Antifungal and antimycobacterial activity of 1-(3,5-diaryl-4,5-dihydro-1H-pyrazol-4-yl)-1H-imidazole derivatives

Article abstract of DOI:10.1016/j.bmc.2008.02.055

1-(3,5-Diaryl-4,5-dihydro-1H-pyrazol-4-yl)-1H-imidazole derivatives were synthesized and tested for their in vitro antifungal and antimycobacterial activities. These imidazole derivatives showed an excellent antifungal activity against a clinical strain of Candida albicans and an interesting antitubercular activity againstMycobacterium tuberculosis H₃₇Rv reference strain.

Full text of DOI:10.1016/j.bmc.2008.02.055

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4516–4522
Antifungal and antimycobacterial activity of 1-(3,5-diaryl-4,5-
dihydro-1H-pyrazol-4-yl)-1H-imidazole derivativesq
Daniele Zampieri,a Maria Grazia Mamolo,a,* Erik Laurini,a
Giuditta Scialino,b Elena Banfib and Luciano Vioa
aDepartment of Pharmaceutical Sciences, Piazzale Europa 1, University of Trieste, I-34127 Trieste, Italy
bDepartment of Biomedical Sciences, Microbiology Section, Via Alexander Fleming 22, University of Trieste, I 34127 Trieste, Italy
Received 20 November 2007; revised 11 February 2008; accepted 15 February 2008
Available online 21 February 2008
Abstract—1-(3,5-Diaryl-4,5-dihydro-1H-pyrazol-4-yl)-1H-imidazole derivatives were synthesized and tested for their in vitro anti-
fungal and antimycobacterial activities. These imidazole derivatives showed an excellent antifungal activity against a clinical strain
of Candida albicans and an interesting antitubercular activity againstMycobacterium tuberculosis H37Rv reference strain.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
like gene encodes a bacterial sterol 14a-demethylase
(MT P45014DM) which acts on 14a-methyl sterols and
binds known antifungal azole derivatives.8 On the basis
of these considerations and because many imidazole
derivatives showed potent antifungal activity associated
with good antimycobacterial activity,6,7,9,10 we synthe-
sized a series of compounds 1a–t (Scheme 1) in which
the imidazolyl moiety, present in many azole antifungal
drugs, is directly linked at 4 position of a pyrazoline ring
3,5-disubstituted 1a–e, and 1,3,5-trisubstituted 1f–t
whose importance with respect to the antifungal11–14
and antimycobacterial15 activities has been previously
described.
Considering the increased incidence of severe opportu-
nistic fungal infections in immunocompromised patients
together with the development of resistance among
pathogenic Candida spp., there is a great need for new
antifungal compounds. On the other hand, the increase
of tuberculosis due to emergence of multidrug-resistant
strains of Mycobacterium tuberculosis,1–4 together with
the increased incidence of severe disseminated infections
produced by mycobacteria other than tuberculosis
(MOTT), particularly Mycobacterium avium5 in immu-
nocompromised patients, has prompted the search for
new antimycobacterial drugs.
All the synthesized compounds were tested in vitro for
their antifungal and antitubercular activity towards
Candida albicans 685 and M. tuberculosis H37Rv,
respectively.
Molecular modelling investigations confirmed that anti-
fungal azole derivatives synthesized by us6,7 act interact-
ing at the active site of the fungal cytochrome P450-
dependent lanosterol 14a-demethylase (P45014DM, CYP51
)
but exhibit simultaneously antimycobacterial activity
which may be due to a similar inhibitory interaction
with the corresponding mycobacterial cytochrome. In
effect, it was established, using genomic DNA from a
strain of M. tuberculosis (MT) H37Rv, that a CYP51-
The synthesized compounds showed a high antifungal
activity associated with a remarkable antimycobacterial
activity.
2. Chemistry
Keywords: 1-(3,5-Diaryl-4,5-dihydro-1H-pyrazol-4-yl)-1H-imidazole
derivatives; Antifungal and antimycobacterial activity.
q
A preliminary account of this work was presented at 5th Joint
The synthesis of compounds 1a–t was carried out via a
three-step reaction which involved an N-alkylation of
imidazole with the substituted-2-bromoacetophenones
4a–e to afford the corresponding 1-aryl-2-(1H-imida-
zol-1-yl)-ethanones 3a–e in accordance with the
Meeting on Medicinal Chemistry, June 17–21, 2007, Portoroz,
Slovenia.
Corresponding author. Fax: +39 4052572; e-mail: mamolo@units.it
*
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.055

D. Zampieri et al. / Bioorg. Med. Chem. 16 (2008) 4516–4522
4517
CHO
O
O
N
R
N
N
THF
CH2Br
+
R
HN
R
piperidine
3 a-e
4 a-e
O
N
N
N
R1-NHNH2
N
R
R
R
R
2 a-e
N
N
R
1
1 f-t
NH2NH2
N
N
R= 4-H, 4-Br, 4-Cl, 2,4(Cl)2, 4-CH3
R1= CH3 Ph, 4-F-Ph
,
R
R
N
NH
1 a-e
Scheme 1.
literature procedure6 followed by condensation with
substituted benzaldehydes and the final cyclization of
the obtained a,b-unsatured ketones 2a–e, with hydrazine
hydrate, methylhydrazine sulfate, phenylhydrazine and
4-fluorophenylhydrazine to give the corresponding com-
pounds 1a–e, 1f–j, 1k–o, and 1p–t, respectively.
activities (Table 1) of derivatives 1a–t were tested
against a clinical strain of C. albicans 685 in comparison
with miconazole and amphotericin B, and against a
strain of M. tuberculosis H37Rv in comparison with
isoniazid.
