Novel Tetrazole-Containing Analogues of Itraconazole as Potent Antiangiogenic Agents with Reduced Cytochrome P450 3A4 Inhibition

Article abstract of DOI:10.1021/acs.jmedchem.8b01252

Itraconazole has been found to possess potent antiangiogenic activity, exhibiting promising antitumor activity in several human clinical studies. The wider use of itraconazole in the treatment of cancer, however, has been limited by its potent inhibition of the drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4). In an effort to eliminate the CYP3A4 inhibition while retaining its antiangiogenic activity, we designed and synthesized a series of derivatives in which the 1,2,4-triazole ring is replaced with various azoles and nonazoles. Among these analogues, 15n with tetrazole in place of 1,2,4-triazole exhibited optimal inhibition of human umbilical vein endothelial cell proliferation with an IC₅₀ of 73 nM without a significant effect on CYP3A4 (EC₅₀ > 20 μM). Similar to itraconazole, 15n induced Niemann-Pick C phenotype (NPC phenotype) and blocked AMPK/mechanistic target of rapamycin signaling. These results suggest that 15n is a promising angiogenesis inhibitor that can be used in combination with most other known anticancer drugs.

Full text of DOI:10.1021/acs.jmedchem.8b01252

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Article
Novel Tetrazole-containing Analogs of Itraconazole as Potent
Anti-angiogenic Agents with Reduced CYP3A4 Inhibition
Yingjun Li, Kalyan Kumar Pasunooti, Ruo-Jing Li, Wukun Liu, Sarah A. Head, Wei Q. Shi, and Jun O. Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01252 • Publication Date (Web): 27 Nov 2018
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Novel Tetrazole-containing Analogs of Itraconazole as Potent Anti-
angiogenic Agents with Reduced CYP3A4 Inhibition
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Yingjun Li†,II Kalyan Kumar Pasunooti,†,II Ruo-Jing Li,† Wukun Liu,† Sarah A. Head,† Wei Q.
Shi,† Jun O. Liu*, †, ‡
†Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine,
Baltimore, Maryland 21205, United States
‡Department of Oncology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, United
States
II These authors contributed equally to this work.
Corresponding Author
*J.O.L.: phone, 410-955-4619; fax, 410-955-4520; e-mail, joliu@jhu.edu
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ABSTRACT
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Itraconazole has been found to possess potent anti-angiogenic activity, exhibiting promising
antitumor activity in several human clinical studies. The wider use of itraconazole in the
treatment of cancer, however, has been limited by its potent inhibition of the drug metabolizing
enzyme CYP3A4. In an effort to eliminate the CYP3A4 inhibition while retaining its anti-
angiogenic activity, we designed and synthesized a series of derivatives in which the 1, 2, 4-
triazole ring is replaced with various azoles and non-azoles. Among these analogs, 15n with
tetrazole in place of 1, 2, 4-triazole exhibited optimal inhibition of HUVEC proliferation with
an IC50 of 73 nM without significant effect on CYP3A4 (EC50 > 20 µM). Similar to itraconazole,
15n induced Nieman-Pick C phenotype (NPC phenotype) and blocked AMPK/mTOR
signaling. These results suggest that 15n is a promising angiogenesis inhibitor that can be used
in combination with most other known anticancer drugs.
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Introduction:
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Angiogenesis, the formation of new blood vessels, plays a critical role in the onset and
progression of cancer as well as a number of other human diseases.1 Inhibition of angiogenesis
has become an important strategy to combat cancer as underscored by the clinical introduction
of a number of inhibitors of angiogenesis.2 In an effort to repurpose existing drugs as new
angiogenesis inhibitors, we previously found that the antifungal drug itraconazole (1, Fig. 1)
possessed potent anti-angiogenic activity.3,4 The molecular target of itraconazole underlying
its antifungal activity is lanosterol 14α-demethylase (14-DM).5 However, itraconazole only
shows weak inhibition of human 14-DM, ruling it out as a relevant target for the anti-
angiogenic activity of itraconazole.6 Instead, we have identified voltage-dependent anion
channel (VDAC)1 and Niemann Pick type C (NPC)1 as direct targets of itraconazole.7,8,9 We
have shown that binding of itraconazole to NPC1 leads to inhibition of cholesterol trafficking
out of the endolysosome, which in turn induces NPC1 phenotype. Binding of itraconazole to
VDAC1 blocks ATP biosynthesis in the mitochondria, increasing cytosolic AMP/ATP ratio
and activating AMPK. Inhibition of cholesterol trafficking and activation of AMPK lead to
synergistic inhibition of mTOR signaling.10 The unique mechanism of action of itraconazole
distinguishes it from rapamycin, a direct inhibitor of mTOR, and other azole antifungal drugs
such as ketoconazole (2, Fig. 1), which do not have antiangiogenic activity.11 These new
mechanistic insights, along with other preclinical results, have facilitated the entrance of
itraconazole into multiple phase II clinical trials in the treatment of prostate cancer, non-small
cell lung cancer, basal cell carcinoma and other types of cancers.12,13,14,15
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A major limitation of itraconazole as a novel anticancer drug is its strong inhibition of human
liver cytochrome P450 3A4 (CYP3A4).16 CYP3A4 is a major xenobiotic metabolizing enzyme
and it contributes to the metabolism of approximately 50% of prescribed drugs, including the
majority of anticancer drugs.17 Inhibition of CYP3A4 causes strong drug-drug interaction,
preventing the combination of itraconazole with most known anticancer drugs including
targeted kinase inhibitors that are metabolized by CYP3A4.18,19 Many anticancer drugs,
especially those that inhibit angiogenesis, are most effective when used in combination with
other drugs.20 Thus, there is a need to develop novel itraconazole analogs with reduced or with
no CYP3A4 inhibition while retaining its anti-angiogenic activity.
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Previously, we identified an itraconazole stereoisomer with cis-2S,4R stereochemistry of the
dioxolane moiety with increased anti-angiogenic activity and significantly reduced
hepatotoxicity.21,22 We also found that the sec-butyl or a similar side chain is required for
antiangiogenic activity.23 The triazole moiety of itraconazole is a critical pharmacophore
required for the binding of itraconazole to the heme group of 14-DM as well as the heme group
of CYP3A4.24,25 However, little is known about the importance of the triazole moiety in anti-
angiogenic activity of itraconazole.26
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In an effort to identify novel analogs of itraconazole with reduced or no CYP3A4 inhibition
while retaining its anti-angiogenic potency, we systematically replaced the 1,2,4-triazole
moiety with different non-azoles or azoles. Herein, we report the SAR (structure–activity
relationship) study of novel itraconazole analogs for their anti-angiogenic activity and CYP3A4
inhibition. Further, we identified one compound, 15n, which contains tetrazole in place of
triazole and exhibits improved anti-angiogenic activity and significantly reduced CYP3A4
inhibitory activity compared with itraconazole.
Cl
Cl
Cl
Cl
2
N
1
N
O
N
3
O
O
O
O
O
N
N
N
4
N
N
N
N
5
O
N
N
O
dioxolane ring
side chain
R
Itraconazole (1)
Ketoconazole (2)
Figure 1. Structures of itraconazole (1) and ketoconazole (2).
Results and Discussion
Chemistry:
The synthetic route to the non-azole itraconazole analogs (10a-10e) is outlined in Scheme 1.
The synthesis commenced with the commercially available 1, 3-dichlorobenzene and a series
of different acid chlorides. The intermediates 2, 4-dichloro benzaldehyde (5a) and 2-bromo-
2',4'-dichloroacetophenone (5c) are commercially available. The other intermediates (5b, 5d
and 5e) were efficiently prepared by the acylation of 1, 3-dichlorobenzene with acid chlorides
under Friedel-Craft acylation conditions in satisfactory yields (70-90%). Our previous results
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suggested that 2S,4R-cis-stereochemistry on the 1, 3-dioxalane ring is more potent for anti-
angiogenic activity than alternative stereochemical configurations.21 We thus constructed the
cis-1,3-dioxolane (7a-7e) via acid-assisted ketalization of 2, 4-dichloro acetophenones (5a-5e)
with optically pure glyceryl tosylate 6 in the presence of trifluoromethanesulfonic acid (TfOH)
in toluene, yielding cis and trans diastereomers in the ratio of 3:1 with good yields. Those two
diastereomers were separated by conventional column chromatography. The required phenol
fragment 9 was synthesized with the same method as we reported previously.23 The phenol
fragment 9 was reacted with the O-tosylates (7a-7e) in the presence of sodium hydride (NaH)
to afford final products (10a-10e). Analogs with a tertiary amine group (11a-11d) were
prepared from a nucleophilic substitution reaction of bromo substituted compound 10c with
different secondary amines (Scheme 2).
