inhibitory activity compound with a higher affinity for
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
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,
we also explore the selectivity of their HCN channel subtypes by measuring the
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
2
. Results and discussion
.1 Chemistry
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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
2
a-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
,5-dimethyl-1H-pyrazole intermediates where paraformaldehyde provided the
the
3
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
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
ethanol yield compounds 6a-b, while there wasn’t any TLR4 inhibitory activity been
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).
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2
2
.2. Biological activity.
.2.1 In vitro TLR4 inhibition assay.
In vitro assay was used to screen all the newly synthesized compounds for their
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
specific ligand LPS can activate the NF-κB signaling pathway to induce the SEAP
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
TLR4 with IC of 33.22 µM. Following study observed its linker carbon extension
5
0
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
extending the carbon chain of 7a the inhibitory activity was completely abolished (7a
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,
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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,
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
group of the pyrazole ring significantly reduced the activity (8d vs 8f, Table 1), which
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
(
1
0e. Based on such results, the best compound 8a (IC =12.45±0.29 µM, Fig. 1) was
50
selected out for further evaluation and compound 10a was chosen as a negative
control in the following assay.
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
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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
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).
Compound 10a was used as the negative control in the NO assay. Human
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
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).
These data showed that 8a could inhibit the TLR4-related inflammatory cytokines
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
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TLR4 in a concentration dependent manner. TLR4 protein was suppressed more than
9
0% 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
cells, which further indicated the inhibition of TLR4 protein and SEAP, NO and
TNF-α above mentioned were not caused by the toxicity.
2
.2.4 Molecular docking studies of 8a (SMU-XY3).
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
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
TLR4/MD-2 complex by conducting molecular docking experiments in the human
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
1
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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
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.
2
.2.5 Electro physiological properties of 8a blockage on HCN channels
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
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,
were investigated in the study. Especially HCN4 is highly expressed in the
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/I ) were analyzed. After
0
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/I for
0
HCN2 was 0.71 ± 0.06 (n = 6) and for HCN4 was 0.50 ± 0.04 (n = 7). Then we
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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
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
(Table 2). Besides, 8a slowed the speed of current activation at -120 mV while it had
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).
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
hTLR4 cells and human PBMC. We first found Carvedilol, a classic anti-heart failure
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
1
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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
channel subtypes which could pave the way for future treatment of inflammatory
disease, heart disease and others.
4
4
. Experimental section
.1. Chemistry
Chemicals were purchased from Macklin or Aladdin Biochemical Technology
1
and used without further purification. Hydrogen nuclear magnetic resonance ( H
1
3
NMR) spectra and carbon nuclear magnetic resonance ( C NMR) spectra were
recorded on a Bruker AM-400 spectrometer in CDCl or DMSO-d , and chemical
3
6
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
compounds were routinely checked by thin-layer chromatography (TLC), and the
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 CH Cl . The reaction mixture was stirred for
2
2
2
4h. 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
1
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by H O until the aqueous washing reached pH=7. The organic phase was dried over
2
Na SO and the solvent removed under reduced pressure. Yield 90% (1020 mg) of
2
4
compound 1a as clear oil. Generation of the compound 1b was accomplished by using
similar reaction conditions.
General procedure for the synthesis of compound 2a-g.
4
2
-Ethoxyphenol (500mg, 3.62 mmol), anhydrous potassium carbonate (366mg,
.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
with water, 5% aqueous NaOH, and water again before being dried with Na SO and
2
4
concentrated under reduced pressure. The resulting liquid was purified by flash
chromatography on silica-gel with 20% ethyl acetate in petroleum. Yielding 83% of
compound 2a (585 mg) as a clear liquid. Compounds 2b-g were synthesized following
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
o
h at 80 C 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
1
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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
CHCl . The combined organic layers were washed with 100ml of water, dried with
3
anhydrous Na SO and concentrated under reduced pressure. The residue was purified
2
4
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
synthesized following the procedure of preparation 3a.
General procedure for the synthesis of compound 4a-d.
