Design, synthesis, and biological evaluation of 4-chloro-2H-thiochromenes featuring nitrogen-containing side chains as potent antifungal agents

Article abstract of DOI:10.1016/j.bmcl.2018.06.035

A series of 4-chloro-2H-thiochromenes featuring nitrogen-containing side chains were designed, synthesized and tested in vitro for their antifungal activities. The results of preliminary antifungal tests showed that most target compounds exhibited good inhibitory activities against Candida albicans, Cryptococcus neoformans, Candida tropicalis. Notably, compounds 10e and 10y showed most potent activity in vitro against a variety of fungal pathogens with low MICs. Meanwhile, low cytotoxicity on mammalian cells has been observed for compounds 10e and 10y in the tested concentrations by the MTT assay. Therefore, the 4-chloro-2H-thiochromenes with nitrogen-containing groups provide new lead structures in the search for novel antifungal agents.

Full text of DOI:10.1016/j.bmcl.2018.06.035

Accepted Manuscript
Design, synthesis, and biological evaluation of 4-chloro-2H-thiochromenes fea-
turing nitrogen-containing side chains as potent an agents
Dan-Jiao Wang, Zhuang Hou, Hang Xu, Ran An, Xin Su, Chun Guo
PII:
S0960-894X(18)30524-9
https://doi.org/10.1016/j.bmcl.2018.06.035
BMCL 25916
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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20 April 2018
31 May 2018
17 June 2018
Please cite this article as: Wang, D-J., Hou, Z., Xu, H., An, R., Su, X., Guo, C., Design, synthesis, and biological
evaluation of 4-chloro-2H-thiochromenes featuring nitrogen-containing side chains as potent an agents, Bioorganic
& Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.06.035
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Design, synthesis, and biological evaluation of 4-chloro-2H-thiochromenes
featuring nitrogen-containing side chains as potent an agents
Dan-Jiao Wanga,1, Zhuang Houa, 1, Hang Xua, Ran Ana, Xin Sub,*, Chun Guoa,*
a
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR
China
b
School of Life Science and Biopharmaceutical, Shenyang Pharmaceutical University, Shenyang 110016,
PR China
* Corresponding author: Chun Guo. E-mail address: chunguo@syphu.edu.cn (C. Guo), Xin Su. E-mail:
suxin1966@163.com (X. Su)
1 These authors contributed equally to this work.
Abstract
A series of 4-chloro-2H-thiochromenes featuring nitrogen-containing side chains were
designed, synthesized and tested in vitro for their antifungal activities. The results of
preliminary antifungal tests showed that most target compounds exhibited good inhibitory
activities against Candida albicans, Cryptococcus neoformans, Candida tropicalis. Notably,
compounds 10e and 10y showed most potent activity in vitro against a variety of fungal
pathogens with low MICs. Meanwhile, low cytotoxicity on mammalian cells has been
observed for compounds 10e and 10y in the tested concentrations by the MTT assay.
Therefore, the 4-chloro-2H-thiochromenes with nitrogen-containing groups provide new lead
structures in the search for novel antifungal agents.
Keywords: 4-Chloro-2H-thiochromenes; Antifungal activity; Nitrogen-containing group;
Cytotoxicity
Invasive fungal infections (IFIs) present serious public health concerns and the incidence of
their associated mortality has been increasing dramatically, especially in
immunocompromised patients, patients with acquired immune deficiency syndrome (AIDS),
and patients undergoing cancer chemotherapy.1,2 Cryptococcus, Candida, Aspergillus, and
Pneumocystis account for a large percentage of the main pathogens that lead to the deaths of
invasive fungal infections.3 Several classes of antifungal agents such as polyenes, azoles,
echinocandins are widely used clinically, but some available antifungal drugs just have

