Synthesis and antifungal activity of the novel triazole compounds

Article abstract of DOI:10.1039/c3md20086h

A series of 1-(1H-1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-substituted- 2-propanols (1a-o) which are analogues of fluconazole, have been designed and synthesized for the first time by the click reaction 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 HRMS. The in vitro antifungal activities of all the target compounds were evaluated against eight human pathogenic fungi.

Full text of DOI:10.1039/c3md20086h

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Synthesis and antifungal activity of the novel triazole
compounds†
Cite this: DOI: 10.1039/c3md20086h
Shichong Yu,‡a Xiaoyun Chai,‡a Nan Wang,ab Hong Cui,a Qingjie Zhao,a
a
a
a
a
*
*
Honggang Hu, Yan Zou, Qingyan Sun and Qiuye Wu
A series of 1-(1H-1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-substituted-2-propanols (1a-o) which are
analogues of fluconazole, have been designed and synthesized for the first time by the click reaction on
the basis of computational docking experiments to the active site of the cytochrome P450 14a-
Received 6th April 2012
Accepted 9th January 2013
1
DOI: 10.1039/c3md20086h
demethylase (CYP51). Their structures were characterized by 1H NMR, 3C NMR and HRMS. The in vitro
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antifungal activities of all the target compounds were evaluated against eight human pathogenic fungi.
Some studies10,11 had revealed a pharmacophore of anti-
fungal triazoles, which contains a triazole ring linked to a
Introduction
Fungal infections represent a serious problem for patients with
immune systems compromised either by HIV infection or
administration of immunosuppressive drugs during cancer
therapy and organ transplantation.1,2 Many microbes, including
fungi those were previously thought to be nonpathogenic, have
emerged as signicant pathogens in immunosuppressed pop-
ulations. Clinically, Candidosis, Aspergillosis and Cryptococcosis
are three major fungal infections in immunocompromized
patients.3,4 Currently, triazole agents (uconazole (FCZ), itra-
conazole (ICZ), voriconazole (VCZ) and posaconazole, Fig. 1) are
the most frequently used antifungals in clinic.5 However, FCZ is
not effective against invasive Aspergillosis and has suffered
severe drug resistance.6,7 This has led to an interest to develop
new triazole derivatives possessing broader antifungal spectra
and higher therapeutic indexes for pathogens that affect
patients with impaired immunity.
Azole antifungals act by competitive inhibition of CYP51, the
enzyme that catalyzes the oxidative removal of the 14a-methyl
group of lanosterol to give O14,15-desaturated intermediates in
ergosterol biosynthesis.8 In general, the active site of CYP51 for
ligand binding can be divided into four subsites: a coordination
bond with iron of the heme group, the hydrophilic H-bonding
region, the hydrophobic region, and the narrow hydrophobic
cle formed by the residues in the helix B0-meander 1 loop and
N-terminus of helix I.9
dihalophenyl ring through a two carbon chain. In addition, the
carbon alpha to the phenyl ring bears a hydroxyl group. More-
over the side chain located in the narrow hydrophobic cle was
also very important.10 We intended to alter the side chain to nd
potent systemic antifungal compounds with a broad antifungal
spectrum and less potential to develop resistance.
According to the above characteristics of target enzyme
CYP51 and the previous research results,12–17 we here designed a
new series of 1-(1H-1,2,4-triazole-1-yl)-2-(2,4- diuorophenyl)-3-
substituted-2-propanols (1 Fig. 2) containing a triazole ring, a
diuorophenyl group, a hydroxyl group and a side chain. In our
design, We systematically altered the structure of FCZ as a
platform and tried to insert a 1,2,3-triazole group into the side
chain for the rst time.
Compounds 1a-o were synthesized according to a very effi-
cient and straightforward synthetic route outlined in Scheme 1.
Aer the key intermediate oxirane 5 was synthesized by a known
procedure,18 compound 6 was synthesized by ring-opening
reaction of oxirane 5 with methylamine and then in the pres-
ence of KI and K2CO3 in acetonitrile at room temperature to
obtain compound 7. The target compounds were obtained for
the rst time using a click reaction19 with various substituted
benzyl azides.
