Synthesis and the selective antifungal activity of 5,6,7,8-tetrahydroimidazo[1,2-a]pyridine derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.12.023

Even though there are new classes of compounds now frequently used in treatment of fungal infections, the density of deeply invasive candidiasis has increased at least 10-fold during the past decade. Furthermore, many infections due to Candida species are actually refractory to antifungal therapy. In this present study, it was aimed to synthesize, new hydrazide derivatives of tetrahydroimidazo[1,2-a]pyridine and evaluate their anticandidal activity and cytotoxicity in vitro. The reaction of tetrahydroimidazo[1,2-a]pyridine-2-carboxylic acid hydrazides with various benzaldehydes gave tetrahydroimidazo[1,2-a]pyridine-2-carboxylic acid benzylidene hydrazide derivatives. The chemical structures of the compounds were elucidated and confirmed by IR, ¹H NMR, MS-FAB⁺ spectroscopy and elemental analyses. Eight new tetrahydroimidazo[1,2-a]pyridine derivatives were synthesized and screened for their antifungal effects against a panel of ten human pathogenic Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Candida utilis, and Candida zeylanoides using agar diffusion and broth microdilution assays, respectively. Furthermore, their cytotoxicity was tested against six mammalian cell lines. Among the analogues, the compound 5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-2-carboxylic acid-(4-cyanobenzylidene) showed very strong inhibitory activity (up to MIC 0.016?mg/mL) against the screened Candida species. The same compound showed no in vitro toxicity up to 25?μg/mL concentration suggesting that its antifungal activity (MICs 0.016-1?mg/mL) is selective.

Full text of DOI:10.1016/j.ejmech.2009.12.023

European Journal of Medicinal Chemistry 45 (2010) 2080–2084
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis and the selective antifungal activity of
5,6,7,8-tetrahydroimidazo[1,2-a]pyridine derivatives
a,
a
a
b
c
_
*
¨
Ahmet Ozdemir , Gu¨lhan Turan-Zitouni , Zafer Asım Kaplancıklı , Go¨kalp Is¸can , Shabana Khan ,
Fatih Demirci b
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskis¸ehir, Turkey
b Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, 26470 Eskis¸ehir, Turkey
c National Center for Natural Products Research, School of Pharmacy, University of Mississippi, MS 38677, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Even though there are new classes of compounds now frequently used in treatment of fungal infections,
the density of deeply invasive candidiasis has increased at least 10-fold during the past decade.
Furthermore, many infections due to Candida species are actually refractory to antifungal therapy. In this
present study, it was aimed to synthesize, new hydrazide derivatives of tetrahydroimidazo[1,2-a]pyridine
and evaluate their anticandidal activity and cytotoxicity in vitro. The reaction of tetrahydroimidazo[1,2-
a]pyridine-2-carboxylic acid hydrazides with various benzaldehydes gave tetrahydroimidazo[1,2-
a]pyridine-2-carboxylic acid benzylidene hydrazide derivatives. The chemical structures of the
compounds were elucidated and confirmed by IR, 1H NMR, MS-FABþ spectroscopy and elemental
analyses. Eight new tetrahydroimidazo[1,2-a]pyridine derivatives were synthesized and screened for
their antifungal effects against a panel of ten human pathogenic Candida albicans, Candida glabrata,
Candida krusei, Candida parapsilosis, Candida tropicalis, Candida utilis, and Candida zeylanoides using agar
diffusion and broth microdilution assays, respectively. Furthermore, their cytotoxicity was tested against
six mammalian cell lines. Among the analogues, the compound 5,6,7,8-tetrahydroimidazo[1,2-a]pyri-
dine-2-carboxylic acid-(4-cyanobenzylidene) showed very strong inhibitory activity (up to MIC
0.016 mg/mL) against the screened Candida species. The same compound showed no in vitro toxicity up
Received 12 January 2009
Received in revised form
1 December 2009
Accepted 10 December 2009
Available online 21 December 2009
Keywords:
Tetrahydroimidazo[1,2-a]pyridine
In vitro antifungal activity
Candida spp.
Cytotoxicity
to 25
m
g/mL concentration suggesting that its antifungal activity (MICs 0.016–1 mg/mL) is selective.