Paraformaldehyde (820mg, 27.2mmol) and methylamine hydrochloride (920mg,
1
3.6mmol) were dissolved in ethanol and stirred for 1h, then 3a (1000mg, 4.53mmol)
was added and the reaction mixture was stirred at reflux for 16h. The mixture liquor
then cooled to room temperature and quenched with aqueous NaHCO (15 mL). The
3
aqueous layer was extracted 3 times with chloroform (3×15 mL) and the combined
organic layers were washed with brine (30 mL). The organic layer was dried with
Na SO and concentrated under reduced pressure. The resulting yellow oil was
2
4
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.
1
a (897 mg, 6.55 mmol) and NaOH (222 mg, 5.55 mmol) solution were added to a
1
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solution of 4-hydroxycarbazole (1000 mg, 5.55 mmol) in DMSO. The reaction
o
o
mixture was stirred for 24h at 60 C. It was then cooled to 20 C, diluted with 25ml of
water and extracted with ethyl acetate three times (3×20mL). The collected organic
layers were washed with brine, dried with Na SO and concentrated. The residue was
2
4
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
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
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 68% compound 6a (128 mg) as a white solid. Compound 6b was
synthesized following the procedure of preparation 6a.
General procedure for the synthesis of compound 7a-j.
2
a (100mg, 0.52mol) and 4a (163 mg, 0.62 mmol) were dissolved in ethanol and
o
warmed to 75 C 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
1
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bicarbonate, and the organic layer was dried with Na SO and the solvent was
2
4
removed under reduced pressure. The resulting oil was purified using flash column
chromatography with 2% methanol in dichloromentane as an eluting solvent. Yielding
8
4% of 7a (200 mg) as a white solid. Compounds 7b-g were synthesized following the
procedure of preparation 7a.
General procedure for the synthesis of compound 8a-f.
5
a (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
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
preparation 8a.
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
1
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synthesized following the procedure of preparation 9a.
General procedure for the synthesis of compound 10a-g.
5
a (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
concentrated under reduced pressure. The residue was purified by flash
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.
2
-(bromomethyl)oxirane. (1a)
1
Light yellow liquid, yield: 92%. H NMR (CDCl , 400 MHz) δ 2.86 – 2.77 (m, 1H),
3
1
3
2
.75 – 2.67 (m, 2H), 2.30 – 2.24 (m, 1H), 2.03 – 1.95 (m, 1H). C NMR (CDCl , 101
3
+
MHz) δ 51.10, 48.30, 33.20. ESI-MS: m/z calcd for C H BrO (M + H ) 137.0, found
3
5
1
37.0. Anal calcd for C H O: C, 26.31; H, 3.68; O, 11.68. Found C, 26.29; H, 3.70; O,
3 5
1
1.71.
2
-(2-bromoethyl)oxirane. (1b)
1
Light yellow liquid, yield: 90%. H NMR (CDCl , 400 MHz) δ 3.69 – 3.39 (m, 2H),
3
3
2
.13 – 3.07 (m, 1H), 2.89 – 2.77 (m, 1H), 2.66 – 2.54 (m, 1H), 2.20 – 2.13 (m, 1H),
1
3
.10 – 2.02 (m, 1H). C NMR (CDCl , 101 MHz) δ 50.58, 46.90 , 35.60 , 28.92.
3
1
9

ACCEPTED MANUSCRIPT
+
ESI-MS: m/z calcd for C H BrO (M + H ) 150.0, found 150.2. Anal calcd for C H O:
4
7
4
7
C, 31.82; H, 4.67; O, 10.60. Found C, 31.87; H, 4.64; O, 10.70.
2
-(2-(4-ethoxyphenoxy)ethyl)oxirane. (2b)
1
Clear liquid, yield: 83%. H NMR (CDCl , 400 MHz) δ 6.85 (s, 4H), 4.10 – 3.96 (m,
3
4
1
1
3
H), 3.19 – 3.15 (m, 1H), 2.86 – 2.83 (m, 1H), 2.62 – 2.58 (m, 1H), 2.14 – 2.05 (m,
1
3
H), 1.98 – 1.90 (m, 1H), 1.44 – 1.38 (m, 3H). C NMR (CDCl , 101 MHz) δ
3
53.19 , 152.76 , 115.38 , 115.38 , 115.34 , 115.34 , 65.19 , 63.89 , 49.69 , 47.01 ,
+
2.50 , 14.87 . ESI-MS: m/z calcd for C H O (M + H ) 208.1, found 208.2. Anal
12
16
3
calcd for C H O : C, 69.21; H, 7.74; O, 23.05. Found C, 69.18; H, 7.62; O, 23.08.
