Discovery of biphenyl imidazole derivatives as potent antifungal agents: Design, synthesis, and structure-activity relationship studies

Article abstract of DOI:10.1016/j.bmc.2016.11.051

Fungal infections have became a serious medical problem due to their high incidence and mortality. We describe the discovery and structure-activity relationships studies (SARs) of a series of novel biphenyl imidazole derivatives with excellent antifungal

Full text of DOI:10.1016/j.bmc.2016.11.051

Accepted Manuscript
Discovery of biphenyl imidazole derivatives as potent antifungal agents: Design,
synthesis, and structure-activity relationship studies
Dongmei Zhao, Shizhen Zhao, Liyu Zhao, Xiangqian Zhang, Peng Wei,
Chunchi Liu, Chenzhou Hao, Bin Sun, Xin Su, Maosheng Cheng
PII:
S0968-0896(16)31019-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.051
BMC 13418
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
19 October 2016
25 November 2016
26 November 2016
Please cite this article as: Zhao, D., Zhao, S., Zhao, L., Zhang, X., Wei, P., Liu, C., Hao, C., Sun, B., Su, X., Cheng,
M., Discovery of biphenyl imidazole derivatives as potent antifungal agents:Design, synthesis, andstructure-activity
relationship studies, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.11.051
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Discovery of biphenyl imidazole derivatives as potent antifungal
agents: Design, synthesis, and structure-activity relationship studies
a,
a
a
a
a
Dongmei Zhao *, Shizhen Zhao , Liyu Zhao , Xiangqian Zhang , Peng Wei , Chunchi
a
a
c
b,
a
Liu , Chenzhou Hao , Bin Sun , Xin Su *, Maosheng Cheng
a
Key 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, PR China
b
The School of life Science and Biopharmaceutical, Shenyang Pharmaceutical University, 103
Wenhua Road, Shenhe District, Shenyang, 110016, PR China
c
Institute of BioPharmaccutical Research, Liaocheng University, 1 Hunan Road, Liaocheng 252000,
PR China
*
Corresponding author.
E-mail address: medchemzhao@163.com (D.-M. Zhao), suxin1966@163.com (X. Su)
ABSTRACT:
Fungal infections have became a serious medical problem due to their high incidence and
mortality. We describe the discovery and structure-activity relationships studies (SARs) of a series
of novel biphenyl imidazole derivatives with excellent antifungal activities against Candida
albicans and Cryptococcus neoformans. The most promising compounds 12f-g and 19a-b exhibited
excellent activity with minimum inhibitory concentration (MIC) values in the range of 0.03125 to 2
µg/mL. Preliminary mechanism studies showed that the potent antifungal activity of compound 12g
stemed from inhibition of CYP51 in Candida albicans. Furthermore, compounds 12g and 19b
exhibited low inhibition profiles for various human cytochrome P450 isoforms. The SARs and
binding mode established in this study will be useful for further lead optimization.
KEYWORDS:
Antifungal activity, CYP51, Structure-activity relationship, Azole antifungals
1
. Introduction
Fungal infections have been increasing dramatically and present a serious threat to human
health, especially in immunocompromised patients. The occurrence of these infections have

increased since the early 1980s and have resulted from many factors, such as patients undergoing
organ transplants or anticancer chemotherapy and patients with AIDS as well as patients given
1
-4
potent pharmacologic immunomodulators or broad-spectrum antibiotics . Clinically, the three
major pathogens Candida spp., Cryptococcus neoformans and Aspergillus spp. account for most
fungal infections. Currently, the conventional antifungal agents to treat fungal infections can be
divided into four categories based on their mode of action, including the polyenes (e.g.,
5
6
amphotericin B and nystatin) , echinocandins (e.g., caspofungin and micafungin) , azoles(e.g.,
7
8
fluconazole, voriconazole and itraconazole) , and antimetabolites (e.g., 5-fluorocytosine) . Among
9
these agents, azoles are most widely used in front-line antifungal therapy .
Azole antifungal agents inhibit the activity of lanosterol 14-demethylase (CYP51), a member
5
of the CYP51 class of cytochrome P450 enzymes . The inhibition of CYP51 enzymes reduces
endogenous concentrations of ergosterol, a significant cellular membrane component, and causes
5
, 10-12
the accumulation of 14α-methylsterols (e.g., lanosterol and 14α-methyl-3-6-diol)
. The active
site of CYP51 contains a heme cofactor that can coordinate to an imidazole ring or the triazole ring
13
of azole drugs . Not only are these drugs essential for the clinical treatment of systemic fungal
1
4-16
infections, but they are also a major focus in the search for new antifungals
. Several azole
1
7
antifungal agents, such as itraconazole, voriconazole and bifonazole have been approved (Fig. 1) .
However, these antifungal agents exist many problems, such as drug resistance, narrow antifungal
1
2, 18
spectrum and low bioavailability, which negatively affect their clinical efficacy
. Therefore, it is
necessary to develop novel antifungal compounds with potent activity, broad spectrum and low
resistance.
Based on results obtained from structure-activity relationships of azole antifungal agents and
computational docking experiments, we performed a cell-based antifungal screening of an in-house
1
9-21
library
using standard NCCLS (National Committee for Clinical Laboratory Standards)
protocols. Compound 5, which features biphenyl and imidazole scaffolds, showed modest
antifungal activities, with an MIC value of 2 μg/mL against Candida albicans. The common
phamacophore of classic azole antifungal drugs include a N-(phenethyl) azole skeleton and a
2
2
tertiary alcohol group, both of which contribute to their antifungal activities . Compound 5
contains an imidazole group but no tertiary alcohol group or N-(phenethyl) azole skeleton,
rendering the chemical scaffold of compound 5 a novel structure compared to the classic azole

antifungal agents and necessitating the investigation of its structure-activity relationships (SARs) to
design a novel antifungal lead compound.
Figure 1. Chemical structures azole antifungal agents and lead compounds.
2
. Results and discussion
2.1 Chemistry
The synthetic routes of the key intermediates 8a-m are illustrated in Scheme 1. Commercially
available 4-bromobenzoic acid 6 and the substituted phenylboronic acids 7a-m were subjected to a
Suzuki coupling in the presence of Pd(PPh ) to afford the key intermediates 8a-m.
3
4
Scheme 1. Synthesis of the intermediates 8a-m.Reagents and conditions: (a) Pd(PPh ) , K CO , reflux, 5h.
3
4
2
3
The target compounds 12a-v were synthesized according to our previously reported procedure
13, 21
(Scheme 2)
. L-serine 9 was treated with an alcohol (methanol, ethanol, propanol, isopropyl

alcohol, or isobutyl alcohol) in the presence of SOCl to afford serine ester 10a-e. The serine esters
2
10a-e were then treated with 8a-m in the presence of a condensing agent to give intermediates 11a-v.
These compounds were treated with imidazole in the presence of CDI, to afford the target
compounds 12a-v. Subsequently, compounds 13a-b were synthesized from compounds 12a-v via
hydrolyzation and amidation.
Compounds 15a-b were prepared according to the procedure in Scheme 3. Alcohols 11a-b
were chlorinated with SOCl2 to afford intermediate 14a-b, which were then subjected to
nucleophilic substitution with triazole to yield the target compounds.
The synthetic procedure for compounds 19a-b was similar to the synthesis of compounds
12a-v, except that D-serine was used instead of L-serine(Scheme 4).
Scheme 2. General synthesis of the target compounds 12a-v and 13a-b.Reagents and conditions: (a) alcohol
reagent, SOCl , reflux, 1–2 h; (b) EDCI, HOBt, DIEA, r.t., 7 h; (c) CDI, imidazole, CH CN, reflux, 7 h; (d)
2
3
1
)NaOH, MeOH/H O; 2)R NH , EDCI, HOBt, DIEA, r.t., 7 h.
2
3
2

