Synthesis and biological evaluation of vinyl ether-containing azole derivatives as inhibitors of Trichophyton rubrum

Article abstract of DOI:10.1016/j.bmcl.2012.05.070

In an attempt to search for many target compounds with excellent activities, a series of vinyl ether-containing azole derivatives were designed, synthesized, and evaluated as antifungal agents. Results of preliminary antifungal tests against Trichophyton rubrum in vitro indicated that most of the synthesized compounds showed excellent activities. In comparison with fluconazole, itraconazole, voriconazole, omoconazole and amphotericin B, several compounds (such as 7d, 7g and 7h) exhibited more potent inhibitory activities, suggesting that they were promising leads for the development of novel antifungal agents.

Full text of DOI:10.1016/j.bmcl.2012.05.070

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4887–4890
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of vinyl ether-containing azole
derivatives as inhibitors of Trichophyton rubrum
Lulu Wang a, Wenge Yang b, Kai Wang a, Jing Zhu b, Fei Shen c, Yonghong Hu a,
⇑
a College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, China
b School of Pharmaceutical Sciences, Nanjing University of Technology, Nanjing 210009, China
c Jiangsu Engineering Technology Research Center of Polypeptide Pharmaceutical, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 February 2012
Revised 3 May 2012
Accepted 7 May 2012
Available online 24 May 2012
In an attempt to search for many target compounds with excellent activities, a series of vinyl ether-con-
taining azole derivatives were designed, synthesized, and evaluated as antifungal agents. Results of pre-
liminary antifungal tests against Trichophyton rubrum in vitro indicated that most of the synthesized
compounds showed excellent activities. In comparison with fluconazole, itraconazole, voriconazole,
omoconazole and amphotericin B, several compounds (such as 7d, 7g and 7h) exhibited more potent
inhibitory activities, suggesting that they were promising leads for the development of novel antifungal
agents.
Keywords:
Azole
Synthesis
Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
Antifungal activity
Rational design
Trichophyton rubrum
CYP51
During the past several decades, fungal infections have become
a continuous and serious threat to human health and life particu-
larly in immunocompromised individuals.1–3 Fungal infections
include superficial and systemic fungal infections. The anthropo-
philic species Trichophyton rubrum is an important etiologic agent
of tinea (ringworm) infections, including onychomycosis (nail
infection), tinea cruris (groin infection), and tinea pedis (‘athlete’s
foot’).4 It is reported that about 90% of the chronic dermatophyte
infections are caused by T. rubrum. These conditions are relatively
benign and easy to treat, except in immunocompromised patients,
but therapy is costly and can fail in a significant proportion of
cases.5
Currently, numerous antifungal drugs such as amphotericin B,
5-fluorocytosine, azoles, and echinocandins (including caspofungin
and micafungin) spring up.6 However, because of the emergence of
drug resistance, undesirable side effects, high risk of toxicity and
insufficiencies in their antifungal activity, the clinical uses of most
agents have been limited. For this reason, it is necessary to develop
and extend the safe and efficient chemotherapeutic agents with
potent antifungal activities.7
membranes, by the heterocyclic nitrogen atom (N-3 of imidazole,
N-4 of triazole) binding to the heme iron atom located in the active
site of the cytochrome P450 14a-demethylase (CYP51), which may
disrupt the close packing of acyl chains of phospholipids, impairing
the functions of certain membrane bound enzymes, such as ATPase
and enzymes of the electron transport system. Together, these
actions inhibit fungal growth.8–10 However, the extensive use of
such antifungal agents has led to the development of severe
resistance, which significantly reduced their efficacy.11,12 Survey
reveals some clinically used drugs, such as fluconazole, may also
cause resistance to new structurally related azoles, for example,
voriconazole and ravuconazole.13–15 Hence, the discovery of novel
and potent antifungal azoles is very important and necessary to
overcome this situation and develop effective therapies.
Up to the present, the structurally and functionally important
regions have been recognized accurately. 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 hydro-
phobic cleft formed by the residues in the helix B’-meander 1 loop
and N-terminus of helix I.16 Important residues involved in azole
binding have been investigated by flexible molecular docking17–
Among these antifungal drugs, azoles (such as fluconazole,
itraconazole and voriconazole) are the most widely used antifungal
agents due to their high therapeutic index. Azole antifungals act by
inhibiting the biosynthesis of ergosterol, the bulk sterol in fungal
and site-directed mutagenesis.20 The molecular modeling that
19
gives the utilization of structural information of fungal CYP51s
greatly facilitates the process of rational antifungal agents design.
