Synthesis, characterization and antifungal evaluation of novel thiochromanone derivatives containing indole skeleton

Article abstract of DOI:10.1248/cpb.c16-00366

Invasive fungal disease constitutes a growing health problem and development of novel antifungal drugs with high potency and selectivity against new fungal molecular targets are urgently needed. In order to develop potent antifungal agents, a novel series of 6-alkyl-indolo[3,2-c]-2H-thiochroman derivatives were synthesized. Microdilution broth method was used to investigate antifungal activity of these compounds. Most of them showed good antifungal activity in vitro. Compound 4o showed the best antifungal activity, which (inhibition of Candida albicans and Cryptococcus neoformans) can be achieved at the concentration of 4 μg/mL. Compounds 4b (inhibition of Cryptococcus neoformans), 4j (inhibition of Cryptococcus neoformans), 4d (inhibition of Candida albicans) and 4h (inhibition of Candida albicans) also showed the best antifungal activity at the concentrations of 4 μg/mL. The molecular interactions between 4o and the N-myristoyltransferase of Candida albicans (PDB ID: 1IYL) were finally investigated through molecular docking. The results indicated that these thiochromanone derivatives containing indole skeleton could serve as promising leads for further optimization as novel antifungal agents.

Full text of DOI:10.1248/cpb.c16-00366

Vol. 64, No. 9
Chem. Pharm. Bull. 64, 1411–1416 (2016)
1411
Note
Synthesis, Characterization and Antifungal Evaluation of Novel
Thiochromanone Derivatives Containing Indole Skeleton
,b
Xiao-Yan Han,a Yi-Fan Zhong,a Sheng-Bin Li,a Guo-Chao Liang,a Guan Zhou,a Xiao-Ke Wang,*
,a
Bao-Hua Chen,a and Ya-Li Song*
a Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmaceutical Sciences, Hebei
b
University; Baoding, Hebei 071002, China: and Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of
Ministry of Education, College of Chemistry and Environmental Science, Hebei University; Baoding, Hebei 071002,
China.
Received May 1, 2016; accepted June 20, 2016; advance publication released online July 2, 2016
Invasive fungal disease constitutes a growing health problem and development of novel antifungal drugs
with high potency and selectivity against new fungal molecular targets are urgently needed. In order to de-
velop potent antifungal agents, a novel series of 6-alkyl-indolo[3,2-c]-2H-thiochroman derivatives were syn-
thesized. Microdilution broth method was used to investigate antifungal activity of these compounds. Most of
them showed good antifungal activity in vitro. Compound 4o showed the best antifungal activity, which (in-
hibition of Candida albicans and Cryptococcus neoformans) can be achieved at the concentration of 4µg/mL.
Compounds 4b (inhibition of Cryptococcus neoformans), 4j (inhibition of Cryptococcus neoformans), 4d (in-
hibition of Candida albicans) and 4h (inhibition of Candida albicans) also showed the best antifungal activity
at the concentrations of 4µg/mL. The molecular interactions between 4o and the N-myristoyltransferase of
Candida albicans (PDB ID: 1IYL) were finally investigated through molecular docking. The results indicated
that these thiochromanone derivatives containing indole skeleton could serve as promising leads for further
optimization as novel antifungal agents.
