2-Hydroxyphenacyl azoles and related azolium derivatives as antifungal agents

Article abstract of DOI:10.1016/j.bmcl.2007.10.111

2-Hydroxyphenacyl azole and 2-hydroxyphenacyl azolium compounds have been described as a new class of azole antifungals. Most target compounds showed significant in vitro antifungal activities against tested fungi (Candida albicans, Saccharomyces cerevisiae, Aspergillus niger, and Microsporum gypseum) with low MICs values included in the range of 0.25-32 μg/mL comparable to reference drug fluconazole. The most active compounds were also assessed for their cytotoxicity using MTT colorimetric assay on normal mouse fibroblast (NIH/3T3) cells. The results of antifungal activity and toxicity tests indicated that these compounds display antifungal activity at non-cytotoxic concentrations.

Full text of DOI:10.1016/j.bmcl.2007.10.111

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 141–146
2-Hydroxyphenacyl azoles and related azolium derivatives
as antifungal agents
Saeed Emami,a,* Alireza Foroumadi,b,d Mehraban Falahati,c Ensieh Lotfali,c
Saeed Rajabalian,d Soltan-Ahmed Ebrahimi,c Shirin Farahyarc and Abbas Shafieeb
aDepartment of Medicinal Chemistry, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari 48175, Iran
bDepartment of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center,
Tehran University of Medical Sciences, Tehran 14174, Iran
cDepartment of Parasitology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran
dKerman Neuroscience Research Center, Kerman University of Medical Sciences, Kerman, Iran
Received 31 May 2007; revised 11 October 2007; accepted 31 October 2007
Available online 4 November 2007
Abstract—2-Hydroxyphenacyl azole and 2-hydroxyphenacyl azolium compounds have been described as a new class of azole anti-
fungals. Most target compounds showed significant in vitro antifungal activities against tested fungi (Candida albicans, Saccharo-
myces cerevisiae, Aspergillus niger, and Microsporum gypseum) with low MICs values included in the range of 0.25–32 lg/mL
comparable to reference drug fluconazole. The most active compounds were also assessed for their cytotoxicity using MTT color-
imetric assay on normal mouse fibroblast (NIH/3T3) cells. The results of antifungal activity and toxicity tests indicated that these
compounds display antifungal activity at non-cytotoxic concentrations.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The incidence of invasive fungal infections caused by
opportunistic pathogens, often characterized by high
mortality rates, has been increasing over the past two
decades. Patients that become severely immunocompro-
mised because of underlying diseases such as leukemia
or, recently, acquired immunodeficiency syndrome or
patients who undergo cancer chemotherapy or organ
transplantation are particularly susceptible to opportu-
nistic fungal infections.1,2 A matter of concern in the
treatment of fungal infections is the limited number of
efficacious antifungal drugs. Many of the currently
available drugs are toxic, produce recurrence because
they are fungistatic and not fungicides or lead to the
development of resistance due in part to the prolonged
periods of administration of the available antifungal
drugs. There is, therefore, a clear need for the discovery
of new structures with antifungal properties, which
could lead to the development of new drugs for the man-
agement of fungal infections and treatment of systemic
mycoses.3
Up to only 30 years ago the choice of systemically avail-
able antimycotics was between two drugs, amphotericin
B and flucytosine, neither of which was satisfactory.
Then a series of inhibitors of the biosynthesis of ergos-
terol, the major sterol of the fungal cell membrane, were
found to have excellent antifungal activity, improved
safety, and some were also active after oral or parenteral
application. Starting with miconazole and ketoconazole,
and improving through fluconazole and itraconazole,
the imidazole and triazole inhibitors of lanosterol l4a-
demethylation have been the most successful. Although
the use of a new generation of triazoles, the available
polyenes in lipid formulations, the use of echinocandins
or the combination therapy have been introduced as
alternatives in the last 10 years, the number of available
preparations to treat systemic fungal infections is still
limited and more alternatives are needed, particularly
with improved efficacy against emerging pathogens with
limited susceptibility to the available preparations.4,5
In order to seek new antifungal agents we synthesized a
series of 2-hydroxyphenacyl-azole and 2-hydroxyphena-
cyl-azolium compounds depicted in Figure 1. We pre-
sumed that the azole portion of the molecule is
essential to the high antifungal activity. It is a common
Keywords: Azole; Imidazole; 1,2,4-Triazole; 2-Hydroxyacetophenone;
Antifungal activity; Cytotoxicity.