Omoconazole (Fig. 1) which developed by Siegfrieds’s research
laboratories is an imidazole antifungal and has been widely used
⇑
Corresponding author. Tel./fax: +86 25 83587108.
E-mail address: yonghonghu09@gmail.com (Y. Hu).
0960-894X/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.070

4888
L. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4887–4890
Cl
F
Modified
Cl
F
α
O
α
O
O
CH2
O
n
β
β
R
N
N
Cl
N
N
N
Figure 1. Chemical structure of omoconazole and modified positions of the compounds.
for the treatment of superficial fungal infections, including dermat-
ophytoses, pityriasis versicolor, molds, and cutaneous and vaginal
candidiasis. It also has bacteriostatic effect on Gram-positive bac-
teria such as Staphylococcus aureus and Group A and D Strepto-
cocci.10 The spectrum of antifungal activity of omoconazole has
been reported to be quite similar to that of the imidazole family,
such as tioconazole and econazole. A number of clinical studies
show that omoconazole has exhibited excellent tolerance and
highly effective in the treatment of vaginal candidiasis as vaginal
ovules, in the treatment of dermatomycoses and tinea versicolor
as a 1% cream formulation and spray, and in the treatment of can-
didal balanitis as a 1% spray powder formulation.21 Researches
indicated that the imidazole ring, the dichlophenyl group and the
vinyl ethers were the pharmacophores of omoconazole. Further-
more, it is reported that the vinyl ethers structures were very
important and helpful for optimal antifungal activity, stability
and toxicological properties. In addition, introduction of a double
In our compounds design, we systematically altered the struc-
ture of omoconazole using the strategy of structure-based rational
drug design. The modification was focused mainly on the side
chains at the
a-position of 1-(1H-imidazolyl)methylarylketones.
The side chains which located in the narrow hydrophobic cleft
were also important.16 And the optimization of the side chain at-
tached to the pharmacophore is attractive to the current re-
searches. Besides, some researchers suggested that triazole
antifungals, such as fluconazole, voriconazole and posaconazole,
were more efficient than imidazole antifungals and the activity
of a drug with the diflurophenyl group at the pharmacophore
was higher than that with the dichlophenyl group.23,24 To sum
up, we intended to find highly potent new antifunal drugs with
excellent antifungal activity, low toxicity and less potential to de-
velop resistance.
The general synthetic methodology for the preparation of the
target compounds 6a–6e and 7a–7j was accomplished using chem-
istry illustrated in Scheme 1. As a key intermediate of our designed
vinyl ether-containing azole antifungals, the compound 3 was syn-
thesized with known procedures.19 Compound 5 were another key
intermediates and they can normally be prepared from alkylation
bond between
a and b carbon atoms leads to stereoisomers differ-
ing in physicochemical and biological parameters and increasing
the spatial demand at the
sired properties.22
a
carbon by alkylation improved the de-
F
F
F
F
a
F
b
HNO3
O
O
F
Cl
N
N
N
3
1
2
OH
O
Br
c
n
R
R
O
4
5
F
F
d
O
HNO3
n
R
N
N
N
6a-6e
7a-7j
Scheme 1. Synthesis route to the title compounds. Reagents and conditions: (a) CH3CHClCOCl, AlCl3, 55 °C, in 91% yield; (b) 1H-1,2,4-triazole, K2CO3, CH2Cl2, rt, 24 h, HNO3,
55%; (c) 1,n-dibromoalkane, NaOH, tetrabutyl ammonium bromide(TBAB), DMF, reflux, 8 h, 66–80%; (d) 3, NaOH, tetrabutylammonium hydroxide(10% in water), toluene,
50 °C, 20 h, 65% HNO3, 40–62%.27,28

L. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4887–4890
4889
Table 1
Antifungal activities of the title compounds in vitro (MIC80, l
g/mL)a
Compound
n
R
T. rubrum
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
7f
7g
2
3
4
5
6
3
3
4
4
4
4
4
4
3
4
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Br
4-F
2-Cl
3-Cl
2,4-Cl
2,4,6-Cl
4-Br
4-F
4-CH3
4-CH3
0.2
0.125
0.0625
0.25
2.5
0.25
0.1
0.1
0.025
2
>20
0.05
0.025
2.5
7h
7i
7j
0.2
FCZ
ICZ
VCZ
OCZ
AMB
4
0.125
0.0625
0.1
2
a
Abbreviations: T. rubrum, Trichophyton rubrum; FCZ, fluconazole; ICZ, itraco-
nazole; VCZ, voriconazole; OCZ, omoconazole; AMB, amphotericin B.
Figure 2. X-ray analysis of compound 6c.
