Synthesis and evaluation of novel benzene-ethanol bearing 1,2,4-triazole derivatives as potential antimicrobial agents

Article abstract of DOI:10.1007/s00044-016-1724-6

The azole pharmacophore is still regarded as a viable lead structure for the synthesis of more efficacious and broad-spectrum antimicrobial agents. In this study, a novel series of triazole derivates that are structurally related to the famous antimicrobial azole pharmacophore were synthesized and the structures of them were characterized by spectral (IR, ¹H NMR, ¹³C NMR, and MS spectra) analysis. Antimicrobial activity was measured against both bacteria and fungus. In vitro antimicrobial evaluation showed that five compounds had growth inhibitory effects on the tested Gram-positive bacteria and fungus with special efficacy. Potential antibacterial and antifungal activities are incorporated in these triazole compounds. Results of antimicrobial activities also revealed that compounds (5a–i) were the potent antibacterial and antifungal agents as compared to standard drugs (ciprofloxacin and itraconazole), and thus could be promising new lead molecules.

Full text of DOI:10.1007/s00044-016-1724-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1724-6
ORIGINAL RESEARCH
Synthesis and evaluation of novel benzene-ethanol bearing
1,2,4-triazole derivatives as potential antimicrobial agents
Bochao Li1 Dawei Zhang2 Yumin Zhang2 Dan Jiang1 Shuang Li1 Wei Lei1
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Huiying Wang1 Feng Lin1
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Received: 29 April 2016 / Accepted: 7 September 2016
© Springer Science+Business Media New York 2016
Abstract The azole pharmacophore is still regarded as a
viable lead structure for the synthesis of more efficacious
and broad-spectrum antimicrobial agents. In this study, a
novel series of triazole derivates that are structurally related
to the famous antimicrobial azole pharmacophore were
synthesized and the structures of them were characterized
by spectral (IR, 1H NMR, 13C NMR, and MS spectra)
analysis. Antimicrobial activity was measured against both
bacteria and fungus. In vitro antimicrobial evaluation
showed that five compounds had growth inhibitory effects
on the tested Gram-positive bacteria and fungus with special
efficacy. Potential antibacterial and antifungal activities are
incorporated in these triazole compounds. Results of anti-
microbial activities also revealed that compounds (5a–i)
were the potent antibacterial and antifungal agents as
compared to standard drugs (ciprofloxacin and itracona-
zole), and thus could be promising new lead molecules.
Introduction
Infectious diseases have increased in frequency and severity
for the past few years resulting from not only clinical use
and misuse antimicrobial agents, corticosteroids or immune
inhibitors, but also organ transplantations and intervention
techniques are also widely used in clinic (Genin et al., 2000;
Sheng et al., 2006; Sheehan et al., 1999). In particular,
much more attention has been focused on the problem of
multi-drug resistant bacteria and fungi (Akbas and Berber,
2005). For these reasons, the development of new potent
antimicrobial drugs becomes even more urgent. Novel azole
compounds are the candidates for potent and effective
antimicrobial activities.
In general, azole compounds have better biological
activities, high efficiency, low toxicity, broad antimicrobial
spectrum, and stereoselectivity characteristics (Karyotakis
and Anaissie, 1994). Thus, azole compounds are better
clinical drugs, such as antibacterial (Plech et al., 2015;
Mathew et al., 2006), antifungal (Grant and Clissold, 1990;
Wong-Beringer and Kriengkauykiat, 2003; Sun et al.,
2007), antituberculosis (Kucukguzel et al., 2001), antic-
ancer (Brueggemeir et al., 2005; Bordie, 2002), herbicide
(Zhang and Shi, 2014) and anti-inflammatory (Amir and
Shikha, 2004; Navidpour et al., 2006; Metwally et al., 2007;
Kumar et al., 2008; Abdel-Megeed et al., 2009), and so on.
Well-known azole derivates are the most widely used
antifungal drugs for systemic fungal infection (Genin et al.,
2000; Sheng et al., 2006; Sheehan et al., 1999). At present,
antifungal azole derivates such as econazole, miconazole,
isoconazole, and ketoconazole were still widely used in
clinic (Fig. 1). Comparing with imidazole derivatives, tria-
zole compounds such as fluconazole and itraconazole
(Fig. 1) are less toxic.
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Keywords Benzene-ethanol 1,2,4-Triazole Antibacterial
Antifungal
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1724-6) contains supplementary material,
which is available to authorized users.
