Molecular docking, design, synthesis and antifungal activity study of novel triazole derivatives containing the 1,2,3-triazole group

Article abstract of DOI:10.1039/c3ra41310a

A series of 1-(1H-1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-[N-propyl-N- ((1-substituted-1H-1,2,3-triazol-4-yl)methyl)amino]-2-propanols which are analogues of fluconazole, have been designed and synthesized on the basis of computational docking experiments to the active site of the cytochrome P450 14α-demethylase (CYP51). Their structures were characterized by ¹H NMR, ¹³C NMR and HR ESI MS. The MIC₈₀ values indicate that the target compounds 1a-o showed higher activities against nearly all the fungi tested to some extent except against A. fum. and T. rub. All of the target compounds exhibited higher activities against C. alb. SC5314 and C. alb. Y0109 than all of the six positive controls.

Full text of DOI:10.1039/c3ra41310a

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Molecular docking, design, synthesis and antifungal
activity study of novel triazole derivatives containing
the 1,2,3-triazole group3
Cite this: DOI: 10.1039/c3ra41310a
Shichong Yu,{a Lunuan Wang,{b Yanwei Wang,b Yang Song,b Yongbing Cao,c
Yuanying Jiang,c Qingyan Sun*a and Qiuye Wu*a
A series of 1-(1H-1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-[N-propyl-N-((1-substituted-1H-1,2,3-triazol-4-
yl)methyl)amino]-2-propanols which are analogues of fluconazole, have been designed and synthesized
on the basis of computational docking experiments to the active site of the cytochrome P450
14a-demethylase (CYP51). Their structures were characterized by 1H NMR, 13C NMR and HR ESI MS. The
MIC80 values indicate that the target compounds 1a–o showed higher activities against nearly all the fungi
tested to some extent except against A. fum. and T. rub. All of the target compounds exhibited higher
activities against C. alb. SC5314 and C. alb. Y0109 than all of the six positive controls.
Received 19th March 2013,
Accepted 30th May 2013
DOI: 10.1039/c3ra41310a
www.rsc.org/advances
subsites: a coordination bond with the iron in the heme group,
the hydrophilic H-bonding region, the hydrophobic region,
Introduction
During the past several decades, the number of invasive deep
fungal infections has continued to increase, particularly in
immunocompromised individuals such as patients under-
going organ transplantation, anticancer chemotherapy, or
those suffering from AIDS.1,2 Three major fungal infections,
including candidosis, aspergillosis and cryptococcosis, are
responsible for clinical infections in immunocompromised
patients.3,4 Currently, triazole agents (fluconazole (FCZ),
itraconazole (ICZ), voriconazole (VCZ), and posaconazole) are
the most frequently used antifungals in clinical use Fig. 1.5
However, FCZ is not effective against invasive aspergillosis and
has suffered from severe drug resistance.6,7 This situation,
thus, highlights the need for new triazole derivatives posses-
sing broader antifungal spectra and higher therapeutic
indexes.
and the narrow hydrophobic cleft formed by the residues in
the helix B9-meander 1 loop and the N-terminus of helix I.9
Some studies10,11 have revealed
a pharmacophore of
antifungal triazoles, which contains a triazole ring linked to
a dihalophenyl ring through a two carbon chain. In addition,
the carbon alpha to the phenyl ring bears a hydroxyl group. We
intended to alter the side chains to find potent systemic
antifungal compounds with a broad antifungal spectrum and
less potential to develop resistance.
To design the new antifungal compounds, firstly we did a
molecular docking investigation, we constructed a 3D Candida
Azole antifungals act by competitive inhibition of CYP51,
the enzyme that catalyzes the oxidative removal of the
14a-methyl group of lanosterol to give D14,15-desaturated
intermediates in the ergosterol biosynthesis.8 In general, the
active site of CYP51 for ligand binding can be divided into four
aDepartment of Organic Chemistry, College of Pharmacy, Second Military Medical
University, 325 Guohe Road, Shanghai 200433, People’s Republic of China.