First of all, we substituted the triazole ring and the diflurophenyl
group for imidazole ring and dichlophenyl group at the pharmaco-
phore respectively (compound 6a). The MIC values of compound
6a and omoconazole were almost the same, which indicated that
these modifications could not improve the inhibitory activity
against T. rubrum effectively. We noticed that the side chains at
of substituted phenols 4 by using 1,n-dibromoalkanes in the pres-
ence of NaOH, catalytic amount of tetrabutyl ammonium bromide
(TBAB) in DMF. To a stirred mixture of 3 and 5 in the presence of
50% aqueous soda lye, tetrabutylammonium hydroxide(10% in
water) as phase transfer catalyst in toluene and then mixed with
65% aqueous nitric acid, we can afford the compounds 6a–6e.
The target compounds 7a–7j were synthesized in the same way
as 6a–6e. All the new compounds described above were character-
ized by LC–MS, and NMR spectroscopic analysis.25 Moreover, we
did X-ray analysis of some compounds, for example, compound
6c (Fig. 2).26
The in vitro minimal inhibitory concentrations (MICs) of the
compounds were determined by the micro-broth dilution method
in 96-well microtestplates according to the methods defined by the
National Committee for Clinical Laboratory Standards (NCCLS).29
The MIC80 was defined as the first 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 di-
methyl sulfoxide (DMSO), serially diluted in growth medium, inoc-
ulated and incubated at 35 °C. Growth MIC was determined at 72 h
for T. rubrum. Fluconazole(FCZ), itraconazole(ICZ), voriconazole
(VCZ), omoconazole(OCZ), and amphotericin B(AMB) were ob-
tained from their respective manufacturers served as the positive
control. The results of assays were summarized in Table 1. The data
were obtained from the mean of replicates. All of our susceptibility
tests were performed three times.
In vitro antifungal activity assay (Table 1) indicates that most of
the synthesized compounds (6a–6e, 7a–7j) show moderate to
excellent activity against T. rubrum. From the MIC80 values of 6a–
6e series, we can see that compounds 6b (n = 3) and 6c (n = 4)
show higher antifungal activities than those of the others. Espe-
cially, the activity of compound 6c is comparable to that of voric-
onazole and superior to that of omoconazole and itraconazole. So
we choose n = 3,4 for the synthesis of the 7a–7j series, particularly
n = 4. Noticeably, compounds 7d, 7g and 7h show higher inhibitory
activities than those of all the positive controls. The MIC80 values of
compounds 7d and 7h are 160 times lower than that of fluconazole
against T. rubrum in vitro, indicating that they are promising leads
for the discovery of novel antifungal agents.
the a-position of 1-(1H-triazolyl)methylarylketones which located
in the narrow hydrophobic cleft of CYP51 were very important to
the activity. So the modification was focused mainly on the side
chains. We wanted to find out how chain length and the substitu-
tions on the phenyl ring could influence the inhibitory activity.
Having a number of reactive reagents compound 5, we tried these
on compound 3, which led us to obtain the target compounds (6a–
6e,7a–7j) containing vinyl ether structure. Compared with omoco-
nazole, increasing the spatial demand at the
a carbon by alkylation
could improve the desired properties. From the MIC80 values of
6a–6e series, we could find that the alkyl of 4 carbon atoms (com-
pound 6c) exhibited higher inhibitory activities than that of the
other lengths of carbon chain. In addition, the substitutions on
the phenyl ring which could form strong hydrophobic interactions
with the active site of CYP51 played an important role for the anti-
fungal activities. In general, halogens group (e.g. compounds 6b,
6c, 7d, 7g and 7h) were favorable for the antifungal activity. On
the contrary, the introduction of methyl (compounds 7i and 7j)
did not show improved antifungal activity, which might indicate
that the alkyl groups on the phenyl ring could not effectively im-
prove the inhibitory activity in this study. For compounds 6c, 7c
and 7d, the position of the substitutions also had influence on
the antifungal activity. The phenyl group substituted by chlorine
in the 3 position (compound 7d) showed higher inhibitory activity
against T. rubrum than that in the 4 or 2 position. Besides, from the
MIC80 values of 6c, 7g and 7h, we could obtain that the phenyl
substituted by fluorine showed better antifungal activity than that
by chlorine or bromine in the same position. In comparison with
the mono-chlorine substituted compounds (6c, 7c and 7d), the
di- and tri-substituted derivatives (7e and 7f) led to the decrease
of the antifungal activity to a large extent. Because it is a new type
of side chain in azole antifungal agents, futher structural modifica-
tion is essential to obtain more information about SARs.
