Design, synthesis, and structure-activity relationship studies of L-amino alcohol derivatives as broad-spectrum antifungal agents

Article abstract of DOI:10.1016/j.ejmech.2019.05.047

To discover broad spectrum antifungal agents, two strategies were applied, and a novel class of L-amino alcohol derivatives were designed and synthesized. 3-F substituted compounds 14i, 14n, 14s and 14v exhibited excellent antifungal activities with broad antifungal spectra against C. albicans and C. tropicalis, with MIC values in the range of 0.03–0.06 μg/mL, and against A. fumigatus and C. neoformans, with MIC values in the range of 1–2 μg/mL. Notably, Compounds 14i, 14n, 14s and 14v also displayed moderate activities against fluconazole-resistance strains 17# and CaR that were isolated from AIDS patients. Moreover, only compounds in the S-configuration showed antifungal activity. Preliminary mechanistic studies showed that the potent antifungal activity of compound 14v stemmed from inhibition of C. albicans CYP51. Compounds 14n and 14v were almost nontoxic to mammalian A549 cells, and their stability in human plasma was excellent.

Full text of DOI:10.1016/j.ejmech.2019.05.047

Accepted Manuscript
Design, synthesis, and structure-activity relationship studies of l-amino alcohol
derivatives as broad-spectrum antifungal agents
Liyu Zhao, Linfeng Tian, Nannan Sun, Yin Sun, Yixuan Chen, Xinran Wang, Shizhen
Zhao, Xin Su, Dongmei Zhao, Maosheng Cheng
PII:
S0223-5234(19)30457-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.047
EJMECH 11357
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 April 2019
Revised Date: 16 May 2019
Accepted Date: 16 May 2019
Please cite this article as: L. Zhao, L. Tian, N. Sun, Y. Sun, Y. Chen, X. Wang, S. Zhao, X. Su, D. Zhao,
M. Cheng, Design, synthesis, and structure-activity relationship studies of l-amino alcohol derivatives
as broad-spectrum antifungal agents, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.05.047.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis, and structure-activity relationship studies of
L-amino alcohol derivatives as broad-spectrum antifungal agents
Liyu Zhaoa, Linfeng Tiana, Nannan Suna, Yin Suna, Yixuan Chena, Xinran Wanga, Shizhen Zhaob,
Xin Suc, Dongmei Zhaoa,*, Maosheng Chenga
aKey Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe
District, Shenyang 110016, China
bKey Laboratory of Receptors-Mediated Gene Regulation and Drug Discovery, School of
Medicine, Henan University, Kaifeng 475004, China
cThe School of life Science and Biopharmaceutical, Shenyang Pharmaceutical University, 103
Wenhua Road, Shenhe District, Shenyang, 110016, China
*Corresponding author: Dongmei Zhao, E-mail: medchemzhao@163.com
ABSTRACT:
To discover broad spectrum antifungal agents, two strategies were applied, and a novel class
of L-amino alcohol derivatives were designed and synthesized. 3-F substituted compounds 14i,
14n, 14s and 14v exhibited excellent antifungal activities with broad antifungal spectra against C.
albicans and C. tropicalis, with MIC values in the range of 0.03 - 0.06 ꢀg/mL, and against A.
fumigatus and C. neoformans, with MIC values in the range of 1 - 2 ꢀg/mL. Notably, Compounds
14i, 14n, 14s and 14v also displayed moderate activities against fluconazole-resistance strains 17#
and CaR that were isolated from AIDS patients. Moreover, only compounds in the S-configuration
showed antifungal activity. Preliminary mechanistic studies showed that the potent antifungal
activity of compound 14v stemmed from inhibition of C. albicans CYP51. Compounds 14n and
14v were almost nontoxic to mammalian A549 cells, and their stability in human plasma was
excellent.
Keywords:
CYP51; Azole antifungals; Structure-activity relationship; Fluconazole-resistance.
1. Introduction
Over the past few decades, the incidence of systemic fungal infections has increased
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markedly, especially in immunocompromised hosts, such as patients undergoing organ transplants
or anticancer chemotherapy and patients with AIDS [1-4]. Candida spp., Cryptococcus
neoformans and Aspergillus spp. are the three major pathogenic fungi that threaten human health
[5]. Approximately 72.8 million people suffer from Candida spp. infections, 65.5 million people
suffer from Cryptococcus spp. infections and 12.4 million people suffer from Aspergillus spp.
infections each year [6]. Currently, clinical antifungal agents can be divided into four classes:
polyenes (such as Amphotericin B and Nystatin) [7], azoles (such as Fluconazole and Itraconazole)
[8], echinocandins (such as Caspofungin and Micafungin) [9] and antimetabolites (such as
5-fluorocytosine) [10].
Azoles, inhibitors of fungal lanosterol 14α-demethylase (CYP51), can prevent the
biosynthesis of ergosterol, which is a very important component of biofilms [11, 12]. Fluconazole
is a typical azole antifungal agent that has been used clinically for almost 30 years. Due to the
high therapeutic index, azole drugs have been used as first-line antifungal agents, and some azole
derivatives have been developed, such as itraconazole, voriconazole, ketoconazole (Fig. 1).
However, some problems remain, such as severe drug resistance and cytotoxicity and a narrow
antifungal spectrum [13, 14]. Therefore, an urgent clinical need exists to develop broad spectrum
azole antifungal agents with novel structures, low toxicity and high efficiency.
Figure 1 Chemical structures of azole antifungal agents and lead compound 5
.
In our previous studies [15-17], a series of azole derivatives with an ester group were
designed and synthesized. Most of the compounds exhibited excellent in vitro antifungal activity
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against Candida albicans. However, these compounds were inactive against Aspergillus fumigatus.
The potent compound 5 displayed excellent antifungal activity against Candida albicans (SC5314)
with an MIC value 0.03 ꢀg/mL but was inactive against Aspergillus fumigatus. To discover
antifungal agents with a broad spectrum, the Candida albicans CYP51 (PDB: 5TZ1) [18] and
Aspergillus fumigatus CYP51 (PDB: 4UYM) [19] were overlaid, and we found that the amino
acid residues Leu503 and Tyr122 made the cavity of Aspergillus fumigatus CYP51 more narrow than
that of Candida albicans CYP51 (Fig. 2A). The steric clash between the phenyl group of ester
compound 5 with Leu503 may be responsible for the inactivity against Aspergillus fumigatus (Fig.
2B). To avoid the clash and increase the compound’s activity against Aspergillus fumigatus, two
strategies were applied: (1) replacing the ester group with small alkyl side chains to reduce the
steric hindrance, and (2) conformational restriction of the phenyl group of the compound by
interacting with surrounding amino acid residues to prevent rotation (Fig. 3). A series of L-amino
alcohol derivatives were designed and synthesized.
Figure 2 (A) Overlay of Candida albicans CYP51 (green, PDB: 5TZ1) and Aspergillus fumigatus CYP51 (yellow,
PDB: 4UYM). (B) Predicted binding mode of 5 in Aspergillus fumigatus CYP51. The distances are shown as red
dashed lines.
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Figure 3 Design strategies of novel L-amino alcohol derivatives. C. alb., Candida albicans (CPCC400523); A.
fum., Aspergillus fumigatus (cgmcc 3.7795).
2. Results and discussion
2.1. Chemistry
The key intermediates 8a-n were prepared conveniently via palladium-catalyzed Suzuki
coupling of commercially available substituted 4-bromobenzoic acid 6a-h and substituted
phenylboronic 7a-d (Scheme 1).
Scheme 1 Synthesis of intermediates 8a-n. Reagents and conditions: (a) Pd(PPh3)4, K2CO3, dioxane, reflux, 5 h.
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Scheme 2 General synthesis of the target compounds 14a-ac. Reagents and conditions: (a) (Boc)2O, TEA, DCM,
rt.; (b) TsCl, TEA, DMAP, DCM, rt.; (c) imidazole, NaH, DMF; (d) HCl-EtOH, rt.; (e) EDCI, HOBt, DIEA, r.t., 4
h.
The alkyl compounds 14a-ac were prepared according the procedures shown in Scheme 2.
Various L-amino alcohol derivatives 9a-g were protected with (Boc)2O to afford compounds
10a-g, which were converted to 11a-g through substitution reactions. Treatment of compounds
11a-g with imidazole and NaH in dry DMF afforded 12a-g, and then cleavage of the Boc
protecting group with hydrochloric acid gave the key intermediates 13a-g. The target compounds
14a-ac were synthesized via amide reaction of amino 13a-g and acid 8a-n in DMF at room
temperature.
Scheme 3 Synthesis of the target compound 17. Reagents and conditions: (a) triazole, NaH, DMF; (b) EtOH-HCl,
rt.; (c) EDCI, HOBt, DIEA, 4 h, rt.
As depicted in Scheme 3, the triazole derivative 17 was prepared by using procedures similar
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to those described above, except that triazole was used instead of imidazole. The synthetic
procedures for compound 23 of R-configuration were similar to the synthesis of compounds
14a-ac (Scheme 4).
Scheme 4 Synthesis of the target compound 23. Reagents and conditions: (a) (Boc)2O, TEA, DCM, rt.; (b) TsCl,
TEA, DMAP, DCM, rt.; (c) imidazole, NaH, DMF; (d) EtOH-HCl, rt.; (e) EDCI, HOBt, DIEA, r.t., 4 h.
2.2. In vitro antifungal activity
The target compounds were evaluated for their in vitro antifungal activity against five
pathogenic fungi according to the protocols of the National Committee for Clinical Laboratory
Standards (NCCLS). Fluconazole (FCZ) and itraconazole (ICZ) were used as the reference drugs.
The results of in vitro antifungal activities are summarized in Table 1.
Compounds 14a-f with different alkyl side chain lengths exhibited moderate to good
antifungal activities. Compound 14a with a methyl substitution increased the activity against
Aspergillus fumigatus with an MIC value of 1 ꢀg/mL. However, the antifungal activities of 14a
against Candida albicans, Cryptococcus neoformans and Candida tropicalis were moderate or
even disappeared. Compounds 14e and 14f with bulk substitutions displayed excellent activities
against Candida albicans and Candida tropicalis with MIC values < 0.03 ꢀg/mL but were inactive
against Aspergillus fumigatus.
Most of the compounds 14h-x with fluorine substitution maintained excellent antifungal
activities against Candida albicans, Cryptococcus neoformans and Candida tropicalis. Moreover,
the 3-F substitution compounds 14i, 14n, 14s and 14v increased the antifungal activities against
Aspergillus fumigatus with MIC values in the range of 1 - 2 ꢀg/mL. Substitution of -Cl, -CH3 and
-OCH3 (14y-14ac) led to disappearance of the antifungal activity against Aspergillus fumigatus.
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Replacing the imidazole group (14n) with a triazole group (17) resulted in a slight decrease
in antifungal activities against Candida albicans, Candida tropicalis and Aspergillus fumigatus.
Notably, the compound of R-configuration (23) displayed no antifungal activity compared to the
S-configuration (14d). It turns out that the S-configuration is the pharmacodynamic configuration.
Table 1
In vitro antifungal activities of target compounds (MIC, ꢀg/mL)a.
Compd.
14a
14b
14c
14d
14e
14f
R1
R2
H
R3
H
C. alb.(I)
4
C. alb.(II)
0.5
C. neo.
C. tro.
0.25
A. fum.
1
-CH3
>64
16
2
-CH2CH3
H
H
0.25
0.06
0.06
<0.03
<0.03
0.125
0.06
0.06
0.06
0.06
0.5
<0.06
<0.03
<0.03
<0.03
<0.03
<0.03
<0.03
<0.03
<0.03
<0.03
0.25
0.125
<0.03
<0.03
<0.03
<0.03
<0.03
0.06
4
-(CH2)2CH3
H
H
4
-CH(CH3)2
H
H
1
8
-CH(CH3)CH2CH3
-CH2CH(CH3)2
-CH2Ph
H
H
0.5
1
>64
>64
>64
>64
1
H
H
H
H
0.25
2
14g
14h
14i
-CH(CH3)2
2-F
3-F
2'-F
3'-F
4'-F
2-F
3-F
2'-F
3'-F
4'-F
2-F
3-F
2-F
3-F
3-F
3-F
3-F
2-Cl
3-Cl
H
-CH(CH3)2
H
2
<0.03
0.06
-CH(CH3)2
H
1
>64
>64
32
14j
-CH(CH3)2
H
2
0.06
14k
14l
-CH(CH3)2
H
1
0.25
-CH(CH3)CH2CH3
-CH(CH3)CH2CH3
-CH(CH3)CH2CH3
-CH(CH3)CH2CH3
-CH(CH3)CH2CH3
-CH2CH(CH3)2
-CH2CH(CH3)2
-CH2Ph
H
0.06
<0.03
0.06
0.125
0.25
0.06
0.06
0.06
0.06
0.06
0.06
0.25
4
<0.03
<0.03
<0.03
<0.03
0.125
<0.03
<0.03
<0.03
<0.03
<0.03
<0.03
0.125
0.5
2
0.06
>64
2
14m
14n
14o
14p
14q
14r
14s
H
1
<0.03
0.06
H
1
>64
>64
>64
>64
2
H
2
0.06
H
1
0.125
<0.03
<0.03
<0.03
<0.03
<0.03
<0.03
0.125
0.25
H
4
H
2
H
0.5
>64
1
>64
>64
2
14t
-CH2Ph
H
14u
14v
14w
14x
14y
14z
-CH(CH3)CH2CH3
-CH(CH3)CH2CH3
-CH(CH3)CH2CH3
-CH(CH3)CH2CH3
-CH(CH3)CH2CH3
2'-F
3'-F
4'-F
H
4
4
4
8
4
>64
>64
H
0.25
0.06
>64
0.125
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-CH(CH3)CH2CH3
2-CH3
H
H
H
-
>16
1
>16
0.25
0.25
0.125
>16
>64
>64
>64
>64
>64
2
2
0.5
>64
>64
>64
2
14aa
14ab
14ac
17
-CH(CH3)CH2CH3
3-CH3
-CH(CH3)CH2CH3
3-OCH3
1
0.125
0.25
8
-
-
-
-
-
-
-
-
-
-
0.25
>16
0.25
1
-
>64
>64
>64
<0.125
23
-
<0.03