modest effects on the treatment of invasive fungal infections caused by Candidiasis and
Cryptococcosis.4 There is still an urgent need to develop a new generation of antifungal
agents with novel mechanism of action to meet the increasing requirements for managing the
fungal infections.
Thiochroman-4-ones which are analogs of chromones possess several biological activities,
and serve as building blocks in synthetic chemistry.5 Since 1980s, thiochroman-4-one
derivatives with good antifungal activities were reported by the structural modification. A
substituent at 3-position of thiochroman-4-ones can effectively increase their antifungal
activities. Nakib et al.6 reported that 3-benzylidenethiochroman-4-ones (1, Fig. 1) exhibited
considerable antifungal activities against Candida species, Cryptococcus strains, Torulopsis
glabrata and Trichosporon cutaneum. In our previous works,7,8 a series of 3-substituted
thiochroman-4-one derivatives such as 3-mannich bases (2, Fig. 1), 3-bromos (3, Fig. 1) were
synthesized and showed antifungal activities against C. albicans, C. neoformans, E. floccosum,
T. rubrum, and M. gypseum. Based on results obtained from structure-activity relationships
(SARs) of thiochroman-4-one derivatives,6-9 the thiochroman-4-one attached
a
nitrogen-containing moiety increase antifungal activity to some extent. In particular,
3-substituted thiochroman-4-one analogues such as 3-mannich bases, exhibited better
antifungal activities.8 Thiochromanone moieties found in 3- substituted analogs have shown
in Fig. 1.
Fig. 1. The structures of 3-substituted thiochroman-4-one derivatives
Compounds 4-chloro-2H-thiochromene-3-carbaldehydes have been identified as modest
antifungal agents against a varieties of pathogens in our lab (not published). When the
carbonyl groups were substituted with chloro groups of thiochroman-4-ones, it appears that
position 4 were tolerable. (Data shown in Fig. 2 and Table 1). This work is a continuation of

our study on the chemical modification and biological evaluation of
4-chloro-2H-thiochromene-3-carbaldehydes.
Aliphatic amine moieties (e.g., diethylamine, piperidine, pyrrolidine) are important parts of
medicinal compounds with various biological properties such as anti-Alzheimer's disease,10
anticancer11 and antifungal activities.12 In addition, azole moieties are important
pharmacophores in drug design13 and widely distributed in antifungal agents such as
voriconazole, fluconazole, and itraconazole.14 Although azoles are the most widely used
antifungals as first-line antifungal therapy, the search for novel azoles with broadest activity,
low toxicity, and low resistance still remains an attractive subject.15 The widespread
applications of azole and aliphatic amine moieties in medicinal chemistry encourage us to
explore more excellent antifungals of 4-chloro-2H-thiochromenes featuring these fragments.
Thus, we designed and synthesized a new series of compounds by incorporating
4-chloro-2H-thiochromene-3-carbaldehydes with a nitrogen-containing side chain in a single
molecule (Fig. 2).
Fig. 2. Rationale design of the title compounds

Thichroman-4-ones
6a-6d7
and
the
key
intermediates
4-chloro-2H-thiochromene-3-carbaldehydes 7a-7d16 were synthesized according to literature
procedures (Scheme 1). Firstly, compounds 5a-5d were obtained by reactions of
commercially available substituted thiophenols 4a-4d and β-chloropropionic acid, and then
were cyclized in the presence of concentrated H2SO4 to afford thiochroman-4-ones 6a-6d via
intramolecular Friedel-Crafts. Subsequently, ketones 6a-6d were under Vilsmeier–Haack
conditions to generate the key intermediates 7a-7d.
Scheme 1. Reagents and conditions: (a) (i) KOH, H2O, EtOH, 60 oC; (ii) β-chloropropionic acid, 30%
K2CO3 aqueous solution, reflux; (b) H2SO4; (c) POCl3, DMF, 50 oC.
The synthesis of target compounds 10a–10z were illustrated in Scheme 2. Aldehydes 7a-7d
and amines (diethylamine, piperidine, pyrrolidine, N-methyl piperazine) were treated with
NaBH(OAc)3 in 1,2-dichloroethane at room temperature to give compounds 10a-10n via
reductive aminations.17 Aldehydes 7a-7d were treated with NaBH4 to afford alcohols 8a-8d,
which were then chlorinated with SOCl2 to afford compounds 9a-9d. Subsequently,
nucleophilic substitutions of compounds 9a-9d with aliphatic amines (diisopropylamine and
isopropylamine) or azoles (imidazole and 1,2,4-triazole) at the presence of K2CO3/KI in DMF
gave 10o-10z.

