Novel potentially antifungal hybrids of 5-flucytosine and fluconazole: Design, synthesis and bioactive evaluation

Article abstract of DOI:10.1016/j.bmcl.2017.10.020

A series of novel potentially antifungal hybrids of 5-flucytosine and fluconazole were designed, synthesized and characterized by ¹H NMR, ¹³C NMR, IR and HRMS spectra. Bioactive assay manifested that some prepared compounds showed moderate to good antifungal activities in comparison with fluconazole and 5-flucytosine. Remarkably, the 3,4-dichlorobenzyl hybrid 7h could inhibit the growth of C. albicans ATCC 90023 and clinical resistant strain C. albicans with MIC values of 0.008 and 0.02 mM, respectively. The active molecule 7h could not only rapidly kill C. albicans but also efficiently permeate membrane of C. albicans. Molecular docking study revealed that compound 7h could interact with the active site of CACYP51 through hydrogen bond. Quantum chemical studies were also performed to explain the high antifungal activity. Further preliminary mechanism research suggested that molecule 7h could intercalate into calf thymus DNA to form a steady supramolecular complex, which might block DNA replication to exert the powerful bioactivities.

Full text of DOI:10.1016/j.bmcl.2017.10.020

Accepted Manuscript
Novel potentially antifungal hybrids of 5-flucytosine and fluconazole: Design,
synthesis and bioactive evaluation
Xian-Fu Fang, Di Li, Vijai Kumar Reddy Tangadanchu, Lavanya Gopala, Wei-
Wei Gao, Cheng-He Zhou
PII:
S0960-894X(17)30993-9
https://doi.org/10.1016/j.bmcl.2017.10.020
BMCL 25349
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
11 August 2017
6 October 2017
8 October 2017
Please cite this article as: Fang, X-F., Li, D., Tangadanchu, V.K.R., Gopala, L., Gao, W-W., Zhou, C-H., Novel
potentially antifungal hybrids of 5-flucytosine and fluconazole: Design, synthesis and bioactive evaluation,
Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.10.020
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Bioorganic & Medicinal Chemistry Letters
jour nal homepage: w ww.elsevier. com
Novel potentially antifungal hybrids of 5-flucytosine and fluconazole: Design,
synthesis and bioactive evaluation
†
‡
Xian-Fu Fang, Di Li, Vijai Kumar Reddy Tangadanchu , Lavanya Gopala , Wei-Wei Gao, Cheng-He
*
Zhou
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing Municipality, School of Chemistry and Chemical
Engineering, Southwest University, Chongqing 400715, PR China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of novel potentially antifungal hybrids of 5-flucytosine and fluconazole were
1
13
designed, synthesized and characterized by H NMR, C NMR, IR and HRMS spectra.
Bioactive assay manifested that some prepared compounds showed moderate to good
antifungal activities in comparison with fluconazole and 5-flucytosine. Remarkably, the 3,4-
dichlorobenzyl hybrid 7h could inhibit the growth of C. albicans ATCC 90023 and clinical
resistant strain C. albicans with MIC values of 0.008 and 0.02 mM, respectively. The active
molecule 7h could not only rapidly kill C. albicans but also efficiently permeate membrane of
C. albicans. Molecular docking study revealed that compound 7h could interact with the active
site of CACYP51 through hydrogen bond. Quantum chemical studies were also performed to
explain the high antifungal activity. Further preliminary mechanism research suggested that
molecule 7h could intercalate into calf thymus DNA to form a steady supramolecular complex,
which might block DNA replication to exert the powerful bioactivities.
Keywords:
5
-Flucytosine
Fluconazole
Antifungal
Molecular modeling
Quantum chemical studies
DNA
2017 Elsevier Ltd. All rights reserved.
3
The development of drug-resistant fungal strains presents a
serious threat to human health worldwide because conventional
antifungal agents are becoming weak efficacy in treating fungal
infections. Therefore, the increasingly active effort is to discover
and investigate their antifungal potencies.