The results of antifungal activities in vitro showed that all the
15 target compounds (1a-o) were active against nearly all fungi
tested to some extent except against Aspergillus fumigatus
(A. fum.). Most of the target compounds exhibited higher
activities against Candida albicans SC5314 (C. alb SC5314) and
Candida albicans Y0109 (C. alb Y0109) than all six positive
controls. The MIC80 value of compound 1k is 32 times lower
than that of FCZ against C. alb. SC5314 in vitro (with the MIC80
value of 0.0156 mg mLꢀ1), and 64 times lower than that of FCZ
against Candida kefyr (C. kef.) (with the MIC80 value of 0.0156 mg
mLꢀ1). The MIC80 value of compound 1o is 8 times lower than
aDepartment of Organic Chemistry, College of Pharmacy, Second Military Medical
University, Guohe Road 325, Shanghai 200433, People's Republic of China. E-mail:
wuqy6439@sohu.com; sqy_2000@163.com; Fax: +86 21 81871225; Tel: +86 21
81871225
bCollege of Pharmacy, Yantai University, Qingquan Road 30, Yantai 264005, People's
Republic of China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3md20086h
‡ Shichong Yu and Xiaoyun Chai contributed equally to this work.
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Fig. 1 Triazole antifungal agents used in clinical therapy.
further study, and are expected to be developed into new anti-
fungal drugs.
To explain the results, we proposed a likely binding mode for
1k to the active site of CYP51 based on computational docking
results (Fig. 3). As usual, the triazole interacts with iron of the
heme group, while the 2,4-diuorophenyl group in the designed
compound could be placed into the hydrophobic pocket formed
by Leu121, Phe126, Phe228, Met306, Gly307 and Gly308. The 3-
nitrobenzyl of the side chain would generate p–p stacking
interactions with the Phe380. The side chain incorporated to
adjust the overall physical-chemical properties of the molecules
and orientation of aromatic rings. It would also be oriented to
interact with a hydrophobic pocket formed by Gly65, Met92,
Ala117, Tyr118, Pro375, Leu376, His377, Ser378, Ile379 and
Met508.
Fig. 2 Generic structure of the designed fluconazole analogues.
that of FCZ against C. alb. SC5314 in vitro (with the MIC80 value
of 0.0625 mg mLꢀ1), and 16 times lower than that of FCZ against
C. kef. in vitro (with the MIC80 value of 0.0625 mg mLꢀ1). and 32
times lower than that of FCZ against Trichophyton rubrum
(T. rub.) in vitro (with the MIC80 value of 0.0625 mg mLꢀ1). The
MIC80 value of compound 1o is the same as ICZ against all 8
fungi test except A. fum. Compounds 1k and 1o are worthy of
In addition, the side chains were the pharmacophores, and
the spatial orientations of the pharmacophores were just
Scheme 1 Synthesis of the target compounds 1a-o Conditions: (a) ClCH2COCl, AlCl3, 50 ꢁC, 5 h, 80%; (b) 1H-1,2,4-triazole, NaHCO3, toluene, reflux, 5 h, 42%; (c)
(CH3)3SOI, NaOH, cetylmethylammonium bromide, toluene, 60 ꢁC, 3 h, 53%; (d) CH3SO3H, 0 ꢁC, 1 h, 89%; (e) Et3N, methylamine, EtOH, reflux, 6 h, 80%; (f) propargyl
bromide, KI, K2CO3, CH3CN, rt, 5–6 h, 70%; (g) NaN3, substituted benzyl bromide, DMSO, CuSO4$5H2O, sodium ascorbate, rt, 12 h, 60–70%.
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dened by the National Committee for Clinical Laboratory
Standards (NCCLS).20 The MIC80 was dened as the rst well
with an approximate 80% reduction in growth compared to the
growth of 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 C
Growth MIC was determined at 24 h for C. alb. and at 72 h for
C. neo. The results of assays are summarized in Table 1. The
data points form the mean of replicates. All of our susceptibility
tests were performed three times by each antifungal agent.
Molecular docking
In our studies, we constructed a 3D model of Candida albicans
CYP51 on the basis of Ji et al.21 All the molecular modeling
calculations were performed using SYBYL 6.9 version. And the
structures of the compounds were assigned with Gasteiger-
¨
Huckle partial atomic charges. Energy minimization was per-
formed using the Tripos force eld, Powell optimization
Fig. 3 Computed binding geometry of the new inhibitor 1k in the active site of
method, and MAXIMIN2 minimizer with a convergence crite-
CYP51.
rion 0.001 kcal molꢀ1 A. Simulated annealing was then per-
˚
formed. The system was heated to 1000 K for 1.0 ps and then
annealed to 250 K for 1.5 ps. The annealing function was
exponential; 50 such cycles of annealing were run and the
resulting 50 conformers were optimized using methods
described above. The lowest energy conformation was selected.