*
Corresponding author. E-mail: sd_emami@yahoo.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.111

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S. Emami et al. / Bioorg. Med. Chem. Lett. 18 (2008) 141–146
derivatives 3.12,13 Compound 3a was converted to the
oxime derivative 4a by stirring with excess of HON-
H2ÆHCl in methanol at room temperature.14
OH
O
Az
R
Several routes have been developed for the regiospecific
synthesis of 1-substituted and 4-substituted 1,2,4-tria-
zoles.15,16 The thermodynamically less stable regioisom-
er 5, can be obtained by blocking the more reactive
N-atom of 1,2,4-triazole, followed by quaternization
and cleavage of the protecting group.17 Thus, quatern-
ization of cyanoethyl modified triazole (1H-1,2,4-tria-
zole-1-propanenitrile)17 could be achieved with
bromoacetophenones 2 and subsequent treatment with
aqueous sodium hydroxide gave the expected product
5.18 For preparation of 1-substituted 1,2,4-triazoles,
4-amino-1,2,4-triazole,15 a protected, directing synthetic
equivalent of 1,2,4-triazole can be utilized in cases where
regiospecificity under mild nonbasic conditions is
required. Thus, the quaternization of 4-amino-1,2,4-tri-
azole with 2 was utilized to form pure triazolium salts
6,19 which on subsequent deamination with nitrous acid
yielded exclusively the 1-substituted product 7.20,21
Compounds 2 were converted to bis(substituted)triazoli-
um bromide derivative 8 by stirring with 0.5 equiv of
1,2,4-triazole in DMF at room temperature.22
R = H, Cl, OMe
Az = 1H-imidazol-1-yl; 1H-1,2,4-triazol-1-yl;
4H-1,2,4-triazol-4-yl; 1H-1,2,4-triazolium;
4-amino-4H-1,2,4-triazoliumyl
Figure 1. General structure of 2-hydroxyphenacyl azole and
2-hydroxyphenacyl azolium compounds.
substructure seen in many other azole antifungals. The
substitution pattern of the azole ring was explored using
1H-imidazole, 1,2,4-1H-triazole, 1,2,4-4H-triazole, 4-
aminotriazolium, and 1,4-disubstituted triazolium in
the (un)substituted 2-hydroxy acetophenone skeleton
(Fig. 1). On the other hand, several studies reveal the
importance of phenolic groups for the antifungal activ-
ity of acetophenone derivatives such as chalcones
against various fungi. In addition, the antifungal activity
of these hydroxylated acetophenone derivatives related
to the location of the phenolic group in the aryl ring
and compounds possessing 2-hydroxyl function showed
better activity.6–9 Accordingly, we wish to report here
the synthesis and antifungal activity of 2-hydroxyphena-
cyl-azole and 2-hydroxyphenacyl-azolium derivatives.
The in vitro antifungal activities of the synthesized com-
pounds 3–8 were investigated against several representa-
tive pathogenic fungi as yeast (Candida albicans PTCC
5027 and Saccharomyces cerevisiae PTCC 5177), derma-
tophyte (Microsporum gypseum PTCC 5070), and mould
(Aspergillus niger PTCC 5012). The minimum inhibitory
concentrations (MICs) were determined by agar dilution
method.23 Sabouraud Dextrose Agar was employed for
fungal growth. Stock solutions of tested compounds
Our synthetic pathway to target compounds 3–8 is pre-
sented in Scheme 1. The starting 2-hydroxyacetophenon-
es 1 were obtained from the corresponding phenols
according to the method reported in the literature.10 Ke-
tone 1 was brominated with copper (II) bromide in reflux-
ing CHCl3–AcOEt to give corresponding a-bromoketone
2.11 Reaction of imidazole with a-bromoketone 2 in
DMF, at 40–45 ꢁC, afforded the corresponding imidazole
R
OH
O
Br
OH
a: R= H
N
N
O
CH3
O
OH
b: R= Cl
c: R= CH3O
N
R
1a-c
a
8a-c
R
h
NH2
OH
O
N
N
Br
OH
O
Br
OH
O
N
N
b
f
N
R
R
R
2a-c
6a-c
3a-c
d, e
g
c
N
N
N
OH NOH
OH
O
OH
O
N
N
N
N
N
R
R
4a
5a, b
7a-c
Scheme 1. Reagents and conditions: (a) CuBr2, CHCl3–EtOAc, reflux; (b) imidazole, DMF, 40–45 ꢁC; (c) HONH2ÆHCl, MeOH, rt; (d) 1H-1,2,4-
triazole-1-propanenitrile, CH3CN; (e) aq NaOH; (f) 4-amino-1,2,4-triazole, 2-propanol, reflux; (g) NaNO2, HCl; (h) 1H-1,2,4-triazole 0.5 equiv,
DMF, rt.