Fluconazole as one of the most important triazole antifungal
drugs plays a significant role in prophylaxis and treatment of
both superficial and invasive yeast fungal infections. It is a potent
inhibitor of cytochrome P450 for the biosynthesis of ergosterol
which is an essential component of the yeast cell membrane. Due
to the good absorption and high oral bioavailability, fluconazole
is the first choice of antifungal agent for treating infections
caused by Candida albicans and Cryptococcus neoformans and
has an exceptional therapeutic record including potent activity,
excellent safety profile and favorable pharmacokinetic
new antifungal agents. 5-Fluorocytosine is
a synthetic
antimycotic drug in basic health systems, however, 5-
fluorocytosine has no intrinsic antifungal activity indeed, only
after uptake by the fungal cells, this compound can be converted
into 5-fluorouracil first, and further turned into metabolite 5-
fluorodeoxyuridine, which inhibits the synthesis of RNA and
1
DNA. Unfortunately, 5-fluorocytosine as a single antifungal
agent in clinical use is problematic owing to its relatively weak
antifungal efficacy and fast development of resistance. Thus, the
combinational use of 5-fluorocytosine with clinical antifungal
drugs is a prevalent therapy for fungal infection. Amphotericin B
with 5-fluorocytosine can treat severe systemic mycoses, such as
4
characteristics. However, the excessive use of fluconazole has
resulted in the increasing emergency of fluconazole-resistant C.
albicans strains. Therefore, the development of resistant strains,
the observed toxicity and the limited antifungal spectrum of
fluconazole have created an urgent need to develop novel
antifungal drugs with ameliorated potency, reduced adverse
effects and completely novel targets. Currently, a large amount of
effort has been directed towards structural modification of
fluconazole to exploit fluconazole-based drugs with good
efficiency, broad 5–a6ntimicrobial spectrum and amendatory
therapeutic indexes.
cryptococcosis,
candidosis,
chromoblastomycosis
and
aspergillosis. Particularly, the combination of 5-fluorocytosine
with azole-type antifungal agents such as fluconazole,
itraconazole or ketoconazole exhibits excellent antifungal
2
effects. Attracted and inspired by such features, it is of great
interest for us to employ 5-fluorocytosine as a constructing block
to develop a series of hybrids of 5-fluorocytosine and fluconazole,
—
——
In the design of new drugs, the hybridization approach is a
practical strategy worldwide to discover molecules with better
activity and less drug resistance due to the possibly different
action mechanisms of hybrids. Our previous work has reported
some successful hybridization: a) clinafloxacin-fluconazole
*
Corresponding author. Tel./fax: +86-23-68254967.
E-mail: zhouch@swu.edu.cn (C.-H. Zhou)
Postdoctoral fellow from CSIR-Indian Institute of Chemical Technology,
†
7–8
Hyderabad 500007, India.
‡
Postdoctoral fellow from Sri Venkateswara University, India.

derivatives displayed broad antimicrobial spectrum, good
antibacterial and antifungal activities with low MIC values
ranging from 0.25 to 2 µg/mL; b) metronidazole-quinolones
exhibited good or even stronger antimicrobial activities in
comparison with reference drugs and were proven that the
possible antibacterial mechanism might be in relation with
multiple binding sites between bioactive molecules and topo IV–
DNA complex.
derivatives 6a–k with yields ranging from 19.4 to 30.6%. The
reaction of molecule 5 with halobenzyl halides produced the
halophenyl derivatives 7a–h in 20.3–30.8% yields. All the new
compounds were characterized by H NMR, C NMR, IR and
HRMS spectra.
1
13
All the newly prepared compounds were screened for their
antifungal efficacies. Their minimum inhibitory concentration
(
MIC) in vitro was determined according to the method
recommended by the National Committee for Clinical Laboratory
Substitution
N
Standards (NCCLS) using the two folds serial dilution method in
13
F
F
N
N
96-well microtest plates. The tested fungi included two standard
fungal strains (Candida albicans ATCC 90023, Candida
parapsilosis ATCC 22019) and three clinical fungal strains
N
NH2
HO
F
(
Candida albicans, Aspergillus fumigatus, Candida tropicals)
HN
N
N
with the positive controls of clinical antifungal fluconazole and
5
-flucytosine. The antifungal data were summarized in Table 1.
O
N
Antimycotic drug
Antifungal fluconazole
The antifungal evaluation in vitro displayed that some
prepared hybrids showed good bioactivities against the tested
fungal strains (Table 1). Remarkably, 5-flucytosine-fluconazole
hybrid 5 exhibited moderate to good antifungal activities with
MIC values ranging from 0.04 to 0.17 mM in comparison with
clinical drugs, which suggested that the hybridization of 5-
flucytosine and fluconazole was favorable for the antifungal
potentiality. According to this evidence, various substituents
including alkyl and aryl groups were further introduced to
modify 5-flucytosine-fluconazole hybrid 5 in order to illuminate
the structure-activity relationships.