Bochao Li and Dawei Zhang contributed equally to this work
* Feng Lin
linfeng@jlu.edu.cn
1
School of Life Sciences, Jilin University, 2699 Qianjin Street,
Changchun 130012, People’s Republic of China
2
School of Chemistry, Jilin University, 2699 Qianjin Street,
Changchun 130012, People’s Republic of China

Med Chem Res
Cl
Cl
N
N
Cl
Cl
Cl
N
Cl
Cl
F
N
OH
N
Cl
O
O
N
O
N
N
N
N
N
N
F
Cl
Cl
Cl
Econazole
N
Miconazole
N
Isoconazole
Fluconazole
N
N
N
O
N
N
N
O
O
O
O
H
Cl
Cl
N
O
O
N
N
N
N
Cl
O
Cl
Ketoconazole
Fig. 1 Structure of some antifungal azoles
Itraconazole
The antifungal mechanism of azoles is related to the
ergosterols, which plays an important role in the main-
tenance of membrane organization and functions. 14α-
Lanosterol demethylase (CYP51, P-45014DM) is a key
enzyme in the pathway of ergosterol biosynthesis. It is a
member of the cytochrome P450 superfamily containing an
iron protoporphyrin unit located in its active site catalyzing
the oxidative removal of the 14α-methyl group of lanosterol
to give Δ14,15-desaturated intermediates. Azoles, including
imidazole and triazole derivatives, inhibit the synthesis of
sterols in fungi by inhibiting cytochrome P450-dependent
14α-lanosterol demethylase (P-45014DM), which removes
the methyl group on C-14 of lanosterol, a key intermediate
step in the formation of ergosterol in the fungal cell mem-
brane (Grillot and Lebeau, 2005).
Usually, the nitrogen (N-3 in the imidazole or N-4 in the
triazole) is to bind the heme iron of enzyme-bound cyto-
chrome P450 to prevent oxidation of steroidal substrates by
the enzyme. The elementary structure for the antimicrobial
azole class has a weakly basic imidazole or triazole ring
bonded by a nitrogen–carbon linkage to the rest of the
structure. Besides, they often have aromatic rings to mimic
the corresponding non-polar steroidal portion of the sub-
strate for the enzyme, which contribute to high degree
lipophilicity (Watkins and Renau, 2003). However, the
increasing incidence of Candida species infections coupled
with the emergence of azole-resistant fungal strains have
stimulated the search for novel antifungal drugs. The
synthesis of a new class of antibacterial and antifungal
agents against especially Gram-positive drug-resistant bac-
teria and some fungi is urgent need nowadays, since these
types of microorganisms are responsible for some infections
in the acute and long-term care units in hospitals.
CYP51 plays an important role in ergosterol biosynth-
esis, which is related to antifungal activity. The antifungal
mechanism indicates that azole derivates is an efficient
group for antifungal activities. The bioisosteric replacement
of imidazole and triazole is an efficient concept for the
discovery and development of novel drug molecules pos-
sessing enhanced antifungal or antibacterial activity (Wahbi
et al., 1995; Guven et al., 2007; Marrapu et al., 2011).
Besides, the available azole antifungal drugs usually contain
halo-substituted aryl groups, such as 2-chloro, 2-fluoro, 2,4-
dichloro-substituted benzyl rings. Herein, we combined the
main structure of CYP51 inhibition and halo-substituted
rings into 1,2,4-triazole to get novel target molecules. Based
on these, we designed and synthesized a series of com-
pounds with the 1,2,4-triazole ring as the efficacy groups.
Then we elevated the antimicrobial activity of the com-
pounds against both bacteria and fungus. Results suggested
that five compounds possessed antimicrobial activity
against tested fungus and Gram-positive bacteria, indicating
that the compounds could be potent antimicrobial agents for
therapy.
Material and methods
Chemistry
Various m-dichlorobenzene, 1,2,4-triazol and substituted
benzyl chlorides were of analytical-reagent grade from

Med Chem Res
Aladdin reagent Co. and used without further purification.
The other solvents and reagents used were supplied by
Tianjin Tiantai Chemical Co. Ltd (China) and Beijing
Chemical Plant (China). All melting points were determined
on a XT−4 melting point apparatus and were uncorrected.
1H and 13C NMR spectra were measured using a Bruker
AVANCE-500 or 600 NMR spectrometer and with TMS as
an internal standard. MS was collected with an Agilent
HP1100/6890 LC/MS spectrometer and an Agilent 1290-
micrOTOF Q II spectrometer, respectively. FT-IR spectra
were obtained as KBr pellets using IRAffinity-1 instrument
in the range of 500–3500 cm–1.
Generation of α-(1H-1,2,4-triazol-1-yl)-2,4-
dichlorobenzene ethanol (4)
According to the literature (Guven et al., 2007), α-(1-tria-
zole)-2,4-dichloroacetophenone 5.12 g (0.02 mol) and 10 mL
methanol was added into a 100 mL round flask under ice
bath. Sodium borohydride 0.91 g (24 mmol) was added
dropwise into the flask under stirring and then reacted for 6 h.
