Design, synthesis, and antifungal activity of novel conformationally restricted triazole derivatives

Article abstract of DOI:10.1002/ardp.200900103

A series of new triazole derivatives were designed and synthesized on the basis of the active site of lanosterol 14α-demethylase from Candida albicans (CACYP51). 2-(2,4-Difluorophenyl)-3-(methyl-(3-phenoxyalkyl)amino)-1- (1H-1,2,4-triazol-1-yl)propan-2-ols show excellent in-vitro activity against most of the tested pathogenic fungi. The MIC₈₀ value of compound 8a against Candida albicans is 0.01 μM, which provides a good starting template for further structural optimization. The binding modes of the designed compounds were investigated by flexible molecular docking. The compounds interacted with CACYP51 through hydrophobic, van-der-Waals, and hydrogenbonding interactions.

Full text of DOI:10.1002/ardp.200900103

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Arch. Pharm. Chem. Life Sci. 2009, 342, 732–739
Full Paper
Design, Synthesis, and Antifungal Activity of Novel
Conformationally Restricted Triazole Derivatives
Wenya Wang1, Chunquan Sheng1, Xiaoying Che1, Haitao Ji2, Zhenyuan Miao1, Jianzhong Yao1,
and Wannian Zhang1
1 School of Pharmacy, Second Military Medical University, Shanghai, P.R. China
2 Department of Chemistry, Department of Biochemistry, Molecular Biology, and Cell Biology and Center for
Drug Discovery and Chemical Biology, Northwestern University, Evanston, Illinois, USA
A series of new triazole derivatives were designed and synthesized on the basis of the active site
of lanosterol 14a-demethylase from Candida albicans (CACYP51). 2-(2,4-Difluorophenyl)-3-(methyl-
(3-phenoxyalkyl)amino)-1-(1H-1,2,4-triazol-1-yl)propan-2-ols show excellent in-vitro activity
against most of the tested pathogenic fungi. The MIC80 value of compound 8a against Candida
albicans is 0.01 lM, which provides a good starting template for further structural optimization.
The binding modes of the designed compounds were investigated by flexible molecular docking.
The compounds interacted with CACYP51 through hydrophobic, van-der-Waals, and hydrogen-
bonding interactions.
Keywords: Antifungal agents / Lanosterol demethylase / Molecular docking / Restricted analogues / Triazoles /
Received: May 7, 2009; Accepted: July 7, 2009
DOI 10.1002/ardp.200900103
clinic because of their safety profile and high therapeutic
Introduction
index. Unfortunately, the broad use of azoles has led to
the development of severe resistance [7, 8], which signifi-
cantly reduced the efficacy of them. This situation has
led to an ongoing search for new azoles and several new
drugs, such as voriconazole [9], posaconazole [10], ravu-
conazole [11], and albaconazole [12], which are marketed
or currently in the late stages of clinical trials.
Azole antifungal agents are competitive inhibitors of
the lanosterol 14a-demethylase (CYP51) [13], which is the
key enzyme in sterol biosynthesis of fungi. As an impor-
tant target in antifungal chemotherapy, it is of great sig-
nificance to know the three-dimensional (3D) structures
of fungal CYP51s. However, they are membrane-associ-
ated proteins, and solving their crystal structures
remains a challenge. In our continual interest in rational
antifungal drug design, a 3-D model of CYP51 from Can-
dida albicans (CACYP51) has been constructed through
homology modeling [14, 15]. The mechanism of azoles
binding with CACYP51 has been investigated by flexible
molecular docking [14, 16] and site-directed mutagenesis
[17]. The CACYP51 model has accelerated the discovery of
novel CYP51 inhibitors [16, 18]. In this paper, structure-
based rational drug design was used leading to the dis-
During the past two decades, fungal infection has
become an important complication and a major cause of
morbidity and mortality in immunocompromized indi-
viduals such as patients undergoing anticancer chemo-
therapy or organ transplants and patients with AIDS [1].
Clinically, candidosis, aspergillosis, and cryptococcosis
have been identified as three major opportunistic patho-
gens in the etiology of fungal infections [2, 3]. However,
there is a limited number of antifungal agents that can
be used for life-threatening fungal infections. These are
amphotericin B [4], 5-fluorocytosine, azoles (such as
fluconazole and itraconazole) [5], and echinocandins
(such as caspofungin and micafungin) [6]. Among those,
azoles are the most widely used antifungal agents in the
Correspondence: W. N. Zhang and C. Q. Sheng, School of Pharmacy,
Second Military Medical University, 325 Guohe Road, Shanghai 200433,
P.R. China.
E-mail: (W.N.Z) zhangwnk@hotmail.com; (C.Q.S): shengcq@hotmail.
com
Fax: (W.N.Z) +86 21 818 71 -243; (C.Q.S) -233
Abbreviation: p-toluenesulfonic acid (PTSA)
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2009, 342, 732–739
Conformationally Restricted Triazole Derivatives
733
Reagents and conditions: a) ClCH2COCl, AlCl3, CH2Cl2, 408C, 3 h, 50%; b) triazole, K2CO3, CH2Cl2, r.t., 24 h, 70.0%; c)
(CH3)3SOI, NaOH, toluene, 608C, 3 h, 62.3%; d) phenol, K2CO3, DMF, 708C, 2 h, 91.8 to 92.3%; e) methylamine alcohol
solution, r.t., 12 h, 97 to 98%; f) 4, Et3N, EtOH, reflux, 9 h, 51.2 to 60.3%.