Hybridization
F
NH
OH
N
N
F
N
R
N
O
N
R = H, alkyl, aryl
Target molecules
The replacement of hydrogen atom in the NH bridge of
compound 5 with ethyl group led to compound 6a with lower
antifungal efficacy. Further extension of the carbon chain gave a
series of derivatives 6b–i with weak or no obviously antifungal
activities in comparison with compound 5 and fluconazole, while
the antifungal activity of ethyl analogue 6b was superior to that
of 5-flucytosine towards all the tested fungal pathogen. Specially,
alkyl compounds 6a–i showed good anti-C. tropicals and anti-A.
fumigatus activities (except for hybrids 6f and 6g) in contrast
with 5-flucytosine. These results revealed that the length of alkyl
chain seemed to have weak effect on biological activities. Propyl
analogue 6b had moderate bioactivities against the tested fungal
strains with MIC values ranging from 0.31 to 0.62 mM.
Meanwhile, propyl chain was replaced by the unsaturated alkyl
groups to produce compounds 6j and 6k, which exerted
approximately the same antifungal activity in comparison with
propyl hybrid 6b, while their antifungal activities against three
clinical fungal strains were better than those of 5-fluorocytosine
and fluconazole (except for fluconazole aganist C. albicans).
These data suggested that the introduction of saturated or
unsaturated alkyl substituents exhibited weak effect on the
antifungal potencies.
F
Figure 1. Design of novel target hybrids of 5-flucytosine and fluconazole.
Based on the above mentions and as an extension of our
studies on the development of azole compounds,
9
–11
we would
like to combine the clinical antimycotic 5-fluorocytosine and
antifungal fluconazole into one molecule for the first time to
generate a series of novel hybrids 5–7 as potentially antifungal
agents. Various alkyl or aryl groups were introduced into the
target hybrids to investigate the effects of different substituents
on biological activities. Their antifungal activities were evaluated
in vitro against five fungi strains. The fungicidal kinetic assay
and membrane permeabilization of the highly active molecule
were also evaluated. Moreover, the molecular modeling and
experimental investigation of the highly active 5-fluorocytosine-
fluconazole hybrid with DNA were further studied to explore the
possible antifungal mechanism.
The synthetic route of target hybrids of 5-flucytosine and
fluconazole was outlined in Scheme 1. The desired molecules
were prepared via multistep reactions starting from commercially
available materials 2,4-difluorobenzene, triazole and 5-
flucytosine. The chloroacetylated intermediate 2 could be
efficiently prepared in satisfactory yield of 65.8% by the
acetylation of 2,4-difluorobenzene 1 with chloroacetyl chloride,
and was then N-alkylated with 1,2,4-triazole in acetonitrile in the
presence of potassium carbonate to afford the triazolyl ethanone
In comparison with alkyl derivatives 6a–k, most of
halobenzyl compounds 7a–h gave relatively stronger activities in
inhibiting the growth of the tested strains. Noticeably, in this
halobenzyl series, compound 7h with 3,4-dichlorobenzyl group
exerted the best activities with MIC values of 0.008–0.03 mM,
especially, it could inhibit the growth of C. albicans ATCC
3
in good yield of 70.4%. Further epoxidation of compound 3 in
9
0
0023 and clinical resistant strain C. albicans with MIC values of
.008 and 0.02 mM, respectively. Moreover, its anti-C. tropicals
dichloromethane by trimethyl sulfoxonium iodide (TMSOI),
cetyltrimethylammonium bromide (CTAB) and 40% potassium
o
(MIC = 0.03 mM) and anti-A. fumigatus (MIC = 0.03 mM)
activities were superior to those of fluconazole and 5-
fluorocytosine. For clinical C. albicans strains, it also showed
eight times more active than fluconazole and 5-fluorocytosine.
The substitution of 3,4-dichlorobenzyl moiety by 4-chlorobenzyl
group yielded analogue 7d, which showed stronger bioactivities
against three clinical fungal strains with MIC values of 0.13 mM
hydroxide at 40–45 C produced the triazolyl oxirane 4 in 68.2%
11
yield. The target molecule 5 was conveniently obtained in yield
of 40.1% by the reaction of 5-flucytosine with compound 4 in
ethanol using potassium carbonate as base at 80 C. The N-
alkylation of compound 5 with a series of saturated and
unsaturated alkyl bromides in ethanol at 65 C in the presence of
o
o
potassium carbonate as base respectively afforded alkyl

than fluconazole and 5-fluorocytosine. Furthermore, 2,4-
dichlorobenzyl derivative 7g gave MIC value of 0.12 mM against
C. tropicals, which exhibited much more potent than reference
fluconazole (MIC = 0.83 mM) and 5-flucytosine (MIC = 1.98
mM). However, the replacement of 3,4-dichlorobenzyl fragment
in compound 7h by fluorobenzyl or chlorobenzyl group afforded
derivatives 7b–c and 7e–f with weaker activities. Additionally,
compound 7a without substituents on the phenyl ring displayed
moderate antifungal efficiencies with MIC values of 0.14–0.28
mM towards the corresponding fungi.
