Design, synthesis, and structure-activity relationship studies of novel thienopyrrolidone derivatives with strong antifungal activity against Aspergillus fumigates

Article abstract of DOI:10.1016/j.ejmech.2015.08.023

In order to further enhance the anti-Aspergillus efficacy of our previously discovered antifungal lead compounds (I), two series of novel azoles featuring thieno[2,3-c]pyrrolidone and thieno[3,2-c]pyrrolidone nuclei were designed and evaluated for their in vitro activity on the basis of the binding mode of albaconazole using molecular docking, along with SARs of antifungal triazoles. Most of target compounds exhibited excellent activity against Candida and Cryptococcus spp., with MIC values in the range of 0.0625 μg/ml to 0.0156 μg/ml. The thieno[3,2-c]pyrrolidone unit was more suited for improving activity against Aspergillus spp. The most potent compound, 18a, was selected for further development due to its significant in vitro activity against Aspergillus spp. (MIC Combining double low line 0.25 μg/ml), as well as its high metabolic stability in human liver microsomes.

Full text of DOI:10.1016/j.ejmech.2015.08.023

Accepted Manuscript
Design, synthesis, and structure-activity relationship studies of novel
thienopyrrolidone derivatives with strong antifungal activity against Aspergillus
fumigates
Xufeng Cao, Yuanyuan Xu, Yongbing Cao, Ruilian Wang, Ran Zhou, Wenjing Chu,
Yushe Yang
PII:
S0223-5234(15)30207-5
10.1016/j.ejmech.2015.08.023
EJMECH 8059
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 May 2015
Revised Date: 6 August 2015
Accepted Date: 9 August 2015
Please cite this article as: X. Cao, Y. Xu, Y. Cao, R. Wang, R. Zhou, W. Chu, Y. Yang, Design,
synthesis, and structure-activity relationship studies of novel thienopyrrolidone derivatives with strong
antifungal activity against Aspergillus fumigates, European Journal of Medicinal Chemistry (2015), doi:
10.1016/j.ejmech.2015.08.023.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis, and structure-activity relationship studies of novel
thienopyrrolidone derivatives with strong antifungal activity against Aspergillus
fumigates
Xufeng Caoa,†, Yuanyuan Xua,†, Yongbing Caob, Ruilian Wangb, Ran Zhouc, Wenjing Chua, and
Yushe Yang a,*
aState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai
Institute for Biological Sciences, Chinese Academy of Sciences, 555 Zuchong Zhi Road, Shanghai
201203, China
bSchool of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433,
China
cDrug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, 555 Zuchong Zhi Road, Shanghai 201203,
China
∗Corresponding author: Yushe Yang. Tel: 86-21-5080 6786. Fax: 86-21-5080 6786. E-mail:
ysyang@simm.ac.cn.
†These authors contributed equally to this work.
Abstract
In order to further enhance the anti-Aspergillus efficacy of our previously discovered antifungal
lead compounds (I) , two series of novel azoles featuring thieno[2,3-c]pyrrolidone and
thieno[3,2-c]pyrrolidone nuclei were designed and evaluated for their in vitro activity on the basis
of the binding mode of albaconazole using molecular docking, along with SARs of antifungal
triazoles. Most of target compounds exhibited excellent activity against Candida and
Cryptococcus spp., with MIC values in the range of 0.0625 ꢀg/ml to 0.0156 ꢀg/ml. The
thieno[3,2-c]pyrrolidone unit was more suited for improving activity against Aspergillus spp. The
most potent compound, 18a, was selected for further development due to its significant in vitro
activity against Aspergillus spp. (MIC = 0.25 ꢀg/ml), as well as its high metabolic stability in
human liver microsomes.
Keywords: Azoles, Synthesis, Antifungal activity, CYP51, Molecular docking
During the past two decades, there is no denying the fact that the incidence of systemic fungal
infections, with an enormous growth, has led to high morbidity and mortality in
immunocompromised patients, such as oncology patients undergoing chemotherapy, HIV-infected
individuals, patients given potent pharmacologic immunomodulators and broad-spectrum
antibiotics [1-4]. Clinically, the three major pathogens, including the Candida spp. (mortality rate:
20%~40%), Cryptococcus neoformans (mortality rate: 20%~70%), and Aspergillus spp. (mortality
rate: 20%~70%) account for most of systemic fungal infections [5]. Currently, 4 different classes
of antifungal agents, which include the polyenes (such as amphotericin B and nystatin), azoles,
echinocandins (such as caspofungin and micafungin), and antimetabolites (such as
5-fluorocytosine), are commonly used in clinic.
Among these antifungal drugs, azoles exert activity by selective inhibition of the lanosterol
14α-demethylase (P45014DM, CYP51), the key enzyme in sterol biosynthesis, which would cause
the growth inhibition of fungal cells [6]. Based on their broader antifungal spectrum, higher
1

ACCEPTED MANUSCRIPT
efficacy, and lower toxicity, azoles, including fluconazole, itraconazole, voriconazole, and
posaconazole (Fig. 1) are most widely used in antifungal chemotherapy [7]. Besides that,
albaconazole (Fig. 1), one of the new generation azoles, has been investigated in clinical trials due
to its strong therapeutic efficacy for systemic aspergillosis [8, 9].
In recent years, the incidence of Aspergillus infections among immunocompromised patients
has increased dramatically [10]. Unfortunately, this pathogen is intrinsically resistant to
fluconazole. The broader-spectrum azoles, such as itraconazole, voriconazole, and posaconazole
are active against Aspergillus, however, the mortality rate related to aspergillosis still remains
unacceptably high [10]. Therefore, there is still a pressing need for novel antifungal azoles with
excellent activity against a variety of clinical pathogens, particularly, Aspergillus spp.
O
N
N
N
N
N
O
N
N
N
N
N
N
N
OH
O
N
N
O
F
Cl
Itraconazole
F
Fluconazole
Cl
O
N
N
OH
N
N
N
N
N
N
F
O
O
N
N
N
N
N
O
F
N
OH
OH
N
N
Cl
N
F
F
F
N
Voriconazole
Albaconazole
Posaconazole
F
F
Figure 1. Antifungal azoles (names refer to single enantiomers, except itraconazole, which is racemic).
In this line, we have recently reported the discovery and development of several series of novel
fused heterocycles-linked azoles (depicted by general formula I, the most potent compound from
each classes of azoles, 19, 20, 21 and 22 were taken as examples) with high potency against
Candida and Cryptococcus spp. Unfortunately, almost all of the target compounds discussed
above showed only moderate or low potency (MIC = 2.0 ꢀg/ml to 32.0 ꢀg/ml) against Aspergillus
fumigatus compared with albaconazole (MIC = 0.25 ꢀg/ml), the highly potent lead compound [11].
Thus, this has prompted us to continue to undertake structural modification of potent original
compounds in search of novel azoles with improved anti-Aspergillus efficacy.
To further explore the potential mechanism of azoles and provide guidance for new
scaffold design, albaconazole was docked into the active site of Candida albicans CYP51
(CACYP51) [12]. As shown in Fig. 2, the triazole ring and difluorophenyl group of albaconazole
formed a coordination bond and hydrophobic interactions with heme group (Fe atom) and
hydrophobic pocket (F126, I104 and I131), respectively. The C2 hydroxyl group of albaconazole
might form hydrogen-bonding interaction with Y132 mediated by crystal waters [13, 14]. Most
importantly, the quinazolinone ring of albaconazole formed hydrophobic interactions with Y118,
L376 and F380.
2

ACCEPTED MANUSCRIPT
Figure 2. The predicted binding mode of albaconazole and 18a complex (A), albaconazole (B)
and compound 18a (C) in the active site of CACYP51.
Figure 3. Previous selected compounds and new structures.
Based on the structural features of albaconazole and fused heterocycles-linked azoles (I), along
with the binding mode of albaconazole (Fig. 3) [15-17], we aimed to develop a new scaffold (II)
derived from azoles (I), in which the piperazine or piperidine ring (A ring of I) will be replaced by
3

ACCEPTED MANUSCRIPT
a pyrrolidone ring (A ring of II) (Fig. 3). It was our hope that, introduction of a five-membered
heterocyclic ring (B ring of II) fused to the pyrrolidone ring (A ring of II) as a linker, which was
purposely designed to mimic the hydrophobic interactions of quinazolinone ring of albaconazole,
might result in development of more potent compounds with higher anti-Aspergillus efficacy.
Then, during the comprehensive surveys of the B ring of II (such as thiophene ring, thiazole ring,
etc.), as well as the R1 and R2 groups attached to the B ring of II，two more potent classes of novel
azoles, 10a–i and 18a–u, which contained thieno[2,3-c]pyrrolidone and thieno[3,2-c]pyrrolidone
units respectively, caught our interest. This paper describes the synthesis and SAR studies of the
novel antifungal azoles and the selection of 18a for further development.
As shown in Scheme 1, side chain 5 was obtained by slight modifications of published
procedures [18]. Thiophene-3-carboxylic acid 1 was methylated with methyl iodide and
n-butyllithium in THF to yield compound 2, which was converted to bromide-substituted
compound 4 through methyl esterification and bromination reactions. Treatment of 4 with
N-bromosuccinimide (NBS) and benzoyl peroxide (BPO) in CH3CN afforded compound 5 as side
chain.
Scheme 1. Synthetic route of intermediate 5. Reagents and conditions: (a) THF, -78 °C, n-butyl
lithium, 1h, CH3I, -78 °C then room temp, 2h; (b) MeOH, Conc. H2SO4, 0 °C then reflux, 4h; (c)
DMF/AcOH (3:2, v/v), NBS, room temp, overnight; (d) CCl4, BPO, NBS, 80 °C, 12h.
Synthesis of the title compounds 10a–i was carried out according to Scheme 2. The
ring-opening reaction of oxirane 6 [19] with NaN3 followed by catalytic hydrogenation gave
amino 7 [20, 21], which was subsequently treated with compound 5 to afford intermediate 8 [22].
Thus, compound 8 was subjected to hydrolysis reaction and cyclization [23] to obtain
bromine-substituted compound 10a, which was considered for use as a pivotal precursor in Suzuki
coupling to efficiently prepare R2-substituted title compounds 10b–i.
4