S. Emami et al. / Bioorg. Med. Chem. Lett. 18 (2008) 141–146
143
were prepared in dimethylsulfoxide (DMSO). Inocula
containing approximately 106 CFUs/mL of fungi were
prepared from broth cultures in log phase growth. Fun-
gal plates were made in triplicate and incubated at 30 ꢁC
about 24–48 h for yeast, about 72 h for moulds and
about 168 h for dermatophytes. The MIC value was de-
fined as lowest concentration of the antifungal agent at
which there was no growth. The MIC values (in lg/mL)
for different pathogenic fungi, in comparison with
fluconazole and miconazole, are summarized in Table 1.
Among the compounds used, bisphenacyl triazolium
derivative 8a showed greater toxicity with IC50 values
of 47 5 lg/mL. Also bis(4-methoxyphenacyl)triazoli-
um derivative 8c showed moderate cytotoxic effect on
NIH/3T3 cell line at concentration <100 lg/mL. On
the other hand, mono-phenacyl azoles 3c, 6b, 6c, and
7c showed no significant toxicity at the concentrations
used (IC50 values = 221–370 lg/mL). The results re-
corded in Table 2 showed compound 6b to be signifi-
cantly less toxic than other compounds and there was
no significant deference between the cytotoxicity of com-
pound 6b and reference drug fluconazole.
The results of in vitro antifungal activities showed that
the title compounds were active against nearly all fungi
tested to some extent. Most derivatives showed signifi-
cant in vitro antifungal activities against tested fungi
with low MIC values included in the range of
0.25–32 lg/mL.
In comparison of the results of toxicity and antifungal
activity tests, it is seen that these compounds display
antifungal activity at non-cytotoxic concentrations thus
the antifungal activity of the compounds is not due to
their general toxicity effect, however their antifungal
activity can be possibly because of their selective anti-
fungal effect.
Compound 6c followed by compound 6b was the most
potent against C. albicans, with MIC values of 2 lg/mL
and 4 lg/mL, respectively. These compounds possessed
superior activity with respect to the reference drug fluco-
nazole (MIC = 8 lg/mL) against this yeast. In addition,
methoxy analogs 3c, 7c, and 8c were equivalent in anti-
Candida activity with MIC values of 8 lg/mL and their
activity was found to be comparable to reference drug
fluconazole. The MIC values of the test derivatives
against S. cerevisiae indicate that most compounds pos-
sessed a comparable or better activity with respect to
fluconazole. In fact, the most active compounds were
3b, 3c, and 6b (MIC = 0.25 lg/mL) being 128-fold more
active than fluconazole and four times more active than
miconazole against S. cerevisiae. Furthermore, com-
pound 8a with MIC value of 1 lg/mL showed good activ-
ity against S. cerevisiae comparable to miconazole and
32-fold more potent than fluconazole. Among the com-
pounds tested, compounds 3c, 5a, 6c, 7b, and 8c showed
highest activity against A. niger and their MIC values
were determined to be 16 lg/mL, comparable to that of
fluconazole. Compounds 3c and 6c also inhibited more
significantly the growth of M. gypseum (MIC = 8 lg/
mL) equipotent to miconazole and 4-fold more potent
than fluconazole. Moreover, compounds 5a, 6b, 7b, 7c,
8a and 8c showed considerable activity against M. gypse-
um with MIC values of 16 lg/mL.