1
2
16
3
1
-(2-chlorobenzyl)-3,5-dimethyl-1H-pyrazole. (3a)
1
Clear liquid, yield: 87%. H NMR (CDCl , 400 MHz) δ 7.37 (d, J = 7.6 Hz, 1H), 7.22
3
–
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
1
3
(
s, 3H). C NMR (CDCl , 101 MHz) δ 156.58 , 129.98 , 129.86 , 114.32 , 64.62 ,
3
4
9.70 , 47.03 , 46.93 , 35.65 , 32.48 , 28.90 , 20.41 . ESI-MS: m/z calcd for
+
C H ClN (M + H ) 220.1, found 220.4. Anal calcd for C H N : C, 65.31; H, 5.94;
12
13
2
12 13
2
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)
1
Yellow oil, yield: 77%. H NMR (CDCl , 400 MHz) δ 7.40 – 7.36 (m, 1H), 7.24 –
3
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,
2
0

ACCEPTED MANUSCRIPT
1
3
3
6
H), 2.28 (s, 3H). C NMR (CDCl , 101 MHz) δ 156.40 , 143.49 , 126.18 , 113.91 ,
3
4.52 , 50.65 , 49.74 , 47.06 , 46.96 , 35.64 , 34.01 , 32.47 , 31.47 , 28.87 . ESI-MS:
+
m/z calcd for C H ClN (M + H ) 263.1, found 263.3. Anal calcd for C H N : C,
1
4
18
3
14 18
3
6
3.75; H, 6.88; N, 15.93. Found C, 63.81; H, 6.84; N, 15.98.
4
-(oxiran-2-ylmethoxy)-9H-carbazole. (5a)
o
1
White solid, yield: 60%, m.p. 135–138 C. H NMR (DMSO-d , 400 MHz) δ 11.33 (s,
6
1
H), 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,
1
1
1
6
H), 4.57 – 4.51 (m, 1H), 4.11 – 4.05 (m, 1H), 3.56 – 3.51 (m, 1H), 2.96 – 2.92 (m,
1
3
H), 2.87 – 2.83 (m, 1H).
C NMR (DMSO-d , 101 MHz) δ 154.93 , 141.59 ,
6
39.42 , 126.87 , 125.10 , 122.75 , 122.04 , 119.11 , 111.98 , 110.91 , 104.69 , 101.12 ,
+
9.16 , 50.36 , 44.20 . ESI-MS: m/z calcd for C H NO (M + H ) 239.1, found 239.3.
1
5
13
2
Anal calcd for C H NO : C, 75.30; H, 5.48; N, 5.85. Found C, 75.31; H, 5.42; N,
1
5
13
2
5
.88.
1
-(4-ethoxyphenoxy)-3-(((2-methoxyphenoxy)methyl)amino)propan-2-ol. (6a)
o
1
Yellow solid, yield: 66%, m.p. 78–81 C. H NMR (CDCl , 400 MHz) δ 6.98 – 6.93
3
(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
13
–
3.95 (m, 5H), 3.85 – 3.81 (m, 2H), 3.20 – 2.80 (m, 4H), 1.44 – 1.37 (m, 3H). C
NMR (CDCl , 101 MHz) δ 153.20 , 152.77 , 149.30 , 147.81 , 121.56 , 120.85 ,
3
1
15.40 , 115.31 , 113.60 , 113.42 , 111.50 , 70.58 , 68.28 , 67.38 , 63.91 , 59.56 ,
2
1

ACCEPTED MANUSCRIPT
+
5
5.56 , 54.92 , 14.88 . ESI-MS: m/z calcd for C H NO (M + H ) 346.2, found 346.2.
