Design, synthesis, and biological evaluation of novel 1-(1H-1,2,4-triazole-1-yl)-2-(2,4-difluorophenyl)-3-substituted benzylamino-2-propanols

Article abstract of DOI:10.1016/j.bmcl.2009.01.048

Based on the results of computational docking to the active site of the cytochrome P450 14α-demethylase (CYP51), a series of 1-(1H-1,2,4-triazole-1-yl)-2-(2,4-difluorophenyl)-3-substituted benzylamino-2-propanols as analogs of fluconazole were designed, synthesized, and evaluated as antifungal agents. Results of preliminary antifungal tests against eight human pathogenic fungi in vitro showed that all the title compounds exhibited excellent activities with broad spectrum.

Full text of DOI:10.1016/j.bmcl.2009.01.048

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1811–1814
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and biological evaluation of novel 1-(1H-1,2,4-triazole-1-yl)-
2-(2,4-difluorophenyl)-3-substituted benzylamino-2-propanols
Xiaoyun Chai, Jun Zhang, Shichong Yu, Honggang Hu, Yan Zou, Qingjie Zhao, Zhigang Dan,
*
Dazhi Zhang, Qiuye Wu
Department of Organic Chemistry, College of Pharmacy, Second Military Medical University, Guohe Road 325, Shanghai 200433, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the results of computational docking to the active site of the cytochrome P450 14a-demethylase
Received 24 September 2008
Revised 19 December 2008
Accepted 9 January 2009
Available online 21 January 2009
(CYP51), a series of 1-(1H-1,2,4-triazole-1-yl)-2-(2,4-difluorophenyl)-3-substituted benzylamino-2-prop-
anols as analogs of fluconazole were designed, synthesized, and evaluated as antifungal agents. Results of
preliminary antifungal tests against eight human pathogenic fungi in vitro showed that all the title com-
pounds exhibited excellent activities with broad spectrum.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Triazole
Synthesis
Antifungal activity
CYP51
Molecular docking
During the past several decades, systemic fungal infection had
become a momentous complication and a major cause of morbidity
and mortality in immunocompromised individuals.1,2 Currently,
several clinical drugs such as azoles, amphotericin B, 5-fluorocyto-
sine, and caspofungin have been developed to reduce the impact of
fungal diseases infected by candidosis, aspergillosis, and crypto-
coccosis.3,4 Among these antifungal agents, azoles were used
widely and efficiently, especially triazole, such as fluconazole,
voriconazole, and itraconazole, now play a leading role in the treat-
ment of invasive fungal infections. These antifungal drugs act by
inhibiting CYP51 in the process of biosynthesis of ergosterol
through a mechanism in which the heterocyclic nitrogen atom
(N-4 of triazole) binds to the heme iron atom.5 However, the
increasing administration of such antifungal agents has led to the
development of fungal resistance. Survey reveals genetic muta-
tions that result in resistance to clinically used drugs, especially
fluconazole, may also result in resistance to new structurally re-
lated azoles such as voriconazole and ravuconazole.6–8 The emer-
gence of resistance shows the need of the discovery of new
antifungal compounds. And so, there is a must need for broad spec-
trum and low-toxicity antifungal agents genuinely, although it is
developed rapidly recently.
into four subsites: a coordination bond with iron of the heme
group, the hydrophilic H-bonding region, the hydrophobic region,
and the narrow hydrophobic cleft formed by the residues in the he-
lix B0-meander 1 loop and N-terminus of helix I.10 Based on the re-
sults of Ji et al., we constructed a 3D model of CYP51 from C.
albicans and analyzed the binding between fluconazole and
CYP51. As shown in Figure 2, fluconazole binds to the active site
of CYP51 via coordination of the N atom of the triazole nucleus
with iron of the heme group. The diflurophenyl group is located
in the hydrophobic binding cleft lined with Ala114, Phe126,
Leu139, Met140, Phe145, Ile304, Met306, and Gly307. Several res-
idues lined with Leu121, Thr122, Phe228, Thr311, Pro375, Leu376,
His377, Ser378, Met508, Val509, and Val510 were observed to
form indirect nonbonding interactions with another triazole ring.11
Researches indicated that the triazole ring, the diflurophenyl
group and the hydroxyl group were the pharmacophores of anti-
fungal agents. But the side chains which located in the narrow
The crystal structure of CYP51 of fungi has not been obtained. Ji
et al. constructed a 3D model of CYP51 from Candida albicans.9 In
general, the active site of CYP51 for ligand binding can be divided
* Corresponding author. Tel./fax: +86 21 25070381.
