Design, synthesis and evaluation of aromatic heterocyclic derivatives as potent antifungal agents

Article abstract of DOI:10.1016/j.ejmech.2017.05.043

To further enhance the anti-Aspergillus efficacy of our previously discovered antifungal lead compounds (1), a series of aromatic heterocyclic derivatives were designed, synthesized and evaluated for in vitro antifungal activity. Many of the target compounds showed good inhibitory activity against Candida albicans and Cryptococcus neoformans. In particular, the isoxazole nuclei were more suited for improving the activity against Aspergillus spp. Among these compounds, 2-F substituted analogues 23g and 23h displayed the most remarkable in vitro activity against Candida spp., C. neoformans, A. fumigatus and fluconazole-resistant C.alb. strains, which is superior or comparable to the activity of the reference drugs fluconazole and voriconazole. Notably, the compounds 23g and 23h exhibited low inhibition profiles for various isoforms of human cytochrome P450 and excellent blood plasma stability.

Full text of DOI:10.1016/j.ejmech.2017.05.043

Accepted Manuscript
Design, synthesis and evaluation of aromatic heterocyclic derivatives as potent
antifungal agents
Shizhen Zhao, Xiangqian Zhang, Peng Wei, Xin Su, Liyu Zhao, Mengya Wu,
Chenzhou Hao, Chunchi Liu, Dongmei Zhao, Maosheng Cheng
PII:
S0223-5234(17)30404-X
10.1016/j.ejmech.2017.05.043
EJMECH 9473
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 April 2017
Revised Date: 16 May 2017
Accepted Date: 20 May 2017
Please cite this article as: S. Zhao, X. Zhang, P. Wei, X. Su, L. Zhao, M. Wu, C. Hao, C. Liu, D. Zhao,
M. Cheng, Design, synthesis and evaluation of aromatic heterocyclic derivatives as potent antifungal
agents, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.05.043.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and evaluation of aromatic heterocyclic derivatives
as potent antifungal agents
Shizhen Zhaoa, Xiangqian Zhanga, Peng Weia, Xin Sub, Liyu Zhaoa, Mengya Wua,
Chenzhou Haoa, Chunchi Liua, 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, PR China
bThe School of life Science and Biopharmaceutical, Shenyang Pharmaceutical University, 103
Wenhua Road, Shenhe District, Shenyang, 110016, PR China
*Corresponding author: Dongmei Zhao, E-mail: medchemzhao@163.com
ABSTRACT:
To further enhance the anti-Aspergillus efficacy of our previously discovered antifungal lead
compounds (1), a series of aromatic heterocyclic derivatives were designed, synthesized and
evaluated for in vitro antifungal activity. Many of the target compounds showed good inhibitory
activity against Candida albicans and Cryptococcus neoformans. In particular, the isoxazole
nuclei were more suited for improving the activity against Aspergillus spp. Among these
compounds, 2-F substituted analogues 23g and 23h displayed the most remarkable in vitro activity
against Candida spp., C. neoformans, A. fumigatus and fluconazole-resistant C.alb. strains, which
is superior or comparable to the activity of the reference drugs fluconazole and voriconazole.
Notably, the compounds 23g and 23h exhibited low inhibition profiles for various isoforms of
human cytochrome P450 and excellent blood plasma stability.
Keywords:
Antifungal activity, Azole antifungals, CYP51, Structure-activity relationship
1. Introduction
The incidence of fungal infections has steadily increased over the past few decades and
presents a serious threat to human health, especially in immunocompromised patients, such as
those undergoing organ transplants or anticancer chemotherapy and patients with AIDS[1-3].

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Candida spp., Cryptococcus neoformans and Aspergillus spp. are still the three main pathogens of
fungal infections[4]. The clinically available antifungal agents can be divided into four different
classes based on their mode of action, including the polyenes (e.g., amphotericin B and
nystatin)[4], echinocandins (e.g., caspofungin and micafungin)[5], azoles(e.g., fluconazole,
voriconazole and itraconazole)[6], and antimetabolites (e.g., 5-fluorocytosine)[7].
Azole antifungal agents are currently most widely used in first-line antifungal therapy. They
prevent the synthesis of ergosterol biosynthesis in the cell by inhibiting the activity of lanosterol
14-demethylase (CYP51), a member of the CYP51 class of cytochrome P450 enzymes[8, 9]. The
imidazole or triazole ring of azole antifungals agents is an essential pharmacophoric portion,
which can coordinate with the heme iron atom cofactor of the CYP51[10]. During the past 35
years, azole drugs, such as fluconazole, itraconazole, voriconazole and posaconazole, have made a
significant impact on the management of systemic fungal infections[11]. However, several factors
have limited their practical applications, such as drug resistance, narrow antifungal spectrum, and
low bioavailability[12, 13]. Therefore, there is still an urgent need for developing novel azole
antifungal agents with potent activity, broad spectrum, low toxicity, and low resistance.
Figure 1. Chemical structures of azole antifungal agents and lead compound
.
We have previously reported the synthesis and in vitro evaluation of antifungal activity of a
new series of biphenyl imidazole derivatives[14, 15]. Most compounds displayed strong
antifungal activities with MIC values in the range of 0.03125 µg/mL to 2 µg/mL against Candida
albicans and Cryptococcus neoformans. However, almost all of the target compounds were

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inactive against Aspergillus fumigatus, which prompted us to continue with studying the structural
modification of potent original compounds in search of novel compounds with potent activity and
broad spectrum.
A published crystal structure of voriconazole bound within the active site cavity of CYP51B
of Aspergillus fumigatus (PDB ID:4UYM) served as a useful template for the rational
structure-based design of novel drugs[16]. Voriconazole has much higher antifungal potency
against Aspergillus fumigatus than fluconazole and itraconazole. The crystal structure suggests
that the reason may be the formation of the hydrogen bonds between the 5-fluoropyrimidine ring
of voriconazole and A. fumigatus CYP51 Tyr122 (Figure 2A).
Figure 2. Crystal structure of sterol 14-alpha demethylase (CYP51B) from a pathogenic filamentous fungus
Aspergillus fumigatus in complex with voriconazole (A and B) and the binding mode of compound 1 (B) in the
active site.
To further explore the potential binding mode of biphenyl imidazole derivatives and guide
the design of new compounds, compound 1 was docked into the active site of A. fumigatus
CYP51B (PDB ID:4UYM, Figure 2B). Based on the interactions between compound 1 and A.
fumigatus CYP51, along with the binding mode of voriconazole, we aimed to develop new
heterocycles-linked compounds from biphenyl imidazole derivatives to formation hydrogen bonds
interactions with Tyr122 of CYP51. The benzene ring (A ring of compound 1) would be replaced
by a five or six- membered heterocyclic ring, which might result in the development of more
potent compounds with higher antifungal activities, especially the anti-Aspergillus efficacy.

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Figure 3. Our strategy for the formation hydrogen bonds interactions with CYP51B Tyr122 of A. fumigatus by
replacing the middle benzene ring with 10 heteroaromatics.
2. Results and discussion
2.1 Chemistry
The synthetic routes of the key intermediates 1a-j are illustrated in Scheme 1 and 2.
Acetophenone 3 was used as the starting material, and treated with diethyl oxalate in the presence
of NaOEt by Claisen condensation to obtain intermediate 4, which was used in a ring-closure
reaction with hydroxylamine hydrochloride to form isoxazole 2a. Replacing the oxygen atoms of
benzamide 5 with sulphur via Lawesson’s Reagent yielded thiobenzamide 7. Then, benzamide 5
and thiobenzamide 7 were treated with ethyl bromopyruvate by intermolecular cyclization to
obtain thiazole 2b and oxazole 2c, respectively. The thiazole 2d was obtained from
intermolecular cyclization between 2-Bromoacetophenone 8 and ethyl thiooxamate 9.The ethyl
2-chloroacetoacetate 10 was condensed with thiobenzamide 6 to form thiazole 2e.
The intermediate 2f was obtained by ring-closure reaction between phenylhydrazine 11 and
ethyl 2,4-dioxopentanoate 12. Ethyl 3,3-diethoxy propionate 13 was treated with Ethyl formate in
the presence of NaH by Claisen condensation to form intermediate 14, which were combined with
benzamidine hydrochloride hydrate in an intermolecular cyclization to yield intermediates
pyrimidine 2g. Commercially available 16,17 and 18 and phenylboronic acids 15 were subjected
to Suzuki coupling in the presence of Pd(PPh3)4 to form the intermediates 2h, 2i and 1j. Finally,
intermediates 2a-i were saponified with 2 N NaOH to obtain the key intermediates 1a-i.

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Scheme 1. Synthesis of intermediates 1a-e. Reagents and conditions:(a) Diethyl oxalate, NaOEt, 18 h;(b)
Hydroxylamine hydrochloride, EtOH, reflux, 2 h; (c) Hydrazine hydrate, EtOH, reflux, 2 h; (d) NaOH,
MeOH/H2O; (e) EtOH, reflux; (f) Lawesson’s reagent, toluene, reflux, 7 h.
Scheme 2. Synthesis of intermediates 1f-j. Reagents and conditions: (a) EtOH, reflux; (b) NaOH, MeOH/H2O; (c)
ethyl formate, NaH, THF; (d) benzamidine hydrochloride hydrate, K2CO3, DMF, 100oC ; (e) Pd(PPh3)4, K2CO3,
reflux, 5h.