In conclusion, a new class of vinyl ether-containing azole deriv-
atives had been synthesised. Results of preliminary antifungal test
against T. rubrum in vitro showed that these analogs exhibited
From the antifungal activity data, preliminary structure-activity
relationships (SARs) of the synthesized compounds were obtained.

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L. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4887–4890
18. Sheng, C.; Miao, Z.; Ji, H.; Yao, J.; Wang, W.; Che, X.; Dong, G.; Lu, J.; Guo, W.;
Zhang, W. Antimicrob. Agents Chemother. 2009, 53, 3487.
19. Sheng, C.; Zhang, W.; Ji, H.; Zhang, M.; Song, Y.; Xu, H.; Zhu, J.; Miao, Z.; Jiang,
Q.; Yao, J.; Zhou, Y.; Lu, J. J. Med. Chem. 2006, 49, 2512.
20. Chen, S. H.; Sheng, C. Q.; Xu, X. H.; Jiang, Y. Y.; Zhang, W. N.; He, C. Biol. Pharm.
Bull. 2007, 30, 1246.
21. Castar, J.; Prous, J. Drugs Future 1988, 13, 624.
22. Zirngibl, L.; Fischer, J.; Jahn, U.; Thiele, K. Ann. N.Y. Acad. Sci. 1988, 544, 63.
23. Wang, S.; Wang, Z.; Cai, Z.; Zhou, W. Chin. J. Pharm. 2007, 38, 49.
24. Wang, R. Clin. Med. J. 2007, 5, 1.
excellent activities. The vitro antifungal assay also indicated that a
proper length of side chain and the substitutions on the phenyl
ring played very important roles for the antifungal activities. Sev-
eral compounds, such as 7d and 7h, were promising leads for the
discovery of novel antifungal agents. Effort aimed at further opti-
mization, as well as in-depth biological investigations, of the iden-
tified lead compounds is continuing in our laboratories, and results
will be reported in due course.
25. Experimental: Representative analytical data for compound 6a. A mixture of 1-
(2,4-difluorophenyl)-2 -(1,2,4-triazol)-1-yl)propan-1-one (3.0 g, 0.01 mol),
10 g of a 50% aqueous soda lye and 1.5 mL tetrabutylammonium hydroxide
as phase transfer catalyst in toluene (15 mL) was stirred and heated to 50 °C.
Then 1-bromo-2-(4-chlorophenoxy)-ethane (2.4 g, 0.01 mol) dissolved in
10 mL toluene, was instilled into the reaction solution. The mixture was
subsequently stirred for another 20 h at 50 °C (monitored by TLC, eluent, ethyl
acetate/petroleum ether, 1/1, v/v). Then the solvents were evaporated under
reduced pressure, and the residue was treated with water (100 mL) and
extracted with chloroform (3 Â 100 mL). The organic layers were combined,
dried with anhydrous Na2SO4, and concentrated under reduced pressure. The
remaining residue was a dark oil that was diluted with 10 ml 2-propanol and
then adjusted to a pH-value of 2 by 65% HNO3. The thus derived nitric acid
solution was then cooled in the refrigerator. The impure precipitated product
herein was subsequently crystallized from the mixture solvent of ethyl acetate/
ethanol (1:1, v/v) to afford compound 6a (2.8 g) as white solid. Yield: 62%; mp:
127–129 °C; 1H NMR (500 MHz, CDCl3) d: 9.71 (1H, s, TriazC3-H), 8.31 (1H, s,
TriazC5-H), 7.46 (1H, s, Ar-H), 7.23 (2H, m, Ar-H), 6.99–7.09 (2H, m, Ar-H), 6.74
(2H, m, Ar-H), 4.00 (2H, m, –CH2–), 3.93 (2H, m, –CH2–), 2.12 (3H, s, –CH3); LC–
MS, m/z: 392.1 (M+H)+. Compound 7a: Prepared according to the procedure
described for the preparation of compound 6a, starting from a mixture of 1-
(2,4-difluorophenyl)-2-(1,2,4-triazol)-1-yl)propan-1-one (3.0 g, 0.01 mol), 10 g
of a 50% aqueous soda lye and 1.5 mL tetrabutylammonium hydroxide in
15 mL toluene was stirred and heated to 50 °C. Then 1-bromo-3-(4-
bromophenoxy)-propane (3.0 g, 0.01 mol) dissolved in 10 mL toluene, was
instilled into the reaction solution. The mixture was subsequently stirred for
20 h at 50 °C (monitored by TLC, eluent, ethyl acetate/petroleum ether, 1/1, v/
v). Post-treatment process of the reaction solution can according to the
procedure described for the preparation of compound 6a. The impure
precipitated product was subsequently crystallized from the mixture solvent
of ethyl acetate/petroleum ether (2:1, v/v) to afford compound 7a (2.5 g) as
white solid. Yield: 48.7%; mp: 120–122 °C; 1H NMR (500 MHz, CDCl3) d: 9.57
(1H, s, TriazC3-H), 8.35 (1H, s, TriazC5-H), 6.92–7.37 (7H, m, Ar-H), 3.88 (2H, m,
–CH2–), 3.75 (2H, m, –CH2–), 2.08 (3H, s, –CH3), 2.01 (2H, m, –CH2–); LC–MS,
m/z: 450.1 (M+H)+.