0.5
<0.06
1
5
-
4
FCZ
ITZ
-
<0.03
<0.03
<0.125
<0.03
aAbbreviations: C. alb.(I), Candida albicans (CPCC400523); C. alb.(II), Candida albicans (ATCC SC5314); C.
neo., Cryptococcus neoformans (cgmcc 2.3161); C. tro., Candida tropicalis (cgmcc 2.3739); A. fum., Aspergillus
fumigatus (cgmcc 3.7795); FCZ: Fluconazole; ITZ: Itraconazole.
2.3. In vitro antifungal activity against fluconazole-resistant strains of Candida albicans.
Azole antifungal agents have been used clinically for many years, especially fluconazole,
which has been applied in the clinic for approximately 30 years. The widespread use of azole
antifungal agents has led to severe drug-resistance, which has been a major clinical problem for
treating systemic fungal infection. Therefore, it is necessary to further evaluate the antifungal
activity of the compounds against fluconazole-resistant strains when developing innovative drugs.
The potent compounds 14i, 14n, 14s and 14v were selected to evaluate their antifungal activity
against fluconazole-resistant strains of C. albicans (strain 17# and strain CaR), which were
isolated from AIDS patients. The results are summarized in Table 2. The compounds showed
moderate antifungal activities against strain 17# with MIC values in the range of 1 - 4 ꢀg/mL and
against strain CaR with MIC values in the range of 4 - 32 ꢀg/mL.
Table 2
In vitro antifungal activities of the target compounds (MIC, ꢀg/mL)a.
Candida albicans
Compd.
Strain 17#
Strain CaR
14i
14n
14s
4
2
32
4
1
4
14v
2
8
FCZ
>64
>64
aAbbreviations: strain 17#, fluconazole-resistant strain of Candida albicans; strain CaR, fluconazole-resistant
strain of Candida albicans; FCZ: Fluconazole. Strain 17# and strain CaR were provided by Institute of
Microbiology, Chinese Academy of Sciences.
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2.4. GC-MS analysis of sterol composition in Candida albicans (SC5314)
L-amino alcohol derivatives are a novel class of antifungal agents, and only compounds in
the S-configuration showed antifungal activity. To investigate the antifungal mechanism of
compound 14v, gas chromatography-mass spectrometry (GC-MS) was used to analyze the sterol
composition in cells using fluconazole as the reference drug. The sterol profile results are
summarized in Table 3. Ergosterol was the major sterol (100%) in cells, and other sterols were not
detected in the absence of drug. When Candida albicans were cultured for 16 h with different
concentrations of fluconazole, the sterol components of the cells changed in
a
concentration-dependent manner. The content of ergosterol decreased sharply from 97.7% to 0%
with increasing fluconazole concentration, and the obtusifoliol, lanosterol and eburicol content
increased dramatically. These changes in sterol contents were caused by inhibiting the sterol
14α-demethylase enzyme (CYP51) of the ergosterol biosynthetic pathway. The same changes in
sterol content were shown when Candida albicans was cultured with different concentrations of
compound 14v. The ergosterol content decreased and the obtusifoliol, lanosterol, eburicol contents
increased with increasing concentration of 14v. The results indicate that the antifungal mechanism
of compound 14v is similar to fluconazole, and the target is fungal lanosterol 14α-demethylase
(CYP51).
Table 3
Analysis of sterol composition in C. albicans by GC-MS.a
% of total sterols (C. alb.a)
concentration
Compd.
(ꢀg/mL)
Ergosterol
98.0
36.0
6.5
Obtusifoliol
Lanosterol
2.0
Eburicol
0
0.13
0.5
2
0
12.2
23.8
31.3
0
21.0
20.0
23.8
2.3
30.8
49.7
44.9
0
14vb
8
0
0.13
0.5
2
97.7
90.1
17.2
0
0
9.9
0
FLCc
17.7
37.2
0
28.0
22. 9
0
37.1
39.9
0
8
Controld
-
100
aAbbreviations: C. alb., Candida albicans (ATCC SC5314); bTreated with compound 14v; cTreated with FLC;
dControl (no drug).
2.5. In vitro cytotoxicity assay
Fungus is a type of eukaryote that is similar to mammalian cells. The compounds designed as
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antifungal agents may cause side-effects on mammalian cells. Therefore, it is necessary to
consider the toxicity of the compounds to mammalian cells. The potent compounds 14n and 14v
were selected to evaluate their toxicity against A549 cells (human lung carcinoma) according to
the protocol described previously [20], and voriconazole was selected as a control. The results are
shown in Table 4. Compounds 14n and 14v displayed 14% and 13.9% inhibition, respectively,
against A549 cells at a concentration of 50 ꢀM/L, and these results were superior to voriconazole,
which displayed 37% inhibition. Therefore, compounds 14n and 14v were almost nontoxic to
A549 cells.
Table 4
In vitro cytotoxicity of compounds in A549 cells.
Inhibition (ꢀM/L)
Compd.
0.08
1.2%
5.4%
17.1%
0.40
1.0%
6.6%
20.3%
2
10
50
1.2%
7.3%
25.5%
3.1%
8.3%
32%
14.0%
13.9%
36.7%
14n
14v
voriconazole
2.6. In vitro human plasma stability assay
The stability of compounds in human plasma is an important property that must be
considered in the development of new drugs. In general, ester groups are unstable in human
plasma and can be easily hydrolyzed. Replacing ester groups with alkyl side chains, the potent
alkyl derivatives 14n and 14v with broad antifungal spectra were selected to evaluate their human
plasma stability. The results are summarized in Table 5 and show that 14n and 14v both exhibited
excellent stability in human plasma, and 113.1% and 113.6% of each compound remained,
respectively, when incubated in plasma for 60 min.
Table 5
In vitro human plasma stability of compounds 14n and 14v.
Stability in human blood plasma
Compd.
% Remaining at 60 min
% Remaining at 120 min
113.1
113.6
86.3
92.6
14n
14v
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2.7. Structure-activity relationships
Compounds 14a-f with different lengths of alkyl side chains were investigated. The results
showed that the activities of 14a-f against Candida albicans, Cryptococcus neoformans and
Candida tropicalis increased with extension of the alkyl side chains. However, the activities
against Aspergillus fumigatus decreased with extension of the alkyl side chains.
Fluorine substituted compounds 14h-x with bulky alkyl side chains maintained excellent
activities against Candida albicans, Cryptococcus neoformans and Candida tropicalis. Moreover,
compounds of 3-F substitution on the biphenyl group displayed activity against Aspergillus
fumigatus, such as the compounds 14i, 14n, 14s and 14v. Compounds with -Cl, -Me or -OCH3
substitution on the biphenyl group showed moderate or even disappearing antifungal activity.
In addition, the influence of the stereochemical configuration was also investigated.
Compared to the S-configured compound 14d, compound 23 in its R-configuration lost all activity
against the fungi. It turns out that the S-configuration is the pharmacodynamic configuration.
2.8. Molecular docking model analysis of compounds 14n and 14v in the active site of CYP51
To better understand the binding mode of compounds with Aspergillus fumigatus CYP51 and
provide information for further optimization, compounds 14n and 14v were docked into the active
site of Aspergillus fumigatus CYP51 by using the CDOCKER program in the Discovery Studio
3.0 software. The published crystal structure of Aspergillus fumigatus CYP51 (PDB ID: 4UYM)
served as a useful template for generating the binding mode [18]. As shown in Fig. 4A, the
imidazole of compound 14n binds to the heme group through formation of a coordination bond
with the iron atom. The alkyl side chain forms hydrophobic interactions with Thr126, Phe130 and
Tyr136. The biphenyl group extends into the CYP51 channel and mainly forms hydrophobic and
Van der Waals interactions with the surrounding residues such as Leu91, Phe234, Ile377 and Leu503
.
Moreover, the 3-F atom on the biphenyl group can form a halogen (fluorine) bond with Leu503 and
a hydrogen bond with Ile373 (Ile373 as the H-donor and fluorine as the H-acceptor). Therefore, the
conformation of the phenyl of compound 14n can be restricted to avoid the steric clash with
Leu503. The binding mode of compound 14v with Aspergillus fumigatus CYP51 is similar to that
of 14n (Fig. 4B).
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Figure 4 (A) Predicted binding mode of 14n in the active site of Aspergillus fumigatus CYP51. (B) Predicted
binding mode of 14v in the active site of Aspergillus fumigatus CYP51. The distances are shown as red dashed
lines. The halogen bonds are shown as blue dashed lines. The hydrogen bonds are shown as yellow dashed lines.
Figures were generated using PyMOL.
3. Conclusions
The steric clash with Leu503 is the main reason that caused ester compound 5 to be inactive
against A. fumigatus. To avoid this clash and increase the activity against A. fumigatus, the bulky
ester group was replaced with small alkyl side chains. Compounds with different lengths of alkyl
side chains were synthesized. The in vitro activities showed that the activities against A. fumigatus
increased when the alkyl side chains were shortened, but the activities against Candida albicans,
Cryptococcus neoformans and Candida tropicalis decreased when the alkyl side chains were
shortened. Compound 14a with methyl substitution increased the antifungal activity against A.
fumigatus with an MIC value of 1 ꢀg/mL. However, the activities against Candida albicans,
Cryptococcus neoformans and Candida tropicalis were moderate or even lost.
To maintain excellent activities against Candida albicans, Cryptococcus neoformans and
Candida tropicalis, the isopropyl, sec-butyl and isobutyl substitutions were selected. Moreover, in
order to be effective against A. fumigatus, the strategy of conformational restriction of the
compound was applied. A series of fluorine-substituted compounds were synthesized. Of these,
3-F substitution compounds 14i, 14n, 14s and 14v exhibited the excellent activities against
Candida albicans, Cryptococcus neoformans and Candida tropicalis and displayed antifungal
activity against A. fumigatus with MIC values in the range of 1 - 2 ꢀg/mL.
Notably, compounds 14n, 14s and 14v showed moderate activities against the fluconazole
resistant strains of C. albicans (strain 17# and strain CaR) which were isolated from AIDS
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patients. In addition, the configuration of the compounds had a great influence on the antifungal
activity, and only compounds of the S-configuration showed antifungal activity. Preliminary
mechanistic studies showed that the potent antifungal activity of compound 14v stemmed from
inhibition of C. albicans CYP51. Compounds 14n, 14v were almost nontoxic to A549 cells at 50
ꢀM/L and exhibited excellent stabilities in human plasma. Molecular docking results showed that
the 3-F can interact with Leu503 and Ile373, which is very important for the conformational
restriction of the compounds.
4. Experimental section
4.1. Chemistry
All reagents used were commercially available without further purification. Solvent were
purified according to standard procedures. The reactions were monitored by TLC, which was
performed on silica gel plates with fluorescence F-254 and visualized with UV light. Silica gel of
200-300 mesh was used for column chromatography. The melting points of the compounds were
determined on a BüCHI Melting Point B-540 melting point apparatus and were uncorrected. Mass
spectrometry was performed on an Agilent 1200 LC-MS (Agilent, Palo Alto, CA, USA).
High-resolution mass spectra (HRMS) were performed on an Agilent 6530 accurate-mass Q-TOF
LC-MS system. GC−MS analysis was performed on an Agilent 1200 LC−MS using ESI mode.
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III-600 or Bruker
Avance III-400 instruments (600 or 400 MHz for 1H and 150 or 100 MHz for 13C) with TMS as an
internal standard. The coupling constants (J) are reported in hertz and the peak multiplicities were
described as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad peak.
4.2. General procedure for the synthesis of intermediates (8a-n)
Under an argon atmosphere, substituted 4-bromobenzoic acid (1 equiv), benzoic acid (1.1
equiv), K2CO3 (2 equiv), Pd[P(Ph)3]4 (0.1 equiv) were added to dioxane/H2O (10:1). The mixture
o
was stirred in an 110 C oil bath for 5 h. The reaction mixture was cooled to room temperature,
and the solvent was removed by rotary evaporation. The residue was dissolved in water, and the
solution was acidified with 3 N HCl until the product precipitated. The precipitate was filtered to
afford compounds 8a-n.
4.3. General procedure for the synthesis of intermediates (10a-g)
L-amino alcohol (1 equiv), (Boc)2O (1.2 equiv) and trimethylamine (2 equiv) were added to
13