After the reaction was over, the mixture was cooled by ice
water for 20 min. The filter cake was washed with distilled
water. The pale yellow powder solid (4) in yield of 82.7 %
(4.3 g) was got after the mixture was recrystallized with 95 %
ethanol-hexane (1:1, V/V). The m.p. of 4 was 86–88 °C.
General procedures for the preparation of
intermediates 2–4
General synthesis produce for 1-(2-(substituted
benzyloxy)-2-(2,4-dichlorophenyl)ethyl) -1H-1,2,4-
triazoles (5)
Synthesis of 2-chloro-(2,4-dichlorobenzene) ethanone (2)
Referring to the literature (Pericherla et al., 2012), a mixture
of m-dichlorobenzene 14.70 g (0.1 mol) and alchlor 21.40 g
(0.16 mol) were added in a 250 mL three-necked bottle with
a drying device and a condenser. Chloracetyl chloride 9.8
mL (130 mmol) was dripped into the above slurry under
stirring and the reaction temperature 30 °C. After chlor-
acetyl chloride was completely dripped into the slurry, the
reaction was on with stirring under 40–50 °C for 9 h. The
reaction mixture was poured into 5 % hydrochloric acid,
then was filtered and washed with distilled water. Light
yellow solid 2 in yield of 88.5 % (18.62 g) was obtained
after recrystallizing in hexane. The detected m.p. of the
target product 2 was 52–54 °C.
1-(2,4-Dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethanol
(1.00 mmol, 0.26 g) were dissolved into THF (5 mL). Tet-
rabutylammonium bromide (TBAB) (0.99 mmol, 0.032 g)
and NaOH (2 mmol, 0.08 g) were added into THF slurry at
room temperature. After 10 min of stirring, substituted
benzyl chlorides (1.01 mmol) was added into the reaction
mixture. The reaction process was monitored at reflux by
TLC. After the reaction was over, the slurry was extracted
with edichloromethane. The organic layers were dried over
anhydrous sodium sulfate, filtrated, and then evaporated
under a vacuum to provide the crude product which was
purified by recrystallization after column chromatography
(silica gel, 200–300 mesh) to furnish the product.
Preparation of α-(1H-1,2,4-triazol-1-yl)-2,4-
dichloroacetophenone (3)
1-(2-(benzyloxy)-2-(2,4-dichlorophenyl)ethyl)-1H-1,2,4-
triazole (5a)
Compound 3 was synthesized by referring the literature
(Yin et al., 2014). 2-Chloride-1-(2,4-dichlorphenyl)-etha-
none 4.47 g (0.02 mol) was dissolved with acetonitrile in a
250 mL three-necked bottle. Then, the dissolved 1,2,4-
triazole and potassium carbonate 3.31 g (24 mmol) was
slowly added into the above solution, stirred and refluxed
for 12 h. The mixture was diluted with ethyl acetate after
cooling, was extracted with distilled water and washed with
saturated sodium chloride. The organic layer was dried with
anhydrous sodium sulfate and concentrated by rotary eva-
poration. Brown viscous liquid 3 with yield of 68.2 % (3.50
g) was obtained after the mixture was purified by silica gel
column chromatography.
Yield:71.9 %; light yellow solid; IR (KBr) (ν/cm–1) 3129,
3084, 3066, 3028 (Ar–H), 2951, 2913, 2875, (CH2), 1585,
1562, 1508 (Ar–C=C), 1470, 1450 (CH2), 1092 (Ar–Cl),
1
1042, 1014 (C–O–C); H NMR (600 MHz, CDCl3) δ ppm:
8.14 (s, 1H, triazole–H5), 7.92 (s, 1H, triazole–H3), 7.44
(dd, J = 7.0, 5.3 Hz, 2H, Ar–H), 7.34–7.27 (m, 4H, Ar–H),
7.10–7.08 (m, 2H, Ar–H), 5.16 (dd, J = 8.5, 2.9 Hz, 1H,
CH), 4.48–4.42 (m, 2H, OCH2), 4.29–4.18 (m, 2H, CH2);
13C NMR (151 MHz, CDCl3) δ ppm: 151.8, 144.2
(triazole–C3 and C5), 136.6, 135.0, 134.0, 133.7, 129.8,
128.6, 128.2, 127.9, 127.9 (Ar–C), 75.5 (CH), 71.7
(OCH2), 53.9 (CH2); HRMS m/z (pos): 348.0663
C17H15Cl2N3O [M + H]+ (Calcd, 348.0670).