Scheme 1. Synthesis of compounds 8a–c.
covery of 2-(2,4-difluorophenyl)-3-(methyl-(3-phenoxyal-
were treated with piperidin-4-one hydrochloride and
kyl)amino)-1-(1H-1,2,4-triazol-1-yl)propan-2-ols as a novel K2CO3 to give side chains 11a–c. The target compounds
class of antifungal triazoles. The binding mode of the 12a–c were synthesized by treating amino compound 10
designed compounds was investigated by molecular with side chains 11a–c and a catalytic amount of p-tolu-
docking.
enesulfonic acid (PTSA) in MeOH at 708C with moderate
yields (Scheme 2). Compounds 14a–c were synthesized
via two steps from 6a–c. Excess propane-1,3-diamine was
treated with 6a–c to give phenoxyalkyl-substituted dia-
mines 13a–c. Compounds 13a–c were subsequently
treated with concentrated HCl and formaldehyde at
Results and discussion
Chemistry
As a key intermediate of our designed compounds, the room temperature to give phenoxyalkyl-substituted
oxirane compound 4 was synthesized by our reported hexahydropyrimidines 14a–c. The target products 15a–c
procedure (Scheme 1) [16]. The N-methyl-phenoxyalkan-1- were obtained by the same method for 8a–c (Scheme 3).
amine side chains 7a–c were synthesized via two steps. All the target compounds were obtained as racemates.
Excess dibromoalkanes were treated with phenol to give
bromoalkoxy benzenes 6a–c. Compounds 6a–c were sub- Inhibitor design
sequently reacted with methylamine in EtOH at room As discussed earlier [18], the active site of CACYP51 can be
temperature to afford side chains 7a–c. The target com- divided into four pockets. The triazole ring (located in
pounds 8a–c were obtained as racemates by treating pocket S2), the difluorophenyl group (located in pocket
epoxide 4 with side chains 7a–c in the presence of tri- S3), and the C2-hydroxyl group (located in pocket S1) are
ethylamine and EtOH at 808C with moderate to high a common skeleton of triazole antifungal agents. The
yields (Scheme 1). Starting from the epoxide 4, 1-amino-2- fundamental purpose of the present drug design is to
(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol 10 find a new type of side chain, which can be well accom-
was obtained via two steps. First, compound 4, NaN3, and modated in the S4 pocket. Considering the hydrophobic
NH4Cl in MeOH were refluxed to give the azido com- nature of the S4 pocket of CACYP51, a hydrophobic side
pound 9. Then, the azido group of compound 9 was chain is necessary, while an additional hydrogen-bond-
reduced to the amino group by the hydrogenation in the ing interaction will further improve the affinity and spe-
presence of Pd/C in EtOH to give 10. Compounds 6a–c cificity of the inhibitors. Therefore, a series of substituted
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W. Wang et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 732–739
Reagents and conditions: a) NaN3, NH4Cl, MeOH, reflux, 8 h, 98 to 99.8%; b) H2, Pd/C, EtOH, 4 h, 98 to 99.8%; c) piper-
idin-4-one hydrochloride, K2CO3, MeOH, 608C, 7 h, 89.5 to 99.2%; d) 10, PTSA, MeOH, reflux, 0.5 h, 35.5 to 42.6%.
Scheme 2. Synthesis of compounds 12a–c.
Reagents and conditions: a) H2N(CH2)3NH2, MeOH, r.t., 5 h, 68.8 to 76.4%; b) HCHO, conc. HCl, r.t., 1 h, 16.8 to 29.8%;
c) 4, Et3N, EtOH, reflux, 9 h, 37.3 to 50.8%.
Scheme 3. Synthesis of compounds 15a–c.
alkoxybenzene side chains were designed, which were the range of 0.04 to 0.01 lM. Compound 8a also shows a
supposed to form hydrophobic, van-der-Waals, and higher activity than the newly marketed voriconazole.
hydrogen-bonding interactions with the S4 pocket of On the C. neoformans strain, compounds 8a–c are more
CACYP51 (Fig. 1).
active than fluconazole, and compounds 8b and 8c are
comparable to voriconazole. Compounds 8a–c also show
good activity against dermatophytes (i.e., Trichophyton
In-vitro antifungal activities
The in-vitro antifungal activity assay (Table 1) indicates rubrum) with their MIC80 values in the range of 2.32 to
that compounds 8a–c show excellent potency against C. 0.62 lM. However, they are not very sensitive against
albicans and Cryptococcus neoformans. The compounds are Aspergillus Fumigatus and only compound 8c shows mod-
almost 5 to 320-folds more potent against C. albicans than erate inhibitory activity (MIC80 = 37.19 lM). The replace-
fluconazole and itraconazole with their MIC80 values in ment of the N-methyl group of compounds 8a–c by the
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Conformationally Restricted Triazole Derivatives
735
Table 1. In-vitro antifungal activities of the compounds (MIC80, in lM).