Scheme 3. General synthesis of the target compounds 15a-b. Reagents and conditions: (a) SOCl , DMF, r.t., 2 h;
2
(
b) 1H-1,2,4-triazole, Triethylamine, DMF, r.t. 12 h.
Scheme 4. General synthesis of the target compounds 19a-b. Reagents and conditions: (a) alcohol reagent, SOCl2,
reflux, 1-2 h; (b) EDCI, HOBt, DIEA, r.t., 7 h; (c) CDI, imidazole, CH CN, reflux, 7 h
3
2.2 In vitro antifungal activity
The target compounds were evaluated for their antifungal activity against five main fungal
pathogens according to the protocols from the NCCLS. Broth microdilution methods was used to
determine the minimum inhibitory concentrations (MICs) of the target compounds in 96-well
16
microtest plates .
The in vitro antifungal activities of the target compounds are listed in Table 1. Fluconazole
FLC) and Itraconazole (ITR) were used as reference drugs. Most of the synthesized compounds
exhibited moderate to good antifungal properties with broad spectrums of activity. Of these,
(

compounds 12a-b, 12f-g, 12q, and 19a-b showed the most potent activity against C. albicans, C.
neoformans, and C. tropicalis with MIC values in the range of 0.03125 to 2 µg/mL, which are
superior or comparable to those of the reference drugs FLC and ITR. Substitution in the alkyl ester
side chain (12a-b) by an amide group (15a-b) led to obvious decrease of antifungal activity.
Replacing the imidazole group (16b) with a triazole group (16f) resulted in a slight decrease in
activity. The R-enantiomers(19a-b) and S-enantiomers(14o-p) show similar antifungal activities.
However, most of the compounds were inactive against Aspergillus fumigatus.
Table 1
a
In vitro antifungal activities of the target compounds (MIC, μg/mL) .
Compd.
R
1
R
2
/ R
-CH(CH
-CH CH(CH
3
C. alb.(Ⅰ)
C. alb(Ⅱ).
C. neo.
C.tro.
A. fum.
1
2a
2b
H
H
3
)
2
0.03125
0.03125
16
0.25
0.25
16
2
1
0.0625
0.03125
4
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
1
2
3
)
2
1
2c
2d
2e
2f
2-F
-CH
3
2
1
2-F
-CH
-CH
-CH(CH
-CH CH(CH
-CH(CH
-CH CH(CH
-CH(CH
-CH CH(CH
2
CH
3
0.25
1
1
0.125
0.0625
0.0625
0.03125
0.5
1
2-F
2
CH
2
CH
3
0.125
0.0625
0.03125
0.25
0.25
0.25
0.125
1
0.5
1
1
2-F
)
3 2
1
2g
2h
2-F
2
3
)
2
2
2
0.5
16
16
16
>16
>16
16
4
1
2-Cl
2-Cl
2-CH
2-CH
4-F
)
3 2
1
2i
2j
2
3
)
)
0.5
2
0.5
1
3
3
3
)
2
0.5
2
2
1
2k
2l
2m
2
3
>16
>16
8
4
1
-CH(CH
-CH(CH
-CH(CH
-CH(CH
3
3
3
3
)
)
)
)
2
1
0.5
1
4-Cl
2
2
2
0.25
1
0.25
0.0625
2
1
2n
2o
4-CH
3
0.0625
>16
0.5
>16
1
4-CF
3
>16

1
1
2p
2q
4-OCF
3
-CH(CH
-CH(CH
-CH(CH
-CH(CH
-CH(CH
-CH(CH
-CH(CH
3
3
3
3
3
3
3
)
)
)
)
)
)
)
2
2
2
2
2
2
2
0.125
0.0625
>16
1
0.125
0.25
>16
2
16
16
0.5
0.0625
8
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
4
3-F
1
1
2s
3-CN
>16
8
2t
2,4-F
2
0.5
1
1
1
1
1
1
2u
2v
3a
3b
5a
5b
3,4-F
2
>16
0.25
>16
>16
0.25
1
>16
1
>16
16
8
2,5-F
2
0.0625
>16
>16
2
H
>16
>16
4
>16
>16
16
H
-CH
-CH(CH
-CH CH(CH
-CH(CH3)2
-CH CH(CH )
CH(CH
)
2
3
2
2
H
)
3 2
H
)
8
16
4
2
3
1
9a
9b
2-F
2-F
-
0.125
0.0625
2
0.5
0.5
2
>16
>16
>16
4
0.25
0.125
0.25
1
1
2
3 2
5
-
-
-
FCZ
ITZ
-
0.5
1
-
0.0625
0.25
1
0.5
a
Abbreviations: C.alb.(I), Candida albicans (ATCC SC5314); C.alb.(II), Candida albicans (CPCC400523); C. neo.,
Cryptococcus neoformans (cgmcc 2.3161); A.fum., Aspergillus fumigatus (cgmcc 3.7795); C.tro., Candida
tropicalis (cgmcc 2.3739); FCZ: Fluconazole; ITZ: Itraconazole.
2
.2 In vitro antifungal activity against fluconazole-resistant strains of C. alb.
Fluconazole is widely used today and can seriously increase the problem of drug-resistance .
Based on the results of in vitro antifungal activity assays, the most potent compounds 12f-g and
2
3
19a-b were further evaluated against fluconazole-resistant strains of C. alb. (strain 100 and strain
103). As shown in Table 2, compounds 12f-g and compounds 19a-b showed moderate antifungal
activities against strain 100 and strain 103, with MIC values in the range of 2 to 8 µg/mL.
Table 2
a
In vitro antifungal activities of the target compounds (MIC, μg/mL) .
C. alb.
Compd.
R1
R2
Strain100
Strain103
1
2f
2-F
2-F
-CH(CH
3
)
2
8
2
4
4
1
2g
-CH CH(CH )
2 3 2

1
9a
9b
2-F
2-F
-
-CH(CH
3
)
2
4
4
8
4
1
-CH
2
CH(CH
)
3 2
FCZ
-
>64
>64
a
Abbreviations: C.alb., Candida albicans; strain 100, fluconazole-resistant strains of Candida albicans; strain 103,
fluconazole-resistant strains of Candida albicans; FCZ: Fluconazole.Strain 100 and strain 103 were provided by
The Second Military Medical University.
2.3 Analysis of sterol composition
To confirm the antifungal mechanism of compound 12g, we analysed sterol composition in C.
albicans by GC-MS. GC-MS methods have been successfully used to analyse the differences in
sterol composition in C. albicans cells, to elucidate the mechanism of action of antifungal agents on
2
4, 25
sterol biosynthesis pathways
. FLC was used as a reference drug and cholesterol was added as an
internal standard. The GC-MS analysis results are shown in Table 3. In the untreated control,
ergosterol was the major sterol, accounting for 89.4% of the total sterol content. While lanosterol
comprised only 3.5% and other 14-methylated sterols (obtusifoliol and eburicol) were not observed.
When the C. albicans was treated with FLC at 8 μg/mL for 16 h, the content of ergosterol was
reduced to 2.0% of the total amount of sterol. In contrast, the content of lanosterol and obtusifoliol
were increased to 18.2% and 6.8%, respectively. Especially, eburicol content was increased to 68.1%
of the total sterol content. These changes in sterol composition were caused by the inhibition of
2
1, 26, 27
CYP51 in C. albicans by FLC and are consistent with the earlier reports
. Treatment with 12g
also resulted in a marked increase in the lanosterol and eburicol contents and a reduction of the
ergosterol content (reduced to 2.5%). The GC-MS results suggest that the novel compound 12g
might have a similar mechanism with FLC by inhibiting fungal lanosterol 14-demethylase (CYP51)
in C. albicans.
Table 3
a
Analysis of sterol composition in C.albicans by GC-MS .
%
of total sterols(Candida albicans)
sterol
a
b
c
control
89.4
--
FLC
12g
Ergosterol
Obtusifoliol
Lanosterol
2.0
6.8
2.5
6.6
3.5
18.2
19.6