Acknowledgments
This research work was supported by the Production Teaching
Research Joint Innovation Fund Projects of Jiangsu Province in Chi-
na (Grant No. BY2011106). This research work was also supported
by the National Natural Science Foundation of China (Grant No.
31171644).
We also thank the editors and the anonymous reviewers.
References and notes
1. Georgopapadakou, N.; Tkacz, J. Trends Microbiol. 1995, 3, 98.
2. Nagiec, M.; Nagiec, E.; Baltisberger, J.; Wells, G.; Lester, R.; Dickson, R. J. Biol.
Chem. 1997, 272, 9809.
3. Walsh, T. J.; Groll, A.; Hiemenz, J.; Fleming, R.; Roilides, E.; Anaissie, E. Clin.
Microbiol. Infect. 2004, 10, 48.
4. Jackson, C. J.; Barton, R. C.; Kelly, S. L.; Evans, E. G. V. J. Clin. Microbiol. 2000, 38,
4527.
5. Blake, J. S.; Dahl, M. V.; Herron, M. J.; Nelson, R. D. J. Invest. Dermatol. 1991, 96,
657.
6. Xu, Y.; Sheng, C.; Wang, W.; Che, X.; Cao, Y.; Dong, G.; Wang, S.; Ji, H.; Miao, Z.;
Yao, J.; Zhang, W. Bioorg. Med. Chem. Lett. 2010, 20, 2942.
7. Johnson, D. S.; Li, J. J. Art Drug Synth. 2007, 71.
8. Vanden, B. H.; Willemsens, G.; Marichal, P. Crit. Rev. Microbiol. 1987, 15, 57.
9. Georgopapadakou, N. H.; Walsh, T. J. Antimicrob. Agents Chemother. 1996, 40,
279.
10. Waugh, C. D. Xpharm: Compr. Pharmacol. Ref. 2008, 1.
11. Casalinuovo, I. A.; Di Francesco, P.; Garaci, E. Eur. Rev. Med. Pharmacol. Sci. 2004,
8, 69.
12. Hoffman, H. L.; Ernst, E. J.; Klepser, M. E. Expert Opin. Investig. Drugs 2000, 9,
593.
13. Pfaller, M. A.; Messer, S. A.; Hollis, R. J.; Jones, R. N. Antimicrob. Agents
Chemother. 2001, 45, 2862.
26. The crystal structure of 6c is well documented in our reported literature: Chen,
J.; Wang, K.; Shen, Y.; Ren, L.; Chen, G. Acta Crystallogr., Sect. E 2011, E67,
o2800. Full crystallographic details of 6c have been deposited at the Cambridge
Crystallographic Data Center and allocated the deposition number CCDC
872815.
27. Thiele, K.; Zirngibl, L.; Pfenninger, M. H. Helv. Chim. Acta 1987, 70, 441.
28. Ludwig, Z.; Kurt, T. U.S. Patent 4,554,356, 1985.
29. National Committee for Clinical Laboratory Standards, reference method for
broth dilution antifungal susceptibility testing of yeasts approved standard.
Document M27-A2, National Committee for Clinical Laboratory Standards,
Wayne, PA, 2002.
14. Jeu, L.; Piacenti, F. J.; Lyakhovetskiy, A. G.; Fung, H. B. Clin. Pharmacol. Ther.
2003, 25, 1321.
15. Lepesheva, G. I.; Zaitseva, N. G.; Nes, W. D.; Zhou, W.; Arase, M.; Liu, J.; Hill, G.
C.; Waterman, M. R. J. Biol. Chem. 2006, 281, 3577.
16. Sheng, C.; Zhang, W.; Ji, H. Chin. Chem. Lett. 2004, 15, 404.
17. Ji, H.; Zhang, W.; Zhou, Y.; Zhang, M.; Zhu, J.; Song, Y.; Lu, J. J. Med. Chem. 2000,
43, 2493.