o
Scheme 1. Reagents and conditions: i) ClCH
2
COCl, AlCl
3
, CH
2
Cl
2
, rt; ii) 1,2,4-triazole, K
2
CO
3
, CH
3
CN, rt–80 C; iii) TMSOI, 40% KOH, CTAB,
o
o
dichloromethane, 40–45 C; iv) 5-fluorocytosine, K CO , C
2
3
2
H
5
OH, reflux; v) alkyl bromide, K
2
CO , C
3
2
H
5
OH, 65–75 C; vi) halobenzyl halide, K
2
CO
3
, C
2
H
5
OH,
o
6
5–75 C.
experimental results manifested that this compound had rapidly
killing effect against C. albicans.
Hydrophobic/lipophilic properties possess remarkable effects
on biological activities. Liposome/water partition coefficients
14
1
1
1
4
2
0
8
6
4
2
(
ClogP) have been extensively employed to predict the
Control
Compound 7h
bioactivity of target molecules. The theoretically calculated value
of logP (ClogP) for all the new compounds was calculated using
the commercial ChemBioOffice 2014 (Cambridge Soft,
Massachusettes, USA), and the obtained results were shown in
Table 1. The ClogP values of the alkyl derivatives 6a−i were
related to the length of alkyl chain, displaying that the longer the
alkyl chain was, the higher the ClogP value was, and the
biological activity of these derivatives was weaker. However, for
derivatives 7a–h, the ClogP values varied with the change of the
position and number of fluorine or chlorine atoms on the benzene
ring. The ClogP values followed an order of 2,4-dichlorobenzyl
(
7g) > 3,4-dichlorobenzyl (7h) > 2-chlorobenzyl (7f), 3-
0
2
4
6
8
10
12
chlorobenzyl (7e) and 4-chlorobenzyl (7d) > 3-fluorobenzyl (7c)
and 4-fluorobenzyl (7b). In comparison with other halobenzyl
derivatives, the 3,4-dichlorobenzyl compound 7h showed good
biological activity, which might be explained by the possibility
that compound with appropriate ClogP value was favorable for
being delivered to the binding sites in organism, and manifested
the significant role of suitable lipophilicity in drug design.
Time (h)
Figure 2. Representative time-kill studies of compound 7h at 4 × MIC
against C. albicans. The data obtained from two independent experiments
performed in triplicate.
Fungal cell membrane is an important factor to induce severe
resistance towards clinically available antifungal agents.
Destroying fungal cell membrane through small molecules
represents a promising new strategy to develop novel antifungal
agents. Herein, the permeabilization ability of analogue 7h to
the fungi cell membrane was tested using propidium iodide (PI),
a common dye that can pass through the membrane of
compromised fungi cells and fluoresces upon binding to the
DNA. As can be seen in Fig. 3, the increased fluorescence
intensity within 105 min might be resulted from the formation of
The active molecule 7h was further evaluated the fungicidal
activity against C. albicans via time-kill kinetic assay. The time-
kill kinetic experiment suggested that compound 7h showed
rapidly fungicidal potency. As depicted in Fig. 2, it revealed
more than 3 log (CFU/mL) reduction in the number of viable
fungi within an hour at a concentration of 4 × MIC. The
1
5

PI–DNA complexes as the gradual damage of fungal membrane
demonstrated that compound 7h could efficiently permeate the
in the presence of the active molecule 7h at 12 × MIC, which
membrane of C. albicans.
a, b, c
Table 1 ClogP values and MIC values (mM) of compounds 5, 6a–k, 7a–h, fluconazole and 5-flucytosine against fungal strains.
C. albicans
ATCC 90023
C. parapsilosis
ATCC 22019
Compds
ClogP
C. albicans C. tropicals A. fumigatus
5
–0.52
0.18
0.71
1.24
1.77
2.30
3.36
4.42
5.47
7.59
0.07
0.43
1.37
1.51
1.51
2.08
2.08
2.08
2.80
2.68
–0.44
–1.63
0.17
0.32
0.31
0.30
0.58
0.56
0.53
2.02
1.91
1.73
1.26
0.62
0.28
0.53
0.53
0.13
0.13
0.26
0.12
0.008
0.003
0.03
0.17
0.32
0.62
0.30
1.17
0.28
0.53
1.01
0.95
0.86
1.26
1.25
0.28
0.53
0.53
0.13
0.26
0.52
0.48
0.03
0.006
0.99
0.17
0.16
0.31
0.30
0.58
0.56
1.06
1.01
0.95
0.86
0.31
0.31
0.28
0.26
0.26
0.13
0.13
0.13
0.24
0.02
0.20
0.49
0.04
0.64
0.62
0.60
0.29
1.13
1.06
1.01
0.95
0.86
0.63
0.62
0.28
0.53
0.53
0.13
0.52
0.26
0.12
0.03
0.83
1.98
0.08
0.64
0.62
0.60
0.58
0.56
2.13
2.02
1.91
1.77
0.63
0.62
0.14
0.26
0.53
0.13
0.13
0.26
0.24
0.03
1.65
1.98
6
6
a
b
6
c
6
d
6
e
6
f
6
6
g
h
6
i
6
j
6k
7a
7b
7
c
7
d
7
e
7
f
7
7
g
h
Fluconazole
5
-Fluorocytosine
a
b
Minimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates.