All the other parameters were default value.
oriented in the hydrophobic pocket. They played a role in
adjusting the physico-chemical properties of the whole mole-
cule to avoid some dissatisfying side effects and improve their
pharmacokinetic and pharmacodynamic behavior.
In conclusion, an efficient method using the click reaction
has been developed for the synthesis of a diverse series of novel
triazole derivatives. All the target compounds are reported for
the rst time. Results of preliminary antifungal tests against
eight human pathogenic fungi in vitro showed that these
analogs exhibited excellent activities with broad spectrum. The
obtained results indicated that for antifungal activity of these
novel triazole derivatives it is very helpful to introduce the 1,2,3-
triazole group and the substituted benzyl as side chains. The
research has led to the discovery of a series of compounds for
further optimization.
General procedures
Melting points were measured on a Yamato MP-21 melting-
1
point apparatus and are uncorrected. H and 13C NMR spectra
were recorded in CDCl3 unless otherwise indicated with a
Bruker AC- 300P spectrometer or a Bruker Avance II 600 spec-
trometer, using TMS as internal standard. High resolution
electron spray ionization mass spectra (HR ESI MS) were per-
formed on an Agilent 6538 Q-TOF mass spectrometer. The
solvents and reagents were used as received or dried prior to use
as needed.
Experimental
Pharmacology
1-(1H-1,2,4-triazole-1-yl)-2-(2,4-diuorophenyl)-3-(N-
methylamino)-2-propanol (6)
The in vitro antifungal activities of all the target compounds
were evaluated against eight human pathogenic fungi,
C. alb.SC5314, Cryptococcus neoformans (C. neo.), Candida para-
psilosis (C. par.), C. alb.Y0109, Candida tropicalis (C. tro.), T. rub.,
C. kef., A. fum., which are oen encountered clinically, and were
compared with ICZ, terbinane (TRB), ketoconazole (KCZ),
amphotericin B (AMB), VCZ and FCZ. C. alb. SC5314 and C. neo.
were provided by Shanghai Changzheng Hospital; C. par., C. alb.
Y0109, C. tro., T. rub., C. kef. 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.
FCZ, ICZ, KCZ, VCZ, AMB and TRB served as the positive control
were obtained from their respective manufacturers.
A mixture of compound 5 (33.3 g, 0.10 mol), CH3CH2OH
(500 mL) and Et3N (50 mL), methylamine (4.7 g, 0.15 mol) was
stirred and reuxed for 6 h. The reaction was monitored by TLC.
Aer ltration, the ltrate 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 (ref. 22) (21.0 g, 79%).
1-(1H-1,2,4-triazole-1-yl)-2-(2,4-diuorophenyl)-3-(N-methyl-N-
propargyl amino)-2- propanol (7)
A mixture of compound 6 (2.68 g, 0.01 mol), propargyl bromide
The in vitro minimal inhibitory concentrations (MICs) of the (2.36 g, 0.02 mol), KI (166 mg, 0.001 mol), K2CO3 (3.45 g, 0.025
compounds were determined by the micro-broth dilution mol), and CH3CN (100 mL) was stirred at room temperature for
method in 96-well microtestplates according to the methods 6 h. The reaction was monitored by TLC. Aer reaction, ltrated
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Table 1 Antifungal activities of the target compounds in vitro (MIC80, mg mLꢀ1
)
Compd
R
C. alb SC5314
C. alb Y0109
C. kef
C. neo
T. rub
C. tro
C. par
A. fum
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
ICZ
TBR
KCZ
AMB
VCZ
FCZ
3 F
4 F
0.25
1
0.25
0.5
0.25
<0.125
0.25
<0.125
<0.125
0.25
<0.125
<0.125
<0.125
0.25
<0.125
0.5
<0.0125
0.0625
2
<0.125
4
64
16
16
0.25
0.0625
0.25
1
0.25
0.25
0.25
2
1
16
4
4
4
0.25
1
4
4
0.5
0.25
1
0.5
0.25
0.25
0.25
0.25
0.0625
0.25
0.0625
0.0625
0.25
0.0625
0.25
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
2
2-Cl
4-Cl
2-Br
3-Br
4-Br
2-CH3
4-CH3
2-NO2
3-NO2
4-NO2
2-CN
3-CN
4-CN
—
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.0156
0.25
0.25
1
0.0625
<0.0625
16
<0.125
8
0.25
1
0.25
0.25
1
0.25
1
1
0.25
0.25
0.25
4
0.25
0.125
8
0.5
4
<0.125
8
1
0.25
0.25
0.25
0.25
1
1
4
1
0.0156
0.25
0.25
0.0625
1
1
0.0625
0.25
0.25
1
0.0625
<0.0625
<0.125
<0.125
0.25
<0.125
<0.125
0.0625
0.25
1
1
0.0625
0.0625
0.0625
0.0625
0.25
0.0625
0.0625
<0.125
<0.125
0.125
<0.125
2
0.0156
0.0625
<0.125
<0.125
1
—
—
—
—
0.25
0.125
32
<0.125
>64
32
0.5
<0.125
0.5
0.0039
1
0.25
<0.125
—
off the solid, washed with CH3CN, the ltrate was concentrated 104.1, 72.6, 60.3, 56.1, 56.0, 53.4, 44.1; HR ESI MS: calcd. for
in a vacuum. Column chromatography of the residue afforded
compound 7 as a brown oil (1.9 g, 62%). H NMR (300 MHz,
C
22H23F3N7O [M + H]+ m/z: 458.1911; found: 458.1918.