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Itraconazole analogs (15a-15q) were synthesized using a similar route (Scheme 3). The
intermediates 12a-12q were prepared by N-alkylation of 2-bromo-2',4'-dichloroacetophenone
(5c) with a series of azoles in DCM at room temperature. Ketalization of 2, 4-dichloro
acetophenones 12a-12q with glyceryl tosylate 6 gave 13a-13q as cis diastereoisomers with low
to moderate yields (10% - 65%). Finally, O-tosylates (13a-13q) were treated with fragment 9
to yield the desired products 15a-15q.27
Scheme 1:
Cl
Cl
OHC
5a
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
AlCl3, rt, 12h
Cl
TfOH, toluene, rt, 60h
R
R
O
R
O
O
O
R
OH
6
HO
OTs
OTs
O
OTs
3
5b-5e
O
(Cis)
7a-7e
(Trans)
8a-8e
4b-4e
O
NaH, DMF, 50-90 oC, 4 h
N
N
N
N
HO
N
9
Cl
Cl
R
O
O
Br
H
O
N
N
N
R =
N
O
10a
10b
10c
10d
10e
N
10a-10e
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Scheme 2:
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Cl
Cl
Cl
Cl
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R
O
amine, THF, 120 oC, 16 h
O
Br
O
O
O
O
N
N
N
N
N
N
N
O
N
N
N
O
10c
11a-11d
N
N
N
N
O
N
HN
R
=
11a
11b
11d
11c
Scheme 3:
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
R
TfOH, toluene, rt, 60h
R
O
O
azoles, K2CO3, DCM, rt, 12h
O
O
R
Br
OTs
OTs
OH
HO
O
(Trans)
O
OTs
(Cis)
13a 13q
12a 12q
-
5c
14a 14q
-
-
6
NaH, DMF, 50-90 oC, 12h
9
Cl
Cl
R
O
N
O
O
N
N
N
N
O
15a-15q
Ph
N
N
N
N
N
N
F3C
N
N
N
N
N
N
N
N
N
N
N
N
N
15a
15c
15d
15e
15g
15h
15b
15f
R =
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Ph
15q
F3C
15i
15j
15k
15l
15m
15o
15p
15n
SAR studies of new itraconazole analogs using HUVEC proliferation and CYP3A4
enzymatic assays
The anti-angiogenic activity of new itraconazole analogs was determined using HUVEC
proliferation assay with [3H] thymidine incorporation as a readout (Table 1).3 CYP3A4
inhibition was determined using a cell-free fluorescence-based assay at 1 µM of each analog
after concentration optimization of itraconazole (Fig. S1).28
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To assess the importance of the triazole moiety for the anti-angiogenic activity of
itraconazole and CYP3A4 inhibition, we removed the 1,2,4-triazolyl-methyl group (10a) or
replaced it with ethyl (10b), bromo methyl (10c), cyclopentyl-methyl (10d), or phenyl-methyl
group (10e). As expected, 10a-10e without a nitrogen atom in the R position exhibited no
CYP3A4 inhibition at 1 µM. Further dose response assays showed that 10b completely lost
CYP3A4 inhibition (Fig. 2). In contrast, the same group of analogs only suffered a 2-3 fold
decrease in anti-proliferative activity against HUVEC, suggesting differential dependence of
the two activities on the presence of the triazole moiety.
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Next, a tertiary amine was introduced to 1,2,4-triazole position (11a-11d) with
dimethylamine, morpholine, piperidyl or N-methyl piperidyl group, respectively. Analogs 11a-
11d have the added advantage of having improved water solubility compared with itraconazole.
The analogs containing an aliphatic amine in place of 1,2,4-triazole inhibited CYP3A4 at 1 µM
except for 11a. Structurally, 11b-11d have a nucleophilic nitrogen atom or oxygen atom in a
position comparable to N4 of 1,2,4-triazole in itraconazole, suggesting that the aliphatic
nitrogen and oxygen are also capable of interacting with the heme group in CYP3A4.
Compared to itraconazole, 11a-11d had a 2-9 fold decrease in anti-proliferative activity in
HUVEC. Together, the results confirmed that CYP3A4 inhibition of itraconazole is highly
dependent on 1,2,4-triazole moiety, in particular the basic N4 atom of triazole and suggested
that replacing the triazole moiety with non-azoles is not sufficient to decrease CYP3A4
inhibition without compromising the anti-angiogenic activity.
Given the importance of the triazole moiety in the anti-angiogenic activity of itraconazole,
we decided to make less drastic structural alterations of the triazole by increasing the steric
hindrance around the nitrogen atom at 4 position that might decrease its access to the heme
iron in CYP3A4. To start with, we introduced methyl or trifluoromethyl substitution on 1,2,4-
triazole ring. Analogs 15a with 3-methyl-1H-1,2,4-triazole substitution and 15b with 3,5-
dimethyl-1H-1,2,4-triazole substitution showed reduced potency in CYP3A4 inhibition. The
trifluoromethyl substituted compound 15c showed no CYP3A4 inhibition at 1 µM and weak
inhibition at higher concentrations (Fig. 2). The IC50 values of 15a-15c for inhibition of
HUVEC proliferation were 0.27, 0.22, and 0.27 µM, respectively, slightly higher than that of
itraconazole (0.17 µM). Compound 15d contains an imidazole in place of the 1,2,4-triazole
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and exhibited higher potency against HUVEC proliferation than itraconazole. We synthesized
a series of analogs containing imidazole group with various substitutions (15e-15l). Similar to
15c, compound 15l with 2-methyl-4-trifluoromethyl-1H-imidazol-yl moiety suffers from both
steric hindrance and electron withdrawing effect exerted by the trifluoromethyl group,
rendering the N3 nitrogen unreactive towards heme. As expected, 15l had no inhibitory effect
on CYP3A4 at 1 µM. However, the dual substituted compounds (15j-15l) also suffered a 2-3
fold decrease in anti-angiogenic activity. Interestingly, among the single substituted imidazol-
yl compounds, the 2-isopropyl-1H-imidazol-yl analog 15g had the most potent HUVEC
inhibition activity, with an IC50 of 0.084 µM. Compounds with substituents smaller or larger
than the isopropyl group were all less potent than 15g. On the other hand, CYP3A4 inhibition
showed an opposite trend—the larger the substituents on the imidazole, the less inhibition of
CYP3A4.
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As coordination of the basic nitrogen to heme iron is required for the inhibitory activity of
azoles against CYP3A4, we reasoned that replacement of triazole with tetrazole that has
reduced basicity compare to imidazole and triazole, should has reduced CYP3A4 inhibition.29
The 1-tetrazole-yl analog 15n showed improved anti-proliferative activity in HUVEC with an
IC50 of 0.073 µM and significantly reduced CYP3A4 inhibition with an EC50 above 20 µM
(Fig. 2). In contrast, the structurally related 2-tetrazole-yl analog 15o is less potent in HUVEC
and exhibited greater CYP3A4 inhibition than 15n. Addition of a 5-methyl or 5-phenyl
substituents to the tetrazole group did not lead to further improvement over analog 15n,
rendering 15n the optimal compound among all itraconazole analogs made to date. Thus, we
selected 15n for further biological evaluation.
Inhibition of Tube Formation
To further assess the anti-angiogenic activity of 15n, we employed an in vitro tube formation
assay. In this assay, HUVEC were seeded on matrigel, cells migrate and elongate to form
tubule-like networks after 20 hours, which is reminiscent of new blood vessel formation.30 As
showed in Fig. 3, treatment of HUVEC with 5 µM itraconazole inhibited 45% of HUVEC tube
formation as judged by the total tube length. At the same concentration, 15n inhibited 61% of
HUVEC tube formation, confirming that 15n is a more potent anti-angiogenesis inhibitor.
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Table 1: HUVEC Anti-proliferation Activities and CYP3A4 Enzyme Inhibition of
Itraconazole Analogs
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8
Cl
Cl
9
R
O
O
10
11
12
13
14
15
16
17
18
19
20
21
22
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27
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60
O
N
N
N
N
N
O
CYP3A4
inhibition at
1µM b
HUVEC
IC50 (µM) a
Compounds
R
1
0.170 ± 0.013
0.328 ± 0.070
0.353 ± 0.033
0.569 ± 0.200
84% ± 6%
NI
10a
10b
10c
H
NI
3% ± 1%
Br
10d
10e
0.512 ± 0.177
0.513 ± 0.125
NI c
NI
N
11a
11b
11c
11d
1.397 ± 0.165
1.175 ± 0.231
0.608 ± 0.260
0.396 ± 0.168
NI
N
16% ± 3%
91% ± 1%
18% ± 7%
O
HN
N
N
N
N
N
15a
15b
0.272 ± 0.112
0.221 ± 0.008
30% ± 7%
27% ± 4%
N
N
N
N
N
F3C
N
N
15c
15d
0.270 ± 0.084
0.085 ± 0.014
NI
N
N
99% ± 1%
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15e
15f
0.173 ± 0.063
71% ± 13%
7% ± 4%
N
N
N
0.224 ± 0.064
N
10
11
12
13
14
15
16
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15g
0.084 ± 0.016
6% ± 3%
N
N
Ph
15h
15i
0.136 ± 0.070
0.102 ± 0.015
23% ± 11%
98% ± 2%
N
N
N
N
N
N
N
N
15j
15k
15l
0.492 ± 0.053
0.765 ± 0.072
0.441 ± 0.134
38% ± 4%
23% ± 7%
NI
N
N
F3C
N
N
N
15m
15n
0.307 ± 0.045
0.073 ± 0.017
NI
N
N
2% ± 1%
N
N
N
N
15o
15p
15q
0.124 ± 0.049
0.101 ± 0.047
0.119 ± 0.011
17% ± 4%
7% ± 5%
18% ± 5%
N
N
N
N
N
N
N
N
N
Ph
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a IC50 in HUVEC were evaluated using 3H thymidine incorporation assay. b CYP3A4 enzyme
inhibition was evaluated using Vivid® CYP3A4 green screening assay. Values indicated are %
enzyme inhibition at 1 µM. Values represent the mean ± SD in three independent experiments
carried out in triplicate. c No Inhibition.
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itraconazole
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Figure 2. CYP3A4 enzyme activity in the presence of different concentrations of
itraconazole (1), 10b, 15c, 15g and 15n.
Figure 3. Compound 15n inhibits HUVEC tube formation. HUVECs were plated on Matrigel
and treated with 5μM itraconazole (1), 1μM 15n, 5μM 15n or DMSO for 20h. (A) Cells were
stained with Calcein-AM and vascular networks were imaged using fluorescence microscopy.