ACCEPTED MANUSCRIPT
Scheme 2. Synthetic route of the title compounds 10a-i. Reagents and conditions: (a) NaN3,
NH4Cl, DMF, 100 °C, 6 h; (b) H2, Pd/C, room temp, overnight; (c) K2CO3, CH3CN, 0 °C then
room temp, overnight; (d) NaOH, MeOH, THF, 0 degree then room temp, 8h; (e) CH2Cl2, EDCI,
HOBt, Et3N, 0 °C then room temp, 8h; (f) R2B(OH)2 or R2B(pin), Cs2CO3, Pd(PPh3)4,
1,4-dioxane/H2O, 80 °C, 10h.
To further investigate the effect of different types of linkers (fused heterocycles of II) on their
antifungal activity, many novel azoles 18a–u featuring R1 and R2-substituted
thieno[3,2-c]pyrrolidone side chains were synthesized. As outlined in Scheme 3, intermediate 15
was synthesized via four steps [24]. Bromidation of the starting material 11 with Br2 in CHCl3,
followed by oxidation with NaH2PO4/H2O2 and methyl esterification afforded methyl ester 14. In
the presence of BPO, compound 14 was subsequently reacted with NBS in CCl4 to yield side
chain 15.
Scheme 3. Synthetic route of intermediate 15. Reagents and conditions: (a) Br2, CHCl3, 0 °C then
reflux, 4h; (b) CH3CN, NaH2PO4, H2O2, 0 °C then room temp, 8h; (c) MeOH, Conc. H2SO4, 0 °C
then reflux, 4h; (d) CCl4, BPO, NBS, 80 °C, 12h.
As shown in Scheme 4, another pivotal precursor, 18a was prepared from amino 7 by using
procedures similar to those described above. Afterward, the title compounds 18b–u were prepared
conveniently by Suzuki coupling of compound 18a with various boric acid esters or boric acid. All
the new compounds described above were characterized by NMR and ESI-MS spectroscopic
analysis.
The in vitro minimum inhibitory concentration (MIC) of all title compounds were determined
by the method recommended by the National Committee for Clinical Laboratory Standards
5

ACCEPTED MANUSCRIPT
(NCCLS) using the serial dilution method in 96-well microtest plates [25, 26]. All of target
compounds were evaluated for their antifungal activity against a panel of pathogenic fungi
obtained from the American Type Culture Collection (ATCC) or clinical isolates. Fluconazole,
voriconazole, and albaconazole purchased from respective manufacturers, as well as previously
prepared azoles 19–22 [11], were served as the positive control drugs. The results of in vitro
antifungal activities are summarized in Tables 1–2.
Scheme 4. Synthetic route of the title compounds 18a-u. Reagents and conditions: (a) K2CO3,
CH3CN, 0 °C then room temp, overnight; (b) NaOH, MeOH, THF, 0 degree then room temp, 6h;
(c) CH2Cl2, EDCI, HOBt, Et3N, 0 °C then room temp, 8h; (d) R2B(OH)2 or R2B(pin), Cs2CO3,
Pd(PPh3)4, 1,4-dioxane/H2O, 80 °C, 10h.
As summarized in Table 1, almost all of thieno[2,3-c]pyrrolidone derivatives 10a–I exhibited
remarkable activity against all of the tested strains, with the exception of Aspergillus fumigatus,
which are superior or comparable to those of the reference drugs, including compounds 19–22,
voriconazole and albaconazole. Generally, the aryl-substituted analogs 10b–I were more potent
than bromide-substituted compound 10a. In addition, these analogs substituted with phenyl ring
(10b), as well as some different types of nitrogen aromatic heterocycles (10c–i), showed similar
activity.
Table 1 In vitro antifungal activity of thieno[2,3-c]pyrrolidone derivatives. (MIC, ꢀg/ml)a
6

ACCEPTED MANUSCRIPT
S
S
OH
OH
R2
Br
N
N
N
F
N
F
N
N
N
N
O
O
F
F
10b-i
10a
C. alb
SC5314
C. par
22019
0.0625
C.neo
32609
0.125
C.gla
A.fum
compd
R2
-
Y0109
537
7544
10a
0.03125 0.0625
0.125
32
32
10b
phenyl-
0.0156
0.0156
0.0156
0.0156
0.0156
0.0156
0.0078
0.0156
0.0156
0.03125 0.03125 0.0625
0.0625
0.0625
0.0156
0.0156
0.0625
0.0156
10c
4-pyridyl-
3-pyridyl-
0.0156
0.0156
0.0156
0.0156
0.0156
0.0156
0.0156
0.0156
0.03125 0.0156
>64
16
10d
0.0156
0.0156
0.0156
0.0156
0.0156
0.0156
0.0156
0.0625
0.125
0.5
0.0156
0.0078
0.0156
0.0156
0.0078
0.0156
0.0625
0.0625
0.125
0.5
10e
5-pyrimidinyl-
32
10f
2-CN-5-pyridyl-
32
10g
2-methoxy-5-pyridyl-
64
10h
3-F-5-pyridyl-
0.0625 >64
10i
2-methoxy-5-pyrimidinyl-
0.0156
0.0156
0.0625
1
16
2
19
-
-
-
-
-
-
-
20
0.03125 0.0625
2
21
0.25
0.125
0.5
32
>32
>64
22
0.25
4
fluconazole
voriconazole
albaconazole
0.25
0.25
1
4
1
0.0156
0.0078
0.0156
0.0156
0.0156
0.0156
0.0156
0.0078
0.0156
0.0156
0.25
0.25
aAbbreviations: C.alb., Candida albicans; C.par., Candida parapsilosis; C.neo., Cryptococcus
neoformans; C.gla., Candida glabrata; A.fum., Aspergillus fumigatus.
Apparently, as all of the target compounds 10a–i discussed above (Table 1) were ineffective
against Aspergillus fumigatus, a series of novel azole 18a–u were prepared through the
bioisosteric approach reported previously [11]. Similar to compounds 10a–i, most of
thieno[3,2-c]pyrrolidone derivatives showed excellent in vitro activity (Table 2) against Candida
spp. and Cryptococcus neoforman. Most importantly, some azoles also had high or moderate
potency against Aspergillus fumigatus. Compared with the most potent compound 18a (R2 = Br ),
introduction of several types of phenyl groups (18b–l) on position 2 of the thiophene ring of
thieno[3,2-c]pyrrolidone derivatives resulted in a slight decrease in activity. Substitution within
this phenyl ring was also investigated. We found that the in vitro antifungal activity of
para-substituted derivatives were stronger than those of meta-substituted compounds. (18c and
18d, 18e and18f, 18g and 18h, 18i and 18j, 18k and 18l ). Compounds (18g, 18i, 18k or 18h, 18j,
18l ) with a phenyl ring substituted with an electron-withdrawing group (–F, –Cl, –CN) exhibited
activity similar to those observed in electron-donating groups (–CH3, –CH3O) substituted phenyl
analogs (18c, 18e or 18d, 18f). In addition, most of the pyridine and pyrimidine ring analogs
(18m–u) were tested and proved to be comparable to those of the phenyl analogs, except for 18p
and18s, which featured an electron-withdrawing group (–CN, –F) at the ortho-position of the
meta-pyridyl ring. Compared with the pyridine ring analogs 18p–t (–CN, –CH3O, –F) and
pyrimidine ring analog 18u (–CH3O), unsubstituted aryl analogs 18m–o showed relatively
stronger activity.
Table 2 In vitro antifungal activity of thieno[3,2-c]pyrrolidone derivatives. (MIC, ꢀg/ml)a
7

ACCEPTED MANUSCRIPT
aAbbreviations: C.alb., Candida albicans; C.par., Candida parapsilosis; C.neo., Cryptococcus
neoformans; C.gla., Candida glabrata; A.fum., Aspergillus fumigatus.
In order to further investigate the binding mode and the SARs of thieno[3,2-c]pyrrolidone
derivatives 18a–u, a representative compound, 18a, was docked into the active site of Candida
albicans CYP51 (CACYP51) [12]. Compound 18a and albaconazole exhibited similar
conformations as their triazole ring and difluorophenyl moiety superimposed well (Fig. 2). A
conserved water molecule in CYP51 [27] formed three hydrogen bonds with C2 hydroxyl group of
18a, propionate of porphyrin ring D and Y132. The water-mediated hydrogen bond network
among 18a, heme group and CYP51 plays important roles in strengthening their binding affinity.
The variable part, bromine-substituted thieno[3,2-c]pyrrolidone nuclei formed hydrophobic
interactions with Y118, L376 and F380, similar to the quinazolinone ring of albaconazole.
Moreover, the bromine atom of compound 18a formed additional hydrophobic interactions with
M508. These results may provide a good explanation for the excellent in vitro activity of
compound 18a.
Because of its significant activity and broad antifungal spectrum, compound 18a was evaluated
for prediction of its metabolic stability using human liver microsomes (Table 3). With high
metabolic bioavailability (MF =80% ), a long half-life (t1/2 = 420.1 min) and low in vitro liver
8

ACCEPTED MANUSCRIPT
microsomal clearance (Clint = 5.0 ꢀL/min/mg) in human liver microsomes , compound 18a is
regarded metabolically sufficiently stable [28-30].
Table 3 Half-lives and intrinsic clearances of compounds 18a in human liver microsomesa
a0.33 mg/mL microsomal protein, NADP+-regenerating system, [inhibitor], 0.1 ꢀM, incubation
at 37 °C, samples taken at 0, 7, 17, 30, and 60 min, determination of parent compound by MS. bt1/2:
c
d
elimination half-life in human liver microsomes. Clint: intrinsic body clearance. MF: metabolic
bioavailability
In conclusion, we identified two series of novel azoles (II) as potent antifungal agents
containing a thienopyrrolidone nucleus. Summarizing the results of Tables 1–2, we can conclude
that both potent in vitro antifungal activity and broad spectrum were well attained when different
types of fused heterocycles linkers of I (19–22) were replaced with thienopyrrolidone nucleus. In
addition, the linker of II, thieno[3,2-c]pyrrolidone nucleus has been identified for anti-Aspergillus
activity. Among these compounds, bromide-substituted analog 18a displayed the most remarkable
in vitro activity against Candida spp., Cryptococcus neoformans and Aspergillus fumigatus, which
is superior or comparable to those of the reference drugs voriconazole and albaconazole. The
observation described above was also clarified by a computational molecular docking of azoles
with CYP51. Additionally, 18a was found to be stable metabolically in human liver microsomes.
Further developments of compound 18a are ongoing in our laboratory.
Acknowledgment
The authors thank Prof. Cheng Luo and Ran Zhou from Drug Discovery and Design Center,
Shanghai Institute of Materia Medica, for molecular docking experiments; We also thank Ran
Zhou for writing the script for the molecular docking analysis presented in the paper. We also
thank Dr. Jia Liu for performing the metabolic stability assay. We are grateful to the National
Science and Technology Major Project for the support of this research. The project described was
supported by Key New Drug Creation and Manufacturing Program, China (No.
2014ZX09101004-003). This work was also supported by the National Natural ScienceFoundation
of China (No. 21402223).
Supplementary data
Experimental details and spectroscopic characterization of compounds involved in the synthesis
are available online.
References and notes
[1] M.K. Kathiravan, A.B. Salake, A.S. Chothe, P.B. Dudhe, R.P. Watode, M.S. Mukta, S. Gadhwe,
The biology and chemistry of antifungal agents: A review, Bioorg. Med. Chem. 20 (2012)
5678-5698.
9