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The target compounds 22a-v were formed according to the reaction pathways illustrated in
Scheme 3. L-serine 19 was treated with an alcohol (isopropanol or isobutanol) and refluxed with
SOCl2 to give the serine esters 20a-b. The serine esters 20a-b reacted with the key intermediates
1a-j in the presence of EDCI and HOBt, to give compounds 21a-t. Finally, the compounds 21a-t
were treated with imidazole/CDI, to obtain the target compounds 22a-t.
Scheme 3. General synthesis of the target compounds 22a-t. Reagents and conditions: (a) alcohol reagent, SOCl2,
reflux, 1–2 h; (b) EDCI, HOBt, DIEA, r.t., 7 h; (c) CDI, imidazole, CH3CN, reflux, 7 h.
2.2 In vitro antifungal activity
To explore an optimal polar heteroaromatic to replace the middle phenyl of compound 1, we
evaluated the in vitro antifungal activities of the synthesized 19 heteroaromatic compounds
according to the protocols from the NCCLS[17]. Broth microdilution methods were used to
determine the minimum inhibitory concentrations (MICs) of the target compounds in 96-well
microtest plates. Fluconazole (FLC) and Itraconazole (ITR) were used as reference drugs.
The in vitro antifungal activities of the target compounds are listed in Table 1. The MIC
values revealed that most compounds showed a decrease in antifungal activity compared with the
lead compound 1. Interestingly, the heteroatom compounds such as thiophene (22a-b),
2-Methylthiazole (22m-n), isoxazole (22e-f), pyrimidine(22q-s) and pyridine(22t) exhibited
moderate to good antifungal properties. Of these, isoxazole (22e-f) showed the most potent
activity against C. albicans, C. neoformans, and C. tropicalis with MIC values in the range of
0.03125 to 1 µg/mL, which are superior or comparable to those of the reference drugs FLC and
ITR. Interestingly, the isoxazole core (22e-f) showed excellent antifungal activities against
Aspergillus fumigatus with an MIC value of 4 µg/mL, which was intended to increase the
antifungal activities against Aspergillus fumigatus. Moreover, other combinations of heteroatoms

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such as furans (22c-d), oxazole (22g-h) and thiazole(22k-l) did not show the desired potency,
further confirming the importance of the electrical and hydrophobic effect in ligands.
Based on the results above, we selected a scaffold of isoxazole core (22e-f) as our starting
point for further modification. Our optimized efforts were directed toward replacing the terminal
benzene ring with various substituents to expand the SAR studies (Table 2). Most of the
compounds(23a-h) exhibited excellent antifungal properties with broad spectrums of activity. The
results showed that the structure-activity relationships of these compounds were identical to our
previously reported biphenyl imidazole derivatives[15]. Of these, compounds 23g-h with 2-F
substituents showed the best antifungal activity.
Table 1
In vitro antifungal activities of the target compounds (MIC, µg/mL)a.
Compd.
22a
22b
22c
22d
22e
22f
Het
R2
C. alb.(Ⅰ)
8
C. alb(Ⅱ).
C. neo.
16
C.tro.
1
A. fum.
>16
>16
>16
>16
4
8
-CH(CH3)2
2
4
>16
>16
>16
0.5
0.25
>16
>16
>16
>16
2
0.125
8
-CH2CH(CH3)2
-CH(CH3)2
>16
>16
0.25
0.0625
>16
>16
>16
>16
>16
>16
2
>16
>16
0.5
1
4
-CH2CH(CH3)2
-CH(CH3)2
0.0625
0.03125
8
4
-CH2CH(CH3)2
-CH(CH3)2
>16
>16
>16
>16
>16
>16
2
>16
>16
>16
>16
>16
>16
>16
>16
22g
22h
22i
4
-CH2CH(CH3)2
-CH(CH3)2
>16
>16
0.0625
0.125
0.25
0.125
-CH2CH(CH3)2
-CH(CH3)2
22j
22k
22l
2
-CH2CH(CH3)2
-CH(CH3)2
16
22m
22n
0.25
1
8
-CH2CH(CH3)2

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>16
>16
0.25
0.25
>16
>16
1
>16
>16
8
4
4
>16
>16
>16
>16
-CH(CH3)2
-CH2CH(CH3)2
-CH(CH3)2
22o
22p
22q
22s
0.0625
0.125
0.5
2
-CH2CH(CH3)2
2
4
16
1
>16
-CH(CH3)2
22t
0.0625
1
0.25
2
1
4
1
0.0625
0.25
>16
16
1
1
FCZ
ITZ
-
-
-
-
0.03125
0.125
0.03125
aAbbreviations: C.alb.(I), Candida albicans (ATCC SC5314); C.alb.(II), Candida albicans (CPCC400523);
C. neo., Cryptococcus neoformans (cgmcc 2.3161); A.fum., Aspergillus fumigatus (GIM 3.524); C.tro., Candida
tropicalis (cgmcc 2.3739); FCZ: Fluconazole; ITZ: Itraconazole.
Table 2
In vitro antifungal activities of the target compounds (MIC, µg/mL)a.
Compd.
R1
R2
C. alb.(Ⅰ)
0.25
C. alb(Ⅱ).
C. neo.
0.5
0.25
2
C.tro.
0.0625
A. fum.
0.5
1
4
4
-CH(CH3)2
-CH2CH(CH3)2
-CH(CH3)2
-CH2CH(CH3)2
-CH(CH3)2
-CH2CH(CH3)2
-CH(CH3)2
-CH2CH(CH3)2
-CH(CH3)2
-CH2CH(CH3)2
-
22e
22f
H
0.0625
0.25
0.03125
0.03125
0.03125
0.03125
0.03125
0.03125
0.03125
0.03125
0.03125
0.25
0.5
0.5
2
8
23a
23b
23c
23d
23e
23f
4-CH3
4-F
0.125
1
0.5
2
8
8
0.5
2
0.5
2
8
0.0625
0.03125
0.0625
0.03125
1
0.25
0.125
0.25
0.125
2
>16
>16
4
4-Br
2
0.5
0.25
4
23g
23h
FCZ
2-F
-
4
>16

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ITZ
-
-
0.03125
0.125
1
0.03125
1
aAbbreviations: C.alb.(I), Candida albicans (ATCC SC5314); C.alb.(II), (CPCC400523); C. neo.,
Cryptococcus neoformans (cgmcc 2.3161); A.fum., Aspergillus fumigatus (GIM 3.524); C.tro., Candida tropicalis
(cgmcc 2.3739); FCZ: Fluconazole; ITZ: Itraconazole.
2.3 In vitro antifungal activity against fluconazole-resistant strains of C. alb.
Currently, the widespread use of Fluconazole, especially for prolonged treatment periods, has
led to a severe increase in drug-resistance, which has become a major clinical problem in fungal
infection therapy. Therefore, there is an urgent need to find new types of inhibitors that could be
effective against fluconazole-resistant strains of C. albicans. The most potent compounds 23g and
23h were further evaluated against fluconazole-resistant strains of C.alb. (strains 100 and 103).
Compounds 23g and 23h displayed strong antifungal activities against strains 100 and 103, with
MIC values in the range from 0.125 to 1 µg/mL (Table 3).
Table 3
In vitro antifungal activities of the target compounds (MIC, µg/mL)a.
C. alb.
Compd.
R1
R2
Strain100
1
Strain103
2-F
2-F
-
-CH(CH3)2
1
23g
23h
-CH2CH(CH3)2
-
0.125
>64
0.5
>64
FCZ
aAbbreviations: 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.
2.4 Dose-dependent effect on sterol composition in Candida albicans (ATCC SC5314)
The antifungal mechanism of compound 23g was confirmed by analysing the change in sterol
composition in 23g-treated C. albicans SC5314 cells by GC-MS, which was compared to that of
untreated or fluconazole-treated C. albicans SC5314. The assay has been successfully used in
studying the mechanism of action of antifungal agents on the sterol biosynthesis pathways[18-20].
FLC was used as a reference drug and cholesterol was added as an internal standard. As shown in
Fig. 4, 23g and FLC reduced the content of ergosterol and increased the content of lanosterol and
eburicol in a dose-dependent manner. The GC-MS analysis results are shown in Table 4. In the
untreated control, the sterol fraction contained 98.7% ergosterol, while lanosterol was not

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observed and other 14-methylated sterols (obtusifoliol and eburicol) contained only 1.0 %. When
C. albicans was treated with FLC at 0.03125-8 µg/mL for 16 h, the content of ergosterol was
reduced to 1.7% from 98.7% of the total amount of sterol fraction, whereas the lanosterol and
eburicol contents were increased to 34.8 % and 37.2%, respectively. These changes were caused
by the competitive inhibition of CYP51 in C. albicans by FLC and are consistent with previously
published studies[15, 21-23]. Interestingly, treatment with 23g also resulted in noticeable
accumulation of eburicol and lanosterol and a significant reduction in the ergosterol content
(reduced to 0.7%). These results suggested that the novel compound 23g caused the potent
disruption of sterol biosynthesis by inhibiting CYP51 activity in C. albicans, similar to the
accepted mechanism of FLC.
Figure 4. Sterol composition in 23g or fluconazole-treated SC5314 C. albicans cells by GC-MS.
Table 4.
Analysis of sterol composition in C.albicans by GC-MS.a
% of total sterols (C. alb.a)
concentration
Compd.
4,4-dimethyl
(µg/mL)
Ergosterol
Obtusifoliol
Lanosterol
Eburicol
zymosterol
0.03125
93.9
85.9
30.8
5.4
4.3
6.9
6.9
9.2
8.9
3.6
8.9
6.9
16.1
13.3
0.6
-
1.7
4.9
-
0.125
2.2
16.7
22.0
26.7
-
-
23gb
0.5
45.6
63.3
63.7
0.8
-
2
-
8
0.7
-
0.03125
95.6
88.7
81.4
18.2
1.7
-
0.125
0.4
4.5
23.0
34.8
-
2.0
-
-
FLCc
0.5
2
7.2
27.0
37.2
0.4
15.7
13.0
-
8
Controld
-
98.7
b
aAbbreviations: C.alb., Candida albicans (ATCC SC5314); Treated with compound 23g; cTreated with FLC;
dControl (no drug).

