Design, synthesis and evaluation of benzoheterocycle analogues as potent antifungal agents targeting CYP51

Article abstract of DOI:10.1016/j.bmc.2018.04.054

To further enhance the anti-Aspergillus efficacy of our previously discovered antifungal lead compound 1, a series of benzoheterocycle analogues were designed, synthesized and evaluated for their in vitro antifungal activity. The most promising compounds 13s and 14a exhibited excellent antifungal activity against C. albicans, C. neoformans, A. fumigatus and fluconazole-resistant C. albicans strains, that was superior or comparable to those of the reference drugs fluconazole and voriconazole. GC–MS analyses suggested that the novel compound 13s might have a similar mechanism to fluconazole by inhibiting fungal lanosterol 14α-demethylase (CYP51). Furthermore, compounds 13s and 14a exhibited low inhibition profiles for various human cytochrome P450 isoforms as well as excellent blood plasma stability.

Full text of DOI:10.1016/j.bmc.2018.04.054

Accepted Manuscript
Design, synthesis and evaluation of benzoheterocycleanalogues as potent anti-
fungal agents targeting CYP51
Shizhen Zhao, Peng Wei, Mengya Wu, Xiangqian Zhang, Liyu Zhao, Xiaolin
Jiang, Chenzhou Hao, Xin Su, Dongmei Zhao, Maosheng Cheng
PII:
S0968-0896(18)30132-9
https://doi.org/10.1016/j.bmc.2018.04.054
BMC 14337
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
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22 January 2018
25 April 2018
26 April 2018
Please cite this article as: Zhao, S., Wei, P., Wu, M., Zhang, X., Zhao, L., Jiang, X., Hao, C., Su, X., Zhao, D.,
Cheng, M., Design, synthesis and evaluation of benzoheterocycleanalogues as potent antifungal agents targeting
CYP51, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.04.054
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Design, synthesis and evaluation of benzoheterocycle analogues as
potent antifungal agents targeting CYP51
Shizhen Zhaoa, Peng Weia, Mengya Wua, Xiangqian Zhanga, Liyu Zhaoa, Xiaolin Jianga,
Chenzhou Haoa, Xin Sub, 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
compound 1, a series of benzoheterocycle analogues were designed, synthesized and evaluated for
their in vitro antifungal activity. The most promising compounds 13s and 14a exhibited excellent
antifungal activity against C.albicans, C.neoformans, A.fumigatus and fluconazole-resistant
C.albicans strains, that was superior or comparable to those of the reference drugs fluconazole and
voriconazole. GC-MS analyses suggested that the novel compound 13s might have a similar
mechanism to fluconazole by inhibiting fungal lanosterol 14α-demethylase (CYP51). Furthermore,
compounds 13s and 14a exhibited low inhibition profiles for various human cytochrome P450
isoforms as well as excellent blood plasma stability.
Keywords:
Antifungal activity, Azole antifungals, CYP51, Structure-activity relationship
1. Introduction
Fungal infections have been increasing dramatically and are currently estimated to directly
affect approximately 1.2 billion people globally1. The incidence of invasive fungal infections (IFIs)
and the emergence of resistant fungal pathogens have increased markedly, leading to high
morbidity and mortality in immunocompromised patients, such as patients receiving organ

transplants, patients undergoing anticancer chemotherapy and patients with AIDS2, 3. Clinically,
the three fungal genera Aspergillus, Candida, and Cryptococcus account for most fungal
infections4. The common antifungal agents currently used in the clinic can be divided into four
different classes based on their mode of action: polyenes (e.g., amphotericin B and nystatin)5,
echinocandins (e.g., caspofungin and micafungin)6, azoles(e.g., fluconazole, voriconazole and
itraconazole)7, and antimetabolites (e.g., 5-fluorocytosine)8. Among these agents, azoles are most
widely used as first-line antifungal therapy.
Figure 1. Chemical structures of azole antifungal agents and lead compound.
Azole antifungal agents inhibit fungal lanosterol 14α-demethylase (CYP51), which is
involved in the biosynthesis of ergosterol, a significant cellular membrane component9. The
inhibition of CYP51 would cause a reduction of the endogenous concentrations of ergosterol and
an accumulation of lanosterol and other 14-methyl sterols, thus inhibiting the growth of fungal
cells10. Azole antifungal drugs, such as fluconazole, itraconazole, voriconazole and posaconazole,
have had a significant impact on the treatment of systemic fungal infections11, 12. However, many
of the marketed azole drugs are limited in practical applications, due to their drug resistance,
narrow antifungal spectrum, and low bioavailability13, 14. Therefore, there is still an urgent need
for developing novel azole antifungal agents with potent activity, broad spectrum, low toxicity,
and low resistance.
In our previous studies, a new series of benzothiazole derivatives were designed, synthesized
and evaluated for their in vitro antifungal activity15, 16. Most of the compounds showed excellent

antifungal activity against Candida albicans and Cryptococcus neoformans, with MIC values in
the range of 0.25 µg/mL to 2 µg/mL. Unfortunately, almost all of these target compounds were
inactive against Aspergillus fumigatus. Therefore, this prompted us to continue studying the
structural modification of our potent original compounds in search of novel compounds with
improved anti-Aspergillus fumigatus efficacy.
The structure of the voriconazole-A. fumigatus CYP51B complex (PDB ID:4UYM) (Figure
2A) suggests that hydrogen bonds formed between the 5-fluoropyrimidine ring of voriconazole
and A. fumigatus CYP51 Tyr122. This could explain why voriconazole has much higher antifungal
potency against Aspergillus fumigatus than fluconazole does 17.
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 potent and broad antifungal spectrum of imidazole derivatives,
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, the benzothiazole ring
was replaced by benzoheterocycles to facilitate the formation of hydrogen bond interactions with
Tyr122 of CYP51. This might result in the development of more potent compounds with higher
antifungal activities and a broader antifungal spectrum, especially with improved anti-Aspergillus
efficacy.

Figure 3. Our strategy to facilitate the formation of hydrogen bond interactions with CYP51B Tyr122 of A.
fumigatus by replacing the benzothiazole ring with 12 benzoheterocycles.
2. Results and discussion
2.1 Chemistry
The synthetic routes of the key intermediates 1a-f are shown in Scheme 1. The
benzothiophene 2a was obtained from the intermolecular cyclization between 2-nitrobenzaldehyde
and methyl 2-mercaptoacetate. The salicylaldehyde was treated with methyl bromoacetate in the
presence of K2CO3, and cyclization afforded the benzofuran 2b. 1-tetralone 6 was treated with
diethyl oxalate in the presence of LiHMDS by a Claisen condensation to afford intermediate 7,
which was then subjected to a ring-closure reaction with hydroxylamine hydrochloride to form
isoxazole 2c. The intermediate 2d was obtained by DDQ dehydrogenation reaction from 2c. The
4-amino-3-hydroxybenzoic acid was condensed with the substituted methyl orthoformate 9 to
form the benzo[d]oxazole acids 1e-f. Finally, intermediates 2a-d were saponified with 2 N NaOH
to obtain the key intermediates 1a-d.

Scheme 1. Synthesis of intermediates 1a-f. Reagents and conditions: (a) K2CO3, DMF; (b) NaOH, MeOH/H2O, r.t.,
7 h; (c) K2CO3, DMF, 80 °C, 7 h; (d) Diethyl oxalate, LiHMDS; (e) Hydroxylamine hydrochloride, EtOH, reflux,
2 h; (f) DDQ, 1,4-dioxane; (g) 80 °C, 2 h.
The syntheses of target compounds 13a-v were carried out according to our previously
reported procedure, as shown in Scheme 2. L-serine 10 was dissolved in alcohol (isopropanol or
isobutanol) and refluxed with SOCl2 to afford the serine esters 11a-b. The serine esters 11a-b were
then treated with intermediates 1a-j in the presence of a condensing reagent to give compounds
12a-u. Finally, the target compounds 13a-u were successfully obtained by introducing the
imidazole group using CDI/imidazole.