ACCEPTED MANUSCRIPT
dichloromethane. The mixture was stirred under ice bath conditions. The reaction was followed by
TLC and diluted with dichloromethane. The organic phase was washed with citric acid (1 N) and
saturated NaHCO3 then dried over Na2SO4 and filtered. The filtrate was concentrated and purified
by a silica column to afford compounds 10a-g.
4.3.1. Tert-butyl ((2S,3S)-1-hydroxy-3-methylpentan-2-yl)carbamate (10e)
Light yellow oil; yield: 91%; 1H NMR (400 MHz, DMSO) δ 6.36 (d, J = 8.6 Hz, 1H), 4.38 (t,
J = 5.4 Hz, 1H), 3.43 – 3.28 (m, 3H), 1.55 – 1.48 (m, 1H), 1.41 – 1.36 (m, 10H), 1.09 – 1.00 (m,
1H), 0.85 – 0.78 (m, 6H).
4.4. General procedure for the synthesis of intermediates (11a-g)
To a solution of intermediates 10a-g (1 equiv) in dichloromethane, TsCl (1.2 equiv), DMAP
o
(0.2 equiv) and trimethylamine (3 equiv) were added. The solution was stirred at -20 C. The
reaction was followed by TLC and quenched with water. The organic phase was washed with
citric acid (1 N) and saturated NaHCO3 then dried over Na2SO4 and filtered. The filtrate was
concentrated and purified by a silica column to afford compounds 11a-g as a white solid.
4.4.1. (S)-2-((tert-butoxycarbonyl)amino)propyl 4-methylbenzenesulfonate (11a)
Light white solid; yield 85.1%; 1H NMR (600 MHz, CDCl3) δ 7.79 (d, J = 8.2 Hz, 2H), 7.35
(d, J = 8.0 Hz, 2H), 7.26 (s, 1H), 4.03 – 3.88 (m, 3H), 2.45 (s, 3H), 1.40 (s, 9H), 1.16 (d, J = 6.8
Hz, 3H).
4.5. General procedure for the synthesis of intermediates (12a-g)
Under an argon atmosphere, the imidazole (2 equiv) and NaH (3 equiv) were added to dry
DMF at 0 oC. The mixture was stirred for 1 h. Then, intermediates 11a-g (1 equiv) was added and
stirred at room temperature for 3 - 4 h, controlled by TLC. Upon completion of the reaction, the
reaction was quenched with water and extracted with ethyl acetate three times. The combined
organic layers were dried over Na2SO4 and evaporated in a vacuum. The crude product was
purified by silica gel column chromatography (CH3OH:CH2Cl2 = 40:1).
4.5.1. tert-butyl ((2S,3S)-1-(1H-imidazol-1-yl)-3-methylpentan-2-yl)carbamate (12e)
1
Light white solid; yield 35.3%; H NMR (400 MHz, DMSO) δ 7.08 (s, 1H), 6.83 (m, 2H),
4.07 (dd, J = 13.9, 3.8 Hz, 1H), 3.82 (dd, J = 13.8, 10.3 Hz, 1H), 3.56 (m, 1H), 1.48 – 1.42 (m,
2H), 1.30 (s, 9H), 1.14 – 1.07 (m, 1H), 0.90 – 0.85 (m, 6H).
4.6. General procedure for the synthesis of intermediates (13a-g)
14