Compounds
C. albicans§
C. neoformans$
T. rubrum&
A. fumigatus#
8a
8b
8c
0.01
0.04
0.04
0.62
0.15
0.15
0.62
0.60
2.32
>159.13
>153.77
37.19
12a
12b
12c
15a
15b
15c
FLZ*
ITZp
VOR{
>136.39
>132.44
>128.71
2.19
2.12
2.06
3.27
0.18
0.04
>136.39
>132.44
>128.71
139.97
33.95
32.97
3.27
>136.39
132.44
128.71
>139.97
135.81
131.89
13.06
0.18
>136.39
>132.44
>128.71
>139.97
>135.81
>131.89
>208.97
2.83
0.18
0.18
0.18
2.86
Abbreviations: § C. Albicans: Candida albicans, $ C. Neoforman: Cryptococcus neoformans, & T. Rubrum: Trichophyton rubrum, # A. fumiga-
tus: Aspergillus fumigatus, * FLZ: Fluconazole, p ITZ: Itraconazole, { VOR: Voriconazole.
Figure 1. Illustration of the expected interactions between the
designed azoles and the active site of CACYP51 strucure.
Important residues interacting with the compounds are shown and hydrogen bonds
are displayed through red dotted lines. The images were generated using the InsightII
2000 software package.
piperidin-4-imino group (compounds 12a–c) leads to the
loss of the antifungal activity. Only compounds 12b and
12c show marginal activity against T. rubrum, with their
Figure 2. The docking conformation of compound 8a (A) and 8b
(B) in the active site of CACYP51.
MIC80 values on a level of 130 lM. If the N-methyl group
of compounds 8a–c is substituted by the hexahydropyri- obtained docking model of fluconazole with CACYP51
midinyl group (compounds 15a–c), the antifungal activ- [16], compounds 8a and 8b show similar conformation in
ity is decreased to a large extent. Compounds 15a–c show the active site. The triazole ring, difluorophenyl group,
a comparative activity against C. albicans as fluconazole and C2-hydroxyl group are identical substructures of
with their MIC80 range from 2.06 to 2.19 lM. However, compounds 8a–c and fluconazole and they also form sim-
they only show moderate activity against C. neoformans.
ilar interactions with CACYP51. The triazole ring of the
compounds binds to the S2 pocket through the forma-
tion of a coordination bond with the iron of the heme
Binding mode of the azoles
Flexible molecular docking was used to clarify the bind- group. The difluorophenyl group is located in the S3
ing mode of the compounds and provide straightforward pocket and forms a hydrophobic interaction with
information for further structural optimization. In our Phe126, Met306, and Phe145. No interaction was
previous studies [16], the affinity method within the observed between the hydroxyl group attached to C2 and
InsightII 2000 software package [19] has been proven to the active site of CACYP51. From the docking model it is
be a powerful docking tool to identify key residues possible that the water molecules in the active site medi-
involved in azole binding. Figures 2A and 2B show the ate the interaction between the hydroxyl group and the
binding mode of compounds 8a and 8b with the active vicinal H310 of the S1 subsite, which is a highly con-
site of CACYP51. As compared with our previously served functional residue in the CYP51 family.
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W. Wang et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 732–739
Table 2. Calculated interaction energies (kcal/mol) for the com-
plexes of compounds 8a–c and fluconazole with the active site
of CACYP51.
fungi and dermatophytes. Compound 8a shows higher
activity against C. albicans (MIC80 = 0.01 lM) than flucon-
azole, itraconaozle, and voriconazole. The N-methyl
group of compounds 8a–c is important for their antifun-
gal activity. When the N-methyl group was replaced by
the hexahydropyrimidyl or piperidin-4-imino group, the
antifungal activity was decreased to a large extent. Flexi-
ble molecular docking was used to clarify the binding
mode of the compounds. Compounds 8a–c interacted
with CACYP51 through hydrophobic, van-der-Waals, and
Compounds
Evdw
Eelect
Etotal
8a
8b
8c
–62.2
–62.8
–63.1
–54.8
–9.4
–5.8
–5.3
–3.2
–71.6
–68.6
–68.4
–58.0
Fluconazole
The substituted alkoxybenzene side chain was ori- hydrogen-bonding interactions. Compounds 8a–c pro-
ented into the S4 pocket with an extended conformation. vide a good starting point for the further structural opti-
The N-methyl group of the side chain forms hydrophobic mization.