The antifungal activity of compounds 1g, 1h, 1k and 1p
was remarkable reaching MIC values of 0.06 lg/mL
even at 48 h (1g, 1h). Their activities were superior than
that of the reference drugs miconazole and amphotericin
B. From the obtained data it seems that the presence of
the electron-accepting chloro or bromo groups in the
para position at the phenyl residues enhance the activity
(1g, 1h), as same as the electron-donating methyl group
at N-1 position of the pyrazoline moiety, even if the 3,5-
diphenyl derivatives 1k and 1p, bearing a phenyl or 4-
fluorophenyl substituent at the N-1 position of the pyr-
azoline cycle maintain a very high potency. On the other
hand, the presence of the electron-donating methyl
group at the para position of phenyl residues, reduces
the activity of compounds 1e, 1j, 1o and 1t. Moreover,
compounds 1a–t were tested against a strain of M. tuber-
culosis H37Rv and showed a good antimycobacterial
activity reaching MIC values of 4 lg/mL for 6 com-
pounds of the series. In this case the substitution at
1
From the H NMR spectra of the obtained compounds
it appears that only the derivatives 1f–j show a cis isom-
erism while all the other compounds (1a–e, 1k–t) show a
trans isomerism. The values of the coupling constants
for the H4 and H5 pyrazoline protons for the trans iso-
mers and for the cis isomers are in the range of 3.30–
8.06 Hz and 10.37–10.99 Hz, respectively, according to
the values of the coupling constants reported in the lit-
erature for other pyrazoline derivatives.16–18
3. Results and discussion
A series of 1-(3,5-diaryl-4,5-dihydro-1H-pyrazol-4-yl)-
1H-imidazole 1a–e and 1-[(1-aralkyl)-3,5-diaryl-4,5-
dihydro-1H-pyrazol-4-yl]-1H-imidazole derivatives 1f–t
were synthesized with the aim to evaluate their anti-
fungal and antimycobacterial activities. The in vitro

4518
D. Zampieri et al. / Bioorg. Med. Chem. 16 (2008) 4516–4522
Table 1. Activity of compounds 1 a-t against Candida albicans 685 clinical strain and against Mycobacterium tuberculosis reference strain H37Rv
N
N
R
R
N
N
R1
1 a-t
Compound
R
R1
C. albicans 685
H37Rv
MIC lg/mL after 24 h
MIC lg/mL after 48 h
MIC lg/mL
Miconazole
Amphotericin B
—
—
—
—
0.06
1
0.12
1
—
—
0.5
16
8
Isoniazid
1a
1b
1c
—
—
H
—
16
4
—
H
Br
>64
8
H
Cl
H
4
16
64
8
1d
1e
2,4-(Cl)2
CH3
H
H
8
8
H
8
64
64
16
8
1f
CH3
CH3
CH3
CH3
CH3
Ph
1
1g
1h
1i
Br
Cl
0.06
0.06
0.12
4
0.06
0.06
32
8
8
2,4-(Cl)2
CH3
H
16
16
8
1j
64
1k
1l
0.06
1
1
64
Br
Cl
Ph
Ph
8
1m
1n
1o
1p
1q
1r
2
64
4
2,4-(Cl)2
CH3
H
Ph
Ph
2
>64
64
4
8
4
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
0.06
8
1
8
Br
Cl
>64
>64
>64
64
4
2
4
1s
2,4-(Cl)2
CH3
1
64
16
4
1t
N-1 position of the pyrazoline ring with a phenyl or 4-
fluorophenyl moiety, rather than a methyl or hydrogen,
improves the antimycobacterial activity of compounds
1m, 1n, 1q, 1r and 1t. The substitution at the phenyl
groups at 3, 5 position of the pyrazole ring, did not ap-
pear to be influent for the activity.
as d (ppm) in CDCl3 solution (0.05% v/v TMS). Reac-
tion courses and product mixtures were routinely
monitored by thin-layer chromatography (TLC) on
silica gel precoated F254 Merck plates. ESI-MS spectra
were obtained on a PE-API I spectrometer by infusion
of a solution of the sample in MeOH. Elemental anal-
yses (C, H, N) were performed on a Carlo Erba ana-
lyzer and were within 0.3 of the theoretical value.
According to the results obtained for other imidazole
derivatives,6,7,9,10,19 the newly synthesized imidazole
derivatives showed both in vitro antifungal and antimy-
cobacterial property, suggesting an inhibitory interac-
tion of these compounds with similar enzymatic sites
present in fungi and mycobacteria.