Med Chem Res
1-(2-((2-chlorobenzyl)oxy)-2-(2,4-dichlorophenyl)ethyl)-
1-(2-((2,4-dichlorobenzyl)oxy)-2-(2,4-dichlorophenyl)
1H-1,2,4-triazole (5b)
ethyl)-1H-1,2,4-triazole (5e)
Yield: 40.7 %; light yellow solid; IR (KBr) (ν/cm–1) 3128,
3067 (Ar–H), 2959, 2919, 2869 (CH2), 1582, 1512
(Ar–C=C), 1470 (CH2), 1099 (Ar–Cl), 1045, 1020
(C–O–C); 1H NMR (600 MHz, CDCl3) δ ppm: 8.12 (d, J =
12.0 Hz, 1H, triazole–H5), 7.90 (s, 1H, triazole–H3),
7.47–7.42 (m, 2H, Ar–H), 7.33–7.30 (m, 2H, Ar–H),
7.24–7.21 (m, 2H, Ar–H), 7.20–7.17 (m, 1H, Ar–H), 5.21
(dt, J = 14.9, 7.4 Hz, 1H, CH), 4.55–4.44 (m, 2H, OCH2),
4.36–4.25 (m, 2H, CH2); 13C NMR (151 MHz, CDCl3) δ
ppm: 151.8, 144.3 (triazole–C3 and C5), 135.1, 134.4,
133.8, 133.7, 133.4, 129.8, 129.6, 129.5, 129.4, 128.5,
127.9, 126.9 (Ar–C), 76.1 (CH), 68.9 (OCH2), 53.9 (CH2);
HRMS m/z (pos): 382.0236 C17H14Cl3N3O [M + H]+
(Calcd, 382.0281).
Yield: 39.7 %; light yellow solid; IR (KBr) (ν/cm–1) 3101
(Ar–H), 2943, 2919 (CH2), 1589, 1563, 1555, 1505
(Ar–C=C), 1470, 1448 (CH2), 1096 (Ar–Cl), 1038, 1016
(C–O–C); 1H NMR (600 MHz, CDCl3) δ ppm: 8.11 (s, 1H,
triazole–H5), 7.92 (s, 1H, triazole–H3), 7.47–7.40 (m, 2H,
Ar–H), 7.35–7.31 (m, 2H, Ar–H), 7.21 (dd, J = 8.2, 2.0 Hz,
1H, Ar–H), 7.10 (d, J = 8.2 Hz, 1H, Ar–H), 5.21 (dd, J =
8.5, 2.9 Hz, 1H, CH), 4.50–4.43 (m, 2H, OCH2), 4.31–4.25
(m, 2H, CH2); 13C NMR (151 MHz, CDCl3) δ ppm: 151.9,
144.2 (triazole–C3 and C5), 135.2, 134.6, 134.0, 133.7,
133.6, 133.0, 130.3, 129.9, 129.3, 128.4, 128.0, 127.3
(Ar–C), 76.2 (CH), 68.3 (OCH2), 53.8 (CH2); HRMS m/z
(pos): 415.9838 C17H13Cl4N3O [M + H]+ (Calcd,
415.9891).
1-(2-((3-chlorobenzyl)oxy)-2-(2,4-dichlorophenyl)ethyl)-
1H-1,2,4-triazole (5c)
1-(2-(2,4-dichlorophenyl)-2-((2-fluorobenzyl)oxy)ethyl)-
1H-1,2,4-triazole (5f)
Yield: 74.6 %; light yellow solid; IR (KBr) (ν/cm–1) 3132,
3078 (Ar–H), 2947, 2919, 2881 (CH2), 1585, 1560, 1504
(Ar–C=C), 1466 (CH2), 1092 (Ar–Cl), 1042, 1018
(C–O–C); 1H NMR (600 MHz, CDCl3) δ ppm: 8.11 (d, J =
17.1 Hz, 1H, triazole–H5), 7.94 (s, 1H, triazole–H3),
7.47–7.38 (m, 2H, Ar–H), 7.35–7.31 (m, 1H, Ar–H),
7.26–7.18 (m, 2H, Ar–H), 7.06 (d, J = 11.9 Hz, 1H, Ar–H),
6.94 (d, J = 7.4 Hz, 1H, Ar–H), 5.16 (dd, J = 8.5, 3.0 Hz,
1H, CH), 4.47–4.40 (m, 2H, OCH2), 4.25 (ddd, J = 55.7,
22.5, 8.7 Hz, 2H, CH2); 13C NMR (151 MHz, CDCl3) δ
ppm: 152.0, 144.2 (triazole–C3 and C5), 138.7, 135.2, 134.4,
133.8, 133.7, 129.8, 128.5, 128.3, 128.0, 127.7, 125.7
(Ar–C), 75.7 (CH), 70.8 (OCH2), 53.2 (CH2); HRMS m/z
(pos): 382.0292 C17H14Cl3N3O [M + H]+ (Calcd, 382.0281).