and van-der-Waals interactions with Phe228. The alkyl
group of compounds 8a and 8b interacts with surround-
ing hydrophobic residues such as Val509 and Val510. The
terminal phenyl group interacted with surrounding
Experimental
hydrophobic residues lined with Leu376, Phe380,
Leu461, Val404, and Met508. For compound 8a, the oxy-
gen atom forms two hydrogen bonds with His377 and
Ser378. As compared with fluconazole, the long side
chain of compound 8a forms stronger hydrophobic and
hydrogen-bonding interactions with CACYP51. There-
fore, compound 8a shows lower interaction energy with
CACYP51 than fluconazole (Table 2). If the propyl group
of compound 8a is extended to butyl (compound 8b) or
pentyl (compound 8c), the oxygen atom only forms one
hydrogen bond with Ser378, which results in the four-
fold decrease of the antifungal activity. When the N-
methyl group of compounds 8a–c is replaced by the hexa-
hydropyrimidinyl group (compounds 15a–c), the orienta-
tion of alkoxyphenyl groups is largely changed. The loss
of important hydrogen-bonding and hydrophobic inter-
actions of compounds 15a–c with CACYP51 might lead to
the decrease of antifungal activity. Moreover, com-
pounds 12a–c are almost inactive because of their piperi-
din-4-imino group. It is supposed that the double bond of
the piperidin-4-imino group might decrease the flexibil-
ity of the side chain and the compounds could not reach
the active site, because conformational changes are nec-
essary when the azole inhibitor enters the active site
through the substrate-access channel.
Chemistry
Nuclear magnetic resonance (NMR) spectra were recorded on a
Bruker 500 spectrometer (Bruker Bioscience, USA) with TMS as
an internal standard and CDCl3 as solvent. Chemical shifts (d val-
ues) and coupling constants (J values) are given in ppm and Hz,
respectively. High-resolution mass spectra were carried out
using a GAA065 mass spectrometer (Thermo Scientific). TLC
analysis was carried out on silica gel plates GF254 (Qindao
Haiyang Chemical, China). Silica gel column chromatography
was performed with Silica gel 60 G (Qindao Haiyang Chemical).
Commercial solvents were used without any pretreatment.
General procedure for the synthesis of 1-(4-
bromobutyl)benzene 6b
A solution of phenol (9.41 g, 0.10 mol) in 40 mL DMF was added
dropwise to a stirred mixture of 1,4-dibromobutane (43.18 g,
0.20 mol), K2CO3 (20.73 g, 0.15 mol), and DMF (100 mL) at room
temperature. After the addition was completed, the reaction
mixture was stirred at room temperature for 2 h and then
heated to 708C for 2 h. The mixture was filtered and the result-
ing solution was diluted with ethyl acetate (200 mL) and washed
by H2O (36200 mL). The organic layer was separated, dried with
anhydrous Na2SO4, and concentrated under reduced pressure.
The residue was purified by column chromatography (hexane)
to give 6b as white solid: 22.31 g, (91.8%). 1H-NMR d (ppm): 6.87–
7.29 (m, 5H, Ar-H), 3.99 (t, J = 6.0 Hz, 2H, OCH2), 3.48 (t, J = 6.6 Hz,
2H, CH2Br), 2.04–2.10 (m, 2H, OCH2CH2CH2CH2Br), 1.91–1.97 (m,
2H, OCH2CH2CH2CH2Br); MS (ESI) m/z: 229 [M + 1].
The synthetic methods for the following compounds 6a and
6c were similar to the synthesis of compound 6b.
N-Methyl-4-phenoxybutan-1-amine 7b
Conclusions
A solution of compound 6b (5.73 g, 0.025 mol) in EtOH (20 mL)
was added dropwise to a methylamine alcohol solution (50 mL).
The mixture was stirred at room temperature for 12 h and the
reaction was almost completed. The solvent was evaporated
under reduced pressure to give 7b as a white solid: 6.18 g, (97%).
The product was used in the next step without further purifica-
tion. 1H-NMR d (ppm): 6.92–7.30 (m, 5H, Ar-H), 3.98 (t, J = 5.6 Hz,
2H, OCH2), 2.95 (t, J = 7.2 Hz, 2H, CH2Br), 2.56 (s, 3H, NCH3), 1.73–
1.76 (m, 4H, OCH2CH2CH2CH2Br); MS (ESI) m/z: 180 [M + 1].
In response to an urgent need for the discovery of novel
antifungal agents, a series of novel triazole derivatives
were designed and synthesized. The in-vitro antifungal
activity assay indicates that compounds with N-methyl-3-
phenoxyalkyl-1-amine side chains (compounds 8a–c)
show excellent activity against both systemic pathogenic
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Arch. Pharm. Chem. Life Sci. 2009, 342, 732–739
Conformationally Restricted Triazole Derivatives
737
The synthetic methods for the following compounds 7a and
7c were similar to the synthesis of compound 7b.
= 6.1 Hz, 2H, OCH2), 2.77 (br, 4H, N(CH2CH2)2CO), 2.55 (br, 2H,
NCH2CH2CH2CH2O), 2.47 (br, 4H, N(CH2CH2)2CO), 1.84–1.87 (m,
2H, NCH2CH2CH2CH2O), 1.73 (m, 2H, NCH2CH2CH2CH2O); MS (ESI)
m/z: 248 [M + 1].
The synthetic methods for the following compounds 11a and
11c were similar to the synthesis of compound 11b.