ACCEPTED MANUSCRIPT
Imidazole derivatives 12a-g were dissolved in a solution of HCl-EtOH (4 N). The mixture
was stirred at room temperature. The reaction was monitored with TLC. The mixture was filtered
to afford compounds 13a-g as a white solid.
4.7. General procedure for the synthesis of compounds (14a-ac)
To a solution of acid 8a-n (1 equiv) in DMF, HOBt (1.1 equiv), EDCI (1.1 equiv) was added
and the mixture was stirred for 1 h at room temperature. Then, the amino salt 13a-g (1.1 equiv)
and DIEA (2.2 equiv) were added, and the reaction was stirred for 4 h and monitored with TLC.
The reaction was poured into water and extracted with ethyl acetate three times. The combined
organic layers were dried over Na2SO4 and evaporated in vacuum. The residue was purified by
silica gel column chromatography (CH3OH:CH2Cl2 = 40:1) to afford the target compounds
14a-ac.
4.7.1. (S)-N-(1-(1H-imidazol-1-yl)propan-2-yl)-[1,1'-biphenyl]-4-carboxamide (14a)
Light white solid; yield: 59.0%; mp: 237.1 – 239.6 °C. 1H NMR (600 MHz, DMSO) δ 8.43 (d,
J = 8.1 Hz, 1H), 7.89 (d, J = 8.3 Hz, 2H), 7.76 (d, J = 8.3 Hz, 2H), 7.73 (d, J = 7.4 Hz, 2H), 7.60
(s, 1H), 7.50 (t, J = 7.7 Hz, 2H), 7.41 (t, J = 7.3 Hz, 1H), 7.15 (s, 1H), 6.85 (s, 1H), 4.34 – 4.30 (m,
1H), 4.15 – 4.08 (m, 2H), 1.16 (d, J = 6.7 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 166.04, 143.22,
139.66, 138.10, 133.67, 129.50 (2C), 128.61, 128.52, 128.37 (2C), 127.35 (2C), 126.94 (2C),
120.36, 50.62, 46.71, 18.46. HRMS (EI): m/z (M+) for C19H19N3O: calcd. 305.1528; found
305.1531.
4.7.2. (S)-N-(1-(1H-imidazol-1-yl)butan-2-yl)-[1,1'-biphenyl]-4-carboxamide (14b)
Light white solid; yield: 57.2%; mp: 195.8 –197.9 °C. 1H NMR (600 MHz, DMSO) δ 8.31 (d,
J = 8.2 Hz, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 8.4 Hz, 2H), 7.74 – 7.70 (m, 2H), 7.57 (s,
1H), 7.50 (t, J = 7.7 Hz, 2H), 7.41 (t, J = 7.4 Hz, 1H), 7.13 (s, 1H), 6.83 (s, 1H), 4.17 (dd, J = 15.9,
5.2 Hz, 2H), 4.08 (d, J = 5.1 Hz, 1H), 1.60 – 1.56 (m, 1H), 1.53 – 1.49 (m, 1H), 0.92 (t, J = 7.4 Hz,
3H). 13C NMR (150 MHz, DMSO) δ 166.43, 143.18, 139.71, 138.08, 133.71, 129.50 (2C), 128.53,
128.49, 128.40 (2C), 127.35 (2C), 126.93 (2C), 120.35, 52.30, 49.54, 25.38, 10.86. HRMS (EI):
m/z (M+) for C20H21N3O: calcd. 319.1685; found 319.1687.
4.7.3. (S)-N-(1-(1H-imidazol-1-yl)pentan-2-yl)-[1,1'-biphenyl]-4-carboxamide (14c)
Light white solid; yield: 51.6%; mp: 185.0 – 186.6 °C. 1H NMR (600 MHz, DMSO) δ 8.32 (d,
J = 8.5 Hz, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 8.4 Hz, 2H), 7.74 – 7.70 (m, 2H), 7.58 (s,
15

ACCEPTED MANUSCRIPT
1H), 7.50 (t, J = 7.7 Hz, 2H), 7.41 (t, J = 7.4 Hz, 1H), 7.13 (s, 1H), 6.84 (s, 1H), 4.29 – 4.24 (m,
1H), 4.16 (dd, J = 13.9, 4.7 Hz, 1H), 4.07 (dd, J = 13.9, 8.6 Hz, 1H), 1.54 – 1.47 (m, 2H), 1.43 –
1.37 (m, 1H), 1.35 – 1.29 (m, 1H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (150 MHz, DMSO) δ
166.36, 143.22, 139.70, 138.05, 133.71, 129.50 (2C), 128.54, 128.51, 128.34 (2C), 127.36 (2C),
126.96 (2C), 120.37, 50.44, 49.95, 34.31, 19.19, 14.32. HRMS (EI): m/z (M+) for C21H23N3O:
calcd. 333.1841; found 333.1840.
4.7.4. (S)-N-(1-(1H-imidazol-1-yl)-3-methylbutan-2-yl)-[1,1'-biphenyl]-4-carboxamide (14d)
Light white solid; yield: 43.0%; mp: 177.4 – 179.5 °C. 1H NMR (600 MHz, DMSO) δ 8.26 (d,
J = 8.8 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H), 7.73 – 7.70 (m, 2H), 7.59 (s,
1H), 7.50 (t, J = 7.7 Hz, 2H), 7.41 (t, J = 7.4 Hz, 1H), 7.15 (s, 1H), 6.80 (s, 1H), 4.23 (dd, J = 13.2,
3.2 Hz, 1H), 4.16 – 4.13 (m, 1H), 4.12 – 4.09 (m, 1H), 1.87 (td, J = 13.4, 6.7 Hz, 1H), 0.99 (d, J =
6.8 Hz, 3H), 0.96 (d, J = 6.8 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 166.55, 143.19, 139.73,
138.07, 133.82, 129.49 (2C), 128.49 (2C), 128.33 (2C), 127.36 (2C), 126.95 (2C), 120.27, 55.86,
47.80, 31.01, 19.93, 18.89. HRMS (EI): m/z (M+) for C21H23N3O: calcd. 333.1841; found
333.1840.
4.7.5. N-((2S,3S)-1-(1H-imidazol-1-yl)-3-methylpentan-2-yl)-[1,1'-biphenyl]-4-carboxamide
(14e)
Light white solid; yield: 60.9%; mp: 203.2 – 205.7 °C. 1H NMR (600 MHz, DMSO) δ 8.31 (d,
J = 8.8 Hz, 1H), 7.85 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H), 7.73 – 7.70 (m, 2H), 7.62 (s,
1H), 7.49 (t, J = 7.7 Hz, 2H), 7.41 (t, J = 7.4 Hz, 1H), 7.16 (s, 1H), 6.80 (s, 1H), 4.25 (dd, J = 13.5,
3.3 Hz, 1H), 4.20 – 4.16 (m, 1H), 4.10 (dd, J = 13.5, 10.3 Hz, 1H), 1.67 – 1.62 (m, 1H), 1.58 –
1.53 (m, 1H), 1.22 – 1.17 (m, 1H), 0.99 (d, J = 6.8 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H). 13C NMR
(150 MHz, DMSO) δ 166.37, 143.19, 139.73, 138.18, 133.78, 129.49 (2C), 128.49, 128.37,
128.32 (2C), 127.36 (2C), 126.95 (2C), 120.39, 54.93, 47.41, 37.50, 25.54, 15.81, 11.77. HRMS
(EI): m/z (M+) for C22H25N3O: calcd. 347.1998; found 347.2004.
4.7.6. (S)-N-(1-(1H-imidazol-1-yl)-4-methylpentan-2-yl)-[1,1'-biphenyl]-4-carboxamide (14f)
Light white solid; yield: 61.1%. 1H NMR (600 MHz, DMSO) δ 8.30 (d, J = 8.7 Hz, 1H), 7.88
(d, J = 8.4 Hz, 2H), 7.76 (d, J = 8.4 Hz, 2H), 7.74 – 7.71 (m, 2H), 7.56 (s, 1H), 7.50 (t, J = 7.7 Hz,
2H), 7.41 (t, J = 7.4 Hz, 1H), 7.12 (s, 1H), 6.83 (s, 1H), 4.35 – 4.31 (m, 1H), 4.14 (dd, J = 13.9,
4.9 Hz, 1H), 4.04 (dd, J = 13.9, 8.4 Hz, 1H), 1.66 – 1.62 (m, 1H), 1.55 – 1.51 (m, 1H), 1.27 – 1.24
16