Candida albicans ATCC 90023, C. albicans ATCC 90023; Candida parapsilosis ATCC 22019, C. parapsilosis ATCC 22019; Candida albicans, C. albicans;
Candida tropical, C. tropical; Aspergillus fumigatus, A. fumigatus.
c
ClogP values were calculated by ChemBioOffice 2014.
1000
Compound 7h
Control
800
600
400
200
0
Figure 4. The binding mode of compound 7h in the active site of CACYP51.
Computational methods are able to predict important
pharmacokinetic properties and have been used to rationalize
biological activity. Intermolecular interactions were dominated
by the frontier molecular orbitals (FMO), the highest occupied
molecular orbital (HOMO) and the lowest unoccupied molecular
orbital (LUMO). Therefore, plots of HOMO, LUMO and
energy levels of derivatives 5, 6a, 6g, 6h, 7d and 7h calculated
by using the B3LYP/6-31G(d, p) basis set were presented in
Table 2. It was noteworthy that the active molecule 7h had the
lowest energy gap (∆E) of 4.923 eV, which suggested that
compound 7h could lead to greater stabilizing interactions.
Although the energy gap (∆E) of compounds 6g and 6h is close
to that of the high active compound 7h, the antifungal efficacy
0
15
30
45
60
75
90
105
120
135
150
Time (h)
Figure 3. Fungal membrane permeabilization of compound 7h (12 × MIC)
against C. albicans.
1
7
In order to further investigate the antifungal action mechanism
and the binding mode of compound 7h, it was docked into the
active site of C. albicans CYP51 (PDB code: 5EQB). As shown
in Fig. 4, the binding energy of CYP51 with the active molecule
1
6
−1
7
h was −9.58 kcal mol . The hydroxyl group was in close
vicinity to the residue Tyr132 through a hydrogen bond with
distance of 3.1 Å, which played an important role in
strengthening their binding affinity.

for compounds 6g and 6h is poor due to their poor solubility and
investigate the preliminary mechanism of antifungal action, the
high ClogP values.
binding studies in vitro between the active molecule 7h and calf
thymus DNA on molecular level were carried out by UV–vis
Table 2 displayed that HOMO of the most active antifungal
derivative 7h was mainly located in the pyrimidine, triazole and
,4-difluorobenzene ring, revealing that those rings might be
18–19
spectroscopy.
Fig. S1 showed that the maximum absorption
of DNA at 260 nm displayed proportional increase with the
increasing concentration of compound 7h. Simultaneously, the
absorption value of 7h–DNA complex was a little greater than
the absorption values of simply sum of free DNA and compound
2
active sites and biological interactions possibly existed between
positively charged molecules and these sites. It was also found
that substituents on 5-flucytosine moiety did not contribute
directly to HOMOs indicating that these groups might be
primarily used to adjust the physicochemical properties.
However, the LUMOs of these molecules were mainly focused
on the pyrimidine ring in which nucleophilic attacks might be
favorable.
7
h, which was observed in the inset of Fig. S1. These indicated
that a hyperchromic effect existed between DNA and compound
h. Furthermore, the intercalation of the aromatic chromophore
7
of compound 7h into the helix and the large overlap of π-π*
states in the large π-conjugated system with the electronic states
of DNA bases were consistent with the observed spectral changes.
Table 2 Plots of HOMO, LUMO and energy levels of the molecules 5, 6a, 6g,
Table 3 Comparison of the MEP surfaces of compounds 5, 6a, 7d and 7h.
6
h, 7d and 7h.
Compds
MEPs
Compds
HOMOs
LUMOs
5
6
6
a
g
6
7
7
h
d
h
To further understand the interaction between compound 7h
and DNA, the absorption spectra of competitive interaction of
compound 7h with neutral red (NR) were investigated. NR as a
planar phenazine dye is structurally similar to other planar dyes
acridine, thiazine and xanthene. It has been displayed that the
binding of NR with DNA is an intercalative binding type.