E-mail address: wuqy6439@sohu.com (Q. Wu).
Figure 1. Chemical structure of fluconazole and modified positions of the
compounds.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.048

1812
X. Chai et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1811–1814
Figure 2. Computed binding geometry of the clinical used inhibitor fluconazole in the active site of CYP51.
hydrophobic cleft were also important. And the optimization of the
side chain attached to the pharmacophore remains attractive to the
current researches. We intended to alter the side chains to find po-
tent systemic antifungal agents with a broad antifungal spectrum
and less potential to develop resistance.
in the presence of KI, K2CO3 in acetonitrile, we afforded compounds
6An. The target compounds 6Bn–6Cn were synthesized in the same
way as 6An. All the new compounds (6An–6Cn) described above
were characterized by IR, LC–MS, and NMR spectroscopic
analysis.13
In our compounds design, we systematically altered the struc-
ture of fluconazole (Fig. 1) based on the results of computational
docking to the active site of the CYP51, and the modification was
focused on one triazole moiety of fluconazole.
Synthesis of the target compounds 6An–6Cn was accomplished
using chemistry illustrated in Scheme 1. The intermediate oxirane
4 was synthesized with known procedures.12 Oxirane 4 was al-
lowed to react with n-propylamine in the presence of triethyl-
amine in ethanol, and then added HCl gas to formed compounds
5An. To a stirred mixture of 5An and substituted benzyl bromide
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).14 C. albicans and Cryptococcus neoformans are ATCC stan-
dard strains, and others are clinic isolates. Fluconazole, itraconaz-
ole, ketoconazole, voriconazole, amphotericin B, and terbinafine
were obtained from their respective manufacturers served as the
positive control. The minimum 80% inhibitory concentration
(MIC80) values are summarized in Table 1.
Scheme 1. Synthetic route to the title compounds. Reagents and conditions: (a) ClCH2COCl, AlCl3, 50 °C, 5 h, in 87% yield; (b) C6H5CH3, NaHCO3, 1H-1,2,4-triazole, reflux, 5 h,
in 87% yield; (c) C6H5CH3, (CH3)3SOI, NaOH, centylmethylammonium bromide, 60 °C, 3 h, in 86% yield; (d) CH3SO3H, 0 °C, 1 h, in 89%yield; (e) CH3CH2OH, Et3N, primary
amine, reflux, 6 h; (f) HCl(g), in 80–90% yield; (g) CH3CN, KI, K2CO3, substituted benzyl bromide, rt, 5–6 h, in 50–70% yield.

X. Chai et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1811–1814
1813
Table 1
Antifungal activities of the title compounds in vitro (MIC80, lg/mL)