4.1. Synthesis
4.1.1. 1,3-Bis(4-bromophenyl)-2-(1H-imidazol-1-yl)-prop-
2-en-1-one (2b). To a solution of 1-(4-bromophenyl)-2-
(1H-imidazol-1-yl)-ethanone 3b (2.5 g, 9.4 mmol) in
80 mL of toluene, 4-bromobenzaldehyde (1.74 g,
9.4 mmol) and piperidine (0.8 g, 9.4 mmol) were added.
The reaction mixture was stirred under reflux for 16 h.
The solvent was removed under reduced pressure and
the residue was extracted with CHCl3 (3· 100 mL)
and the organic phase was washed with distilled water.
The collected organic phases were dried over sodium
sulfate, filtered and evaporated under reduced pressure.
The residue was crystallized from diethyl ether. Mp
148–150 ꢁC; yield 65%. IR (Nujol, cmÀ1): 1655. 1H
4. Experimental
Mps were determined with a Buchi 510 capillary appa-
ratus, and are uncorrected. Infrared spectra in Nujol
mulls were recorded on a Jasko FT 200 spectropho-
tometer. Proton nuclear magnetic resonance (1H
NMR) spectra and carbon nuclear magnetic resonance
(13C NMR) spectra were determined on a Varian
Gemini 200 spectrometer, chemical shifts are reported

D. Zampieri et al. / Bioorg. Med. Chem. 16 (2008) 4516–4522
4519
NMR (CDCl3/TMS): d 6.84 (s, 1H, @CH–), 6.87
Compounds 1b–e were obtained similarly.
(s, 1H, CH imid.), 6.99–7.88 (m, 10H, arom. and
imidazole). MS: m/z 431 [MH+], 433 [MH++2]. Anal.
Calcd for C18H12N2OBr2 (MW 432.11): C, 50.03: H,
2.80; N, 6.48%; found: C, 49.80; H, 2.95; N, 6.60%.
4.1.7. ( ) trans-1-[3,5-Bis-(4-bromophenyl)-4,5-dihydro-
1H-pyrazol-4-yl]-1H-imidazole (1b). Mp 173–175 ꢁC;
1
yield 71%. IR (Nujol, cmÀ1): 3348. H NMR (CDCl3/
TMS): d 4.79 (dd, 1H, CH, H5 pyrazole, JH5–H4
= 5.86 Hz; JH5–NH = 2.20 Hz); 5.54 (d, 1H, CH, H4 pyr-
azole, JH4–H5 = 5.86 Hz); 6.46 (d, 1H, NH disappearing
on deuteration; JNH–H5 = 2.20 Hz); 6.94–7.64 (m, 11H,
8H arom. and 3H imidazole). 13C NMR (CDCl3/
TMS): d 70.1, 73.2, 116.9, 125.8, 126.4, 127.8, 128.6,
129.5, 129.7, 130.7, 131.8, 136.8, 139.4, 145.7. MS: m/z
445 [MH+], 447 [MH++2]. Anal. Calcd for C18H14N4Br2
(MW 446.14): C, 48.36: H, 3.16; N, 12.56%; found: C,
48.15; H, 3.37; N, 12.58%.
In an analogous way compounds 2a and 2c–e were
obtained.
4.1.2. 2-(1H-Imidazol-1-yl)-1,3-diphenylprop-2-en-1-one
(2a).20 Mp 109 ꢁC; yield 32%. Solvent crystallization:
ethyl acetate. IR (Nujol, cmÀ1): 1665. 1H NMR
(CDCl3/TMS): d 6.84 (s, 1H, @CH–), 6.87 (s, 1H, CH
imid.), 6.99–7.88 (m, 12H, arom. and imid.). MS: m/z
275 [MH+]. Anal. Calcd for C18H14N2O (MW 274.32):
C, 78.81: H, 5.14; N, 10.21%; found: C, 78.70; H, 5.19;
N, 10.07%.
4.1.8. ( ) trans-1-[3,5-Bis-(4-chlorophenyl)-4,5-dihydro-
1H-pyrazol-4-yl]-1H-imidazole (1c). Mp 175–177 ꢁC; yield
15%. IR (Nujol, cmÀ1): 3289. 1H NMR (CDCl3/TMS): d
4.80 (d, 1H, CH, H5 pyrazole, JH5–H4 = 6.10 Hz); 5.54 (d,
1H, CH, H4 pyrazole, JH4–H5 = 6.10 Hz);6.41 (s, broad sig-
nal, 1H, NH disappearing on deuteration); 6.91–7.57 (m,
11H, 8H arom. and 3H imidazole). 13C NMR (CDCl3/
TMS): d 70.0, 73.1, 117.4, 126.4, 127.2, 129.2, 129.5,
129.7, 130.7, 131.8, 134.4, 136.8, 139.4, 145.7. MS: m/z
357 [MH+], 359 [MH++2]. Anal. Calcd for C18H14N4Cl2
(MW 357.24): C, 60.52: H, 3.95; N, 15.68%; found: C,
60.55; H, 3.90; N, 15.40%.
4.1.3. 1,3-Bis(4-chlorophenyl)-2-(1H-imidazol-1-yl)-prop-
2-en-1-one (2c).20 Mp 123 ꢁC; yield 49%. IR (Nujol,
1
cmÀ1): 1656. H NMR (CDCl3/TMS): d 6.84 (s, 1H,
@CH–), 6.87 (s, 1H, CH imid.), 6.99–7.88 (m, 10H,
arom. and imidazole). MS: m/z 343 [MH+], 345
[MH++2]. Anal. Calcd for C18H12N2OCl2 (MW
343.21): C, 62.99: H, 3.52; N, 8.16%; found: C, 62.80;
H, 3.35; N, 8.30%.