Scheme 2. Reagents and conditions: (d) NaBH(OAc)3, amine, DCE, N2, rt. (e) NaBH4, THF, rt; (f) SOCl2,
CHCl2, reflux; (g) aliphatic amine/azole, K2CO3, KI, DMF, 50 oC.
The compounds 10a-10z, 6a-6d, 7a-7d have been screened for their antifungal activities
against Candida albicans (cpcc 400616), fluconazole-sensitive strains of Candida albicans
(ATCC SC5314), fluconazole-resistant strains of Candida albicans (strain 103), Cryptococcus
neoformans (cgmcc 2.3161), Candida tropicalis (cgmcc 2.3739) and Aspergillus fumigatus
(cgmcc 3.7795). According to the National Committee for Clinical Laboratory Standards
(NCCLS), the minimal inhibitory concentrations (MICs) of target compounds were
determined by the broth microdilution method in 96-well plates.18 Fluconazole (FCZ) was
used as reference drug. In brief, tested compounds and fluconzole were dissolved in DMSO,
respectively. Then, these compounds serially diluted by growth medium in 96-well microtest
plates. The inoculum suspension was added to each well of microtiter plate to give the
concentrations (128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μg/mL) and incubated at 32oC. MIC
values were determined by visual observation after 2-7d of incubation.
The in vitro antifungal activities are summarized in Table 1. Most of the synthesized
compounds exhibited moderate to good antifungal activities. Among these compounds,
compound 10e and 10y showed comparable or even better efficacy than fluconazole against
all tested strains, particularly for C. albicans (MICs = 0.5-8 μg/mL) and C. neoformans (MIC
= 0.25-1 μg/mL).

Table 1
In vitro antifungal activities of the target compounds (MIC, μg/mL)a.
MIC
Compd.
10a
10b
10c
R1
F
R2
C. a (I)
C. a (II)
C. a (III)
C. n
C. t
A. f
8
8
64
2
2
>128
F
2
8
8
16
64
2
4
1
1
4
4
4
4
8
8
16
16
32
2
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
F
8
F
16
4
16
0. 5
1
16
10d
10e
Cl
Cl
Cl
Br
Br
Br
Br
8
32
32
16
32
16
16
>128
64
4
10f
16
8
64
64
128
128
16
10g
10h
10i
64
8
64
8
64
10j
8
64
10k

H3C
H3C
H3C
F
32
32
32
16
16
4
32
16
8
64
128
64
8
16
32
32
64
4
64
64
>128
>128
>128
>128
>128
>128
64
10l
10m
10n
10o
10p
10q
10r
10s
8
4
64
8
F
4
32
8
F
4
64
2
F
8
2
64
4
2
Cl
Cl
Cl
Cl
Br
Br
Br
32
16
4
16
8
64
8
64
32
64
32
>128
>128
>128
64
32
4
>128
>128
64
10t
4
64
64
0.5
8
10u
10v
10w
10x
10y
32
64
>128
0.5
16
16
64
0.5
32
>128
>128
0.5
>128
>128
>128
8
0.25