Yield: 95.3 %; light yellow solid; IR (KBr) (ν/cm–1) 3130,
3088, 3063 (Ar–H), 2953, 2924, 2856 (CH2), 1584, 1560,
1508 (Ar–C=C), 1458 (CH2), 1236 (Ar–F), 1090 (Ar–Cl),
1
1045, 1018 (C–O–C); H NMR (500 MHz, CDCl3) δ ppm:
8.12 (s, 1H, triazole–H5), 7.90 (s, 1H, triazole–H5),
7.46–7.42 (m, 2H, Ar–H), 7.30 (ddd, J = 9.7, 5.4, 1.9 Hz,
2H, Ar–H), 7.11 (dtd, J = 15.3, 7.7, 1.1 Hz, 2H, Ar–H),
7.04–6.98 (m, 1H, Ar–H), 5.18 (dd, J = 8.5, 2.8 Hz, 1H,
CH), 4.52 (d, J = 11.6 Hz, 1H, OCH2), 4.45 (dd, J = 14.3,
2.9 Hz, 1H, OCH2), 4.31–4.22 (m, 2H, CH2); 13C NMR
(151 MHz, CDCl3) δ ppm: 161.8 (2-fluorophenyl C2),
151.7, 144.2 (triazole–C3 and C5), 135.1, 133.8, 133.7,
130.3, 130.2, 129.7, 128.5, 127.9, 124.2, 124.2, 115.5
(Ar–C), 75.9 (CH), 65.5 (OCH2), 53.9 (CH2); HRMS m/z
(pos): 366.0571 C17H14Cl2FN3O [M + H]+ (Calcd,
366.0576).
1-(2-((4-chlorobenzyl)oxy)-2-(2,4-dichlorophenyl)ethyl)-
1H-1,2,4-triazole (5d)
1-(2-(2,4-dichlorophenyl)-2-((4-fluorobenzyl)oxy)ethyl)-
1H-1,2,4-triazole (5g)
Yield: 37.1 %; light yellow solid; IR (KBr) (ν/cm–1) 3132,
3075, 3046 (Ar–H), 2957, 2926, 2894 (CH2), 1585, 1559,
1501, 1489 (Ar–C=C), 1462 (CH2), 1096 (Ar–Cl), 1038,
Yield: 96.4 %; light yellow solid; IR (KBr) (ν/cm–1) 3129,
3078, 3040 (Ar–H), 2945, 2924, 2875 (CH2), 1585, 1560,
1508 (Ar–C=C), 1467 (CH2), 1223 (Ar–F), 1092 (Ar–Cl),
1043, 1015 (C–O–C);1H NMR (600 MHz, CDCl3) δ ppm:
8.12 (s, 1H, triazole–H5), 7.92 (s, 1H, triazole–H3),
7.47–7.40 (m, 2H, Ar–H), 7.33 (dd, J = 8.3, 1.9 Hz, 1H,
Ar–H), 7.04 (dd, J = 8.3, 5.6 Hz, 2H, Ar–H), 6.98 (t, J =
8.6 Hz, 2H, Ar–H), 5.14 (dd, J = 8.6, 2.9 Hz, 1H, CH),
4.45–4.39 (m, 2H, OCH2), 4.28–4.13 (m, 2H, CH2); 13C
NMR (151 MHz, CDCl3) δ ppm: 163.4 (2-fluorophenyl C2),
151.9, 144.2 (triazole–C3 and C5), 135.1, 133.8, 133.7,
132.4, 132.4, 129.7, 129.7, 128.5, 128.0, 115.5, 115.4
1
1016 (C–O–C); H NMR (600 MHz, CDCl3) δ ppm: 8.11
(d, J = 11.2 Hz, 1H, triazole–H5), 7.93 (s, 1H, triazole–H3),
7.43 (dt, J = 14.2, 3.9 Hz, 2H, Ar–H), 7.35–7.31 (m, 1H,
Ar–H), 7.27 (s, 1H, Ar–H), 7.26 (s, 1H, Ar–H), 7.00 (d, J =
8.3 Hz, 2H, Ar–H), 5.14 (dt, J = 17.8, 8.9 Hz, 1H, CH),
4.46–4.39 (m, 2H, OCH2), 4.28–4.14 (m, 2H, CH2); 13C
NMR (151 MHz, CDCl3) δ ppm: 151.9, 144.2 (triazole–C3
and C5), 135.1, 135.1, 134.0, 133.8, 133.7, 129.8, 129.1,
128.7, 128.5, 128.0 (Ar–C), 75.5 (CH), 70.8 (OCH2), 53.8
(CH2); HRMS m/z (pos): 382.0289 C17H14Cl3N3O [M +
H]+ (Calcd, 382.0281).

Med Chem Res
(Ar–C), 75.4 (CH), 70.9 (OCH2), 53.9 (CH2); HRMS m/z
(pos): 366.0488 C17H14Cl2FN3O [M + H]+ (Calcd,
366.0576).
and itraconazole were used as reference antibiotics against
bacteria and yeasts, respectively.