4.1.4. 1,3-Bis(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)-
prop-2-en-1-one (2d). Mp 119 ꢁC; yield 41%. IR (Nujol,
4.1.9. ( ) trans-1-[3,5-Bis-(2,4-dichlorophenyl)-4,5-dihy-
dro-1H-pyrazol-4-yl]-1H-imidazole (1d). Mp 155–157 ꢁC;
1
cmÀ1): 1678. H NMR (CDCl3/TMS): d 6.84 (s, 1H,
@CH–), 6.87 (s, 1H, CH imid.), 6.99–7.88 (m, 8H, arom.
and imidazole). MS: m/z 411 [MH+], 413 [MH++2].
Anal. Calcd for C18H10N2OCl4 (MW 412.11): C,
52.46: H, 2.45; N, 6.80%; found: C, 52.40; H, 2.35; N,
6.80%.
1
yield 13%. IR (Nujol, cmÀ1): 3330. H NMR (CDCl3/
TMS): d 5.34 (dd, 1H, CH, H5 pyrazole, JH5–
H4 = 8.06 Hz; JH5–NH = 2.93 Hz); 6.32 (d, 1H, CH, H4
pyrazole, JH4–H5 = 8.06 Hz); 6.41 (d, 1H, NH disappear-
ing on deuteration, JNH–H5 = 2.93 Hz); 6.56–7.75 (m,
9H, 6H arom. and 3H imidazole). 13C NMR (CDCl3/
TMS): d 62.4, 65.5, 116.1, 127.6, 127.8, 128.0, 129.3,
129.7, 129.9, 130.3, 130.6, 130.8, 131.4, 131.7, 136.3,
136.4, 140.7, 152.7. MS: m/z 425 [MH+], 427 [MH++2].
Anal. Calcd for C18H12N4Cl4 (MW 426.13): C, 50.73:
H, 2.84; N, 13.15%; found: C, 50.55; H, 2.90; N, 13.40%.
4.1.5. 1,3-Bis(4-methylphenyl)-2-(1H-imidazol-1-yl)-prop-
2-en-1-one (2e). Mp 122 ꢁC; yield 40%. IR (Nujol,
1
cmÀ1): 1648. H NMR (CDCl3/TMS): d 2.26 (s, 3H,
CH3), 2.36 (s, 3H, CH3), 6.60–7.75 (m, 12H, @CH–,
arom. and imidazole). MS: m/z 303 [MH+]. Anal. Calcd
for C20H18N2O (MW 302.37): C, 79.44: H, 6.00; N,
9.26%; found: C, 79.50; H, 6.05; N, 9.00%.
4.1.10. ( ) trans-1-[4,5-Dihydro-3,5-bis-(4-methylphenyl)-
1H-pyrazol-4-yl]-1H-imidazole (1e). Mp 158–160 ꢁC;
1
yield 58%. IR (Nujol, cmÀ1): 3318. H NMR (CDCl3/
4.1.6. ( ) trans-1-(4,5-Dihydro-3,5-diphenyl-1H-pyrazol-
4-yl)-1H-imidazole (1a). To an ethanolic (20 mL) solu-
tion of 2a (0.6 g, 2.1 mmol), hydrazine hydrate (98%
sol., 0.13 g, 2.6 mmol) was added under stirring. The
reaction was allowed to stirring at room temperature
and monitored by TLC. The solvent was evaporated un-
der reduced pressure and the solid obtained was crystal-
lized from diethyl ether. Mp 124–126 ꢁC; yield 32%. IR
(Nujol, cmÀ1): 3350. 1H NMR (CDCl3/TMS): d 4.75 (d,
1H, CH, H5 pyrazole, JH5–H4 = 4.70 Hz); 5.58 (d, 1H,
CH, H4 pyrazole, JH4–H5 = 4.70 Hz); 6.50 (s, broad sig-
nal, 1H, NH disappearing on deuteration); 7.09–8.44
(m, 13H, 10H arom. and 3H imidazole). 13C NMR
(CDCl3/TMS): d 69.4, 72.5, 117.2, 125.6, 126.2, 129.0,
129.1, 129.2, 129.4, 130.7, 132.0, 136.8, 139.4, 145.7.
MS: m/z 289 [MH+]. Anal. Calcd for C18H16N4 (MW
288.35): C, 74.98: H, 5.59; N, 19.43%; found: C, 75.14;
H, 5.37; N, 19.58%.