(B) Total tube lengths from the fluorescence images were quantified using the ImageJ software
and plotted using GraphPad Prism. Data, mean ± SD of three independent experiments. (* p <
0.01)
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Figure 4. Itraconazole (1), 10b and 15n induce NPC phenotype at 0.2μM. HUVECs were
treated with 0.2 μM itraconazole (1), 0.2 μΜ 15n or DMSO for 24 h. Intracellular cholesterol
was visualized using filipin staining and fluorescent images were captured under a confocal
microscope.
Figure 5. 15n dose-dependently activates AMPK and inhibits mTOR in HUVECs. (A)
HUVECs were treated with 0.01μM, 0.03μM, 0.1μM, 0.3μM, 1μM of 15n or DMSO for 24 h.
Cell lysates were subjected to Western Blot. (B) The mean gray value and area of the blots
were quantified using the ImageJ software and plotted using GraphPad Prism. Data given as
mean ± SD of three independent experiments.
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Nieman-Pick C phenotype and mTOR inhibition
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We have previously shown that the mechanism underlying the anti-angiogenic activity of
itraconazole is mediated in part through inhibition of endolysosomal cholesterol trafficking and
mTOR inhibition. We thus determined whether compound 15n shared the same mechanism
with itraconazole. Intracellular cholesterol was detected using the cholesterol-binding
fluorescent dye filipin.10 Similar to itraconazole, 15n induced NPC phenotype at a
concentration of 0.2 µM as judged by the accumulation of cholesterol in the late
endosome/lysosome (Fig. 4). Compound 10b, which was less potent against HUVEC, showed
reduced activity in NPC phenotype induction. Other compounds 15c and 15g also induced
cholesterol accumulation (Fig. S2). Analog 15n also increased the phosphorylation of the 
subunit of AMPK at Thr172 in a dose-dependent manner (Fig. 5). The phosphorylation of the
AMPK substrate acetyl CoA carboxylase 1 (ACC1) was also increased as expected.31,32 In
addition, treatment of HUVEC with 15n led to a decrease in the phosphorylation of mTOR and
its substrate p70 S6 Kinase (S6K). A significant change was seen in AMPK signaling at 0.1µM
of 15n and in mTOR signaling at 0.3µM of 15n (Fig. 5B, Fig. S3). Moreover, pretreatment
with 15h and 15n was able to compete the crosslinking of the itraconazole photoaffinity probe
(Fig. S5) to VDAC1 similar to itraconazole (Fig. S4), indicating direct binding of 15h and 15n
to VDAC1. Together, these results suggested that 15n inhibited HUVEC proliferation and
angiogenesis with the same targets and underlying mechanism as itraconazole.
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Conclusions
The antifungal drug was identified and validated as a promising anti-angiogenic agent,
demonstrating efficacy against different types of cancer in multiple Phase 2 human clinical
studies. The inhibition of CYP3A4 by itraconazole, however, severely limits its wider use as
an anticancer agent due to interactions with most other known anticancer drugs. To overcome
this limitation, we have synthesized a series of azole and non-azole derivatives of itraconazole
in attempts to maintain their anti-angiogenic activity while reducing or eliminating their effects
on CYP3A4. Given that the CYP3A4 inhibition is highly dependent on the coordination of
1,2,4-triazole to the heme iron in CYP3A4, we carried out a series of chemical modifications
of triazole moiety to reduce binding of itraconazole analogs to CYP3A4. In addition to
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replacing the triazole with alkyl or alkylamine substituents, we also incorporated methyl and
trifluoromethyl substituents to azoles to decrease the azole-heme iron interaction through steric
hindrance or reduction of basicity of the azole nitrogen. While many of the analog showed
significantly reduced or no inhibition of CYP3A4, their inhibitory activity against HUVEC was
also decreased in comparison to itraconazole. Eventually, we attempted to replace triazole with
tetrazole and one of the analogs, 15n, not only has significantly reduced CYP3A4 inhibition
with an EC50 of above 20 µM, but also exhibited more potent anti-proliferative activity against
HUVEC than itraconazole. The angiogenic potency of 15n was further confirmed by HUVEC
tube formation. Like itraconazole, 15n bound to VDAC1 as judged by its competition for
VDAC1 binding against the itraconazole photoaffinity probe, activated AMPK pathway,
induced NPC1 phenotype and inhibited mTOR, which are hallmarks of the mechanism of
inhibition of angiogenesis by itracoazole. Together, our results strongly suggest that the
tetrazole-containing analog 15n is a promising new lead for the development of the next
generation itraconazole analogs that can be used in combination with most other known
anticancer drugs.
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Experimental Section:
Chemistry. Reactions were carried out in oven-dried glassware. All reagents were purchased
from commercial sources and were used without further purification unless noted. Unless
otherwise stated, all reactions were carried out under argon atmosphere, monitored by Merck
pre-coated silica gel 60F-254 plates and visualized using 254 nm UV light. Column
chromatography was performed on normal-phase silica flash columns (RediSepRf). NMR data
were collected on Bruker Avance III (500 MHz 1H, 125 MHz 13C) machine in the Department
of Pharmacology and Molecular Sciences, the Johns Hopkins University, School of Medicine.
Chemical shifts () are reported in ppm. 1H NMR spectra and 13C NMR spectra were obtained
1
in deuteriochloroform (CDCl3) with tetramethylsilane (TMS,  = 0.00 for H) as an internal
reference. Data are presented in the form: chemical shift (multiplicity, coupling constants, and
integration). Low resolution ESI-MS and HPLC purity were recorded on an Agilent 6120
quadrupole LC/MS. The reported purity values were obtained with a Pursuit XRs Diphenyl
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column (150×4.5mm) and a diode array detector (DAD). A flow rate of 1.0 ml/min was used
with a mobile phase of solvent A (H2O with a 0.1% formic acid (v/v)) and solvent B
(acetonitrile with a 0.1% formic acid (v/v)). A gradient elution method were used: started with
40% solvent B, changed to 95% over 14 min, maintained at 95% for 3min and then change to
40% over 0.5 min, maintained at 40% for 2.5 min. The absorbance was detected under UV at
250nm and area of peaks were quantified to calculate the compound purity. The purity of all
compounds are ≥95%.
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General experimental procedure for 10a-10e and 15a-15q
To a solution of tosylates 7a-7e and 13a-13q (1 eq) in anhydrous DMF was added sodium
hydride (NaH, 60% dispersion in mineral oil, (1.5 eq) under argon atmosphere. After tiring at
50℃ for 1 hour, a solution of 9 (1.2 eq) in DMF was added slowly at the same temperature.
Then the temperature was increased to 90℃ and the reaction was stirred for another 3 hours.
The reaction mixture was quenched by the saturated sodium chloride, and the resulting mixture
was extracted twice with dichloromethane. The organic fractions were dried over Na2SO4,
filtered and concentrated under vacuum to yield the crude product which was purified by
column chromatography to afford the desired product 10a-10e and 15a-15q in moderate to
good yields.
1-sec-butyl-4-(4-(4-(4-(((4R)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-5(4H)-one (Mixture of trans
and cis) (10a). Trans compound: 1H NMR (500 MHz, CDCl3, δH): 7.62 (s, 1H), 7.57 (d, J =
8.5 Hz, 1H), 7.43 (d, J = 8.5 Hz, 2H), 7.40 (t, J = 2.0 Hz, 1H), 7.28 (t, J = 2.0 Hz, 1H), 7.27(bs,
1H), 7.03 (d, J = 9.0 Hz, 2H), 6.95 (bs, 1H), 6.92-6.88 (m, 2H), 6.27 (s, 1H), 4.66-4.60 (m,
1H), 4.34 (dd, J = 8.2, 6.7 Hz, 1H), 4.31-4.27 (m, 1H), 4.18-4.15 (m, 1H), 4.14-4.07 (m, 1H),
4.03 (dd, J = 8.5, 6.5 Hz, 1H), 3.37 (bs, 4H), 3.25 (bs, 4H), 1.90 – 1.84 (m, 1H), 1.75 – 1.69
(m, 1H), 1.39 (d, J = 6.5 Hz, 3H), 0.91 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3, δC):
152.1, 135.8, 135.7, 134.4, 134.3, 133.9, 129.6, 128.8, 127.4, 127.2, 123.6, 118.5, 116.7, 115.5,
100.7, 76.5, 74.7, 68.7, 68.6, 67.9, 52.7, 50.7, 28.5, 19.3, 10.8. Cis compound: 1H NMR (500
MHz, CDCl3, δH): 7.62 (s, 1H), 7.57 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 8.5 Hz, 2H), 7.40 (t, J =
2.0 Hz, 1H), 7.29 (d, J = 2.0 Hz, 1H), 7.27(bs, 1H), 7.03 (d, J = 9.0 Hz, 2H), 6.95 (bs, 1H),
6.92-6.88 (m, 2H), 6.16 (s, 1H), 4.66-4.60 (m, 1H), 4.31-4.27 (m, 1H), 4.21 (dd, J = 8.2, 6.7
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Hz, 1H), 4.18-4.15 (m, 1H), 4.14-4.07 (m, 1H), 4.03 (dd, J = 8.5, 6.5 Hz, 1H), 3.37 (bs, 4H),
3.25 (bs, 4H), 1.90 – 1.84 (m, 1H), 1.75 – 1.69 (m, 1H), 1.39 (d, J = 6.5 Hz, 3H), 0.91 (t, J =
7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3, δC): 152.1, 135.8, 135.7, 134.4, 134.3, 133.9, 129.6,
128.8, 127.4, 127.2, 123.6, 118.5, 116.7, 115.5, 100.7, 76.5, 74.7, 68.7, 68.6, 67.9, 52.7, 50.7,
28.5, 19.3, 10.8. HRMS (ESI) calcd for C32H35Cl2N5O4: 624.2144; found 624.2114. HPLC
Purity: 95.9%, tR = 13.7 min.