Ó 2009 Published by Elsevier Masson SAS.
1. Introduction
chronically subjected to antimycotic therapy, i.e. those treated with
broad-spectrum antibiotics, immunosuppressive agents, anti-
Even though Candida spp. are commensally present in the
human flora of about 30–60% of healthy individuals, where they
may become pathologic depending on predisposing circumstances
related to the host, as an example of systemic disease leading to
immunosuppression, and local destructive conditions. In fact, the
pathogenicity of Candida spp. is affected by several virulence
factors, such as the ability to adhere to epithelial and endothelial
cells, germination, extracellular proteinases and phospholipases,
and phenotypic switching [1,2].
cancer, and anti-AIDS drugs [3,4].
Azole and non-azole antifungal agents are usually used to treat
Candida infections, but despite the good antifungal activities
observed in vitro, candidemia is still a major cause of death [4].
Many valid therapy programs fail because of widespread secondary
C. albicans infections. New effective anticandida agents are needed
to combat the drug-resistant strains and widespread diffusion of C.
albicans, Candida glabrata, Candida utilis, Candida tropicalis, Candida
krusei, Candida zeylanoides, Candida parapsilosis.
In recent years, fungal infections have increased remarkably and
one of the main targets is the opportunistic pathogen Candida
albicans. A major obstacle in the treatment of C. albicans infections
is the spread of antifungal drug resistance mainly in patients
Imidazoles, the first group to be developed in azole antifungals
also inhibit the accumulation of methylated sterols disrupts the
organization of the lipidic bilayer of membranes. Furthermore, some
imidazole drugs, at high concentrations, could exert direct inhibitory
effects on membranes, without interference with sterols and sterol
esters [5,6]. The literature survey reveals that, although there are
several synthetic study examples of the imidazole ring, there are
none on the imidazo[1,2-a]pyridine ring as an imidazole fused six-
membered ring system, to the best of our knowledge. Furthermore, it
* Corresponding author. Tel.: þ90 222 335 05 80/37 74; fax: þ90 222 335 07 50.
¨
E-mail address: ahmeto@anadolu.edu.tr (A. Ozdemir).
0223-5234/$ – see front matter Ó 2009 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2009.12.023

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A. Ozdemir et al. / European Journal of Medicinal Chemistry 45 (2010) 2080–2084
2081
Table 1
is well known that hydrazide-hydrazone group plays an important
role for antimicrobial activity of such compounds [7–10].
In the design of new drugs, the development of hybrid mole-
cules through the combination of different pharmacophores in one
frame may lead to compounds with interesting biological and
pharmacological profiles.
Prompted by these observations, in the present study, the
synthesis of 8 new hydrazide-hydrazone derivatives of tetrahy-
droimidazo[1,2-a]pyridines as hybrid molecules including different
pharmacophores were aimed initially for in vitro anticandidal as
well as cytotoxic screenings.
Some characteristics of the tetrahydroimidazo[1,2-a]pyridine hydrazine derivatives.
Comp.
R1
R2
M.p. (ꢁC)
Yield (%)
Formula
MW
2a
2b
2c
2d
2e
2f
H
H
H
CH3
N(CH3)2
CH(CH3)2
212–214
276–278
188–190
208–210
266–268
240–242
236–238
230–232
80
90
77
75
92
73
84
81
C16H18N4O
C17H21N5O
C18H22N4O
C16H16N4O3
C15H15N5O3
C15H15BrN4O
C15H15ClN4O
C16H15N5O
282
311
310
312
313
347
302
293
O–CH2–O
H
H
H
H
NO2
Br
Cl
2g
2h
CN
4. Results, discussion and conclusion
2. Chemistry
Among several clear synthetic routes to derivatives 2a–h, we
decided to explore in our synthetic approach the acylhydrazine
derivative 1 as a key intermediate. This compound could be
transformed in 2a–h using classical functional group interconver-
sion, i.e. CONHNH2 / CONHN]CH-Ar. Finally, the new target
compounds 2a–h were obtained, in good yields (73–92%), by
condensing 1 with the corresponding aromatic aldehydes in
ethanol, as illustrated in Table 1 and Scheme 1.