2-(2,4-Difluorophenyl)-3-[methyl-(4-phenoxybutyl)-
amino]-1-(1H-1,2,4-triazol-1-yl)propan-2-ol 8b
A solution of epoxide 4 (1.67 g, 0.005 mol), 7b (1.08 g, 0.006 mol),
triethylamine (3 mL), and EtOH (30 mL) was heated to reflux for
9 h. The solvent was evaporated under reduced pressure. The res-
idue was purified by silica gel column chromatography (CH2Cl2/
MeOH, 100:2, v/v) to give 8b as pale yellow oil: 1.15 g (55.2%). 1H-
NMR d (ppm): 8.16 (s, 1H, TriazC3-H), 7.77 (s, 1H, TriazC5-H), 6.77–
7.57 (m, 8H, Ar-H), 5.29 (br, 1H, OH), 4.49 (d, J = 14.2 Hz, 2H, C1-
HaHb), 3.86 (t, J = 6.2 Hz, 2H, OCH2), 3.03 (d, J = 13.6 Hz, 1H, C3-
Ha), 2.73 (d, J = 13.6 Hz, 1H, C3-Hb), 2.31–2.36 (m, 2H,
NCH2CH2CH2CH2O), 2.04 (s, 3H, NCH3), 1.48–1.64 (m, 2H,
NCH2CH2CH2CH2O), 1.45–1.48 (m, 2H, NCH2CH2CH2CH2O); 13C-
NMR d (ppm): 162.56, 158.84, 158.78, 150.77, 144.57, 129.37,
126.39, 120.52, 114.38, 111.29, 104.02, 71.72, 67.17, 62.00, 58.56,
56.34, 43.43, 26.62, 23.77; HMRS m/z calcd. for C22H26F2N4O2:
416.2024; found: 416.2010.
2-(2,4-Difluorophenyl)-3-[1-(4-phenoxybutyl)piperidin-4-
ylideneamino]-1-(1H-1,2,4-triazol-1-yl)propan-2-ol 12b
A mixture of compound 10 (1.27 g, 0.005 mol), 11b (2.47 g, 0.010
mol), and PTSA (0.38 g, 0.002 mol) was dissolved in methanol (20
mL). The reaction mixture was heated to reflux for 0.5 h. Then
the solvent was evaporated under reduced pressure. The residue
was purified by silica gel column chromatography (CH2Cl2/
MeOH, 100:2, v/v) to give 12b as yellow oil: 0.86 g (35.5%). 1H-NMR
d (ppm): 8.13 (s, 1H, TriazC3-H), 7.92 (s, 1H, TriazC5-H), 6.82–7.60
(m, 8H, Ar-H), 5.30 (s, 1H, OH), 4.54 (d, J = 14.2 Hz, 1H, C1-Ha), 4.24
(d, J = 14.2 Hz, 1H, C1-Hb), 4.00 (d, J = 13.3 Hz, 1H, C3-Ha), 3.84 (t, J
= 6.2 Hz, 2H, OCH2), 3.03 (d, J = 13.2 Hz, 1H, C3-Hb), 2.47 (br, 4H,
N(CH2)2), 1.77–1.82 (m, 4H, piperidin-(CH2)2), 1.58 (br, 2H,
NCH2CH2CH2CH2O), 1.32 (br, 4H, NCH2CH2CH2CH2O); HMRS m/z
calcd. for C26H31F2N5O2: 483.2446; found: 483.2411.
The synthetic methods for the target compounds 8a and 8c
were similar to the synthesis of compound 8b.
The synthetic methods for the target compounds 12a and 12c
were similar to the synthesis of compound 12b.
1-Azido-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-
yl)propan-2-ol 9
N1-(4-Phenoxybutyl)propane-1,3-diamine 13b
A solution of 4 (16.65 g, 0.05 mol), sodium azide (9.75 g, 0.15 mol),
and ammonium chloride (6.36 g, 0.12 mol) in MeOH (150 mL) was
heated to reflux for 8 h, and the reaction was almost completed.
The cooled mixture was diluted with H2O (100 mL) and extracted
with dichloromethane (36200 mL). The organic layer was sepa-
rated, dried with anhydrous Na2SO4, and concentrated under
reduced pressure to give 9 as yellow oil: 13.86 g, (99.0%), which
was used in the next step without further purification. 1H-NMR d
(ppm): 8.03 (s, 1H, TriazC3-H), 7.82 (s, 1H, TriazC5-H), 6.50–7.90 (m,
3H, Ar-H), 4.72 (br, 2H, C1-H2), 3.70 (d, J = 13 Hz, 1H, C3-Ha), 3.56 (d, J
= 13 Hz, 1H, C3-Hb); MS (ESI) m/z: 281 [M + 1].
A solution of compound 6b (4.58 g, 0.02 mol) in methanol (20
mL) was added dropwise to a stirred solution of propane-1,3-di-
amine (7.41 g, 0.10 mol) in methanol (50 mL). The mixture was
stirred at room temperature for 5 h. Then, the solvent was evapo-
rated under reduced pressure. The residue was diluted with
water (100 mL) and extracted with ethyl acetate (36100 mL).