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1-sec-butyl-4-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-ethyl-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-5(4H)-one (10b). 1H NMR
(500 MHz, CDCl3, δH): 7.61 (s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 9.0 Hz, 2H), 7.40
(d, J = 2.0 Hz, 1H), 7.23 (dd, J = 8.5, 2.0 Hz, 1H), 7.03 (d, J = 9.0 Hz, 2H), 6.95 (bs, 2H), 6.89
(d, J = 9.0 Hz, 2H), 4.33-4.27 (m, 2H), 4.12 (dd, J = 9.5, 5.0 Hz, 1H), 3.98 (dd, J = 8.5, 4.5 Hz,
1H), 3.96 (dd, J = 9.5, 6.5 Hz, 1H), 3.86 (dd, J = 8.5, 7.0 Hz, 1H), 3.37 (bs, 4H), 3.24 (bs, 4H),
2.18 – 2.09 (m, 2H), 1.89 – 1.83 (m, 1H), 1.75 – 1.69 (m, 1H), 1.39 (d, J = 7.0 Hz, 3H), 0.90
(t, J = 7.5 Hz, 3H), 0.89 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3, δC): 152.1, 137.4,
134.5, 133.9, 132.8, 131.2, 129.8, 126.8, 123.6, 118.5, 116.7, 115.2, 111.2, 73.7, 69.3, 67.1,
52.7, 50.7, 49.3, 30.7, 28.5, 19.3, 10.8, 7.7. HRMS (ESI) calcd for C34H39Cl2N5O4: 652.2457;
found 652.2426. HPLC Purity: 95.0%, tR = 13.4 min.
4-(4-(4-(4-(((2S,4R)-2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1-sec-butyl-1H-1,2,4-triazol-5(4H)-one (10c).
1H NMR (500 MHz, CDCl3, δH): 7.65 (d, J = 8.5 Hz, 1H), 7.61 (s, 1H), 7.43-7.42 (m, 3H), 7.27
(dd, J = 8.5, 2.0 Hz, 1H), 7.03 (d, J = 9.0 Hz, 2H), 6.95 (bs, 2H), 6.90 (d, J = 9.0 Hz, 2H), 4.46-
4.42 (m, 1H), 4.31-4.27 (m, 1H), 4.22 (dd, J = 9.5, 5.0 Hz, 1H), 4.14 (dd, J = 8.5, 5.5 Hz, 1H),
4.09 (dd, J = 8.5, 6.5 Hz, 1H), 4.02 (dd, J = 8.5, 7.0 Hz, 1H), 3.96 (d, J = 11.5, 1H), 3.86 (d, J
= 11.5 Hz, 1H), 3.37 (bs, 4H), 3.25 (bs, 4H), 1.89 – 1.84 (m, 1H), 1.75 – 1.69 (m, 1H), 1.39 (d,
J = 7.0 Hz, 3H), 0.90 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3, δC): 152.1, 135.7, 134.7,
133.9, 133.0, 131.3, 120.0, 127.0, 123.6, 118.5, 116.7, 115.5, 107.8, 74.8, 68.6, 68.4, 52.7,
50.7, 49.3, 35.4, 28.5, 19.3, 10.8. HRMS (ESI) calcd for C33H36BrCl2N5O4: 716.1406; found
716.1422. HPLC Purity: 95.7%, tR = 13.3 min.
1-sec-butyl-4-(4-(4-(4-(((2R,4R)-2-(cyclopentylmethyl)-2-(2,4-dichlorophenyl)-1,3-
dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-5(4H)-one (10d).
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1H NMR (500 MHz, CDCl3, δH): 7.62 (s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 9.0 Hz,
2H), 7.39 (d, J = 2.5 Hz, 1H), 7.22 (dd, J = 8.5, 2.0 Hz, 1H), 7.03 (d, J = 9.0 Hz, 2H), 6.95 (bs,
2H), 6.89 (d, J = 9.0 Hz, 2H), 4.31-4.27 (m, 2H), 4.12 (dd, J = 9.2, 4.5 Hz, 1H), 3.99-3.95 (m,
2H), 3.84 (dd, J = 8.0, 7.0 Hz, 1H), 3.37 (bs, 4H), 3.24 (bs, 4H), 2.24– 2.16 (m, 2H), 1.90 –
1.81 (m, 2H), 1.75 – 1.69 (m, 3H),1.57 – 1.53 (m, 2H), 1.44 – 1.41 (m, 2H), 1.39 (d, J = 7.0
Hz, 3H), 1.14 – 1.06 (m, 2H), 0.90 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3, δC): 152.1,
137.8, 134.5, 133.9, 133.0, 131.2, 129.8, 126.6, 123.6, 118.5, 116.7, 115.5, 111.1, 73.6, 69.1,
66.8, 52.7, 50.7, 49.3, 43.6, 35.4, 33.6, 33.5, 28.5, 25.1, 25.0, 19.3, 10.8. HRMS (ESI) calcd
for C38H45Cl2N5O4: 706.2927; found 706.2915. HPLC Purity: 95.4%, tR = 15.2 min.
4-(4-(4-(4-(((2R,4R)-2-benzyl-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-
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yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1-sec-butyl-1H-1,2,4-triazol-5(4H)-one (10e).
1H NMR (500 MHz, CDCl3, δH): 7.62 (s, 1H), 7.46-7.42 (m, 4H), 7.43-7.42 (m, 3H), 7.22 (bs,
5H), 7.16 (dd, J = 8.5, 2.0 Hz, 1H), 7.03 (d, J = 9.0 Hz, 2H), 6.93 (bs, 2H), 6.73 (d, J = 9.0 Hz,
2H), 4.32-4.26 (m, 2H), 3.79-3.77 (m, 2H), 3.68 (dd, J = 9.5, 5.0 Hz, 1H), 3.45 (d, J = 14.0,
1H), 3.37 (bs, 4H), 3.34 (d, J = 14.0 Hz, 1H), 3.32-3.28 (m, 1H), 3.25 (bs, 4H), 1.89 – 1.84 (m,
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1H), 1.75 – 1.69 (m, 1H), 1.39 (d, J = 6.5 Hz, 3H), 0.90 (t, J = 7.2 Hz, 3H). C NMR (125
MHz, CDCl3, δC): 152.1, 137.7, 135.2, 134.6, 133.9, 132.9, 131.3, 131.1, 129.5, 127.7, 123.6,
116.7, 115.3, 110.1, 74.0, 68.4, 67.2, 52.7, 49.2, 43.7, 36.5 28.5, 19.3, 10.8. HRMS (ESI) calcd
for C39H41Cl2N5O4: 714.2614; found 714.2590. HPLC Purity: 95.1%, tR = 14.8 min.
1-(sec-butyl)-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-((dimethylamino)methyl)-1,3-
dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-5(4H)-one (11a).
1H NMR (500 MHz, CDCl3, δH): 8.41 (s, 1H), 7.74-7-75 (m, 2H), 7.59 (d, J = 9 Hz, 2H), 7.19
(d, J = 9 Hz, 2H), 7.06 (d, J = 9 Hz, 2H), 7.00 (d, J = 9 Hz, 2H), 4.53-4.48 (m, 1H), 4.23-4.18
(m, 3H), 4.10-4.03 (m, 4H), 3.43-3.38 (m, 4H), 3.27-3.24 (m, 4H), 2.59 (s, 6H), 1.84-1.78 (m,
1H), 1.76-1.70 (m, 1H), 1.38 (d, J = 6.5 Hz, 3H), 0.88 (t, J = 6.5 Hz, 3H). 13C NM R (125
MHz, CDCl3, δC): 152.8, 150.2, 146.5, 135.5, 134.2, 130.2, 127.0, 126.6, 124.3, 119.3, 117.5,
116.2, 77.2, 72.2, 68.4, 60.7, 50.4, 47.6, 29.9, 20.8, 12.4.
HRMS (ESI) calcd for
C36H40Cl2N8O4: 681.2723; found 681.2714. HPLC Purity: 97.1%, tR = 4.7 min.
1-(sec-butyl)-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-(morpholinomethyl)-1,3-
dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-5(4H)-one (11b).
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1H NMR (500 MHz, CDCl3, δH): 7.63 (s, 1H), 7.61(d, J = 9.0 Hz, 1H), 7.45 (d, J = 8.5 Hz,
2H), 7.41(d, J = 1.5 Hz, 1H), 7.25(dd, J = 8, 1.5 Hz, 1H), 7.06(d, J = 9.0 Hz, 2H), 6.98(d, J =
9.0 Hz, 2H), 6.92(d, J = 8.5 Hz, 2H), 4.36-4.41 (m, 1H), 4.28-3.34 (m, 1H), 4.14-4.18 (m, 1H),
4.05-4.10 (m, 2H), 3.89 (t, J = 7.0 Hz, 1H), 3.57 (t, J = 4 Hz, 4H), 3.36-3.40 (m, 4H), 3.26 (t,
J = 5 Hz, 4H), 2.59 (t, J = 4 Hz, 4H), 1.95-1.85 (m, 1H), 1.79-1.70 (m, 1H), 1.42 (d, J = 7 Hz,
3H), 0.93 (t, J = 7 Hz, 3H). 13C NMR (125 MHz, CDCl3, δC): 152.6, 151.1, 146.5, 135.3, 133.6,
131.6, 130.6, 127.2, 126.6, 124.3, 119.3, 117.5, 116.2, 75.2, 68.4, 56.0, 53.9, 51.9, 50.5, 31.1,
29.9, 20.8, 12.4. HRMS (ESI) calcd mass for C37H44Cl2N6O5: 723.2828; found 723.2826.
HPLC Purity: 96.2%, tR = 5.0 min.
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1-(sec-butyl)-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-(piperazin-1-ylmethyl)-1,3-
dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-5(4H)-one (11c).