The synthetic route of compounds is outlined in Scheme 1. To
the best of our knowledge, in the present work, tetrahy-
droimidazo[1,2-a]pyridine-2-carboxylic acid hydrazide (1) was
prepared for the first time, in accordance with the method
described in literature [11,12]. The condensation of the acid
hydrazides with appropriate benzaldehydes resulted in the
formation of new tetrahydroimidazo[1,2-a]pyridine-2-carboxylic
acid arylidene hydrazide derivatives such as 2a–h.
Formulas of compounds (2a–h) were found by elemental and
spectroscopic analyses and their structures were determined by IR,
1H NMR, and FABþ-MS spectral data, respectively. IR data provided
functional group evidence for the formation of the expected
structures. In the IR spectra, some significant stretching bands due
to N–H, C]O, C]N, C]N–Ph and N]C–H were at 3389–
3. Biology
3.1. Antifungal activity
3158 cmꢀ1, 1640–1630 cmꢀ1, 1610–1590 cmꢀ1, 1549–1485 cmꢀ1
,
The antifungal properties of compounds 2a–h were evaluated
by the broth microdilution method according to the NCCLS refer-
ence document M27-A2 [13] against C. albicans (isolate, obtained
from Osmangazi University, Faculty of Medicine, Department of
Microbiology, Eskisehir, Turkey), C. albicans (ATCC 90028), C. glab-
rata (isolate-1 obtained from Osmangazi University, Faculty of
Medicine, Department of Microbiology, Eskisehir, Turkey), C. utilis
(NRRL Y-900), C. tropicalis (NRRL Y-12968), C. krusei (NRRL Y-7179),
C. zeylanoides (NRRL Y-1774), C. parapsilosis (NRRL Y-12696),
C. albicans (NRRL Y-12983), C. glabrata (isolate-2 obtained from
Osmangazi University, Faculty of Medicine, Department of Micro-
biology, Eskisehir, Turkey). Ketoconazole, clotrimazole and griseo-
fulvin were used as positive controls.
and 760–750 cmꢀ1, respectively. The 1H NMR data were also
consistent with the assigned structures. In 1H NMR spectrum of
compounds; we observed paired peaks for each of the protons
N]CH (8.35–8.65 ppm), N–NH (11.10–11.75 ppm) corresponding to
(E)- and (Z)-forms of the compounds, respectively. All other
aromatic and aliphatic protons were observed at expected regions.
The mass spectra of compounds showed [M þ 1] peaks, in agree-
ment with their molecular formula. All compounds gave satisfac-
tory elemental analysis results in correlation with the calculations.
The antifungal activity of the compounds was studied with ten
pathogenic fungi. Ketoconazole, griseofulvin and clotrimazole were
used as reference agents for inhibitory activity against the tested
fungi. Minimal inhibitory concentrations (MIC) were recorded as
the minimum concentration of a compound that inhibits the
growth of tested microorganisms. All of the compounds tested
illustrated medium to very good anticandidal inhibitory activity
when compared with the reference agents. The MIC values were
found within the range of 0.016–1 mg/mL against all evaluated
strains. Some of the compound inhibitory concentrations were
expressed as >0.25 where some solubility problems were
observed. The results are summarized in Table 2.
3.2. Cytotoxicity
The compounds synthesized (2a–h) were tested for their initial
in vitro cytotoxicity against a panel of human solid tumor cells (SK-
MEL: malignant melanoma; KB: oral epidermal carcinoma; BT-549:
breast ductal carcinoma, and SK-OV-3: ovary carcinoma) as well as
noncancerous kidney fibroblast (Vero) and kidney epithelial cells
(LLC-PK11) [14].
N
N
Ethanol
NH-NH2
H2N-NH2
+
O
N
N
H
O
O
1
H
N
O
R1
R2
Absolute
Ethanol
N
N
N
1
+
O
R2
2a-h
R1
Scheme 1. The general synthetic reaction (see Table 1 for 2a–h, R1–R2 details).

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A. Ozdemir et al. / European Journal of Medicinal Chemistry 45 (2010) 2080–2084
2082
Table 2
Anticandidal evaluation of tetrahydroimidazo[1,2-a]pyridine hydrazine derivatives (MIC values in mg/mL).