Scheme 2. Reagents and conditions: (a) isopropanol or isobutanol, 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
The in vitro minimum inhibitory concentration (MIC) of all target compounds was
determined using the method recommended by the National Committee for Clinical Laboratory
Standards (NCCLS) and the serial dilution method in 96-well microtest plates18, 19. All of the
target compounds were evaluated for their antifungal activity against five important fungal
pathogens. Fluconazole (FLC) and itraconazole (ITR) were used as reference drugs. The in vitro
antifungal activity results are summarized in Table 1.
As shown in Table 1, the introduction of different benzoheterocycle rings had a notable
influence on the antifungal activity. Many of these modifications showed a decrease in antifungal
activity compared with the lead compound 1. Interestingly, the benzoheterocycle compounds such
as benzothiophene (13a-b), benzofuran (13c-d), N-methylindole (13j-k), and
4,5-dihydronaphtho[2,1-d]isoxazole (13s-t) exhibited moderate to good antifungal properties with
a broad spectrum of activity. Among these, 4,5-dihydronaphtho[2,1-d] isoxazole (13s-t) showed
the most potent activity against all of the tested strains, with the exception of Aspergillus
fumigatus, and was superior or comparable to those of the reference drugs FLC and ITR. When
the middle six-membered ring of 13s was changed to the seven-membered ring (13q) showed
decreased antifungal activity.

Table 1
In vitro antifungal activities of the target compounds (MIC, μg/mL)a.
Compd.
13a
13b
13c
R1
R2
C. alb.(Ⅰ)
C. alb(Ⅱ).
C. neo.
4
C.tro.
0.25
0.25
0.5
A. fum.
8
-CH(CH3)2
1
0.5
16
8
2
-CH2CH(CH3)2
-CH(CH3)2
1
1
8
32
8
4
32
32
64
64
64
64
-CH2CH(CH3)2
-CH(CH3)2
4
0.25
32
13d
13e
64
64
64
64
64
64
64
64
64
64
64
64
-CH2CH(CH3)2
-CH(CH3)2
32
13f
16
13g
13h
-CH2CH(CH3)2
16
-CH(CH3)2
32
32
8
0.5
64
13i
-CH(CH3)2
8
8
8
8
64
4
0.5
1
64
64
13j
-CH2CH(CH3)2
13k
-CH(CH3)2
64
64
64
64
64
13l
-CH(CH3)2
64
64
64
64
64
64
32
16
64
64
13m
13n
-CH2CH(CH3)2
-CH(CH3)2
-CH(CH3)2
-CH(CH3)2
64
64
16
64
64
2
64
64
64
32
64
1
64
64
64
13o
13p
13q
-CH(CH3)2
0.25
0.25
0.5
8
4
0.125
0.125
8
8
13s
13t
-CH2CH(CH3)2
0.125

1
0.125
0.5
0.5
1
2
4
1
1
>64
>64
4
-
-
FCZ
ITZ
VRC
0.125
0.125
0.5
0.25
1
0.5
0.125
0.5
2
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 (cgmcc 3.7795); C.tro., Candida
tropicalis (cgmcc 2.3739); FCZ: Fluconazole; ITZ: Itraconazole, VRC: Voriconazole.
Based on the results above, we selected a scaffold of 4,5-dihydronaphtho[2,1-d]isoxazole
core (13s-t) as our starting point for further modification. When substituents were introduced at
the 7 or 8 position of the 4,5-dihydronaphtho[2,1-d] isoxazole scaffold , the resulting compounds
(14c-g) showed decreased in vitro antifungal activity. Especially, Compound 14c with -Br groups
at the 8-position showed no antifungal activity. Notably, the 4,5-dihydronaphtho[2,1-d] isoxazole
nucleus, when replaced with a naphtho[2,1-d] isoxazole nucleus, led to improved antifungal
activity in compounds 14a-b. Of these, compound 14a showed the best antifungal activity.
Table 2
In vitro antifungal activities of the target compounds (MIC, μg/mL)a.
Compd.
R1
R2
C.
C.
alb(Ⅱ).
0.25
C. neo.
C.tro.
A. fum.
alb.(Ⅰ)
0.125
0.125
-CH(CH3)2
0.25
0.125
0.125
4
14a
14b
-CH2CH(CH3)2
0.25
0.125
16
-CH(CH3)2
64
64
64
64
64
14c
-CH(CH3)2
-CH2CH(CH3)2
-CH(CH3)2
-CH2CH(CH3)2
-
0.25
0.25
0.25
0.25
0.5
16
32
4
64
64
64
64
4
2
4
64
64
14d
14e
8
64
14f
64
1
32
1
64
14g
FCZ
-
>64

-
-
0.125
0.125
0.5
1
0.5
4
2
ITZ
VRC
0.25
0.5
0.125
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 (cgmcc 3.7795); C.tro., Candida
tropicalis (cgmcc 2.3739); FCZ: Fluconazole; ITZ: Itraconazole, VRC: Voriconazole.
2.3 In vitro antifungal activity against fluconazole-resistant strains of C. alb.
The wide use of fluconazole has greatly increased the fluconazole resistance of C. alb. strains,
which has become a major clinical problem for treating fungal infections. Therefore, there is an
urgent need to find a new type of inhibitor that is effective against fluconazole-resistant strains of
C. alb. Based on the results of the in vitro antifungal activity assays, the most potent
compounds 13s-t and 14a-b were further evaluated against fluconazole-resistant strains of C. alb.
(strain 100 and strain 103). As shown in Table 3, compounds 13s-t and 14a-b displayed strong
antifungal activity against strain 100 and strain 103, with MIC values in the range from 0.5 to 2
µg/mL.
Table 3
In vitro antifungal activities of the target compounds (MIC, μg/mL)a.
C. alb.
Compd.
Strain100
Strain103
2
1
0.5
0.5
1
13s
13t
2
14a
14b
FCZ
2
2
>64
>64
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 GC-MS analysis of sterol composition
To confirm the antifungal mechanism of compound 13s, we analysed the sterol composition
of C. albicans (ATCC SC5314) cells by using GC-MS methods. GC-MS methods have been
successfully used to reflect the effect on C. albicans membrane and elucidate the mechanism of
antifungal agent16, 20-22. FLC was used as a reference drug and cholesterol was added as an internal

standard. The GC-MS analysis results are shown in Table 4 and Figure 4. The untreated cell sterol
fraction contained 98.7% ergosterol, whereas lanosterol was not observed, and other
14-methylated sterols (obtusifoliol and eburicol) were found at only 1.0%. When C. albicans was
treated with 13s and FLC at 0.03125-8 μg/mL for 16 h, they acted in a dose-dependent fashion to
decrease the ergosterol content and increase the lanosterol and eburicol contents. The decrease of
ergosterol and the concomitant accumulation of lanosterol and eburicol provide indirect evidence
that compound 13s might have a similar mechanism as FLC by inhibiting fungal lanosterol
14α-demethylase (CYP51), which catalyses the oxidative removal of the 14-methyl group of
lanosterol or eburicol.
Figure 4. Sterol composition in 13s 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
95.6
85.2
10.7
2.3
3.1
5.3
-
1.3
5.3
-
0.125
4.2
31.2
34.1
23.6
-
-
13sb
0.5
12.4
9.4
36.8
49.5
65.1
0.8
8.9
2
4.7
8
1.3
10.0
3.6
-
0.03125
95.6
88.7
81.4
18.2
1.7
-
0.125
8.9
0.4
4.5
23.0
34.8
-
2.0
-
-
FLCc
0.5
2
6.9
7.2
16.1
13.3
0.6
27.0
37.2
0.4
15.7
13.0
-
8
Controld
-
98.7
b
c
aAbbreviations: C.alb., Candida albicans (ATCC SC5314); Treated with compound 13s; Treated with FLC;
dControl (no drug).