19 25 5
Anal calcd for C H NO : C, 65.69; H, 7.25; N, 4.03. Found C, 65.70; H, 7.33; N,
1
9
25
5
4
.05.
1
-(4-ethoxyphenoxy)-3-((2-(2-methoxyphenoxy)ethyl)amino)propan-2-ol.(6b)
o
1
Yellow solid, yield: 60%, m.p. 55–58 C. H NMR (CDCl , 400 MHz) δ 6.98 – 6.89
3
(m, 4H), 6.86 – 6.81 (m, 4H), 4.15 – 4.06 (m, 4H), 4.01 – 3.92 (m, 3H), 3.86 (s, 3H),
3
1
1
6
.10 – 3.05 (m, 2H), 2.88 – 2.84 (m, 1H), 2.68 – 2.62 (m, 1H), 1.98 – 1.81 (m, 2H),
13
.42 – 1.38 (m, 3H).
C NMR (CDCl , 101 MHz) δ 153.01 , 152.97 , 152.93 ,
3
49.38 , 147.86 , 121.57 , 120.79 , 115.42 , 115.33 , 113.75 , 113.58 , 111.49 , 67.40 ,
5.54 , 65.04 , 63.93 , 61.58 , 55.55 , 34.09 , 14.89 . ESI-MS: m/z calcd for
+
C H NO (M + H ) 361.2, found 361.4. Anal calcd for C H NO : C, 66.46; H,
20
27
5
20 27
5
7
.53; N, 3.88. Found C, 66.44; H, 7.54; N, 3.85.
1
-(((1-(2-chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)methyl)(methyl)amino)-3-(
4
-ethoxyphenoxy)propan-2-ol. (7a T5342126)
o
1
White solid, yield: 85%.m.p. 61–63 C. H NMR (CDCl , 400 MHz) δ7.38 (d, J =
3
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,
H), 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
2
=
13.2 Hz, 2H), 3.35 (d, J = 13.2 Hz, 1H), 2.66 – 2.61 (m, 1H), 2.53 – 2.49 (m, 1H),
1
3
2
.29 (s, 6H), 2.15 (s, 3H), 1.41 (t, J = 7.0 Hz, 3H).
C NMR (CDCl , 101 MHz)δ
3
1
53.25 , 152.83 , 147.79 , 138.39 , 135.09 , 131.69 , 129.18 , 128.53 , 127.37 , 127.25 ,
2
2

ACCEPTED MANUSCRIPT
1
15.42 , 115.42 , 115.31 , 115.31 , 113.48 , 71.06 , 66.18 , 63.91 , 59.30 , 51.49 ,
+
5
0.05 , 41.88 , 14.89 , 12.12 , 9.58 . ESI-MS: m/z calcd for C H ClN O Na (M +
2
5
32
3
3
+
Na ) 480.2, found 480.2. Anal calcd for C H N O : C, 65.56; H, 7.04; N, 9.17.
2
5
32
3
3
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-(
4
-ethoxyphenoxy)butan-2-ol. (7b)
1
Yellow oil, yield: 84%. H NMR (CDCl , 400 MHz) δ 7.37 (d, J = 8.0 Hz, 1H), 7.23 –
3
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,
13
3
H), 1.93 – 1.78 (m, 2H), 1.42 – 1.37 (m, 3H).