ACCEPTED MANUSCRIPT
(m, 1H), 0.92 – 0.87 (m, 6H). 13C NMR (150 MHz, DMSO) δ 166.25, 143.22, 139.71, 138.06,
133.69, 129.50 (2C), 128.52, 128.51, 128.34 (2C), 127.36 (2C), 126.96 (2C), 120.38, 50.39, 48.85,
41.14, 24.81, 23.80, 22.20. HRMS (EI): m/z (M+) for C22H25N3O: calcd. 347.1998; found
347.2008.
4.7.7. (S)-N-(1-(1H-imidazol-1-yl)-3-phenylpropan-2-yl)-[1,1'-biphenyl]-4-carboxamide (14g)
Light white solid; yield: 55.7%; mp: 204.9 – 206.5 °C. 1H NMR (600 MHz, DMSO) δ 8.41 (d,
J = 8.6 Hz, 1H), 7.78 (d, J = 8.3 Hz, 2H), 7.74 – 7.69 (m, 4H), 7.58 (s, 1H), 7.49 (t, J = 7.7 Hz,
2H), 7.40 (t, J = 7.3 Hz, 1H), 7.29 – 7.24 (m, 4H), 7.20 – 7.16 (m, 1H), 7.15 (s, 1H), 6.84 (s, 1H),
4.52 – 4.48 (m, 1H), 4.24 (dd, J = 13.9, 4.5 Hz, 1H), 4.15 (dd, J = 13.9, 8.7 Hz, 1H), 2.91 – 2.86
(m, 2H). 13C NMR (100 MHz, DMSO) δ 166.24, 143.22, 139.69, 138.72, 138.11, 133.67, 129.60
(2C), 129.48 (2C), 128.67 (2C), 128.49, 128.23 (2C), 127.34 (2C), 126.93 (2C), 126.68, 120.37,
52.23, 49.81, 37.99. HRMS (EI): m/z (M+) for C25H23N3O: calcd. 381.1841; found 381.1863.
4.7.8. (S)-N-(1-(1H-imidazol-1-yl)-3-methylbutan-2-yl)-2-fluoro-[1,1'-biphenyl]-4-carboxamide
(14h)
Light white solid; yield: 52.3%; mp: 160.4 – 162.7 °C. 1H NMR (600 MHz, DMSO) δ 8.34 (d,
J = 8.8 Hz, 1H), 7.74 – 7.67 (m, 2H), 7.63 (t, J = 8.0 Hz, 1H), 7.61 – 7.55 (m, 3H), 7.51 (t, J = 7.6
Hz, 2H), 7.45 (t, J = 7.3 Hz, 1H), 7.16 (s, 1H), 6.81 (s, 1H), 4.25 (dd, J = 13.5, 3.3 Hz, 1H), 4.18 –
4.13 (m, 1H), 4.09 (dd, J = 13.4, 10.3 Hz, 1H), 1.90 – 1.84 (m, 1H), 1.00 (d, J = 6.8 Hz, 3H), 0.96
(d, J = 6.8 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 165.21, 159.97, 158.33, 138.09, 136.01,
135.96, 134.79, 131.37, 131.28, 131.26, 131.23, 129.32, 129.31, 129.18 (2C), 128.83, 128.52,
124.19, 124.17, 120.27, 115.39, 115.22, 56.04, 47.81, 30.95, 19.93, 18.85. HRMS (EI): m/z (M+)
for C21H22FN3O: calcd. 351.1747; found 351.1746.
4.7.9. (S)-N-(1-(1H-imidazol-1-yl)-3-methylbutan-2-yl)-3-fluoro-[1,1'-biphenyl]-4-carboxamide
(14i)
Light white solid; yield: 56.8%; mp: 161.2 – 163.0 °C. 1H NMR (600 MHz, DMSO) δ 8.25 (d,
J = 9.1 Hz, 1H), 7.77 – 7.71 (m, 2H), 7.63 – 7.56 (m, 3H), 7.50 (t, J = 7.6 Hz, 2H), 7.43 (t, J = 7.6
Hz, 2H), 7.16 (s, 1H), 6.86 (s, 1H), 4.21 (dd, J = 13.8, 3.9 Hz, 1H), 4.16 – 4.11 (m, 1H), 4.02 (dd,
J = 13.7, 10.1 Hz, 1H), 1.84 (dd, J = 13.1, 6.6 Hz, 1H), 1.00 (d, J = 6.8 Hz, 3H), 0.96 (d, J = 6.8
Hz, 3H). 13C NMR (150 MHz, DMSO) δ 163.77, 160.07, 158.43, 144.05, 143.99, 138.03, 137.66,
130.18, 130.15, 129.10 (2C), 128.52, 128.08, 126.92 (2C), 123.38, 123.28, 122.49, 122.48, 119.78,
17

ACCEPTED MANUSCRIPT
114.08, 113.92, 55.32, 47.44, 30.25, 19.52, 18.01. HRMS (EI): m/z (M+) for C21H22FN3O: calcd.
351.1747; found 351.1755.
4.7.10. (S)-N-(1-(1H-imidazol-1-yl)-3-methylbutan-2-yl)-2'-fluoro-[1,1'-biphenyl]-4-carboxamide
(14j)
Light white solid; yield: 51.9%; mp: 159.8 – 161.4 °C. 1H NMR (600 MHz, DMSO) δ 8.29 (d,
J = 8.8 Hz, 1H), 7.86 (d, J = 8.3 Hz, 2H), 7.66 – 7.59 (m, 3H), 7.57 (td, J = 7.8, 1.4 Hz, 1H), 7.46
(ddd, J = 9.2, 7.2, 1.6 Hz, 1H), 7.37 – 7.31 (m, 2H), 7.16 (s, 1H), 6.81 (s, 1H), 4.24 (dd, J = 13.3,
3.2 Hz, 1H), 4.18 – 4.13 (m, 1H), 4.10 (dd, J = 13.3, 10.3 Hz, 1H), 1.90 – 1.84 (m, 1H), 1.00 (d, J
= 6.8 Hz, 3H), 0.96 (d, J = 6.8 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 166.53, 160.36, 158.73,
138.17, 138.06, 134.31, 131.30, 131.28, 130.62, 130.56, 129.15, 129.13, 128.42, 127.99, 127.95
(2C), 127.90, 125.53, 125.51, 120.30, 116.74, 116.59, 55.88, 47.85, 30.99, 19.94, 18.87. HRMS
(EI): m/z (M+) for C21H22FN3O: calcd. 351.1747; found 351.1753.
4.7.11. (S)-N-(1-(1H-imidazol-1-yl)-3-methylbutan-2-yl)-3'-fluoro-[1,1'-biphenyl]-4-carboxamide
(14k)
Light white solid; yield: 50.9%; mp: 161.2 – 163.5 °C. 1H NMR (600 MHz, DMSO) δ 8.29 (d,
J = 8.8 Hz, 1H), 7.87 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.3 Hz, 2H), 7.62 – 7.56 (m, 3H), 7.56 –
7.51 (m, 1H), 7.25 (td, J = 8.3, 1.8 Hz, 1H), 7.15 (s, 1H), 6.81 (s, 1H), 4.24 (dd, J = 13.3, 3.2 Hz,
1H), 4.18 – 4.14 (m, 1H), 4.10 (dd, J = 13.2, 10.3 Hz, 1H), 1.87 (dt, J = 13.3, 6.7 Hz, 1H), 1.00 (d,
J = 6.8 Hz, 3H), 0.96 (d, J = 6.8 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 166.42, 163.98, 162.37,
142.17, 142.11, 141.70, 141.69, 138.07, 134.36, 131.47, 131.41, 128.50, 128.35 (2C), 127.13 (2C),
123.45, 123.43, 120.26, 115.28, 115.14, 114.17, 114.03, 55.89, 47.80, 31.00, 19.93, 18.90. HRMS
(EI): m/z (M+) for C21H22FN3O: calcd. 351.1747; found 351.1747.
4.7.12. (S)-N-(1-(1H-imidazol-1-yl)-3-methylbutan-2-yl)-4'-fluoro-[1,1'-biphenyl]-4-carboxamide
(14l)
Light white solid; yield: 55.7%; mp: 198.4 – 200.1 °C. 1H NMR (600 MHz, DMSO) δ 8.27 (d,
J = 8.8 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.79 – 7.75 (m, 2H), 7.74 (d, J = 8.4 Hz, 2H), 7.59 (s,
1H), 7.33 (t, J = 8.9 Hz, 2H), 7.15 (s, 1H), 6.80 (s, 1H), 4.24 (dd, J = 13.2, 3.1 Hz, 1H), 4.18 –
4.13 (m, 1H), 4.10 (dd, J = 13.2, 10.3 Hz, 1H), 1.87 (dq, J = 13.3, 6.7 Hz, 1H), 0.99 (d, J = 6.8 Hz,
3H), 0.96 (d, J = 6.8 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 166.49, 163.49, 161.86, 142.12,
138.08, 136.20, 136.18, 133.77, 129.45, 129.39, 128.50, 128.34 (2C), 126.90 (2C), 120.26, 116.38,
18

ACCEPTED MANUSCRIPT
116.24, 55.86, 47.80, 31.00, 19.93, 18.89. HRMS (EI): m/z (M+) for C21H22FN3O: calcd. 351.1747;
found 351.1752.
4.7.13. N-((2S,3S)-1-(1H-imidazol-1-yl)-3-methylpentan-2-yl)-2-fluoro-[1,1'-biphenyl]-4-carboxa
mide (14m)
Light white solid; yield: 53.6%; mp: 153.8 – 155.7 °C. 1H NMR (600 MHz, DMSO) δ 8.38 (d,
J = 8.8 Hz, 1H), 7.73 – 7.66 (m, 2H), 7.63 (t, J = 8.0 Hz, 1H), 7.61 – 7.56 (m, 3H), 7.51 (t, J = 7.6
Hz, 2H), 7.45 (t, J = 7.3 Hz, 1H), 7.15 (s, 1H), 6.81 (s, 1H), 4.25 (dd, J = 13.7, 3.4 Hz, 1H), 4.20 –
4.15 (m, 1H), 4.09 (dd, J = 13.6, 10.3 Hz, 1H), 1.67 – 1.62 (m, 1H), 1.58 – 1.53 (m, 1H), 1.22 –
1.18 (m, 1H), 0.99 (d, J = 6.8 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, DMSO) δ
163.93, 158.89, 157.26, 137.06, 134.90, 134.85, 133.71, 130.30, 130.21, 130.18, 130.16, 128.25,
128.23, 128.10 (2C), 127.76, 127.45, 123.10, 123.08, 119.24, 114.30, 114.14, 54.03, 46.28, 36.34,
24.43, 14.74, 10.66. HRMS (EI): m/z (M+) for C22H24FN3O: calcd. 365.1903; found 365.1915.
4.7.14. N-((2S,3S)-1-(1H-imidazol-1-yl)-3-methylpentan-2-yl)-3-fluoro-[1,1'-biphenyl]-4-carboxa
mide (14n)
Light white solid; yield: 53.0%; mp: 169.8 – 171.7 °C. 1H NMR (600 MHz, DMSO) δ 8.28 (d,
J = 9.0 Hz, 1H), 7.73 (d, J = 7.4 Hz, 2H), 7.62 – 7.56 (m, 3H), 7.50 (t, J = 7.6 Hz, 2H), 7.43 (td, J
= 7.6, 2.3 Hz, 2H), 7.16 (s, 1H), 6.85 (s, 1H), 4.22 (dd, J = 13.8, 3.6 Hz, 1H), 4.18 – 4.14 (m, 1H),
4.02 (dd, J = 13.8, 10.1 Hz, 1H), 1.62 – 1.55 (m, 2H), 1.23 – 1.18 (m, 1H), 0.99 (d, J = 6.7 Hz,
3H), 0.90 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 164.05, 160.55, 158.90, 144.53,
144.47, 138.49, 138.16, 130.66, 130.63, 129.56, 128.99, 128.54, 127.38, 123.78, 123.68, 122.95,
120.30, 114.54, 114.39, 54.93, 47.45, 37.24, 25.19, 15.84, 11.71. HRMS (EI): m/z (M+) for
C22H24FN3O: calcd. 365.1903; found 365.1917.
4.7.15. N-((2S,3S)-1-(1H-imidazol-1-yl)-3-methylpentan-2-yl)-2'-fluoro-[1,1'-biphenyl]-4-carbox
amide (14o)
Light white solid; yield: 52.5%; mp: 172.8 – 174.5 °C. 1H NMR (600 MHz, DMSO) δ 8.34 (d,
J = 8.8 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 7.0 Hz, 2H), 7.60 (s, 1H), 7.58 – 7.55 (m,
1H), 7.48 – 7.44 (m, 1H), 7.37 – 7.31 (m, 2H), 7.15 (s, 1H), 6.81 (s, 1H), 4.24 (dd, J = 13.6, 3.3
Hz, 1H), 4.21 – 4.16 (m, 1H), 4.10 (dd, J = 13.5, 10.3 Hz, 1H), 1.67 – 1.62 (m, 1H), 1.59 – 1.53
(m, 1H), 1.22 – 1.17 (m, 1H), 1.02 – 0.96 (m, 3H), 0.90 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz,
DMSO) δ 166.32, 160.36, 158.72, 138.17, 138.12, 134.27, 131.30, 131.28, 130.61, 130.56, 129.15,
19