Br
8
4
16
4
>128
>128
10z
F
Cl
Br
CH3
F
-
-
-
-
-
-
-
-
-
32
16
32
16
64
64
32
32
4
8
4
64
16
64
64
64
64
64
64
32
64
32
64
32
32
32
32
64
1
16
8
64
6a
6b
>128
>128
>128
>128
>128
>128
>128
>128
8
16
4
6c
8
6d
64
32
16
16
1
16
32
64
64
2
7a
Cl
Br
CH3
-
7b
7c
7d
FCZ
aAbbreviations: C. a (I): Candida albicans (cpcc 400616); C. a (II): Candida albicans (ATCC SC5314); C.
a (III): strain 103, fluconazole-resistant strains of Candida albicans; C. n: Cryptococcus neoformans
(cgmcc 2.3161); C. t: Candida tropicalis (cgmcc 2.3739); A. f: Aspergillus fumigatus (cgmcc 3.7795); FCZ:
fluconzole.
The in vitro antifungal activities of compounds 10a-10z are summarized in Table 1, and the
SARs of these compounds were also discussed.
Firstly, the introduction of acyclic aliphatic amine moieties on the 3-position of 7a–7d
afforded more potent compounds (eg., 10a, 10e) than those (eg., 10d, 10g) containing the
cyclic aliphatic amine moieties on the 3-position. Secondly, compounds (e.g., 10w-10x) with
imidazole or 1,2,4-triazole moieties showed lower activities than the compounds (e.g.,
10y-10z) with aliphatic amine moieties. Meanwhile, the relationship between various
substituents on the 6-position of benzene ring were investigated. These compounds showed
broad antifungal activities when 6-position of the benzene ring was substituted with a fluoro
(e.g., 10a-10c, 10q-10r) or chloro group (e.g., 10e-10f). On the contrary, when 6-position was
substituted with an electron-donating group such as methyl (e.g., 10l-10n), the antifungal

activities markedly decreased. Moreover, most of the target compounds showed improved
antifungal activities against tested fungal strains compared with the intermediates 6a-6d and
7a-7d. It can be seen that 4-chloro-2H-thiochromenes attached a nitrogen-containing side
chain, their antifungal activities obviously increased. This finding demonstrates that the
3-position of 4-chloro-2H-thiochromenes attached a nitrogen-containing side chain can
effectively enhance antifungal activities.
In order to ensure selectivity of the antifungal effects, the cytotoxicity of compounds 10e
and 10y were evaluated by MTT assay using four mammalian cell lines, including A549
(human lung carcinoma), HepG2 (human liver carcinoma), HT-29 (human colorectral cancer)
and H9C2 (normal, rat myocytes). As presented in Table 2, the IC50 values of compound 7b
were 21.53 μg/mL in HepG2 and 18.44 μg/mL in HT-29, respectively. Compound 7c gave
IC50 values of 26.61 μg/mL against HepG2 and 28.79 μg/mL against HT-29. Compound 10e
and 10y showed low toxicities in four cell lines in tested concentrations. We concluded that
introduction of nitrogen-containing side chain at the 3-position of 4-chloro-2H-thiochromenes
favorably reduces its cytotoxic activities.
Table 2 Cytotoxicity of tested compounds on mammalian cells (IC50, μg/mL).
A549
>100
36.04
>100
>100
>100
1.77
HepG2
>100
>100
21.53
26.61
>100
2.90
HT-29
>100
>100
18.44
28.79
>100
1.44
H9C2
>100
>100
>100
>100
>100
0.29
10e
10y
7b
7c
FCZ
DOX
In conclusion, we have synthesized a series of 4-chloro-2H-thiochromenes featuring
nitrogen-containing side chains as antifungal agents. Among these compounds, compounds
10e and 10y exhibited excellent antifungal activities against tested fugal strains, and no
cytotoxic activities against mammalian cells. The investigation of antifungal mechanism of
these compounds are underway.

Acknowledgements
We greatly appreciate the funding support for this research provided by the National
Natural Science Foundation of China (Grant No.81573292).
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Highlights
1. Twenty-six 4-chloro-2H-thiochromenes were designed and synthesized.
2. Most compounds showed good antifungal activities against tested fungal strains.
3. Compound 10e and 10y showed better efficacy than fluconazole against strain 103.