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2.5 Cytochrome P450 Inhibition Assay
Cytochrome P450s (CYP) comprise a superfamily of enzymes that catalyse the oxidation of a
wide variety of drugs. Drug-drug interactions (DDI) caused by inhibiting cytochrome P450s
enzymes can result in dangerous side effects. However, many azole antifungals, such as
ketoconazole and itraconazole, had greater inhibitory effects on Cytochrome P450 (CYP)
enzymes[24]. For example, the IC50 of ketoconazole and itraconazole for CYP3A4 inhibition were
25 and 32.6 nM, respectively[25]. The DDI potential for compounds 23g and 23h were tested
against the five major human CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and
CYP3A4-M). As shown in Table 5, the compounds 23g and 23h showed weak inhibitory activity
against CYP1A2, CYP2C9, CYP2C19, and CYP2D6 with IC50 values of ≥50µM, while
compounds 23g and 23h exhibited moderate activity against CYP3A4 with IC50 values of 5.81
µM and 8.38 µM, respectively. The results showed that compounds 23g and 23h had a low
potential for causing DDI.
Table 5.
In Vitro CYP Inhibition assessment of compounds.
IC50 (µM)
Compd.
CYP1A2
>50
CYP2C9
>50
CYP2C19
>50
CYP2D6
>50
CYP3A4-M
5.81
23g
23h
>50
>50
44.1
>50
8.38
2.6 In vitro human Plasma Stability Assay
The stability of compounds in human plasma is an important consideration in drug discovery.
Based on their in vitro antifungal activities, compounds 23g and 23h were incubated with human
Plasma. As shown in Table 6, compounds 23g and 23h exhibited excellent metabolic profiles in
human plasma at 120 min (remaining 91.8% and 61.4%, respectively).
Table 6.
In Vitro human Plasma Stability of compounds 23g and 23h.
Stablity in Human Blood Plasma
Compd.
% Remaining at 60 min
% Remaining at 120 min
95.3
74.6
91.8
61.4
23g
23h

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3. Summary
A major focus of our optimization effort was to increase the anti-Aspergillus efficacy of our
previous series of biphenyl imidazole derivatives to formation hydrogen bond interactions with
Tyr122 of CYP51. A series of aromatic heterocyclic derivatives was designed, synthesized and
evaluated for in vitro antifungal activity. Among these compounds, the pyrimidine and isoxazole
nuclei was more suited for improving activity against Candida spp. and Cryptococcus ssp.
Specifically, isoxazole nuclei were identified for anti-Aspergillus activity. Among these, 2-F
substituted analogues 23g and 23h displayed the most remarkable in vitro activity against Candida
spp., C. neoformans, A. fumigatus and fluconazole-resistant C.alb. strains, which was superior or
comparable to those of the reference drugs fluconazole and voriconazole. Notably, the compounds
23g and 23h exhibited low inhibition profiles for various human cytochrome P450 isoforms and
excellent blood plasma stability. Further developments of compounds 23g and 23h are ongoing in
our laboratory.
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 carried out 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 Labortechnik, Flawil, Switzerland) and were uncorrected. Nuclear magnetic
resonance (1H-NMR and 13C-NMR )spectra were recorded on a Bruker 400 MHz NMR
spectrometer with TMS as an internal standard. The chemical shifts were reported in parts per
million (ppm), the coupling constants (J) were expressed in hertz (Hz). Peak multiplicities were
described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m) and broad (br).
4.1 ethyl 2,4-dioxo-4-phenylbutanoate (4)

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Na (5.2 g, 224.7 mmol) was was dissolved in 80 mL ethanol, cooled to < 0 °C using a salted
ice bath. Diethyl oxalate (18.2 g, 124.8 mmol) and acetophenone (6.0 g, 49.9 mmol) were added
to a stirred solution of NaOEt, and the resulting mixture was stirred for 8 h at ambient temperature.
After confirming that the reaction was complete by using TLC analysis, the reaction was
quenched with aqueous 3N HCl and the ethanol was removed under reduced pressure. The mixture
was extracted with EtOAc, and then with brine. The organic phase was dried over Na2SO4
overnight and the solvent was removed in vacuo to give the target product 4.
4.2 ethyl 5-phenylisoxazole-3-carboxylate (2a)
Hydroxylamine hydrochloride (1.26 g, 18.2 mmol)was added to a solution of ethyl 2,
4-dioxo-4-phenylbutanoate (2.00 g, 9.1 mmol) in EtOH (40 mL) at ambient temperature. The
reaction was refluxed for 6 h and concentrated under reduced pressure. The reaction mixture was
extracted with EtOAc followed by extraction with brine. The organic phase was dried over
Na2SO4 overnight and the solvent was removed in vacuo. The crude product was purified by silica
gel column chromatography to yield the target product 2a.1H NMR (600 MHz, DMSO-d6) δ 7.98
(dd, J = 7.5, 1.9 Hz, 2H), 7.61 – 7.55 (m, 3H), 7.53 (s, 1H), 4.41 (q, J = 7.1 Hz, 2H), 1.35 (t, J =
7.1 Hz, 3H).
4.3 ethyl 2-phenyloxazole-4-carboxylate(2b)
Ethyl bromopyruvate (3.86 g, 19.8 mmol) was added to a solution of benzamide 5 (2.00 g,
16.5 mmol) in ethanol at ambient temperature. The solution was refluxed for 6 h. After confirming
the reaction was complete by using TLC analysis, the solution was cooled to room temperature.
The solvent was removed under reduced pressure, and the residue was extracted with ethyl acetate
and washed with brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The
crude product was purified by silica gel column chromatography to yield the target product 2b.
4.4 benzothioamide(7)
A solution of benzamide 5 (5.00 g, 41.3 mmol) and Lawesson’s reagent (8.35 g, 20.6 mmol)
in toluene (60 mL) was stirred at 70 °C for 3 h. After confirming that the reaction was complete by
using TLC analysis, the crude mixture was concentrated under reduced pressure. The resulting
residue was subjected to flash chromatography purification to give the titled compound 7 as a red
solid.
4.5 ethyl 2-phenylthiazole-4-carboxylate(2c)

ACCEPTED MANUSCRIPT
Ethyl bromopyruvate (3.41 g, 17.5 mmol) was added to a solution of benzothioamide 7 (2.00
g, 14.6 mmol) in ethanol at ambient temperature. The solution was refluxed for 6 h. After
confirming that the reaction was complete by using TLC analysis, the solution was cooled to room
temperature. The solvent was removed under reduced pressure, and the residue was extracted with
ethyl acetate and washed with brine. The organic layer was dried over anhydrous Na2SO4 and
concentrated. The crude product was purified using silica gel column chromatography to yield the
target product 2c.
4.6 ethyl 4-phenylthiazole-2-carboxylate（2d）
A solution of ethyl thiooxamate (2.00 g, 10.1 mmol) and 2-Bromoacetophenone (1.29 g, 11.1
mmol) in 60 mL ethanol was heated to reflux for 8 h. After confirming that the reaction was
complete by using TLC analysis, the solution was cooled to room temperature. The solvent was
removed under reduced pressure, and the residue was extracted with ethyl acetate and washed
with brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude
product was purified by silica gel column chromatography to yield the target product 2d as a white
solid.
4.7 ethyl 4-methyl-2-phenylthiazole-5-carboxylate (2e)
A solution of benzothioamide 7 (2.0 g, 14.6 mmol) and ethyl 2-chloroacetoacetate (2.6 g,
16.1 mmol) in 50 mL ethanol was heated to reflux for 10 h. After confirming that the reaction was
complete by using TLC analysis, the solvent was removed under reduced pressure, and the residue
was extracted with ethyl acetate and washed with brine. The organic layer was dried over
anhydrous Na2SO4 and concentrated. The crude product was purified by silica gel column
chromatography to yield the target product 2e as a white solid.
4.8 ethyl 5-methyl-1-phenyl-1H-pyrazole-3-carboxylate (2f)
A solution of phenylhydrazine hydrochloride (1.64 g, 15.18 mmol) and ethyl 2,
4-dioxopentanoate (2.19 g, 12.65 mmol) in 50 mL ethanol was heated to reflux for 4 h. At the end
of the reaction, the solution was cooled to room temperature. The solvent was removed under
reduced pressure, and the residue was extracted with ethyl acetate and washed with brine. The
organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified
by silica gel column chromatography to yield the target product 2f as a white solid.
4.9 ethyl 2-formyl-3-oxopropanoate(14)

ACCEPTED MANUSCRIPT
Sodium hydride (2.00 g, 50.6 mmol, 60%) was added to a solution of ethyl formate (10.13 g,
168.7 mmol) in tetrahydrofuran while maintaining the internal temperature below 0˚C. Then, ethyl
3,3-diethoxy propionate (5.00 g, 33.75 mmol) was added dropwise to the reaction mixture and
stirred for 24 h at ambient temperature. After confirming that the reaction was complete by using
TLC analysis, the reaction was quenched with 20 mL water and the tetrahydrofuran was removed
under reduced pressure. The mixture was extracted with ethyl acetate and then brine. The organic
phase was dried over Na2SO4 overnight and the solvent was removed in vacuo to give the title
compound 14.
4.10 ethyl 2-phenylpyrimidine-5-carboxylate(2g)
K2CO3 (2.1 g, 15.4 mmol) was added to a mixture of benzamidine hydrochloride hydrate
(1.55 g, 10.0 mmol) and 14 (1.0 g, 7.7 mmol) in DMF at ambient temperature. The the solution
was heated at 100°C for 4 h. After confirming that the reaction was complete by using TLC
analysis, the solution was cooled to room temperature and water (15 mL) is added. The reaction
mixture was extracted with EtOAc and brine. The organic phase was dried over Na2SO4 overnight
and the solvent was removed under vacuum. The crude product was purified by silica gel column
chromatography to give the target product 2g.1H NMR (600 MHz, DMSO-d6) δ 9.31 (s, 2H), 8.49
– 8.44 (m, 2H), 7.59 (ddd, J = 15.9, 7.7, 3.6 Hz, 3H), 4.40 (q, J = 7.1 Hz, 2H), 1.37 (t, J = 7.1 Hz,
3H).
4.11 General procedure for the synthesis of compounds (2h,2i and 1j)
Under an argon atmosphere, compound (16, 17, 18) (1 equiv.), boronic acid (1.2 equiv.) and
Pd[P(C6H5)3]4(1 mol%) were dissolved in a mixed solution of dioxane/H2O (10:1). K2CO3 (2
equiv.) was added and the mixture was heated under reflux for 6 h. The reaction mixture was
cooled to room temperature and the dioxane was removed by rotary evaporation. H2O was 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.
4.11.1 methyl 5-phenylfuran-2-carboxylate (2h)
Light white solid; yield: 71.7%; 1H NMR (600 MHz, DMSO-d6) δ 7.85 – 7.80 (m, 2H), 7.50
(dd, J = 10.6, 4.8 Hz, 2H), 7.42 (dd, J = 10.6, 5.5 Hz, 2H), 7.19 (d, J = 3.6 Hz, 1H), 3.85 (s, 3H).
4.11.2 6-phenylnicotinic acid(1j)

