Design, synthesis, and biological evaluation of novel 1, 2, 4-triazole derivatives as antifungal agent

Article abstract of DOI:10.1007/s12272-012-1105-8

A series of novel 1, 2, 4-triazole derivatives (9a-p) have been designed and synthesized as the potential antifungal agents. All compounds were characterized by ¹H-NMR, ¹³C-NMR, and LCMS. Their antifungal activities against seven human pathogenic fungi were evaluated in vitro by measuring the minimal inhibitory concentrations. Most of the tested compounds were found to be more potent against Candida albicans than the control drug fluconazole.

Full text of DOI:10.1007/s12272-012-1105-8

Arch Pharm Res Vol 35, No 11, 1895-1901, 2012
DOI 10.1007/s12272-012-1105-8
Design, Synthesis, and Biological Evaluation of Novel 1, 2, 4-Triazole
Derivatives as Antifungal Agent
Xiaoyun Chai1,*, Shichong Yu1,*, Yongwei Jiang1, Yan Zou1, Qiuye Wu1, Dazhi Zhang1, Yuanying Jiang2,
Yongbing Cao2, and Qingyan Sun1
1Department of Organic Chemistry, School of Pharmacy, Second Military Medical University, Guohe Road 325, Shang-
2
hai 200433, China and Drug Research Center, School of Pharmacy, Second Military Medical University, Guohe Road
325, Shanghai 200433, China
(Received April 6, 2012/Revised June 14, 2012/Accepted June 25, 2012)
A series of novel 1, 2, 4-triazole derivatives (9a-p) have been designed and synthesized as the
potential antifungal agents. All compounds were characterized by 1H-NMR, 13C-NMR, and LC-
MS. Their antifungal activities against seven human pathogenic fungi were evaluated in vitro
by measuring the minimal inhibitory concentrations. Most of the tested compounds were found
to be more potent against Candida albicans than the control drug fluconazole.
Key words: Triazole, Synthesis, Antifungal activity, CACYP51, Molecular docking
infections, have been shown to inhibit lanosterol 14
demethylase (CYP51), leading to block ergosterol syn-
α-
INTRODUCTION
There has been tremendous increase in the frequen- thesis, and thereby, fungal growth by binding in the
cy of fungal infections during the past four decades active-site cavity of the enzyme and ligating the iron
(Warnock, 2007). As we all know, Candida albicans, atom of the heme cofactor through a nitrogen atom of
Cryptococcus neoformans, and Aspergillus fumigatus the azole (Trösken et al., 2006). From previous re-
are the most dreadful human pathogens (Walsh et al., searches (Chai et al., 2009, 2011; Yu et al., 2010; Yan
2004; Vonberg and Gastmeier, 2006). And for the et al., 2011), we concluded that the triazole ring, the
treatment of these infections, azoles (such as flucona- diurophenyl group and the hydroxyl group were very
zole and itraconazole) are available in clinical use due indispensable for the antifungal activity. However,
to their broad antifungal spectrum, high potency and the side chains were also very important to the effect
low toxicity (Maertens, 2004). However, azoles’ clinical of antifungal agent.
uses have been limited by the emergence of drug re-
On the basis of these observations, we thought of an
sistance recently (Mayr and Lass-Flörl, 2011). Hence, alteration of the side chains with a triazolone side
it is now widely recognized that there is still a need to chain. In this case, the N atom of the target triazole
screen for safe and efficient chemotherapeutic agents antifungal compounds may be coordinated to the iron
with potent antifungal activities.
atom of the heme, the diuorophenyl group may be lo-
Azoles, which is used as the first choice of fungal cated into the hydrophobic pocket, and the triazolone
side chain may be interacted with the residues of the
*These authors contributed equally to this work.
Correspondence to: Qingyan Sun, Department of Organic Chem-
istry, School of Pharmacy, Second Military Medical University,
Guohe Road 325, Shanghai 200433, China
Tel, Fax: 86-21-8187-1228
narrow hydrophobic cleft, according to our hypothesis.
We hope the introduction of the side chains can help
us find potent systemic antifungal agents with a broad
antifungal spectrum and less potential to develop
resistance.
E-mail: sqy_2000@163.com
Yongbing Cao, Drug Research Center, School of Pharmacy, Sec-
ond Military Medical University, Guohe Road 325, Shanghai
200433, China
MATERIALS AND METHODS
Tel, Fax: 86-21-8187-1275
E-mail: ybcao@vip.sina.com
1H- and 13C-NMR spectra were recorded in CDCl3,
1895

1896
X. Chai et al.
room temperature for 6 h led to the synthesis of com-
pounds 9a-p (Scheme 2). All the target compounds
were obtained as racemates.