In terms of structure–activity relationships (SARs), the
substitution pattern of azole ring was explored using
1H-imidazole, 1,2,4-1H-triazole, 1,2,4-4H-triazole, 4-
aminotriazolium, and 1,4-disubstituted triazolium in
(un)substituted 2-hydroxyacetophenone skeleton. Gen-
erally, the type of azole ring and chlorine atom linked
to the 4-position of the phenacyl moiety seemed to have
different influence on the antifungal activity against var-
ious fungi strains. Yeasts (C. albicans and S. cerevisiae),
in particular, seemed to be more sensitive toward both
changes in the substitution pattern of the azole ring,
and phenacyl moiety. In 1H-imidazole, 1,2,4-1H-tria-
zole and 4-aminotriazolium series, 4-methoxyphenacyl
derivatives 3c, 6c, and 7c clearly showed superior activ-
ity compared to their analogs bearing a hydrogen or
chlorine atom at position 4. Furthermore, in 1,4-disub-
stituted triazolium series, 4-methoxy analog 8c exhibited
better profile of antifungal activity with respect to 4-
chloro analog 8b. Therefore, it can be concluded that
the methoxy group of phenacyl residue improves anti-
fungal activity. Surprisingly, 4-aminosubstitution of
the triazole core (compounds 6b and 6c) resulted in
superior activity compared to that of 7b and 7c against
yeasts (see Table 1). Furthermore, bisphenacyl triazoli-
um 8a showed comparable or superior activity com-
pared to its related azoles 5a and 7a. Notably,
compound 8a could be considered as a hybrid of 5a
and 7a. Results from compounds 6 and 8 demonstrate
that quaternization of the N-4 nitrogen of the phenacyl
triazoles also is possible without loss of potency, and
even in some cases provides an improvement of activity.
Because most of antifungal azoles are too lipophilic for
parenteral formulation and cannot attain reliable blood
levels after administration, quaternary azolium salts
such as 6b and 6c could be useful and promising
water-soluble antifungals, although it seems that these
In general, the results of antifungal evaluation of test
compounds in comparison with reference drugs indi-
cated that all methoxy analogs 3c, 6c–8c along with 6b
and 8a showed comparable or more potent antifungal
activity with respect to the reference drug fluconazole
against all tested fungal species.
The compounds 3c, 6b, 6c, 7c, 8a, and 8c that were most
active (MIC 6 16 lg/mL) as anti-Candida agents were
also assessed for their cytotoxicity using MTT colori-
metric assay on normal mouse fibroblast (NIH/3T3)
cells.24,25 The IC50 values obtained for the six com-
pounds in comparison with reference drug fluconazole
are shown in Table 2.
ionic compounds maybe have
bioavailability.
a unsuitable oral
In conclusion, 2-hydroxyphenacyl-azole and 2-hydrox-
yphenacyl-azolium compounds have been identified as
a new class of azole antifungal agents with a good
As can be seen from the results, there was no significant
cytotoxicity at the concentrations below 47 lg/mL.

144
S. Emami et al. / Bioorg. Med. Chem. Lett. 18 (2008) 141–146
Table 1. Chemical structures and in vitro antifungal activity of compounds 3–8
Compound
Chemical structure
Tested fungi (MICs in lg/mL)a
C. albicans
S. cerevisiae
A. niger
M. gypseum
N
OH
O
N
3a
64
64
64
64
N
OH
O
N
3b
3c
4
64
0.25
0.25
32
64
16
64
32
Cl
N
OH
O
N
8
8
MeO
OH
OH
N
O
N
32
64
N
N
OH
N
N
5a
32
16
16
16
N
OH
O
N
N
5b
6a
6b
16
64
4
16
32
32
32
64
32
16
Cl
NH2
Br
OH
O
N
32
N
N
NH2
Br
OH
O
N
0.25
N
N
N
Cl
NH2
Br
OH
O
N
6c
2
4
16
8
N
MeO
OH
N
O
N
7a
7b
7c
64
32
8
64
64
8
32
16
32
64
16
16
N
N
OH
O
N
N
Cl
N
OH
O
N
N
MeO

S. Emami et al. / Bioorg. Med. Chem. Lett. 18 (2008) 141–146
145
Table 1 (continued)
Compound
Chemical structure
Tested fungi (MICs in lg/mL)a
C. albicans
S. cerevisiae
A. niger
M. gypseum
OH
Br
O
8a
8b
8c
16
1
32
16
32
16
N
N
O
OH
N
OH
Cl
Br
O
32
8
4
64
16
Cl
N
N
O
OH
N
OH
OMe
Br
O
8
MeO
N
N
O
OH
N
Fluconazole
Miconazole
8
1
32
1
32
2
32
8
a Minimum inhibitory concentrations were determined by agar dilution method for duplicate determinations.