Therefore, this work employed NR as a spectral probe to
investigate the binding mode of 7h with DNA. The absorption
spectra of NR upon the addition of DNA were shown in
Supporting Information. It was apparent that the absorption peak
of the NR at about 460 nm decreased gradually with the
increasing concentration of DNA, and a new band developed
near 530 nm. This was attributed to the formation of the new
DNA–NR complex. An isosbestic point at 504 nm provided the
evidence of the formation of DNA–NR complex (Fig. S3). The
competitive binding between NR and 7h with DNA was
observed in Fig. S4. With the increasing concentration of
compound 7h, a significant increase in intensity was observed
near 460 nm, which was opposite with the absorption of NR–
DNA complex at the same wavelength. The various spectral
changes suggested that compound 7h could substitute NR in the
DNA–NR complex, which revealed that the active molecule
could intercalate DNA.
The molecular electrostatic potential (MEP) surface gives an
indication of the charged surface area, a concept for explaining
the hydrophilicity of compounds and the orientation of drug
candidates for their activities. Thus, the MEPs were performed to
examine the similarities and differences in electrostatic binding
characteristic of the surface of molecules (Table 3). The
comparison of the electrostatic maps of hybrids 5, 6a, 7d and 7h
revealed that molecule 7h possessed more negative charged
regions (in red) on the hydroxyl group, oxygen atom of 5-
flucytosine and nitrogen atoms of triazole ring. It indicates the
capability of forming hydrogen bonds may be related to hydroxyl
group, oxygen atom of 5-flucytosine and nitrogen atoms of
triazole ring. This was in agreement with the binding mode from
docking study (Figure 4).
DNA is a significant informational molecule encoding the
genetic instructions and has been widely researched for the
advisable design and development of efficient drugs. Due to its
biological importance and commercial available property, calf
thymus DNA has always been employed as a model. To
In conclusion, a novel series of hybrids of 5-flucytosine and
fluconazole have been successfully developed starting from
commercially available 2,4-difluorobenzene, triazole and 5-
1
flucytosine. All the new compounds were confirmed by H NMR,

1
3
C NMR, IR and HRMS spectra. The 3,4-dichlorobenzyl
7. (a) Cui SF, Addla D, Zhou CH. J. Med. Chem., 2016, 59, 4488–
510; (b) Cheng Y, Avula SR, Gao WW, Addla D, Tangadanchu
VKR, Zhang L, Lin JM, Zhou CH. Eur. J. Med. Chem., 2016, 124,
4
substituted hybrid 7h could effectively inhibit the growth of C.
albicans ATCC 90023 and clinical strain C. albicans with MIC
values of 0.008 and 0.02 mM, respectively. The active molecule
9
1
35–945; (c) Gao WW, Zhou CH. Future Med. Chem., 2017, 9,
461–1464.
7
h could not only rapidly kill C. albicans but also efficiently
8. (a) Cui SF, Peng LP, Zhang HZ, Rasheed S, Kumar KV, Zhou
CH. Eur. J. Med. Chem., 2014, 86, 318–334; (b) Jeyakkumar P,
Liu HB, Gopala L, Cheng Y, Peng XM, Geng RX, Zhou CH.
Bioorg. Med. Chem. Lett., 2017, 27, 1737–1743; (c) Zhang FF,
Gan LL, Zhou CH. Bioorg. Med. Chem. Lett., 2010, 20, 1881–
permeate membrane of C. albicans. Molecular docking study
revealed that compound 7h could interact with CACYP51 mainly
through hydrogen bond. Quantum chemical studies indicated the
capability of forming hydrogen bond with share of the hydroxyl
group, which was in agreement with the binding mode in docking
study. Further preliminary research revealed that compound 7h
could effectively intercalate into calf thymus DNA to form 7h–
DNA complex, which might block DNA replication to exert
powerful antifungal activity. These results manifested that the
active molecule 7h should be worthy to be further investigated as
a promising starting point for the treatment of fungal infections.
Further researches, including the in vivo bioactive evaluation,
toxicity, resistance study and introduction of various functional
substituents into the hybrid backbone to investigate the effects on
antifungal activities are now in progress in our group.
1
884.
9
.
(a) Zhang L, Peng XM, Damu GLV, Geng RX, Zhou CH. Med.
Res. Rev., 2014, 34, 340–437; (b) Zhang L, Addla D, Ponmani J,
Wang A, Xie D, Wang YN, Zhang SL, Geng RX, Cai GX, Zhou
CH. Eur. J. Med. Chem., 2016, 111, 160–182; (c) Wen SQ,
Jeyakkumar P, Avula SR, Zhang L, Zhou CH. Bioorg. Med. Chem.