2.5 Cytochrome P450 inhibition assay
Cytochrome P450 (CYP) enzymes are heme-thiolate proteins that are responsible for the
oxidative metabolism of a wide variety of drugs. Inhibiting cytochrome P450 enzymes is one of
the most common mechanisms leading to drug-drug interactions (DDI)23. Many azole antifungal
agents have great inhibitory effects of CYP enzymes. For example, ketoconazole and itraconazole
inhibit CYP3A4, a major drug-metabolizing P450 isoform in the human liver24. The compounds
13s and 14a were tested against the five major human CYP isoforms (CYP1A2, CYP2C9,
CYP2C19, CYP2D6, and CYP3A4-M). As shown in Table 4, compounds 13s and 14a showed no
inhibitory activity against CYP1A2, CYP2C9, CYP2C19, and CYP2D6 with IC50 values of ≥50
μM, whereas compounds 13s and 14a exhibited weak activity against CYP3A4 with IC50 values
of 7.27 μM and 14.6 μM, respectively. The results showed that compounds 13s and 14a had a low
potential for causing DDI (for more information, see Supporting Information).
Table 5.
In vitro CYP inhibition assessment of compounds.
IC50 (μM)
Compd.
CYP1A2
CYP2C9
CYP2C19
CYP2D6
CYP3A4-M
>50
>50
>50
>50
7.27
13s
14a
>50
>50
>50
>50
14.6
2.6 In vitro human Plasma Stability Assay
Plasma stability plays an important role in drug discovery and development. Based on their in
vitro antifungal activities, compounds 13s and 14a were selected for an in vitro plasma stability
study. As shown in Table 6, compounds 13s and 14a exhibited excellent metabolic profiles in
human plasma.
Table 6.
In vitro Stability in Human Blood Plasma.
Stability in Human Blood Plasma
Compd.
Time Point(min)
%Remaining
Time Point(min)
%Remaining
60
95.0
120
90.5
13s
14a
60
95.8
120
91.7
2.7 Molecular docking model analysis of compound 13s in the active site of CYP51

To investigate the binding mode of compound 13s and 14a, two highly active compounds,
they were docked into the active site of CYP51 by using the CDOCKER program in the Discovery
Studio 3.0 software. The published crystal structure of C. albicans CYP51(PDB ID:5FSA, Figure
5)25 and A. fumigatus CYP51(PDB ID:4UYM, Figure 6) 17served as a useful template for
generating binding modes. As shown in Figure 5, the imidazole ring of compounds 13s and 14a
coordinated the iron in the heme group, and the alkyl ester formed a hydrophobic interaction with
Phe126 and Gly303. The 4,5-dihydronaphtho[2,1-d] isoxazole and naphtho[2,1-d] isoxazole side
chains extended into the CYP51 channel to form van der Waals and hydrophobic interactions with
the surrounding residues Ala62, Gly65, Leu88, Leu121, Pro230, Phe233, Phe380 and Met508.
Figure 5. The binding mode of compounds 13s (A) and 14a (B) in the active site of CYP51of C. albicans(PDB ID:
5FSA).
As shown in Figure 6, the imidazole ring of compounds 13s and 14a coordinated the iron in
the heme group, and the alkyl ester formed a hydrophobic interaction with Phe130, Val135 and
Ala303. The 4,5-dihydronaphtho[2,1-d] isoxazole side chain extended into the CYP51 channel to
form van der Waals and hydrophobic interactions with the surrounding residues Leu125, Phe229,
Phe234, Ile373 and Leu503. In addition, hydrogen bonds formed between the isoxazole ring of
compound 13s and A. fumigatus CYP51 Tyr122.These results may provide a good explanation for
the excellent in vitro antifungal activity of compounds 13s and 14a.

Figure 6. The binding mode of compounds 13s (A) and 14a (B) in the active site of A. fumigatus CYP51 (PDB ID:
4UYM).
3. Conclusions
To further enhance the anti-Aspergillus efficacy of our previous compounds, a novel class of
benzoheterocycles ring derivatives were designed, synthesized and evaluated for their in vitro
antifungal activity. Among these compounds, the 4,5-dihydronaphtho[2,1-d] isoxazole nucleus
13s and naphtho[2,1-d] isoxazole nucleus 14a exhibited the most remarkable in vitro activity
against a variety of fungal pathogens, including Candida spp., C. neoformans and A. fumigatus
and fluconazole-resistant strains of C. alb., that was superior or comparable to those of the
reference drugs fluconazole and voriconazole. Further mechanistic investigations showed that the
potent antifungal activity of novel compound 13s might act by inhibiting the CYP51 of Candida
albicans. Notably, a CYP enzyme inhibition assay showed that compounds 13s and 14a had weak
inhibition for various human cytochrome P450 isoforms, which indicated they had a low potential
to cause DDI. In addition, compounds 13s and 14a exhibited excellent blood plasma stability.
Further studies on the antifungal mechanism and structural optimization are in progress.
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 Labortechnik, Flawil, Switzerland) and are 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 are reported in parts per
million (ppm), and 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 methyl benzo[b]thiophene-2-carboxylate (2a)
A solution of 2-nitrobenzaldehyde (2.0 g, 13.1 mmol) and anhydrous potassium carbonate
(7.32 g, 52.9 mmol) in DMF (60 mL) was cooled to 0°C. Next, methyl 2-mercaptoacetate (1.40 g,
13.23 mmol) was added dropwise to the reaction mixture. The resulting mixture was stirred for 30
min at 0°C, and then stirred for 4 h at ambient temperature. The reaction mixture was poured into
ice water, and the resulting solid was filtered and dried to give the desired compound.
4.3 methyl benzofuran-2-carboxylate (2b)
A solution of salicylaldehyde (2.0 g, 16.2 mmol) and anhydrous potassium carbonate (9.05 g,
65.5 mmol) in DMF (60 mL) was cooled to 0°C. Then ethyl bromoacetate (5.01 g, 32.8 mmol)
was added dropwise to the reaction mixture. The resulting mixture was stirred at 0°C for 30 min,
and then at 80 °C for 5 h. After confirming that the reaction was complete by TLC analysis, the
solution was cooled to room temperature and water (15 mL) was 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 2b.
4.4 ethyl 2-oxo-2-(1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetate (8)
A solution of 1-tetralone (3.0 g, 20.5 mmol) and diethyl oxalate (3.6 g, 24.6 mmol) in dry
THF (100 mL) was cooled to 0°C under an argon atmosphere. Then LiHMDS (1 M in THF, 24.6
mL, 24.5 mmol) was added dropwise to the reaction mixture, and the resulting mixture was stirred
for 4 h at ambient temperature. The solvent was removed under reduced pressure, and the residue