The target compounds 1b-o were synthesized by the same
1
CDCl3) d: 8.13 (1H, s, triazole-H), 7.78 (1H, s, triazole-H), 7.58- operation procedure of the compound 1a.
7.50 (1H, m, Ar-H), 6.84-6.74 (2H, m, Ar-H), 4.54 (2H, s, CH2),
3.22-3.07 (2H, m, triazole-CH2), 2.73 (1H, d, J ¼ 12.0 Hz, CH2),
Acknowledgements
2.21-2.19 (2H, m, CH2), 2.17 (3H, s, NCH3); 13C NMR (75 MHz,
CDCl3) d: 152.9, 146.5, 131.4, 113.4, 106.1, 79.8, 75.3, 74.5, 62.0,
58.0, 49.1, 45.4; HR ESI MS: calcd. for C15H17F2N4O [M + H]+ m/z:
307.1365; found: 307.1362.
This work was supported by the National Natural Science
Foundation of China (Nos. 20772153, 30300437), Doctoral
Innovation Foundation of Second Military Medical University
and by Shanghai Leading Academic Discipline Project Number:
B906.
General procedure for the preparation of the compounds 1a–o
A mixture of NaN3 (100 mg, 1.4 mmol), 3-uorobenzyl bromide
(200 mg, 1.2 mmol) and DMSO (15 mL) was stirred at room
temperature for 6 h. Then was added the compound 7 (184 mg,
0.6 mmol), sodium ascorbate (20 mg), CuSO4$5H2O (25 mg),
H2O (1 mL), was stirred at room temperature for 2 h, then put
the reaction solution into NH3$H2O, extracted with ethyl-
acetate, the organic layer was acidicated with dilute hydro-
chloric acid, then the aqueous layer was adjusted pH about 7.0
by saturation sodium bicarbonate, extracted with ethyl acetate,
washed with water, dried with Na2SO4. concentrated in a
vacuum to afford compound 1a (186 mg, 68%). White powder,
Mp: 86.6–88.2 ꢁC; 1H NMR (300 MHz, CDCl3) d: 8.10 (1H, s,
triazole-H), 7.77 (1H, s, triazole-H), 7.54-7.62 (1H, m, Ar-H),
7.33-7.40 (1H, m, Ar-H), 7.14 (1H, s, triazole-H), 6.79-7.10 (3H,
m, Ar-H), 6.73-6.78 (2H, m, Ar-H), 5.54 (2H, s, Ar–CH2–), 4.53
(1H, d, J ¼ 13.8 Hz, triazole-CH2), 4.42 (1H, d, J ¼ 13.8 Hz,
triazole-CH2), 3.65 (1H, d, J ¼ 13.8 Hz, triazole-CH2), 3.56 (1H,
d, J ¼ 14.4 Hz, triazole-CH2), 3.03 (1H, d, J ¼ 13.8 Hz, CH2),
2.72 (1H, d, J ¼ 13.5 Hz, CH2), 2.13 (3H, s, NCH3); 13C NMR
(75 MHz, CDCl3) d: 162.7, 159.2, 150.8, 144.6, 137.0, 130.7,
129.6, 126.0, 123.5, 122.2, 115.9, 115.7, 115.1, 114.8, 111.5,
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