Compound
R1
R3
C. alb.
C. neo.
C. par.
C. tro.
T. rub.
C. kru.
M. gyp.
A. fum.
6A1
6A2
6A3
6A4
6A5
6A6
6A7
6A8
6A9
6A10
6A11
6A12
6A13
6A14
6B1
6B2
6B3
6B4
6B5
6B6
6B7
6B8
6B9
6B10
6B11
6B12
6B13
6B14
6C1
6C2
6C3
6C4
6C5
6C6
6C7
6C8
6C9
6C10
6C11
6C12
6C13
6C14
ICZ
KCZ
FCZ
VCZ
AMB
TRB
–(CH2)2CH3
–(CH2)2CH3
–(CH2)2CH3
–(CH2)2CH3
–(CH2)2CH3
–(CH2)2CH3
–(CH2)2CH3
–(CH2)2CH3
–(CH2)2CH3
–(CH2)2CH3
–(CH2)2CH3
–(CH2)2CH3
–(CH2)2CH3
–(CH2)2CH3
–CH(CH3)2
–CH(CH3)2
–CH(CH3)2
–CH(CH3)2
–CH(CH3)2
–CH(CH3)2
–CH(CH3)2
–CH(CH3)2
–CH(CH3)2
–CH(CH3)2
–CH(CH3)2
–CH(CH3)2
–CH(CH3)2
–CH(CH3)2
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
–(CH2)3CH3
—
H
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
2-Br
4-Br
2-CH3
4-CH3
4-NO2
4-CH2CH3
2,4-Cl,Cl
H
2-F
3-F
4-F
2-Cl
0.0156
0.0156
0.0156
0.0625
0.25
0.0625
0.0625
0.0625
0.0625
0.0625
0.0625
0.0156
0.0156
0.25
0.0156
0.0625
0.0625
0.0625
0.0625
0.0156
0.0625
0.25
0.0039
0.0156
1
0.0156
0.0625
0.25
0.0625
0.0625
0.0156
0.25
0.0625
0.25
0.0625
0.0625
0.0625
0.25
0.0625
0.25
0.25
0.25
0.25
0.25
1
0.0625
0.0625
0.0625
0.0625
0.25
0.0625
0.0625
0.25
0.25
0.25
0.25
0.25
0.25
0.0625
0.0625
0.0625
0.25
0.25
4
0.25
0.25
0.0625
0.25
0.25
1
0.25
0.0625
0.0625
0.25
1
0.25
1
1
0.0625
0.25
0.25
0.25
0.25
1
1
0.25
1
0.0625
1
1
1
4
4
1
1
0.25
4
0.25
0.25
1
0.0625
0.0625
0.0625
0.0625
0.25
0.25
0.0625
0.25
0.25
0.0625
0.0625
0.0625
1
0.0156
0.0156
0.0625
0.0039
0.0156
0.0625
0.0625
0.0625
0.0625
0.0039
0.0156
0.0156
0.25
0.0156
0.0156
0.0625
0.0625
0.0625
0.0625
1
>64
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
64
>64
>64
64
>64
64
>64
>64
>64
1
>64
4
>64
64
64
0.25
0.25
0.0039
0.0156
0.0625
0.0625
0.0625
0.25
1
1
1
1
0.25
0.25
0.0625
0.0625
0.25
1
0.25
0.25
0.25
0.25
0.25
0.25
1
0.0625
0.0625
0.0625
0.0625
0.25
0.0625
1
0.25
0.25
0.0625
0.0625
0.0625
0.25
0.0625
0.25
0.25
0.25
0.25
0.0625
1
0.25
0.25
0.25
0.25
0.25
0.0625
0.0625
0.25
0.0625
0.25
1
0.25
0.25
1
1
1
3-Cl
4-Cl
0.25
0.0625
0.0625
0.0625
0.0625
0.25
0.0625
1
1
0.0625
0.25
0.25
0.0625
0.25
0.25
1
0.25
0.25
0.25
1
0.25
4
4
0.0625
0.0156
0.0156
0.0625
0.0625
0.0156
0.25
2-Br
4-Br
2-CH3
4-CH3
4-NO2
4-CH2CH3
2,4-Cl,Cl
H
2-F
3-F
4-F
2-Cl
>64
>64
>64
16
16
1
1
0.0625
0.25
0.25
0.25
0.25
0.25
1
0.25
0.25
0.25
0.25
0.0625
0.25
1
0.25
0.25
1
1
1
1
1
1
1
1
1
1
1
16
0.5
0.25
4
0.0625
2
0.0156
0.25
0.0625
0.0156
0.25
0.25
1
0.25
1
0.0156
0.25
0.0625
1
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
0.5
1
1
1
1
4
1
4
1
1
1
1
3-Cl
4-Cl
2-Br
4-Br
2-CH3
4-CH3
4-NO2
4-CH2CH3
2,4-Cl,Cl
—
—
—
—
—
0.25
1
1
0.25
4
16
0.125
0.0625
4
0.0625
2
0.25
1
0.0625
1
0.0156
0.0039
0.0039
0.0625
0.0156
0.5
0.0156
2
4
1
0.25
0.125
0.0625
1
0.0156
2
0.125
0.0625
1
0.0039
2
0.125
0.0625
1
0.0039
2
0.0625
0.0156
0.25
0.0039
2
—
—
—
—
1
>64
0.25
64
—
—
1
0.25
0.0625
0.0625
16
4
0.0625
Abbreviations: C .alb, Candida albicans; C. neo, Cryptococcus neoformans; C. par, Candida parapsilosis; C. tro, Candida tropicalis; T. rub, Trichophyton rubrum; C. kri, Candida Krusei;
M .gyp, Microsporum gypseum; A. fum, Aspergillus fumigatus.