The organic layer was separated, dried with anhydrous Na2SO4,
and concentrated under reduced pressure to give 13b as white
solid: 3.40 g, (76.4%). The product was used in the next step with-
1
out further purification. H-NMR d (ppm): 6.91–7.30 (m, 5H, Ar-
H), 3.98 (t, J = 6.2 Hz, 2H, OCH2), 3.08–3.12 (m, 2H, CH2NH2), 2.87–
2.93 (m, 4H, CH2NHCH2), 1.81 (br, 2H, NHCH2CH2CH2CH2O), 1.72–
1.77 (m, 4H, NHCH2CH2CH2CH2O, NHCH2CH2CH2NH2); MS (ESI)
m/z: 223 [M + 1].
1-Amino-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-
yl)propan-2-ol 10
A solution of 9 (7.00 g, 0.025 mol) in EtOH (200 mL) was shaken
with 10% palladium-charcoal (1.0 g) under H2 atmosphere at
room temperature for 4 h. The mixture was filtered and the fil-
trate was concentrated under reduced pressure to give 10 as
white solid: 6.34 g, (99.8%), which was used in the next step with-
out further purification. 1H-NMR d (ppm): 8.10 (s, 1H, TriazC3-H),
7.83 (s, 1H, TriazC5-H), 6.50–7.70 (m, 3H, Ar-H), 4.58 (br, 2H, C1-
H2), 3.18 (d, J = 14.0 Hz, 1H, C3-Ha), 2.98 (d, J = 14.0 Hz, 1H, C3-Hb);
MS (ESI) m/z: 455 [M + 1].
The synthetic methods for the following compounds 13a and
13c were similar to the synthesis of compound 13b.
1-(4-Phenoxybutyl)hexahydropyrimidine 14b
A solution of compound 13b (1.78 g, 0.008 mol) in H2O (1.5 mL)
was neutralized with concentrated HCl under ice condition, and
compound 13b (1.55 g, 0.007 mol) was added. An aqueous solu-
tion of 37% formaldehyde (1.22 g, 0.015 mol) was added drop-
wise. The mixture was stirred at room temperature for 1 h and
then NaOH pellets (3.00 g, 0.075 mol) were added. The precipi-
tate was removed by filtration and the filtrate was concentrated
to dryness under reduced pressure. The residue was diluted with
dichloromethane (50 mL) and washed by H2O (3620 mL). The
organic layer was separated, dried with anhydrous Na2SO4, and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (CH2Cl2/MeOH, 100:5, v/v)
to give 14b as yellow oil: 1.05 g, (29.8%). 1H-NMR d (ppm): 6.80–
7.25 (m, 5H, Ar-H), 3.95 (t, J = 6.5 Hz, 2H, OCH2), 3.21 (s, 2H,
NCH2NH), 2.61 (t, J = 5.3 Hz, 2H, NHCH2CH2CH2N), 2.49–2.51 (m,
2H, NCH2CH2CH2NH), 2.20 (t, J = 7.4 Hz, 2H, NCH2CH2CH2CH2O),
1.69–1.72 (m, 2H, OCH2CH2CH2CH2N), 1.49–1.54 (m, 2H,
1-(4-Phenoxybutyl)piperidin-4-one 11b
A solution of compound 6b (6.87 g, 0.03 mol) in 20 mL methanol
was added dropwise to a stirred mixture of piperidin-4-one
hydrochloride (8.14 g, 0.06 mol), K2CO3 (8.29 g, 0.06 mol), and
methanol (50 mL) at room temperature. Then the reaction mix-
ture was heated to reflux for 7 h. The solvent was evaporated
under reduced pressure. The residue was quenched with water
(25 mL), and extracted with dichloromethane (3650 mL). The
organic layer was separated, dried with anhydrous Na2SO4, and
concentrated under reduced pressure to give 11b as yellow oil:
6.64 g, (89.5%). 1H-NMR d (ppm): 6.89–7.30 (m, 5H, Ar-H), 4.00 (t, J
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W. Wang et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 732–739
NCH2CH2CH2NH), 1.44–1.47 (m, 2H, OCH2CH2CH2CH2N). MS (ESI)
m/z: 235 [M + 1].
The synthetic methods for the following compounds 14a and
14c were similar to the synthesis of compound 14b.
2-(2,4-Difluorophenyl)-3-[1-(5-phenoxypentyl)piperidin-4-
ylidenamino]-1-(1H-1,2,4-triazol-1-yl)propan-2-ol 12c
1H-NMR d (ppm): 8.13 (s, 1H, TriazC3-H), 7.92 (s, 1H, TriazC5-H),
6.83–7.60 (m, 8H, Ar-H), 5.30 (s, 1H, OH), 4.54 (d, J = 14.2 Hz, 1H,
C1-Ha), 4.24 (d, J = 14.1 Hz, 1H, C1-Hb), 4.00 (d, J = 13.0 Hz, 1H, C3-
Ha), 3.95 (t, J = 6.4 Hz, 2H, OCH2), 3.35 (d, J = 13.4 Hz, 1H, C3-Hb),
2.35–2.54 (m, 6H, N(CH2)2, NCH2CH2CH2CH2CH2O), 1.79 (2H,
NCH2CH2CH2CH2CH2O), 1.49–1.71 (m, 6H, piperidin-(CH2)2, NCH2-
CH2CH2CH2CH2O), 1.25–1.47 (m, 2H, NCH2CH2CH2CH2CH2O); 13C-
NMR d (ppm): 162.65, 158.96, 158.73, 151.69, 145.05, 129.26,
126.52, 120.40, 114.41, 111.31, 104.20, 97.71, 81.20, 67.57, 58.00,
56.22, 55.63, 54.29, 36.00, 35.10, 29.05, 26.73, 23.98. HMRS m/z
calcd. for C27H33F2N5O2: 497.2602; found: 497.2618.