Synthesis of the intermediate 4-(3-nitrophenyl)-
1H-1, 2, 4-triazol-5(4H)-one (2)
Methyl carbazate (3.90 g, 0.043 mol) and Trimeth-
oxymethane (4.60 g, 0.043 mol) were added to a solu-
tion of 3-Nitroaniline (5.70 g, 0.041 mol) in methanol
Scheme 1. Synthesis of the Intermediate 2. Conditions: (a)
Methyl carbazate, Trimethoxymethane, CH3ONa, CH3OH,
-toluenesulfonic acid, reflux, 5 h.
p
unless otherwise indicated with a Bruker AC-300P (150 mL). After the reaction mixture was refluxed for 2
spectrometer, using TMS as internal standard. The h, 25% sodium methoxide (3.50 g, 0.064 mol) was add-
HPLC-MS were recorded on Agilent 1100 series LC- ed dropwise to the mixture which continued to reflux
MS. The solvents and reagents were purchased from for additional 3 h. The resulting reaction mixture was
commercial vendors and were used as received or concentrated under reduced pressure, then added 200
dried, prior to use as needed.
mL water to the syrup and adjusted the pH value to 1
Synthetic routes of the intermediate
2
and the with concentrated hydrochloric acid. Filtration of the
oxirane intermediate
Scheme 2. The intermediate
3-Nitroaniline to react with Trimethoxymethane, Meth-
yl carbazate and -toluenesulfonic acid under basic
medium (Krishnamurthy and James, 1998). Accord-
ing to a known procedure, the oxirane intermediate
was obtained by Corey-Chaykovsky epoxidation in the
presence of trimethylsulfoxonium iodide and NaOH corresponding intermediate
6
are outlined in Scheme 1 and latter mixture gave the corresponding intermediate
2
2
was generated by using (5.51 g, 65%).
p
Synthesis of 1-(2-(2, 4-difluorophenyl)-2-hydroxy-
3-(1H-1, 2, 4-triazol-1-yl)propyl) -4-(3-nitrophe-
nyl)-1H-1, 2, 4-triazol-5(4H)-one (7)
6
The oxirane intermediate
6
2
(5.00 g, 0.015 mol) and
(2.10 g, 0.010 mol) were
(Richardson, 1983).
The synthetic route of compounds 9a
added to a suspension of potassium carbonate in DMF.
was outlined Then the reaction mixture was refluxed for 6 h. The
-p
in Scheme 2. In the presence of potassium carbonate reaction was monitored by TLC. 5-fold amount water
in DMF, treatment of the oxirane intermediate with was added to the mixture, which was extracted with
the corresponding intermediate
at 80oC for 6 h gave ethyl acetate. The combined organic layer was washed
compound . Reduction of compound with H2, origi- with saturated brine (80 mL), dried over anhydrous
nated compound , which by reacting with the corres- Na2SO4 and concentrated under reduced pressure. The
ponding carboxylic acid in the presence of -(3-di- residue was crystallized from ethanol (5.65 g, 85%).
6
2
7
7
8
N
methylaminopropyl)-N’-ethylcarbodiimide hydrochlo-
ride (EDCI) and 4-dimethyl-aminopyridine (DMAP) at
Scheme 2. Synthesis of the target compounds 9a-p
. Conditions: (a) ClCH2COCl, AlCl3, 50oC, 5 h, 80%; (b) 1H-1,2,4-triazole,
NaHCO3, toluene, reflux, 5 h, 42%; (c) (CH3)3SOI, NaOH, centylmethylammonium bromide, toluene, 60oC, 3 h, 53%; (d)
CH3SO3H, 0oC, 1 h, 89%; (e) Intermediate
DMAP, CH2Cl2, rt, 6 h, 70-80%.
2
, DMF, K2CO3, 80oC, 6 h, 85%; (f) Pd/C, H2, rt, 4 h, 95%; (g) RCOOH, EDCI/

Synthesis of Antifungal Triazole Derivatives
1897
(12H, m, Ar-H), 5.71 (1H, s, OH), 4.76 (2H, s, triazole-
Synthesis of 4-(3-aminophenyl)-1-(2-(2, 4-diflu-
orophenyl)-2-hydroxy-3-(1H-1, 2, 4-triazol-1-yl)
propyl)-1H-1, 2, 4-triazol-5(4H)-one (8)
CH2-), 4.21-4.52 (2H, dd,
J
= 14.7 Hz, -CH2-triazolone);
164.5, 160.7, 159.2, 158.5,
13C-NMR (75 MHz, CDCl3):
δ
Compound
7 (5.65 g, 0.013 mol) was reduced by 151.4, 150.2, 146.2, 143.4, 136.5, 133.6, 132.4, 131.6,
catalytic hydrogenation with H2 and 10% Pd/C (0.20 g) 131.0, 130.5, 129.2, 126.8, 125.0, 123.3, 118.5, 115.2,
in 20 mL methanol to get compound
8
(5.10 g, 95%).