C NMR (CDCl , 101 MHz) δ
3
1
53.08 , 152.88 , 147.83 , 134.98 , 131.72 , 129.21 , 128.57 , 127.38 , 127.24 , 115.42 ,
15.40 , 115.35 , 77.27 , 66.62 , 65.28 , 64.19 , 63.95 , 62.81 , 51.35 , 50.08 , 41.49 ,
1
+
3
4.61 , 32.62 , 14.88 , 12.13 , 9.65 . ESI-MS: m/z calcd for C H ClN O (M + H )
2
6
34
3
3
4
71.2, found 471.3. Anal calcd for C H ClN O : C, 66.16; H, 7.26; N, 8.90. Found
26 34 3 3
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)
1
Yellow oil, yield: 80%. H NMR (CDCl , 400 MHz) δ 7.37 (d, J = 7.6 Hz, 1H), 7.22 –
3
7
2
.12 (m, 2H), 7.04 – 6.92 (m, 2H), 6.90 – 6.82 (m, 2H), 6.53 – 6.50 (m, 1H), 5.32 (s,
H), 4.21 – 4.06 (m, 2H), 4.01 – 3.91 (m, 1H), 3.54 – 3.48 (m, 1H), 3.37 – 3.26 (m,
2
3

ACCEPTED MANUSCRIPT
1
–
1
1
9
H), 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
3
1.87 (m, 1H), 1.85 – 1.75 (m, 1H). C NMR (CDCl , 101 MHz) δ 154.94 , 147.75 ,
3
38.40 , 135.05 , 131.71 , 129.20 , 128.55 , 127.36 , 127.21 , 115.79 , 115.56 , 115.45 ,
15.37 , 113.41 , 65.30 , 64.05 , 62.84 , 62.83 , 51.40 , 50.04 , 41.56 , 34.52 , 12.10 ,
+
.56 . ESI-MS: m/z calcd for C H ClFN O (M + H ) 445.2, found 445.3. Anal
2
4
29
3
2
calcd for C H N O : C, 64.64; H, 6.55; N, 9.42. Found C, 64.73; H, 6.50; N, 9.33.
2
4
29
3
2
1
-(((1-(2-chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)methyl)(methyl)amino)-4-(
p-tolyloxy)butan-2-ol. (7d)
1
Yellow oil, yield: 75%. H NMR(CDCl , 400 MHz) δ 7.38 (d, J = 8.0 Hz, 1H), 7.25 –
3
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,
1
H), 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
3
1
1
6
.98 – 1.79 (m, 2H). C NMR (CDCl , 101 MHz) δ 156.64 , 147.82 , 139.60 , 138.59 ,
3
38.32 , 137.91 , 129.80 , 129.80 , 129.20 , 128.57 , 127.36 , 127.23 , 114.33 , 77.16 ,
4.70 , 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 C H ClN O (M + H ) 441.2, found 441.4. Anal calcd for
25
32
3
2
C H N O : C, 67.93; H, 7.30; N, 9.51. Found C, 67.88; H, 7.39; N, 9.50.
25
32
3
2
1
-(((1-(2-chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)methyl)(methyl)amino)-4-(
4
-ethylphenoxy)butan-2-ol.(7e)
2
4

ACCEPTED MANUSCRIPT
1
Light yellow oil, yield: 79%. H NMR (CDCl , 400 MHz) δ 7.38 (d, J = 7.6 Hz,1H),
3
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),
2
2
1
9
1
.72 – 2.54 (m, 3H), 2.45 – 2.41 (m, 1H), 2.28 (s, 6H), 2.16 (s, 3H), 1.97 – 1.83 (m,
1
3
H), 1.23 (t, J = 7.6 Hz, 3H). C NMR (CDCl , 101 MHz) δ 156.79 , 147.83 , 141.44 ,
3
36.41 , 134.96 , 131.72 , 129.21 , 128.63 , 128.63 , 127.38 , 127.23 , 114.34 , 114.34 ,
8.22 , 64.67 , 64.25 , 62.82 , 51.35 , 50.76 , 50.06 , 41.50 , 34.55 , 27.90 , 15.81 ,
+
2.10 , 9.63 . ESI-MS: m/z calcd for C H ClN O (M + H ) 455.3, found 455.3.
2
6
34
3
2
Anal calcd for C H N O : C, 68.48; H, 7.52; N, 9.21. Found C, 68.36; H, 7.56; N,
2
6
34
3
2
9
.19.