ACCEPTED MANUSCRIPT
Light white solid; yield: 67.1%; 1H NMR (600 MHz, DMSO-d6) δ 13.37 (s, 1H), 9.22 – 9.04
(m, 1H), 8.33 (dd, J = 8.3, 2.2 Hz, 1H), 8.17 (dd, J = 8.2, 1.3 Hz, 2H), 8.14 – 8.09 (m, 1H), 7.53
(tdd, J = 6.9, 4.5, 2.1 Hz, 3H).
4.12 General procedure for the synthesis of compounds (1a-i)
Sodium hydroxide (2N) was added to a solution of intermediate 2a-i (1 equiv.) in methanol at
ambient temperature. The reaction mixture was stirred for 4 h and the methanol was removed by
rotary evaporation. The resultant mixture was adjusted to pH=5-6 with 1 N HCl solution. The
precipitated white solid was collected by filtration and dried to give the carboxylic acid
intermediate (1a-i).
4.12.1 5-phenylisoxazole-3-carboxylic acid(1a)
Light white solid; yield: 91.5%; 1H NMR (600 MHz, DMSO-d6) δ 7.95 (dd, J = 7.8, 1.7 Hz,
2H), 7.58 – 7.53 (m, 3H), 7.41 (s, 1H).
4.12.2 2-phenyloxazole-4-carboxylic acid(1b)
Light white solid; yield: 95.3%; 1H NMR (600 MHz, DMSO-d6) δ 13.20 (s, 1H), 8.84 (s, 1H),
8.03 – 8.01 (m, 2H), 7.58 (dd, J = 5.1, 1.9 Hz, 3H).
4.12.3 2-phenylthiazole-4-carboxylic acid(1c)
Light white solid; yield: 94.9%; 1H NMR (600 MHz, DMSO-d6) δ 13.12 (s, 1H), 8.51 (s, 1H),
7.98 (dd, J = 6.4, 3.2 Hz, 2H), 7.54 (dd, J = 5.0, 1.9 Hz, 3H).
4.12.4 4-phenylthiazole-2-carboxylic acid(1d)
Light white solid; yield: 91.6%; 1H NMR (600 MHz, DMSO-d6) δ 13.12 (s, 1H), 8.51 (s, 1H),
7.99 – 7.97 (m, 2H), 7.54 (dd, J = 5.0, 1.9 Hz, 3H).
4.12.5 4-methyl-2-phenylthiazole-5-carboxylic acid(1e)
Light white solid; yield: 89.3%; 1H NMR (600 MHz, DMSO-d6) δ 13.39 (s, 1H), 7.98 (d, J =
6.7 Hz, 2H), 7.58 – 7.50 (m, 3H), 2.68 (s, 3H).
4.12.6 5-methyl-1-phenyl-1H-pyrazole-3-carboxylic acid(1f)
Light white solid; yield: 90.3%; 1H NMR (600 MHz, DMSO-d6) δ 12.75 (s, 1H), 7.58 – 7.55
(m, 4H), 7.52 – 7.48 (m, 1H), 6.71 (d, J = 0.6 Hz, 1H), 2.33 (s, 3H).
4.12.7 5-phenylthiophene-2-carboxylic acid(1i)

ACCEPTED MANUSCRIPT
Light white solid; yield: 91.7%; 1H NMR (600 MHz, DMSO-d6) δ 13.24 (s, 1H), 8.20 (d, J =
1.5 Hz, 1H), 8.13 (d, J = 1.6 Hz, 1H), 7.78 – 7.73 (m, 2H), 7.43 (t, J = 7.7 Hz, 2H), 7.33 (t, J = 7.4
Hz, 1H).
4.13 General procedure for the synthesis of L-serine ester (20a,20b)
Thionyl chloride (3 equiv.) was added dropwise to a solution of L-serine (1 equiv.) in alcohol
reagent (isopropanol or isobutanol) cooled to < 0 °C. The mixture was heated under reflux for 6
h. The reaction mixture was then concentrated under reduced pressure to yield a white solid.
4.14General procedure for the synthesis of compounds (21a-t)
EDCI (1.1 equiv.) and HOBt (1.1 equiv) were added to a solution of the intermediate acid
compound 1a-j (1 equiv.) in anhydrous DMF. The reaction mixture was stirred for 1h 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, and the resulting solid was filtered and dried to give the desired compound.
4.15 General procedure for the synthesis of compounds (22a-t)
CDI (2 equiv.) and imidazole (1.2 equiv.) were added to a solution of the intermediate 21a-t
(1 equiv.) in CH3CN. The solution was heated to reflux for 6 h. The reaction mixture was
extracted with EtOAc and brine. The organic phase was dried over Na2SO4 overnight and the
solvent was removed under vacuum. The crude product was purified by silica gel column
chromatography to give the target product 22a-t.
4.15.1 isopropyl (S)-3-(1H-imidazol-1-yl)-2-(5-phenylthiophene-2-carboxamido)propanoate(22a)
Light white solid; yield: 69.1%; mp: 126.1-128.9 °C.1H NMR (400 MHz, DMSO-d6) δ 9.01
(d, J = 7.9 Hz, 1H), 8.25 (d, J = 1.2 Hz, 1H), 8.13 (d, J = 1.2 Hz, 1H), 7.70 (d, J = 7.3 Hz, 2H),
7.65 (s, 1H), 7.47 (t, J = 7.7 Hz, 2H), 7.35 (t, J = 7.4 Hz, 1H), 7.23 (s, 1H), 6.86 (s, 1H), 4.95 (dt,
J = 12.5, 6.2 Hz, 1H), 4.77 (td, J = 9.1, 5.3 Hz, 1H), 4.50 (dd, J = 14.1, 5.2 Hz, 1H), 4.36 (dd, J =
14.0, 9.5 Hz, 1H), 1.24 – 1.14 (m, 6H).13C NMR (100 MHz, DMSO-d6) δ 169.02, 161.21, 141.78,
139.22, 137.83, 134.47, 129.07(2C), 128.24, 127.63, 127.49, 126.18, 125.88(2C), 119.95, 68.85,
53.76, 45.98, 21.48, 21.41. HRMS calcd for C20H22N3O3S, [M + H]+, 384.1382; found 384.1385.
4.15.2 isobutyl (S)-3-(1H-imidazol-1-yl)-2-(5-phenylthiophene-2-carboxamido)propanoate(22b)
Light white solid; yield: 65.7%; mp: 102.4-104.5 °C.1H NMR (400 MHz, DMSO-d6) δ 9.05
(d, J = 8.0 Hz, 1H), 8.25 (d, J = 15.0 Hz, 1H), 8.13 (d, J = 1.1 Hz, 1H), 7.69 (d, J = 7.4 Hz, 2H),