ACCEPTED MANUSCRIPT
[2] A.H. Groll, J. Lumb, New developments in invasive fungal disease, Future Microbiol. 7 (2012)
179-184.
[3] O. Turel, Newer antifungal agents, Expert Rev. Anti-Infect. Ther. 9 (2011) 325-338.
[4] L.A. Sorbera, J. Aravamudan, E. Rosa, Therapeutic target for candidiasis, Drugs Future 36
(2011) 627-630.
[5] J.-P. Latgé, Aspergillus fumigatus and Aspergillosis, Clin. Microbiol. Rev. 12 (1999) 310-350.
[6] L. Meerpoel, P. Lewi, Conazoles, Molecules 15 (2010) 4129-4188.
[7] S. Wang, G. Jin, W. Wang, L. Zhu, Y. Zhang, G. Dong, Y. Liu, C. Zhuang, Z. Miao, J. Yao, W.
Zhang, C. Sheng, Design, synthesis and structure–activity relationships of new triazole derivatives
containing N-substituted phenoxypropylamino side chains, Eur. J. Med. Chem. 53 (2012)
292-299.
[8] C. Girmenia, New generation azole antifungals in clinical investigation, Expert Opin. Invest.
Drugs 18 (2009) 1279-1295.
[9] R. Guillon, F. Pagniez, C. Picot, D. Hedou, A. Tonnerre, E. Chosson, M. Duflos, T. Besson, C.
Loge, P. Le Pape, Discovery of a novel broad-spectrum antifungal agent derived from
albaconazole, ACS Med. Chem. Lett. 4 (2013) 288-292.
[10] I. Hadrich, F. Makni, S. Neji, S. Abbes, F. Cheikhrouhou, H. Trabelsi, H. Sellami, A. Ayadi,
Invasive Aspergillosis: Resistance to Antifungal Drugs, Mycopathologia 174 (2012) 131-141.
[11] X. Cao, Z. Sun, Y. Cao, R. Wang, T. Cai, W. Chu, W. Hu, Y. Yang, Design, Synthesis, and
Structure–Activity Relationship Studies of Novel Fused Heterocycles-Linked Triazoles with Good
Activity and Water Solubility, J. Med. Chem. 57 (2014) 3687-3706.
[12] B.C. Monk, T.M. Tomasiak, M.V. Keniya, F.U. Huschmann, J.D. Tyndall, J.D. O'Connell, 3rd,
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. USA 111 (2014) 3865-3870.
[13] C. Sheng, W. Zhang, H. Ji, M. Zhang, Y. Song, H. Xu, J. Zhu, Z. Miao, Q. Jiang, J. Yao, Y.
Zhou, J. Zhu, J. Lü, Structure-Based Optimization of Azole Antifungal Agents by CoMFA,
CoMSIA, and Molecular Docking, J. Med. Chem. 49 (2006) 2512-2525.
[14] H. Ji, W. Zhang, Y. Zhou, M. Zhang, J. Zhu, Y. Song, J. Lu, J. Zhu, A Three-Dimensional
Model of Lanosterol 14α-Demethylase of Candida albicans and Its Interaction with Azole
Antifungals, J. Med. Chem. 43 (2000) 2493-2505.
[15] X. Chai, J. Zhang, Y. Cao, Y. Zou, Q. Wu, D. Zhang, Y. Jiang, Q. Sun, New azoles with
antifungal activity: Design, synthesis, and molecular docking, Bioorg. Med. Chem. Lett. 21 (2011)
686-689.
[16] Y. Zou, Q. Zhao, J. Liao, H. Hu, S. Yu, X. Chai, M. Xu, Q. Wu, New triazole derivatives as
antifungal agents: Synthesis via click reaction, in vitro evaluation and molecular docking studies,
Bioorg. Med. Chem. Lett. 22 (2012) 2959-2962.
[17] Y. Jiang, J. Zhang, Y. Cao, X. Chai, Y. Zou, Q. Wu, D. Zhang, Y. Jiang, Q. Sun, Synthesis, in
vitro evaluation and molecular docking studies of new triazole derivatives as antifungal agents,
Bioorg. Med. Chem. Lett. 21 (2011) 4471-4475.
[18] M. Lang, M.H.J. Seifert, K.K. Wolf, A. Aschenbrenner, R. Baumgartner, T. Wieber, V.
Trentinaglia, M. Blisse, N. Tajima, T. Yamashita, D. Vitt, H. Noda, Discovery and hit-to-lead
optimization of novel allosteric glucokinase activators, Bioorg. Med. Chem. Lett. 21 (2011)
5417-5422.
10

ACCEPTED MANUSCRIPT
[19] J. Pesti, C.-K. Chen, L. Spangler, A.J. DelMonte, S. Benoit, D. Berglund, J. Bien, P.
Brodfuehrer, Y. Chan, E. Corbett, C. Costello, P. DeMena, R.P. Discordia, W. Doubleday, Z. Gao,
S. Gingras, J. Grosso, O. Haas, D. Kacsur, C. Lai, S. Leung, M. Miller, J. Muslehiddinoglu, N.
Nguyen, J. Qiu, M. Olzog, E. Reiff, D. Thoraval, M. Totleben, D. Vanyo, P. Vemishetti, J. Wasylak,
C. Wei, The Process Development of Ravuconazole: An Efficient Multikilogram Scale Preparation
of an Antifungal Agent (1), Org. Process Res. Dev. 13 (2009) 716-728.
[20] T. Konosu, T. Miyaoka, Y. Tajima, S. Oida, Triazole antifungals. III. Stereocontrolled
synthesis of an optically active triazolylmethyloxirane precursor to antifungal oxazolidine
derivatives, Chem. Pharm. Bull. 39 (1991) 2241-2246.
[21] T. Konosu, Y. Tajima, N. Takeda, T. Miyaoka, M. Kasahara, H. Yasuda, S. Oida, Triazole
antifungals. IV. Synthesis and antifungal activities of 3-acylamino-2-aryl-2-butanol derivatives,
Chem. Pharm. Bull. 39 (1991) 2581-2589.
[22] B.S. Freeze, M. Hirose, L. Hong Myung, T.B. Sells, Z. Shi, L.R. Takaoka, S. Vyskocil, T. Xu,
Preparation of heteroaryls as PI3K and mTor kinases inhibitors for treating proliferative,
inflammatory or cardiovascular disorders, in, Millennium Pharmaceuticals, Inc., USA . 2012, pp.
268pp.
[23] M. Prat, M.A. Buil, M.D. Fernandez, J. Castro, J.M. Monleon, L. Tort, G. Casals, M. Ferrer,
J.M. Huerta, S. Espinosa, M. Lopez, V. Segarra, A. Gavalda, M. Miralpeix, I. Ramos, D. Vilella,
M. Gonzalez, M. Cordoba, A. Cardenas, F. Anton, J. Beleta, H. Ryder, Discovery of novel
quaternary ammonium derivatives of (3R)-quinuclidinyl carbamates as potent and long acting
muscarinic antagonists, Bioorg. Med. Chem. Lett. 21 (2011) 3457-3461.
[24] A. Pasternak, R.K. Dejesus, Y. Zhu, L. Yang, S. Walsh, B. Pio, A. Shahripour, H. Tang, K.
Belyk, D. Kim, Inhibitors of the renal outer medullary potassium channel, in, Merck Sharp &
Dohme Corp., USA . 2012, pp. 229pp.
[25] Clinical and Laboratory Standards Institute/National Committee for Clinical Laboratory
Standards: Reference method for broth dilution antifungal susceptibility testing of Yeast. Approved
Standard, edn 3; Document M27-A3. Wayne, PA: Clinical and Laboratory Standards Institute;
2009.
[26] Clinical and Laboratory Standards Institute/National Committee for Clinical Laboratory
Standards: Reference method for broth dilution antifungal susceptibility testing of Yeast. Approved
Standard, edn 3; Document M38-A2. Wayne, PA: Clinical and Laboratory Standards Institute;
2008.
[27] N. Strushkevich, S.A. Usanov, H.W. Park, Structural basis of human CYP51 inhibition by
antifungal azoles, J. Mol. Biol. 397 (2010) 1067-1078.
[28] R.S. Obach, Prediction of human clearance of twenty-nine drugs from hepatic microsomal
intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding to
microsomes, Drug Metab. Dispos. 27 (1999) 1350-1359.
[29] Y.Y. Lau, G. Krishna, N.P. Yumibe, D.E. Grotz, E. Sapidou, L. Norton, I. Chu, C. Chen, A.D.
Soares, C.C. Lin, The use of in vitro metabolic stability for rapid selection of compounds in early
discovery based on their expected hepatic extraction ratios, Pharm. Res. 19 (2002) 1606-1610.
[30] A. Fritsche, A.S. Elfringhoff, J. Fabian, M. Lehr, 1-(2-Carboxyindol-5-yloxy)propan-2-ones
as inhibitors of human cytosolic phospholipase A2α: Synthesis, biological activity, metabolic
stability, and solubility, Bioorg. Med. Chem. 16 (2008) 3489-3500.
11

ACCEPTED MANUSCRIPT
Design, synthesis, and structure-activity relationship studies of novel
thienopyrrolidone derivatives with strong antifungal activity against Aspergillus
fumigates
Xufeng Caoa,†, Yuanyuan Xua,†, Yongbing Caob, Ruilian Wangb, Ran Zhouc, Wenjing Chua, and
Yushe Yang a,*
aState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai
Institute for Biological Sciences, Chinese Academy of Sciences, 555 Zuchong Zhi Road, Shanghai
201203, China
bSchool of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433,
China
cDrug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, 555 Zuchong Zhi Road, Shanghai 201203,
China
∗Corresponding author: Yushe Yang. Tel: 86-21-5080 6786. Fax: 86-21-5080 6786. E-mail:
ysyang@simm.ac.cn.
†These authors contributed equally to this work.
Highlights
• Two series of novel azoles featuring thienopyrrolidone nuclei were designed and synthesized.
• A molecular docking of azoles with CYP51 was conducted to understand the mechanism of
action and provide guidance for new scaffold design.
• The target compounds exhibited excellent activity with a broad spectrum.
• Compound 18a exhibit significant anti-Aspergillus efficacy (MIC = 0.25 µg/ml).
• Compound 18a was metabolically stable in human liver microsomes (MF =80% ).
1