1
-(((1-(2-chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)methyl)(methyl)amino)-4-(
4
-propylphenoxy)butan-2-ol. (7f)
1
Clear oil, yield: 82%. H NMR (CDCl , 400 MHz) δ 7.38 (d, J = 7.6 Hz, 1H), 7.23 –
3
7
1
–
2
1
1
1
3
.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,
H), 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),
.28 – 2.25 (m, 5H), 2.14 (s, 3H), 1.97 – 1.77 (m, 2H), 1.66 – 1.59 (m, 2H), 1.29 –
13
.24 (m, 1H), 0.95 (t, J = 7.3 Hz, 3H). C NMR (CDCl , 101 MHz) δ 156.84 ,
3
47.81 , 138.51 , 135.04 , 134.79 , 131.71 , 129.23 , 129.23 , 129.23 , 129.20 , 128.55 ,
27.37 , 127.23 , 114.24 , 114.17 , 64.67 , 64.25 , 62.85 , 51.37 , 50.06 , 41.52 , 37.09 ,
+
4.58 , 24.71 , 13.72 , 12.12 , 9.60 . ESI-MS: m/z calcd for C H ClN O (M + H )
2
7
36
3
2
2
5

ACCEPTED MANUSCRIPT
4
6
69.3, found 469.5. Anal calcd for C H N O : C, 68.99; H, 7.72; N, 8.94. Found C,
2
7
36
3
2
9.13; H, 7.63; N, 8.90.
4
-(4-(tert-butyl)phenoxy)-1-(((1-(2-chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)
methyl)(methyl)amino)butan-2-ol. (7g)
1
Clear oil, yield: 77%. H NMR (CDCl , 400 MHz) δ 7.39 (d, J = 6.8 Hz, 1H), 7.31 (d,
3
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,
1
3
2
1
6
3
4
7
H), 5.33 (s, 2H), 4.15 (t, J = 5.2 Hz, 2H), 4.01 (s, 1H), 3.58 – 3.52 (m, 1H), 3.40 –
.34 (m, 1H), 2.54 (s, 1H), 2.42 (s, 1H), 2.28 (s, 6H), 2.15 (s, 3H), 1.95 – 1.83 (m,
1
3
H), 1.32 (s, 9H).
C NMR (CDCl , 101 MHz) δ 156.53 , 147.80 , 143.28 , 138.46 ,
3
35.07 , 131.70 , 129.19 , 128.54 , 127.37 , 127.23 , 126.13 , 126.13 , 113.91 , 113.91 ,
4.63 , 64.26 , 62.88 , 51.39 , 50.06 , 41.54 , 34.58 , 33.99 , 31.47 , 31.47 , 31.47 ,
+
1.47 , 12.12 , 9.59 . ESI-MS: m/z calcd for C H ClN O (M + H ) 483.2, found
2
8
38
3
2
83.2. Anal calcd for C H N O : C, 69.47; H, 7.91; N, 8.68. Found C, 69.45; H,
2
8
38
3
2
.88; N, 8.66.
4
-(4-ethoxyphenoxy)-1-(((1-(2-fluorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)methy
l)(methyl)amino)butan-2-ol. (7h)
1
Clear oil, yield: 82%. H NMR (CDCl , 400 MHz) δ 7.27 – 7.22 (m, 1H), 7.11 – 7.01
3
(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
6
.56 – 3.50 (m, 1H), 3.36 – 3.30 (m, 1H), 2.52 (s, 1H), 2.42 – 2.36 (m, 1H), 2.26 (s,
1
3
H), 2.18 (s, 3H), 1.91 – 1.80 (m, 2H), 1.41 (t, J = 7.0 Hz, 3H). C NMR (CDCl ,
3
2
6

ACCEPTED MANUSCRIPT
1
1
5
01 MHz) δ 153.04 , 152.93 , 147.57 , 138.10 , 129.07 , 128.49 , 124.48 , 124.34 ,
15.39 , 115.33 , 115.33 , 115.19 , 114.98 , 113.36 , 65.33 , 64.25 , 63.92 , 62.93 ,
1.40 , 46.11 , 46.06 , 41.53 , 34.61 , 14.89 , 12.09 , 9.51 . ESI-MS: m/z calcd for
+
C H FN O (M + H ) 455.3, found 455.5. Anal calcd for C H N O : C, 68.55; H,
26
34
3
3
26 34
3
3
7
.52; N, 9.22; O, 10.54. Found C, 68.55; H, 7.44; N, 9.23; O, 10.57.