Test substances
Aa
B
Ca
D
E
F
G
H
I
Ka
2a
2b
2c
2d
2e
2f
2g
2h
0.032
0.125
0.125
0.063
0.125
1
0.125
0.032
0.063
0.25
>0.25b
>0.25b
>0.25b
>0.25b
0.5
0.5
0.5
0.063
0.063
0.25
>0.25b
>0.25b
>0.25b
>0.25b
0.5
>0.25b
>0.25b
>0.25b
0.125
0.5
0.125
0.125
0.125
0.125
0.25
>0.25b
>0.25b
>0.25b
>0.25b
0.5
>0.25b
>0.25b
>0.25b
>0.25b
0.25
0.5
>0.25b
>0.25b
>0.25b
>0.25b
0.25
0.125
0.5
0.032
0.032
0.5
>0.25b
>0.25b
0.5
>0.25b
>0.25b
>0.25b
0.25
>0.25b
0.25
0.5
1
0.5
0.25
1
1
0.5
0.032
0.032
0.5
0.25
>0.25b
0.032
0.125
0.25
>0.25b
0.032
0.125
0.5
>0.25b
0.016<
0.125
>0.25b
0.016<
0.125
0.25
>0.25b
0.032
0.5
0.125
0.032
0.032
0.125
0.08<
Ketoconazole
Griseofulvin
Clotrimazole
0.125
0.032
0.5
0.125
0.08<
0.08<
0.08<
0.08<
0.08<
0.08<
0.08<
A: Candida albicans (clinical isolate), B: Candida albicans (ATCC 90028), C: Candida glabrata (clinical isolate-1), D: Candida utilis (NRRL Y-900), E: Candida tropicalis (NRRL
Y-12968), F: Candida krusei (NRRL Y-7179), G: Candida zeylanoides (NRRL Y-1774), H: Candida parapsilosis (NRRL Y-12696), I: Candida albicans (NRRL Y-12983), K: Candida
glabrata (clinical isolate-2).
a
Clinical isolates were obtained from Osmangazi University, Faculty of Medicine, Department of Microbiology, Eskisehir, Turkey.
Represents insolubility of the substance in the medium, with partial inhibition towards the microorganism.
b
In comparing MIC values with the standard reference agents,
5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-2-carboxylic acid-(4-cya-
nobenzylidene) (2h) was in particular strong inhibitory against C.
tropicalis and C. krusei (0.016 < mg/mL), respectively. Compound 2h
showed very strong inhibitory activity (0.016–0.063 mg/mL) towards
all screened Candida species.
Strong inhibitory activity with a MIC value of 0.063 mg/mL was
also observed for compound 2d against the clinical isolate of
C. albicans.
Compounds 2a, 2d, and 2h were also effective against C. albicans
(clinical isolate). Compounds 2a and 2h especially showed strong
inhibitory activity against the tested microorganisms. Compound
2d showed a similar level of activity with the reference agent
whereas 2b, 2c, 2e and 2g showed moderate activity.
In comparing their MIC values with ketoconazole, compounds
2d and 2h were effective against C. utilis (NRRL Y-900). Compound
2h especially showed high activity. Compound 2d showed equal
activity when compared with the reference agent.
2b, 2c, and 2g especially showed strong activity. Compound 2e
showed similar activity, whereas the compound 2f showed
moderate activity when compared with griseofulvin.
From the similar results obtained with C. tropicalis (NRRL
Y–12 968). The compound 2h especially showed strong activity. 2a,
2b, 2c, and 2d showed similar activity and the other compound 2e
indicated moderate activity with reference agent.
In comparing their MIC values with griseofulvin, all of the
compounds were effective against C. zeylanoides (NRLL Y-1774). The
compound 2h, 2e, 2a, 2b, 2c, 2d and 2g especially showed strong
activity. The compound 2f showed moderate activity when
compared with griseofulvin. All of the compounds were effective
against C. parapsilosis (NRRLY-12 696). The compound 2h, 2b, 2c, 2d
and 2e especially showed strong activity. 2a and 2g showed similar
activity and the other compound 2f indicated moderate activity with
reference agent. All of compounds were effective against C. albicans
(NRRL Y-12 983), Compound 2h, 2f, 2e, 2a, 2b, 2c and 2d especially
showed high activity. Compound 2g showed equal activity.