Table 2. In vitro cytotoxic activity of selected compounds against
mouse fibroblast (NIH/3T3) cell line
8. Sato, M.; Tsuchiya, H.; Akagiri, M.; Fujiwara, S.; Fujii,
T.; Takagi, N.; Matsuura, N.; Iinuma, M. Lett. Appl.
Microbiol. 1994, 18, 53.
9. Batovska, D.; Parushev, St.; Slavova, A.; Bankova, V.;
Tsvetkova, I.; Ninova, M.; Najdenski, H. Eur. J. Med.
Chem. 2007, 42, 87.
10. Bryan, J. D.; Goldberg, A. A.; Wragg, A. H. J. Chem. Soc.
1960, 1279.
11. King, L. C.; Ostrum, G. K. J. Org. Chem. 1964, 29,
3459.
Compound
IC50 (lg/mL)a
3c
6b
221 11
370 28
254 12
6c
7c
222
47
7
5
2
8a
8c
98
Fluconazole
387 17
12. Cozzi, P.; Mongelli, N.; Pillan, A. J. Heterocycl. Chem.
1984, 21, 311.
a IC50 is the concentration required to inhibit 50% of cell growth. The
values represent means standard deviation of triplicate
determinations.
13. Typical procedure: the preparation of 1-(4-methoxy-2-
hydroxyphenyl)-2-(1H-imidazol-1-yl)ethanone (3c). A solu-
tion of 2-bromo-40-methoxy-20-hydroxyacetophenone 2c
(490 mg, 2.0 mmol) and imidazole (408 mg, 6.0 mmol) in
DMF (4 mL) was stirred at 40 ꢁC for 4 h. The mixture was
poured into water and then extracted with CHCl3. The
organic phase after washing with H2O was shaken with a
solution of 10% HCl. The aqueous acid solution was
neutralized with NaHCO3 and the precipitate was filtered,
washed with water, and dried to give 3c. Yield 58%; mp
130–132 ꢁC; 1H NMR (80 MHz, CDCl3) d 3.87 (s, 3H,
CH3), 5.33 (s, 2H, CH2), 6.40–6.58 (m, 3H, H-3 and H-5
Ar and H-imidazole), 7.24–7.79 (m, 3H, H-6 Ar and H-
imidazole), 12.05 (br s, 1H, OH). Anal. Calcd for
C12H12N2O3: C, 62.06; H, 5.21; N, 12.06. Found: C,
61.98; H, 5.33; N, 12.20.
spectrum of activity. Most derivatives showed signifi-
cant in vitro antifungal activities against tested fungi
with low MIC values included in the range of 0.25–
32 lg/mL comparable to the reference drug fluconazole.
We concluded from our investigations that 2-hydrox-
yphenacyl-azoles (3c and 7c) and 2-hydroxyphenacyl-
4-aminotriazoliums (6b and 6c) may be considered
promising for the development of new antifungal agents
with their biological activity and toxicity screening.
References and notes
14. The preparation of 1-(2-hydroxyphenyl)-2-(1H-imidazol-1-
yl)ethanone oxime (4a). A solution of ketone 3a (1.0 mmol)
and hydroxylamine hydrochloride (208 mg, 3.0 mmol) in
MeOH (5 mL) was stirred at room temperature for 3 d.
After concentrating the reaction mixture by evaporation
under reduced pressure, MeOH was replaced with water
(20 mL) and neutralized with NaHCO3. The precipitate
was filtered by filtration, washed with water, and dried to
give oxime 4a. Yield 71%; mp 163–165 ꢁC; 1H NMR
(80 MHz, DMSO-d6) d 5.32 (s, 2H, CH2), 6.51–7.99 (m,
7H, phenyl and imidazole), 10.61 (s, 1H, OH), 12.04 (s,
1H, OH). Anal. Calcd for C11H11N3O2: C, 60.82; H, 5.10;
N, 19.34. Found: C, 60.81; H, 5.28; N, 19.52.
1. Kontoyannis, D.; Mantadakis, E.; Samonis, G. J. Hosp.
Infect. 2003, 53, 243.