ICZ, itraconazole; KCZ, ketoconazole; FCZ, fluconazole; VCZ, voriconazole; AMB, amphotericin B; TRB, terbinafine.
Compared to the most prescribed antifungals in clinic vorico-
nazole, these compounds also exhibited excellent activities and
broad spectrum. The MIC80 values indicate that the series of 6An
and 6Bn showed more excellent antifungal activities against the
fungi than that of the 6Cn series. Noticeably, the 6An and 6Bn series
showed higher activity against nearly all the fungi tested except
against Aspergillus fumigatus. Noticeably, the MIC value of com-
pound 6A4 and 6A10 is 64 times lower than that of fluconazole
against fluconazole-resistant strains of C. albicans. The Trichophyton
species Trichophyton rubrum was found to be much less sensitive to
these derivatives, as indicated by their MIC80 values showing mod-
erate activity. As far as we all know A. fumigatus possesses an
intrinsic mechanism resistant to triazole antifungals.15
We proposed a likely binding mode for 6B9 to the active site of
CYP51 based on computational docking results (Fig. 3). In our pre-
vious study, both R and S isomers of the compounds interacted
with CYP51 through a similar binding mode. However, R isomers
showed lower interaction energy with CYP51 than the S isomers,
which indicated that the R isomers might have better antifungal
activity than the S isomers.11 In the following discussion, all the
docked conformations refer to the R configuration of the com-
pounds. As usual, the triazole interacts with iron of the heme
group, while the diflurophenyl group in the designed compound
could be placed into the hydrophobic pocket formed by Ala114,
Phe126, Leu139, Met140, Phe145, Ile304, Met306, and Gly307.
against Microsporum gypseum in vitro. And compound 6B9, 6C13
,
and 6C14 showed 128 times higher activity (with the MIC80 value
of 0.0039 mg/mL) than that of fluconazole against C. albicans and
also showed higher activities than that of the other positive con-
trols. These results clearly indicated that the substituted benzyl
side chain linked to the triazole pharmacophore greatly enhanced
the antifungal activity of these analogs against Candida species, and
that the amine side chain showed higher activity as it is shortened.
In addition, despite the positive control drug itraconazole and
voriconazole showing cross-resistance to fluconazole, almost all
of our target compounds displayed certain extent of activities
And the 4-bromobenzyl group would generate
p–p stacking inter-
actions with the Tyr-118. The N-substituted group of the linker

1814
X. Chai et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1811–1814
Figure 3. Computed binding geometry of the new inhibitor 6B9 in the active site of CYP51.
3. Latge, J. P. Clin. Microbiol. Rev. 1999, 12, 310.
would be oriented to interact with a hydrophobic pocket formed
by Leu121, Thr122, Phe228, Thr311, Pro375, Leu376, His377,
Ser378, Met508, Val509, and Val510.
4. Steenbergen, J. N.; Casadevall, A. J. J. Clin. Microbiol. 2000, 38, 1974.
5. Georgopapadakou, N. H.; Walsh, T. J. Antimicrob. Agents Chemother. 1996, 40,
279.
6. Pfaller, M. A.; Messer, S. A.; Hollis, R. J.; Jones, R. N. Antimicrob. Agents
Chemother. 2001, 45, 2862.
7. Jeu, L.; Piacenti, F. J.; Lyakhovetskiy, A. G.; Fung, H. B. Clin. Pharmaocol. Ther.
2003, 25, 1321.
8. 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.
9. Ji, H.; Zhang, W.; Zhou, Y.; Zhang, M.; Zhu, J.; Song, Y.; Lü, J.; Zhu, J. J. Med. Chem.
2000, 43, 2493.
In addition, all of the side chains were of the pharmacophores,
and the spatial orientations of the pharmacophores were just ori-
ented in the hydrophobic pocket. The side chains of inhibitors were
not the determinants for activity, but were very important. They
played a role in adjusting the physico-chemical properties of the
whole molecule to avoid some dissatisfying side effects and im-
prove their pharmacokinetic and pharmacodynamic behaviors.