113.6, 110.1, 104.5, 63.4, 62.2, 61.6; LC-MS (ESI), m/z
Calcd. for C26H20ClF2N7O3, 551.1, found [M+1]+ 552.2.
General procedure for the target compounds
(9a-p)
N-(3-{1-[2-(2, 4-difluorophenyl)-2-hydroxy-3-(1H-
Substituted Carboxylic Acids (0.0015 mol), EDCI 1, 2, 4-triazol-1-yl)propyl]-5-oxo-1H-1, 2, 4-triazol-
(0.20 g, 0.0013 mol) and DMAP (0.10 g, 0.0008 mol) 4(5H)-yl}phenyl)-4-chlorobenzamide (9d)
was added to a solution of compound
8 δ 8.46 (s, 1H, NH), 8.17
(0.413 g, 0.001 1H-NMR (300 MHz, CDCl3):
mol) in CH2Cl2 (30 mL). Then the reaction mixture was (1H, s, triazole-H), 7.95 (1H, s, triazole-H), 6.78-7.81
stirred at room temperature for 6 h. The mixture was (12H, m, Ar-H), 5.71 (1H, s, OH), 4.71 (2H, s, triazole-
concentrated under reduced pressure, and extracted CH2-), 4.17-4.59 (2H, dd,
J
= 14.7 Hz, -CH2-triazolone);
164.2, 161.0, 159.4, 158.7,
with ethyl acetate. The combined organic layer was 13C-NMR (75 MHz, CDCl3):
δ
washed with 1 N HCl (80 mL), saturated NaHCO3 (80 151.7, 150.6, 146.4, 143.5, 136.3, 133.7, 132.5, 131.3,
mL) and saturated brine (80 mL), then dried over 131.0, 130.2, 129.4, 126.6, 125.3, 123.4, 118.8, 115.9,
anhydrous Na2SO4 and concentrated under reduced 113.5, 110.6, 104.7, 62.2, 61.9, 61.0; LC-MS (ESI), m/z
pressure to give a crude product which was crystallized Calcd. for C26H20ClF2N7O3, 551.1, found [M+1]+ 552.2.
from ethanol.
N-(3-{1-[2-(2, 4-difluorophenyl)-2-hydroxy-3-(1H-
N-(3-{1-[2-(2, 4-difluorophenyl)-2-hydroxy-3-(1H-1, 1, 2, 4-triazol-1-yl)propyl]-5-oxo-1H-1, 2, 4-triazol-
2, 4-triazol-1-yl)propyl]-5-oxo-1H-1, 2, 4-triazol-4 4(5H)-yl}phenyl)-4-methylbenzamide (9e)
(5H)-yl}phenyl)-2-fluorobenzamide (9a)
1H-NMR (300 MHz, CDCl3):
8.45 (s, 1H, NH), 8.19 (1H, s, triazole-H), 7.94 (1H, s, triazole-H), 6.78-7.84
(1H, s, triazole-H), 7.94 (1H, s, triazole-H), 6.78-7.81 (12H, m, Ar-H), 5.74 (1H, s, OH), 4.73 (2H, s, triazole-
(12H, m, Ar-H), 5.75 (1H, s, OH), 4.71 (2H, s, triazole- CH2-), 4.18-4.56 (2H, dd, = 14.4 Hz, -CH2-triazolone),
CH2-), 4.18-4.57 (2H, dd, 165.3,
= 14.7 Hz, -CH2-triazolone); 3.85 (3H, s, CH3); 13C-NMR (75 MHz, CDCl3):
13C-NMR (75 MHz, CDCl3):
164.9, 161.0, 159.8, 158.5, 162.0, 159.3, 158.5, 152.1, 150.6, 146.4, 143.5, 136.3,
1H-NMR (300 MHz, CDCl3):
δ 8.43 (s, 1H, NH), 8.27
δ
J
J
δ
δ
151.7, 150.2, 146.9, 143.2, 136.1, 133.7, 132.9, 131.4, 133.6, 132.8, 131.5, 131.2, 130.1, 129.6, 126.8, 125.5,
131.3, 130.0, 129.5, 126.8, 125.3, 123.5, 118.4, 115.6, 123.4, 118.8, 115.8, 113.4, 110.6, 104.5, 62.2, 61.6, 61.0,
113.7, 110.3, 104.7, 62.5, 62.0, 61.1; LC-MS (ESI), m/z 18.5; LC-MS (ESI), m/z Calcd. for C27H23F2N7O3,
Calcd. for C26H20F3N7O3, 535.2, found [M+1]+ 536.2.