ACCEPTED MANUSCRIPT
7.66 (s, 1H), 7.47 (t, J = 7.6 Hz, 2H), 7.35 (t, J = 7.3 Hz, 1H), 7.23 (s, 1H), 6.86 (s, 1H), 4.83 (td,
J = 9.4, 5.0 Hz, 1H), 4.54 (dd, J = 14.1, 4.9 Hz, 1H), 4.39 (dd, J = 14.0, 9.8 Hz, 1H), 3.97 – 3.81
(m, 2H), 1.95 – 1.77 (m, 1H), 0.87 (d, J = 6.5 Hz, 6H).13C NMR (150 MHz, DMSO-d6) δ 169.41,
161.30, 141.78, 139.17, 137.66, 134.46, 129.11(2C), 127.68, 127.48, 127.30, 126.28, 125.90(2C),
120.26, 70.81, 53.57, 46.16, 27.24, 18.76(2C). HRMS calcd for C21H24N3O3S, [M + H]+, 398.1538;
found 398.1538.
4.15.3 isopropyl (S)-3-(1H-imidazol-1-yl)-2-(5-phenylfuran-2-carboxamido)propanoate(22c)
Light white solid; yield: 70.4%; mp: 131.3-133.7 °C.1H NMR (400 MHz, DMSO-d6) δ 8.93
(d, J = 8.1 Hz, 1H), 7.91 (d, J = 7.4 Hz, 2H), 7.69 (s, 1H), 7.50 (t, J = 7.6 Hz, 2H), 7.40 (t, J = 7.4
Hz, 1H), 7.24 (s, 1H), 7.21 (d, J = 3.6 Hz, 1H), 7.13 (d, J = 3.6 Hz, 1H), 6.87 (s, 1H), 4.96 (dt, J =
12.5, 6.2 Hz, 1H), 4.79 (td, J = 9.5, 5.2 Hz, 1H), 4.54 (dd, J = 14.0, 5.2 Hz, 1H), 4.41 (dd, J = 14.0,
9.7 Hz, 1H), 1.20 (dd, J = 6.2, 4.2 Hz, 6H).13C NMR (100 MHz, DMSO-d6) δ 169.06, 157.70,
154.90, 146.11, 137.76, 129.25, 128.96(2C), 128.77, 128.03, 124.39(2C), 119.97, 116.58, 107.74,
68.88, 53.16, 45.91, 21.48, 21.44. HRMS calcd for C20H22N3O4, [M + H]+, 368.1610; found
368.1608.
4.15.4 isobutyl (S)-3-(1H-imidazol-1-yl)-2-(5-phenylfuran-2-carboxamido)propanoate(22d)
Yellow oil; yield: 62.5%;1H NMR (600 MHz, DMSO-d6) δ 9.10 (d, J = 8.2 Hz, 1H), 7.92 (d,
J = 7.3 Hz, 2H), 7.67 (s, 1H), 7.49 (s, 2H), 7.40 (d, J = 7.3 Hz, 1H), 7.25 (dd, J = 16.0, 5.5 Hz,
2H), 7.13 (dd, J = 3.5, 2.2 Hz, 1H), 6.83 (s, 1H), 4.86 (dd, J = 8.5, 4.9 Hz, 1H), 4.57 – 4.54 (m,
1H), 4.50 – 4.49 (m, 1H), 3.90 (dd, J = 6.5, 1.8 Hz, 2H), 1.91 – 1.84 (m, 1H), 0.87 (dd, J = 6.7, 2.2
Hz, 6H).13C NMR (101 MHz, DMSO-d6) δ 169.96, 158.22, 155.36, 146.56, 138.17, 129.70,
129.42(2C), 129.24, 128.36, 124.84(2C), 120.46, 117.03, 108.21, 68.20, 53.47, 46.34, 27.69,
19.59, 19.19. HRMS calcd for C21H24N3O4, [M + H]+, 382.1767; found 382.1768.
4.15.5 isopropyl(S)-3-(1H-imidazol-1-yl)-2-(5-phenylisoxazole-3-carboxamido)propanoate(22e)
Light white solid; yield: 63.8%; mp: 171.4-173.3 °C.1H NMR (400 MHz, DMSO-d6) δ 9.31
(d, J = 8.1 Hz, 1H), 7.99 – 7.90 (m, 2H), 7.62 (s, 1H), 7.56 (dd, J = 5.2, 1.7 Hz, 3H), 7.36 (s, 1H),
7.21 (s, 1H), 6.85 (s, 1H), 4.96 (dt, J = 12.5, 6.2 Hz, 1H), 4.89 – 4.78 (m, 1H), 4.52 (dd, J = 14.0,
5.0 Hz, 1H), 4.41 (dd, J = 14.0, 9.7 Hz, 1H), 1.20 (dd, J = 6.0, 4.8 Hz, 6H). 13C NMR (100 MHz,
DMSO-d6) δ 170.69, 168.45, 158.86, 158.69, 137.80, 130.95, 129.34(2C), 128.34, 126.18,
125.84(2C), 119.84, 99.88, 68.94, 53.42, 45.57, 21.45, 21.42. HRMS calcd for C20H22N5O3, [M +

ACCEPTED MANUSCRIPT
H]+, 380.1723; found 380.1720.
4.15.6 isobutyl (S)-3-(1H-imidazol-1-yl)-2-(5-phenylisoxazole-3-carboxamido)propanoate(22f)
Light white solid; yield: 67.1%; mp: 107.1-109.5 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.37
(d, J = 8.3 Hz, 1H), 7.96 – 7.90 (m, 2H), 7.64 (s, 1H), 7.58 – 7.54 (m, 3H), 7.36 (s, 1H), 7.22 (s,
1H), 6.86 (s, 1H), 4.93 (td, J = 9.8, 4.8 Hz, 1H), 4.56 (dd, J = 14.0, 4.7 Hz, 1H), 4.44 (dd, J = 14.0,
10.1 Hz, 1H), 3.92 (dd, J = 6.5, 2.8 Hz, 2H), 1.88 (dp, J = 13.2, 6.6 Hz, 1H), 0.88 (d, J = 6.7 Hz,
6H).13C NMR (100 MHz, DMSO-d6) δ 171.19, 169.35, 159.31, 159.21, 138.21, 131.43,
129.81(2C), 128.49, 126.62, 126.30(2C), 120.39, 100.32, 71.28, 53.70, 46.05, 27.69, 19.24, 19.19.
HRMS calcd for C21H24N5O3, [M + H]+, 394.1879; found 394.1875.
4.15.7 isopropyl (S)-3-(1H-imidazol-1-yl)-2-(2-phenyloxazole-4-carboxamido)propanoate(22g)
Yellow oil; yield: 67.1%.1H NMR (400 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.75 (s, 1H), 8.04
(dd, J = 6.6, 3.0 Hz, 2H), 7.66 (s, 1H), 7.64 – 7.57 (m, 3H), 7.21 (s, 1H), 6.87 (s, 1H), 4.96 (dt, J =
12.5, 6.3 Hz, 1H), 4.83 (td, J = 8.7, 5.3 Hz, 1H), 4.56 – 4.44 (m, 2H), 1.20 (d, J = 6.2 Hz, 6H).13C
NMR (100 MHz, DMSO-d6) δ 168.82, 160.73, 160.12, 142.71, 137.73, 136.43, 131.33,
129.30(2C), 128.09, 126.33(2C), 126.12, 119.92, 68.92, 53.04, 45.87, 21.47, 21.44. HRMS calcd
for C19H21N4O4, [M + H]+, 369.1563; found 369.1560.
4.15.8 isobutyl (S)-3-(1H-imidazol-1-yl)-2-(2-phenyloxazole-4-carboxamido)propanoate(22h)
Yellow oil; yield: 59.4%.1H NMR (400 MHz, DMSO-d6) δ 8.82 (d, J = 8.3 Hz, 1H), 8.75 (s,
1H), 8.03 (dd, J = 4.4, 2.8 Hz, 2H), 7.65 (s, 1H), 7.60 – 7.58 (m, 3H), 7.21 (s, 1H), 6.86 (s, 1H),
4.91 (td, J = 9.0, 5.1 Hz, 1H), 4.59 – 4.47 (m, 2H), 3.91 (d, J = 6.5 Hz, 2H), 1.91 – 1.83(m, 1H),
0.88 (dd, J = 6.7, 1.0 Hz, 6H).13C NMR (100 MHz, DMSO-d6) δ 169.28, 160.75, 160.20, 142.74,
137.71, 136.45, 131.36, 129.32(2C), 128.00, 126.33(2C), 126.12, 119.94, 70.79, 52.91, 45.82,
27.24, 18.75(2C). HRMS calcd for C20H23N4O4, [M + H]+, 383.1719; found 383.1716.
4.15.9 isopropyl (S)-3-(1H-imidazol-1-yl)-2-(2-phenylthiazole-4-carboxamido)propanoate(22i)
Light white solid; yield: 58.9%; mp: 128.8-131.6 °C.1H NMR (400 MHz, DMSO-d6) δ 8.88
(d, J = 8.2 Hz, 1H), 8.34 (s, 1H), 8.06 (dd, J = 6.5, 3.0 Hz, 2H), 7.63 (s, 1H), 7.60 – 7.53 (m, 3H),
7.20 (s, 1H), 6.84 (s, 1H), 4.97 (dt, J = 12.5, 6.2 Hz, 1H), 4.85 (td, J = 8.6, 5.3 Hz, 1H), 4.59 –
4.47 (m, 2H), 1.21 (d, J = 6.3 Hz, 6H).13C NMR (100 MHz, DMSO-d6) δ 168.95, 167.40, 160.52,
149.58, 137.79, 132.34, 130.89, 129.30(2C), 128.32, 126.49(2C), 125.03, 119.86, 68.96, 53.34,
45.87, 21.48, 21.45. HRMS calcd for C19H21N4O3S, [M + H]+, 385.1334; found 385.1332.