Eburicol
--
68.1
4.9
65.1
6.2
unidentified
7.1
a
b
c
Abbreviations: C.alb., Candida albicans (ATCC SC5314); Control (no drug). Treated with FLC at 8 μg/ mL.
d
Treated with compound 12g at 8 μg/ mL.
2.4 Cytochrome P450 Inhibition Assay
Cytochrome P450 (CYP) enzymes play an important role in drug metabolism. However, many
azole antifungals interfere with cytochrome P450 enzymes, such as CYP3A4 and CYP2D6, while
maintaining a high activity against for fungal CYP51. For example, the IC50 of ketoconazole and
2
8
itraconazole for inhibition of CYP3A4 are 31.7 and 32.6 nM , respectively. The compounds 12g
and 19b were tested against the five major human CYP isoforms (CYP1A2, CYP2C9, CYP2C19,
CYP2D6, and CYP3A4-M). As shown in Table 4, compounds 12g and 19b showed
weak-to-moderate inhibition against CYP1A2, CYP2C9, CYP2C19, and CYP2D6, while
compounds 12g and 19b exhibited strong inhibition against CYP3A4 with IC50 values 0.155 μM
and 0.224 μM, respectively. The R-enantiomer 19b showed higher selectivity than the S-enantiomer
12g.
Table 4.
In Vitro CYP Inhibition assessment of compounds.
IC50 (μM)
CYP2C19
1.16
Compd.
CYP1A2
21.0
CYP2C9
2.36
CYP2D6
7.59
CYP3A4-M
0.155
1
2g
9b
1
41.4
3.22
1.89
10.2
0.227
2.5 Structure-activity relationships
The relationship between various substituents on the aromatic ring was investigated
(
compounds 12a-v). Compounds with electron-donating groups at the 4-position, such as -CH and
3
-
OCF moieties, exhibited higher antifungal activity than those with electron-withdrawing groups
3
such as -CF and halogens such as -F and -Cl. On the contrary, electron-donating groups such as
3
methyl at the 2-position resulted in weaker activity compared to single halogens such as -F and -Cl.
Among these electron-poor aryls, compounds 12f-g with 2-F substituents showed the best
antifungal activity. Moreover, Compounds 12t-v with multi-halogens substituents showed no
advantage.

The antifungal activities of compounds 12c-g, which have differing alkyl ester side chains
methyl ester, ethyl ester, propyl ester, isopropyl ester and isobutyl ester) were investigated. The in
(
vitro antifungal activity data showed that bulky groups such as isopropyl ester and isobutyl ester
have the potential for higher antifungal activity. Replacement of the alkyl ester side chain by an
amide group (13a-b) resulted in a significant decrease in antifungal activity. In addition, replacing
the imidazole group (12a-b) with a triazole group (15a-b) resulted in a slight decrease in activity.
The influence of the stereochemical configuration was examined by synthesizing novel
R-configured compounds (19a-b) and studying their antifungal activities. The results show that R-
or S-configured compounds have similar antifungal activity.
2.6 Molecular docking model analysis of compound 12g in the active site of CYP51
A molecular docking study was performed to investigate interactions between CYP51 and 12g
and to provide a basis for further structure-based drug design. The first full-length structure of a
2
9
fungal CYP51 was from Saccharomyces cerevisiae and was determined by X-ray crystallography .
A published crystal structure of ITZ bound within the active site cavity of CYP51(PDB ID:5EQB,
Figure 2A) served as a useful template for generating binding modes. Compound 12g was docked
into the active site using the CDOCKER program in Discovery Studio 3.0. Images depicting the
proposed binding modes were generated using PyMOL. As shown in Figure 2B, the imidazole ring
of compound 12g coordinated the iron in the heme group, and the alkyl ester formed a hydrophobic
interaction with Met313. Like the side chain of ITZ, the biphenyl side chain extended into CYP51
channel to form van der Waals and hydrophobic interactions with the surrounding residues Ala69,
Leu95, Pro238, Phe241 and His381.

Figure 2. The binding mode of ITZ (A) and compound 12g (B) in the active site of CYP51.
3
. Conclusion
In summary, we discovered a novel class of compounds with excellent antifungal activities.
Further examination of their antifungal activity and structure-activity relationship(SAR) led to a
series of novel biphenyl imidazole derivatives that were designed, synthesized and evaluated for in
vitro antifungal activity. Most compounds displayed moderate or strong antifungal activities with
MIC values in the range of 0.03125 µg/mL to 2 µg/mL against Candida albicans and Cryptococcus
neoformans. Compounds 12f-g and 14a-b displayed moderate antifungal activities against
fluconazole-resistant strains of C.alb. Notably, compounds 12g and 19b were selective for fungal
targets compared to human CYP1A2, CYP2C9, CYP2C19, and CYP2D6 (IC50>1 µM).Preliminary
mechanism studies showed that the potent antifungal activity of novel compound 12g stems from
inhibition of CYP51 in Candida albicans. Based on the SAR and a molecular docking study,
compound 12g was identified as a promising antifungal agent. Further optimization on these
compounds is underway.
4
. Experimental section
4.1 General procedure for the synthesis of compounds
Unless otherwise noted, all reagents and solvents were obtained from commercially available
sources and were used without purification. TLC analysis was performed on GF254 silica gel plates
Jiangyou, Yantai). Column chromatography was performed with silica gel (200-300 mesh) from
(

Qingdao Haiyang Chemicals (Qingdao, Shandong, China). Mass spectrometry was performed using
ESI mode on an Agilent 1200 LC-MS (Agilent, Palo Alto, CA, USA). High-resolution accurate
mass determinations (HRMS) were recorded on a Bruker Micromass Time of Flight mass
spectrometer equipped with electrospray ionisation (ESI). Melting points (mp.) were determined
using glass capillary tubes on a BüCHI Melting Point B-540 melting point apparatus (Büchi
1
Labortechnik, Flawil, Switzerland) and are uncorrected. Nuclear magnetic resonance ( H-NMR and
1
3
C-NMR )spectra were recorded on a Bruker 400 MHz NMR spectrometer with TMS as an internal
standard. The chemical shifts are reported in parts per million (ppm), the coupling constants (J) are
expressed in hertz (Hz). Peak multiplicities are described as singlet (s), doublet (d), triplet (t),
quartet (q), multiplet (m) and broad (br).
4.2 General procedure for the synthesis of intermediates (8a-m)
Under an argon atmosphere, 4-bromobenzoic acid (1 equiv.), substituted boronic acid (1.2
equiv.) and Pd[P(C H ) ] (1 mol%) were dissolved in a mixed solution of dioxane/H2O (10:1).
6
5 3 4
K CO (2 equiv.) was added and the mixture was heated under reflux for 6 h. The reaction mixture
2
3
was cooled to room temperature and the dioxane was removed by rotary evaporation. H O was
2
added and the solution was adjusted to pH=1-3 with 2 N HCl. The white solid precipitate was
collected by filtration and dried to give the desired compound.
4.3 General procedure for the synthesis of L-serine ester (10a–e)
To a solution of L-serine (1 equiv.) in alcohol reagent (methanol, ethanol, propanol,
isopropanol or isobutanol) was added dropwise thionyl chloride (3 equiv.) at less then 0 °C
using a salted ice bath. The mixture was heated under reflux for 2-10 h. The reaction mixture was
then concentrated under reduced pressure to yield a white solid.
4.4 General procedure for the synthesis of compounds (11a-v)
To a solution of the intermediate acid compound (1 equiv.) in anhydrous DMF was added
EDCI(1.1 equiv) and HOBt(1.1 equiv), respectively.The reaction mixture was stirred for 2 h at
ambient temperature, and the L-serine ester (1.1 equiv.) and DIEA (3 equiv.) were added. The
solution was heated to 70 °C for 6 h and then cooled to room temperature. The reaction mixture was
poured into ice water, the resulting solid was filtered and dried to give the desired compound.
4.5 General procedure for the synthesis of compounds (12a-v)
To a solution of the intermediate 11a-v (1 equiv.) in CH CN was added CDI (2 equiv.) and
3