E-mail: qywu2008@163.com; sqy_2000@163.com; Fax: 086 21 8187 1225;
Tel: 086 21 8187 1225
bPharmacy Team, Administrative Brigade of Postgraduate, Second Military Medical
University, 41 Zhengtong Road, Shanghai 200433, People’s Republic of China
cDrug Research Center, School of Pharmacy, Second Military Medical University, 325
Guohe Road, Shanghai 200433, People’s Republic of China
Electronic supplementary information (ESI) available: 1H NMR, 13C NMR and
3
HR ESI MS spectral data of compounds 1b–o. See DOI: 10.1039/c3ra41310a
{ Shichong Yu and Lunuan Wang contributed equally to this manuscript.
Fig. 1 Triazole antifungal agents used in clinical therapy.
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Fig. 3 Generic structure of the designed fluconazole analogues.
Scheme 1. After the key intermediate oxirane 5 was synthesized
by a known procedure,13 compound 6 was synthesized by the
ring-opening reaction of the oxirane 5 with propylamine, and
then in the presence of KI and K2CO3 in acetonitrile at room
temperature compound 7 was prepared. The target com-
pounds were obtained by using a click reaction14 with various
substituted benzyl derivatives.
Fig. 2 Computed compound 1i binding to the heme iron atom at the active site
of CYP51.
Pharmacology
The in vitro antifungal activities of all the target compounds
were evaluated against eight human pathogenic fungi,
Candida albicans SC5314 (C. alb. SC5314), Cryptococcus neofor-
mans (C. neo.), Candida parapsilosis (C. par.), Candida albicans
Y0109 (C. alb. Y0109), Candida tropicalis (C. tro.), Trichophyton
rubrum (T. rub.), Candida krusei (C. kru.), Aspergillus fumigatus
(A. fum.), which are often encountered clinically, and they were
compared with itraconazole (ICZ), voriconazole (VCZ) and
fluconazole (FCZ). C. alb. SC5314 and C. neo. were provided by
Shanghai Changzheng Hospital; C. par., C. alb. Y0109, C. tro.,
T. rub., C. Kru. and A. fum. were provided by Shanghai
Changhai Hospital. C. alb. SC5314 and C. neo. were purchased
from ATCC, and other strains were clinic isolates. Fluconazole
(FCZ), itraconazole (ICZ) and voriconazole (VCZ) served as the
positive controls and were obtained from their respective
manufacturers.
The in vitro minimal inhibitory concentrations (MICs) of the
compounds were determined by the micro-broth dilution
method in 96-well microtest plates according to the methods
defined by the National Committee for Clinical Laboratory
Standards (NCCLS).15 The MIC80 was defined as the first well
with an approximately 80% reduction in growth compared to
the growth in the drug-free well. For assays, the title
compounds to be tested were dissolved in dimethyl sulfoxide
(DMSO), serially diluted in growth medium, inoculated and
incubated at 35 uC. The growth MIC was determined at 24 h for
C. alb. and at 72 h for C. neo. The results of the assays are
summarized in Table 1. The data points are the mean of the
replicates. All of our susceptibility tests were performed three
times with each of the antifungal agents.
albicans CYP51 on the basis of work by Ji et al.12 A likely
binding mode of compound 1i in the active site of CYP51 is
proposed based on computational docking results (Fig. 2).
Sheng et al.10 reported that both the R and S isomers of the
compounds interacted with CYP51 through a similar binding
mode. However, the R isomer had a lower interaction energy
with CYP51 than the S isomer, which indicated that the R
isomer might have better antifungal activity than the S isomer.
In our discussion, the docked conformations refer to the R
configuration of the compounds. As usual, the N(4) atom of
the 1,2,4-triazole moiety of 1i coordinates with the heme Fe-
atom, while the 2,4-difluorophenyl group in the designed
compound could be placed into the hydrophobic pocket
formed by Pro375, Leu376, His377, Ser378, Ile379 and Phe380.
The 1,2,3-triazole group in the side chain would generate p–p
stacking interactions with Tyr118. Finally, the substituted
benzyl could interact with the hydrophobic pocket formed by
Gly303, Met306, Gly307, Gly308 and Tyr311. In addition, the
side chains were the pharmacophores, and they were oriented
in the hydrophobic pocket. The side chains of the inhibitors
were found to be very important.