1
4
-(((3,5-dimethyl-1-(2-methylbenzyl)-1H-pyrazol-4-yl)methyl)(methyl)amino)-4-(
-ethoxyphenoxy)butan-2-ol.(7i)
1
Light yellow oil, yield: 69%. H NMR (CDCl , 400 MHz) δ 7.17 (d, J = 8.4 Hz, 2H),
3
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 –
.47 (m, 1H), 2.40 (s, 1H), 2.35 (s, 3H), 2.27 (s, 6H), 2.12 (s, 3H), 1.97 – 1.80 (m,
(
2
2
1
1
1
1
3
H), 1.41 (t, J = 7.2 Hz, 3H). C NMR (CDCl , 101 MHz) δ 153.05 , 152.97 , 147.34 ,
3
38.35 , 135.41 , 134.61 , 130.08 , 127.19 , 126.34 , 125.67 , 115.41 , 115.41 , 115.35 ,
15.35 , 65.34 , 64.23 , 63.95 , 62.87 , 51.41 , 50.71 , 50.68 , 41.50 , 34.62 , 19.00 ,
+
4.89 , 12.09 , 9.70 . ESI-MS: m/z calcd for C H N O (M + H ) 451.3, found 451.6.
2
7
37
3
3
Anal calcd for C H N O : C, 71.81; H, 8.26; N, 9.30; O, 10.63 Found C, 71.70; H,
2
7
37
3
3
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)
2
7

ACCEPTED MANUSCRIPT
1
Clear oil, yield: 68%. H NMR (CDCl , 400 MHz) δ 7.30 (s, 1H), 7.28 (s, 1H), 7.00
3
(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,
3
1
H), 3.56 – 3.50 (m, 1H), 3.36 – 3.30 (m, 1H), 2.55 – 2.46 (m, 1H), 2.41 – 2.35 (m,
13
H), 2.26 (s, 6H), 2.14 (s, 3H), 1.97 – 1.75 (m, 2H), 1.41 (t, J = 6.8 Hz, 3H). C
NMR (CDCl , 101 MHz) δ 153.07 , 152.90 , 147.51 , 135.73 , 133.29 , 128.84 ,
3
1
6
28.84 , 127.84 , 127.84 , 115.39 , 115.39 , 115.35 , 115.35 , 77.15 , 65.28 , 64.20 ,
3.95 , 62.89 , 52.06 , 51.34 , 41.49 , 34.60 , 14.89 , 12.07 , 9.77 . ESI-MS: m/z calcd
+
for C H ClN O (M + H ) 471.2, found 471.3. Anal calcd for C H N O : C, 66.17;
2
6
34
3
3
26 34
3
3
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)
methyl)(methyl)amino)propan-2-ol.(8a)
o
1
White solid, yield: 42%, m.p. 90–92 C. H NMR (CDCl , 400 MHz) δ 8.28 (d, J = 7.6
3
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
(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),
4
2
.38 – 4.29 (m, 2H), 4.25 – 4.20 (m, 1H), 3.58 – 3.55 (m, 1H), 3.43 – 3.39 (m, 1H),
13
.83 – 2.70 (m, 2H), 2.36 (s, 3H), 2.30 (s, 3H), 2.14 (s, 3H), 1.30 – 1.26 (m, 1H). C
NMR (CDCl , 101 MHz) δ 155.34 , 147.81 , 140.90 , 138.69 , 138.53 , 135.04 ,
3
1
1
1
31.72 , 129.19 , 128.55 , 127.39 , 127.23 , 126.67 , 124.79 , 122.84 , 122.66 , 119.41 ,
12.61 , 109.92 , 103.42 , 101.19 , 64.65 , 64.34 , 62.88 , 51.33 , 50.03 , 41.51 , 34.74 ,
+
2.08 , 9.54 . ESI-MS: m/z calcd for C H ClN O (M + H ) 503.0, found 503.1.
2
9
31
4
2
2
8