imidazole (1.2 equiv.). The solution was heated at reflux for 6 h. The reaction mixture was extracted
with EtOAc and brine. The organic phase was dried over Na SO overnight and the solvent was
2
4
removed under vacuum. The crude product was purified by silica gel column chromatography
(
CH OH : CH Cl = 1:50, v/v) to give the target product 12a-v.
3 2 2
4.5.1 isopropyl(S)-2-([1,1'-biphenyl]-4-carboxamido)-3-(1H-imidazol-1-yl)propanoate(12a)
1
Light white solid; yield: 53.3%; mp: 92.7-94.9 °C. H NMR (400 MHz, DMSO-d ) δ 9.02 (d, J
6
=
7.9 Hz, 1H), 7.90 (d, J = 8.3 Hz, 2H), 7.80 (d, J = 8.3 Hz, 2H), 7.74 (d, J = 7.3 Hz, 2H), 7.65 (s,
1
1
H), 7.50 (t, J = 7.5 Hz, 2H), 7.42 (t, J = 7.3 Hz, 1H), 7.24 (s, 1H), 6.87 (s, 1H), 4.96 (m, J = 18.7 Hz,
1
3
H), 4.85 – 4.71 (m, 1H), 4.76 - 4.38 (m, 2H), 1.20 (t, J = 5.9 Hz, 6H). C NMR (100 MHz,
DMSO-d ) δ 169.14, 166.25, 143.19, 139.09, 137.81, 132.29, 129.01(2C), 128.29, 128.10,
6
127.98(2C), 126.89(2C), 126.61(2C), 119.87, 68.63, 53.89, 45.77, 21.47, 21.42. HRMS calcd for
+
C H N O , [M + H] , 378.1818; found 378.1812.
2
2
24
3
3
4.5.2 isobutyl (S)-2-([1,1'-biphenyl]-4-carboxamido)-3-(1H-imidazol-1-yl)propanoate (12b)
1
Yellow oil; yield: 59.1%; H NMR (400 MHz, DMSO-d ) δ 9.04 (d, J = 8.0 Hz, 1H), 7.89 (d, J
6
=
8.4 Hz, 2H), 7.88 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.65 (s, 1H), 7.50 (t, J = 7.7 Hz, 2H),
7.42 (t, J = 7.3 Hz, 1H), 7.23 (s, 1H), 6.86 (s, 1H), 4.84 (m, 1H), 4.58 – 4.40 (m, 2H), 3.91 (d, J = 6.1
1
3
Hz, 2H), 1.87 (m, 1H), 0.87 (d, J = 6.7 Hz, 6H). C NMR (100 MHz, DMSO-d ) δ 169.60, 166.29,
6
143.19, 139.07, 137.78, 132.25, 129.02(2C), 128.19, 128.12, 127.96(2C), 126.90(2C), 126.62(2C),
+
1
19.88, 70.64, 53.79, 45.72, 27.23, 18.74(2C). HRMS calcd for C H N O , [M + H] , 392.1974;
2
3
26
3
3
found 392.1967.
4
)
.5.3 methyl(S)-2-(2'-fluoro-[1,1'-biphenyl]-4-carboxamido)-3-(1H-imidazol-1-yl)propanoate(12c
1
Light white solid; yield: 58.1%; mp: 199.8-201.3 °C. H NMR (600 MHz, DMSO-d ) δ 9.06
6
(d, J = 8.0 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.69 – 7.66 (m, 2H), 7.65 (s, 1H), 7.59 (td, J = 7.9,
1
9
3
1
4
4
.5 Hz, 1H), 7.50 – 7.44 (m, 1H), 7.37 – 7.32 (m, 2H), 7.22 (s, 1H), 6.86 (s, 1H), 4.87 (ddd, J =
.9, 8.1, 4.7 Hz, 1H), 4.56 (dd, J = 14.1, 4.7 Hz, 1H), 4.42 (dd, J = 14.1, 10.0 Hz, 1H), 3.70 (s,
1
3
H). C NMR (150 MHz, DMSO-d ) δ 170.10, 166.14, 159.90, 158.26, 138.31, 137.85, 132.59,
6
30.84, 130.21, 129.43, 128.86, 128.38, 127.65(2C), 127.37, 125.08, 119.89, 116.14, 53.68, 52.33,
+
5.76.HRMS calcd for C H FN O , [M + H] , 368.1410; found 368.1414.
20
19
3
3
.5.4 ethyl(S)-2-(2'-fluoro-[1,1'-biphenyl]-4-carboxamido)-3-(1H-imidazol-1-yl)propanoate(12d)

1
Light white solid; yield: 61.7%; mp: 109.5-110.8 °C. H NMR (400 MHz, DMSO-d ) δ 9.06 (d,
6
J = 8.0 Hz, 1H), 7.92-7.90 (d, J = 8.0Hz, 2H), 7.69 – 7.60 (m, 3H), 7.58 – 7.48 (m, 1H), 7.48 (t, J =
12 Hz, 1H), 7.44-7.32 (m, 2H), 7.23 (s, 1H), 6.87 (s, 1H), 4.85 (m, 1H), 4.58 – 4.39 (m, 2H), 4.19 (q,
1
3
J = 7.1 Hz, 2H), 1.21 (t, J = 14.2 Hz, 3H). C NMR (100 MHz, DMSO-d ) δ 169.57, 166.16, 160.29,
6
1
1
3
4
57.83, 138.25, 137.81, 132.67, 130.82, 130.24, 128.83, 128.80, 128.28, 127.61(2C), 127.26,
+
25.05, 125.02, 119.88, 116.30, 61.05, 53.75, 45.78, 13.99.HRMS calcd for C H FN O , [M + H] ,
2
1
21
3
3
82.1567; found 382.1570.
.5.5 propyl(S)-2-(2'-fluoro-[1,1'-biphenyl]-4-carboxamido)-3-(1H-imidazol-1-yl)propanoate(12e)
1
Light white solid; yield: 49.5%; mp: 67.9-71.3 °C. H NMR (400 MHz, DMSO-d ) δ 9.09 (d, J
6
=
8.4 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.68 – 7.60 (m, 3H), 7.60 – 7.58 (m, 1H), 7.57-7.56 (m, 1H),
7
2
1
1
.47-7.31 (m, 2H), 7.24 (s, 1H), 6.88 (s, 1H), 4.87 (m, 1H), 4.59 – 4.42 (m, 2H), 4.07 (t, J = 10.8 Hz,
1
3
H), 1.61-1.56 (m, 2H), 0.87 (t, J = 7.5 Hz, 3H). C NMR (100 MHz, DMSO-d ) δ 169.61, 166.20,
6
60.29, 157.84, 138.24, 137.78, 132.69, 130.81, 130.23, 128.83, 128.80, 128.15, 127.59(2C),
27.25, 125.05, 119.91, 116.29, 66.41, 53.77, 45.77, 21.44, 10.16. HRMS calcd for C H FN O ,
2
2
23
3
3
+
[
M + H] , 396.1723; found 396.1720.
4
2
.5.6 isopropyl(S)-2-(2'-fluoro-[1,1'-biphenyl]-4-carboxamido)-3-(1H-imidazol-1-yl)propanoate(1
f)
1
Light white solid; yield: 58.8%; mp: 75.4-78.4 °C. H NMR (400 MHz, DMSO-d ) δ 9.02 (d, J
6
=
7.9 Hz, 1H), 7.91 (d, J = 8.3 Hz, 2H), 7.69 (d, J = 7.2 Hz, 2H), 7.66 (s, 1H), 7.61 (s, 1H), 7.49-7.34
(
(
m, 3H), 7.23 (s, 1H), 6.86 (s, 1H), 4.99-4.95 (m, 1H), 4.78 - 4.76 (m, 1H), 4.55- 4.37 (m, 2H), 1.20
13
t, J = 5.9 Hz, 6H). C NMR (100 MHz, DMSO-d ) δ 169.09, 166.21, 160.29, 157.84, 138.22,
6
137.82, 132.76, 130.79, 130.24, 128.83, 128.81, 128.31, 127.59(2C), 127.27, 125.02, 119.87,
+
1
16.30, 68.65, 53.89, 45.76, 21.48, 21.43. HRMS calcd for C H FN O , [M + H] , 396.1723;
2
2
23
3
3
found 396.1716.
4.5.7 isobutyl(S)-2-(2'-fluoro-[1,1'-biphenyl]-4-carboxamido)-3-(1H-imidazol-1-yl)propanoate(12
g)
1
Light white solid; yield: 63.1%; mp: 117.2-119.7 °C. H NMR (400 MHz, DMSO-d ) δ 9.13 (d,
6
J = 8.0 Hz, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.68 (t, J = 9.1 Hz, 3H), 7.59 - 7.55 (m, 1H), 7.46 -7.45 (m,
H), 7.35 -7.30 (m, 2H), 7.27 (s, 1H), 6.90 (s, 1H), 4.92 - 4.87 (m, 1H), 4.62 - 4.45 (m, 2H), 3.92 (t,
1
1
3
J = 6.1 Hz, 2H), 1.89 – 1.84 (m, 1H), 0.87 (d, J = 6.8 Hz, 6H). C NMR (100 MHz, DMSO-d ) δ
6