Key words antifungal activity; antifungal lead compound; thiochromanone; indole; molecular docking
Over the past two decades, the incidence of invasive fun- targeting and the function of many proteins in a variety of
gal infections and associated mortality has been increasing signal transduction cascades and other critical cellular func-
dramatically, due to a huge increase in the number of patients tions.14) Moreover, NMTs are striking in their remarkable
undergoing organ transplants, anticancer chemotherapy, or diversity of peptide substrates and diversity in the peptide-
other procedures that cause potential hosts to become immu- binding groove has offered potential of developing species-
nocompromised.1) However, effective and low toxic antifungal specific inhibitors.14) As a result, NMT has been identified
agents are limited. Clinically, Aspergillus fumigatus (mortal- as a potential chemotherapeutic target enzyme for antifungal
ity rate: 50–90%), Cryptococcus neoformans (mortality rate: agents.1)
20–70%), and Candida albicans (mortality rate: 20–40%) have
Previously, Sheng and colleagues12) had prepared tetrahy-
been identied as the most common causes of fungal infec- drocarbazole derivatives (Fig. 1) and tested their antimicro-
tions.2–4) For the treatment of these infections, orally active bial activity (minimal inhibitory concentration (MIC) range:
polyenes (e.g., amphotericin B),5) fluorinated pyrimidines (e.g., 0.0156 to 64μg/mL). It had broad-spectrum inhibitory activity
5-fluorocytosine), azoles (e.g., fluconazole and voriconazole),6) against a wide range of fungal pathogens. Because the chemi-
and echinocandins (e.g., caspofungin and micafungin),7) have cal scaffold of this compound differs from that of all reported
been widely used in clinic use8) (Fig. 1). However, several antifungal agents, it is interesting to investigate its structure–
factors have limited their practical applications. For example, activity relationships (SARs) and discover the novel antifungal
amphotericin B has significant nephrotoxicity and many other lead compound which has a similar skeleton structure to tetra-
side effects. Severe resistance to azoles is increasingly being hydrocarbazole derivatives.
reported. Echinocandins cannot be orally administrated and
Thiochromanone is a versatile reagent that has been ex-
are not active against Cryptococcus neoformans.9–11) To sum tensively utilized in heterocyclic synthesis.15) It had been
up, these antifungal agents have achieved limited success in reported to possess important biological activities. Hoettecke
terms of severe resistance, limited efficacy and spectrum, et al.16) had prepared 2-alkenylthiochroman-4-ones and
drug related toxicity, non-optimal pharmacokinetics, and other tested their antimicrobial activity. Moreover, in our previous
problems.12) Hence, it is urgent to elaborate new, highly potent work,17) 1-(4-phenylthiazol-2-yl)-1,4-dihydrothiochroman[4,3-
antibiotics with alternative modes of actions. In this regard, c]pyrazole was synthesized and showed good inhibition for
the myristoyl coenzyme A (MCoA)/protein N-myristoyltrans- Cryptococcus neoformans. Therefore, the biological activity
ferase (NMT) is an attractive target.13)
of thiochromanone is well documented and thiochromanone
NMT is a ubiquitous enzyme present in eukaryotes such as derivatives with antifungal18,19) and antitumor20) activity have
fungi, protozoa and mammals. It catalyzes the transfer of the been synthesized. These results promoted us to synthesize
14-carbon saturated fatty acid myristate from myristoyl-CoA novel derivatives substituted cyclohexane of tetrahydrocarba-
to the N-terminal glycine residue of a variety of eukaryotic zole with thiochroman.
cellular and viral proteins.13) NMT participates in membrane
Continuing our efforts on the discovery of thiochroma-
*To whom correspondence should be addressed. e-mail: wxkhbu@163.com; yalisong@hbu.edu.cn
© 2016 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 64, No. 9 (2016)
Fig. 1. Chemical Structures of Representative Antifungal Agents
Reagents and conditions: (a) EtOH/reflux/6h/66–75% (b) CH3CN/NaOH/reflux/6min/70–78% (c) CH3CN/K2CO3/KI/reflux/2h/70–91%.
Chart 1. Reagents and Conditions of the Synthetic Compounds 4a–6e
none,19) novel 6-alkyl-indolo[3,2-c]-2H-thiochroman structures 3 in 70–78% yield. Compounds 4–6 was then obtained by
were designed and synthesized. We studied their antifungal treating 3 with secondary amine (R3H), potassium iodide and
activities against Cryptococcus neoformans (C. neo.), Candida anhydrous potassium carbonate in acetonitrile under reflux
albicans (C. alb.), Epidermophyton floccosum (E. flo.), Mucor for about 2h. After completion of the reaction as indicated by
racemosus (M. rac.) and Candida krusei (C. kru.). Crystal TLC, the reaction mixture was purified by column chromatog-
structures of C. alb. NMT complexed with two classes of in- raphy (Chart 1).
hibitors competitive for peptide substrates have been present-
The structures of all compounds are shown in Table 1. All
ed.13) One is a nonpeptidic inhibitor having a benzofuran core the compounds were elucidated by high resolution mass spec-
(Fig. 1). Molecular docking was performed using the X-ray troscope (HR-MS), H-NMR, and 13C-NMR.