ACCEPTED MANUSCRIPT
Design, synthesis, and structure-activity relationship studies of novel thienopyrrolidone
derivatives with strong antifungal activity against Aspergillus fumigates
Xufeng Caoa,†, Yuanyuan Xua,†, Yongbing Caob, Ruilian Wangb, Ran Zhouc, Wenjing Chua, and
Yushe Yanga,*
aState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai
Institute for Biological Sciences, Chinese Academy of Sciences, 555 Zuchong Zhi Road,
Shanghai 201203, China
bSchool of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433,
China
cDrug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, 555 Zuchong Zhi Road, Shanghai 201203,
China
∗Corresponding author: Yushe Yang. Tel: 86-21-5080 6786. Fax: 86-21-5080 6786. E-mail:
ysyang@simm.ac.cn.
†These authors contributed equally to this work.
Experimental details
I. Biological test
Minimum Inhibitory Concentration Testing. The in vitro minimum inhibitory concentration
(MIC) of all title compounds were determined by the method recommended by the National
Committee for Clinical Laboratory Standards (NCCLS) using the serial dilution method in 96-
well microtest plates [1, 2]. The tested fungi species included six pathogenic fungi. Candida
albicans, Candida parapsilosis 22019 and Cryptococcus neoformans 32609 were purchased
from the American Type Culture Collection (ATTC), and other strains were provided by
Shanghai Changhai Hospital. Candida parapsilosis 22019 was used as the quality-controlled
strain, and tested in each assay. Fluconazole, voriconazole, and albaconazole purchased from
respective manufacturers, as well as previously prepared azoles 19–21, were served as the
positive control drugs. All of test compounds were dissolved in dimethyl sulfoxide (DMSO),
serially diluted in growth medium. The strains were incubated at 35 °C. The MIC values was
determined at 24 h for Candida spp., at 48 h for Aspergillus fumigatus, and 72 h for
Cryptococcus neoformans 32609.
S1

ACCEPTED MANUSCRIPT
Metabolic Stability Assay. The assay was performed with liver microsomes pooled from
human (adult male & female, catalogue no. 99268, BD Gentest, USA). The incubation was
performed as follows: microsomes in 0.1 M trishydroxymethyl aminomethane/hydrochloric acid
buffer pH 7.4 (0.33 mg/mL microsomal protein), cofactor MgCl2 (5 mM), tested compound
(final concentration 0.1 ꢀM, cosolvent (0.01% DMSO), 0.005% bovine serum albumin), and
then NADPH (1 mM) at 37 °C for 60 min. The reaction can be started by the addition of liver
microsomes or the tested compound or NADPH. Aliquots were sampled at 0, 7, 17, 30, and 60
min incubation, and enzymatic reaction was stopped by protein precipitation in methanol. After
centrifugation, samples were then analyzed by LC/MS/MS. The assay evaluated the metabolic
stability of compounds by measuring the in vitro half-life (t1/2) and human liver microsomal
clearance (Clint) as previously described [3-5]
Molecular Docking. Structure of Candia albicans CYP51 was built based on crystal structure
of S. cerevisiae CYP51 [6] (PDB code: 4K0F) using SWISS-MODEL [7]. GOLD Suite v5.0 was
utilized to perform docking analysis. Iron atom of heme group was treated as metal ion to predict
the coordination geometry. The position of C1 atom of the ligand in template CYP51 was
selected as the centroid of the binding site. GoldScore was employed to evaluate the fitness.
II. Chemistry
General Chemical Methods. Compounds not described below were purchased from
commercial vendors. Provided samples were of greater than 95% purity, as determined by the
suppliers, via NMR. Melting points (uncorrected) were determined on an X-4 melting point
apparatus. Optical rotations were determined with a Perkin-Elmer 241 polarimeter at 22 °C and
at 589 nm using a sodium lamp and a 1 ml cell. Data are reported as follows: [α]22
D
1
(concentration g/100ml, solvent). H NMR spectral data and 13C NMR spectral data were
recorded on a Bruker 300 NMR or a Bruker 400 NMR or a Bruker 500 NMR spectrometer using
TMS as an internal standard, chemical shifts are given in parts per million (d) values and
coupling constants (J) in Hertz. EI-MS spectra were obtained on a Finnigan MAT 95 mass
spectrometer and ESI-MS spectra were obtained on a Krats MS 80 mass spectrometer. Elemental
analysis was obtained via a vario EL spectrometer. HPLC analysis was conducted for all assayed
compounds on an Agilent 1100 series LC system (PLATISILTM ODS 5ꢀm 250 × 4.6 mm) with
two solvent systems (acetonitrile/water or methanol/buffer (0.1% CF3COOH in water)). All the
S2

ACCEPTED MANUSCRIPT
assayed compounds possess ≥ 95% purity. Silica gel thin-layer chromatography was performed
on precoated plates GF254 (Qindao Haiyang Chemical, China). Column chromatography was
performed on silica gel H (200-300 mesh) , and the solvent proportions were expressed on a
volume:volume basis. Chemicals and solvents used were commercially available without any
pretreatment.
2-methylthiophene-3-carboxylic acid (2). 1 (15.00 g, 0.12 mol) was dissolved in THF (50
ml). Under argon protection, n-butyl lithium (2.0 M THF solution, 120 ml) was added dropwise
at -78 °C. The reaction mixture was stirred for 1 h at -78 °C. To this mixture was added CH3I
(8.7 ml, 0.14 mol), then the reaction mixture was allowed to warm to room temperature and
stirred for 2 hours. Slowly quenched the reaction with hydrogen chloride (2.0 M ethyl acetate
solution), then stirred the mixture at room temperature for 30 min before concentrating in vacuo.
The residue was diluted with H2O (100 ml) and extracted with ethyl acetate (200 ml). The
combined organic layers were washed with H2O (50 ml ×2) and brine (50 ml ×2), dried over
anhydrous Na2SO4, and filtrated, then the solvent was evaporated under reduced pressure to give
a yellow crude solid, which was purified by recrystallization from acetic acid/H2O to afford 2
(14.00 g, 82.1% ) as a white solid: mp 108-110 °C. 1H NMR (400 MHz, CDCl3)：δ 7.45 (d, J =
5.5 Hz, 1H), 7.01 (d, J = 5.5, 1H), 2.73 (s, 3H). MS (EI) m/z: 142 (M+).
methyl 2-methylthiophene-3-carboxylate (3). 2 (14.00 g, 98.60 mmol) was dissolved in
methanol (60 ml). Concentrated sulfuric acid (30 ml) was added dropwise at 0 °C. The reaction
mixture was stirred for 4 h at reflux, then was cooled to room temperature, and the solvent was
evaporated under reduced pressure. The residue was partitioned between CH2Cl2 (200 ml) and
H2O (60 ml). The organic phase was washed with H2O (50 ml), saturated aqueous sodium
bicarbonate solution (50 ml) and saturated brine solution (50 ml), dried over Na2SO4, and
filtrated, then the solvent was evaporated under reduced pressure to give 3 (12.00 g, 78.0%) as a
colorless viscous oil. 1H NMR (400 MHz, CDCl3)：δ 7.38 (d, J = 5.5 Hz, 1H), 6.98 (d, J = 5.1,
1H), 3.85 (s, 3H), 2.73 (s, 3H). MS (EI) m/z: 156 (M+).
methyl 5-bromo-2-methylthiophene-3-carboxylate (4). 3 (12.00 g, 76.82 mmol) was
dissolved in 100 ml DMF/acetic acid (3:2). N-bromosuccinimide (13.70 g, 77.00 mmol) was
added at room temperature and stirred overnight. The reaction mixture was diluted with H2O (30
S3

ACCEPTED MANUSCRIPT
ml) and extracted with ethyl acetate (60 ml ×2). The organic phase was washed with saturated
aqueous sodium bisulfate solution (30 ml) and saturated brine solution (30 ml), dried over
Na2SO4, and filtrated, then the solvent was evaporated under reduced pressure to give 4 (16.10 g,
1
789.1%) as a pale yellow viscous oil. H NMR (400 MHz, CDCl3)：δ 7.33 (s, 1H), 3.84 (s, 3H),
2.67 (s, 3H). MS (EI) m/z: 235 (M+).
methyl 5-bromo-2-(bromomethyl)thiophene-3-carboxylate (5). 4 (10.00 g, 42.53 mmol)
and benzoyl peroxide (1.10 g, 4.53 mmol) was dissolved in 150 ml CCl4. N-bromosuccinimide
(7.96 g, 44.72 mmol) was added in batches at room temperature. The reaction mixture was
allowed to warm to 80 °C and stirred for 12 hours. After cooling, the solvent was evaporated
under reduced pressure. The residue was partitioned between ethyl acetate (200 ml) and H2O (80
ml). The organic phase was washed with H2O (60 ml) and saturated brine solution (60 ml), dried
over Na2SO4, and filtrated, then the solvent was evaporated under reduced pressure. The residue
was purified by silica gel column chromatography (petroleum: ethyl acetate 80:1~60:1) to give 5
1
(8.23 g, 61.6%) as a white solid: mp 95-96 °C. H NMR (400 MHz, CDCl3)：δ 7.35 (s, 1H),
5.00 (s, 2H), 3.86 (s, 3H). MS (EI) m/z: 312 (M+).
(2R,3R)-3-amino-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol (7). A mixture
of epoxide 6 (10.00 g, 40.00 mmol), sodium azide (3.90 g, 60.00 mmol), ammonium chloride
(2.73 g, 51.00 mmol) and DMF(60.0 ml) was stirred at 80 °C for 10h. After cooling, the mixture
was partitioned between ethyl acetate (200 ml ×2) and brine (80 ml). The organic phase was
washed with H2O (60 ml ×2) and saturated brine solution (60 ml ×2), dried over Na2SO4, and
filtrated, then the solvent was evaporated under reduced pressure to afford dark brown oil, which
was used for the following reaction without further purification.
The crude solid was dissolved in methanol (40 ml), to the solution was added 10% palladium
on carbon (1.60 g) at room temperature. The mixture was stirred under hydrogen at 1 atm
overnight and then filtered, concentrated under reduced pressure to give 7 (7.35 g, 68.8%) as a
1
white solid: mp 146-148°C. [α]22D –71.0°(c 0.5, CHCl3). H NMR (300 MHz, DMSO) δ 8.25 (s,
1H), 7.63 (s, 1H), 7.25 (dd, J = 16.0, 8.9 Hz, 1H), 7.17-7.06 (m, 1H), 6.86 (td, J = 8.5, 2.3 Hz,
1H), 5.48 (s, 1H), 4.65 (q, J = 14.4 Hz, 2H), 3.41 (dd, J = 6.5, 2.7 Hz, 1H), 0.69 (d, J = 6.5 Hz,
3H). MS (EI) m/z: 268(M+).
S4