531.2, found [M+1]+ 532.3.
N-(3-{1-[2-(2, 4-difluorophenyl)-2-hydroxy-3-(1H- N-(3-{1-[2-(2, 4-difluorophenyl)-2-hydroxy-3-(1H-
1, 2, 4-triazol-1-yl)propyl]-5-oxo-1H-1, 2, 4-triazol- 1, 2, 4-triazol-1-yl)propyl]-5-oxo-1H-1, 2, 4-triazol-
4(5H)-yl}phenyl)-3-fluorobenzamide (9b)
4(5H)-yl}phenyl)-3, 4-dimethylbenzamide (9f)
1H-NMR (300 MHz, CDCl3): 8.46 (s, 1H, NH), 8.18 1H-NMR (300 MHz, CDCl3):
δ
δ
8.41 (s, 1H, NH), 8.35
(1H, s, triazole-H), 7.95 (1H, s, triazole-H), 6.79-7.82 (1H, s, triazole-H), 7.96 (1H, s, triazole-H), 6.75-7.84
(12H, m, Ar-H), 5.71 (1H, s, OH), 4.72 (2H, s, triazole- (11H, m, Ar-H), 5.68 (1H, s, OH), 4.75 (2H, s, triazole-
CH2-), 4.18-4.58 (2H, dd,
J
= 14.7 Hz, -CH2-triazolone); CH2-), 4.20-4.54 (2H, dd,
δ
J = 14.7 Hz, -CH2-triazolone),
13C-NMR (75 MHz, CDCl3):
165.2, 161.4, 159.6, 158.2, 2.30 (6H, m, 2×CH3); 13C-NMR (75 MHz, CDCl3):
δ
151.3, 150.5, 146.6, 143.2, 136.1, 133.5, 132.2, 131.4, 164.5, 161.8, 159.5, 158.3, 151.8, 150.9, 146.6, 143.3,
131.0, 130.1, 129.3, 126.6, 125.5, 123.8, 118.2, 115.2, 136.5, 133.7, 132.9, 131.5, 131.1, 130.3, 129.6, 126.7,
113.5, 110.9, 104.0, 62.4, 62.1, 61.0; LC-MS (ESI), m/z 125.5, 123.8, 118.6, 115.5, 113.4, 110.8, 104.7, 62.4,
Calcd. for C26H20F3N7O3, 535.2, found [M+1]+ 536.2.
62.0, 61.2, 18.7, 18.5; LC-MS (ESI), m/z Calcd. for
C28H25F2N7O3, 545.2, found [M+1]+ 546.2.
N-(3-{1-[2-(2, 4-difluorophenyl)-2-hydroxy-3-(1H-
1, 2, 4-triazol-1-yl)propyl]-5-oxo-1H-1, 2, 4-triazol- N-(3-{1-[2-(2, 4-difluorophenyl)-2-hydroxy-3-(1H-
4(5H)-yl}phenyl)-3-chlorobenzamide (9c) 1, 2, 4-triazol-1-yl)propyl]-5-oxo-1H-1, 2, 4-triazol-
1H-NMR (300 MHz, CDCl3):
8.72 (s, 1H, NH), 8.41 4(5H)-yl}phenyl)-4-methoxybenzamide (9g)
(1H, s, triazole-H), 7.94 (1H, s, triazole-H), 6.76-7.83 1H-NMR (300 MHz, CDCl3):
8.27 (s, 1H, NH), 8.15
δ
δ

1898
X. Chai et al.
(1H, s, triazole-H), 7.97 (1H, s, triazole-H), 6.79-7.86 found [M+1]+ 574.3.