ACCEPTED MANUSCRIPT
129.13, 128.51, 127.99, 127.94 (2C), 127.90, 125.53, 125.50, 120.31, 116.73, 116.59, 54.98,
47.37, 37.48, 25.52, 15.82, 11.77. HRMS (EI): m/z (M+) for C22H24FN3O: calcd. 365.1903; found
365.1907.
4.7.16. N-((2S,3S)-1-(1H-imidazol-1-yl)-3-methylpentan-2-yl)-3'-fluoro-[1,1'-biphenyl]-4-carbox
amide (14p)
Light white solid; yield: 69.8%; mp: 170.0 – 172.3 °C. 1H NMR (600 MHz, DMSO) δ 8.33 (d,
J = 8.8 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.62 (s, 1H), 7.60 – 7.57 (m,
2H), 7.55 – 7.51 (m, 1H), 7.25 (td, J = 8.0, 1.5 Hz, 1H), 7.16 (s, 1H), 6.81 (s, 1H), 4.25 (dd, J =
13.5, 3.3 Hz, 1H), 4.20 – 4.16 (m, 1H), 4.11 (dd, J = 13.5, 10.3 Hz, 1H), 1.67 – 1.62 (m, 1H), 1.58
– 1.53 (m, 1H), 1.22 – 1.18 (m, 1H), 0.99 (d, J = 6.8 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H). 13C NMR
(150 MHz, DMSO) δ 166.22, 163.98, 162.37, 142.16, 142.11, 141.71, 138.06, 134.30, 131.47,
131.41, 128.34 (3C), 127.13 (2C), 123.45, 123.43, 120.36, 115.28, 115.14, 114.17, 114.02, 54.96,
47.40, 37.47, 25.54, 15.82, 11.76. HRMS (EI): m/z (M+) for C22H24FN3O: calcd. 365.1903; found
365.1904.
4.7.17. N-((2S,3S)-1-(1H-imidazol-1-yl)-3-methylpentan-2-yl)-4'-fluoro-[1,1'-biphenyl]-4-carbox
amide (14q)
Light white solid; yield: 48.6%; mp: 187.5 – 189.4 °C. 1H NMR (400 MHz, DMSO) δ 8.29 (d,
J = 8.4 Hz, 1H), 7.85 (d, J = 8.5 Hz, 2H), 7.79 – 7.71 (m, 4H), 7.60 (s, 1H), 7.32 (t, J = 8.9 Hz,
2H), 7.14 (s, 1H), 6.80 (s, 1H), 4.24 (dd, J = 12.8, 2.9 Hz, 1H), 4.20 – 4.14 (m, 1H), 4.10 (dd, J =
12.6, 9.8 Hz, 1H), 1.68 – 1.51 (m, 2H), 1.24 – 1.17 (m, 1H), 0.99 (d, J = 6.8 Hz, 3H), 0.90 (t, J =
7.4 Hz, 3H). 13C NMR (100 MHz, DMSO) δ 166.30, 163.89, 161.46, 142.12, 138.07, 136.21,
136.18, 133.76, 129.45, 129.37, 128.43, 128.32 (2C), 126.89 (2C), 120.31, 116.41, 116.19, 54.95,
47.39, 37.48, 25.54, 15.82, 11.75. HRMS (EI): m/z (M+) for C22H24FN3O: calcd. 365.1903; found
365.1906.
4.7.18. (S)-N-(1-(1H-imidazol-1-yl)-4-methylpentan-2-yl)-2-fluoro-[1,1'-biphenyl]-4-carboxamid
e (14r)
Light white solid; yield: 46.8%; mp: 150.6 – 152.3 °C. 1H NMR (600 MHz, DMSO) δ 8.38 (d,
J = 8.6 Hz, 1H), 7.75 – 7.69 (m, 2H), 7.64 (t, J = 8.0 Hz, 1H), 7.61 – 7.56 (m, 3H), 7.51 (t, J = 7.6
Hz, 2H), 7.45 (t, J = 7.4 Hz, 1H), 7.13 (s, 1H), 6.84 (s, 1H), 4.36 – 4.31 (m, 1H), 4.15 (dd, J =
13.9, 4.8 Hz, 1H), 4.03 (dd, J = 13.9, 8.4 Hz, 1H), 1.67 – 1.62 (m, 1H), 1.55 – 1.50 (m, 1H), 1.30
20

ACCEPTED MANUSCRIPT
– 1.26 (m, 1H), 0.90 (d, J = 6.7 Hz, 3H), 0.89 (d, J = 6.6 Hz, 3H). 13C NMR (150 MHz, DMSO) δ
164.91, 164.90, 159.99, 158.36, 138.10, 135.91, 135.87, 134.78, 131.40, 131.31, 131.27, 131.25,
129.32, 129.30, 129.18 (2C), 128.84, 128.60, 124.18, 124.16, 120.40, 115.41, 115.25, 50.34,
49.03, 41.02, 24.80, 23.78, 22.15. HRMS (EI): m/z (M+) for C22H24FN3O: calcd. 365.1903; found
365.1910.
4.7.19. (S)-N-(1-(1H-imidazol-1-yl)-4-methylpentan-2-yl)-3-fluoro-[1,1'-biphenyl]-4-carboxamid
e (14s)
Light white solid; yield: 50.2%; mp: 137.3 – 139.3 °C. 1H NMR (600 MHz, DMSO) δ 8.25 (d,
J = 8.6 Hz, 1H), 7.77 – 7.71 (m, 2H), 7.64 – 7.55 (m, 3H), 7.53 – 7.47 (m, 3H), 7.45 – 7.41 (m,
1H), 7.15 (s, 1H), 6.87 (s, 1H), 4.33 – 4.27 (m, 1H), 4.14 (dd, J = 13.9, 4.8 Hz, 1H), 4.01 (dd, J =
13.9, 8.3 Hz, 1H), 1.73 – 1.67 (m, 1H), 1.49 – 1.44 (m, 1H), 1.27 – 1.24 (m, 1H), 0.90 (t, J = 6.4
Hz, 6H). 13C NMR (150 MHz, DMSO) δ 163.98, 160.61, 158.96, 144.60, 144.55, 138.45, 138.13,
130.77, 130.75, 129.57 (2C), 129.01, 128.61, 127.39 (2C), 123.62, 123.51, 122.95, 122.94, 120.38,
114.56, 114.41, 50.27, 48.91, 40.95, 24.73, 23.79, 22.11. HRMS (EI): m/z (M+) for C22H24FN3O:
calcd. 365.1903; found 365.1908.
4.7.20. (S)-N-(1-(1H-imidazol-1-yl)-3-phenylpropan-2-yl)-2-fluoro-[1,1'-biphenyl]-4-carboxamid
e (14t)
Light white solid; yield: 59.4%; mp: 173.5 – 175.4 °C. 1H NMR (600 MHz, DMSO) δ 8.48 (d,
J = 8.5 Hz, 1H), 7.66 – 7.54 (m, 6H), 7.50 (t, J = 7.5 Hz, 2H), 7.44 (t, J = 7.3 Hz, 1H), 7.31 – 7.23
(m, 4H), 7.20 – 7.17 (m, 1H), 7.16 (s, 1H), 6.85 (s, 1H), 4.53 – 4.48 (m, 1H), 4.25 (dd, J = 13.9,
4.3 Hz, 1H), 4.14 (dd, J = 13.9, 8.8 Hz, 1H), 2.92 (dd, J = 13.8, 4.6 Hz, 1H), 2.86 (dd, J = 13.7,
9.4 Hz, 1H). 13C NMR (100 MHz, DMSO) δ 164.89, 160.34, 157.89, 138.61, 138.12, 135.89,
135.82, 134.75, 131.43, 131.26, 131.23, 129.58, 129.31, 129.28, 129.16, 128.83, 128.69, 128.58,
126.72, 124.07, 124.04, 120.41, 115.33, 115.08, 52.36, 49.82, 37.92. HRMS (EI): m/z (M+) for
C25H22FN3O: calcd. 399.1747; found 399.1756.
4.7.21. (S)-N-(1-(1H-imidazol-1-yl)-3-phenylpropan-2-yl)-3-fluoro-[1,1'-biphenyl]-4-carboxamid
e (14u)
Light white solid; yield: 53.9%; mp: 188.9 – 190.8 °C. 1H NMR (600 MHz, DMSO) δ 8.35 (d,
J = 8.6 Hz, 1H), 7.73 (d, J = 7.9 Hz, 2H), 7.61 (s, 1H), 7.59 (d, J = 11.8 Hz, 1H), 7.55 (d, J = 8.0
Hz, 1H), 7.49 (t, J = 7.5 Hz, 2H), 7.43 (t, J = 7.3 Hz, 1H), 7.37 (t, J = 7.7 Hz, 1H), 7.32 – 7.23 (m,
21