ACCEPTED MANUSCRIPT
methyl 5-bromo-2-[{(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)-
butan-2-ylamino}methyl]thiophene-3-carboxylate (8). Under nitrogen flow, K2CO3 (0.84g,
6.05 mmol) was added to a stirred solution of 7 (1.35 g, 5.03 mmol) and 5 (1.74 g, 5.56 mmol) in
CH3CN (50 ml) at 0 °C. After addition, the reaction mixture was was allowed to warm to room
temperature and stirred overnight. The solvent was evaporated under reduced pressure. The
residue was partitioned between ethyl acetate (60 ml) and H2O (30 ml). The organic phase was
washed with saturated brine solution (30 ml), dried over Na2SO4, and filtrated, then the solvent
was evaporated under reduced pressure. The residue was purified by silica gel column
chromatography (petroleum: ethyl acetate 6:1~4:1) to give 8 (1.68 g, 66.6%) as a colorless oil.
1H NMR (400 MHz, CDCl3)：δ 7.83 (s, 1H), 7.77 (s, 1H), 7.38-7.31 (m, 2H), 6.75-6.63 (m, 2H),
5.08 (d, J = 14.3, 1H), 4.89-4.84 (m, 2H), 4.53 (d, J = 17.0, 1H), 4.09 (d, J = 17.0, 1H), 3.80 (s,
3H), 3.26 (q, J = 6.9, 1H), 0.91 (d, J = 6.6 Hz, 3H). MS (EI) m/z: 500 (M+).
5-bromo-2-[{(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)-butan-2-
ylamino}methyl]thiophene-3-carboxylic acid (9). NaOH (1.0 M aqueous solution, 7.85 ml)
was added to a stirred mixture of 8 (786.50 mg，1.57 mmol) and MeOH/THF (10 ml/10 ml) at
0°C. The mixture was stirred for 8 h at room temperature, then the solvent was evaporated under
reduced pressure. The residue was added to ice and acided (PH = 2) by addition of hydrogen
chloride (1.0 M aqueous solution). The aqueous solution was extracted with ethyl
acetate/isopropanol (45 ml/5 ml), the combined organic phase was washed with saturated brine
solution, dried over Na2SO4, and filtrated, the solution was concentrated in vacuo to give 9
1
(650.00 mg, 85.0%) as a colorless viscous oil. H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 7.74
(s, 1H), 7.46-7.36 (m, 2H), 6.83-6.71 (m, 2H), 5.23-5.06 (m, 3H), 4.59 (d, J = 19.1 Hz, 1H), 4.26
(d, J = 14.6 Hz, 1H), 1.13 (d, J = 7.2 Hz, 3H). MS (EI) m/z: 486 (M+).
2-bromo-5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-
yl]-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10a). HOBt (66.00 mg, 0.49 mmol) and
triethylamine (125.00 mg, 1.23 mmol) were added to a stirred mixture of 9 (195.00 mg, 0.40
mmol) and CH2Cl2 (10 ml) at 0°C. The mixture was stirred for 0.5 h at room temperature, then
the solvent was evaporated under reduced pressure. To this mixture was added EDCI (118.00 mg,
S5

ACCEPTED MANUSCRIPT
0.61 mmol), then the reaction mixture was allowed to warm to room temperature and stirred for
8 hours. The organic layers were washed with H2O (20 ml×2) and hydrogen chloride (1.0 M
aqueous solution), then neutralized by addition of saturated aqueous sodium bicarbonate solution.
The organic phase was washed with saturated brine solution (20 ml), dried over Na2SO4, and
filtrated, then the solvent was evaporated under reduced pressure. The residue was purified by
silica gel column chromatography (CH2Cl2: MeOH 200:1~100:1) to give 10a (131.00 mg, 69.3%)
as a pale yellow solid: mp 136-138 °C. 1H NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 7.67 (s, 1H),
7.38 (dd, J = 14.8, 8.0 Hz, 1H), 7.23 (s, 1H), 6.78-6.68 (m, 2H), 5.11 (d, J = 14.2 Hz, 1H), 4.98
(d, J = 17.9 Hz, 2H), 4.55 (d, J = 18.6 Hz, 1H), 4.21 (d, J = 14.2 Hz, 1H), 1.08 (d, J = 7.1 Hz,
3H). 13C NMR (101 MHz, CDCl3): δ 165.44, 158.00, 153.62, 151.52, 140.85, 130.31, 128.30,
123.21, 123.14 (2C, overlap), 118.14, 111.86, 104.35, 79.85, 55.43 (2C, overlap), 24.87, 13.52.
MS (EI) m/z: 468 (M+). HRMS (EI): Anal. Calcd for C18H15BrF2N4O2S: 468.0165, Found:
468.0116.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-phen-
yl-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10b). A mixture of 10a (100.00 mg, 0.21 mmol),
cesium carbonate (139.20 mg, 0.43 mmol), phenylboronic acid (34.00 mg, 0.28 mmol) and
tetrakis(triphenylphosphine)palladium (0) (25.00 mg, 0.02 mmol) in dioxane (15 ml) and H2O (5
ml) was degassed and flushed with argon. The mixture was hearted at 80 °C for 10 h. The
solvent was evaporated under reduced pressure. The residue was diluted with H2O (30 ml) and
extracted with ethyl acetate (40 ml ×2). The combined organic layers were washed with H2O (20
ml ×2) and brine (20 ml ×2), dried over anhydrous Na2SO4, and filtrated, then the solvent was
evaporated under reduced pressure. The residue was purified by silica gel column
chromatography (CH2Cl2: MeOH 200:1~50:1) to give 10b (58.00 mg, 59.2%) as a white solid:
mp 122-124 °C. 1H NMR (500 MHz, DMSO) δ8.27 (s, 1H)，7.81-7.26 (m, 7H), 7.25(d, J = 15.1
Hz, 2H)，6.93 (s, 1H), 5.01 (d, J = 16.1 Hz, 2H), 4.86 (d, J = 13.5 Hz, 1H), 4.71 (d, J = 12.6 Hz,
1H), 4.39 (d, J = 12.9 Hz, 1H), 1.16 (d, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 165.12,
150.68, 150.40, 148.51, 144.68, 139.78, 132.00, 131.41, 129.22, 125.55, 124.43, 124.31 (2C,
overlap), 110.81, 103.96, 78.65, 55.09 (2C, overlap), 39.75, 13.37. MS (EI) m/z: 466 (M+).
HRMS (EI): Anal. Calcd for C24H20F2N4O2S: 466.1318, Found: 466.1329.
S6

ACCEPTED MANUSCRIPT
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(pyri-
din-4-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10c). 10c (63.00 mg, 63.3%) was
prepared from 10a (100.00 mg, 0.21 mmol) and 4-pyridinylboronic acid (37.82 mg, 0.31 mmol)
1
in the same manner as described for 10b. white solid: mp 136-139 °C. H NMR (500 MHz,
CDCl3) δ 8.78 (d, J = 6.6 Hz, 2H), 7.86 (s, 1H), 7.78 (s, 1H), 7.38 (dd, J = 14.8, 8.0 Hz, 1H),
7.32 (d, J = 6.6 Hz, 2H), 7.18 (s, 1H), 6.83-6.76 (m, 2H), 5.31-5.06 (m, 3H), 4.68 (d, J = 18.5
13
Hz, 1H), 4.37 (d, J = 14.3 Hz, 1H), 1.06 (d, J = 6.8 Hz, 3H). C NMR (101 MHz, CDCl3): δ
161.32, 151.42, 150.97, 149.96, 145.91, 143.64, 140.57, 140.08, 129.91, 122.84, 122.70 (2C,
overlap), 119.43, 117.65, 111.36, 103.83, 79.43, 54.90 (2C, overlap), 47.49, 13.00. MS (EI) m/z:
467 (M+). HRMS (EI): Anal. Calcd for C23H19F2N5O2S: 467.1218, Found: 467.1229.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(pyri-
din-3-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10d). 10d (71.12 mg, 71.2%) was
prepared from 10a (100.00 mg, 0.21 mmol) and 3-pyridinylboronic acid pinacol ester (62.78 mg,
0.31 mmol) in the same manner as described for 10b. white solid: mp 125-127 °C. 1H NMR (500
MHz, CDCl3) δ 8.89 (s, 1H), 8.60 (s, 1H), 7.90 (d, J = 8.1 Hz, 2H), 7.74 (s, 1H), 7.52 (s, 1H),
7.45 (d, J = 7.1 Hz, 1H), 7.38 (dd, J = 7.6, 4.8 Hz, 1H), 6.88-6.70 (m, 2H), 5.20 (d, J = 12.3 Hz,
1H), 5.17-5.08 (m, 2H), 4.71 (d, J = 18.5 Hz, 1H), 4.32 (d, J = 14.3 Hz, 1H), 1.16 (d, J = 7.0 Hz,
3H). 13C NMR (101 MHz, CDCl3): δ 165.82, 163.87, 161.79, 159.15, 157.09, 151.89, 150.56,
149.30, 146.95, 140.48, 133.23, 133.08, 123.32, 123.21 (2C, overlap), 116.96, 111.43, 104.30,
79.74, 55.39 (2C, overlap), 29.70, 13.48. MS (EI) m/z: 467 (M+). HRMS (EI): Anal. Calcd for
C23H19F2N5O2S: 467.1236, Found: 467.1218.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(pyri-
midin-5-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10e). 10e (56.00 mg, 56.6%) was
prepared from 10a (100.00 mg, 0.21 mmol) and 5-pyrimidinylboronic acid pinacol ester (52.82
mg, 0.26 mmol) in the same manner as described for 10b. white solid: mp 138-140 °C. 1H NMR
(500 MHz, CDCl3) δ 9.18 (s, 1H), 8.96 (s, 2H), 7.89 (s, 1H), 7.74 (s, 1H), 7.55 (s, 1H), 7.43-7.36
(m, 1H), 6.86-6.73 (m, 2H), 5.21-5.12 (m, 3H), 4.72 (d, J = 18.7 Hz, 1H), 4.30 (d, J = 14.3 Hz,
13
1H), 1.15 (d, J = 7.1 Hz, 3H). C NMR (101 MHz, CDCl3): δ 165.44, 161.92, 158.00, 153.62,
151.52, 144.18, 141.46, 130.38, 128.30, 123.24, 123.14 (2C, overlap), 118.14, 111.72, 104.35,
S7