According to the above results, we designed a series of 1-
(1H-1,2,4-triazole-1-yl)-2-(2,4-difluorophenyl)-3-substituted-2-
propanols (1, Fig. 3) containing a triazole ring, a difluorophe-
nyl group, a hydroxyl group and a side chain. In our design, we
systematically altered the structure of the fluconazole platform
and inserted a 1,2,3-triazole group into the side chain.
The in vitro antifungal activities are summarized in Table 1.
The MIC values (in mg mL21) are presented against different
pathogenic fungi, in comparison with ICZ, VCZ and FCZ.
The results of the antifungal activities in vitro showed that
all of the target compounds were active against nearly all of the
fungi tested to some extent except against A. fum. and T. rub.
Results and discussion
Chemistry
The target compounds 1a–o were synthsized according to the
very efficient and straightforward synthetic route outlined in
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Scheme 1 Synthesis of the target compounds 1a–o. Conditions: (a) ClCH2COCl, AlCl3, 50 uC, 5 h, in 80% yield; (b) C6H5CH3, NaHCO3, 1H-1,2,4-triazole, reflux, 5 h, in
42% yield; (c) C6H5CH3, (CH3)3SOI, NaOH, cetylmethylammonium bromide, 60 uC, 3 h, in 86% yield; (d) CH3SO3H, 0 uC, 1 h, in 89% yield; (e) CH3CH2OH, Et3N,
propylamine, reflux, 6 h, in 80% yield; (f) CH3CN, KI, K2CO3, propargyl bromide, rt, 5–6 h, in 70% yield; (g) NaN3, substituted benzyl bromide, DMSO, CuSO4?5H2O,
sodium ascorbate, rt, 12 h, in 60–70% yield.
All of the target compounds exhibited higher activities against
C. alb. SC5314 and C. alb. Y0109 than all of the three positive
controls. Obviously, the MIC80 value of compound 1i is 128
times lower than that of FCZ against C. alb. Y0109 in vitro (with
an MIC80 value of 0.0039 mg mL21), 16 times lower than that of
ICZ against C. alb. Y0109 in vitro; the MIC80 values of
Table 1 Antifungal activities of the target compounds in vitro (MIC80, mg mL21
)
Compd
C. alb. SC5314
C. alb. Y0109
C. neo.
A. fum.
T. rub.
C. tro.
C. pra.
C. kef.
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
4
2
1
0.5
1
0.25
2
0.25
0.25
0.5
,0.125
,0.125
0.5
0.0156
0.0625
0.25
0.0625
0.0625
0.0625
0.0625
0.0156
0.0039
0.25
0.0156
0.25
0.25
0.25
0.25
8
.64
.64
.64
.64
.64
.64
.64
.64
64
.64
.64
.64
.64
.64
.64
2
4
4
1
4
4
4
8
4
4
4
1
2
4
4
4
4
8
32
8
8
8
16
4
16
1
0.25
1
4
4
4
,0.125
0.5
4
1
1
1
2
0.5
2
4
0.25
2
4
0.5
1
0.0625
0.25
,0.125
0.25
0.25
0.25
0.25
16
64
32
8
16
32
32
16
32
8
1
0.25
0.0625
1
0.0625
0.0625
0.25
4
0.25
1
0.0625
0.0039
1
1k
1l
8
8
4
1m
1n
1o
ICZ
VCZ
FCZ
,0.125
0.25
,0.0625
32
64
16
0.125
,0.125
8
0.0625
,0.125
0.5
0.0625
,0.125
2
,0.0625
,0.125
,0.125
,0.125
.64
0.5
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compounds 1h, 1j, and 1k are 16 times lower than that of FCZ
against C. kef. in vitro (with an MIC80 value of 0.0625 mg mL21).
The MIC80 value of compounds 1a and 1k are 32 times lower
than that of FCZ against C. alb. Y0109 in vitro (with an MIC80
value of 0.0156 mg mL21).