1
1
69.56, 166.25, 160.29, 157.84, 138.24, 137.81, 132.74, 130.82, 130.16, 128.84, 128.31,
27.58(2C), 127.25, 125.05, 125.02, 119.86, 116.30, 116.07, 70.66, 53.81, 45.69, 27.23, 18.73.
+
HRMS calcd for C H FN O , [M + H] , 410.1880; found 410.1876.
23
25
3
3
4.5.8 isopropyl(S)-2-(2'-chloro-[1,1'-biphenyl]-4-carboxamido)-3-(1H-imidazol-1-yl)propanoate(
12h)
1
Light white solid; yield: 57.3%; mp: 122.8-124.2 °C. H NMR (400 MHz, DMSO-d ) δ 9.02
6
(d, J = 7.9 Hz, 1H), 7.87 (d, J = 8.3 Hz, 2H), 7.64 (s, 1H), 7.62 – 7.57 (m, 1H), 7.55 (d, J = 8.3 Hz,
2
4
H), 7.49 – 7.41 (m, 3H), 7.23 (s, 1H), 6.86 (s, 1H), 5.02 – 4.90 (m, 1H), 4.79 – 4.75 (m, 1H),
1
3
.52 (dd, J = 14.0, 5.0 Hz, 1H), 4.40 (dd, J = 14.0, 9.9 Hz, 1H), 1.20 (t, J = 5.8 Hz, 6H). C NMR
(
150 MHz, DMSO-d ) δ 169.11, 166.32, 141.86, 138.98, 137.86, 132.81, 131.44, 131.20, 129.92,
6
129.68, 129.35(2C), 128.37, 127.63, 127.26(2C), 119.90, 68.68, 53.91, 45.77, 21.51, 21.46.
+
HRMS calcd for C H ClN O , [M + H] , 412.1428; found 412.1419.
22
23
3
3
4.5.9 isobutyl(S)-2-(2'-chloro-[1,1'-biphenyl]-4-carboxamido)-3-(1H-imidazol-1-yl)propanoate(12
i)
1
Yellow oil; yield: 59.1%. H NMR (400 MHz, DMSO-d ) δ 9.06 (d, J = 8.0 Hz, 1H), 7.86 (d,
6
J = 8.3 Hz, 2H), 7.64 (s, 1H), 7.63 – 7.58 (m, 1H), 7.55 (d, J = 8.3 Hz, 2H), 7.48 – 7.41 (m, 3H),
7
1
.23 (s, 1H), 6.86 (s, 1H), 4.91 – 4.76 (m, 1H), 4.55 (dd, J = 14.0, 4.8 Hz, 1H), 4.43 (dd, J = 14.0,
1
3
0.1 Hz, 1H), 3.96 – 3.87 (m, 2H), 1.91 – 1.85 (m, 1H), 0.88 (d, J = 6.7 Hz, 6H). C NMR (150
MHz, DMSO-d ) δ 169.58, 166.36, 141.86, 138.96, 137.85, 132.79, 131.44, 131.20, 129.92,
6
129.68, 129.35(2C), 128.39, 127.63, 127.23(2C), 119.87, 70.69, 53.82, 45.69, 27.26, 18.77(2C).
+
HRMS calcd for C H ClN O , [M + H] , 426.1584; found 426.1581.
23
25
3
3
4.5.10 isopropyl(S)-3-(1H-imidazol-1-yl)-2-(2'-methyl-[1,1'-biphenyl]-4-carboxamido)propanoate
(12j)
1
Light white solid; yield: 56.9%; mp: 80.1-82.9 °C. H NMR (400 MHz, DMSO-d ) δ 8.98 (d,
6
J = 7.9 Hz, 1H), 7.85 (d, J = 8.3 Hz, 2H), 7.72 (d, J = 1.0 Hz, 1H), 7.64 (s, 1H), 7.46 (d, J = 8.3
Hz, 2H), 7.34 – 7.25 (m, 3H), 7.23 (s, 1H), 6.86 (s, 1H), 4.99 – 4.92 (m, 1H), 4.82 – 4.74 (m, 1H),
4
6
1
2
.51 (dd, J = 14.0, 5.0 Hz, 1H), 4.39 (dd, J = 14.1, 9.8 Hz, 1H), 2.24 (s, 3H), 1.20 (t, J = 6.1 Hz,
1
3
H). C NMR (150 MHz, DMSO-d ) δ 169.16, 166.44, 144.57, 140.39, 137.85, 134.69, 132.04,
6
30.46, 129.42, 129.03(2C), 128.37, 127.77, 127.30(2C), 126.05, 119.88, 68.66, 53.91, 45.77,
+
1.51, 21.46, 20.13. HRMS calcd for C H N O , [M + H] , 392.1974; found 392.1975.
23
26
3
3

4.5.11 isobutyl(S)-3-(1H-imidazol-1-yl)-2-(2'-methyl-[1,1'-biphenyl]-4-carboxamido)propanoate(
12k)
1
Yellow oil; yield: 51.4%. H NMR (400 MHz, DMSO-d ) δ 9.03 (d, J = 8.0 Hz, 1H), 7.85 (d,
6
J = 8.3 Hz, 2H), 7.72 (d, J = 1.2 Hz, 1H), 7.66 (s, 1H), 7.46 (d, J = 8.3 Hz, 2H), 7.34 – 7.27 (m,
3
1
H), 7.24 (s, 1H), 6.87 (s, 1H), 4.85 (ddd, J = 10.1, 8.1, 4.9 Hz, 1H), 4.56 (dd, J = 14.0, 4.8 Hz,
H), 4.44 (dd, J = 14.1, 10.1 Hz, 1H), 3.92 – 3.89 (m, 2H), 2.24 (s, 3H), 1.94 – 1.81 (m, 1H), 0.88
13
(
d, J = 6.7 Hz, 6H). C NMR (150 MHz, DMSO-d ) δ 169.64, 166.51, 144.59, 140.39, 137.86,
6
1
5
4
4
1
34.70, 132.03, 130.46, 129.42, 129.04(2C), 128.38, 127.77, 127.29(2C), 126.05, 119.87, 70.69,
+
3.84, 45.73, 27.27, 20.12, 18.77(2C). HRMS calcd for C H N O , [M + H] , 406.2131; found
2
4
28
3
3
06.2130.
.5.12 isopropyl(S)-2-(4'-fluoro-[1,1'-biphenyl]-4-carboxamido)-3-(1H-imidazol-1-yl)propanoate(
2l)
1
Light white solid; yield: 62.6%; mp: 98.4-100.1 °C. H NMR (400 MHz, DMSO-d ) δ 9.02 (s,
6
1H), 7.91 (d, J = 8.4 Hz, 2H), 7.69 – 7.68(m, 2H), 7.66 (d, J = 7.7 Hz, 1H), 7.57 (s, 1H), 7.49 - 7.34
(m, 3H), 7.23 (s, 1H), 6.86 (s, 1H), 4.99 – 4.95 (m, 1H), 4.78 - 4.76(m, 1H), 4.55 – 4.37 (m, 2H),
13
1
.21 (t, J = 5.9 Hz, 6H). C NMR (100 MHz, DMSO-d ) δ 169.14, 166.22, 163.49, 161.04, 142.13,
6
137.81, 135.08, 132.25, 129.02, 128.94, 128.18, 128.01, 127.94, 126.58, 121.50, 119.93, 115.96,
+
1
15.75, 68.66, 53.89, 45.81, 21.48, 21.44. HRMS calcd for C H FN O , [M + H] , 396.1723;
2
2
23
3
3
found 396.1719.
4.5.13 isopropyl(S)-2-(4'-chloro-[1,1'-biphenyl]-4-carboxamido)-3-(1H-imidazol-1-yl)propanoate
(12m)
1
Light white solid; yield: 54.8%; mp: 171.9-173.3 °C. H NMR (400 MHz, DMSO-d ) δ 9.00
6
(d, J = 7.9 Hz, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.79 (dd, J = 11.1, 8.5 Hz, 4H), 7.64 (s, 1H), 7.56 (d,
J = 8.6 Hz, 2H), 7.22 (s, 1H), 6.86 (s, 1H), 4.98 – 4.92 (m, 1H), 4.81 – 4.71 (m, 1H), 4.51 (dd, J =
1
3
1
4.0, 5.1 Hz, 1H), 4.39 (dd, J = 14.0, 9.8 Hz, 1H), 1.21 – 1.18 (m, 6H). C NMR (100 MHz,
DMSO-d ) δ 169.11, 166.13, 141.80, 137.87, 137.81, 133.04, 132.58, 128.97(2C), 128.67(2C),
6
128.32, 128.03(2C), 126.59(2C), 119.85, 68.63, 53.89, 45.75, 21.47, 21.42. HRMS calcd for
+
C H ClN O , [M + H] , 412.1428; found 412.1423.
2
2
23
3
3
4.5.14 isopropyl(S)-3-(1H-imidazol-1-yl)-2-(4'-methyl-[1,1'-biphenyl]-4-carboxamido)propanoate
(12n)