1H NMR (500 MHz, CDCl3, δH): 7.62 (s, 1H), 7.60 (d, J = 8.5 Hz, 2H), 7.44 (d, J = 9 Hz, 2H),
7.39 (d, J = 1.5 Hz, 1H), 7.24 (dd, J = 8.5, 2.0 Hz, 1H), 7.04 (d, J = 8.5 Hz, 2H), 6.96 (d, J = 9
Hz, 2H), 6.90 (d, J = 9 Hz, 2H), 4.39-4.34 (m, 1H), 4.32-4.27 (m, 1H), 4.16-4.13 (m, 1H), 4.08-
4.03 (m, 2H), 3.89 (t, J = 7.5 Hz, 1H), 3.56 (t, J = 4.5 Hz, 4H), 3.39-3.35 (m, 4H), 3.26-3.23
(m, 4H), 2.99 (d, J = 3 Hz, 3H), 2.57 (t, J = 4.5 Hz, 4H), 2.19 (s, 1H), 1.90-1.85 (m, 1H), 1.76-
1.70 (m, 1H), 1.41 (d, J = 6.5 Hz, 3H), 0.92 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3,
δC): 153.5, 151.1, 146.5, 137.4, 135.2, 133.6, 131.6, 130.6, 127.2, 126.6, 124.3, 119.3, 117.5,
116.2, 75.2, 70.0, 68.4, 68.2, 64.4, 56.0, 53.9, 51.9, 50.5, 29.9, 20.8, 12.4. HRMS (ESI) calcd
for C37H45Cl2N7O4: 722.7049; found 722.7043. HPLC Purity: 95.1%, tR = 5.1 min.
1-(sec-butyl)-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-((4-methylpiperazin-1-
yl)methyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-
5(4H)-one (11d). 1H NMR (500 MHz, CDCl3, δH): 8.03 (s, 1H), 7.62 (s, 1H), 7.58 (d, J = 8.5
Hz, 1H), 7.43 (d, J = 7.5 Hz, 2H), 7.38 (s, 1H), 7.23 (d, J = 8.5 Hz, 1H), 7.04 (d, J = 8 Hz, 2H),
6.96 (d, J = 8 Hz, 2H), 6.90 (d, J = 8 Hz, 2H), 4.36-4.34 (m, 1H), 4.32-4.28 (m, 1H), 4.15-4.12
(m, 1H), 4.06-4.02 (m, 2H), 3.87 (t, J = 7.5 Hz, 1H), 3.60-3.58 (m, 4H), 3.37-3.36 (m, 4H),
3.25-3.22 (m, 4H), 3.02 (s, 2H), 2.70-2.67 (m, 4H), 2.33 (s, 3H), 1.89-1.84 (m, 1H), 1.75-1.69
(m, 1H), 1.41 (d, J = 6.5 Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H). 13C NM R (125 MHz, CDCl3, δC):
153.5, 152.6, 151.1, 146.5, 137.4, 135.2, 133.7, 131.6, 130.6, 127.2, 126.6, 124.3, 119.3, 117.5,
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116.2, 75.2, 70.1, 68.2, 63.6, 55.8, 54.3, 53.9, 51.9, 50.5, 46.7, 29.9, 20.8, 12.4. HRMS (ESI)
calcd for C38H47Cl2N7O4: 736.7291; found 736.7252. HPLC Purity: 96.3%, tR = 7.5 min.
1-sec-butyl-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-((3-methyl-1H-1,2,4-triazol-1-
yl)methyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-
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5(4H)-one (15a). H NMR (500 MHz, CDCl3, δH): 8.08 (bs, 1H), 7.62 (s, 1H), 7.60 (s, 1H),
7.46 (d, J = 2.0 Hz, 1H), 7.43 (d, J = 9.0 Hz, 2H), 7.26 (dd, J = 8.0, 2.0 Hz, 1H), 7.03 (d, J =
9.5 Hz, 2H), 6.98-6.97 (m, 2H), 6.80 (d, J = 9.0 Hz, 2H), 4.75 (d, J = 15.0 Hz, 1H), 4.65 (d, J
= 15.0 Hz, 1H), 4.36 (dt, J = 6.5, 5.0, 2.5 Hz, 1H), 4.29 (dd, J = 8.5, 6.5 Hz, 1H), 3.90 (dd, J =
8.5, 6.5 Hz, 1H), 3.83 (dd, J = 8.5, 4.5 Hz, 1H), 3.77 (dd, J = 9.5, 5.0 Hz, 1H), 3.43 (dd, J =
9.5, 6.5 Hz, 1H), 3.39 (m, 4H), 3.25 (t, J = 5.0 Hz, 4H), 2.35 (s, 3H), 1.89 – 1.83 (m, 1H), 1.74
– 1.69 (m, 1H), 1.39 (d, J = 6.5 Hz, 3H), 0.90 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3,
δC): 152.1, 145.3, 136.0, 134.3, 134.0, 133.2, 131.5, 129.7, 127.3, 123.7, 123.6, 118.7, 116.8,
115.3, 107.7, 74.7, 67.7, 67.5, 53.3, 52.7, 50.9, 49.2, 28.5, 19.3, 13.9, 10.8. HRMS (ESI) calcd
for C36H40Cl2N8O4: 719.2628; found 719.2614. HPLC Purity: 97.4%, tR = 9.2 min.
1-sec-butyl-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-((3,5-dimethyl-1H-1,2,4-triazol-
1-yl)methyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-
5(4H)-one (15b). 1H NMR (500 MHz, CDCl3, δH): 7.67 (d, J = 8.5 Hz, 1H), 7.61 (s, 1H), 7.47
(d, J = 2.0 Hz, 1H), 7.43 (d, J = 9.0 Hz, 2H), 7.29 (dd, J = 8.5, 2.0 Hz, 1H), 7.03 (d, J = 9.5
Hz, 2H), 6.97 (d, J = 9.5 Hz, 2H), 6.77 (dd, J = 7.0, 2.5 Hz, 2H), 4.64 (d, J = 15.0 Hz, 1H),
4.54 (d, J = 15.0 Hz, 1H), 4.36 (dt, J = 6.5, 5.0, 2.0 Hz, 1H), 4.29 (qt, J = 8.5, 6.5, 4.0 Hz, 1H),
3.84 (t, J = 5.0 Hz, 1H), 3.67 (dd, J = 5.0, 6.5 Hz, 1H), 3.39 (t, J = 5.0 Hz, 4H), 3.29-3.38 (m,
1H), 3.24 (t, J = 5.0 Hz, 4H), 2.51 (s, 3H), 2.30 (s, 3H), 1.89 – 1.83 (m, 1H), 1.74 – 1.69 (m,
1H), 1.39 (d, J = 6.5 Hz, 3H), 0.90 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3, δC): 159.0,
154.5, 152.1, 150.5, 136.0, 134.7, 134.0, 133.1, 131.4, 129.8, 127.3, 127.1, 126.0, 123.6, 123.6,
118.7, 116.8, 116.2, 115.2, 108.6, 74.6, 67.6, 67.3, 52.7, 51.9, 50.8, 49.2, 28.5, 19.3, 13.7, 10.8.
HRMS (ESI) calcd for C37H42Cl2N8O4: 733.2784; found 733.2764. HPLC Purity: 95.4%, tR =
7.9 min.
1-sec-butyl-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-((3-(trifluoromethyl)-1H-1,2,4-
triazol-1-yl)methyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-
triazol-5(4H)-one (15c). 1H NMR (500 MHz, CDCl3, δH): 8.32 (s, 1H), 7.62 (bs, 1H), 7.60 (s,
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1H), 7.49 (d, J = 2.0 Hz, 1H), 7.43 (d, J = 8.5 Hz, 2H), 7.29 (dd, J = 8.5, 2.0 Hz, 1H), 7.03 (d,
J = 9.0 Hz, 2H), 6.94-6.91 (m, 2H), 6.79 (d, J = 8.0 Hz, 2H), 4.87 (d, J = 15.0 Hz, 1H), 4.80
(d, J = 15.0 Hz, 1H), 4.37 (t, J = 5.0 Hz, 1H), 4.29 (dd, J = 8.5, 6.5, 2.0 Hz, 1H), 3.93 (dd, J =
8.5, 7.0 Hz, 1H), 3.82 (dd, J = 8.5, 5.0 Hz, 1H), 3.80 (dd, J = 10.0, 4.5 Hz, 1H), 3.49 (dd, J =
9.5, 6.0 Hz, 1H), 3.37 (bs, 4H), 3.25 (bs, 4H), 1.89 – 1.83 (m, 1H), 1.74 – 1.69 (m, 1H), 1.39
(d, J = 7.0 Hz, 3H), 0.90 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3, δC): 162.6, 152.1,
146.6, 136.3, 134.0, 133.6, 133.2, 131.5, 129.7, 127.4, 123.6, 116.7, 115.2, 107.2, 74.7, 67.4,
67.4, 54.1, 52.7, 49.2, 36.5, 28.5, 19.3, 10.8. HRMS (ESI) calcd for C36H37Cl2F3N8O4:
773.2345; found 773.2357. HPLC Purity: 95.5%, tR = 12.2 min.
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4-(4-(4-(4-(((2S,4R)-2-((1H-imidazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1-sec-butyl-1H-1,2,4-triazol-5(4H)-one (15d).