General procedure for the preparation of the compounds 1a–o
A mixture of NaN3 (100 mg, 1.4 mmol), 4-Cl benzyl bromide
(200 mg, 1.2 mmol), and DMSO (15 mL) was stirred at room
temperature for 6 h. Then compound 7 (200 mg, 0.6 mmol),
sodium ascorbate (20 mg), CuSO4?5H2O (25 mg), and H2O(1
mL) were added, the solution was stirred at room temperature
for another 2 h. The reaction was monitored by TLC. The
reaction solution was then added to 10 mL NH3?H2O, and
extracted with ethyl acetate. The organic layer was acidified
with dilute hydrochloric acid, then the aqueous layer was
adjusted to around pH 7 using saturated sodium bicarbonate
solution, and extracted with ethyl acetate, the organic layer
was washed with water and brine, and dried with Na2SO4.
Concentration in vacuum afforded compound 1a (201 mg,
Conclusions
In summary, a novel series of antifungal agents have been
designed and synthesized. In vitro antifungal activity assays
indicate that most of the compounds showed moderate
antifungal activities against both systemic pathogenic fungi
and dermatophytes. The activity did not differ significantly in
terms of the strength of the electron-withdrawing or donating
groups substituted on the benzene ring. The disubstituted
activity was not as good as the monosubstituted activity.
Several of the compounds showed high in vitro antifungal
activity with a broad spectrum, and are valuable for further
evaluation.
1
67%). Mp: 94.9–96.4 uC; H NMR (300 MHz, CDCl3) d: 8.09 (1
H, s, triazole–H), 7.77 (1 H, s, triazole–H), 7.19–7.60 (5 H, m,
Ar–H), 7.04 (1 H, s, triazole–H), 6.73–6.81 (2 H, m, Ar–H), 5.50
(2 H, s, Ar–CH2–), 5.46 (1 H, s, OH), 4.52 (1 H, d, J = 13.8 Hz,
triazole–CH2–), 4.39 (1 H, d, J = 14.1 Hz, triazole–CH2–), 3.61 (1
H, d, J = 14.4 Hz, triazole–CH2–), 3.54 (1 H, d, J = 12.9 Hz,
triazole–CH2–), 3.15 (1 H, d, J = 13.8 Hz, –CH2–), 2.70 (1 H, d, J
= 13.8 Hz, –CH2–), 2.11–2.29 (2 H, m, NCH* CH2CH3), 1.24–
2
1.30 (2 H, m, NCH2CH* CH3), 0.65–0.70 (3 H, m,
2
Experimental part
NCH2CH2CH* ); 13C NMR (75 MHz, CDCl3) d: 164.7, 160.8,
3
Melting points were measured on a Yamato MP-21 melting-
point apparatus and are uncorrected. 1H and 13C NMR spectra
were recorded in CDCl3 unless otherwise indicated with a
Bruker AC-300P spectrometer or a Bruker Avance II 600
spectrometer, using TMS as the internal standard. High
resolution electron spray ionization mass spectra (HR ESI
MS) were recorded on an Agilent 6538 Q-TOF mass spectro-
meter. The solvents and reagents were used as received or
dried prior to use as needed.
152.7, 146.5, 135.6, 134.1, 131.7, 131.6, 128.4, 124.8, 123.8,
113.3, 105.9, 74.1, 74.0, 60.0, 60.0, 59.3, 58.0, 57.9, 55.3, 51.2,
22.1, 13.1; HR ESI MS: calcd for C24H26ClF2N7O, [M + H]+ m/z:
502.1928; found: 502.1938.
The target compounds 1b–o were synthesized by the same
synthetic procedure that was used for compound 1a.
Acknowledgements
1-(1H-1,2,4-Triazole-1-yl)-2-(2,4-difluorophenyl)-3-
propylamino-2-propanol (6)
This work was supported by the National Natural Science
Foundation of China (No. 20772153), Shanghai Leading
Academic Discipline Project (No. B906) and by Creativity and
Innovation Training Program of Second Military Medical
University (No. MS2012047).
A mixture of compound 5 (33.3 g, 0.10 mol), CH3CH2OH (500
mL), Et3N (50 mL), and propylamine (8.85 g, 0.15 mol) was
stirred and refluxed for 6 h. The reaction was monitored by
TLC. After filtration, the filtrate was evaporated under reduced
pressure. Water was added to the residue, extracted with ethyl
acetate twice, the organic layers were combined, washed with
saturated NaCl solution twice, dried over anhydrous Na2SO4
and evaporated to get compound 6 (23.4 g, 79%).
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