2-(2,4-Difluorophenyl)-3-[3-(4-phenoxybutyl)tetrahydro-
pyrimidin-1(2H)-yl]-1-(1H-1,2,4-triazol-1-yl)propan-2-ol
15b
A solution of epoxide 4 (1.20 g, 3.6 mmol), 14b (1.01 g, 4.3 mmol),
triethylamine (2.5 mL), and EtOH (30 mL) was heated to reflux
for 9 h. The solvent was evaporated under reduced pressure. The
residue was purified by silica gel column chromatography
(CH2Cl2/MeOH, 100:2, v/v) to give 15b as pale yellow oil: 0.63 g
1
(37.3%). H-NMR d (ppm): 8.15 (s, 1H, TriazC3-H), 7.76 (s, 1H, Tri-
azC5-H), 6.76–7.30 (m, 8H, Ar-H), 5.30 (s, 1H, OH), 4.52 (s, 2H,
NCH2N), 3.92 (t, J = 6.3 Hz, 2H, OCH2), 3.29 (d, J = 14.0 Hz, 1H, C1-
Ha), 2.95 (br, 2H, C3-H2), 2.73 (d, J = 14.0 Hz, 1H, C1-Hb), 2.44–2.45
(m, 4H, NCH2CH2CH2N), 2.23 (br, 2H, NCH2CH2CH2CH2O ), 1.69–
1.75 (m, 2H, NCH2CH2CH CH2O), 1.55 (br, 2H, NCH2CH2CH2N),
1.44 (br, 2H, NCH2CH2C2H2CH2O). 13C-NMR d (ppm): 162.43,
158.77, 158.75, 150.61, 144.38, 129.37, 125.85, 120.35, 114.29,
111.16, 103.86, 75.74, 72.34, 67.22, 58.80, 55.99, 54.19, 53.56,
52.08, 26.90, 23.22, 22.74; HMRS m/z calcd. for C25H31F2N5O2:
471.2446; found: 471.2460.
2-(2,4-Difluorophenyl)-3-[3-(3-phenoxypropyl)tetra-
hydropyrimidin-1(2H)-yl]-1-(1H-1,2,4-triazol-1-yl)propan-
2-ol 15a
1H-NMR d (ppm): 8.14 (s, 1H, TriazC3-H), 7.75 (s, 1H, TriazC5-H),
6.74–7.30 (m, 8H, Ar-H), 5.30 (s, 1H, OH), 4.51 (s, 2H, NCH2N), 3.93
(t, J = 6.2 Hz, 2H, OCH2), 3.32 (d, J = 13.8 Hz, 1H, C1-Ha), 2.95 (br,
2H, C3-H2), 2.61 (d, J = 13.8 Hz, 1H, C1-Hb), 2.46 (br, 4H,
NCH2CH2CH2N), 2.23 (br, 2H, NCH2CH2CH2O), 1.71 (br, 2H,
NCH2CH2CH2O), 1.56 (br, 2H, NCH2CH2CH2N); HMRS m/z calcd. for
C24H29F2N5O2: 457.2290; found: 457.2303.
The synthetic methods for the target compounds 15a and 15c
were similar to the synthesis of compound 15b.
2-(2,4-Difluorophenyl)-3-[methyl-(3-phenoxypropyl)-
2-(2,4-Difluorophenyl)-3-[3-(5-phenoxypentyl)tetrahydro-
pyrimidin-1(2H)-yl]-1-(1H-1,2,4-triazol-1-yl)propan-2-ol
15c
amino]-1-(1H-1,2,4-triazol-1-yl)propan-2-ol 8a
1H-NMR d (ppm): 8.09 (s, 1H, TriazC3-H), 7.76 (s, 1H, TriazC5-H),
6.76–7.52 (m, 8H, Ar-H), 5.29 (br, 1H, OH), 4.49 (d, J = 14.2 Hz, 2H,
C1-HaHb), 3.87 (t, J = 6.0 Hz, 2H, OCH2), 3.05 (d, J = 13.6 Hz, 1H, C3-
Ha), 2.76 (d, J = 13.6 Hz, 1H, C3-Hb), 2.52 (t, J = 7.1 Hz, 2H,
NCH2CH2CH2O), 2.09 (s, 3H, NCH3), 1.73–1.79 (m, 2H,
NCH2CH2CH2O); HMRS m/z: calcd. for C21H24F2N4O2: 402.1867;
found: 402.1882.