1H NMR (500 MHz, CDCl3, δH): 7.61 (s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.53 (bs, 1H), 7.46 (d,
J = 2.5 Hz, 1H), 7.42 (d, J = 9.5 Hz, 2H), 7.26 (dd, J = 8.2, 2.2 Hz, 2H), 7.03 (d, J = 9 Hz, 2H),
7.00-6.98 (m, 1H), 6.93 (d, J = 9.0 Hz, 2H), 6.78 (d, J = 9.5 Hz, 2H), 4.51 (d, J = 15.0 Hz, 1H),
4.41 (d, J = 15.0 Hz, 1H), 4.41 (d, J = 15.0 Hz, 1H), 4.34 – 4.28 (m, 2H), 3.87 (dd, J = 8.5, 6.5
Hz, 1H), 3.74 – 3.72 (m, 2H), 3.36 (t, J = 5.0 Hz, 4H), 3.32 – 3.31 (m, 1H), 3.23 (t, J = 5.0 Hz,
4H), 1.89 – 1.83 (m, 1H), 1.74 – 1.71 (m, 1H), 1.39 (d, J = 7.0 Hz, 3H), 0.90 (t, J = 7.0 Hz,
3H). 13C NMR (125 MHz, CDCl3, δC): 152.7, 152.1, 150.6, 146.0, 136.0, 134.6, 134.0, 133.0,
131.4, 129.5, 127.3, 125.9, 123.6, 118.5, 116.7, 115.3, 108.0, 74.8, 67.7, 67.6, 52.7, 50.6, 49.3,
28.5, 19.3, 10.8. HRMS (ESI) calcd for C36H39Cl2N7O4: 704.2519; found 704.2525. HPLC
Purity: 95.3%, tR = 8.3 min.
1-sec-butyl-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-((2-methyl-1H-imidazol-1-
yl)methyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-
5(4H)-one (15e). 1H NMR (500 MHz, CDCl3, δH): 7.63 (d, J = 8.5 Hz, 1H), 7.61 (s, 1H), 7.47
(d, J = 2.0 Hz, 1H), 7.42 (d, J = 9.0 Hz, 2H), 7.29 (dd, J = 8.5, 2.0 Hz, 1H), 7.03 (d, J = 9.0
Hz, 2H), 6.94-6.90 (m, 3H), 6.77 (d, J = 9.5 Hz, 2H), 4.41 (d, J = 15.0 Hz, 1H), 4.34 (d, J =
15.0 Hz, 1H), 4.32 – 4.26 (m, 2H), 3.85 (dd, J = 8.5, 6.5 Hz, 1H), 3.76-3.73 (m, 1H), 3.66 (dd,
J = 9.5, 5.0 Hz, 1H), 3.36 (t, J = 4.5 Hz, 4H), 3.24-3.22 (m, 5H), 2.49 (s, 3H), 1.89 – 1.84 (m,
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1H), 1.74 – 1.69 (m, 1H), 1.39 (d, J = 6.5 Hz, 3H), 0.90 (t, J = 7.5 Hz, 3H). C NMR (125
MHz, CDCl3, δC): 152.7, 152.1, 150.6, 146.0, 136.0, 134.0, 133.1, 131.6, 129.6, 127.3, 125.9,
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123.6, 118.5, 116.7, 115.3, 108.6, 74.8, 67.6, 67.4, 52.7, 50.6, 49.3, 43.2, 28.5, 19.3, 10.8.
HRMS (ESI) calcd for C37H41Cl2N7O4: 718.2675; found 718.2645. HPLC Purity: 95.0%, tR =
5.3 min
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yl)methyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-
5(4H)-one (15f).
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7.65 (d, J = 8.5 Hz, 1H), 7.62 (s, 1H), 7.48 (d, J = 2 Hz, 1H), 7.43 (d, J = 9.0 Hz, 2H), 7.30
(dd, J = 8.5, 2.0 Hz, 1H), 7.04 (d, J = 9 Hz, 2H), 6.95-6.92 (m, 4H), 6.77 (d, J = 9.0 Hz, 2H),
4.51 (d, J = 15.0 Hz, 1H), 4.44 (d, J = 15.0 Hz, 1H), 4.38 (d, J = 15.0 Hz, 1H), 4.35 – 4.27 (m,
2H), 3.86 (dd, J = 8.5, 6.5 Hz, 1H), 3.76 (dd, J = 8.5, 4.5 Hz, 1H), 3.65 (dd, J = 9.5, 5.2 Hz,
1H), 3.37 (t, J = 5.0 Hz, 4H), 3.24 (t, J = 5.0 Hz, 4H), 3.15 (t, J = 8.5 Hz, 1H), 2.8(q, J = 7.5
Hz, 2H), 1.90 – 1.84 (m, 1H), 1.75 – 1.70 (m, 1H), 1.41 (d, J = 6.5 Hz, 3H), 1.34 (3t, J = 7.5
Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3, δC): 153.2, 152.6, 151.3, 151.1,
146.4, 136.5, 135.7, 133.8, 132.1, 130.2, 128.0, 127.2, 126.6, 124.3, 122.3, 119.2, 117.4, 116.0,
109.5, 75.9, 68.7, 68.6, 53.9, 51.9, 50.8, 50.5, 29.9, 21.5, 20.8, 13.4, 12.4. HRMS (ESI) calcd
for C38H43Cl2N7O4: 732.2832; found 732.2855. HPLC Purity: 98.2%, tR = 5.6 min.
1-sec-butyl-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-((2-isopropyl-1H-imidazol-1-
yl)methyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-
5(4H)-one (15g). 1H NMR (500 MHz, CDCl3, δH): 7.63 (d, J = 8.5 Hz, 1H), 7.61 (s, 1H), 7.48
(d, J = 2.5 Hz, 1H), 7.42 (d, J = 9.0 Hz, 2H), 7.29 (dd, J = 8.5, 2.0 Hz, 1H), 7.03 (d, J = 9 Hz,
2H), 6.97 (bs, 1H), 6.94 (s, 1H), 6.93-6.92 (m, 2H), 6.75 (d, J = 9.0 Hz, 2H), 4.51 (d, J = 15.0
Hz, 1H), 4.44 (d, J = 15.0 Hz, 1H), 4.38 (d, J = 15.0 Hz, 1H), 4.35 – 4.27 (m, 2H), 3.85 (dd, J
= 8.5, 6.5 Hz, 1H), 3.76 (dd, J = 8.5, 4.5 Hz, 1H), 3.66 (dd, J = 9.5, 5.2 Hz, 1H), 3.36 (t, J =
5.2 Hz, 4H), 3.26 (t, J = 7.0 Hz, 1H), 3.23 (t, J = 5.0 Hz, 4H), 1.89 – 1.83 (m, 1H), 1.74 – 1.69
(m, 1H), 1.39 (d, J = 6.5 Hz, 3H), 1.34 (d, J = 7.0 Hz, 3H), 1.31 (d, J = 6.5 Hz, 3H), 0.90 (t, J
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= 7.5 Hz, 3H). C NMR (125 MHz, CDCl3, δC): 154.4, 152.6, 152.0, 150.6, 146.0, 136.0,
133.9, 133.1, 131.5, 129.6, 127.4, 125.9, 123.6, 121.3, 118.5, 116.7, 115.2, 108.3, 74.8, 67.6,
67.6, 52.7, 50.6, 49.3, 28.5, 25.5, 22.0, 21.6, 19.3, 10.8. HRMS (ESI) calcd for C39H45Cl2N7O4:
746.2988; found 746.2996. HPLC Purity: 95.8%, tR = 6.0 min.
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1-sec-butyl-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-((2-phenyl-1H-imidazol-1-
yl)methyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-
5(4H)-one (15h). 1H NMR (500 MHz, CDCl3, δH): 7.61 (s, 2H), 7.60 (d, J = 3.5 Hz, 1H), 7.51
(d, J = 8.5 Hz, 1H), 7.43-7.40 (m, 5H), 7.30 (d, J = 2.5 Hz, 1H), 7.25 (d, J = 1.5 Hz, 1H), 7.22
(dd, J = 8.5, 2.5 Hz, 1H), 7.13 (s, 1H), 7.03 (d, J = 9.5 Hz, 2H), 6.94 (d, J = 9.5 Hz, 2H), 6.79
(d, J = 9.5 Hz, 2H), 4.56 (s, 2H), 4.38 – 4.27 (m, 2H), 3.89-3.87 (m, 2H), 3.80 (dd, J = 9.5, 5.0
Hz, 1H), 3.50 (dd, J = 9.5, 6.5 Hz, 1H), 3.37-3.34 (m, 5H), 3.23 (t, J = 5.5 Hz, 4H), 3.20-3.19
(m, 1H), 1.88 – 1.83 (m, 1H), 1.74 – 1.70 (m, 1H), 1.39 (d, J = 6.5 Hz, 3H), 0.90 (t, J = 7.5 Hz,
3H). 13C NMR (125 MHz, CDCl3, δC):152.6, 152.0, 150.6, 146.0, 133.9, 131.4, 129.6, 129.5,
128.5, 127.1, 125.9, 123.6, 123.0, 118.9, 116.0, 115.3, 108.4, 74.7, 67.9, 67.3, 52.7, 50.6, 49.3,
28.5, 19.3, 10.8. HRMS (ESI) calcd for C42H43Cl2N7O4: 780.2832; found 780.2819. HPLC
Purity: 95.6%, tR = 6.5 min.