ACCEPTED MANUSCRIPT
4H), 7.21 (t, J = 6.9 Hz, 1H), 7.18 (s, 1H), 6.89 (s, 1H), 4.50 – 4.46 (m, 1H), 4.25 (dd, J = 13.9,
4.3 Hz, 1H), 4.15 – 4.11 (m, 1H), 2.90 (dd, J = 13.8, 4.5 Hz, 1H), 2.81 (dd, J = 13.7, 9.5 Hz, 1H).
13C NMR (100 MHz, DMSO) δ 163.87, 161.02, 158.55, 144.68, 144.60, 138.52, 138.42, 138.11,
130.73, 130.69, 129.63, 129.55, 129.00, 128.65, 128.49, 127.37, 126.73, 123.34, 123.19, 122.87,
122.85, 120.41, 114.59, 114.36, 52.31, 49.77, 37.90. HRMS (EI): m/z (M+) for C25H22FN3O: calcd.
399.1747; found 399.1759.
4.7.22. N-((2S,3S)-1-(1H-imidazol-1-yl)-3-methylpentan-2-yl)-2',3-difluoro-[1,1'-biphenyl]-4-car
boxamide (14v)
Light white solid; yield: 51.5%; mp: 123.0 – 125.0 °C. 1H NMR (400 MHz, DMSO) δ 8.32 (d,
J = 8.8 Hz, 1H), 7.61 – 7.56 (m, 2H), 7.50 – 7.45 (m, 2H), 7.45 – 7.41 (m, 2H), 7.38 – 7.31 (m,
2H), 7.16 (s, 1H), 6.86 (s, 1H), 4.23 (dd, J = 13.4, 3.6 Hz, 1H), 4.19 – 4.13 (m, 1H), 4.02 (dd, J =
13.3, 9.8 Hz, 1H), 1.63 – 1.53 (m, 2H), 1.24 – 1.18 (m, 1H), 0.99 (d, J = 6.8 Hz, 3H), 0.90 (t, J =
7.4 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 162.90, 159.21, 158.84, 157.58, 157.19, 138.15,
138.09, 137.06, 130.17, 130.16, 130.00, 129.95, 129.15, 129.12, 127.39, 125.76, 125.68, 124.52,
124.50, 124.22, 123.37, 123.27, 119.25, 115.74, 115.59, 53.85, 46.41, 36.14, 24.08, 14.76, 10.62.
HRMS (EI): m/z (M+) for C22H23F2N3O: calcd. 383.1809; found 383.1812.
4.7.23. N-((2S,3S)-1-(1H-imidazol-1-yl)-3-methylpentan-2-yl)-3',3-difluoro-[1,1'-biphenyl]-4-car
boxamide (14w)
Light white solid; yield: 62.0%; mp: 130.1 – 132.2 °C. 1H NMR (400 MHz, DMSO) δ 8.28 (d,
J = 8.8 Hz, 1H), 7.69 – 7.52 (m, 6H), 7.42 (t, J = 7.8 Hz, 1H), 7.29 – 7.23 (m, 1H), 7.15 (s, 1H),
6.85 (s, 1H), 4.23 (dd, J = 13.4, 3.5 Hz, 1H), 4.19 – 4.12 (m, 1H), 4.02 (dd, J = 13.3, 9.8 Hz, 1H),
1.64 – 1.53 (m, 2H), 1.24 – 1.19 (m, 1H), 0.99 (d, J = 6.8 Hz, 3H), 0.90 (t, J = 7.3 Hz, 3H). 13C
NMR (100 MHz, DMSO) δ 164.37, 163.95, 161.95, 160.91, 158.44, 142.99, 142.91, 140.90,
138.15, 131.55, 131.47, 130.66, 130.63, 128.52, 124.42, 124.26, 123.49, 123.12, 123.09, 120.28,
115.81, 115.60, 114.86, 114.63, 114.32, 114.10, 54.94, 47.47, 37.23, 25.19, 15.84, 11.69. HRMS
(EI): m/z (M+) for C22H23F2N3O: calcd. 383.1809; found 383.1811.
4.7.24. N-((2S,3S)-1-(1H-imidazol-1-yl)-3-methylpentan-2-yl)-4',3-difluoro-[1,1'-biphenyl]-4-car
boxamide (14x)
Light white solid; yield: 61.5%; mp: 132.4 – 134.8 °C. 1H NMR (400 MHz, DMSO) δ 8.25 (d,
J = 8.7 Hz, 1H), 7.82 – 7.75 (m, 2H), 7.63 – 7.52 (m, 3H), 7.41 (t, J = 7.8 Hz, 1H), 7.33 (t, J = 8.9
22

ACCEPTED MANUSCRIPT
Hz, 2H), 7.17 (s, 1H), 6.87 (s, 1H), 4.23 (dd, J = 13.4, 3.5 Hz, 1H), 4.19 – 4.12 (m, 1H), 4.03 (dd,
J = 13.3, 9.8 Hz, 1H), 1.64 – 1.52 (m, 2H), 1.24 – 1.18 (m, 1H), 0.99 (d, J = 6.8 Hz, 3H), 0.90 (t, J
= 7.4 Hz, 3H). 13C NMR (100 MHz, DMSO) δ 164.15, 164.01, 161.70, 160.95, 158.49, 143.46,
143.38, 138.05, 134.96, 130.69, 130.65, 129.56, 129.47, 128.30, 123.73, 123.58, 122.89, 122.86,
120.41, 116.49, 116.27, 114.56, 114.33, 54.90, 47.57, 37.25, 25.20, 15.84, 11.68. HRMS (EI): m/z
(M+) for C22H23F2N3O: calcd. 383.1809; found 383.1798.
4.7.25. N-((2S,3S)-1-(1H-imidazol-1-yl)-3-methylpentan-2-yl)-2-chloro-[1,1'-biphenyl]-4-carbox
amide (14y)
Light white solid; yield: 73.7%; mp: 189.1 – 191.0 °C. 1H NMR (400 MHz, DMSO) δ 8.40 (d,
J = 8.6 Hz, 1H), 7.93 (d, J = 1.7 Hz, 1H), 7.80 (dd, J = 8.0, 1.7 Hz, 1H), 7.58 (s, 1H), 7.52 – 7.43
(m, 6H), 7.14 (s, 1H), 6.81 (s, 1H), 4.25 (dd, J = 13.1, 3.1 Hz, 1H), 4.20 – 4.14 (m, 1H), 4.09 (dd,
J = 13.1, 10.0 Hz, 1H), 1.69 – 1.62 (m, 1H), 1.59 – 1.51 (m, 1H), 1.24 – 1.17 (m, 1H), 1.00 (d, J =
6.8 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, DMSO) δ 164.97, 142.76, 138.49,
138.11, 135.49, 131.95, 131.79, 129.57 (2C), 128.82, 128.77 (2C), 128.61, 128.54, 126.76, 120.29,
55.11, 47.40, 37.38, 25.49, 15.84, 11.70. HRMS (EI): m/z (M+) for C22H24ClN3O: calcd. 381.1608;
found 381.1608.
4.7.26. N-((2S,3S)-1-(1H-imidazol-1-yl)-3-methylpentan-2-yl)-3-chloro-[1,1'-biphenyl]-4-carbox
amide (14z)
Light white solid; yield: 65.0%; mp: 178.3 – 180.9 °C. 1H NMR (400 MHz, DMSO) δ 8.41 (d,
J = 8.8 Hz, 1H), 7.75 (d, J = 1.5 Hz, 1H), 7.74 – 7.63 (m, 3H), 7.60 (s, 1H), 7.49 (t, J = 7.4 Hz,
2H), 7.42 (t, J = 7.3 Hz, 1H), 7.23 – 7.11 (m, 2H), 6.90 (s, 1H), 4.25 – 4.12 (m, 2H), 3.97 (dd, J =
13.3, 9.8 Hz, 1H), 1.66 – 1.55 (m, 2H), 1.27 – 1.22 (m, 1H), 1.00 (d, J = 6.7 Hz, 3H), 0.91 (t, J =
7.3 Hz, 3H). 13C NMR (100 MHz, DMSO) δ 166.51, 142.97, 138.53, 138.26, 136.46, 130.79,
129.57 (2C), 129.43, 128.84, 128.59, 127.83, 127.37 (2C), 125.75, 120.35, 54.64, 47.52, 37.33,
25.15, 15.90, 11.72. HRMS (EI): m/z (M+) for C22H24ClN3O: calcd. 381.1608; found 381.1616.
4.7.27. N-((2S,3S)-1-(1H-imidazol-1-yl)-3-methylpentan-2-yl)-2-methyl-[1,1'-biphenyl]-4-carbox
amide (14aa)
Light white solid; yield: 55.7%; mp: 181.2 – 183.5 °C. 1H NMR (400 MHz, DMSO) δ 8.23 (d,
J = 8.4 Hz, 1H), 7.69 (s, 1H), 7.67 – 7.63 (m, 1H), 7.58 (s, 1H), 7.49 – 7.44 (m, 2H), 7.41 – 7.33
(m, 3H), 7.27 (d, J = 7.9 Hz, 1H), 7.14 (s, 1H), 6.80 (s, 1H), 4.24 (dd, J = 12.8, 2.9 Hz, 1H), 4.20
23