TMS): d 2.31 (s, 3H, CH3); 2.35 (s, 3H, CH3); 4.75 (d,
1H, CH, H5 pyrazole, JH5–H4 = 4.88 Hz); 5.58 (d, 1H,
CH, H4 pyrazole, JH4–H5 = 4.88 Hz); 6.30 (s, broad sig-
nal, 1H, NH disappearing on deuteration); 6.90–7.65
(m, 11H, 8H arom. and 3H imidazole). 13C NMR
(CDCl3/TMS): d 21.3, 21.6, 70.3, 72.9, 117.8, 125.6,
127.4, 128.1, 129.5, 129.6, 130.0, 134.4, 136.7, 138.7,
139.8, 145.5. MS: m/z 317 [MH+]. Anal. Calcd for
C20H20N4 (MW 316.13): C, 75.92: H, 6.87; N, 17.71%;
found: C, 75.80; H, 6.20; N, 17.65%.
4.1.11. ( ) cis-1-(4,5-Dihydro-3,5-diphenyl-1-methyl-1H-
pyrazol-4-yl)-1H-imidazole (1f). To an ethanolic (60 mL)
solution of 2a (0.5 g, 1.8 mmol), triethylamine (0.18 g,
1.8 mmol) and methylhydrazine sulfate (0.26 g,
1.8 mmol) were added under stirring. The reaction mix-
ture was allowed to reflux for 3 h. The solvent was evap-

4520
D. Zampieri et al. / Bioorg. Med. Chem. 16 (2008) 4516–4522
orated under reduced pressure and the residue was ex-
tracted with CHCl3 and washed with distilled water.
The collected organic phases were dried over sodium
sulfate, filtered and evaporated under reduced pressure.
The residue was then purified by column chromatogra-
phy (eluted with CH2Cl2) to afford an oil. Yield 43%.
1H NMR (CDCl3/TMS): d 2.99 (s, 3H, CH3); 4.20 (d,
1H, CH, H5 pyrazole, JH5–H4 = 10.98 Hz); 5.63 (d, 1H,
CH, H4 pyrazole, JH4–H5 = 10.98 Hz); 6.90–7.52 (m,
13H, 10H arom. and 3H imidazole). 13C NMR
(CDCl3/TMS): d 41.3, 71.5, 80.6, 117.7, 125.5, 126.4,
128.8, 129.0, 129.2, 129.4, 130.6, 132.3, 136.8, 139.4,
146.2. MS: m/z 303 [MH+]. Anal. Calcd for C19H18N4
(MW 302.40): C, 74.47: H, 6.00; N, 18.53%; found: C,
74.24; H, 6.17; N, 18.48%.
11H, 8H arom. and 3H imidazole). 13C NMR (CDCl3/
TMS): d 21.4, 21.6, 41.4, 71.3, 80.7, 117.7, 125.4,
127.1, 128.1, 129.5, 129.6, 130.0, 130.8, 134.4, 136.7,
138.7, 144.5. MS: m/z 331 [MH+]. Anal. Calcd for
C21H22N4 (MW 330.41): C, 76.33; H, 6.71; N, 19.96%;
found: C, 76.45; H, 6.80; N, 20.07%.
4.1.16. ( ) trans-(4,5-Dihydro-1,3,5-triphenyl-1H-pyra-
zol-4-yl)-1H-imidazole (1k). To an ethanolic (60 mL)
solution of 2a (0.3 g, 1.1 mmol), phenylhydrazine
(0.14 g, 1.3 mmol) was added under stirring. The reac-
tion mixture was refluxed for 3 h and monitored by
TLC. The mixture was allowed to crystallized from eth-
anol to afford a white solid. Mp 183–185 ꢁC; yield 38%.
1H NMR (CDCl3/TMS): d 5.16 (d, 1H, CH, H5 pyra-
zole, JH5–H4 = 3.63 Hz); 5.66 (d, 1H, CH, H4 pyrazole,
JH4–H5 = 3.63 Hz); 6.82–7.70 (m, 18H, 15H phen. and
3H imidazole). 13C NMR (CDCl3/TMS): d 70.0, 73.3,
113.7, 117.1, 120.8, 125.7, 125.8, 128.9, 129.1, 129.3,
129.4, 129.9, 130.7, 131.3, 135.9, 138.3, 142.5, 143.1.
MS: m/z 365 [MH+]. Anal. Calcd for C24H20N4 (MW
364.44): C, 79.10: H, 5.53; N, 15.37%; found: C, 78.94;
H, 5.37; N, 15.48%.
Compounds 1g–j were obtained similarly.
4.1.12. ( ) cis-1-[3,5-Bis(4-bromophenyl)-(4,5-dihydro-1-
methyl-1H-pyrazol-4-yl)-1H-imidazole (1g). Mp 148–
150 ꢁC; yield 34%. 1H NMR (CDCl3/TMS): d 2.96
(s, 3H, CH3); 4.16 (d, 1H, CH, H5 pyrazole, JH5–H4
= 10.98 Hz); 5.55 (d, 1H, CH, H4 pyrazole, JH4–H5
= 10.98 Hz); 6.92–7.60 (m, 11H, 8H arom. and 3H imid-
azole). 13C NMR (CDCl3/TMS): d 41.5, 71.3, 80.7,
117.3, 125.8, 126.3, 127.7, 128.7, 129.5, 129.8, 130.9,
131.9, 136.7, 139.4, 145.9. MS: m/z 359 [MH+], 361
[MH++2]. Anal. Calcd for C19H16N4Br2 (MW 360.17):
C, 49.59: H, 3.50; N, 12.18%; found: C, 49.30; H, 3.33;
N, 12.05%.