1
Light white solid; yield: 52.3%; mp: 121.0-123.6 °C. H NMR (400 MHz, DMSO-d ) δ(ppm)：
6
8
7
.96 (d, J = 7.9 Hz, 1H),7.98 (d, J = 8.4 Hz, 2H), 7.88 (d, J = 8.4Hz, 2H), 7.81 (s, 1H), 7.76 (s, 1H),
.66-7.63 (m, 3H), 7.22 (s, 1H), 6.86 (s, 1H), 4.99 – 4.94 (m, 1H), 4.79 - 4.74 (m, 1H), 4.54 – 4.37
1
3
(
m, 2H), 2.51 (s, 3H), 1.21 (d, J = 5.8 Hz, 6H). C NMR (100 MHz, DMSO-d ) δ 169.15, 166.25,
6
143.09, 137.79, 137.56, 136.16, 131.96, 129.60(2C), 128.27, 127.95(2C), 126.70(2C), 126.27(2C),
+
1
19.86, 68.61, 53.88, 45.77, 21.47, 21.42, 20.68. HRMS calcd for C H N O , [M + H] , 392.1974;
2
3
26
3
3
found 392.1971.
4.5.15 isopropyl(S)-3-(1H-imidazol-1-yl)-2-(4'-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxamido)p
ropanoate(12o)
1
Light white solid; yield: 58.0%; mp: 173.5-175.2 °C. H NMR (400 MHz, DMSO-d ) δ 9.03
6
(d, J = 7.9 Hz, 1H), 8.00 – 7.82 (m, 8H), 7.64 (s, 1H), 7.22 (s, 1H), 6.85 (s, 1H), 4.99 – 4.93 (m,
1
1
1
H), 4.83 – 4.71 (m, 1H), 4.52 (dd, J = 14.0, 5.0 Hz, 1H), 4.40 (dd, J = 14.0, 9.8 Hz, 1H), 1.21 –
13
.18 (m, 6H). C NMR (100 MHz, DMSO-d ) δ 169.09, 166.10, 143.11, 141.54, 137.83, 133.20,
6
28.56, 128.33, 128.10, 127.76, 127.10, 125.86, 119.87, 68.66, 53.90, 45.75, 21.48, 21.43. HRMS
+
calcd for C H F N O , [M + H] , 446.1692; found 446.1688.
23
23
3
3
3
4.5.16 isopropyl(S)-3-(1H-imidazol-1-yl)-2-(4'-(trifluoromethoxy)-[1,1'-biphenyl]-4-carboxamido)
propanoate(12p)
1
Light white solid; yield: 57.1%; mp: 140.9-142.1 °C. H NMR (400 MHz, DMSO-d ) δ 9.01
6
(d, J = 7.9 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.87 (d, J = 8.8 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H),
7.64 (s, 1H), 7.49 (d, J = 8.1 Hz, 2H), 7.22 (s, 1H), 6.86 (s, 1H), 5.00 – 4.91 (m, 1H), 4.84 – 4.71
1
3
(
m, 1H), 4.52 (dd, J = 14.0, 5.0 Hz, 1H), 4.40 (dd, J = 14.0, 9.8 Hz, 1H), 1.21 – 1.18 (m, 6H). C
NMR (100 MHz, DMSO-d ) δ 169.12, 166.18, 148.31, 141.71, 138.41, 137.83, 132.70, 128.88,
6
128.26, 128.05, 126.82, 121.50, 119.91, 68.66, 53.90, 45.80, 21.47, 21.43. HRMS calcd for
+
C H F N O , [M + H] , 462.1641; found 462.1642.
2
3
23
3
3
4
4.5.17 isopropyl(S)-2-(3'-fluoro-[1,1'-biphenyl]-4-carboxamido)-3-(1H-imidazol-1-yl)propanoate(
12q)
1
Light white solid; yield: 48.8%; mp: 122.7-124.8 °C. H NMR (400 MHz, DMSO-d ) δ 9.02
6
(d, J = 7.9 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.67 – 7.48 (m, 4H), 7.31 –
7
1
.18 (m, 2H), 6.86 (s, 1H), 5.01 – 4.91 (m, 1H), 4.81 – 4.73 (m, 1H), 4.52 (dd, J = 14.0, 5.1 Hz,
1
3
H), 4.40 (dd, J = 14.0, 9.8 Hz, 1H), 1.19 (t, J = 5.7 Hz, 6H). C NMR (100 MHz, DMSO-d ) δ
6

1
1
69.14, 166.17, 163.93, 161.51, 141.73, 141.48, 137.85, 132.85, 131.03, 128.36, 128.03(2C),
26.83(2C), 123.01, 119.88, 114.74, 113.77, 68.67, 53.92, 45.77, 21.49, 21.44. HRMS calcd for
+
C H FN O , [M + H] , 396.1723; found 396.1722.
2
2
23
3
3
4.5.18 isopropyl(S)-2-(3'-cyano-[1,1'-biphenyl]-4-carboxamido)-3-(1H-imidazol-1-yl)propanoate(
12s)
1
Yellow oil; yield: 57.4%; H NMR (400 MHz, DMSO-d ) δ 9.08 (d, J = 7.9 Hz, 1H), 8.26 (s,
6
1
1
H), 8.10 (d, J = 8.4 Hz, 1H), 7.94 – 7.88 (m, 5H), 7.71 (t, J = 7.8 Hz, 1H), 7.65 (s, 1H), 7.23 (s,
H), 6.85 (s, 1H), 5.00 - 4.91 (m, 1H), 4.82 – 4.73 (m, 1H), 4.52 (dd, J = 14.0, 5.1 Hz, 1H), 4.42
1
3
(
dd, J = 14.0, 9.8 Hz, 1H), 1.19 (t, J = 5.7 Hz, 6H). C NMR (100 MHz, DMSO-d ) δ 169.10,
6
1
1
66.07, 140.92, 140.19, 137.86, 133.09, 131.70, 131.65, 130.50, 130.23, 128.30, 128.13(2C),
26.94(2C), 119.89, 118.69, 112.20, 68.65, 53.93, 45.73, 21.48, 21.44. HRMS calcd for
+
C H N O , [M + H] , 403.1770; found 403.1764.
2
3
23
4
3
4.5.19 isopropyl(S)-2-(2',4'-difluoro-[1,1'-biphenyl]-4-carboxamido)-3-(1H-imidazol-1-yl)propan
oate(12t)
1
Light white solid; yield: 50.7%; mp: 118.6-120.3 °C. H NMR (400 MHz, DMSO-d ) δ 9.01
6
(d, J = 7.9 Hz, 1H), 7.88 (d, J = 8.3 Hz, 2H), 7.74 – 7.54 (m, 4H), 7.48 – 7.35 (m, 1H), 7.28 – 7.18
(m, 2H), 6.85 (s, 1H), 4.99 – 4.92 (m, 1H), 4.80 – 4.74 (m, 1H), 4.51 (dd, J = 14.0, 5.0 Hz, 1H),
1
3
4
1
1
.39 (dd, J = 14.0, 9.9 Hz, 1H), 1.21 – 1.18 (m, 6H). C NMR (100 MHz, DMSO-d ) δ 169.09,
6
66.18, 163.36, 163.24, 160.46, 160.33, 137.83, 137.39, 132.83, 132.00, 128.80, 128.34,
27.64(2C), 124.05, 119.87, 112.08, 104.62, 68.66, 53.90, 45.76, 21.47, 21.43. HRMS calcd for
+
C H F N O , [M + H] , 414.1629; found 414.1626.
2
2
22
2
3
3
4.5.20 isopropyl(S)-2-(3',4'-difluoro-[1,1'-biphenyl]-4-carboxamido)-3-(1H-imidazol-1-yl)propan
oate(12u)
1
Light white solid; yield: 63.5%; mp: 104.2-106.2 °C. H NMR (400 MHz, DMSO-d ) δ 9.01
6
(d, J = 7.9 Hz, 1H), 7.93 – 7.77 (m, 5H), 7.64 – 7.52 (m, 3H), 7.22 (s, 1H), 6.86 (s, 1H), 5.00 –
4.91 (m, 1H), 4.82 – 4.72 (m, 1H), 4.52 (dd, J = 14.0, 5.1 Hz, 1H), 4.40 (dd, J = 14.0, 9.9 Hz, 1H),
13
1
.21 – 1.18 (m, 6H). C NMR (100 MHz, DMSO-d ) δ 169.10, 166.07, 151.07, 150.63, 148.63,
6
148.29, 140.86, 137.81, 136.67, 132.73, 128.32, 127.99(2C), 126.72(2C), 123.79, 119.85, 118.09,
+
1
16.11, 68.64, 53.89, 45.75, 21.46, 21.42. HRMS calcd for C H F N O , [M + H] , 414.1629;
2
2
22
2
3
3
found 414.1627.