The test compounds (5a–f) were dissolved in dimethyl
sulfoxide (DMSO) at the highest concentration to enhance
the solubility. Tests were conducted in sterile 96-well
plates, into which 100 μL of prepared inoculum were added
to each well. Another 100 μL inoculum containing 2 μL of
test compound were also added into the first well. Then 100
μL mixed inoculum was transferred into the second well.
This twofold dilution was continued from second to the
ninth well and then 100 μL inoculum of the ninth column
was discarded. The tenth column of the plate was reserved
for blank control (without inoculation), the eleventh column
was reserved for negative control (with standard anti-
bacterial drug), and the twelfth column was reserved for
positive growth control wells (without drugs or test com-
pounds). The final concentrations of test compounds were
ranged from 64 μg/mL to 0.125 μg/mL and the final con-
centration of DMSO was not more than 1 % V/V in each
well mircoplate. Plates covered and incubated for 18 h at 37 °C
for all bacteria strains, while 24 h at 30 °C for yeast. The
minimum inhibitory concentration (MIC) was defined as the
lowest concentration, which inhibits the visible growth of a
microbe. The end-points were determined when no turbidity
in the well was observed.
4-((1-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethoxy)
methyl)benzonitrile (5h)
Yield: 62.8 %; light yellow solid; IR (KBr) (ν/cm–1) 3121,
3078, 3052, 3021 (Ar–H), 2954, 2932 (CH2), 2222 (C≡N),
1607, 1585, 1563, 1504 (Ar–C=C), 1474, 1452 (CH2),
1
1084 (Ar–Cl), 1046, 1015 (C–O–C); H NMR (600 MHz,
CDCl3) δ ppm: 8.14 (s, 1H, triazole–H5), 7.95 (s, 1H,
triazole–H3), 7.59 (d, J = 8.1 Hz, 2H, Ar–H), 7.47 (d, J =
2.0 Hz, 1H, Ar–H), 7.40 (t, J = 7.0 Hz, 1H, Ar–H), 7.34 (dd,
J = 8.3, 2.0 Hz, 1H, Ar–H), 7.18 (d, J = 8.1 Hz, 2H, Ar–H),
5.19 (dd, J = 8.6, 3.0 Hz, 1H, CH), 4.52–4.44 (m, 2H,
OCH2), 4.33–4.24 (m, 2H, CH2); 13C NMR (151 MHz,
CDCl3) δ ppm: 152.0, 144.2 (triazole–C3 and C5), 142.0,
135.4, 133.8, 133.3, 132.4, 129.9, 128.3, 128.1, 127.8
(Ar–C), 118.5 (CN), 111.9 (Ar–C), 76.1 (CH), 70.1
(OCH2), 53.8 (CH2); HRMS m/z (pos): 373.0616
C18H14Cl2N4O [M + H]+ (Calcd, 373.0623).
1-(2-(2,4-dichlorophenyl)-2-((4-methylbenzyl)oxy)ethyl)-
1H-1,2,4-triazole (5i)
Yield: 81.2 %; light yellow solid; IR (KBr) (ν/cm–1) 3125,
3091, 3046, 3032, 3013 (Ar–H), 2946(CH3), 2926, 2920
(CH2), 1615, 1585, 1563, 1504 (Ar–C=C), 1470 (CH2),
1377 (CH3), 1092 (Ar–Cl), 1042, 1015 (C–O–C); 1H NMR
(600 MHz, CDCl3) δ ppm: 8.13 (s, 1H, triazole–H5), 7.91
(s, 1H, triazole–H3), 7.43 (dd, J = 13.7, 3.8 Hz, 2H, Ar–H),
7.31 (dd, J = 8.3, 1.9 Hz, 1H, Ar–H), 7.11 (d, J = 7.8 Hz,
2H, Ar–H), 6.98 (d, J = 7.8 Hz, 2H, Ar–H), 5.14 (dd, J =
8.5, 2.9 Hz, 1H, CH), 4.45–4.40 (m, 2H, OCH2), 4.27–4.13
(m, 2H, CH2), 2.36–2.32 (m, 3H, CH3); 13C NMR (151
MHz, CDCl3) δ ppm: 151.7, 144.2 (triazole–C3 and C5),
138.0, 134.9, 134.0, 133.7, 133.6, 129.7, 129.2, 128.6,
128.0, 127.9 (Ar–C), 75.3 (CH), 71.5 (OCH2), 53.9 (CH2),
21.2 (CH3); HRMS m/z (pos): 362.0810 C18H17Cl2N3O [M
+ H]+ (Calcd, 362.0827).