2. Garber, G. Drugs 2001(Suppl. 1), 1.
3. Fostel, J. M.; Lartey, P. A. Drug Discovery Today 2000, 5,
25.
4. White, T. C.; Marr, K. A.; Bowden, R. A. Clin. Microbiol.
Rev. 1998, 11, 382.
5. Denning, D. W.; Kibler, C. C.; Barnes, R. A. Lancet
Infect. Dis. 2003, 3, 230.
6. Dimmock, J. R.; Elias, D. W.; Beazely, M. A.; Kandepu,
N. M. Curr. Med. Chem. 1999, 6, 1125.
7. Tsuchiya, H.; Sato, M.; Akagiri, M.; Takagi, N.; Tanaka,
T.; Iinuma, M. Pharmazie 1994, 49, 756.
15. Astleford, B. A.; Goe, G. L.; Keay, J. G.; Scriven, E. F. V.
J. Org. Chem. 1989, 54, 731.

146
S. Emami et al. / Bioorg. Med. Chem. Lett. 18 (2008) 141–146
1
16. Balasubramanian, M.; Keay, J. G.; Scriven, E. F. V.
Heterocycles 1994, 37, 1951.
129 ꢁC; IR (KBr) 3421, 1639, 1378, 1244, 1132 cmꢀ1; H
NMR (500 MHz, DMSO-d6) d 3.84 (s, 3 H, CH3), 5.84 (s,
2H, CH2), 6.55 (d, 1H, J = 2.27 Hz, H-3 Ar), 6.60 (dd, 1H,
J = 8.91 and 2.33 Hz, H-5 Ar), 7.87 (d, 1H, J = 8.93 Hz,
H-6 Ar), 8.01 (s, 1H, triazole), 8.52 (s, 1H, triazole), 11.58
(s, 1H, OH). Anal. Calcd for C11H11N3O3: C, 56.65; H,
4.75; N, 18.02. Found: C, 56.76; H, 4.81; N, 17.93.
22. Typical procedure: the preparation of 1,4-bis(4-methoxy-2-
hydroxyphenacyl)-1H-1,2,4-triazolium bromide (8c). To a
solution of 1,2,4-triazole (69 mg, 1.0 mmol) in DMF
(3 mL) was added 2-bromo-40-methoxy-20-hydroxyaceto-
phenone 2c (2.4 mmol). The mixture was stirred at room
temperature for 3 d. The resulting solution was poured
into ice-water (20 mL) and the precipitate was filtered,
washed with water (3· 5 mL) and diethyl ether (4· 5 mL)
to give 8c. Yield 49%; mp 215–217 ꢁC; IR (KBr) 3431,
1644, 1239, 1127 cmꢀ1; 1H NMR (500 MHz, DMSO-d6) d
3.84 (s, 6H, CH3), 6.00 (s, 2H, CH2), 6.16 (s, 2H, CH2),
6.59 (d, 1H, J = 2.11 Hz, H-3 Ar), 6.60 (d, 1H,
J = 2.21 Hz, H-30 Ar), 6.61–6.65 (m, 2H, H-5 Ar and H-
50 Ar), 7.84 (d, 1H, J = 8.95 Hz, H-6 Ar), 7.85 (d, 1H,
J = 8.90 Hz, H-60 Ar), 9.21 (s, 1H, triazole), 10.04 (s, 1H,
triazole), 11.50 (br s, 2H, OH). Anal. Calcd for
C20H20BrN3O6: C, 50.22; H, 4.21; N, 8.79. Found: C,
50.38; H, 4.18; N, 8.61.
23. Massa, S.; Di Santo, R.; Retico, A.; Simonetti, N.;
Fabrizi, G.; Lamba, D. Eur. J. Med. Chem. 1992, 27, 495.
24. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
25. The in vitro cytotoxic activity of the test compounds
against normal mouse fibroblast (NIH/3T3) cell line was
investigated using MTT colorimetric assay. Briefly, cul-
tures in the exponential growth phase were trypsinized and
diluted in complete growth medium to give a total cell
count of 5 · 104 cells/mL. One hundred microliters of
suspension was added to wells of sterile 96-well plates.
After plating, 50 lL of a serial dilution of every agent was
added. Each compound dilution was assessed in triplicate.