was used for the next step without further purification.
4.5 ethyl 4,5-dihydronaphtho[2,1-d]isoxazole-3-carboxylate(2c)
To a solution of the residue 8 in AcOH (40 mL) was added hydroxylamine hydrochloride (1.9
g, 27.3 mmol) at ambient temperature. The reaction was stirred at 80°C for 8 h. The reaction
mixture was extracted with EtOAc and then 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 2c.
4.6 ethyl naphtho[2,1-d]isoxazole-3-carboxylate(2d)
2,3-Dicyano-5,6-dichlorobenzoquinone (6.10 g, 27.3 mmol) was added to a solution of ethyl
4,5-dihydronaphtho[2,1-d]isoxazole-3-carboxylate 2c (2.20 g, 9.1 mmol) in ethanol at ambient
temperature. The solution was refluxed for 12 h. After confirming that the reaction was complete
by TLC analysis, the reaction was quenched with aqueous NaHCO3. The reaction mixture was
extracted with EtOAc and then brine. The organic phase was dried over Na2SO4 overnight and the
solvent was removed in vacuum. The crude product was purified by silica gel column
chromatography to yield the target product 2d.
4.6 General procedure for the synthesis of compounds (1a-c)
To a solution of intermediate 2a-c (1 equiv.) in methanol was added 2N sodium hydroxide 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. The precipitated
white solid was collected by filtration and dried to give the carboxylic acid intermediate (1a-c).
4.6.1 benzo[b]thiophene-2-carboxylic acid (1a)
1H-NMR (600 MHz, DMSO-d6) δ 13.48 (s, 1H), 8.12 (s, 1H), 8.05 (d, J = 8.1 Hz, 1H), 8.01
(d, J = 7.9 Hz, 1H), 7.53 – 7.49 (m, 1H), 7.48 – 7.44 (m, 1H).
4.6.2 benzofuran-2-carboxylic acid (1b)
1H-NMR (600 MHz, DMSO-d6) δ 13.58 (s, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.70 (dd, J = 8.4,
0.7 Hz, 1H), 7.67 (d, J = 0.9 Hz, 1H), 7.52-4.49 (m, 1H), 7.38 – 7.33 (m, 1H).
4.6.3 4,5-dihydronaphtho[2,1-d]isoxazole-3-carboxylic acid (1c)
1H-NMR (600 MHz, DMSO-d6) δ 7.63 (d, J = 6.5 Hz, 1H), 7.14 – 7.08 (m, 2H), 7.03 (d, J =
6.8 Hz, 1H), 2.64 (t, J = 7.0 Hz, 2H), 2.32 – 2.27 (m, 2H).
4.6.4 naphtho[2,1-d]isoxazole-3-carboxylic acid(1d)

1H-NMR (600 MHz, DMSO-d6) δ 8.11 (d, J = 8.1 Hz, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.32 –
7.25 (m, 1H), 7.11 (t, J = 7.5 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 6.25 (d, J = 8.4 Hz, 1H).
4.7 General procedure for the synthesis of compounds (1e-f)
A solution of 4-amino-3-hydroxybenzoic acid (1 equiv.) and substituted trimethyl
orthoformate (3 equiv.) was heated to 100°C for 3 h. At the end of the reaction, the solution was
cooled to room temperature and diluted with MeOH. The solvent was removed under reduced
pressure to give the target product 1e-f.
4.8 General procedure for the synthesis of L-serine ester (11a–b)
To a solution of L-serine (1 equiv.) in alcohol reagent (isopropanol or isobutanol), cooled to <
0 °C, was added dropwise thionyl chloride (3 equiv.). The mixture was heated under reflux for 6 h.
The reaction mixture was then concentrated under reduced pressure to yield a white solid.
4.9 General procedure for the synthesis of compounds (12a-t)
To a solution of the intermediate acid compound 1a-f (1 equiv.) in anhydrous DMF was
added EDCI(1.1 equiv) and HOBt(1.1 equiv), respectively. The reaction mixture was stirred for 1
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, and the resulting solid was filtered and dried to give the desired
compound.
4.10 General procedure for the synthesis of compounds (13a-t and 14a-g)
To a solution of the intermediate 11a-v (1 equiv.) in CH3CN was added CDI (2 equiv.) and
imidazole (1.2 equiv.). 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 12a-v.
4.10.1 isopropyl (S)-2-(benzo[b]thiophene-2-carboxamido)-3-(1H-imidazol-1-yl)propanoate (13a)
Light white solid; yield:71.3 %. mp: 151.5-158.4 °C.1H-NMR (600 MHz, DMSO-d6) δ 9.24
(d, J = 7.9 Hz, 1H), 8.12 (s, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.99 (dd, J = 7.0, 1.3 Hz, 1H), 7.65 (s,
1H), 7.53 – 7.39 (m, 2H), 7.23 (d, J = 1.0 Hz, 1H), 6.86 (s, 1H), 4.99 – 4,94 (m, 1H), 4.80 – 4.76
(m, 1H), 4.52 (dd, J = 14.1, 5.1 Hz, 1H), 4.39 (dd, J = 14.1, 9.6 Hz, 1H), 1.19 (dd, J = 9.5, 6.2 Hz,
6H).13C-NMR (100 MHz, DMSO-d6) δ 168.89, 161.82, 140.30, 138.98, 138.55, 137.78, 127.96,

126.50, 125.66, 125.39, 125.05, 122.85, 120.03, 68.88, 53.88, 45.91, 21.48, 21.43. HRMS calcd
for C18H20N3O3S, [M + H]+, 358.1225; found 358.1254.
4.10.2 isobutyl (S)-2-(benzo[b]thiophene-2-carboxamido)-3-(1H-imidazol-1-yl)propanoate (13b)
Light white solid; yield:69.5%. mp: 140.1-144.7 °C.1H-NMR (600 MHz, DMSO-d6) δ 9.27
(d, J = 8.0 Hz, 1H), 8.10 (s, 1H), 8.03 (d, J = 7.9 Hz, 1H), 7.99 (d, J = 7.2 Hz, 1H), 7.66 (s, 1H),
7.51 – 7.43 (m, 2H), 7.23 (s, 1H), 6.86 (s, 1H), 4.85 – 4.81 (m, 1H), 4.55 (dd, J = 14.1, 4.8 Hz,
1H), 4.42 (dd, J = 14.1, 10.0 Hz, 1H), 3.94 – 4.87 (m, 2H), 1.90 – 1.84 (m, 1H), 0.86 (d, J = 6.7
Hz, 6H).13C-NMR (100 MHz, DMSO-d6) δ 169.37, 161.86, 140.31, 138.96, 138.55, 137.83,
128.36, 126.50, 125.59, 125.39, 125.04, 122.86, 119.87, 70.77, 53.86, 45.69, 27.23, 18.73(2C).
HRMS calcd for C19H22N3O3S, [M + H]+, 372.1382; found 372.1413.
4.10.3 isopropyl (S)-2-(benzofuran-2-carboxamido)-3-(1H-imidazol-1-yl)propanoate (13c)
Light white solid; yield:72.9%. mp: 54.1-59.4 °C.1H-NMR (600 MHz, DMSO-d6) δ 9.18 (d,
J = 8.1 Hz, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.64 (s, 1H), 7.58 (d, J = 0.7
Hz, 1H), 7.52 – 7.46 (m, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.22 (s, 1H), 6.85 (s, 1H), 4.99 – 4.92 (m,
1H), 4.84 – 4.80 (m, 1H), 4.53 (dd, J = 14.1, 5.1 Hz, 1H), 4.42 (dd, J = 14.1, 9.7 Hz, 1H), 1.19 (t,
J = 6.1 Hz, 6H).13C-NMR (100 MHz, DMSO-d6) δ 169.21, 158.71, 154.75, 148.58, 138.21,
128.43, 127.65, 127.45, 124.32, 123.41, 120.44, 112.34, 110.84, 69.37, 53.78, 46.27, 21.92, 21.89.
HRMS calcd for C18H20N3O4, [M + H]+, 342.1454; found 342.1474.
4.10.4 isobutyl (S)-2-(benzofuran-2-carboxamido)-3-(1H-imidazol-1-yl)propanoate (13d)
1
Light white solid; yield:68.1%. H-NMR (600 MHz, DMSO-d6) δ 9.23 (d, J = 8.2 Hz, 1H),
7.79 (d, J = 7.7 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.64 (s, 1H), 7.57 (d, J = 0.6 Hz, 1H), 7.52 –
7.46 (m, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.22 (s, 1H), 6.84 (s, 1H), 4.91 – 4.87 (m, 1H), 4.56 (dd, J
= 14.1, 4.8 Hz, 1H), 4.45 (dd, J = 14.1, 10.0 Hz, 1H), 3.95 – 3.85 (m, 2H), 1.90 – 1.84 (m, 1H),
0.86 (dd, J = 6.7, 1.4 Hz, 6H).13C-NMR (100 MHz, DMSO-d6) δ 169.24, 158.31, 154.31, 148.15,
137.80, 128.35, 127.19, 126.98, 123.86, 122.96, 119.84, 111.87, 110.36, 70.78, 53.25, 45.62,
27.23, 18.72(2C). HRMS calcd for C19H22N3O4, [M + H]+, 356.1610; found 356.1639.
4.10.5 isopropyl (S)-3-(1H-imidazol-1-yl)-2-(quinoline-3-carboxamido)propanoate (13e)
Light white solid; yield:69.7%. mp: 79.0-104.0 °C. 1H-NMR (600 MHz, DMSO-d6) δ 9.32 (d,
J = 7.8 Hz, 1H), 9.21 (d, J = 2.2 Hz, 1H), 8.79 (d, J = 2.0 Hz, 1H), 8.11 (dd, J = 14.4, 8.1 Hz, 2H),
7.89 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.76 – 7.70 (m, 1H), 7.67 (s, 1H), 7.25 (s, 1H), 6.87 (s, 1H),