Lett., 2016, 26, 2768–2773; (d) Fang XJ, Jeyakkumar P, Avula SR,
Zhou Q, Zhou CH. Bioorg. Med. Chem. Lett., 2016, 26, 2584–
2588.
10. (a) Peng XM, Cai GX, Zhou CH. Curr. Top. Med. Chem., 2013,
1
3, 1963–2010; (b) Zhang HZ, Damu GLV, Cai GX, Zhou CH.
Curr. Org. Chem., 2014, 18, 359–406; (c) Zhang HZ, Gan LL,
Wang H, Zhou CH. Mini-Rev. Med. Chem., 2017, 17, 122–166; (d)
Peng LP, Nagarajan S, Rasheed S, Zhou CH. Med. Chem.
Commun., 2015, 6, 222–229.
Acknowledgments
11. (a) Zhang L, Kumar KV, Rasheed S, Zhang SL, Geng RX, Zhou,
CH. Med. Chem. Commun. , 2015, 6, 1303–1310; (b) Yin BT, Yan
CY, Peng XM, Zhang SL, Rasheed S, Geng RX, Zhou CH. Eur. J.
Med. Chem., 2014, 71, 148–945; (c) Shi Y, Zhou CH. Bioorg.
Med. Chem. Lett., 2011, 21, 956–960; (d) Zhang SL, Chang JJ,
Damu GLV, Fang B, Zhou XD, Geng RX, Zhou CH. Bioorg. Med.
Chem. Lett., 2013, 23, 1008–1012.
This work was partially supported by National Natural
Science Foundation of China (Nos. 21672173 and 21372186), the
Research Fund for International Young Scientists from
International (Regional) Cooperation and Exchange Program of
NSFC (No. 81650110529), Chongqing Special Foundation for
Postdoctoral Research Proposal (No. Xm2016039), and Program
for Overseas Young Talents from State Administration of
Foreign Experts Affairs, China (No. WQ2017XNDX047).
1
2. Borate HB, Maujan SR, Sawargave SP, et al. Bioorg. Med. Chem.
Lett., 2010, 20, 722–725.
1
3. (a) Clinical and Laboratory Standards Institute/National
Committee for Clinical Laboratory Standards: Reference Method
for Broth Dilution Antifungal Susceptibility Testing of Yeast,
third ed., Clinical and Laboratory Standards Institute, Wayne, PA,
2
009. Approved Standard, Document M27-a3; (b) Clinical and
Supplementary data
Laboratory Standards Institute/National Committee for Clinical
Laboratory Standards: Reference Method for Broth Dilution
Antifungal Susceptibility Testing of Yeast, third ed., Clinical and
Laboratory Standards Institute, Wayne, PA, 2008. Approved
Standard, Document M38-A2.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/
1
4. (a) Sundriyal S, Moniot S, Mahmud Z, et al. J. Med. Chem., 2017,
References and notes
6
0, 1928–1945; (B) Duan JR, Liu HB, Jeyakkumar P, Gopala L,
Li S, Geng RX, Zhou CH. Med. Chem. Commun., 2017, 8, 907–
916; (C) Dai LL, Zhang HZ, Nagarajan S, Rasheed S, Zhou CH.
Med. Chem. Commun., 2015, 6, 147–154.
1
.
(a) Perfect JR. Nat. Rev. Drug Discovery, 2017, 16, 603–616; (b)
May RC, Stone NR, Wiesner DL, Bicanic T, Nielsen K. Nat. Rev.
Microbiol., 2016, 14, 106–117; (c) Jung KW, Yang DH, Maeng S,
et al. Nat. Commun., 2015, 6, 1–14; (d) Vermes A, Guchelaar HJ,
Dankert J. J. Antimicrob. Chemother., 2000, 46, 171–179.
15. (a) Wu SC, Wang Y, Liu N, Dong GQ, Sheng CQ. J. Med. Chem.,
2017, 60, 2193–2211; (b) Gong HH, Baathulaa K, Lv JS, Cai GX,
Zhou CH. Med. Chem. Commun., 2016, 7, 924–931; (c) Kang J,
Tangadanchu VKR, Gopala L, Gao WW, Cheng Y, Liu HB, Geng,
RX, Li S, Zhou CH. Chin. Chem. Lett., 2017, 28, 1369–1374.
16. Doğan İS, Saraç S, Sari S, Kart D, Gökhan ŞE., Vural İ, Dalkara S.
Eur. J. Med. Chem., 2017, 130, 124–138.