ACCEPTED MANUSCRIPT
4.15.10 isobutyl (S)-3-(1H-imidazol-1-yl)-2-(2-phenylthiazole-4-carboxamido)propanoate(22j)
Yellow oil; yield: 63.7%.1H NMR (600 MHz, DMSO-d6) δ 9.39 (d, J = 8.6 Hz, 1H), 8.49 (s,
1H), 8.09 (d, J = 7.3 Hz, 2H), 7.63 (s, 1H), 7.51 (t, J = 7.7 Hz, 2H), 7.42 (t, J = 7.3 Hz, 1H), 7.22
(d, J = 11.0 Hz, 1H), 6.83 (d, J = 4.0 Hz, 1H), 5.00 – 4.85 (m, 1H), 4.63 – 4.49 (m, 2H), 3.92 (d, J
= 6.3 Hz, 2H), 1.88 (m, 1H), 0.82 (d, J = 6.7 Hz, 6H).13C NMR (100 MHz, DMSO-d6) δ 169.94,
167.45, 160.56, 149.52, 137.78, 132.35, 130.93, 129.32(2C), 128.21, 126.53(2C), 125.16, 119.93,
67.76, 52.45, 45.92, 27.25, 19.15, 18.76. HRMS calcd for C20H23N4O3S, [M + H]+, 399.1491;
found 399.1491.
4.15.11 isopropyl (S)-3-(1H-imidazol-1-yl)-2-(4-phenylthiazole-2-carboxamido)propanoate(22k)
Light white solid; yield: 65.2%; mp: 130.7-133.7 °C.1H NMR (400 MHz, DMSO-d6) δ 9.29
(d, J = 8.3 Hz, 1H), 8.46 (s, 1H), 8.08 (d, J = 7.4 Hz, 2H), 7.66 (s, 1H), 7.51 (t, J = 7.6 Hz, 2H),
7.42 (t, J = 7.4 Hz, 1H), 7.22 (s, 1H), 6.85 (s, 1H), 5.01 – 4.95 (m, 1H), 4.86 (td, J = 8.9, 5.2 Hz,
1H), 4.58 (dd, J = 14.0, 5.1 Hz, 1H), 4.50 (dd, J = 14.0, 9.5 Hz, 1H), 1.21 (d, J = 6.2 Hz, 6H).13C
NMR (100 MHz, DMSO-d6) δ 168.55, 162.19, 159.32, 155.42, 137.80, 133.33, 128.87(2C),
128.70, 128.25, 126.31(2C), 120.08, 119.89, 69.08, 53.57, 45.69, 21.44(2C). HRMS calcd for
C19H21N4O3S, [M + H]+, 385.1334; found 385.1334.
4.15.12 isobutyl (S)-3-(1H-imidazol-1-yl)-2-(4-phenylthiazole-2-carboxamido)propanoate(22l)
Yellow oil; yield: 58.7%.1H NMR (400 MHz, DMSO) δ 8.95 (t, J = 9.0 Hz, 1H), 8.33 (d, J =
4.9 Hz, 1H), 8.10 – 8.03 (m, 2H), 7.63 (s, 1H), 7.58 – 7.50 (m, 3H), 7.20 (d, J = 6.0 Hz, 1H), 6.84
(s, 1H), 4.97 – 4.94 (m, 1H), 4.61 – 4.50 (m, 2H), 3.92 (d, J = 6.5 Hz, 2H), 1.97 – 1.80 (m, 1H),
0.82 (d, J = 6.7 Hz, 6H).13C NMR (100 MHz, DMSO-d6) δ 169.44, 162.63, 159.87, 155.89,
138.19, 133.79, 129.34(2C), 129.18, 128.35, 126.77(2C), 120.57, 120.47, 71.35, 53.88, 46.18,
27.68, 19.18(2C). HRMS calcd for C20H23N4O3S, [M + H]+, 399.1491; found 399.1489.
4.15.13 isopropyl(S)-3-(1H-imidazol-1-yl)-2-(4-methyl-2-phenylthiazole-5-carboxamido)propanoa
te(22m)
Light white solid; yield: 66.9%; mp: 149.4-157.4 °C.1H NMR (400 MHz, DMSO-d6) δ 8.81
(d, J = 7.7 Hz, 1H), 7.95 (dd, J = 6.5, 3.0 Hz, 2H), 7.64 (s, 1H), 7.57 – 7.46 (m, 3H), 7.22 (s, 1H),
6.89 (s, 1H), 5.00 - 4.92 (m, 1H), 4.79 – 4.66 (m, 1H), 4.50 (dd, J = 14.0, 5.0 Hz, 1H), 4.37 (dd, J
= 14.0, 9.8 Hz, 1H), 2.52 (s, 3H), 1.21 (t, J = 5.7 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ
168.75, 166.41, 161.34, 155.54, 137.84, 132.30, 131.02, 129.39(2C), 128.36, 126.31(2C), 125.25,

ACCEPTED MANUSCRIPT
119.86, 68.79, 54.00, 45.59, 21.49, 21.46, 16.90. HRMS calcd for C20H23N4O3S, [M + H]+,
399.1491; found 399.1490.
4.15.14 isobutyl(S)-3-(1H-imidazol-1-yl)-2-(4-methyl-2-phenylthiazole-5-carboxamido)propanoat
e(22n)
Light white solid; yield: 63.9%; mp: 83.7-85.9 °C.1H NMR (400 MHz, DMSO-d6) δ 8.86 (d,
J = 7.8 Hz, 1H), 7.94 (dd, J = 6.4, 3.0 Hz, 2H), 7.64 (s, 1H), 7.57 – 7.49 (m, 3H), 7.23 (s, 1H),
6.88 (s, 1H), 4.80 (td, J = 9.9, 4.9 Hz, 1H), 4.53 (dd, J = 14.0, 4.8 Hz, 1H), 4.41 (dd, J = 14.0, 10.1
Hz, 1H), 3.98 – 3.85 (m, 2H), 2.52 (s, 3H), 1.94 – 1.84 (m, 1H), 0.89 (d, J = 6.7 Hz, 6H).13C NMR
(100 MHz, DMSO-d6) δ 169.25, 166.44, 161.38, 155.73, 137.83, 132.29, 131.05, 129.41(2C),
128.23, 126.32(2C), 125.14, 119.90, 70.77, 53.88, 45.56, 27.27, 18.78(2C), 16.96. HRMS calcd
for C21H25N4O3S, [M + H]+, 413.1647; found 413.1644.
4.15.15 isopropyl(S)-3-(1H-imidazol-1-yl)-2-(5-methyl-1-phenyl-1H-pyrazole-3-carboxamido)pro
panoate(22o)
Yellow oil; yield: 51.5%.1H NMR (400 MHz, DMSO-d6) δ 8.58 (d, J = 8.2 Hz, 1H), 7.60 –
7.55 (m, 5H), 7.53 – 7.46 (m, 1H), 7.15 (s, 1H), 6.83 (s, 1H), 6.64 (s, 1H), 4.94 (dt, J = 12.5, 6.2
Hz, 1H), 4.78 (td, J = 8.7, 5.2 Hz, 1H), 4.51 – 4.39 (m, 2H), 2.32 (s, 3H), 1.19 (dd, J = 6.2, 3.3 Hz,
6H).13C NMR (100 MHz, DMSO-d6) δ 169.09, 161.48, 145.70, 140.95, 138.87, 137.76,
129.30(2C), 128.47, 128.27, 125.07(2C), 119.83, 107.16, 68.77, 53.05, 45.88, 21.49, 21.45, 12.00.
HRMS calcd for C20H23N5O3, [M + Na]+,404.1699; found 404.1688.
4.15.16 isobutyl(S)-3-(1H-imidazol-1-yl)-2-(5-methyl-1-phenyl-1H-pyrazole-3-carboxamido)prop
anoate(22p)
Yellow oil; yield: 54.9%.1H NMR (600 MHz, DMSO-d6) δ 8.70 (d, J = 8.4 Hz, 1H), 7.62 –
7.55 (m, 5H), 7.54 – 7.45 (m, 1H), 7.17 (s, 1H), 6.82 (s, 1H), 6.65 (d, J = 0.7 Hz, 1H), 4.86 (td, J
= 9.1, 5.0 Hz, 1H), 4.52 – 4.46 (m, 2H), 3.93 – 3.84 (m, 2H), 2.32 (s, 3H), 1.86 (dt, J = 13.3, 6.7
Hz, 1H), 0.87 (dd, J = 6.7, 2.1 Hz, 6H).13C NMR (100 MHz, DMSO-d6) δ 169.56, 161.56, 145.72,
140.93, 138.87, 137.74, 129.29(2C), 128.46, 128.22, 125.06(2C), 119.84, 107.15, 67.75, 52.93,
45.80, 27.25, 19.14, 18.77, 12.00. HRMS calcd for C21H26N5O3, [M + H]+, 396.2036; found
396.2027.
4.15.17 isopropyl(S)-3-(1H-imidazol-1-yl)-2-(2-phenylpyrimidine-5-carboxamido)propanoate(22q
)

ACCEPTED MANUSCRIPT
Light white solid; yield: 71.2%; mp: 148.2-150.9 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.34
(d, J = 7.8 Hz, 1H), 9.19 (s, 2H), 8.45 (dd, J = 8.1, 1.5 Hz, 2H), 7.68 (s, 1H), 7.58 (ddd, J = 16.1,
7.7, 3.6 Hz, 3H), 7.25 (s, 1H), 6.88 (s, 1H), 5.02 – 4.91 (m, 1H), 4.84 (ddd, J = 9.2, 8.0, 5.2 Hz,
1H), 4.53 (dd, J = 14.2, 5.2 Hz, 1H), 4.40 (dd, J = 14.2, 9.3 Hz, 1H), 1.21 (dd, J = 8.6, 6.3 Hz,
6H).13C NMR (100 MHz, DMSO-d6) δ 168.62, 165.11, 163.51, 156.74(2C), 137.73, 136.27,
131.76, 128.94(2C), 128.29(2C), 127.18, 124.75, 120.43, 69.02, 53.68, 46.23, 21.48, 21.44.
HRMS calcd for C19H21N4O4, [M + H]+, 369.1563; found 369.1565.
4.15.18 isobutyl(S)-3-(1H-imidazol-1-yl)-2-(2-phenylpyrimidine-5-carboxamido)propanoate(22s)
Yellow oil; yield: 61.1%. 1H NMR (600 MHz, DMSO-d6) δ 9.92 (s, 1H), 9.33 (s, 2H), 8.46 (d,
J = 6.9 Hz, 2H), 7.77 (s, 1H), 7.61 – 7.56 (m, 3H), 7.32 (s, 1H), 6.85 (s, 1H), 4.91 (d, J = 6.7 Hz,
1H), 4.57 (t, J = 6.6 Hz, 2H), 3.91 (qd, J = 10.5, 6.6 Hz, 2H), 1.87 (td, J = 13.3, 6.6 Hz, 1H), 0.87
(d, J = 6.7 Hz, 6H).13C NMR (100 MHz, DMSO-d6) δ 169.13, 165.13, 163.49, 156.68(2C), 137.82,
136.24, 131.74, 128.90(2C), 128.28(2C), 127.88, 124.71, 120.11, 70.84, 53.69, 45.93, 27.21,
18.73. HRMS calcd for C20H22N4O4, [M + Na]+, 405.1539; found 405.1536.
4.15.19 isopropyl (S)-3-(1H-imidazol-1-yl)-2-(6-phenylnicotinamido)propanoate(22t)
Light white solid; yield: 64.9%; mp: 60.9-62.8 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.32 (d,
J = 7.8 Hz, 1H), 9.04 (d, J = 1.8 Hz, 1H), 8.26 (dd, J = 8.3, 2.2 Hz, 1H), 8.20 – 8.14 (m, 2H), 8.11
(d, J = 8.3 Hz, 1H), 7.73 (s, 1H), 7.53 (t, J = 7.3 Hz, 2H), 7.50 (d, J = 7.1 Hz, 1H), 7.28 (s, 1H),
6.88 (s, 1H), 4.96 (dt, J = 12.5, 6.2 Hz, 1H), 4.84 – 4.76 (m, 1H), 4.54 (dd, J = 14.1, 4.6 Hz, 1H),
4.45 (dd, J = 14.0, 9.8 Hz, 1H), 1.20 (dd, J = 8.0, 6.4 Hz, 6H).13C NMR (150 MHz, DMSO-d6) δ
168.93, 165.03, 158.45, 148.67, 137.71, 136.26, 134.09, 129.86, 128.91(2C), 127.43, 127.34,
126.96(3C), 119.78, 68.77, 53.87, 48.58, 21.50, 21.46. HRMS calcd for C21H23N4O3, [M + H]+,
379.1770; found 379.1767.
4.15.20 isopropyl(S)-3-(1H-imidazol-1-yl)-2-(5-(p-tolyl)isoxazole-3-carboxamido)propanoate(23a
)
Light white solid; yield: 72.7%; mp: 168.4-170.5 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.28
(d, J = 8.1 Hz, 1H), 7.83 (d, J = 8.1 Hz, 2H), 7.61 (s, 1H), 7.37 (d, J = 8.1 Hz, 2H), 7.28 (s, 1H),
7.20 (s, 1H), 6.84 (s, 1H), 4.96 (dt, J = 12.5, 6.2 Hz, 1H), 4.83 (td, J = 9.4, 5.0 Hz, 1H), 4.52 (dd, J
= 14.0, 5.0 Hz, 1H), 4.40 (dd, J = 14.0, 9.7 Hz, 1H), 2.38 (s, 3H), 1.20 (d, J = 1.4 Hz, 6H).13C
NMR (100 MHz, DMSO-d6) δ 170.85, 168.46, 158.81, 158.74, 140.94, 137.80, 129.87(2C),