5.00 – 4.94 (m, 1H), 4.87 – 4.83 (m, 1H), 4.55 (dd, J = 14.1, 5.2 Hz, 1H), 4.43 (dd, J = 14.1, 9.5
Hz, 1H), 1.20 (dd, J = 10.2, 6.3 Hz, 6H).13C-NMR (100 MHz, DMSO-d6) δ 168.92, 165.26,
148.67, 148.64, 137.81, 135.84, 131.50, 129.20, 128.81, 127.82, 127.59, 126.42, 126.16, 120.16,
68.85, 53.91, 46.04, 21.49, 21.44. HRMS calcd for C19H21N4O3, [M + H]+, 353.1614; found
353.1630.
4.10.6 isobutyl (S)-3-(1H-imidazol-1-yl)-2-(quinoxaline-2-carboxamido)propanoate (13f)
Light white solid; yield:70.3%. mp: 115.2-128.0 °C.1H-NMR (600 MHz, DMSO-d6) δ 9.36
(d, J = 7.9 Hz, 1H), 9.20 (d, J = 2.2 Hz, 1H), 8.78 (d, J = 1.9 Hz, 1H), 8.11 (dd, J = 13.8, 8.4 Hz,
2H), 7.91 – 7.88 (m, 1H), 7.73 – 7.69 (m, 1H), 7.67 (s, 1H), 7.24 (d, J = 9.6 Hz, 1H), 6.86 (s, 1H),
4.93 – 4.89 (m, 1H), 4.58 (dd, J = 14.1, 4.9 Hz, 1H), 4.46 (dd, J = 14.1, 9.8 Hz, 1H), 3.95 – 3.88
(m, 2H), 1.91 – 1.85 (m, 1H), 0.87 (d, J = 6.7 Hz, 6H).13C-NMR (100 MHz, DMSO-d6) δ 169.43,
165.27, 148.63(2C), 137.90, 135.82, 131.51, 129.20, 128.81, 128.42, 127.61, 126.41, 126.14,
119.93, 70.76, 53.90, 45.78, 27.23, 18.74(2C). HRMS calcd for C20H23N4O3, [M + H]+, 367.1770;
found 367.1788.
4.10.7 isopropyl (S)-3-(1H-imidazol-1-yl)-2-(quinoxaline-2-carboxamido)propanoate (13g)
1
Light white solid; yield:65.3%. H-NMR (600 MHz, DMSO-d6) δ 9.43 (d, J = 7.8 Hz, 2H),
8.26 – 8.20 (m, 2H), 8.05 – 7.99 (m, 2H), 7.65 (s, 1H), 7.21 (s, 1H), 6.84 (s, 1H), 4.99 (dd, J =
12.5, 6.3 Hz, 1H), 4.96 – 4.93 (m, 1H), 4.62 – 4.54 (m, 2H), 1.22 (dd, J = 6.2, 3.4 Hz,
6H).13C-NMR (100 MHz, DMSO-d6) δ 168.50, 163.43, 143.59, 143.40, 143.16, 139.75, 137.54,
132.27, 131.55, 129.47, 129.18, 126.52, 120.54, 69.15, 53.37, 46.35, 21.46(2C). HRMS calcd for
C18H19N5O3, [M + Na]+, 376.1386; found 376.1404.
4.10.8 isobutyl (S)-3-(1H-imidazol-1-yl)-2-(quinoxaline-2-carboxamido)propanoate (13h)
Light white solid; yield:68.7%. mp: 97.2-98.0 °C.1H-NMR (600 MHz, DMSO-d6) δ 9.51 (d,
J = 8.3 Hz, 1H), 9.43 (s, 1H), 8.26 – 8.19 (m, 2H), 8.06 – 7.98 (m, 2H), 7.68 (s, 1H), 7.23 (s, 1H),
6.85 (s, 1H), 5.05 – 5.01 (m, 1H), 4.66 – 4.57 (m, 2H), 3.93 (dd, J = 6.5, 1.8 Hz, 2H), 0.87 (dd, J =
6.7, 2.5 Hz, 6H).13C-NMR (100 MHz, DMSO-d6) δ 169.09, 163.45, 143.56, 143.43, 143.14,
139.75, 137.81, 132.25, 131.55, 129.44, 129.18, 128.10, 119.98, 70.88, 53.44, 45.83, 27.21,
18.72(2C). HRMS calcd for C19H22N5O3, [M + H]+, 368.1723; found 368.1741.
4.10.9 isopropyl (S)-2-(6-fluoro-1H-indole-2-carboxamido)-3-(1H-imidazol-1-yl)propanoate (13i)
Light white solid; yield:62.6%. mp: 88.0-91.1°C.1H-NMR (600 MHz, DMSO-d6) δ 11.74 (s,