In conclusion, an efficient method which depended on compu-
tational docking experiments has been developed for the synthesis
of diversified novel triazole derivatives. Results of preliminary
antifungal tests against eight human pathogenic fungi in vitro
showed that these analogs exhibited excellent activities with
broad spectrum. The research has led to the discovery of a series
of compounds for further optimization.
10. Sheng, C.; Zhang, W.; Ji, H. Chin. Chem. Lett. 2004, 15, 404.
11. Sheng, C.; Zhang, W.; Ji, H.; Zhang, M.; Song, Y.; Xu, H.; Zhu, J.; Miao, Z.; Jiang,
Q.; Yao, J.; Zhou, Y.; Zhu, J.; Lü, J. J. Med. Chem. 2006, 49, 2512.
12. Richardson, K. US Patent 4404216, 1983.
13. Representative analytical data for compound 6A1. 1H NMR (400 MHz, CDCl3) d:
8.05 (1H, s, triazole-H), 7.73 (1H, s, triazole-H), 6.74–7.61 (8H, m, Ar-H), 5.37
(1H, br, OH), 4.36–4.47 (2H, dd, J = 14.4 Hz, triazole-CH2–), 3.30–3.51 (2H, dd,
J = 14.0 Hz, Ar-CH2–), 2.83–3.17 (2H, dd, J = 14.0 Hz, CCH2N), 2.23–2.27 (2H, t,
*
*
NCH 2CH2CH3), 1.26–1.33 (2H, m, NCH2CH 2CH3), 0.67–0.71 (3H, t, CH3); IR
(KBr): 3200, 3173, 3066, 2952, 2869, 1614, 1535, 1477, 1251, 1106, 750, 695;
LC–MS, m/z: 387.2 (M+H)+.
Compound 6B9. 1H NMR (400 MHz,CDCl3) d: 8.07 (1H, s, triazole-H), 7.77 (1H, s,
triazole-H), 6.73–7.53 (7H, m, Ar-H), 5.34 (1H, br, OH), 4.35–4.51 (2H, dd,
J = 14.0 Hz, triazole-CH2–), 3.21–3.37 (2H, dd, J = 14.0 Hz, Ar-CH2–), 2.83–3.06
(2H, dd, J = 14.0 Hz, CH2), 2.59–2.62 (1H, m, CH), 0.81–0.88 (6H, dd, J = 6.8 Hz,
2ꢀ CH3); IR (KBr): 3197, 3108, 3002, 2985, 2822, 1650, 1545, 1407, 1384, 1035,
957, 780; LC–MS, m/z: 456.1 (M+H)+.
Acknowledgments
This work was supported by the National Natural Science Foun-
dation of China (No. 20772153) and by Shanghai Leading Academic
Discipline Project No. B906. We thank Dr. Wannian Zhang for his
advice and discussions.
Compound 6C12.
1H NMR (400 MHz,CDCl3) d: 8.04 (1H, s, triazole-H), 7.76 (1H,
s, triazole-H), 6.75–8.14 (7H, m, Ar-H), 5.35 (1H, br, OH), 4.42–4.57 (2H, dd,
J = 14.4 Hz, triazole-CH2–), 3.49–3.61 (2H, dd, J = 14.4 Hz, Ar-CH2–), 2.85–3.19
*
(2H, dd, J = 14.0 Hz, CCH2N), 2.27–2.31 (2H, t, NCH 2CH2CH2CH3), 1.22–1.34
*
*
Supplementary data
(2H, m, NCH2CH 2CH2CH3), 1.06–1.14 (2H, m, NCH2CH2CH 2CH3), 0.75–0.79
(3H, t, CH3); IR (KBr): 3105, 2984, 2833, 1615, 1518, 1497, 1260, 1109, 960,
845; LC–MS, m/z: 446.2 (M+H)+.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.01.048.
14. 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.
15. Mellado, E.; Garcia-Effron, G.; Alcázar-Fuoli, L.; Melchers, W. J. G.; Verweij, P.
E.; Cuenca-Estrella, M.; Rodriguez-Tudela, J. L. Antimicrob. Agents Chemother.
2007, 51, 1897.
References and notes
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