ACCEPTED MANUSCRIPT
128.37, 125.79(2C), 123.53, 119.83, 99.23, 68.93, 53.41, 45.57, 21.45, 21.42, 21.04. HRMS calcd
for C20H22N4O4, [M + Na]+, 405.1539; found 409.1537.
4.15.21 isobutyl(S)-3-(1H-imidazol-1-yl)-2-(5-(p-tolyl)isoxazole-3-carboxamido)propanoate(23b)
Light white solid; yield: 69.5%; mp: 112.7-114.9 °C. 1H NMR (400 MHz, DMSO) δ 9.34 (d,
J = 8.3 Hz, 1H), 7.83 (d, J = 8.1 Hz, 2H), 7.63 (s, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.28 (s, 1H), 7.21
(s, 1H), 6.85 (s, 1H), 4.92 (td, J = 9.8, 4.8 Hz, 1H), 4.55 (dd, J = 14.0, 4.7 Hz, 1H), 4.43 (dd, J =
14.0, 10.0 Hz, 1H), 3.97 – 3.86 (m, 2H), 2.38 (s, 3H), 1.88 (dp, J = 13.3, 6.6 Hz, 1H), 0.88 (d, J =
6.7 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ 171.34, 169.36, 159.25, 141.42, 138.21,
130.34(2C), 128.57, 126.25(2C), 123.98, 120.36, 99.66, 71.27, 53.70, 46.02, 27.70, 21.50, 19.24,
19.19. HRMS calcd for C21H25N4O4, [M + H]+, 397.1876; found 397.1875.
4.15.22 isopropyl(S)-2-(5-(4-fluorophenyl)isoxazole-3-carboxamido)-3-(1H-imidazol-1-yl)propan
oate(23c)
Light white solid; yield: 64.9%; mp:196.2-199.3 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.31
(d, J = 8.1 Hz, 1H), 8.01 (dd, J = 8.7, 5.4 Hz, 2H), 7.61 (s, 1H), 7.42 (t, J = 8.8 Hz, 2H), 7.36 (s,
1H), 7.20 (s, 1H), 6.85 (s, 1H), 4.96 (dt, J = 12.5, 6.2 Hz, 1H), 4.83 (td, J = 9.1, 5.1 Hz, 1H), 4.52
(dd, J = 14.1, 5.0 Hz, 1H), 4.40 (dd, J = 14.0, 9.7 Hz, 1H), 1.20 (dd, J = 6.0, 4.6 Hz, 6H).13C NMR
(100 MHz, DMSO-d6) δ 169.78, 168.44, 164.67(0.5C), 162.19(0.5C), 158.91, 158.64, 137.81,
128.50, 128.41, 128.38, 122.93(0.5C), 122.91(0.5C), 119.82, 116.61, 116.39, 99.85, 68.94, 53.42,
45.56, 21.44, 21.41. HRMS calcd for C19H19FN4O4, [M + H]+, 409.1288; found 409.1287.
4.15.23 isobutyl(S)-2-(5-(4-fluorophenyl)isoxazole-3-carboxamido)-3-(1H-imidazol-1-yl)propano
ate(23d)
Light white solid; yield: 72.4%; mp: 136.2-138.0 °C. 1H NMR (400 MHz, DMSO) δ 9.36 (d,
J = 8.3 Hz, 1H), 8.02 (dd, J = 8.8, 5.4 Hz, 2H), 7.63 (s, 1H), 7.43 (d, J = 8.9 Hz, 2H), 7.36 (s, 1H),
7.21 (s, 1H), 6.85 (s, 1H), 4.93 (td, J = 9.9, 4.8 Hz, 1H), 4.56 (dd, J = 14.0, 4.7 Hz, 1H), 4.44 (dd,
J = 14.0, 10.1 Hz, 1H), 3.92 (dd, J = 6.5, 2.8 Hz, 2H), 1.87 (td, J = 13.3, 6.6 Hz, 1H), 0.88 (d, J =
6.7 Hz, 6H).13C NMR (100 MHz, DMSO-d6) δ 169.83, 168.91, 164.69(0.5C), 162.22(0.5C),
158.92, 158.71, 137.78, 128.53, 128.44, 128.20, 122.93(0.5C), 122.90(0.5C), 119.89, 116.64,
116.42, 99.84, 70.83, 53.27, 45.55, 27.25, 18.78, 18.74. HRMS calcd for C20H22FN4O4, [M + H]+,
401.1625; found 401.1619.
4.15.24 isopropyl(S)-2-(5-(4-bromophenyl)isoxazole-3-carboxamido)-3-(1H-imidazol-1-yl)propan

ACCEPTED MANUSCRIPT
oate(23e)
Light white solid; yield: 67.3%; mp: 135.0-136.8 °C. 1H NMR (400 MHz, DMSO) δ 9.33 (d,
J = 8.1 Hz, 1H), 7.90 (d, J = 8.6 Hz, 2H), 7.78 (d, J = 8.6 Hz, 2H), 7.63 (s, 1H), 7.43 (s, 1H), 7.21
(s, 1H), 6.85 (s, 1H), 4.96 (dt, J = 12.5, 6.2 Hz, 1H), 4.88 – 4.79 (m, 1H), 4.52 (dd, J = 14.0, 5.0
Hz, 1H), 4.41 (dd, J = 14.0, 9.7 Hz, 1H), 1.20 (dd, J = 6.1, 4.3 Hz, 6H). 13C NMR (100 MHz,
DMSO-d6) δ 169.66, 168.41, 158.94, 158.56, 137.79, 132.36(2C), 128.30, 127.81(2C), 125.35,
124.45, 119.85, 100.51, 68.95, 53.42, 45.58, 21.44, 21.41. HRMS calcd for C19H20BrN4O4, [M +
H]+, 447.0668; found 447.0679.
4.15.25 isobutyl(S)-2-(5-(4-bromophenyl)isoxazole-3-carboxamido)-3-(1H-imidazol-1-yl)propano
ate(23f)
Light white solid; yield: 64.5%; mp: 123.7-126.6 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.39
(d, J = 8.3 Hz, 1H), 7.90 (d, J = 8.6 Hz, 2H), 7.77 (d, J = 8.5 Hz, 2H), 7.63 (s, 1H), 7.43 (s, 1H),
7.21 (s, 1H), 6.86 (s, 1H), 4.93 (td, J = 9.8, 4.7 Hz, 1H), 4.56 (dd, J = 14.0, 4.7 Hz, 1H), 4.44 (dd,
J = 14.0, 10.1 Hz, 1H), 3.97 – 3.87 (m, 2H), 1.93 – 1.83 (m, 1H), 0.88 (d, J = 6.7 Hz, 6H).13C
NMR (100 MHz, DMSO-d6) δ 169.72, 168.87, 158.95, 158.63, 137.73, 132.40(2C), 127.94(2C),
125.34, 124.49, 120.01, 100.51, 70.85, 53.24, 45.65, 27.25, 18.75. HRMS calcd for C20H22BrN4O4,
[M + H]+, 461.0824; found 461.0831.
4.15.26 isopropyl(S)-2-(5-(2-fluorophenyl)isoxazole-3-carboxamido)-3-(1H-imidazol-1-yl)propan
oate(23g)
Light white solid; yield: 59.9%; mp: 151.3-154.0 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.45
(d, J = 8.1 Hz, 1H), 8.00 (td, J = 7.7, 1.5 Hz, 1H), 7.69 – 7.60 (m, 2H), 7.46 (ddd, J = 16.1, 9.7,
4.7 Hz, 2H), 7.23 (s, 1H), 7.20 (d, J = 2.9 Hz, 1H), 6.86 (s, 1H), 4.96 (dt, J = 12.5, 6.2 Hz, 1H),
4.89 – 4.81 (m, 1H), 4.53 (dd, J = 14.1, 5.0 Hz, 1H), 4.43 (dd, J = 14.0, 9.7 Hz, 1H), 1.20 (dd, J =
6.1, 4.5 Hz, 6H).13C NMR (100 MHz, DMSO-d6) δ 168.41, 165.07, 158.77, 158.39, 157.22,
137.83, 133.15, 128.27, 127.95, 125.47, 119.88, 116.63, 114.23, 102.74, 68.96, 53.48, 45.54,
21.45, 21.41. HRMS calcd for C19H20FN4O4, [M + H]+, 387.1469; found 387.1463.
4.15.27 isobutyl(S)-2-(5-(2-fluorophenyl)isoxazole-3-carboxamido)-3-(1H-imidazol-1-yl)propano
ate(23h)
Light white solid; yield: 64.7%; mp: 85.5-87.9 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.43 (d,
J = 8.2 Hz, 1H), 8.00 (td, J = 7.8, 1.5 Hz, 1H), 7.64 (d, J = 11.0 Hz, 2H), 7.52 – 7.39 (m, 2H), 7.23