17. (a) Pomastowski P, Sprynskyy M, Zuvela P, et al. J. Am. Chem.
Soc., 2016, 138, 7899−7909; (b) Gao WW, Rasheed S,
Tangadanchu V, Sun Y, Peng XM, Cheng Y, Zhang FX, Lin JM,
Zhou CH. Sci. China Chem., 2017, 60, 769–785.
18. (a) Husain MA, Ishqi HM, Sarwar T, et al. Med. Chem. Commun.,
2017, 8, 1283–1296; (b) Liu HB, Gopala L, Avula SR,
Jeyakkumar P, Peng XM, Zhou CH, Geng RX. Chin. J. Chem.,
2017, 35, 483–496; (c) Zhang HZ, He SC, Peng YJ, Zhang HJ,
Gopala L, Tangadanchu VKR, Gan LL, Zhou CH. Eur. J. Med.
Chem., 2017, 136, 165–183; (d) Cao QC, Wang H, Tangadanchu
VKR, Gopala L, Zhou CH. Sci. Sin. Chim., 2017, 47, 844–858.
19. (a) Jeyakkumar P, Zhang L, Avula SR, Zhou CH. Eur. J. Med.
Chem., 2016, 122, 205–215; (b) Peng XM, Peng LP, Li S, Avula
SR, Kannekanti VK, Zhang SL, Tam KY, Zhou CH. Future Med.
Chem., 2016, 8, 1927–1940; (c) Addla D, Wen SQ, Gao WW,
Maddili SK, Zhang L, Zhou CH. Med. Chem. Commun., 2016, 7,
1988–1994; (d) Peng XM, Kumar KV, Damu GLV, Zhou CH. Sci.
China Chem., 2016, 59, 878–894.
2
.
(a) Edlind TD, Katiyar SK. Antimicrob. Agents Chemother., 2010,
5
4, 4733–4738; (b) Chapeland-Leclerc F, Hennequin C, Papon N,
et al. Antimicrob. Agents Chemother., 2010, 54, 1360–1362.
Somlai C, Correche E, Olivella M, et al. Bioorg. Med. Chem. Lett.,
3
4
.
.
2
012, 22, 4233–4237.
(a) Shekhar-Guturja T, Gunaherath GMKB, Wijeratne EMK, et al.
Nat. Chem. Biol., 2016, 12, 867–875; (b) Zhou CH, Wang Y.
Curr. Med. Chem., 2012, 19, 239–280; (c) Pore V S, Agalave S G,
Singh P, et al. Org. Biomol. Chem., 2015, 13, 6551–6561; (d)
Wang XL, Wan K, Zhou CH. Eur. J. Med. Chem., 2010, 45,
4
631–4639.
5
6
.
.
(a) Cao XF, Sun ZS, Cao YB, Wang RL, Cai TK, Chu WJ, Hu
WH Yang YS. J. Med. Chem., 2014, 57, 3687–3706; (b) Zhang
HZ, Damu GLV, Cai GX, Zhou CH. Eur. J. Med. Chem., 2013,
6
4, 329–344; (c) Gong HH, Addla D, Lv JS, Zhou CH. Curr. Top.
Med. Chem., 2016, 16, 3303–3364; (d) He SC, Jeyakkumar P,
Avula SR, Wang XL, Zhang HZ, Zhou CH. Sci. Sin. Chim., 2016,
4
6, 823–847.
(a) Shrestha SK, Garzan A, Garneau-Tsodikova S. Eur. J. Med.
Chem., 2017, 133, 309–318; (b) Xie F, Ni TJH, Zhao J, Pang L, Li
R, Cai Z, Ding ZC, Wang T, Yu SC, Jin YS. Bioorg. Med. Chem.
Lett., 2017, 27, 2171–2173; (c) Wang Y, Damu GLV, Lv JS, Geng
RX, Yang DC, Zhou CH. Bioorg. Med. Chem. Lett., 2012, 22,
5
363–5366; (d) Benhamou RI, Bibi M, Steinbuch KB, et al. ACS
Chem. Biol., 2017, 12, 1769–1777; (e) Zhang YY, Mi JL, Zhou
CH, Zhou XD. Eur. J. Med. Chem., 2011, 46, 4391–4402.

Graphical Abstract
Novel potentially antifungal hybrids of 5-
flucytosine and fluconazole: Design,
synthesis and bioactive evaluation
Leave this area blank for abstract info.
†
‡
Xian-Fu Fang, Di Li, Vijai Kumar Reddy Tangadanchu , Lavanya Gopala , Wei-Wei Gao, Cheng-He Zhou*