ACCEPTED MANUSCRIPT
79.85, 55.41 (2C, overlap), 24.56, 13.52. MS (EI) m/z: 468 (M+). HRMS (EI): Anal. Calcd for
C22H18F2N6O2S: 468.1216, Found: 468.1228.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(2-cy-
anopyridin-5-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10f). 10f (61.00 mg, 58.0%) was
prepared from 10a (100.00 mg, 0.21 mmol) and 2-cyanopyridine-5-boronic acid pinacol ester
(64.40 mg, 0.28 mmol) in the same manner as described for 10b. white solid: mp 101-103 °C. 1H
NMR (500 MHz, CDCl3) δ 8.50 (d, J = 1.9 Hz, 1H), 7.89 (s, 1H), 7.78 (d, J = 2.3 Hz, 1H), 7.72
(s, 1H), 7.56 (dd, J = 8.1, 2.2 Hz, 1H), 7.56-7.43 (m, 1H), 7.18 (s, 1H), 6.82-6.73 (m, 2H), 5.37-
5.29 (m, 3H), 4.72 (d, J = 18.8 Hz, 1H), 4.26 (d, J = 14.3 Hz, 1H), 1.03 (d, J = 7.1 Hz, 3H). 13C
NMR (101 MHz, CDCl3): δ 165.44, 161.92, 158.00, 153.62, 151.52, 144.18, 141.46, 137.73,
136.58, 132.19, 130.66, 130.38, 128.30, 123.24, 123.14 (2C, overlap), 118.14, 111.72, 104.35,
79.88, 55.43 (2C, overlap), 24.38, 13.32. MS (EI) m/z: 492(M+). HRMS (EI): Anal. Calcd for
C24H18F2N6O2S: 492.1236, Found: 492.1218.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(2-me-
thoxypyridin-5-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10g). 10g (67.00 mg, 63.1%)
was prepared from 10a (100.00 mg, 0.21 mmol) and 2-methoxy-5-pyridinylboronic acid (62.50
mg, 0.28 mmol) in the same manner as described for 10b. white solid: mp 114-116 °C. 1H NMR
(500 MHz, CDCl3) δ 8.39 (d, J = 2.3 Hz, 1H), 7.86 (s, 1H), 7.75 (dd, J = 8.6, 2.5 Hz, 1H), 7.71
(s, 1H), 7.43 (d, J = 7.1 Hz, 1H), 7.33 (s, 1H), 6.82-6.71 (m, 3H), 5.17 (d, J = 13.7 Hz, 1H), 5.06
(d, J = 14.3 Hz, 2H), 4.66 (d, J = 18.4 Hz, 1H), 4.29 (d, J = 14.3 Hz, 1H), 3.95 (s, 3H), 1.13 (d, J
= 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 166.09, 164.10, 151.83, 149.44, 146.22, 144.19,
140.25, 136.56, 130.43, 130.20, 123.48, 123.37, 123.26 (2C, overlap), 115.48, 111.79, 111.31,
104.28, 79.68, 55.46 (2C, overlap), 53.75, 29.70, 13.44. MS (EI) m/z: 497(M+). HRMS (EI):
Anal. Calcd for C24H21F2N5O3S: 497.1316, Found: 497.1328.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(3-flu-
oropyridin-5-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10h). 10h (66.18 mg, 63.8%) was
prepared from 10a (100.00 mg, 0.21 mmol) and 3-fluoropyridine-5-boronic acid pinacol ester
(63.68 mg, 0.29 mmol) in the same manner as described for 10b. white solid: mp 103-106 °C. 1H
S8

ACCEPTED MANUSCRIPT
NMR (500 MHz, CDCl3) δ 8.69 (s, 1H), 8.43 (d, J = 18.2 Hz, 1H), 7.86 (s, 1H), 7.73 (s, 1H),
7.59 (d, J = 9.1 Hz, 1H), 7.53 (s, 1H), 7.49-7.38 (m, 1H), 6.85-6.74 (m, 2H), 5.32-5.11 (m, 3H),
13
4.70 (d, J = 18.6 Hz, 1H), 4.30 (d, J = 14.3 Hz, 1H), 1.15 (d, J = 7.0 Hz, 3H). C NMR (101
MHz, CDCl3): δ 165.64, 163.81, 161.82, 151.91, 151.12, 143.99, 142.71, 140.56, 137.51, 131.32,
130.33, 123.28, 123.14 (2C, overlap), 119.76, 117.91, 111.69, 104.34, 79.86, 55.39 (2C, overlap),
29.71, 13.51. MS (EI) m/z: 485(M+). HRMS (EI): Anal. Calcd for C23H18F3N5O2S: 485.1136,
Found: 485.1128.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(2-me-
thoxypyrimidin-5-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10i). 10i (57.15 mg, 53.8%)
was prepared from 10a (100.00 mg, 0.21 mmol) and 2-methoxypyrimidine-5-boronic acid
pinacol ester (68.80 mg, 0.29 mmol) in the same manner as described for 10b. white solid: mp
120-122 °C. 1H NMR (500 MHz, CDCl3) δ 8.72 (s, 2H), 7.87 (s, 1H), 7.72 (s, 1H), 7.46-7.38 (m,
2H), 6.82-6.72 (m, 2H), 5.18 (d, J = 14.1 Hz, 1H), 5.14-5.03 (m, 2H), 4.68 (d, J = 18.5 Hz, 1H),
4.29 (d, J = 14.3 Hz, 1H), 4.05 (s, 3H), 1.14 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ
165.33, 163.79, 161.79, 156.49, 151.95, 150.34, 141.94, 140.60, 130.38, 123.31, 123.18, 122.32
(2C, overlap), 116.71, 111.82, 104.31, 79.81, 55.34 (2C, overlap), 55.16, 24.85, 13.49. MS (EI)
m/z: 498 (M+). HRMS (EI): Anal. Calcd for C23H20F2N6O3S: 498.1226, Found: 498.1312.
5-bromo-3-methylthiophene-2-carbaldehyde (12). 11 (50.00 g, 0.40 mol) was dissolved in
chloroform (300 ml). Bromine (66.50 g, 0.42 mol) was slowly added dropwise at 0 °C. The
resulting mixture was heated under reflux for 4 hours. The reaction mixture was diluted with
CH2Cl2 (60 ml). The organic phase was washed with H2O (50 ml) and saturated aqueous sodium
bicarbonate solution (50 ml), dried over Na2SO4, and filtrated, then the solvent was evaporated
under reduced pressure. The residue was purified by silica gel column chromatography
(petroleum: ethyl acetate 30:1~20:1) to give 12 (51.80 g, 63.1%) as a brown solid: mp 55-57 °C.
1H NMR (400 MHz, CDCl3) δ 9.89 (s, 1H), 6.95 (s, 1H), 2.52 (s, 3H). MS (EI) m/z: 204 (M+).
5-bromo-3-methylthiophene-2-carboxylic acid (13). 12 (10.00 g, 48.80 mmol) was dissolved
in CH3CN (150 ml). Saturated aqueous monosodium phosphate solution (7.80 g, 50.20 mmol)
was slowly added dropwise at 0 °C, followed by 10% aqueous hydrogen peroxide solution (5.10
S9

ACCEPTED MANUSCRIPT
ml, 48.86 mmol), and stirred for 0.5 h. Then, saturated aqueous sodium chlorite solution (4.41 g,
48.80 mmol) was slowly added, and stirred for 8 h at room temperature. The mixture was diluted
with aqueous sodium hydrogen carbonate (50 ml) and stirred for 0.5 h at 0 °C. The residue was
acided (PH = 2) by addition of hydrogen chloride (1.0 M aqueous solution).The resultant
precipitate was collected by filtration, washed with diethyl ether then dried under vacuum to
1
afford 13 (7.00 g, 65.0%) as a white solid: mp 121-122 °C. H NMR (400 MHz, CD3OD) δ
13.99 (s, 1H), 8.28-7.54 (m, 1H), 3.26 (m, 3H). MS (EI) m/z: 220 (M+).
methyl 5-bromo-3-methylthiophene-2-carboxylate (14). 14 (8.70 g, 75.8%) was prepared
from 13 (10.80 g, 48.85 mmol) in the same manner as described for 3. As a colorless viscous oil.
1H NMR (400 MHz, CDCl3) δ 6.89 (s, 1H), 3.84 (s, 3H), 2.50 (s, 3H). MS (EI) m/z: 234 (M+).
methyl 5-bromo-3-(bromomethyl)thiophene-2-carboxylate (15). 14 (5.00 g, 21.27 mmol)
and benzoyl peroxide (0.50 g, 2.00 mmol) was dissolved in 200 ml CCl4. N-bromosuccinimide
(3.90 g, 21.91 mmol) was added in batches at room temperature. The reaction mixture was
allowed to warm to 80 °C and stirred for 12 hours. After cooling, the solvent was evaporated
under reduced pressure. The residue was partitioned between ethyl acetate (300 ml) and H2O
(100 ml). The organic phase was washed with H2O (80 ml) and saturated brine solution (80 ml),
dried over Na2SO4, and filtrated, then the solvent was evaporated under reduced pressure. The
residue was purified by silica gel column chromatography (petroleum: ethyl acetate 80:1~60:1)
1
to give 15 (4.00 g, 60.0%) as a white solid: mp 81-82°C. H NMR (400 MHz, CDCl3) δ 7.16 (s,
1H), 4.85 (s, 2H), 3.88 (s, 3H). MS (EI) m/z: 312 (M+).
methyl 5-bromo-3-[{(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)-
butan-2-ylamino}methyl]thiophene-2-carboxylate (16). 16 (4.86 g, 80.7%) was prepared from
7 (3.21 g, 12.00 mmol) and 15 (4.14 g, 13.18 mmol) in the same manner as described for 8. As a
colorless viscous oil 1H NMR (400 MHz, CDCl3) δ 7.89 (s, 1H), 7.75 (s, 1H), 7.36 (dd, J = 15.6,
8.8 Hz, 1H), 7.17 (s, 1H), 6.73 (t, J = 8.4 Hz, 2H), 4.93 (d, J = 14.3 Hz, 1H), 4.77 (d, J = 14.5 Hz,
1H), 4.26 (d, J = 14.1 Hz, 1H), 3.97 (d, J = 14.1 Hz, 1H), 3.85 (s, 3H), 3.23-3.10 (m, 1H), 0.92
(d, J = 6.4 Hz, 3H). MS (EI) m/z: 500 (M+).
S10