Results and discussion
Synthesis
The synthetic route of 1-(2-(substituted benzyloxy)-2-(2,4-
dichlorophenyl)ethyl)-1H-1,2,4- triazoles (5a–i) was out-
lined in Scheme 1. The desired target compounds were
synthesized via multistep reactions from commercially
available 1,3-dichlorobenzene, chloroacetyl chloride, 1,2,4-
triazole, and substituted benzyl chloride. α-Chlor-
oacetophenone (2) was prepared by Friedele-Crafts acyla-
tion of 1,3-dichlorobenzene (1) with chloroacetyl chloride
(Pericherla et al., 2012), and then reacted with 1,2,4-triazole
by N-alkylation to produce 2,4-dichloroacetophenone deri-
vative (3) (Yin et al., 2014) in moderate to good yields of
69.1–95.3 %. Reduction (Guven et al., 2007) of inter-
mediate 3 using sodium borohydride at room temperature
resulted in the formation of the corresponding 2,4-dichlor-
obenzene ethanol derivative (4) in excellent yields. The
reaction of intermediate 4 with substituted benzyl chlorides
was synthesized via alcohol etherification in THF and phase
transfer catalyzed by TBAB to afford the target compounds
5a–i (Scheme 1).
In vitro antimicrobial activity assay
The antibacterial activity was carried out in accordance with
the method of National Committee for Clinical Laboratory
(NCCLS) (Vilanova 2000) in vitro. Bacteria strains were
cultured for 6–8 h at 37 °C in MHB and yeast strains were
cultured for 24 h at 35 °C in YEPD, the inoculum density
was adjusted to be equivalent to 2–4 × 108 CFU/mL (twice
the final inoculum size) for bacteria in RPMI-1640 and 1 ×
103–5 × 103 CFU/mL (twice the final inoculum size) for
yeast in RPMI-1640 with haemocytometer. Ciprofloxacin
The available azole antifungal drugs usually contain
halo-substituted aryl groups, such as 2-chloro, 2-fluoro, 2,4-
dichloro-substituted benzyl rings. Herein, we mainly

Med Chem Res
Scheme 1 Synthesis of
benzene-ethanol 1,2,4-triazole
derivatives. (i) Chloroacetyl
chloride, AlCl3, 40–50 °C, 5 h.
(ii) 1,2,4-triazole, K2CO3(aq.),
CH3CN, reflux, 12 h. (iii)
NaBH4, CH3OH, r.t., 6 h. (iv)
Substituted benzyl chloride,
NaOH, TBAB, reflux, 7–10 h
Table 1 Synthesis of benzene-ethanol 1,2,4-triazole derivatives 5a–i
Ciprofloxacin (antibiotic) and itraconazole (antifungals)
were used as reference drugs. The results were recorded for
each tested compound as the average diameter of inhibition
zones (IZ) of bacterial or fungal growth around the discs in
mm (Data not shown). After that, the MIC measurement
was determined with the two-fold serial dilution by the
method of NCCLS. All compounds were evaluated at the
concentrations ranging from 0.125 to 64 μg/mL and scored
for MIC as the level of growth inhibition of the micro-
organisms compared with ciprofloxacin and itraconazole.
The data of antibacterial and antifungal activities are
depicted in Table 2.
a
Entry
R
Product Reaction time Yield /% M.P./°C
1
2
3
4
5
6
7
8
9
a
H
5a
5b
5c
5d
5e
5f
8 h
9 h
8 h
10 h
9 h
7 h
7 h
8 h
8 h
71.9
40.7
74.6
37.1
40.7
95.3
96.4
62.8
81.2
106–108
56–58
2-Cl
3-Cl
4-Cl
2,4-Cl
2-F
55–57
100–102
73–75
128–130
92–94
4-F
5g
5h
4-CN
88–90
4-CH3 5i
51–53
Seen from the antifungal activity of the tested com-
pounds, C. albicans, certain sensitivity against all of the
tested compounds, which was coincidence the triazole
derivates possessed antifungal activities (Genin et al., 2000;
Sheng et al., 2006; Sheehan et al., 1999; Plech et al., 2015;
Mathew et al., 2006). Among them, three compounds,
namely, 5a, 5g, and 5i exhibited much higher growth
inhibitory on C. albicans with MIC of 32 μg/mL, while
compared with itraconazole MIC 1 μg/mL, the positive
antifungal agents used in this assay. The antifungal activity
is usually related to the structure of CYP51 of fungus.
However, there is no three dimensional structure of CYP51
of any fungus, the structure of some bacteria is obtained.
The basic structure to inhibit CYP51 enzymes, includes
three parts: (a) iron coordinating group, consisting of tria-
zole ring, able to interact with the heme iron, (b) a first
hydrophobic moiety (typically aromatic) near the iron
coordinating group, and (c) a second aromatic region (De
Vita et al., 2012). The designed triazole derivates in this
study contained the main structure of CYP51 inhibition.
Moreover, halogen is an important efficiency increas-
ing group, which was introduced into triazole of anti-
bacterial and antifungal drugs, and could improve
pharmacological activity (Guven et al., 2007; Lebouvier
Isolated yields
focused to get target molecules of halo-substituted rings.