4.5.21 isopropyl(S)-2-(2',5'-difluoro-[1,1'-biphenyl]-4-carboxamido)-3-(1H-imidazol-1-yl)propan
oate(12v)
1
Light white solid; yield: 55.7%; mp: 107.8-110.5 °C. H NMR (400 MHz, DMSO-d ) δ 9.04
6
(d, J = 7.9 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H), 7.64 (s, 1H), 7.52 – 7.47 (m,
1
–
6
1
H), 7.45 – 7.41 (m, 1H), 7.35- 7.29 (m, 1H), 7.22 (s, 1H), 6.86 (s, 1H), 5.01 - 4.91 (m, 1H), 4.82
4.72 (m, 1H), 4.52 (dd, J = 14.0, 5.0 Hz, 1H), 4.39 (dd, J = 14.0, 9.8 Hz, 1H), 1.21 – 1.18 (m,
1
3
H). C NMR (100 MHz, DMSO-d ) δ 169.06, 166.10, 159.56, 157.17, 156.47, 154.04, 137.82,
6
37.07, 133.20, 128.90, 128.87, 128.28, 127.63(2C), 119.89, 117.90, 117.06, 116.53, 68.67, 53.89,
+
4
5.77, 21.47, 21.43. HRMS calcd for C H F N O , [M + H] , 414.1629; found 414.1630.
22
22
2
3
3
4.6 General procedure for the synthesis of compounds (13a-b)
To a solution of intermediate 12a-v (1 equiv.) in methanol was added 2N sodium hydroxide at
ambient temperature. The reaction mixture was stirred for 3 h and the methanol was removed by
rotary evaporation. The resultant mixture was adjusted to pH=5-7 with 1 N HCl solution. The
precipitated white solid was collected by filtration and dried to give the carboxylic acid
intermediate.
To a solution of the carboxylic acid intermediate (1 equiv.) in anhydrous DMF was added
EDCI (1.1 equiv.) and HOBt (1.1 equiv.), respectively.The reaction mixture was stirred for 2 h at
ambient temperature, and the aliphatic amine (1.1 equiv.) and DIEA (3 equiv.) were added. The
solution was heated to 70 °C for 6 h and then cooled to room temperature. The reaction mixture was
poured into ice water, and the resulting solid was filtered and dried to give the titled compound.
4.6.1 (S)-N-(3-(1H-imidazol-1-yl)-1-(isopropylamino)-1-oxopropan-2-yl)-[1,1'-biphenyl]-4-carbox
amide(13a)
1
Light white solid; yield: 71.1%; mp: 220.5-224.4 °C. H NMR (400 MHz, DMSO-d ) δ 8.70
6
(d, J = 8.1 Hz, 1H), 8.06 (d, J = 7.1 Hz, 1H), 7.93 (d, J = 7.9 Hz, 2H), 7.79 – 7.73 (m, 4H), 7.62 (s,
1
4
6
1
H), 7.58 – 7.48 (m, 2H), 7.42 (d, J = 7.1 Hz, 1H), 7.17 (s, 1H), 6.83 (s, 1H), 4.83 (s, 1H), 4.39 –
.36 (m, 1H), 4.33 – 4.21 (m, 1H), 3.87 (dd, J = 13.3, 6.6 Hz, 1H), 1.08 (dd, J = 13.7, 6.4 Hz,
1
3
H). C NMR (150 MHz, DMSO-d ) δ 167.97, 166.07, 143.07, 139.16, 137.72, 132.56,
6
29.09(2C), 128.20, 128.16(2C), 126.93(2C), 126.54(2C), 119.81, 54.45, 47.15, 40.80, 22.26(2C).
+
HRMS calcd for C H N O , [M + H] , 377.1978; found 377.1981.
22
25
4
2
4.6.2 (S)-N-(3-(1H-imidazol-1-yl)-1-(isobutylamino)-1-oxopropan-2-yl)-[1,1'-biphenyl]-4-carboxa

mide(13b)
1
Light white solid; yield: 69.5%; mp: 176.5-178.7 °C. H NMR (600 MHz, DMSO-d ) δ 8.78
6
(d, J = 8.5 Hz, 1H), 8.18 (t, J = 5.7 Hz, 1H), 7.92 (d, J = 8.3 Hz, 2H), 7.79 – 7.73 (m, 5H), 7.50 (t,
J = 7.6 Hz, 2H), 7.41 (t, J = 7.4 Hz, 1H), 7.24 (s, 1H), 6.91 (s, 1H), 4.90 – 4.86 (m, 1H), 4.45 (dd,
J = 13.9, 4.4 Hz, 1H), 4.33 (dd, J = 13.8, 10.3 Hz, 1H), 2.94 (t, J = 6.4 Hz, 2H), 1.73 – 1.68 (m,
1
3
1
1
4
H), 0.84 (dd, J = 6.6, 1.2 Hz, 6H). C NMR (150 MHz, DMSO-d ) δ 168.85, 166.12, 143.05,
6
39.13, 137.55, 132.51, 129.04(2C), 128.14(2C), 128.11, 126.90(2C), 126.48(2C), 120.01, 54.44,
+
7.19, 46.17, 28.01, 20.06(2C). HRMS calcd for C H N O , [M + H] , 391.2134; found
2
3
27
4
2
3
91.2129.
.7 General procedure for the synthesis of compounds (15a-b)
To a solution of intermediate 11a-b (1 equiv.) in anhydrous DMF was added thionyl chloride (2
4
equiv) dropwise. The reaction mixture was stirred for 2 h at ambient temperature, and
H-1,2,4-triazole (4 equiv.) and triethylamine (3 equiv.) were added.The mixture was stirred at
1
room temperature overnight. When the reaction was complete, as determined by TLC, the reaction
mixture was extracted with EtOAc and washed with brine. The organic phase was dried over
Na SO overnight and the solvent was removed in vacuo. The crude product was purified by silica
2
4
gel column chromatography (CH OH : CH Cl = 1:40, v/v) to yield the target product 15a-b.
3
2
2
4.7.1 isopropyl(S)-2-([1,1'-biphenyl]-4-carboxamido)-3-(1H-1,2,4-triazol-1-yl)propanoate(15a)
1
Light white solid; yield: 47.4%; mp: 132.5-135.2 °C. H NMR (600 MHz, DMSO-d ) δ 8.97
6
(d, J = 7.8 Hz, 1H), 8.51 (s, 1H), 7.99 (s, 1H), 7.88 (d, J = 8.5 Hz, 2H), 7.79 (d, J = 8.5 Hz, 2H),
7
1
.76 – 7.71 (m, 2H), 7.53 – 7.47 (m, 2H), 7.45 – 7.40 (m, 1H), 4.98 – 4.92 (m, 1H), 4.87 – 4.82 (m,
H), 4.71 (dd, J = 14.0, 5.3 Hz, 1H), 4.65 (dd, J = 14.0, 8.9 Hz, 1H), 1.18 (dd, J = 6.2, 3.3 Hz, 6H).
1
3
C NMR (150 MHz, DMSO-d ) δ 168.84, 166.19, 151.66, 144.91, 143.24, 139.08, 132.18,
6
129.04(2C), 128.14, 127.98(2C), 126.91(2C), 126.64(2C), 68.73, 52.83, 48.56, 21.46, 21.42.
+
HRMS calcd for C H N O , [M + Na] , 401.1590; found 401.1595.
21
22
4
3
4.7.2 isobutyl(S)-2-([1,1'-biphenyl]-4-carboxamido)-3-(1H-1,2,4-triazol-1-yl)propanoate(15b)
1
Light white solid; yield: 50.5%; mp: 108.4-111.9 °C. H NMR (600 MHz, DMSO-d ) δ 9.03
6
(d, J = 7.8 Hz, 1H), 8.52 (s, 1H), 8.00 (s, 1H), 7.88 (d, J = 8.3 Hz, 2H), 7.79 (d, J = 8.3 Hz, 2H),
7
4
.74 (d, J = 7.4 Hz, 2H), 7.50 (t, J = 7.7 Hz, 2H), 7.42 (t, J = 7.4 Hz, 1H), 4.93 – 4.90 (m, 1H),
.75 (dd, J = 14.0, 5.2 Hz, 1H), 4.70 (dd, J = 14.0, 9.1 Hz, 1H), 3.94 – 3.86 (m, 2H), 1.86 (dt, J =