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1-sec-butyl-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-((4-methyl-1H-imidazol-1-
yl)methyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-
5(4H)-one (15i). 1H NMR (500 MHz, CDCl3, δH): 7.61 (s, 1H), 7.58 (d, J = 8.5 Hz, 1H), 7.49
(bs, 1H), 7.46 (d, J = 2.0 Hz, 1H), 7.42 (d, J = 9.0 Hz, 2H), 7.27-7.25 (m, 1H), 7.03 (d, J = 9.0
Hz, 2H), 6.94 (dd, J = 9.0, 2.0 Hz, 2H), 6.78 (d, J = 9.0 Hz, 2H), 6.69 (bs, 1H), 4.43 (d, J =
15.0 Hz, 1H), 4.34 (d, J = 15.0 Hz, 1H), 4.33 – 4.27 (m, 2H), 3.86 (dd, J = 8.5, 6.5 Hz, 1H),
3.79 (dd, J = 8.5, 4.5 Hz, 1H), 3.71 (dd, J = 9.5, 5.0 Hz, 1H), 3.36 (t, J = 4.5 Hz, 4H), 3.29 (dd,
J = 9.5, 6.5 Hz, 1H), 3.23 (t, J = 5.0 Hz, 4H), 2.18 (s, 3H), 1.89 – 1.83 (m, 1H), 1.74 – 1.69 (m,
1H), 1.39 (d, J = 6.5 Hz, 3H), 0.90 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3, δC): 152.7,
152.1, 150.6, 146.0, 136.0, 134.6, 134.0, 133.0, 131.4, 129.6, 127.3, 125.9, 123.6, 118.5, 116.7,
115.2, 108.0, 74.8, 67.7, 67.5, 52.7, 51.4, 50.7, 49.3, 28.5, 19.3, 10.8. HRMS (ESI) calcd for
C37H41Cl2N7O4: 718.2675; found 718.2697. HPLC Purity: 95.7%, tR = 5.3 min.
1-(sec-butyl)-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-((2,4-dimethyl-1H-imidazol-
1-yl)methyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-
5(4H)-one (15j). 1H NMR (500 MHz, CDCl3, δH): 7.65 (d, J = 8.5 Hz, 1H), 7.61 (s, 1H), 7.43-
7.40 (m, 3H), 7.24 (s, 1H), 7.03 (d, J = 8.5 Hz, 2H), 6.89 (d, J = 8.5 Hz, 2H), 6.73 (s, 1H), 6.66
(d, J = 8.5 Hz, 2H), 4.35-4.24 (m, 3H), 4.18-4.15 (m, 1H), 3.94 (t, J = 7.5 Hz, 1H), 3.89-3.86
(m, 1H), 3.82-3.79 (m, 1H), 3.75 (t, J = 7.5 Hz, 1H), 3.33-3.37 (m, 4H), 3.20-3.22 (m, 4H),
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2.50 (s, 3H), 2.20 (s, 3H), 1.90-1.82 (m, 1H), 1.75-1.70 (m, 1H), 1.40 (d, J = 6.5 Hz, 3H), 0.91
(t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3, δC): 152.6,152.0, 150.2, 146.9, 136.2, 143.0,
134.1, 133.8, 131.2, 129.6, 127.2, 125.5, 122.5, 118.2, 117.5, 115.2, 108.2, 75.3, 67.9, 67.4,
52.5, 50.7, 49.4, 28.7, 15.6, 14.2, 10.8. HRMS (ESI) calcd for C38H43Cl2N7O4: 732.2832; found
732.2803. HPLC Purity: 94.8%, tR = 5.0 min
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1-(sec-butyl)-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-((2,5-dimethyl-1H-imidazol-
1-yl)methyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-
5(4H)-one (15k). 1H NMR (500 MHz, CDCl3, δH): 7.73 (d, J = 8.5 Hz, 1H), 7.61 (s, 1H), 7.45-
7.41 (m, 3H), 7.24 (s, 1H), 7.03 (d, J = 8.5 Hz, 2H), 6.89 (d, J = 8.5 Hz, 2H), 6.67-6.65 (m,
3H), 4.33-4.27 (m, 2H), 4.24-4.21 (m, 1H), 4.02-3.98 (m, 1H), 3.85-3.84 (d, J = 4.5 Hz, 1H),
3.81-3.76 (m, 2H), 3.72 (t, J = 1.5 Hz, 1H), 3.37-3.34 (m, 4H), 3.24-3.20 (m, 4H), 2.51 (s, 3H),
2.30 (s, 3H), 1.90-1.85 (m, 1H), 1.75-1.70 (m, 1H), 1.41 (d, J = 6.5 Hz, 3H), 0.93 (t, J = 7.5
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Hz, 3H). C NMR (125 MHz, CDCl3, δC): 152.7,152.0, 150.2, 146.9, 136.2, 142.7, 134.1,
133.8, 131.2, 128.0, 127.2, 125.5, 122.0, 118.2, 117.5, 115.2, 108.2, 75.3, 67.9, 67.4, 52.5, 50.7
49.7, 28.7, 14.0, 10.8. HRMS (ESI) calcd for C38H43Cl2N7O4: 732.2832; found 732.2837.
HPLC Purity: 95.0%, tR = 5.2 min.
1-(sec-butyl)-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-((2-methyl-4-
(trifluoromethyl)-1H-imidazol-1-yl)methyl)-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-5(4H)-one (15l). 1H NMR
(500 MHz, CDCl3, δH): 7.64 (d, J = 8.5 Hz, 2H), 7.61 (s, 1H), 7.48 (d, J = 1.5 Hz, 1H), 7.42
(d, J = 9 Hz, 2H), 7.31-7.29 (m, 2H), 7.03 (d, J = 9 Hz, 2H), 6.93 (d, J = 9 Hz, 2H), 6.75 (d, J
= 9 Hz, 2H), 4.44-4.41 (m, 1H), 4.37-4.26 (m, 3H), 3.85 (t, J = 8.5 Hz, 1H), 3.78-3.76 (m, 1H),
3.65 (q, J = 4.5 Hz, 1H), 3.32-3.38 (m, 4H), 3.40-3.20 (m, 4H), 2.48 (s, 3H), 1.90-1.84 (m,
1H), 1.75-1.69 (m, 1H), 1.40 (d, J = 6.5 Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz,
CDCl3, δC): 152.7, 152.1, 150.6, 146.0, 136.1, 134.5, 133.0, 132.0, 129.6, 127.2, 125.9, 123.6,
121.9, 118.6, 116.5, 107.2, 74.7, 67.8, 52.7, 50.8, 49.3, 28.5, 19.3, 14.1, 10.8. HRMS (ESI)
calcd for C38H40Cl2F3N7O4: 786.2549; found 786.2562. HPLC Purity: 95.7%, tR = 11.9 min.
4-(4-(4-(4-(((2S,4R)-2-((1H-pyrazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1-sec-butyl-1H-1,2,4-triazol-5(4H)-one (15m).
1H NMR (500 MHz, CDCl3, δH): 7.62 (s, 1H), 7.58 (d, J = 8.5 Hz, 1H), 7.49 (t, J = 2.2 Hz,
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2H), 7.45 (d, J = 2.0 Hz, 1H), 7.43 (d, J = 9.0 Hz, 2H), 7.22 (dd, J = 9.5, 2.0 Hz, 1H), 7.03 (d,
J = 9.5 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 6.77 (d, J = 9.0 Hz, 2H), 6.22 (t, J = 2.0 Hz, 1H),
4.79 (d, J = 14.5 Hz, 1H), 4.68 (d, J = 15.0 Hz, 1H), 4.36-4.33 (m, 1H), 4.31-4.27 (m, 1H),
3.87 (dd, J = 8.5, 6.5 Hz, 1H), 3.80 (dd, J = 8.5, 4.5 Hz, 1H), 3.77 (dd, J = 8.5, 5.0 Hz, 1H),
3.37-3.31 (m, 5H), 3.24 (bs, 4H), 1.90 – 1.84 (m, 1H), 1.74 – 1.70 (m, 1H), 1.39 (d, J = 6.5 Hz,
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3H), 0.90 (t, J = 7.2 Hz, 3H). C NMR (125 MHz, CDCl3, δC): 152.1, 139.4, 135.6, 135.0,
134.0, 133.2, 133.3, 131.2, 129.7, 127.1, 123.6, 118.5, 116.7, 115.3, 108.5, 105.8, 74.6, 68.0,
67.6, 56.1, 52.7, 50.7, 49.3, 28.5, 19.3, 10.8. HRMS (ESI) calcd for C36H39Cl2N7O4: 704.2519;
found 704.2542. HPLC Purity: 95.9%, tR = 11.6 min.
4-(4-(4-(4-(((2S,4R)-2-((1H-tetrazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1-sec-butyl-1H-1,2,4-triazol-5(4H)-one (15n).
1H NMR (500 MHz, CDCl3, δH): 8.46 (s, 1H), 7.61 (s, 1H), 7.55 (d, J = 8.5 Hz, 1H), 7.48 (d, J
= 2.0 Hz, 1H), 7.43 (d, J = 9 Hz, 2H), 7.24 (dd, J = 8.5, 2.0 Hz, 1H), 7.03 (d, J = 9.0 Hz, 2H),
6.81 (d, J = 9.0 Hz, 2H), 5.36 (d, J = 14.0 Hz, 1H), 5.27 (d, J = 14.0 Hz, 1H), 4.38 (t, J = 5.0
Hz, 1H), 4.31-4.27 (m, 1H), 3.95 (dd, J = 8.5, 6.5 Hz, 1H), 3.88 – 3.83 (m, 2H), 3.53 (dd, J =
9.5, 6.5 Hz, 1H), 3.38 (bs, 4H), 3.26 (bs, 4H), 1.89 – 1.83 (m, 1H), 1.74 – 1.69 (m, 1H), 1.39
(d, J = 7.0 Hz, 3H), 0.90 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3, δC): 162.5, 152.8,
152.7, 152.0, 136.3, 133.9, 133.3, 131.5, 130.1, 129.6, 127.2, 123.6, 116.8, 115.4, 107.4, 74.8,
67.9, 67.6, 56.6, 52.7, 36.5, 31.0, 28.5, 19.2, 10.8. HRMS (ESI) calcd for C34H37Cl2N9O4:
706.2424; found 706.2425. HPLC Purity: 95.9%, tR = 10.5 min.