1H), 9.01 (d, J = 8.0 Hz, 1H), 7.63 (s, 1H), 7.44 (dd, J = 9.8, 2.5 Hz, 1H), 7.40 (dd, J = 8.9, 4.6 Hz,
1H), 7.21 (s, 1H), 7.15 (d, J = 1.5 Hz, 1H), 7.06 (td, J = 9.2, 2.6 Hz, 1H), 6.83 (s, 1H), 4.99 – 4.90
(m, 1H), 4.81 – 4.78 (m, 1H), 4.51 (dd, J = 14.1, 5.2 Hz, 1H), 4.38 (dd, J = 14.1, 9.7 Hz, 1H), 1.19
(dd, J = 11.5, 6.2 Hz, 6H).13C-NMR (100 MHz, DMSO-d6) δ 169.62, 161.43, 158.79(0.5C),
156.48(0.5C), 138.25, 133.78, 132.76, 128.64, 127.41, 120.40, 114.05, 113.00, 106.41, 103.86,
69.24, 53.99, 46.38, 21.93, 21.88. MS (ESI) m/z 359.3 [M+H]+.
4.10.10 isopropyl
(S)-3-(1H-imidazol-1-yl)-2-(1-methyl-1H-indole-2-carboxamido)propanoate
(13j)
1
Light white solid; yield:72.9%. H-NMR (600 MHz, DMSO-d6) δ 8.94 (d, J = 8.0 Hz, 1H),
7.69 – 7.63 (m, 2H), 7.52 (d, J = 8.4 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.23 (s, 1H), 7.11 (t, J = 7.4
Hz, 1H), 7.07 (s, 1H), 6.86 (s, 1H), 4.99 – 4.95 (m, 1H), 4.77 – 4.73 (m, 1H), 4.51 (dd, J = 14.1,
5.0 Hz, 1H), 4.38 (dd, J = 14.0, 9.9 Hz, 1H), 3.90 (s, 3H), 1.21 (dd, J = 9.4, 6.3 Hz, 6H).13C-NMR
(100 MHz, DMSO-d6) δ 169.47, 162.41, 138.95, 138.27, 131.57, 128.25, 125.91, 124.33, 122.16,
120.73, 120.59, 111.01, 105.49, 69.18, 53.84, 46.41, 31.72, 21.96, 21.93. HRMS calcd for
C19H23N4O3, [M + H]+, 355.1770; found 355.1799.
4.10.11 isobutyl (S)-3-(1H-imidazol-1-yl)-2-(1-methyl-1H-indole-2-carboxamido)propanoate(13k)
Light white solid; yield:71.4%. mp: 58.1-61.6 °C.1H-NMR (600 MHz, DMSO-d6) δ 8.98 (d,
J = 8.1 Hz, 1H), 7.69 – 7.62 (m, 2H), 7.52 (d, J = 8.4 Hz, 1H), 7.31 – 7.27 (m, 1H), 7.23 (s, 1H),
7.11 (t, J = 7.4 Hz, 1H), 7.07 (s, 1H), 6.86 (s, 1H), 4.85 – 4.81 (m, 1H), 4.55 (dd, J = 14.1, 4.7 Hz,
1H), 4.40 (dd, J = 14.0, 10.2 Hz, 1H), 3.90 (s, 3H), 3.79 – 3.69 (m, 2H), 1.88 (d, J = 6.6 Hz, 1H),
0.88 (d, J = 6.7 Hz, 6H).13C-NMR (100 MHz, DMSO-d6) δ 169.96, 162.43, 138.97, 138.21,
131.49, 128.25, 125.89, 124.35, 122.16, 120.73, 120.54, 111.02, 105.49, 71.15, 53.72, 46.34,
31.72, 27.73, 19.21, 19.16. HRMS calcd for C20H25N4O3, [M + H]+, 369.1927; found 369.1955.
4.10.12 isopropyl (S)-2-(1H-benzo[d]imidazole-2-carboxamido)-3-(1H-imidazol-1-yl)propanoate
(13l)
Light white solid; yield:67.4%. mp: 100.6-103.2 °C.1H-NMR (600 MHz, DMSO-d6) δ 13.30
(s, 1H), 9.36 (d, J = 8.4 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.63 (s, 1H), 7.53 (d, J = 8.0 Hz, 1H),
7.33 (dd, J = 11.2, 4.0 Hz, 1H), 7.29 (dd, J = 11.2, 4.0 Hz, 1H), 7.20 (s, 1H), 6.82 (s, 1H), 4.99 –
4.95 (m, 1H), 4.92 – 4.89 (m, 1H), 4.55 (dd, J = 14.1, 5.0 Hz, 1H), 4.49 (dd, J = 14.1, 9.5 Hz, 1H),
1.20 (dd, J = 6.2, 4.0 Hz, 6H).13C-NMR (100 MHz, DMSO-d6) δ 168.54, 159.00, 144.67, 142.43,

137.70, 134.49, 127.55, 124.45, 122.76, 120.12, 120.02, 112.66, 68.97, 53.23, 45.98, 21.47, 21.45.
HRMS calcd for C17H20N5O3, [M + H]+, 342.1566; found 342.1589.
4.10.13 isopropyl (S)-2-(benzo[d][1,3]dioxole-5-carboxamido)-3-(1H-imidazol-1-yl)propanoate
(13m)
Light white solid; yield:74.1%. mp: 60.9-63.3 °C.1H-NMR (600 MHz, DMSO-d6) δ 8.74 (d,
J = 7.9 Hz, 1H), 7.61 (s, 1H), 7.39 (dd, J = 8.1, 1.8 Hz, 1H), 7.31 (d, J = 1.7 Hz, 1H), 7.19 (s, 1H),
7.00 (d, J = 8.1 Hz, 1H), 6.84 (s, 1H), 6.10 (s, 2H), 4.96 – 4.90 (mz, 1H), 4.72 – 4.68 (m, 1H),
4.47 (dd, J = 14.0, 5.1 Hz, 1H), 4.35 (dd, J = 14.0, 9.8 Hz, 1H), 1.17 (dd, J = 8.2, 6.3 Hz,
6H).13C-NMR (100 MHz, DMSO-d6) δ 169.22, 165.70, 150.08, 147.38, 137.78, 128.11, 127.40,
122.48, 119.95, 107.96, 107.30, 101.79, 68.62, 53.92, 45.83, 21.50, 21.44. HRMS calcd for
C17H19N3O5, [M + Na]+, 368.1222; found 368.1243.
4.10.14 isobutyl
(S)-2-(benzo[d][1,3]dioxole-5-carboxamido)-3-(1H-imidazol-1-yl)propanoate
(13n)
1
Light white solid; yield:73.9%. mp: 109.4-113.0 °C. H-NMR (600 MHz, DMSO-d6) δ 8.78
(d, J = 7.9 Hz, 1H), 7.61 (s, 1H), 7.38 (dd, J = 8.2, 1.7 Hz, 1H), 7.30 (d, J = 1.7 Hz, 1H), 7.19 (s,
1H), 7.00 (d, J = 8.1 Hz, 1H), 6.84 (s, 1H), 6.10 (d, J = 1.0 Hz, 2H), 4.77 – 4.74 (m, 1H), 4.51 (dd,
J = 14.0, 4.9 Hz, 1H), 4.38 (dd, J = 14.0, 10.0 Hz, 1H), 3.91 – 3,84 (m, 2H), 1.88 – 1.82 (m, 1H),
0.85 (dd, J = 6.7, 1.1 Hz, 6H).13C-NMR (100 MHz, DMSO-d6) δ 170.14, 166.19, 150.54, 147.83,
138.26, 128.79, 127.84, 122.90, 120.30, 108.41, 107.71, 102.25, 71.07, 54.32, 46.14, 27.69, 19.20.
HRMS calcd for C18H21N3O5, [M + Na]+, 382.1379; found 382.1402.
4.10.15 isopropyl (S)-2-(benzo[d]oxazole-6-carboxamido)-3-(1H-imidazol-1-yl)propanoate(13o)
Light white solid; yield:71.5%. mp: 71.9-75.4 °C. 1H-NMR (600 MHz, DMSO-d6) δ 9.30 (d,
J = 7.8 Hz, 1H), 8.92 (s, 1H), 8.28 (d, J = 0.7 Hz, 1H), 7.91 (dt, J = 17.6, 4.9 Hz, 2H), 7.69 (s, 1H),
7.26 (s, 1H), 6.83 (s, 1H), 4.97 – 4.93 (m, 1H), 4.83 – 4.76 (m, 1H), 4.55 – 4.48 (m, 2H), 1.19 (dd,
J = 8.9, 6.3 Hz, 6H).13C-NMR (100 MHz, DMSO-d6) δ 169.08, 165.90, 156.41, 149.08, 142.30,
137.91, 131.05, 128.29, 124.33, 119.92, 119.86, 110.55, 68.66, 54.14, 45.68, 21.50, 21.45. HRMS
calcd for C17H19N4O4, [M + H]+, 343.1406; found 343.1427.
4.10.16 isopropyl
(S)-3-(1H-imidazol-1-yl)-2-(2-methylbenzo[d]oxazole-6-carboxamido)
propanoate(13p)