Compounds 1l–o were obtained similarly.
4.1.17. ( ) trans-1-[3,5-Bis-(4-bromophenyl)-4,5-dihydro-
1-phenyl-1H-pyrazol-4-yl]-1H-imidazole (1l). Mp 182–
1
184 ꢁC; yield 34%. H NMR (CDCl3/TMS): d 5.13 (d,
1H, CH, H5 pyrazole, JH5–H4 = 3.30 Hz); 5.61 (d, 1H,
CH, H4 pyrazole, JH4–H5 = 3.30 Hz); 6.85–7.70 (m, 16H,
13H arom. and 3H imidazole). 13C NMR (CDCl3/
TMS): d 70.2, 73.4, 114.0, 117.1, 120.6, 125.8, 126.4,
128.2, 128.6, 129.2, 129.5, 129.7, 130.7, 131.8, 136.8,
139.4, 142.3, 145.7. MS: m/z 521 [MH+], 523 [MH++2].
Anal. Calcd for C24H18N4Br2 (MW 522.23): C, 55.20:
H, 3.47; N, 10.73%; found: C, 55.15; H, 3.37; N, 10.68%.
4.1.13. ( ) cis-1-[3,5-Bis(4-chlorophenyl)-(4,5-dihydro-1-
methyl-1H-pyrazol-4-yl)-1H-imidazole (1h). Mp 134–
1
136 ꢁC; yield 89%. H NMR (CDCl3/TMS): d 2.95 (s,
3H, CH3); 4.17 (d, 1H, CH, H5 pyrazole,
JH5–H4 = 10.98 Hz); 5.54 (d, 1H, CH, H4 pyrazole,
JH4–H5 = 10.98 Hz); 6.88–7.50 (m, 11H, 8H arom. and
3H imidazole). 13C NMR (CDCl3/TMS): d 41.5, 71.3,
80.7, 117.4, 126.4, 127.0, 129.3, 129.5, 129.7, 130.7,
131.9, 134.5, 136.8, 139.9, 145.8. MS: m/z 371 [MH+],
373 [MH++2]. Anal. Calcd for C19H16N4Cl2 (MW
371.26): C, 61.47: H, 4.34; N, 15.09%; found: C, 61.35;
H, 4.27; N, 15.00%.
4.1.18. ( ) trans-1-[3,5-Bis-(4-chlorophenyl)-4,5-dihydro-
1-phenyl-1H-pyrazol-4-yl]-1H-imidazole (1m). Mp 183–
1
185 ꢁC; yield 28%. H NMR (CDCl3/TMS): d 5.18 (d,
1H, CH, H5 pyrazole, JH5–H4 = 3.40 Hz); 5.64 (d, 1H,
CH, H4 pyrazole, JH4–H5 = 3.40 Hz); 6.85–7.72 (m,
16H, 13H arom. and 3H imidazole): d 71.0, 73.3,
114.3, 117.7, 120.6, 125.8, 128.2, 128.6, 129.2, 129.5,
129.7, 130.7, 131.8, 132.5, 136.8, 139.5, 142.3, 145.7.
MS: m/z 433 [MH+], 435 [MH++2]. Anal. Calcd for
C24H18N4Cl2 (MW 433.33): C, 66.52: H, 4.19; N,
12.93%; found: C, 66.75; H, 4.42; N, 12.88%.
4.1.14. ( ) cis-1-[3,5-Bis(2,4-dichlorophenyl)-4,5-dihydro-
1-methyl-1H-pyrazol-4-yl]-1H-imidazole (1i). Mp 173–
1
175 ꢁC; yield 44%. H NMR (CDCl3/TMS): d 3.01 (s,
3H, CH3); 4.87 (d, 1H, CH, H5 pyrazole, JH5–H4
= 10.37 Hz); 6.00 (d, 1H, CH, H4 pyrazole, JH4–H5
= 10.37 Hz); 6.80–7.60 (m, 9H, 6H arom. and 3H imid-
azole). 13C NMR (CDCl3/TMS): d 41.3, 70.9, 80.9,
116.8, 127.6, 127.9, 128.1, 129.4, 129.7, 130.0, 130.3,
130.6, 130.7, 131.4, 131.8, 136.0, 136.4, 140.8, 152.5.
MS: m/z 439 [MH+], 441 [MH++2]. Anal. Calcd for
C19H14N4Cl4 (MW 440.15): C, 58.85: H, 3.21; N,
12.73%; found: C, 58.75; H, 3.20; N, 12.50%.
4.1.19. ( ) trans-1-[3,5-Bis-(2,4-dichlorophenyl)-4,5-dihy-
dro-1-phenyl-1H-pyrazol-4-yl]-1H-imidazole (1n). Mp
163–165 ꢁC; yield 16%. 1H NMR (CDCl3/TMS): d
5.70 (d, 1H, CH, H5 pyrazole, JH5–H4 = 3.50 Hz); 5.97
(d, 1H, CH, H4 pyrazole, JH4–H5 = 3.50 Hz); 6.83–7.77
(m, 14H, 11H arom. and 3H imidazole). 13C NMR
(CDCl3/TMS): d 66.4, 69.5, 114.9, 117.4, 120.0, 125.5,
127.5, 127.9, 128.4, 130.6, 130.9, 131.1, 131.3, 131.5,
132.0, 133.1, 133.5, 135.8, 135.9, 142.0, 155.6, 160.4.