1
crystallographic structure of C. alb. NMT in complex with the
Table 2 is shown the reaction conditions exploration of syn-
benzofuran inhibitor 1 (Fig. 1) to explore the binding mode of thetic compound 3. It is clear that the solvent of acetonitrile
the compound at the active site.
and reagent of sodium hydroxide are the best reaction condi-
tions of synthetic compound 3.
Biological Activity In vitro antifungal activity of each
Results and Discussion
Chemistry By means of Fischer indole synthesis, phenyl- compound was expressed as the MIC. As shown in Table
hydrazine hydrochloride and thiochromanone21,22) in an etha- 3, it is clear that target compounds showed weak antifungal
nol solution were heated at reflux for 6h to yield compound activity against E. flo., M. rac. and C. kru., while there is a
2 in 66–75% yield. Compound 2 was then alkylated in aceto- good antifungal activity against C. neo. and C. alb. which are
nitrile solutions under reflux for 6min to obtain compound invasive fungus.

Vol. 64, No. 9 (2016)
Chem. Pharm. Bull.
1413
Table 1. Structure, Yield, Reaction Time and Melting Point of 6-Alkyl-indolo[3,2-c]-2H-thiochroman 4a–6e
No.
R1
R2
R3
n
Time (h)
1.5
Yield (%)
70
mp (°C)
102–104
4a
H
F
2
4b
H
F
2
2
2
1.5
1.5
2
90
85
85
70–72
104–107
84–86
4c
H
H
F
F
4d
4e
4f
H
H
CH3
CH3
2
2
2
88
78
112–115
87–88
1.5
4g
4h
H
H
CH3
CH3
2
2
2.5
1
80
85
133–136
115–118
4i
4j
CH3
CH3
CH3
CH3
2
2
1.5
1.5
70
90
132–134
110–114
4k
4l
CH3
CH3
CH3
CH3
CH3
CH3
2
2
2
2
75
75
70
120–124
138–142
100–103
2.5
2
4m
4n
H
CH3
2
1.5
70
98–103
4o
5a
5b
5c
5d
5e
5f
H
Cl
2
3
3
3
3
3
3
3
3
1
1
1
1
1
1.5
2
91
85
80
80
85
87
83
77
81
70
75
75
70
70
94–96
134–135
122–123
122–124
124–126
82–84
H
CH3
CH3
CH3
CH3
F
H
1.5
2
CH3
CH3
H
1.5
2
H
F
1.5
2
111–113
124–126
97–99
5g
5h
6a
6b
6c
6d
6e
H
Cl
H
Cl
1.5
2
H
F
102–104
98–100
100–102
102–104
99–102
H
Cl
2
H
CH3
CH3
F
1.5
1.5
1.5
CH3
H

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Chem. Pharm. Bull.
Vol. 64, No. 9 (2016)
Table 2. The Influence of Solvent and Reagent on the Yield of Com-
pound 3a)
Solvent
Reagent
Yield (%)
C2H5OH
C2H5OH
C2H5OH
CH3CN
CH3CN
CH3CN
None
K2CO3
(Et)3N
NaOH
K2CO3
(Et)3N
NaOH
NaOH
3
—
11
5
—
76
34
a) All conditions are reflux. “—” represents no reaction.