ACCEPTED MANUSCRIPT
(s, 1H), 7.14 (d, J = 2.9 Hz, 1H), 6.87 (s, 1H), 4.93 (td, J = 9.8, 4.8 Hz, 1H), 4.56 (dd, J = 14.0, 4.7
Hz, 1H), 4.44 (dd, J = 14.0, 10.1 Hz, 1H), 4.03 – 3.83 (m, 2H), 1.92 – 1.85 (m, 1H), 0.88 (d, J =
6.7 Hz, 6H).13C NMR (100 MHz, DMSO-d6) δ 168.85, 165.16, 158.75, 158.44, 157.23, 137.76,
133.27, 133.19, 128.11, 127.96, 125.44, 119.91, 116.84, 116.64, 114.21, 102.67, 70.84, 53.30,
45.55, 27.23, 18.72. HRMS calcd for C20H21FN4O4, [M + Na]+, 423.1445; found 423.1447.
4.16 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 by 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. FLC and ITR
were purchased for use as positive control drugs. All of the compounds were dissolved in DMSO
and serially diluted into the growth medium.
4.17 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).
4.18 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,
30 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 37°C for 10 min, the reaction was initiated with by additing 20 µL NADPH to a final
concentration of 10 mM. The mixture were incubated at 37°C for 10 min and the reaction was
terminated by additing 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.

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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.
References
[1] M. Victoria Castelli, E. Butassi, M. Candida Monteiro, L.A. Svetaz, F. Vicente, S.A. Zacchino, Novel
antifungal agents: a patent review (2011-present), Expert Opin. Ther. Pat., 24 (2014) 323-338.
[2] G.D. Brown, D.W. Denning, N.A.R. Gow, S.M. Levitz, M.G. Netea, T.C. White, Hidden Killers: Human
Fungal Infections, Sci. Transl. Med., 4 (2012) 1-9.
[3] D.W. Denning, W.W. Hope, Therapy for fungal diseases: opportunities and priorities, Trends
Microbiol., 18 (2010) 195-204.
[4] M. Richardson, C. Lass-Flörl, Changing epidemiology of systemic fungal infections, Clin. Microbiol.
Infect., 14 (2008) 5-24.
[5] D.W. Denning, Echinocandin antifungal drugs, Lancet, 362 (2003) 1142-1151.
[6] D. Allen, D. Wilson, R. Drew, J. Perfect, Azole antifungals: 35 years of invasive fungal infection
management, Expert review of anti-infective therapy, 13 (2015) 787.
[7] V. Moudgal, J. Sobel, Antifungals to treat Candida albicans, Expert Opin. Pharmacother., 11 (2010)
2037-2048.
[8] C. Onyewu, J.R. Blankenship, P.M. Del, J. Heitman, Ergosterol biosynthesis inhibitors become
fungicidal when combined with calcineurin inhibitors against Candida albicans, Candida glabrata, and
Candida krusei, Antimicrob. Agents Chemother., 47 (2003) 956-964.
[9] H. Hof, A new, broad-spectrum azole antifungal: posaconazole--mechanisms of action and
resistance, spectrum of activity, Mycoses, 49 (2006) 2-6.
[10] B.C. Monk, T.M. Tomasiak, M.V. Keniya, F.U. Huschmann, J.D.A. Tyndall, J.D. O'Connell, III, R.D.
Cannon, J.G. McDonald, A. Rodriguez, J.S. Finer-Moore, R.M. Stroud, Architecture of a single
membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to
a bilayer, Proc. Natl. Acad. Sci. U. S. A., 111 (2014) 3865-3870.
[11] J. Heeres, L. Meerpoel, P. Lewi, Conazoles, Molecules, 15 (2010) 4129-4188.
[12] G. Rodríguez, Duque, Castellsague, Pérez-Gutthann, Stricker, A cohort study on the risk of acute
liver injury among users of ketoconazole and other antifungal drugs, Br. J. Clin. Pharmacol., 48 (1999)
847.
[13] S. F, P. R, M. PA, M. CA, N. CC, H. R, L. D, B. TA, M. PM, In vitro activities of posaconazole,
fluconazole, itraconazole, voriconazole, and amphotericin B against a large collection of clinically
important molds and yeasts, Antimicrob. Agents Chemother., 50 (2006) 2009-2015.
[14] S. Zhao, L. Zhao, X. Zhang, C. Liu, C. Hao, H. Xie, B. Sun, D. Zhao, M. Cheng, Design, synthesis, and
structure-activity relationship studies of benzothiazole derivatives as antifungal agents, Eur. J. Med.
Chem., 123 (2016) 514-522.
[15] D. Zhao, S. Zhao, L. Zhao, X. Zhang, P. Wei, C. Liu, C. Hao, B. Sun, X. Su, M. Cheng, Discovery of
biphenyl imidazole derivatives as potent antifungal agents: Design, synthesis, and structure-activity

ACCEPTED MANUSCRIPT
relationship studies, Bioorg. Med. Chem., 25 (2016) 750-758.
[16] T.Y. Hargrove, Z. Wawrzak, D.C. Lamb, F.P. Guengerich, G.I. Lepesheva, Structure-Functional
Characterization of Cytochrome P450 Sterol 14α-Demethylase (CYP51B) from Aspergillus fumigatus
and Molecular Basis for the Development of Antifungal Drugs, J. Biol. Chem., 290 (2015) 23916.
[17] Z. Jiang, J. Gu, C. Wang, S. Wang, N. Liu, Y. Jiang, G. Dong, Y. Wang, Y. Liu, J. Yao, Design, synthesis
and antifungal activity of novel triazole derivatives containing substituted 1,2,3-triazole-piperdine side
chains, Eur. J. Med. Chem., 82 (2014) 490-497.
[18] R.-m. Liang, Y.-b. Cao, K.-h. Fan, Y. Xu, P.-h. Gao, Y.-j. Zhou, B.-d. Dai, Y.-h. Tan, S.-h. Wang, H. Tang,
H.-t. Liu, Y.-y. Jiang, 2-Amino-nonyl-6-methoxyl-tetralin muriate inhibits sterol C-14 reductase in the
ergosterol biosynthetic pathway, Acta Pharmacologica Sinica, 30 (2009) 1709-1716.
[19] A.G. Warrilow, C.M. Hull, J.E. Parker, E.P. Garvey, W.J. Hoekstra, W.R. Moore, R.J. Schotzinger, D.E.
Kelly, S.L. Kelly, The clinical candidate VT-1161 is a highly potent inhibitor of Candida albicans CYP51
but fails to bind the human enzyme, Antimicrob. Agents Chemother., 58 (2014) 7121.
[20] I.J. Sud, D.S. Feingold, Mechanisms of action of the antimycotic imidazoles, The Journal of
investigative dermatology, 76 (1981) 438-441.
[21] S.Z. Wang, Y. Wang, W. Liu, N. Liu, Y.Q. Zhang, G.Q. Dong, Y. Liu, Z.G. Li, X.M. He, Z.Y. Miao, J.Z. Yao,
J. Li, W.N. Zhang, C.Q. Sheng, Novel Carboline Derivatives as Potent Antifungal Lead Compounds:
Design, Synthesis, and Biological Evaluation, ACS Med. Chem. Lett., 5 (2014) 506-511.
[22] X. Jia, Y. Wang, Y. Jia, P. Gao, Y. Xu, L. Wang, Y. Cao, Y. Cao, L. Zhang, Y. Jiang, RTA2 is involved in
calcineurin-mediated azole resistance and sphingoid long-chain base release in Candida albicans, Cell.
Mol. Life Sci., 66 (2009) 122-134.
[23] K.S. Barker, S. Crisp, N. Wiederhold, R.E. Lewis, B. Bareither, J. Eckstein, R. Barbuch, M. Bard, P.D.
Rogers, Genome-wide expression profiling reveals genes associated with amphotericin B and
fluconazole resistance in experimentally induced antifungal resistant isolates of Candida albicans, J.
Antimicrob. Chemother., 54 (2004) 376-385.
[24] D.W. Bates, D.T. Yu, Clinical impact of drug-drug interactions with systemic azole antifungals, Drugs
of Today, 39 (2003) 801-813.
[25] T. Niwa, T. Shiraga, A. Takagi, Effect of antifungal drugs on cytochrome P450 (CYP) 2C9, CYP2C19,
and CYP3A4 activities in human liver microsomes, Biol. Pharm. Bull., 28 (2005) 1805-1808.

ACCEPTED MANUSCRIPT
Highlights
ò 27 new compounds with aromatic heterocyclic scaffolds were designed and synthesised.
ò
The isoxazole scaffolds were more suited for improving activity against Aspergillus spp.
ò Compounds 23g and 23h showed better antifungal activity than fluconazole.
ò Compound 23g reduced the content of ergosterol in a dose-dependent manner.
ò Compounds 23g exhibited low inhibition profiles for human cytochrome P450 isoforms.