1H-NMR d (ppm): 8.15 (s, 1H, TriazC3-H), 7.76 (s, 1H, TriazC5-H),
6.78–7.30 (m, 8H, Ar-H), 5.30 (s, 1H, OH), 4.52 (s, 2H, NCH2N), 3.94
(t, J = 6.4 Hz, 2H, OCH2), 3.28 (d, J = 14.0 Hz, 1H, C1-Ha), 2.91 (br,
2H, C3-H2), 2.73 (d, J = 13.5 Hz, 1H, C1-Hb), 2.43 (br, 4H,
NCH2CH2CH2N), 2.17 (br, 2H, NCH2CH2CH2CH2CH2O), 1.71–1.77
(m, 2H, NCH2CH2CH2CH2CH2O), 1.55 (br, 2H, NCH2CH2CH2N), 1.40
(br, 2H, NCH2CH2CH2CH2CH2O), 1.30 (br, 2H, NCH2CH2CH2CH2-
CH2O); HMRS m/z calcd. for C26H33F2N5O2: 485.2602; found:
485.2616.
2-(2,4-Difluorophenyl)-3-[methyl-(5-phenoxypentyl)-
amino]-1-(1H-1,2,4-triazol-1-yl)propan-2-ol 8c
1H-NMR d (ppm): 8.16 (s, 1H, TriazC3-H), 7.77 (s, 1H, TriazC5-H),
6.77–7.58 (m, 8H, Ar-H), 4.49 (d, J = 14.2 Hz, 2H, C1-HaHb), 3.91 (t, J
= 6.3 Hz, 2H, OCH2), 3.03 (d, J = 13.6 Hz, 1H, C3-Ha), 2.73 (d, J = 13.6
Hz, 1H, C3-Hb), 2.69–2.73 (m, 2H, NCH2CH2CH2CH2CH2O), 2.02 (s,
3H, NCH3), 1.67–1.70 (m, 2H, NCH2CH2CH2CH2CH2O), 1.32–1.33
(m, 4H, NCH2CH2CH2CH2CH2O); HMRS m/z calcd. for
C23H28F2N4O2: 430.2180; found: 430.2165.
Flexible docking analysis
The 3-D structures of the azoles were built by the Builder module
within InsightII 2000 software package. Then, the flexible ligand
docking procedure in the Affinity module within InsightII was
used to define the lowest energy position for the substrate using
a Monte-Carlo docking protocol. All the atoms within a defined
radius (8 ꢀ) of the substrate were allowed to move. The solvation
grid supplied with the affinity program was used. If the result-
ing substrate/enzyme system was within a predefined energy tol-
erance of the previous structure, the system was subjected to
minimization. The resulting structure was accepted on the basis
of an energy check, which used the Metropolis criterion, and
also a check of RMS distance of the new structure versus the
structure found so far. The final conformation was obtained
through a simulation annealing procedure from 500 to 300K,
and then 5000 rounds of energy minimization were performed
to reach a convergence, where the resulting interaction energy
values were used to define a rank order.
2-(2,4-Difluorophenyl)-3-[1-(3-phenoxypropyl)piperidin-4-
ylidenamino]-1-(1H-1,2,4-triazol-1-yl)propan-2-ol 12a
1H-NMR d (ppm): 8.14 (s, 1H, TriazC3-H), 7.92 (s, 1H, TriazC5-H),
6.85–7.60 (m, 8H, Ar-H), 4.54 (d, J = 14.2 Hz, 1H, C1-Ha), 4.25 (d, J =
14.2 Hz, 1H, C1-Hb), 4.00 (d, J = 13.0 Hz, 1H, C3-Ha), 3.98 (t, J = 6.2
Hz, 2H, OCH2), 3.36 (d, J = 13.0 Hz, 1H, C3-Hb), 2.54–2.63 (m, 4H,
N(CH2)2), 2.43 (br, 2H, piperidin-2-CH2), 1.88–1.96 (m, 2H, piperi-
din-4-CH2), 1.72 (br, 2H, NCH2CH2CH2O), 1.57 (br, 2H,
NCH2CH2CH2O). HMRS m/z calcd. for C25H29F2N5O2: 469.2289;
found: 469.2273.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 732–739
Conformationally Restricted Triazole Derivatives
739
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In-vitro antifungal activity assays
In-vitro antifungal activity was measured by means of the MIC
using the serial dilution method in 96-well microtest plates. Test
fungal strains were obtained from the ATCC or were clinical iso-
lates. The MIC determination was performed according to the
National Committee for Clinical Laboratory Standards (NCCLS)
recommendations with RPMI 1640 (Sigma) buffered with 0.165
MMOPS (Sigma) as the test medium. The MIC value was defined
as the lowest concentration of test compounds that resulted in a
culture with turbidity less than or equal to 80% inhibition when
compared with the growth of the control. Test compounds were
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were incubated without shaking at 358C for yeasts and at 288C
for dermatophytes. Optical density (OD) was measured at 630
nm and the background OD was subtracted from each well.
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This work was supported in part by the National Natural Science Foun-
dation of China (Grant Nos. 30930107, 30973640), Shanghai Rising-
Star Program (Grant Nos. 09QA1407000) and Shanghai Leading Aca-
demic Discipline Project (Project Nos. B906).
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The authors have declared no conflict of interest.
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www.archpharm.com