Table 3. Minimum Inhibitory Concentrations (MIC µg/mL) of Target
Compoundsa)
MIC (µg/mL)
Fig. 2. MIC (µg/mL) of Target Compounds 4a–o against C. neo. and
C. alb.
Compound
C. neo.
C. alb.
E. flo.
M. rac.
C. kru.
4a
4b
4c
4d
4e
4f
128
4
128
8
128
16
32
8
>128
64
>128
128
>128
64
32
8
16
4
128
64
128
8
128
8
64
16
32
8
64
64
64
64
4g
4h
4i
64
8
32
4
>128
32
128
32
64
4
64
8
128
16
64
32
32
64
16
32
64
16
16
16
32
64
16
64
64
64
32
16
8
128
64
128
64
4j
4k
4l
64
16
32
64
4
64
16
32
32
4
>128
64
128
>128
32
4m
4n
4o
5a
5b
5c
5d
5e
5f
64
32
64
64
32
16
64
8
64
64
16
8
128
>128
64
128
>128
64
Fig. 3. MIC (µg/mL) of Target Compounds That R3 Is Pyrrolidin or N-
Methylpiperazine against C. neo. and C. alb.
16
16
16
32
8
64
64
16
32
64
16
16
16
32
16
16
32
4
128
64
64
128
64
compounds also exceeded the level of fluconazole.
5g
5h
6a
6b
6c
6d
6e
F
128
32
As shown in Fig. 2, the target compounds 4a–o, when R3 is
pyrrolidine, 4b, j, o (MIC=4µg/mL) showed better inhibition
activity to C. neo. When R3 is N-methylpiperazine, the MIC
of 4d and h is 8µg/mL. When R3 is morpholine, piperidine or
diethylamine, the compounds showed weak antifungal activity
(MIC>16 µg/mL).
64
32
16
16
16
32
32
4
64
64
128
128
64
64
>128
64
64
64
64
16
Compounds 4d and h (R3 is N-methylpiperazine) showed
better inhibition activity to C. alb. (MIC=4µg/mL). When
R3 is pyrrolidine, the MIC of 4b, f and j is 8µg/mL. When
R3 is morpholine, piperidine or diethylamine, the compounds
showed weak antifungal activity (MIC>16 µg/mL).
B
2
4
2
a) F, Fluconazole. B, Amphotericin B. C. neo., Cryptococcus neoformans. C. alb.,
Candida albicans. E. flo., Epidermophyton floccosum. M. rac., Mucor racemosus. C.
kru., Candida krusei.
Alkane chain of 4a–o is three carbon chains, 5a–h is four
We mainly discuss the target compounds antifungal activ- carbon chains and 6a–e is two carbon chains. As shown in
ity against C. neo. and C. alb., 4b, d, h, j and o which have Fig. 3, while R3 is pyrrolidin or N-methylpiperazine, the three-
better antifungal activity. Among them, 4o showed the best carbon chain alkane compounds 4a–o showed the highest
antifungal activity. Not only is compound 4o antifungal activ- antifungal activity.
ity against C. neo. and C. alb. superior to the positive control
The inhibition activity to C. neo., when R3 is pyrrolidine
Fluconazole but also surpassed Amphotericin B activity. or N-methylpiperazine, 4b, d, h, j, o (three carbon chains)
Compounds 4b and j (inhibition of C. neo.) can be achieved at >5a–f (four carbon chains) or 6a, b (two carbon chains). The
4µg/mL. Compounds 4d and h (inhibition of C. alb.) can be inhibition activity to C. alb., when R3 is N-methylpiperazine
achieved at 4µg/mL. In addition, antifungal activity of other or pyrrolidine, 4b, d, f, h, j (three carbon chains) >5a–h (four

Vol. 64, No. 9 (2016)
Chem. Pharm. Bull.
1415
Fig. 4. The Predicted Binding Modes of the Benzofuran Inhibitor 1 /4o-N-Myristoyltransferase
A: Predicted binding mode of the benzofuran inhibitor 1 docked into the binding site of N-myristoyltransferase; B: Predicted binding mode of 4o docked into the bind-
ing site of N-myristoyltransferase.
carbon chains) or 6a–e (two carbon chains).