1
Light white solid; yield:70.7%. H-NMR (600 MHz, DMSO-d6) δ 9.01 (d, J = 7.8 Hz, 1H),
8.04 (d, J = 1.1 Hz, 1H), 7.80 (dd, J = 8.3, 1.5 Hz, 1H), 7.74 (d, J = 8.3 Hz, 1H), 7.69 (s, 1H), 7.24
(s, 1H), 6.86 (s, 1H), 4.98 – 4.92 (m, 1H), 4.80 – 4.76 (m, 1H), 4.52 (dd, J = 14.1, 5.1 Hz, 1H),
4.40 (dd, J = 14.0, 9.7 Hz, 1H), 2.65 (s, 3H), 1.18 (dd, J = 8.3, 6.3 Hz, 6H).13C-NMR (100 MHz,
DMSO-d6) δ 169.10, 166.43, 166.06, 150.08, 143.89, 138.00, 130.00, 128.11, 123.83, 120.09,
118.74, 109.57, 68.70, 54.00, 45.89, 21.49, 21.44, 14.32. HRMS calcd for C18H20N4O4, [M + Na]+,
379.1382; found 379.1416.
4.10.17 isopropyl (S)-2-(5,6-dihydro-4H-benzo[3,4]cyclohepta[1,2-d]isoxazole-3-carboxamido)-3
-(1H-imidazol-1-yl)propanoate(13q)
1
Light white solid; yield:68.1%. H-NMR (600 MHz, DMSO-d6) δ 9.23 (d, J = 8.1 Hz, 1H),
7.94 – 7.87 (m, 1H), 7.61 (s, 1H), 7.41 – 7.38 (m, 2H), 7.32 (dd, J = 5.6, 3.3 Hz, 1H), 7.20 (s, 1H),
6.86 (s, 1H), 5.00 – 4.93 (m, 1H), 4.83 – 4.79 (m, 1H), 4.51 (dd, J = 14.1, 4.9 Hz, 1H), 4.39 (dd, J
= 14.1, 9.7 Hz, 1H), 2.93 – 2.87 (m, 2H), 2.86 – 2.73 (m, 2H), 1.91 – 1.88 (m, 2H), 1.21 (t, J = 6.5
Hz, 6H). 13C-NMR (100 MHz, DMSO-d6) δ 168.42, 164.51, 159.72, 157.15, 141.23, 137.82,
130.09, 130.04, 128.32, 126.73, 126.20, 125.70, 119.83, 115.43, 68.90, 53.24, 45.59, 34.53, 24.18,
23.76, 21.44, 21.42. HRMS calcd for C22H25N4O4, [M + H]+, 409.1876; found 409.1907.
4.10.18 isopropyl (S)-2-(4,5-dihydronaphtho[2,1-d]isoxazole-3-carboxamido)-3-(1H-imidazol-1-
yl)propanoate(13s)
1
Light white solid; yield:68.3%. H-NMR (600 MHz, DMSO-d6) δ 9.23 (d, J = 8.1 Hz, 1H),
7.67 (t, J = 8.4 Hz, 1H), 7.62 (s, 1H), 7.42 – 7.36 (m, 3H), 7.20 (s, 1H), 6.85 (s, 1H), 4.99 – 4.93
(m, 1H), 4.84 – 4.80 (m, 1H), 4.51 (dd, J = 14.1, 5.0 Hz, 1H), 4.41 (dd, J = 14.1, 9.7 Hz, 1H), 3.01
(t, J = 7.9 Hz, 2H), 2.89 – 2.79 (m, 2H), 1.20 (t, J = 6.5 Hz, 6H).13C-NMR (100 MHz, DMSO-d6)
δ 168.43, 166.47, 159.34, 154.86, 137.78, 136.86, 130.52, 128.79, 128.00, 127.24, 123.80, 121.47,
119.99, 113.12, 68.97, 53.26, 45.68, 27.82, 21.44(2C), 17.75. HRMS calcd for C21H22N4O4, [M +
Na]+, 417.1539; found 417.1573.
4.10.19 isobutyl (S)-2-(4,5-dihydronaphtho[2,1-d]isoxazole-3-carboxamido)-3-(1H-imidazol-1-
yl)propanoate(13t)
1
Light white solid; yield:67.8%. H-NMR (600 MHz, DMSO-d6) δ 9.29 (d, J = 8.2 Hz, 1H),
7.66 (d, J = 7.3 Hz, 1H), 7.63 (s, 1H), 7.40 (ddd, J = 13.3, 6.9, 3.1 Hz, 3H), 7.21 (s, 1H), 6.86 (s,
1H), 4.92 – 4.88 (m, 1H), 4.55 (dd, J = 14.1, 4.7 Hz, 1H), 4.44 (dd, J = 14.1, 10.0 Hz, 1H), 3.94 –

3.88 (m, 2H), 3.01 (t, J = 7.9 Hz, 2H), 2.83 (dd, J = 16.7, 8.1 Hz, 2H), 1.90 – 1.86 (m, 1H), 0.88
(dd, J = 6.7, 1.6 Hz, 6H).13C-NMR (100 MHz, DMSO-d6) δ 169.39, 166.94, 159.84, 155.28,
138.28, 137.30, 130.98, 129.24, 128.81, 127.69, 124.25, 121.93, 120.31, 113.56, 71.27, 53.63,
45.95, 28.28, 27.70, 19.18(2C), 18.21. HRMS calcd for C22H24N4O4, [M + Na]+, 431.1695; found
431.1727.
4.10.20 isopropyl
(S)-3-(1H-imidazol-1-yl)-2-(naphtho[2,1-d]isoxazole-3-carboxamido)
propanoate(14a)
1
Light white solid; yield:74.1%. H-NMR (600 MHz, DMSO-d6) δ 9.60 (d, J = 8.0 Hz, 1H),
8.45 (dd, J = 6.0, 3.4 Hz, 1H), 8.24 – 8.16 (m, 1H), 7.94 (dd, J = 21.7, 8.7 Hz, 2H), 7.85 – 7.80 (m,
2H), 7.66 (s, 1H), 7.25 (s, 1H), 6.85 (s, 1H), 5.02 – 4.98 (m, 1H), 4.97 – 4.90 (m, 1H), 4.58 (dd, J
= 14.1, 5.0 Hz, 1H), 4.47 (dd, J = 14.1, 9.7 Hz, 1H), 1.22 (t, J = 6.5 Hz, 6H).13C-NMR (150 MHz,
DMSO-d6) δ 168.36, 161.68, 159.03, 152.16, 137.83, 133.59, 129.11, 128.74, 128.29, 128.05,
126.43, 121.14, 119.88, 118.55, 118.11, 115.39, 69.00, 53.45, 45.61, 21.44, 21.42. HRMS calcd
for C21H21N4O4, [M + H]+, 393.1563; found 393.1594.
4.10.21 isobutyl
(S)-3-(1H-imidazol-1-yl)-2-(naphtho[2,1-d]isoxazole-3-carboxamido)
propanoate(14b)
1
Light white solid; yield:73.9%. H-NMR (600 MHz, DMSO-d6) δ 9.65 (d, J = 8.2 Hz, 1H),
8.45 (dd, J = 6.0, 3.5 Hz, 1H), 8.25 – 8.17 (m, 1H), 7.94 (dd, J = 21.7, 8.7 Hz, 2H), 7.83 (dd, J =
6.2, 3.2 Hz, 2H), 7.67 (s, 1H), 7.26 (s, 1H), 6.86 (s, 1H), 5.04 – 5.00 (m, 1H), 4.61 (dd, J = 14.1,
4.8 Hz, 1H), 4.50 (dd, J = 14.1, 10.0 Hz, 1H), 3.98 – 3.91 (m, 2H), 1.91 – 1.87 (m, 1H), 0.88 (dd,
J = 6.7, 1.2 Hz, 6H). 13C-NMR (100 MHz, DMSO-d6) δ 168.83, 161.70, 159.09, 152.13, 137.82,
133.58, 129.12, 128.74, 128.30, 128.05, 126.44, 121.14, 119.85, 118.55, 118.10, 115.37, 70.85,
53.32, 45.53, 27.21, 18.69, 18.66. HRMS calcd for C22H23N4O4, [M + H]+, 407.1719; found
407.1753.
4.10.22 isopropyl
(S)-2-(8-bromo-4,5-dihydronaphtho[2,1-d]isoxazole-3-carboxamido)-3-(1H-
imidazol-1-yl)propanoate(14c)
1
Light white solid; yield:71.7%. H-NMR (600 MHz, DMSO-d6) δ 9.27 (d, J = 8.1 Hz, 1H),
7.80 (d, J = 2.0 Hz, 1H), 7.62 (s, 1H), 7.60 (dd, J = 8.1, 2.1 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.20
(s, 1H), 6.85 (s, 1H), 4.98 – 4.93 (m, 1H), 4.86 – 4.79 (m, 1H), 4.51 (dd, J = 14.1, 5.0 Hz, 1H),
4.40 (dd, J = 14.1, 9.7 Hz, 1H), 2.99 (t, J = 7.9 Hz, 2H), 2.87 – 2.78 (m, 2H), 1.20 (t, J = 6.3 Hz,