ACCEPTED MANUSCRIPT
5-bromo-3-[{(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)-butan-2-
ylamino}methyl]thiophene-2-carboxylic acid (17). 17 (0.88 g, 78.6%) was prepared from 16
(1.16 g, 2.31 mmol) in the same manner as described for 9. As a pale yellow viscous oil 1H NMR
(500 MHz, DMSO) δ 8.38 (s, 1H), 7.74 (s, 1H), 7.61 (s, 1H), 7.54-7.44 (m, 1H), 7.06 (m, 2H),
5.10 (d, J = 14.5 Hz, 1H), 4.74 (d, J = 14.6 Hz, 1H), 4.56 (d, J = 13.6 Hz, 1H), 4.48 (d, J = 13.5
Hz, 1H), 3.60 (d, J = 6.4 Hz, 1H), 1.18 (d, J = 6.1 Hz, 3H). MS (EI) m/z: 486 (M+).
2-bromo-5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-
yl]-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18a). 18a (0.18 g, 62.3%) was prepared from
17 (0.30 g, 0.62 mmol) in the same manner as described for 10a. white solid: mp 146-148 °C. 1H
NMR (400 MHz, CDCl3) δ 7.87 (s, 1H), 7.71 (s, 1H), 7.43-7.38 (m, 1H), 7.11 (s, 1H), 6.82-6.73
(m, 2H), 5.15 (d, J = 14.2 Hz, 1H), 4.90 (d, J = 18.6 Hz, 2H), 4.48 (d, J = 18.5 Hz, 1H), 4.26 (d,
J = 14.2 Hz, 1H), 1.11 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 164.04, 163.97,
161.47, 151.53, 144.19, 135.07, 130.37, 124.20, 123.32, 123.16 (2C, overlap), 111.70, 104.22,
79.62, 55.40 (2C, overlap), 29.78, 13.25. MS (EI) m/z: 468 (M+). HRMS (EI): Anal. Calcd for
C18H15BrF2N4O2S: 468.0136, Found: 468.0128.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-phen-
yl-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18b). A mixture of 18a (100.00 mg, 0.21 mmol),
cesium carbonate (139.20 mg, 0.43 mmol), phenylboronic acid (34.00 mg, 0.28 mmol) and
tetrakis(triphenylphosphine)palladium (0) (25.00 mg, 0.02 mmol) in dioxane (15 ml) and H2O (5
ml) was degassed and flushed with argon. The mixture was hearted at 80 °C for 10 h. The
solvent was evaporated under reduced pressure. The residue was diluted with H2O (20 ml) and
extracted with ethyl acetate (30 ml ×2). The combined organic layers were washed with H2O (20
ml ×2) and brine (20 ml ×2), dried over anhydrous Na2SO4, and filtrated, then the solvent was
evaporated under reduced pressure. The residue was purified by silica gel column
chromatography (CH2Cl2: MeOH 200:1~50:1) to give 18b (61.00 mg, 62.1%) as a white solid:
mp 142-144 °C. 1H NMR (400 MHz, CDCl3) δ 7.87 (s, 1H), 7.68 (d, J = 15.1 Hz, 1H), 7.63 (d, J
= 7.3 Hz, 2H), 7.52-7.32 (m, 4H), 7.27 (s, 1H), 6.84-6.63 (m, 2H), 5.18 (d, J = 13.8 Hz, 1H),
5.08-4.86 (m, 2H), 4.53 (d, J = 18.3 Hz,1H), 4.31 (d, J = 14.3 Hz, 1H), 1.14 (d, J = 7.0 Hz, 3H).
13C NMR (101 MHz, CDCl3): δ 165.67, 155.06, 152.91, 151.76, 144.13, 133.67, 133.09, 129.06,
S11

ACCEPTED MANUSCRIPT
128.96, 126.31, 123.47, 123.33 (2C, overlap), 116.90, 111.58, 104.25, 79.70, 55.50 (2C, overlap),
20.98, 13.40. MS (EI) m/z: 466 (M+). HRMS (EI): Anal. Calcd for C24H20F2N4O2S: 466.1336,
Found: 466.1318.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(4-me-
thylphenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18c). 18c (67.20 mg, 65.6%) was
prepared from 18a (100.00 mg, 0.22 mmol) and 4-methylphenylboronic acid (36.03 mg, 0.26
1
mmol) in the same manner as described for 18b. white solid: mp 173-175 °C. H NMR (400
MHz, CDCl3) δ 7.96 (d, J = 6.9 Hz, 1H), 7.75 (s, 1H), 7.54 (d, J = 8.1 Hz, 2H), 7.43-7.38 (m,
1H), 7.27-7.20 (m, 3H), 6.83-6.74 (m, 2H), 5.37-5.10 (m, 1H), 5.06-4.86 (m, 2H), 4.60 (d, J =
17.5 Hz, 1H), 4.44-4.21 (m, 1H), 2.41 (s, 3H), 1.17 (d, J = 6.9 Hz, 3H). 13C NMR (101 MHz,
CDCl3): δ 162.02, 155.44, 153.01, 151.35, 143.94, 139.17, 134.49, 132.60, 129.88, 128.46,
126.21, 123.37, 123.29 (2C, overlap), 116.37, 111.63, 104.25, 72.80, 55.69 (2C, overlap), 21.75,
21.30, 13.35. MS (EI) m/z: 480 (M+). HRMS (EI): Anal. Calcd for C25H22F2N4O2S: 480.1426,
Found: 480.1435.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(3-me-
thylphenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18d). 18d (58.20 mg, 55.1%) was
prepared from 18a (100.00 mg, 0.22 mmol) and 3-methylphenylboronic acid (36.03 mg, 0.26
1
mmol) in the same manner as described for 18b. white solid: mp 170-171 °C. H NMR (500
MHz, CDCl3) δ 7.88 (s, 1H), 7.70 (s, 1H), 7.48-7.36 (m, 4H), 7.32 (t, J = 7.9 Hz, 1H), 7.19 (d, J
= 7.5 Hz, 1H), 6.82-6.73 (m, 2H), 5.20 (d, J = 12.7 Hz, 1H), 4.91 (d, J = 15.2 Hz, 2H), 4.54 (d, J
= 18.1 Hz, 1H), 4.33 (d, J = 14.3 Hz, 1H), 2.43 (s, 3H), 1.16 (d, J = 6.9 Hz, 3H). 13C NMR (101
MHz, CDCl3): δ 161.80, 155.31, 152.88, 151.76, 144.12, 138.97, 133.69, 133.00, 129.77, 129.08,
126.98, 123.47, 123.39, 123.36 (2C, overlap), 116.76, 111.59, 104.25, 72.83, 55.50 (2C, overlap),
29.66, 21.44, 13.38. MS (EI) m/z: 480 (M+). HRMS (EI): Anal. Calcd for C25H22F2N4O2S:
480.1416, Found: 480.1412.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(4-me-
thoxyphenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18e). 18e (68.00 mg, 62.3%) was
prepared from 18a (100.00 mg, 0.22 mmol) and 4-methoxyphenylboronic acid (38.00 mg, 0.25
S12

ACCEPTED MANUSCRIPT
1
mmol) in the same manner as described for 18b. white solid: mp 107-109 °C. H NMR (400
MHz, CDCl3) δ 7.57 (d, J = 8.6 Hz, 2H), 7.52-7.43 (m, 4H), 6.96 (d, J = 8.7 Hz, 2H), 6.83-6.71
(m, 2H), 5.19 (d, J = 13.8 Hz, 1H), 4.96-4.85 (m, 2H), 4.53 (d, J = 18.2 Hz, 1H), 4.33 (d, J =
14.3 Hz, 1H), 3.86 (s, 3H), 1.16 (d, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 159.86,
152.69, 151.19, 143.68, 135.45, 131.69, 129.97, 127.21, 126.03, 123.06, 122.89 (2C, overlap),
115.38, 114.12, 111.11, 103.79, 72.30, 54.99 (2C, overlap), 54.59, 29.21, 12.92. MS (EI) m/z:
496 (M+). HRMS (EI): Anal. Calcd for C25H22F2N4O3S: 496.1426, Found: 496.1418.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(3-me-
thoxyphenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18f). 18f (75.00 mg, 72.1%) was
prepared from 18a (100.00 mg, 0.22 mmol) and 3-methoxyphenylboronic acid (38.00 mg, 0.25
1
mmol) in the same manner as described for 18b. white solid: mp 106-108 °C. H NMR (500
MHz, CDCl3) δ 7.87 (s, 1H), 7.71-7.61 (m, 2H), 7.56-7.51 (m, 1H), 7.33 (t, J = 8.0 Hz, 1H), 7.27
(d, J = 6.9 Hz, 1H), 7.21 (d, J = 7.7 Hz, 1H), 7.14 (s, 1H), 6.82-6.72 (m, 2H), 5.18 (d, J = 13.0
Hz, 1H), 4.91 (d, J = 15.2 Hz, 2H), 4.52 (d, J = 18.2 Hz, 1H), 4.32 (d, J = 14.2 Hz, 1H), 3.83 (s,
13
3H), 1.14 (d, J = 6.9 Hz, 3H). C NMR (101 MHz, CDCl3): δ 160.07, 152.83, 151.72, 144.12,
135.04, 131.99, 130.26, 128.47, 123.50, 123.40 (2C, overlap), 118.83, 117.12, 114.20, 112.10,
111.54, 104.22, 79.65, 55.55 (2C, overlap), 55.39, 29.69, 13.40. MS (EI) m/z: 496 (M+). HRMS
(EI): Anal. Calcd for C25H22F2N4O3S: 496.1416, Found: 496.1423.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(4-chl-
orophenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18g). 18g (72.00 mg, 68.1%) was
prepared from 18a (100.00 mg, 0.22 mmol) and 4-chlorophenylboronic acid (44.00 mg, 0.28
1
mmol) in the same manner as described for 18b. white solid: mp 133-135 °C. H NMR (400
MHz, CDCl3) δ 7.87-7.79 (m, 1H), 7.76 (s, 1H), 7.70-7.65 (m, 2H), 7.44-7.39 (m, 2H), 7.37-7.33
(m, 1H), 7.27 (s, 1H), 6.89-6.68 (m, 2H), 5.28-4.91 (m, 3H), 4.55 (d, J = 18.0 Hz, 1H), 4.36 (d, J
= 13.7 Hz, 1H), 1.17 (d, J = 6.9 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 162.02, 152.85, 143.84,
135.01, 132.12, 129.41, 128.57, 127.78, 127.49, 123.25, 123.17 (2C, overlap), 117.22, 111.67,
104.29, 72.81, 55.79 (2C, overlap), 29.72, 13.38. MS (EI) m/z: 500 (M+). HRMS (EI): Anal.
Calcd for C24H19ClF2N4O2S: 500.1026, Found: 500.1018.
S13