The synthesized compounds are shown in Table 1. Com-
1
pounds (5a–i) were characterized by FT-IR, H NMR, 13C
NMR, and HRMS spectrometry analysis. The –OCH–
protons of ether and two –CH– protons of 1,2,4-triazole
exhibited resonances at 5.14–5.21 ppm and 7.90–8.14 ppm
using CDCl3 as solvent, while the resonances for the cor-
responding –OCH– and –CH=N–CH– carbon atom were
observed peaks at 75.26–76.17 and 144.2–144.3,
151.7–151.9, respectively.
Biological activity analysis
Compounds 5a–i were screened for antibacterial activities
against Gram-negative Escherichia coli (ATCC 25922) and
Pseudomonas aeruginosa (ATCC 27853, ATCC 9027),
Gram-positive Staphylococcus aureus (ATCC 25923,
ATCC 3933) and Staphylococcus epidermidis (ATCC
12228), and antifungal Candida albicans (ATCC 10231).
Agar-diffusion method was used for determination of the
preliminary antibacterial and antifungal activity.

Med Chem Res
Table 2 Minimal inhibitory
concentrations (MIC, μg/mL) of
compounds 5a–i against some
bacteria and fungus strains
b
Compounds
MIC (μg/mL)
E. coli P. aeruginosa P. aeruginosa S.aureus S.aureus S.epidermidis C. albicans
ATCC ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
25922 27853
9027
>64
>64
>64
>64
>64
>64
>64
>64
>64
0.5
25923
>64
32
3933
64
32
16
16
16
>64
32
>64
16
2
12228
>64
64
10231
32
64
64
64
64
64
32
64
32
/
5a
5b
5c
5d
5e
5f
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
16
>64
>64
32
>64
8
>64
32
>64
64
5g
5h
5i
>64
32
>64
>64
1
Ciprofloxacin 0.125 0.5
Itraconazole
0.5
/
/
/
/
/
/
1
b Minimum inhibitory concentrations (MIC) were defined as the lowest concentration which inhibited 100 %
growth of the tested organism by the micro-broth dilution method
et al., 2007; Wahbi et al., 1995; Marrapu et al., 2011),
lower toxicity (Wu et al., 2016) and drug resistance (Al-
Qawasmeh et al., 2016), and so on. Furthermore, elec-
tronegative substituent groups on 2- and/or 4- position of
benzene ring can be benefit to the antifungal activity of
antifungus (Table 2, Compounds 5b–g). Such groups are
mainly about fluorous or chlorinous substituent, which
could form a function of π-π water repellent. The analysis
of the activity of the synthetic compounds suggested that
substituent containing fluorous or chlorinous groups on
the 2nd and 4th positions of benzene ring show a better
inhibiting effect toward bacteria and fungus compared
with other positions on benzene ring (Table 2, Compound
5e).
Regarding the antibacterial activity, the results revealed
that all compounds displayed variable inhibitory effects
on the growth of the tested gram-positive bacteria. Com-
pounds 5c and 5e exhibited 12.5 % of the potency of
ciprofloxacin (MIC 2 μg/mL) against the species S. aureus
was proved to be the highest sensitive Gram-positive
microorganism, while they displayed totally insensitive to
the Gram-negative microorganism. In addition, 5b, 5g,
and 5i also showed better inhibitory activities against the
test gram-positive bacteria compared with gram-negative
bacteria. The reason might be that the content of the
peptidoglycan in cell wall of gram-positive bacteria was
much more than that of gram-negative bacteria. It can be
speculated that the mechanism of benzene-ethanol triazole
derivatives may be inhibit cross-linking of peptidoglycan,
damage the formation of the bacterial cell wall, resulting
in cell death.
Conclusions
The aim of this study is to find novel 1,2,4-triazole derivates
exhibited higher antibacterial and antifungal activities. The
desired target compounds were synthesized via multistep
reactions from commercially available 1,3-dichlorobenzene,
chloroacetyl chloride, 1,2,4-triazole, and substituted benzyl
chloride. The structures of the synthesized compounds were
confirmed by FT-IR, 1H NMR, 13C NMR, and HRMS
spectra. Results of antifungal and antibacterial activities
showed that five compounds possessed better growth inhi-
bitory effects on the tested Gram-positive bacteria and
fungus. It suggested that compounds (5a–i) had widely
promising application as antibacterial and antifungal agents.
The modification to the compounds for better antimicrobial
activities is being in our laboratory.
Acknowledgments The authors would like to thank the fund for the
12th five-year Scientific and Technical Research Plan of Jilin Province
Department of Education (No. 2014B002).
Compliance with ethical standards
Conflict of interest The authors declare that they have no com-
peting interests.
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