MS: m/z 501 [MH+], 503 [MH++2]. Anal. Calcd for
C24H16N4Cl4 (MW 502.22): C, 57.40: H, 3.21; N,
11.16%; found: C, 57.35; H, 3.42; N, 11.18%.
4.1.15. ( ) cis-1-[4,5-Dihydro-1-methyl-3,5-bis(4-methyl-
phenyl)-1H-pyrazol-4-yl]-1H-imidazole (1j). Oil; yield
35%. 1H NMR (CDCl3/TMS): d 2.31 (s, 3H, CH3);
2.39 (s, 3H, CH3); 2.96 (s, 3H, N–CH3); 4.14 (d, 1H,
CH, H5 pyrazole, JH5–H4 = 10.98 Hz); 5.58 (d, 1H,
CH, H4 pyrazole, JH4–H5 = 10.98 Hz); 6.90–7.54 (m,

D. Zampieri et al. / Bioorg. Med. Chem. 16 (2008) 4516–4522
4521
4.1.20. ( ) trans-1-[4,5-Dihydro-3,5-bis-(4-methyl-
phenyl)-1-phenyl-1H-pyrazol-4-yl]-1H-imidazole (1o).
4.1.24. ( ) trans-1-(3,5-Bis(2,4-dichlorophenyl)-4,5-dihy-
dro-1-(4-fluorophenyl)-1H-pyrazol-4-yl)-1H-imidazole
(1s). Mp 123–125 ꢁC; yield 27%. 1H NMR (CDCl3/
TMS): d 5.66 (d, 1H, CH, H5 pyrazole, JH5–H4
= 4.34 Hz); 5.95 (d, 1H, CH, H4 pyrazole, JH4–H5
= 4.34 Hz); 6.84–7.65 (m, 13H, 10H arom. and 3H imid-
azole). 13C NMR (CDCl3/TMS): d 67.2, 69.6, 114.7,
115.0, 116.2, 116.7, 117.3, 117.5, 127.5, 127.9, 128.5,
130.6, 130.9, 131.1, 131.4, 131.5, 132.0, 133.0, 133.6,
135.7, 136.0, 142.1, 155.7, 160.5. MS: m/z 519 [MH+],
521 [MH++2]. Anal. Calcd for C24H15N4Cl4F (MW
520.21): C, 55.41: H, 2.91; N, 10.77%; found: C, 55.40;
H, 2.88; N, 10.65%.
1
Mp 184–186 ꢁC; yield 25%. H NMR (CDCl3/TMS): d
2.35 (s, 3H, CH3); 2.39 (s, 3H, CH3); 5.13 (d, 1H, CH,
H5 pyrazole, JH5–H4 = 3.45 Hz); 5.65 (d, 1H, CH, H4
pyrazole, JH4–H5 = 3.45 Hz); 6.80–7.70 (m, 16H, 13H
arom. and 3H imidazole). 13C NMR (CDCl3/TMS): d
21.4, 21.6, 70.1, 73.0, 113.7, 117.1, 120.4, 125.5, 125.7,
127.9, 129.3, 129.7, 130.5, 131.2, 135.4, 135.9, 138.7,
139.4, 142.6, 143.3. MS: m/z 393 [MH+]. Anal. Calcd
for C26H24N4 (MW 392.50): C, 79.56: H, 6.16; N,
14.27%; found: C, 79.70; H, 6.12; N, 14.33%.
4.1.21. ( ) trans-1-(4,5-Dihydro-3,5-diphenyl-1-(4-fluoro-
phenyl)-1H-pyrazol-4-yl)-1H-imidazole (1p). To an etha-
nolic (60 mL) solution of 2a (0.4 g, 1.46 mmol),
triethylamine (0.16 g, 1.46 mmol) and 4-fluor-
ophenylhydrazine hydrochloride (0.24 g, 1.46 mmol)
were added under stirring. The reaction mixture was al-
lowed to reflux for 5 h. The solvent was evaporated un-
der reduced pressure and the residue was extracted with
CHCl3 and washed with distilled water. The collected
organic phases were dried over sodium sulfate, filtered
and evaporated under reduced pressure. The residue
was then purified by column chromatography (eluted
with CH2Cl2) and crystallized from diethyl ether to af-
ford a white solid. Mp 173–175 ꢁC; yield 36%. 1H
NMR (CDCl3/TMS): d 5.11 (d, 1H, CH, H5 pyrazole,
JH5–H4 = 4.29 Hz); 5.67 (d, 1H, CH, H4 pyrazole,
JH4–H5 = 4.29 Hz); 6.87–7.60 (m, 17H, 14H phen. and
3H imidazole). 13C NMR (CDCl3/TMS): d 70.3, 73.7,
114.9, 115.1, 115.6, 116.3, 117.2, 125.7, 126.0, 129.1,
129.2, 129.4, 130.0, 130.7, 131.5, 136.0, 138.1, 139.7,
142.7, 155.4, 160.2. MS: m/z 383 [MH+]. Anal. Calcd
for C24H19N4F (MW 382.43): C, 75.37: H, 5.01; N,
14.65%; found: C, 75.54; H, 5.16; N, 14.48%.