Conclusion
Molecular Docking In an attempt to correlate the SARs
In summary, a novel series of thiochromanone derivatives
with the binding mode of the synthesized compounds, the containing indole skeleton were synthesized and evaluated as
benzofuran inhibitor 1 which was reported as NMT inhibi- antifungal inhibitors. Compound 4o (4-chloro-6-(3-pyrrolidin-
tor by Ohtsuka and colleagues23) and 4o were docked into the 1-yl-propyl)-indolo[3,2-c]-2H-thiochroman) showed the best
crystal structure of NMT from C. alb. (CaNMT).13) The dock- antifungal activity in vitro, which (inhibition of C. neo. and C.
ing results of the benzofuran inhibitor 1 and 4o were shown alb.) can be achieved at the concentration of 4µg/mL. More-
in Fig. 4.
over, molecular docking was performed to position compound
The overall conformation of the benzofuran inhibitor 1 in 4o into the C. alb. NMT active site to determine the potential
the active site of CaNMT was shown in Fig. 4A. The benzofu- binding model. Molecular docking studies rationalized the
ran ring was located at the center of the active site, surround- biological properties of compounds by identifying the pos-
ed by some hydrophobic residues, such as Tyr225, Tyr354 and sible interactions with the molecular targets. In conclusion,
Leu394. The phenyl part of the benzofuran ring could form these preliminary results are promising and some of these
π–π interaction with Tyr225. The substituted phenyl group of compounds may be potential candidates for antifungal agents.
C-2 side chain formed hydrophobic interaction with Phe115
and Phe240. The terminal pyridinyl group C-4 side chain
was surrounded by hydrophobic aromatic residues, such as
Experimental
General chemistry methods, synthesis procedures, spec-
Tyr107, Phe117 and Phe176. Four hydrogen bonds were ob- tral data, biological assays, molecular docking are given in
served between the benzofuran inhibitor 1 and CaNMT. The Supplementary materials.
second amine group of C-4 side chain made a hydrogen bond
with C-terminal carboxylate of Leu451, which is an impor-
Acknowledgments We gratefully acknowledge the Open
tant functional residue in the catalytic cycle of CaNMT. The Project Program of Key Laboratory of Pharmaceutical Qual-
oxygen atom of the benzofuran ring and the nitrogen atom of ity Control of Hebei Province, for financial support; the Youth
the methylimidazole ring formed two hydrogen bonds with Foundation of Science and Technology Reasearch in Higher
His227 and Asn392, respectively. Another hydrogen bond was Education Institutions of Hebei Province (QN2015039) for fi-
formed between the pyridine nitrogen of C-4 side chain and nancial support; Hebei University innovative funding projects
Tyr119 hydroxyl group.
for graduate students. We gratefully acknowledge Dr. Carl
The overall conformation of compound 4o in the active site LeBlond (Chemistry Department, College of Natural Sciences
of CaNMT was shown in Fig. 4B. The nitrogen atom of the and Mathematics of IUP) for his helping for grammar and
pyrrolidine ring formed one hydrogen bond with Leu451. The spelling of the paper.
indolo[3,2-c]-2H-thiochroman ring was surrounded by some
hydrophobic residues, such as Tyr225, Tyr354 and Leu394.
Conflict of Interest The authors declare no conflict of
The phenyl part of the thiochroman ring could form π–π in- interest.
teraction with Tyr225. The pyrrolidine ring was surrounded
by hydrophobic aromatic residues, such as Tyr107, Phe117 and
Supplementary Materials The online version of this ar-
Phe176. However, three hydrogen bonds were lost for com- ticle contains supplementary materials.
pound 4o, mainly because of the structure of 4o lacking of a
chain interacting with the residues His227 and Asn392. The
binding mode of compound 4o in the active site of CaNMT
was very similar to that of the benzofuran inhibitor. As a
result, the similar binding mode contributed to the excellent
inhibition activity of compound 4o against C. alb.
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