ACCEPTED MANUSCRIPT
– 4.14 (m, 1H), 4.09 (dd, J = 12.7, 9.8 Hz, 1H), 2.26 (s, 3H), 1.69 – 1.61 (m, 1H), 1.56 (ddd, J =
13.3, 7.4, 4.0 Hz, 1H), 1.24 – 1.17 (m, 1H), 1.02 – 0.97 (m, 3H), 0.90 (t, J = 7.4 Hz, 3H). 13C
NMR (100 MHz, DMSO) δ 166.47, 144.42, 141.01, 138.09, 135.24, 133.84, 129.88, 129.62,
129.27 (2C), 128.78 (2C), 128.49, 127.76, 125.21, 120.29, 54.89, 47.44, 37.49, 25.51, 20.68,
15.85, 11.74. HRMS (EI): m/z (M+) for C23H27N3O: calcd. 361.2154; found 361.2166.
4.7.28. N-((2S,3S)-1-(1H-imidazol-1-yl)-3-methylpentan-2-yl)-3-methyl-[1,1'-biphenyl]-4-carbox
amide (14ab)
Light white solid; yield: 55.5%; mp: 213.0 – 214.4 °C. 1H NMR (400 MHz, DMSO) δ 8.18 (d,
J = 8.8 Hz, 1H), 7.65 (d, J = 7.4 Hz, 2H), 7.58 (s, 1H), 7.51 – 7.44 (m, 4H), 7.38 (t, J = 7.3 Hz,
1H), 7.21 – 7.13 (m, 2H), 6.88 (s, 1H), 4.25 – 4.15 (m, 2H), 3.97 (dd, J = 14.4, 11.6 Hz, 1H), 2.20
(s, 3H), 1.67 – 1.54 (m, 2H), 1.27 – 1.20 (m, 1H), 1.00 (d, J = 6.7 Hz, 3H), 0.92 (t, J = 7.3 Hz, 3H).
13C NMR (100 MHz, DMSO) δ 169.29, 141.33, 140.07, 138.20, 136.90, 136.16, 129.42 (2C),
129.06, 128.53, 128.17, 127.78, 127.20 (2C), 124.14, 120.29, 54.23, 47.59, 37.57, 25.31, 19.63,
15.93, 11.74. HRMS (EI): m/z (M+) for C23H27N3O: calcd. 361.2154; found 361.2159.
4.7.29. N-((2S,3S)-1-(1H-imidazol-1-yl)-3-methylpentan-2-yl)-3-methoxy-[1,1'-biphenyl]-4-carbo
xamide (14ac)
Light white solid; yield: 45.6%; mp: 126.3 – 128.2 °C. 1H NMR (400 MHz, DMSO) δ 8.03 (d,
J = 8.5 Hz, 1H), 7.73 (d, J = 7.3 Hz, 2H), 7.62 – 7.56 (m, 2H), 7.49 (t, J = 7.5 Hz, 2H), 7.41 (t, J =
7.3 Hz, 1H), 7.34 (d, J = 1.1 Hz, 1H), 7.29 (dd, J = 8.0, 1.4 Hz, 1H), 7.15 (s, 1H), 6.85 (s, 1H),
4.24 – 4.16 (m, 2H), 4.10 (dd, J = 14.5, 10.4 Hz, 1H), 3.98 (s, 3H), 1.64 – 1.53 (m, 2H), 1.23 –
1.15 (m, 1H), 0.98 (d, J = 6.7 Hz, 3H), 0.91 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO) δ
165.43, 157.52, 144.32, 139.92, 138.17, 131.01, 129.41 (2C), 128.54 (2C), 127.48 (2C), 123.19,
120.26, 119.29, 110.81, 56.51, 54.58, 47.50, 37.03, 25.07, 15.90, 11.82. HRMS (EI): m/z (M+) for
C23H27N3O2: calcd. 377.2103; found 377.2105.
4.8. Procedures for the synthesis of compounds 17 and 23
The synthetic procedures of compounds 17 and 23 were similar to those used for compounds
14a-ac.
4.8.1. 3-fluoro-N-((2S,3S)-3-methyl-1-(1H-1,2,4-triazol-1-yl)pentan-2-yl)-[1,1'-biphenyl]-4-car
boxamide (17)
24

ACCEPTED MANUSCRIPT
Light white solid; yield: 58.7%; mp: 173.4 – 175.6 °C. 1H NMR (600 MHz, DMSO) δ 8.41 (s,
1H), 8.28 (d, J = 8.3 Hz, 1H), 7.96 (s, 1H), 7.75 – 7.72 (m, 2H), 7.61 – 7.56 (m, 2H), 7.51 – 7.46
(m, 3H), 7.43 (t, J = 7.3 Hz, 1H), 4.41 (dd, J = 13.3, 3.2 Hz, 1H), 4.32 – 4.26 (m, 2H), 1.65 – 1.61
(m, 1H), 1.57 – 1.53 (m, 1H), 1.23 – 1.19 (m, 1H), 1.00 (d, J = 6.8 Hz, 3H), 0.90 (t, J = 7.4 Hz,
3H). 13C NMR (150 MHz, DMSO) δ 163.91, 160.59, 158.94, 151.81, 144.97, 144.61, 144.55,
138.46, 130.75, 130.72, 129.57 (2C), 129.01, 127.39 (2C), 123.57, 123.47, 122.95, 122.93, 114.55,
114.39, 53.85, 50.11, 36.73, 25.23, 15.80, 11.58. HRMS (EI): m/z (M+) for C21H23FN4O: calcd.
366.1856; found 366.1855.
4.8.2. (R)-N-(1-(1H-imidazol-1-yl)-3-methylbutan-2-yl)-[1,1'-biphenyl]-4-carboxamide (23)
Light white solid; yield: 65.0%; mp: 201.3 – 203.3 °C. 1H NMR (600 MHz, DMSO) δ 8.26
(d, J = 8.7 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H), 7.73 – 7.70 (m, 2H), 7.59 (s,
1H), 7.50 (t, J = 7.7 Hz, 2H), 7.41 (t, J = 7.4 Hz, 1H), 7.15 (s, 1H), 6.80 (s, 1H), 4.23 (dd, J = 13.0,
2.9 Hz, 1H), 4.17 – 4.13 (m, 1H), 4.10 (dd, J = 13.1, 10.3 Hz, 1H), 1.89 – 1.85 (m, 1H), 0.99 (d, J
= 6.8 Hz, 3H), 0.96 (d, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO) δ 166.76, 143.22, 139.74,
138.42, 133.78, 129.49 (2C), 128.49, 128.33 (2C), 127.35 (2C), 126.97 (2C), 121.23, 55.62, 48.61,
31.04, 19.87, 18.90. HRMS (EI): m/z (M+) for C21H23N3O: calcd. 333.1841; found 333.1858.
4.9. In vitro antifungal testing
Five sensitive pathogenic fungi and two fluconazole-resistant strains of Candida albicans
were selected to determine the in vitro minimum inhibitory concentrations (MIC) according to the
protocols of the National Committee for Clinical Laboratory Standards (NCCLS). The MIC values
were defined as the lowest concentrations of an antimicrobial that would inhibit the visible growth
of the fungi. Fluconazole and itraconazole were used as positive control drugs. All of the
compounds were dissolved in DMSO and serially diluted into the growth medium.
4.10. GC-MS analysis of sterol composition
Candida albicans (ATCC SC5314) was treated with different concentrations of compound
14v and fluconazole and incubated at 30 °C for ~16 h with continuous agitation (200 rpm). Cells
were harvested by centrifugation 3000 g for 10 min. Then, the cells were washed with PBS three
times and saponified at 80 °C for 60 min with NaOH in ethanol. The nonsaponifiable sterols were
then extracted three times with 6 mL petroleum ether. The combined extracts were evaporated
under vacuum, and the residue was dissolved in hexamethylene. The sterols were analyzed by
25

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GC-MS. The GC-MS data were analyzed using Agilent software (Agilent MSD productivity
ChemStation for GC and GC/MS systems data analysis application) and matched to known MS
data using the NIST Spectrum Database (NIST MS search 2.0).
4.11. In vitro human plasma stability assay
Frozen human plasma was thawed in a water bath at 37 °C prior to experiments. Then, the
human plasma was centrifuged at 4000 rpm for 5 min. Compounds and positive control
propantheline solutions (100 ꢀM) were prepared by diluting. Blank plasma (98 µL) was mixed
with 2 ꢀL of dosing solution (100 ꢀM) to achieve 2 ꢀM of the final concentration in duplicate, and
samples were incubated at 37 °C in a water bath. At each time point (0, 10, 30, 60 and 120 min),
400 µL of stop solution (200 ng/mL tolbutamide and 200 ng/mL labetalol in 50% ACN/MeOH)
was added to terminate the reactions. The sample plates were centrifuged at 4000 rpm for
approximately 10 min. The supernatant (50 ꢀL) was mixed with 100 ꢀL ultrapure water. The
samples were shaken at 800 rpm for 10 min. Samples were analyzed by LC/MS/MS.
Disappearance of test compounds are assessed base on the peak area ratio of analyte/IS (no
standard curve).
4.12. In vitro cytotoxicity assays
A549 cells were plated in 96-well microtiter plates at a concentration of 3000 cells per well.
The 96-well microtiter plates were allowed to incubate at 37 °C with 5% CO2 for 16 h.
Compounds 14n, 14v and voriconazole were dissolved in DMSO and diluted with medium to
achieve final concentrations of 0.08, 0.4, 2.0, 10 and 50 ꢀM/L. Then, the old medium of the tested
well was removed by vacuum suction, and 200 ꢀL of the freshly prepared medium containing the
different concentrations of compounds was added. Afterwards, the 96-well microtiter plates were
incubated at 37 °C with 5% CO2 for 24 h. Finally, MTT was added to the 96-well microtiter plates
and incubated for another 4 h. Observation of each test well was performed at λ490nm
.
Acknowledgements
The authors thank Hongwei Liu from the Institute of Microbiology, Chinese Academy of
Sciences, for providing the fluconazole-resistant strains of Candida albicans(strain 17# and strain
CaR). The authors thank Prof. Yongbing Cao from School of Pharmacy, the Second Military
Medical University, for providing the strains of Candida albicans(SC5314). This work was
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supported by the Program for Innovative Research Team of the Ministry of Education and
Program for Liaoning Innovative Research Team in University.
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