4-(4-(4-(4-(((2S,4R)-2-((2H-tetrazol-2-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1-sec-butyl-1H-1,2,4-triazol-5(4H)-one (15o).
1H NMR (500 MHz, CDCl3, δH): 8.79 (s, 1H), 7.99 (s, 1H), 7.61 (s, 1H), 7.58 (d, J = 8.5 Hz,
1H), 7.48 (d, J = 2.0 Hz, 1H), 7.42-7.40 (m, 2H), 7.28-7.26 (m, 1H), 7.01 (d, J = 9.0 Hz, 2H),
6.79 (d, J = 9.0 Hz, 2H), 5.03 (d, J = 4.0 Hz, 2H), 4.34 (t, J = 9.0 Hz, 1H), 4.29-4.25 (m, 1H),
3.91 (dd, J = 8.5, 7.0 Hz, 1H), 3.77 – 3.74 (m, 2H), 3.56 (dd, J = 10.0, 5.5 Hz, 1H), 3.35 (bs,
4H), 3.23 (d, J = 5.0 Hz, 4H), 1.88 – 1.82 (m, 1H), 1.73 – 1.67 (m, 1H), 1.37 (d, J = 7.0 Hz,
13
3H), 0.88 (t, J = 7.5 Hz, 3H). C NMR (125 MHz, CDCl3, δC): 162.9, 151.9, 151.7, 151.5,
136.3, 133.9, 133.9, 131.5, 131.0, 129.6, 126.9, 123.4, 116.6, 115.4, 107.4, 74.8, 67.9, 67.6,
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56.6, 52.7, 36.5, 31.2, 28.5, 19.2, 10.7. HRMS (ESI) calcd for C34H37Cl2N9O4: 706.2424; found
706.2425. HPLC Purity: 94.6%, tR = 10.4 min.
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1-sec-butyl-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-((5-methyl-1H-tetrazol-1-
yl)methyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-
5(4H)-one (15p). 1H NMR (500 MHz, CDCl3, δH): 7.61 (s, 1H), 7.59 (d, J = 8.5 Hz, 1H), 7.48
(d, J = 2.0 Hz, 1H), 7.43 (d, J = 9 Hz, 2H), 7.26 (dd, J = 8.0, 2.5 Hz, 1H), 7.03 (d, J = 9.0 Hz,
2H), 6.80 (d, J = 8.5 Hz, 2H), 5.27 (d, J = 14.5 Hz, 1H), 5.15 (d, J = 14.5 Hz, 1H), 4.39 (dt, J
= 6.5, 5.0, 1.5 Hz, 1H), 4.31-4.27 (m, 1H), 3.95-3.87 (m, 2H), 3.80 (dd, J = 9.5, 4.7 Hz, 1H),
3.76 (dd, J = 9.0, 7.0 Hz, 1H), 3.38 (bs, 4H), 3.27 (bs, 4H), 2.47 (s, 3H), 1.89 – 1.83 (m, 1H),
1.74 – 1.69 (m, 1H), 1.39 (d, J = 7.0 Hz, 3H), 0.90 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz,
CDCl3, δC): 171.2, 162.9, 152.1, 133.9, 133.5, 131.5, 129.6, 127.2, 123.6, 117.7, 115.4, 107.5,
74.7, 67.9, 67.5, 60.4, 56.4, 52.7, 28.5, 21.1, 19.3, 14.2, 10.8. HRMS (ESI) calcd for
C35H39Cl2N9O4: 720.2580; found 720.2571. HPLC Purity: 94.8%, tR = 11.3 min.
1-sec-butyl-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-((5-phenyl-1H-tetrazol-1-
yl)methyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-
5(4H)-one (15q). 1H NMR (500 MHz, CDCl3, δH): 8.15-8.13 (m, 2H), 8.02 (bs, 1H), 7.63-7.59
(m, 2H), 7.50 (d, J = 2.5 Hz, 1H), 7.48-7.46 (m, 2H), 7.44-7.41 (m, 2H), 7.26 (dd, J = 8.5, 2.0
Hz, 1H), 7.03 (d, J = 9.0 Hz, 2H), 6.67 (d, J = 9.0 Hz, 2H), 5.35 (d, J = 14.5 Hz, 1H), 5.24 (d,
J = 14.5 Hz, 1H), 4.41-4.34 (m, 1H), 4.29 (dd, J = 9.0, 6.5 Hz, 1H), 3.94 (dd, J = 9.0, 6.5 Hz,
1H), 3.88 (dd, J = 8.5, 4.5 Hz, 1H), 3.81 (dd, J = 9.5, 4.5 Hz, 1H), 3.44 (dd, J = 9.5, 6.5 Hz,
1H), 3.35 (bs, 4H), 3.20 (bs, 4H), 1.89 – 1.83 (m, 1H), 1.75 – 1.69 (m, 1H), 1.39 (d, J = 6.5
Hz, 3H), 0.90 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3, δC): 196.5, 133.9, 133.4, 131.5,
129.7, 128.9, 127.0, 123.6, 118.4, 115.3, 107.5, 57.4, 56.7, 52.7, 32.2, 28.5, 19.3, 10.8. HRMS
(ESI) calcd for C40H41Cl2N9O4: 782.2737; found 782.2749. HPLC Purity: 95.7%, tR = 13.7
min.
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HUVEC Culture and Proliferation Assays
HUVEC (Lonza) were grown in EGM-2 bullet kit media (Lonza) and used at passage eight
or lower. The [3H] thymidine incorporation assay was conducted as previously described.32
Briefly, cells were seeded in 96-well plates at a concentration of 2000 cells/well and allowed
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to settle overnight. Drugs were added to each well in triplicate. After 24 h, cells were treated
with 1 μCi of [3H] thymidine for 6 h. Then cells were harvested and transferred to filter mats,
scintillation counted and IC50 were calculated using Graph Pad Prism software (version 6.0)
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CYP3A4 enzyme assay
Inhibition of CYP3A4 enzyme activity was tested using Vivid™ CYP3A4 Green Screening
Kit (ThermoFisher, #P2857) according to the manufacturer`s protocol. Briefly, CYP3A4
baculosomes and drugs were incubated at 37℃ for 10 min. Then Vivid® Substrates were
added and the fluorescence (ex/em:485/520) were read 60 min after substrate added. The
percent inhibition was calculated using 10 μM ketoconazole as 100% inhibition control.
Tube formation assay
Twenty four-well plates were coated with 250 µl matrigel (BD, #CB-40234C) per well.
HUVEC (70,000) were added to the plate with 500 µL media with different drugs. After 24 h,
2 μM calcein AM was added and incubated for 15 min. After replacing with new media, the
tube network was photographed using fluorescent microscopy. The total tube length was
calculated using ImageJ and plotted using GraphPad Prism.
Filipin staining
HUVEC (2,000) were plated in chamber slide with 1ml media and allowed to settle overnight.
Cells were treated with 0.1 µM drug or DMSO for 14 h. The cells were fixed with 4%
paraformaldehyde for 15 min. After washing, cells were incubated with 500 µl 50 µg/ml filipin
solution for 1 h in the dark. Then the cells were washed twice with PBS, mounted and covered
with coverslip. The images were taken using confocal microscope under 360/460 nm.
Western blot
HUVEC were treated with varying concentrations of 15n for 24 h. Cells were lysed using
RIPA buffer and the protein concentration were measured and normalized. After
electrophoresis and transfer onto 0.45 µM nitrocellulose membranes, the membranes were
blocked with 5% BSA for 1 h and then incubated with primary antibody overnight at 4 °C.
Secondary antibody was applied to each membrane for 1 hour. Blots were imaged using
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Syngene PXi imaging system after adding chemiluminescent substrate. The following
antibodies were used for the assay: AMPKα (1:1,000, cell signaling, #2532s), phosphor-
AMPKα (1:1,000, cell signaling #2535s), ACC (1:1,000, cell signaling #3662s), phosphor-
ACC (1:1,000, cell signaling, #3661s), mTOR (1:1,000, cell signaling, #2972S ), phosphor-
mTOR (1:1,000, cell signaling, #9234s), p70 S6 Kinase (1:1,000, santa cruz, #sc-8418),
phosphor- p70 S6 Kinase (1:1,000, cell signaling, #3662s), anti-Rabbit IgG (1:10000, GE
lifesciences, #NA934V).
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Supporting Information: Molecular formula strings (CSV); detail of synthesis procedures;
kenetic curve of CYP3A4 enzyme activities; philipin staining of compound 15c, 15g;
competition assay of itraconazole photoaffinity probe; NMR and HPLC chart of representative
compounds
Corresponding Author
*J.O.L.: phone, 410-955-4619; fax, 410-955-4520; e-mail, joliu@jhu.edu
Author Contributions
II These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
Acknowledgment. This work was supported by the National Cancer Institutes (Grant
R01CA184103) and the Flight Attendant Medical Research Institute.
Abbreviations:
HUVEC, human umbilical vein endothelial cell; NPC1, Niemann-Pick disease, type C1;
VDAC1, voltage-dependent anion channel 1; 14-DM, lanosterol 14-alpha demethylase;
mTORC, mammalian target of rapamycin complex; SAR, structure-activity relationship; IC50,
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half-maximal inhibitory concentration; DMSO, dimethyl sulfoxide; DMF, dimethylformamide;
EC50, half-maximal effective concentration; AMPKα, 5' AMP-activated protein kinase α
subunit; ATP, adenosine triphosphate; AMP, adenosine monophosphate; CYP3A4,
cytochrome P450 3A4; rt, room temperature; THF, tetrahydrofuran; DCM, dichloromethane;
TfOH, trifluoromethanesulfonic acid; NaH, sodium hydride; ACC1, acetyl CoA carboxylase
1; S6K, p70 S6 kinase; , chemical shifts.
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