1
3
1
3.3, 6.7 Hz, 1H), 0.86 (dd, J = 6.7, 1.1 Hz, 6H). C NMR (150 MHz, DMSO-d ) δ 169.35,
6
166.21, 151.66, 144.88, 143.23, 139.06, 132.15, 129.02(2C), 128.13, 127.96(2C), 126.90(2C),
+
1
26.62(2C), 70.73, 52.73, 48.48, 27.19, 18.73. HRMS calcd for C H N O , [M + Na] , 415.1746;
2
2
24
4
3
found 415.1748.
4.8 General procedure for the synthesis of compounds (19a-b)
The synthetic procedure used for compounds 19a-19b was similar to that used for compounds
13a-14v.
4.8.1 isopropyl(R)-2-(2'-fluoro-[1,1'-biphenyl]-4-carboxamido)-3-(1H-1,2,4-triazol-1-yl)propanoa
te(19a)
1
Light white solid; yield: 58.7%; mp: 116.3-119.4 °C. H NMR (400 MHz, DMSO-d ) δ 9.01
6
(d, J = 7.9 Hz, 1H), 7.89 (d, J = 8.3 Hz, 2H), 7.67 (d, J = 7.2 Hz, 2H), 7.64 (s, 1H), 7.61 – 7.56 (m,
1
4
1
1
1
3
4
H), 7.51 – 7.43 (m, 1H), 7.40 – 7.30 (m, 2H), 7.22 (s, 1H), 6.86 (s, 1H), 5.04 – 4.90 (m, 1H),
.80 – 4.74 (td, J = 9.6, 5.2 Hz, 1H), 4.52 (dd, J = 14.0, 5.0 Hz, 1H), 4.40 (dd, J = 14.0, 9.8 Hz,
1
3
H), 1.20 (t, J = 5.7 Hz, 6H). C NMR (150 MHz, DMSO-d ) δ 169.11, 166.21, 159.89, 158.26,
6
38.23, 137.84, 132.76, 130.82, 130.25, 128.83, 128.35, 127.61(2C), 127.30, 125.07, 125.05, 119.89,
+
16.28, 68.65, 53.91, 45.75, 21.49, 21.45. HRMS calcd for C H FN O , [M + H] , 396.1723; found
2
2
23
3
3
96.1723.
.8.2 isobutyl(R)-2-(2'-fluoro-[1,1'-biphenyl]-4-carboxamido)-3-(1H-1,2,4-triazol-1-yl)propanoat
e(19b)
1
Light white solid; yield: 56.6%; mp: 118.6-121.2 °C. H NMR (400 MHz, DMSO-d ) δ 9.05
6
(d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.67 (dd, J = 8.3, 1.3 Hz, 2H), 7.64 (s, 1H), 7.61 –
7
1
.56 (m, 1H), 7.50 -7.44 (m, 1H), 7.40 – 7.30 (m, 2H), 7.22 (s, 1H), 6.86 (s, 1H), 4.88 – 4.78 (m,
H), 4.55 (dd, J = 14.0, 4.8 Hz, 1H), 4.42 (dd, J = 14.0, 10.1 Hz, 1H), 3.96 – 3.85 (m, 2H), 1.87
1
3
(
dp, J = 13.3, 6.7 Hz, 1H), 0.87 (d, J = 6.7 Hz, 6H). C NMR (150 MHz, DMSO-d ) δ 169.58,
6
1
1
66.25, 159.89, 158.26, 138.24, 137.83, 132.73, 130.82, 130.25, 128.85, 128.83, 128.38,
27.59(2C), 127.28, 125.05, 119.86, 116.28, 70.66, 53.82, 45.67, 27.24, 18.75(2C). HRMS calcd
+
for C H FN O , [M + H] , 410.1880; found 410.1880.
2
3
25
3
3
4.9 In vitro antifungal testing
The in vitro minimum inhibitory concentrations (MIC) were determined by serial dilution in
96-well microtiter plates based on the standard guidelines described in the National Committee for

Clinical Laboratory Standards (NCCLS). The MIC values were defined as the lowest concentration
of an antimicrobial that will inhibit the visible growth of the fungi. FLC and ITR were purchased for
use as positive control drugs. All of compounds were dissolved in DMSO and serially diluted into
the growth medium.
4.10 GC-MS analysis of sterol composition
GC-MS was performed with an Agilent Technologies (AT) 6890N Network GC system
equipped with an AT 5975 quadrupole mass selector detector using He as the carrier gas. The sterols
were extracted from C. albicans and analysed by GC-MS. The GC-MS data were analysed 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 Cytochrome P450 Inhibition Assay
Cytochrome P450 inhibition was evaluated in human liver microsomes (0.25 mg/mL) using
five specific probe substrates (CYP1A2, 10 μM phenacetin; CYP2C9, 5 μM diclofenac; CYP2C19,
0 μM S-mephenytoin; CYP2D6, 5 μM dextromethorphan; and CYP3A4, 2 μM midazolam) in the
presence of multiple concentrations of the test compound (0.05-50 μM ). After pre-incubation at
7°C for 10 min, the reaction was initiated with the addition of 20 μL NADPH to a final
3
3
concentration of 10 mM. The mixture were incubated at 37°C for 10 min and the reaction
terminated with the addition of a 400 µL cold stop solution (200 ng/mL tolbutamide and 200 ng/mL
labetalol in acetonitrile). After the reactions were terminated, the plates were centrifuged, and the
supernatants were analysed by LC/MS/MS.
Acknowledgements
The authors thank Prof. Yongbing Cao from School of Pharmacy, the Second Military Medical
University, for providing the fluconazole-resistant strains of Candida albicans(strain 100 and strain
103). This work was supported by Program for Innovative Research Team of the Ministry of
Education and Program for Liaoning Innovative Research Team in University.

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Graphical abstract

Highlights




27 new compounds with biphenyl imidazole scaffolds were designed and synthesized.
Compounds 12f-g and 19a-b showed better antifungal activity than fluconazole.
The structure-activity relationships of compounds were discussed.
Mechanism studies revealed that the 12g might act by inhibiting the CYP51.