6H).13C-NMR (100 MHz, DMSO-d6) δ 168.38, 165.04, 159.11, 154.90, 137.81, 136.07, 133.03,
132.94, 130.92, 128.28, 125.72, 123.84, 119.86, 114.14, 68.94, 53.28, 45.58, 27.29, 21.44, 21.41,
17.52. HRMS calcd for C21H22BrN4O4, [M + H]+, 475.0804; found 475.0843.
4.10.23 isopropyl (S)-3-(1H-imidazol-1-yl)-2-(8-methoxy-4,5-dihydronaphtho[2,1-d]isoxazole-3-
carboxamido)propanoate(14d)
1
Light white solid; yield: 69.3%. H-NMR (600 MHz, DMSO-d6) δ 9.22 (d, J = 8.1 Hz, 1H),
7.61 (s, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 2.7 Hz, 2H), 6.98 (dd, J = 8.4, 2.7 Hz, 1H), 6.85
(s, 1H), 4.99 – 4.93 (m, 1H), 4.83 – 4.79 (m, 1H), 4.51 (dd, J = 14.1, 5.0 Hz, 1H), 4.40 (dd, J =
14.1, 9.7 Hz, 1H), 3.82 (s, 3H), 2.93 (t, J = 7.9 Hz, 2H), 2.86 – 2.76 (m, 2H), 1.20 (t, J = 6.4 Hz,
6H).13C-NMR (150 MHz, DMSO-d6) δ 168.36, 161.68, 159.03, 152.16, 137.83, 133.59, 129.11,
128.74, 128.29, 128.05, 126.43, 121.14, 119.88, 118.55, 118.11, 115.39, 69.00, 53.45, 45.61,
21.44(2C), 21.42. HRMS calcd for C22H25N4O5, [M + H]+, 425.1825; found 425.1859.
4.10.24 isobutyl
(S)-3-(1H-imidazol-1-yl)-2-(8-methoxy-4,5-dihydronaphtho[2,1-d]isoxazole-3-
carboxamido)propanoate(14e)
1
Light white solid; yield: 71.6%. H-NMR (600 MHz, DMSO-d6) δ 9.29 (d, J = 8.2 Hz, 1H),
7.62 (s, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.21 (s, 1H), 7.19 (d, J = 2.7 Hz, 1H), 6.98 (dd, J = 8.4, 2.7
Hz, 1H), 6.85 (s, 1H), 4.98 – 4.88 (m, 1H), 4.55 (dd, J = 14.1, 4.8 Hz, 1H), 4.44 (dd, J = 14.1,
10.0 Hz, 1H), 3.94 – 3.88 (m, 2H), 3.82 (s, 3H), 2.93 (t, J = 7.9 Hz, 2H), 2.85 – 2.75 (m, 2H), 1.91
– 1.85 (m, 1H), 0.88 (dd, J = 6.7, 1.6 Hz, 6H).13C-NMR (100 MHz, DMSO-d6) δ 168.89, 166.45,
159.34, 158.29, 154.87, 137.78, 129.86, 128.66, 128.29, 124.54, 119.81, 116.29, 113.44, 106.56,
70.79, 55.40, 53.15, 45.48, 27.21, 26.98, 18.70, 18.00. HRMS calcd for C23H27N4O5, [M + H]+,
439.1981; found 439.2019.
4.10.25 isopropyl (S)-3-(1H-imidazol-1-yl)-2-(7-methoxy-4,5-dihydronaphtho[2,1-d]isoxazole-3-
carboxamido)propanoate(14f)
1
Light white solid; yield: 73.2%. H-NMR (600 MHz, DMSO-d6) δ 9.17 (d, J = 8.1 Hz, 1H),
7.60 (d, J = 8.7 Hz, 2H), 7.20 (s, 1H), 7.01 (d, J = 2.2 Hz, 1H), 6.93 (dd, J = 8.4, 2.5 Hz, 1H), 6.85
(s, 1H), 5.00 – 4.92 (m, 1H), 4.84 – 4.77 (m, 1H), 4.50 (dd, J = 14.1, 5.0 Hz, 1H), 4.40 (dd, J =
14.1, 9.7 Hz, 1H), 3.81 (s, 3H), 2.98 (t, J = 7.9 Hz, 2H), 2.87 – 2.75 (m, 2H), 1.20 (t, J = 6.4 Hz,
6H).13C-NMR (100 MHz, DMSO-d6) δ 168.44, 166.66, 160.94, 159.43, 154.67, 139.18, 137.78,
128.30, 123.07, 119.82, 116.83, 114.54, 112.45, 111.04, 68.90, 55.36, 53.24, 45.57, 28.24,

21.43(2C), 17.75. HRMS calcd for C22H25N4O5, [M + H]+, 425.1825; found 425.1863.
4.10.26 isobutyl (S)-3-(1H-imidazol-1-yl)-2-(7-methoxy-4,5-dihydronaphtho[2,1-d]isoxazole-3-
carboxamido)propanoate(14g)
1
Light white solid; yield: 76.6%. H-NMR (600 MHz, DMSO-d6) δ 9.23 (d, J = 8.3 Hz, 1H),
7.64 – 7.57 (m, 2H), 7.20 (s, 1H), 7.01 (d, J = 2.4 Hz, 1H), 6.92 (dd, J = 8.5, 2.5 Hz, 1H), 6.85 (s,
1H), 4.91 – 4.87 (m, 1H), 4.54 (dd, J = 14.1, 4.7 Hz, 1H), 4.43 (dd, J = 14.1, 10.0 Hz, 1H), 3.94 –
3.88 (m, 2H), 3.81 (s, 3H), 2.98 (t, J = 7.9 Hz, 2H), 2.86 – 2.74 (m, 2H), 1.91 – 1.84 (m, 1H), 0.88
(dd, J = 6.7, 1.6 Hz, 6H).13C-NMR (100 MHz, DMSO-d6) δ 168.91, 166.69, 160.95, 159.49,
154.63, 139.18, 137.77, 128.32, 123.08, 119.80, 116.82, 114.55, 112.46, 111.03, 70.78, 55.36,
53.12, 45.48, 28.25, 27.21, 18.70(2C), 17.76. HRMS calcd for C23H26N4O5, [M + Na]+, 461.1801;
found 461.1834.
4.11 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 inhibited the visible growth of the fungi. FLC and ITR were
purchased for serve as the positive control drugs. All test compounds were dissolved in DMSO
and serially diluted into the growth medium.
4.12 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 the 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).
4.13 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 the addition of 20 μL NADPH to a final
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.
4.14 Plasma Stability Assay
Test compounds were added to human plasma to a final concentration of 2 μM and incubated
at 37 °C. At each time point (0, 10, 30, 60 and 120 min), the reaction was terminated with the
addition of a 200 µL cold stop solution (200 ng/mL tolbutamide plus 20 ng/mL buspirone in 50%
MeOH/ACN) to precipitate protein. 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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Highlights

26 new compounds with benzoheterocycle scaffolds were designed and synthesized.
 The naphthoisoxazole was selected to improve activity against Aspergillus spp.



Compounds 13s and 14a showed better antifungal activity than fluconazole.
Compound 13s reduced the content of ergosterol in a dose-dependent manner.
Compounds 13s and 14a exhibited low inhibition profiles for human cytochrome P450
isoforms.