(12H, m, Ar-H), 5.82 (1H, s, OH), 4.74 (2H, s, triazole-
CH2-), 4.19-4.60 (2H, dd,
3.87 (3H, s, OCH3); 13C-NMR (75 MHz, CDCl3):
162.2, 159.4, 158.5, 151.9, 150.6, 146.4, 143.4, 136.6, 4(5H)-yl}phenyl)-2-phenylacetamide (9k)
133.2, 132.9, 131.8, 131.2, 130.4, 129.0, 126.5, 125.3, 1H-NMR (300 MHz, CDCl3):
8.35 (s, 1H, NH), 8.20
J
= 14.7 Hz, -CH2-triazolone), N-(3-{1-[2-(2, 4-difluorophenyl)-2-hydroxy-3-(1H-
δ
165.0, 1, 2, 4-triazol-1-yl)propyl]-5-oxo-1H-1, 2, 4-triazol-
δ
123.8, 118.4, 115.5, 113.5, 110.7, 104.4, 62.7, 62.1, 61.4, (1H, s, triazole-H), 7.96 (1H, s, triazole-H), 5.81-7.81
55.6; LC-MS (ESI), m/z Calcd. for C27H23F2N7O4, 547.2, (13H, m, Ar-H), 5.83 (1H, s, OH), 4.72 (2H, s, triazole-
found [M+1]+ 548.2.
CH2-), 4.19-4.57 (2H, dd,
J = 15 Hz, -CH2-triazolone),
3.71 (2H, s, Ar-CH2); 13C-NMR (75 MHz, CDCl3):
δ
N-(3-{1-[2-(2, 4-difluorophenyl)-2-hydroxy-3-(1H- 164.7, 160.5, 159.2, 158.3, 152.6, 150.5, 146.8, 143.3,
1, 2, 4-triazol-1-yl)propyl]-5-oxo-1H-1, 2, 4-triazol- 136.7, 134.6, 133.3, 131.5, 130.9, 130.4, 129.5, 126.4,
4(5H)-yl}phenyl)-4-ethylbenzamide (9h)
125.3, 123.5, 118.2, 115.3, 113.0, 110.8, 104.4, 62.0,
1H-NMR (300 MHz, CDCl3):
δ 8.45 (s, 1H, NH), 8.24 61.7, 61.0, 43.6; LC-MS (ESI), m/z Calcd. for
(1H, s, triazole-H), 7.87 (1H, s, triazole-H), 6.80-7.82 C27H23F2N7O3, 531.2, found [M+1]+ 532.2.
(12H, m, Ar-H), 5.76 (1H, s, OH), 4.71 (2H, s, triazole-
CH2-), 4.16-4.55 (2H, dd,
2.60 (2H, q, CH2), 1.25 (3H, t, CH3); 13C-NMR (75 MHz, 1, 2, 4-triazol-1-yl)propyl]-5-oxo-1H-1, 2, 4-triazol-
CDCl3): 165.0, 161.9, 159.5, 158.2, 152.7, 150.7, 146.5, 4(5H)-yl}phenyl)-pivalamide (9l)
143.6, 136.1, 134.2, 133.5, 131.8, 131.3, 130.6, 129.2, 1H-NMR (300 MHz, CDCl3):
8.41 (s, 1H, NH), 8.17
J = 14.7 Hz, -CH2-triazolone), N-(3-{1-[2-(2, 4-difluorophenyl)-2-hydroxy-3-(1H-
δ
δ
126.8, 125.5, 123.7, 118.6, 115.0, 113.5, 110.2, 104.5, (1H, s, triazole-H), 7.92 (1H, s, triazole-H), 6.78-7.88
62.7, 62.1, 61.1, 27.2, 15.2; LC-MS (ESI), m/z Calcd. for (8H, m, Ar-H), 5.78 (1H, s, OH), 4.77 (2H, s, triazole-
C28H25F2N7O3, 545.2, found [M+1]+ 546.2.
CH2-), 4.20-4.59 (2H, dd,
J = 14.7 Hz, -CH2-triazolone),
1.28 (9H, s, 3×CH3); 13C-NMR (75 MHz, CDCl3):
δ
N-(3-{1-[2-(2, 4-difluorophenyl)-2-hydroxy-3-(1H- 176.7, 163.2, 158.3, 152.0, 150.5, 146.5, 143.7, 138.6,
1, 2, 4-triazol-1-yl)propyl]-5-oxo-1H-1, 2, 4-triazol- 133.3, 131.5, 130.4, 129.5, 123.5, 118.2, 115.3, 110.5,
4(5H)-yl}phenyl)-4-isopropylbenzamide (9i)
104.2, 62.7, 62.4, 61.3, 35.6, 28.2; LC-MS (ESI), m/z
1H-NMR (300 MHz, CDCl3): 8.47 (s, 1H, NH), 8.21 Calcd. for C24H25F2N7O3, 497.2, found [M+1]+ 498.3.