Three wells containing only normal mouse fibroblast
(NIH/3T3) cells suspended in 150 lL of complete medium
were used as control for cell viability. The plates were then
incubated for 72 h. After incubation, 30 lL of a 5 mg/mL
solution of MTT was added to each well and the plate was
incubated for another 1 h. After incubation, the culture
medium was replaced with 100 lL of DMSO. Then, the
absorbance of each well was measured by using a
microplate reader at 492 nm wavelengths. For each
compound, dose–response curve was measured with dif-
ferent drug concentrations, and the concentration causing
50% cell growth inhibition (IC50) compared with the
control was calculated.
´
17. Horvath, A. Synthesis 1995, 1183.
18. Typical procedure: the preparation of 1-(4-chloro-2-
hydroxyphenyl)-2-(4H-1,2,4-triazol-4-yl)ethanone (5b). A
mixture of 1H-1,2,4-triazole-1-propanenitrile (1.0 mmol)
and
2-bromo-40-chloro-20-hydroxyacetophenone
2b
(1.0 mmol) in MeCN (3 mL) was refluxed with stirring
for 9h. The solvent was then evaporated and the residue
was triturated with diethyl ether to give quaternary
triazolium salt. This product was treated with NaOH
(2.0 mmol) in H2O (5 mL) at room temperature with
stirring. After 2 d, the mixture was acidified with 10% aq
HCl and neutralized with NaHCO3. The precipitate was
filtered, washed with water, and crystallized from meth-
anol to afford 5b. Yield 64%; mp 241–243 ꢁC; IR (KBr)
3347, 3128, 1670 cmꢀ1; H NMR (80 MHz, DMSO-d6) d
1
5.66 (s, 2H, CH2), 6.80–7.15 (m, 2H, H-3 and H-5 Ar),
7.84 (d, 1H, J = 8.3 Hz, H-6 Ar), 8.43 (s, 2H, triazole),
11.57 (s, 1H, OH); Anal. Calcd for C10H8ClN3O2: C,
50.54; H, 3.39; N, 17.68. Found: C, 50.81; H, 3.30; N,
17.78.
19. Typical procedure: the preparation of 1-(4-methoxy-2-
hydroxyphenyl)-2-(4-amino-4H-1,2,4-triazoliumyl)ethanone
bromide (6c). A mixture of 4-amino-4H-1,2,4-triazole
(1.51 g, 18.0 mmol) and 2-bromo-40-methoxy-20-hydroxy-
acetophenone 2c (4.41 g, 18.0 mmol) in 2-propanol
(36 mL) was refluxed for 6 h. Upon cooling the colorless
salt was filtered, washed with cold 2-propanol (3· 8 mL)
and diethyl ether (3· 8 mL) to give 6c. Yield 55%; mp 177–
178ꢁC; IR (KBr) 3229, 3114, 2986, 1626, 1255, 1152 cmꢀ1
;
1H NMR (500 MHz, DMSO-d6) d 3.84 (s, 3H, CH3), 6.01
(s, 2H, CH2), 6.58 (d, 1H, J = 2.36 Hz, H-3 Ar), 6.62 (dd,
1H, J = 8.91 and 2.40 Hz, H-5 Ar), 7.17 (br s, 2H, NH2),
7.81 (d, 1H, J = 8.90 Hz, H-6 Ar), 9.30 (s, 1H, triazole),
10.15 (s, 1H, triazole), 11.43 (s, 1H, OH). Anal. Calcd for
C11H13BrN4O3: C, 40.14; H, 3.98; N, 17.02. Found: C,
40.32; H, 4.11; N, 16.87.
20. Emami, S.; Shafiee, A. Heterocycles 2001, 55, 2059.
21. Typical procedure: the preparation of 1-(4-methoxy-2-
hydroxyphenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone (7c). To
a vigorously stirred ice-cold aqueous suspension of 6c
(8.0 mmol in 20 mL) was added concentrated HCl (1.58 g,
16.0 mmol).
A solution of sodium nitrite (0.72 g,
8.4 mmol) in water (4 mL) was added dropwise at a rate
to prevent excessive foaming (30 min). The mixture was
permitted to come to room temperature and was stirred
for another 45 min. Upon neutralization with NaHCO3,
the precipitate was filtered, washed with water, and
crystallized from MeOH to give 7c. Yield 97%; mp 127–