4.1.25. ( ) trans-1-[4,5-Dihydro-1-(4-fluorophenyl)-3,5-
bis-(4-methylphenyl)-1H-pyrazol-4-yl]-1H-imidazole (1t).
Mp 175–178 ꢁC; yield 29%. H NMR (CDCl3/TMS): d
1
2.34 (s, 3H, CH3); 2.36 (s, 3H, CH3); 5.07 (d, 1H, CH,
H5 pyrazole, JH5–H4 = 4.39 Hz); 5.65 (d, 1H, CH, H4
pyrazole, JH4–H5 = 4.39 Hz); 6.85–7.65 (m, 15H, 12H
arom. and 3H imidazole). 13C NMR (CDCl3/TMS): d
21.3, 21.6, 70.1, 72.7, 114.8, 115.1, 116.3, 116.7, 117.3,
128.8, 129.6, 129.7, 130.7, 131.5, 134.4, 137.2, 140.0,
142.4, 155.3, 159.8. MS: m/z 411 [MH+]. Anal. Calcd
for C26H23N4F (MW 410.49): C, 76.08: H, 5.65; N,
13.65%; found: C, 76.00; H, 5.72; N, 13.90%.
4.2. Microbiology
All the new imidazole derivatives were evaluated for
antifungal activity against C. albicans 685, clinical iso-
late; for antitubercular activity against the reference
strain M. tuberculosis H37Rv. Stock solutions of chem-
icals were prepared in DMSO at a concentration of
2 mg/mL. Antifungal activity was always evaluated
by reference methods (NCCLS, 1997); miconazole
and amphotericin B were chosen as a standard in anti-
fungal activity measurements, each MIC was deter-
mined twice in duplicate experiments after 24 and
48 h incubation time. Antitubercular activity was eval-
uated by MRA, a recently developed, 1-week dura-
tion, micro-dilution Resazurin assay.21 The minimum
inhibitory concentration, MIC, was defined as the
lowest drug concentration that prevented Resazurin
colour change from blue to pink and was determined
by visual inspection twice in duplicate experiments;
viable counting from control wells and from test wells
performed onto agar plates confirmed bactericidal and
bacteriostatic activity of compounds. Isoniazid was al-
ways included as a standard in antitubercular activity
measurements, having a MIC of 0.5 lg/mL; DMSO
was also evaluated and was always devoid of inhibit-
ing activity up to the concentration of 2% (v/v). The
antimicrobial activity of the compounds 1a–t is re-
ported in Table 1.
Compounds 1q–t were obtained similarly.
4.1.22. ( ) trans-1-(3,5-Bis(4-bromophenyl)-4,5-dihydro-
1-(4-fluorophenyl)-1H-pyrazol-4-yl)-1H-imidazole (1q).
1
Mp 183–185 ꢁC; yield 30%. H NMR (CDCl3/TMS): d
5.07 (d, 1H, CH, H5 pyrazole, JH5–H4 = 4.27 Hz); 5.61
(d, 1H, CH, H4 pyrazole, JH4–H5 = 4.27 Hz); 6.85–7.72
(m, 15H, 12H arom. and 3H imidazole). 13C NMR
(CDCl3/TMS): d 70.2, 73.1, 115.2, 115.3, 115.6, 116.3,
117.4, 125.8, 126.4, 129.1, 129.5, 129.7, 130.7, 131.8,
136.9, 142.2, 155.4, 160.0. MS: m/z 539 [MH+], 541
[MH++2]. Anal. Calcd for C24H17N4Br2F (MW
540.22): C, 53.36: H, 3.17; N, 10.37%; found: C, 53.34;
H, 3.16; N, 10.37%.
4.1.23. ( ) trans-1-(3,5-Bis(4-chlorophenyl)-4,5-dihydro-
1-(4-fluorophenyl)-1H-pyrazol-4-yl)-1H-imidazole (1r).
1
Mp 180–183 ꢁC; yield 15%. H NMR (CDCl3/TMS): d
5.10 (d, 1H, CH, H5 pyrazole, JH5–H4 = 4.30 Hz); 5.63
(d, 1H, CH, H4 pyrazole, JH4–H5 = 4.30 Hz); 6.85–7.65
(m, 15H, 12H arom. and 3H imidazole). d 70.0, 72.8,
114.8, 115.4, 115.5, 116.6, 117.6, 129.2, 129.5, 129.7,
130.7, 131.5, 131.8, 134.4, 137.2, 142.4, 155.0, 158.9.
MS: m/z 451 [MH+], 453 [MH++2]. Anal. Calcd for
C24H17N4Cl2F (MW 451.32): C, 63.87: H, 3.80; N,
12.41%; found: C, 63.70; H, 3.60; N, 12.35%.
Acknowledgment
This research was carried out with the financial support
of the Italian M.U.R.S.T. (60%) (University of Trieste
2001).

4522
D. Zampieri et al. / Bioorg. Med. Chem. 16 (2008) 4516–4522
10. Biava, M.; Fioravanti, R.; Porretta, G. C.; Sleiter, G.;
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