δ
(1H, s, triazole-H), 7.96 (1H, s, triazole-H), 6.78-7.80
(12H, m, Ar-H), 5.72 (1H, s, OH), 4.71 (2H, s, triazole- N-(3-{1-[2-(2, 4-difluorophenyl)-2-hydroxy-3-(1H-
CH2-), 4.17-4.59 (2H, dd,
2.87 (1H, m, CH), 1.20 (6H, s, 2×CH3); 13C-NMR (75 4(5H)-yl}phenyl)-hexanamide (9m)
MHz, CDCl3): 8.34 (s, 1H, NH), 7.97 (1H,
164.6, 161.4, 159.3, 158.1, 152.4, 150.6, 1H-NMR (300 MHz, CDCl3):
J = 14.7 Hz, -CH2-triazolone), 1, 2, 4-triazol-1-yl)propyl]-5-oxo-1H-1, 2, 4-triazol-
δ
δ
146.2, 143.4, 136.7, 134.5, 133.3, 131.6, 131.1, 130.5, s, triazole-H), 7.85 (1H, s, triazole-H), 6.77-7.61 (8H, m,
129.6, 126.4, 125.2, 123.5, 118.3, 115.8, 113.1, 110.2, Ar-H), 5.92 (1H, s, OH), 4.75 (2H, s, triazole-CH2-),
104.6, 62.3, 61.8, 61.2, 33.5, 23.2, 23.0; LC-MS (ESI), m/ 4.19-4.57 (2H, dd,
J = 15 Hz, -CH2-triazolone), 2.32-
z Calcd. for C29H27F2N7O3, 559.2, found [M+1]+ 560.2.
2.37 (2H, t, CH2CH2CH2CH3), 1.65 (2H, m, CH2CH2CH2
CH3), 1.24 (5H, m, CH2CH2CH2CH3); 13C-NMR (75
MHz, CDCl3):
δ 177.2, 163.7, 158.5, 152.2, 150.8, 146.1,
N-(3-{1-[2-(2, 4-difluorophenyl)-2-hydroxy-3-(1H- 143.3, 138.9, 133.7, 131.4, 130.5, 129.8, 123.2, 118.8,
1, 2, 4-triazol-1-yl)propyl]-5-oxo-1H-1, 2, 4-triazol- 115.6, 110.2, 104.0, 62.4, 62.0, 61.0, 35.3, 32.7, 28.0,
4(5H)-yl}phenyl)-4-n-butylbenzamide (9j)
1H-NMR (300 MHz, CDCl3): 8.36 (s, 1H, NH), 8.22 497.2, found [M+1]+ 498.3.
(1H, s, triazole-H), 7.96 (1H, s, triazole-H), 5.81-7.81
(12H, m, Ar-H), 5.81 (1H, s, OH), 4.72 (2H, s, triazole- N-(3-{1-[2-(2, 4-difluorophenyl)-2-hydroxy-3-(1H-
CH2-), 4.18-4.58 (2H, dd, = 15 Hz, -CH2-triazolone), 1, 2, 4-triazol-1-yl)propyl]-5-oxo-1H-1, 2, 4-triazol-
2.67 (2H, t, Ar-CH2), 1.56 (2H, m, CH2CH2CH3), 1.25 4(5H)-yl}phenyl)-heptanamide (9n)
(2H, m, CH2CH2CH3), 0.89 (3H, t, CH2CH2CH3); 13C- 1H-NMR (300 MHz, CDCl3):
8.24 (s, 1H, NH), 8.20 (1H,
NMR (75 MHz, CDCl3): 165.1, 161.6, 159.8, 158.0, s, triazole-H), 7.84 (1H, s, triazole-H), 6.75-7.82 (8H, m,
152.7, 150.5, 146.3, 143.5, 136.4, 134.5, 133.1, 131.8, Ar-H), 5.58 (1H, s, OH), 4.72 (2H, s, triazole-CH2-), 4.17-
131.5, 130.2, 129.6, 126.5, 125.2, 123.6, 118.5, 115.2, 4.57 (2H, dd, = 14.7 Hz, -CH2-triazolone), 2.31 (2H, t,
22.7, 14.2; LC-MS (ESI), m/z Calcd. for C24H25F2N7O3,
δ
J
δ
δ
J
113.4, 110.5, 104.5, 62.4, 62.0, 61.4, 33.4, 31.7, 23.2, CH2 CH2CH2CH3), 1.67 (2H, m, CH2CH2CH2 CH3), 1.24
14.2; LC-MS (ESI), m/z Calcd. for C30H29F2N7O3, 573.2, (5H, m, CH2CH2CH2CH3); 13C-NMR (75 MHz, CDCl3):

Synthesis of Antifungal Triazole Derivatives
1899
δ
176.5, 164.0, 158.3, 152.7, 150.5, 146.4, 143.7, 138.5, and voriconazole were obtained from their respective
133.6, 131.8, 130.1, 129.5, 123.5, 118.8, 115.3, 110.8, manufacturers. The in vitro minimal inhibitory con-
104.1, 62.6, 62.1, 61.3, 36.5, 33.5, 29.3, 24.4, 22.5, 13.9; centrations (MICs) of the title compounds were deter-
LC-MS (ESI), m/z Calcd. for C25H27F2N7O3, 511.2, mined by the micro-broth dilution method in 96-well
found [M+1]+ 512.3.