ACCEPTED MANUSCRIPT
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(3-chl-
orophenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18h). 18h (72.00 mg, 68.3%) was
prepared from 18a (100.00 mg, 0.22 mmol) and 3-chlorophenylboronic acid (44.00 mg, 0.28
1
mmol) in the same manner as described for 18b. white solid: mp 130-132 °C. H NMR (500
MHz, CDCl3) δ 7.71 (s, 1H), 7.67 (dd, J = 6.4, 5.1 Hz, 2H), 7.56-7.48 (m, 3H), 7.33 (d, J = 9.7
Hz, 1H), 7.29 (s, 1H), , 6.83-6.73 (m, 2H), 5.20 (d, J = 14.0 Hz, 1H), 4.94 (d, J = 16.3 Hz, 2H),
13
4.54 (d, J = 18.2 Hz, 1H), 4.32 (d, J = 14.3 Hz, 1H), 1.15 (d, J = 7.0 Hz, 3H). C NMR (101
MHz, CDCl3): δ 162.16, 152.82, 151.80, 144.11, 132.89, 132.05, 131.95, 130.79, 130.46, 128.47,
123.44, 123.32 (2C, overlap), 122.05, 117.61, 115.65, 113.10, 111.60, 104.26, 72.84, 55.49 (2C,
overlap), 29.70, 13.43. MS (EI) m/z: 500 (M+). HRMS (EI): Anal. Calcd for C24H19ClF2N4O2S:
500.1216, Found: 500.1118.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(4-flu-
orophenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18i). 18i (74.00 mg, 62.6%) was
prepared from 18a (100.00 mg, 0.22 mmol) and 4-fluorophenylboronic acid (39.00 mg, 0.28
1
mmol) in the same manner as described for 18b. white solid: mp 119-120 °C. H NMR (500
MHz, CDCl3) δ 7.88 (s, 1H), 7.72 (s, 1H), 7.70-7.64 (m, 2H), 7.51-7.43 (m, 1H), 7.21 (s, 1H),
7.13 (t, J = 8.6 Hz, 2H), 6.83-6.74 (m, 2H), 5.20 (d, J = 13.2 Hz, 1H), 4.92 (d, J = 16.1 Hz, 2H),
13
4.53 (d, J = 18.1 Hz, 1H), 4.32 (d, J = 14.3 Hz, 1H), 1.15 (d, J = 7.0 Hz, 3H). C NMR (101
MHz, CDCl3): δ 162.48, 160.55, 150.90, 149.74, 147.17, 143.08, 142.09, 136.91, 132.41, 131.78,
121.29, 121.19 (2C, overlap), 116.13, 109.60, 107.93, 102.24, 77.77, 53.40 (2C, overlap), 27.63,
16.37. MS (EI) m/z: 484 (M+). HRMS (EI): Anal. Calcd for C24H19F3N4O2S: 484.1216, Found:
484.1218.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(3-flu-
orophenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18j). 18j (64.70 mg, 63.6%) was
prepared from 18a (100.00 mg, 0.22 mmol) and 3-fluorophenylboronic acid (39.00 mg, 0.28
1
mmol) in the same manner as described for 18b. white solid: mp 114-116 °C. H NMR (500
MHz, CDCl3) δ 7.87 (s, 1H), 7.70 (s, 1H), 7.64 (dd, J = 11.5, 7.7 Hz, 1H), 7.59 (s, 1H), 7.49 (d, J
= 6.9 Hz, 1H), 7.44 (t, J = 7.5 Hz, 2H), 7.37-7.29 (m, 1H), 6.88-6.76 (m 2H), 5.18 (d, J = 13.5
Hz, 1H), 4.92 (d, J = 15.5 Hz, 2H), 4.52 (d, J = 18.2 Hz, 1H), 4.31 (d, J = 14.2 Hz, 1H), 1.14 (d,
S14

ACCEPTED MANUSCRIPT
J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 165.43, 152.81, 151.76, 144.14, 135.45, 135.10,
133.88, 132.02, 130.44, 128.48, 126.18, 124.44, 123.44, 123.31 (2C, overlap), 117.66, 111.60,
104.25,72.56, 55.45 (2C, overlap), 29.35, 13.43. MS (EI) m/z: 484 (M+). HRMS (EI): Anal.
Calcd for C24H19F3N4O2S: 484.1126, Found: 484.1118.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(4-cy-
anophenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18k). 18k (75.00 mg, 72.3%) was
prepared from 18a (100.00 mg, 0.22 mmol) and 4-cyanophenylboronic acid pinacol ester (60.69
mg, 0.26 mmol) in the same manner as described for 18b. white solid: mp 150-152 °C. 1H NMR
(500 MHz, CDCl3) δ 7.87 (s, 1H), 7.77-7.71 (m, 2H), 7.55 (dd, J = 10.6, 4.3 Hz, 2H), 7.47 (td, J
= 7.5, 2.6 Hz, 2H), 7.40 (s, 1H), 6.78-6.66 (m 2H), 5.21 (d, J = 13.9 Hz, 1H), 4.98 (d, J = 18.0
Hz, 2H), 4.57 (d, J = 18.3 Hz, 1H), 4.32 (d, J = 14.3 Hz, 1H), 1.16 (d, J = 7.0 Hz, 3H). 13C NMR
(101 MHz, CDCl3): δ 165.16, 152.77, 151.88, 144.13, 138.01, 135.25, 132.13, 131.94, 128.46,
126.61, 123.37, 123.27 (2C, overlap), 118.74, 118.39, 111.66, 104.30, 79.77, 55.41 (2C, overlap),
29.69, 13.47. MS (EI) m/z: 491 (M+). HRMS (EI): Anal. Calcd for C25H19F2N5O2S: 491.1216,
Found: 491.1226.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(3-cy-
anophenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18l). 18l (67.63 mg, 65.2%) was
prepared from 18a (100.00 mg, 0.22 mmol) and 3-cyanophenylboronic acid (33.11 mg, 0.23
1
mmol) in the same manner as described for 18b. white solid: mp 148-150 °C. H NMR (500
MHz, CDCl3) δ 7.90-7.85 (m, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.70 (s, 1H), 7.69 -7.59 (m, 2H),
7.56-7.51 (m, 2H), 7.33 (s, 1H), 6.85-6.72 (m, 2H), 5.18 (d, J = 13.7 Hz, 1H), 4.95 (d, J = 17.7
Hz, 2H), 4.54 (d, J = 18.3 Hz, 1H), 4.30 (d, J = 14.3 Hz, 1H), 1.14 (d, J = 6.6 Hz, 3H). 13C NMR
(101 MHz, CDCl3): δ 163.74, 152.85, 151.75, 144.12, 135.03, 134.60, 132.08, 130.13, 129.50,
128.61, 123.33, 123.22 (2C, overlap), 120.81, 118.29, 118.14, 113.52, 111.61, 104.26, 79.83,
55.41 (2C, overlap), 29.62, 13.45. MS (EI) m/z: 491 (M+). HRMS (EI): Anal. Calcd for
C25H19F2N5O2S: 491.1226, Found: 491.1221.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(pyr-
idin-4-yl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18m). 18m (64.70 mg, 65.5%) was
S15

ACCEPTED MANUSCRIPT
prepared from 18a (100.00 mg, 0.22 mmol) and 4-pyridinylboronic acid (35.28 mg, 0.29 mmol)
1
in the same manner as described for 18b. white solid: mp 178-180 °C. H NMR (400 MHz,
CDCl3) δ 8.67 (d, J = 5.5 Hz, 2H), 7.85 (s, 1H), 7.72 (s, 1H), 7.52 (d, J = 6.1 Hz, 2H), 7.48 (s,
1H), 7.43 (m, 1H), 6.83-6.74 (m, 2H), 5.20 (d, J = 13.6 Hz, 1H), 4.98 (d, J = 14.3 Hz, 2H), 4.56
(d, J = 14.3 Hz, 1H), 4.30 (d, J = 14.0 Hz, 1H), 1.15 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz,
CDCl3): δ 165.03, 152.62, 151.90, 151.12, 150.69, 144.13, 140.88, 135.44, 130.39, 123.35,
123.26 (2C, overlap), 120.14, 118.96, 111.68, 104.31, 79.92, 55.39 (2C, overlap), 29.70, 13.48.
MS (EI) m/z: 467 (M+). HRMS (EI): Anal. Calcd for C23H19F2N5O2S: 467.1216, Found:
467.1228.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(pyr-
idin-3-yl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18n). 18n (70.00 mg, 71.2%) was
prepared from 18a (100.00 mg, 0.22 mmol) and 3-pyridinylboronic acid pinacol ester (58.85 mg,
0.29 mmol) in the same manner as described for 18b. white solid: mp 156-158 °C. 1H NMR (500
MHz, CDCl3) δ 8.91 (s, 1H), 8.61 (d, J = 4.2 Hz, 1H), 7.91 (d, J = 7.9 Hz, 1H), 7.87 (s, 1H), 7.72
(s, 1H), 7.44 (dd, J = 15.1, 8.0 Hz, 1H), 7.42-7.36 (m, 1H), 7.35 (s, 1H), 6.86-6.78 (m, 2H), 5.20
(d, J = 13.7 Hz, 1H), 4.97 (d, J = 17.6 Hz, 2H), 4.56 (d, J = 18.2 Hz, 1H), 4.32 (d, J = 14.3 Hz,
13
1H), 1.15 (d, J = 7.0 Hz, 3H). C NMR (101 MHz, CDCl3): δ 165.23, 152.87, 151.84, 15059,
149.65, 147.03, 144.12, 134.49, 133.57, 130.40, 129.94, 123.92, 123.41, 123.30 (2C, overlap),
118.05, 111.60, 104.27, 79.75, 55.58 (2C, overlap), 29.68, 13.44. MS (EI) m/z: 467 (M+). HRMS
(EI): Anal. Calcd for C23H19F2N5O2S: 467.1232, Found: 467.1226.
5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(pyri-
midin-5-yl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18o). 18o (63.00 mg, 63.8%) was
prepared from 18a (100.00 mg, 0.21 mmol) and 5-pyrimidinylboronic acid pinacol ester (59.13
mg, 0.29 mmol) in the same manner as described for 18b. white solid: mp 126-128 °C. 1H NMR
(500 MHz, CDCl3) δ 9.22 (s, 1H), 9.00 (s, 2H), 7.89 (s, 1H), 7.74 (s, 1H), 7.49-7.38 (m, 2H),
6.85- 6.71 (m, 2H), 5.21 (d, J = 14.1 Hz, 1H), 5.01 (d, J = 18.5 Hz, 2H), 4.59 (d, J = 18.4 Hz,
1H), 4.32 (d, J = 14.2 Hz, 1H), 1.17 (d, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 164.87,
158.37, 153.82, 152.85, 146.16, 144.10, 135.81, 130.39, 128.29, 123.30, 123.17 (2C, overlap),
S16