microtestplates, according to the methods defined by
the National Committee for Clinical Laboratory Stand-
N-(3-{1-[2-(2, 4-difluorophenyl)-2-hydroxy-3-(1H- ards (NCCLS, 2002). The MIC80 was defined as the first
1, 2, 4-triazol-1-yl)propyl]-5-oxo-1H-1, 2, 4-triazol- well with an approximate 80% reduction in growth,
4(5H)-yl}phenyl)-octanamide (9o)
compared to the growth of the drug-free well. For assays,
1H-NMR (300 MHz, CDCl3):
δ
8.35 (s, 1H, NH), 8.28 (1H, the title compounds to be tested were dissolved in
s, triazole-H), 7.86 (1H, s, triazole-H), 6.79-7.82 (8H, m, dimethyl sulfoxide (DMSO), serially diluted in growth
Ar-H), 5.92 (1H, s, OH), 4.74 (2H, s, triazole-CH2-), medium, inoculated and incubated at 35oC. Growth
4.19-4.59 (2H, dd,
J = 15Hz, -CH2-triazolone), 2.34 (2H, MIC was determined at 24 h for C. albican and at 72
t, CH2CH2 CH2CH2CH2CH3), 1.67 (2H, m, CH2CH2CH2 h for C. neoformans. The results of assays are summa-
CH2 CH2CH3), 1.30 (6H, m, CH2CH2CH2CH2 CH2CH3), rized in Table I. These data are the mean of the three
0.88 (3H, t, CH2CH2CH2CH2CH2 CH3); 13C-NMR (75 replicate tests performed with each antifungal agent.
MHz, CDCl3):
δ 176.3, 164.2, 158.4, 152.4, 150.3, 146.8,
143.4, 138.7, 133.6, 131.6, 130.4, 129.3, 123.8, 118.5,
115.7, 110.6, 104.6, 62.7, 62.3, 61.0, 36.9, 33.3, 29.5,
25.6, 24.0, 21.4, 13.5; LC-MS (ESI), m/z Calcd. for
C26H29F2N7O3, 525.2, found [M+1]+ 526.3.
RESULTS
In our research, the intermediate
via a ‘one-pot’ high-temperature refluxing strategy,
using methanol as the medium. Apparently, the former
2 was synthesized
N-(3-{1-[2-(2, 4-difluorophenyl)-2-hydroxy-3-(1H- method (Hrebabecky and Beranek, 1985) needs three
1, 2, 4-triazol-1-yl)propyl]-5-oxo-1H-1, 2, 4-triazol- steps, while ours is a one step procedure. It is more
4(5H)-yl}phenyl)-nonanamide (9p)
succinct and efficient.
The in vitro antifungal activities of all the target
1H-NMR (300 MHz, CDCl3):
δ
8.34 (s, 1H, NH), 8.26
(1H, s, triazole-H), 7.87 (1H, s, triazole-H), 6.77-7.81 compounds were listed in Table I. The MIC80 values of
(8H, m, Ar-H), 5.92 (1H, s, OH), 4.73 (2H, s, triazole- all the target compounds were determined against
CH2-), 4.20-4.57 (2H, dd,
J = 15 Hz, -CH2-triazolone), seven important fungal pathogens and compared with
2.39 (2H, t, CH2CH2 CH2CH2CH2CH2CH3), 1.67 (2H, itraconazole, voriconazole and fluconazole. The assay
m, CH2CH2CH2CH2CH2CH2CH3), 1.29 (8H, m, CH2CH2 indicated that all the synthesized compounds showed
CH2CH2CH2CH2CH3), 0.86 (3H, t, CH2CH2 CH2CH2CH2 moderate to excellent activity against all the tested
CH2CH3); 13C-NMR (75 MHz, CDCl3):
δ 176.1, 164.2, fungal pathogens, except C. neoformans. The MIC80
158.5, 152.3, 150.6, 146.8, 143.4, 138.2, 133.4, 131.5, value of compound 9a was 32 times lower than that of
130.1, 129.2, 123.4, 118.6, 115.8, 110.5, 104.3, 62.6, 62.0, FCZ against C. albicans. Most of the target compounds
61.1, 36.2, 33.3, 29.7, 24.7, 24.0, 22.2, 13.4; LC-MS 9a-p showed higher activities against C. parasilosis
(ESI), m/z Calcd. for C27H31F2N7O3, 539.3, found [M+1]+ than the standard reference drug FCZ. Additionally,
540.3.
compound 9b and 9e exhibited excellent activities
with an MIC80 value of 0.03125 g/mL against C.
parasilosis versus the MIC80 value of ICZ, which is
g/mL. All the title compounds, except for
µ
Antifungal activity
The in vitro antifungal activities of all the target only 0.125
µ
compounds were evaluated against seven human path- compound 9j exhibited equivalent antifungal activi-
ogenic fungi, Candida albican SC5314 and Y0109, ties of FCZ against C. kefyr with the MIC80 value of 1
Cryptococcus neoformans, Candida parapsilosis, Can-
µg/mL. Besides, compounds 9m, 9n, 9o and 9p, which
dida tropicalis, Trichophyton rubrum, and Candida contained longer alkyl side chains showed poor activity
kefyr, which are frequently encountered in clinic. The against all the tested fungi with the MIC80 value in
results were compared with positive controls itracona- the range of 1-64 µg/mL.
zole, voriconazole and fluconazole. C. albican SC5314
and C. neoformans, purchased from ATCC, were pro-
vided by Shanghai Changzheng Hospital; C. parapsi-
losis, C. albican Y0109, C. tropicalis, T. rubrum and
DISCUSSION
In our paper, a series of novel triazole antifungal
C. kefyr, which are clinic isolates, were provided by agents containing a triazolone side chain were suc-
Shanghai Changhai Hospital. Fluconazole, itraconazole cessfully designed and synthesized. We used a ‘one-

1900
X. Chai et al.
Table I. Antifungal activities of the target compounds in vitro (MIC80,
µg/mL)
C. alb
Compd .
R
C. neo
C. par
C. kef
C. tro
T. rub
Y0109
SC5314
9a
9b
9c
9d
9e
2-FC6H4
3-FC6H4
3-ClC6H4
4-ClC6H4
4-CH3C6H4
3,4-CH3C6H4
4-OCH3C6H4
4-CH2CH3C6H4
4-CH(CH3)2C6H4
4-(CH2)3CH3C6H4
CH2C6H5
0.125
1
1
0.5
1
1
1
4
1
1
32
0.5
32
>64
>64
>64
0.25
0.0625
4
0.25
1
1
1
1
1
1
4
4
1
8
1
>64
>64
>64
>64
0.125
0.0625
4
>64
>64
>64
>64
>64
>64
>64
>64
>64
16
>64
>64
16
16
>64
16
0.0625
0.03125
0.125
0.125
0.03125
0.125
0.125
0.5
4
0.25
16
0.125
>64
32
>64
>64
0.125
0.0039
0.25
1
1
1
1
1
1
1
1
1
4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
8
1
1
8
1
1
1
1
1
>64
1
4
16
8
1
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
ICZ
VCZ
FCZ
>64
>64
0.5
>64
1
>64
>64
>64
>64
0. 25
0.0156
1
C(CH3)3
(CH2)4CH3
(CH2)5CH3
(CH2)6CH3
(CH2)7CH3
0. 25
0.0078
4
0. 5
0.125
1
0. 25
0.0156
1
C. alb, Candida albicans C. tro
;
, Candida tropicalis; C. neo, Cryptococcus neoformans; C. par, Candida parapsilosis; C.
kef Candida kefyr T. rub, Trichophyton rubrum; ICZ, itraconazole; VCZ, voriconazole; FCZ, fluconazole.
,
;
pot’ method to synthesize the side chains and also 81273556), by the Eleventh Five Year Military Medicine
acquired a higher yield. and Public Health Research Projects (Nos. 06MB206)
The antifungal activities of the target compounds were and by Shanghai Leading Academic Discipline Project
screened for seven human pathogenic fungi. In vitro Number: B906.
antifungal activity assay indicated that most of these
compounds showed moderate to excellent antifungal
activities than the reference drug FCZ. Among them,
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