On the other hand the compounds exhibited comparable
activities against C. tropicalis (NRRL Y-12968). The compound 2h
especially showed strong activity. 2a, 2b, 2c, and 2d showed similar
activity and the other compounds were found less active than
ketoconazole.
From the similar results obtained with C. zeylanoides (NRLL
Y-1774), compounds 2a, 2b, 2c, 2d, 2e, 2g, and 2h showed strong
activity, whereas the compound 2f showed moderate activity when
compared with ketoconazole.
When compared with their MIC values against clotrimazole,
compound 2h was effective against C. albicans (ATCC 90028),
C. glabrata (isolate-1), C. utilis (NRRL Y-900), C. tropicalis (NRRL
Y-12968), C. krusei (NRRL Y-7179), C. zeylanoides (NRRL Y-1774),
C. parapsilosis (NRRL Y-12696), C. albicans (NRRL Y-12983), C. glab-
rata (isolate-2). Compound 2h especially showed strong activity.
The other compounds were found less active than clotrimazole.
In comparing their MIC values with clotrimazole, compounds 2a,
2h and 2d were effective against C. albicans (isolate). Compounds 2a,
2h and 2d especially showed strong inhibitory activity.
In comparing their MIC values with griseofulvin, all of
compounds were effective against C. albicans (isolates). The
compound 2f was found inactive against C. albicans (isolate) when
compare with griseofulvin. All of compounds were effective against
C. albicans (ATCC 90028), C. glabrata (isolate-1), and C. krusei (NRRL
Y-7179). Compound 2h especially showed high activity.
Compounds 2a, 2b, 2c, 2d, 2e, 2f, and 2g showed moderate activity
when compared with the reference agent.
In comparing their MIC values with griseofulvin, compounds 2h,
2g, 2e and 2f were effective against C. glabrata (isolate-2), and
among them compound 2h, especially, showed strong activity.
Compound 2g showed
a similar level of inhibitory activity
compared with the reference agent, whereas 2e and 2f showed
moderate activity.
Considering all the results obtained from antifungal screen, in
comparison with reference agents, it can be concluded that 2h
compound is more active than other compounds in the screen
including the reference agents.
All compounds were also tested for their cytotoxicity against
a panel of cancer cell lines (SK-MEL, KB, BT-549, and SK-OV-3) and
noncancer cells (VERO, LLC-PK11). None of the tested compounds
showed any cytotoxicity to any of the cell lines up to a highest
concentration of 25
candidal activity.
mg/mL indicating a high selectivity of anti-
Based on eight compounds evaluated, it appears that 4-cyano
(2h) substitution on the phenyl ring has made a good contribution
to the antifungal activity in this series of imidazopyridinyl–hydra-
zon combination without cell toxicity against selected cell lines.
It is well known that azole antifungal agents act on the synthesis
of the fungal ergosterol by inhibiting the cytochrome P450-
dependent enzyme lanosterol demethylase, which also plays an
important role in cholesterol synthesis in mammals. In therapeutic
concentrations, azole antifungal efficacy is attributed to their
greater affinity and selectivity for fungal P-450DM than for the
mammalian enzyme. Generally, exposure of fungi to an active azole
On the other hand the compounds exhibited comparable
activities against C. utilis (NRRL Y-900). The compound 2h, 2d, 2a,
causes depletion of ergosterol and accumulation of 14
sterols, which interferes with the important functions of ergosterol
a-methylated

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2083
in fungal membranes. Azoles disrupt both the structure of the
membrane and several of its functions such as nutrient transport
and fungal chitin synthesis [3].
and C7 protons of tetrahydroimidazo[1,2-a]pyridine), 2.75 (2H, t, C8
protons of tetrahydroimidazo[1,2-a]pyridine), 3.00 (6H, s, N(CH3)2),
4.00 (2H, t, C5 protons of tetrahydroimidazo[1,2-a]pyridine), 6.75
and 7.45 (4H, 2 d J ¼ 8.60 Hz and J ¼ 8.84 Hz, 1,4-disubstituted
phenyl protons), 7.62 (1H, s, C3 protons of tetrahydroimidazo[1,2-
a]pyridine), 8.35 (1H, s, N]CH), 11.10 (1H, s, N–NH); MS-FABþ: m/z:
311 [M], 312 [M þ 1]. Anal. Calc. for C17H21N5O:C, 65.57; H, 6.80; N,
22.49. Found:C, 65.70; H, 6.84; N, 22.57.
To the best of our knowledge tetrahydroimidazo[1,2-a]pyridine
ring was investigated for the first time for in vitro biological activ-
ities. As the tetrahydroimidazo[1,2-a]pyridine ring in the present
study is a bioisoster of the imidazole ring, which also include
important azole antifungal therapeutics, it can be concluded that
the compounds also display remarkable antifungal activity by using
same mechanism of the current azole antifungals. It is worthwhile
to extend the bioactivity evaluation from in vitro whole cells to
a mechanistic enzymatic level as well as to further in vivo studies.
5.1.2.3. 5,6,7,8-Tetrahydroimidazo[1,2-a]pyridine-2-carboxylic acid-
(4-isopropilbenzylidene) hydrazide (2c). The physical appearance:
shining brown solid. IR (KBr, cmꢀ1): 3195 (NH), 1636 (C]O), 1590
(C]N), 1520 (C]C–H), 1495 (C]N–Ph), 753 (N]C–H); 1H NMR
5. Experimental
(250 MHz) (DMSO-d6) d (ppm): 1.25 (6H, s, 2CH3), 1.80–1.90 (4H, br,
C6 and C7 protons of tetrahydroimidazo[1,2-a]pyridine), 2.73 (2H, t,
C8 protons of tetrahydroimidazo[1,2-a]pyridine), 2.80–3.00 (1H, m,
CH(CH3)2), 4.05 (2H, t, C5 protons of tetrahydroimidazo[1,2-
a]pyridine), 7.30 and 7.60 (4H, 2 d J ¼ 8.18 Hz and J ¼ 8.19 Hz, 1,4-
disubstituted phenyl protons), 7.70 (1H, d J ¼ 6.25 Hz, C3 protons of
tetrahydroimidazo[1,2-a]pyridine), 8.45 (1H, s, N]CH), 11.35 (1H, s,
N–NH); MS-FABþ: m/z: 310 [M], 311 [M þ 1]. Anal. Calc. for
C18H22N4O:C, 69.65; H, 7.14; N, 18.05. Found:C, 69.73; H, 7.19; N,
18.00.
All melting points (m.p.) were determined in open capillaries on
a Gallenkamp apparatus (Weiss-Gallenkamp, Loughborough-UK),
which are uncorrected, given in Table 1. The purity of the
compounds was routinely checked by silica gel thin layer chro-
matography (TLC) plates (60 G, Merck, Darmstadt-Germany).
Spectroscopic data were recorded with the following instruments:
IR; Shimadzu IR-435 spectrophotometer (Shimadzu, Tokyo, Japan);
1H NMR: Bruker 250 MHz spectrometer (Bruker, Billerica, Massa-
chusetts, USA) in DMSO-d6 using TMS as internal standard; and
FAB-MS: VG Quattro Mass spectrometer (Agilent, Minnesota, USA).
Elemental analyses were performed with a Leco CHNS-932 (LECO
Corporation, Michigan, USA) instrument.
5.1.2.4. 5,6,7,8-Tetrahydroimidazo[1,2-a]pyridine-2-carboxylic acid-
(benzo[1,3]dioxol-5-ylmethy-lene)hydrazide
(2d). The
physical
appearance: light brown powder. IR (KBr, cmꢀ1): 3183 (NH), 1631
(C]O), 1592 (C]N), 1549 (C]N–Ph), 755 (N]C–H); 1H NMR
5.1. Chemistry
(250 MHz) (DMSO-d6) d (ppm): 1.80–1.90 (4H, br, C6 and C7 protons
of tetrahydroimidazo[1,2-a]pyridine), 2.75 (2H, t, C8 protons of tet-
rahydroimidazo[1,2-a]pyridine), 3.95 (2H, t, C5 protons of tetrahy-
droimidazo[1,2-a]pyridine), 6.05 (2H, s, O–CH2–O), 6.90–7.25 (3H,
m, phenyl protons), 7.65 (1H, s, C3 protons of tetrahydroimidazo[1,2-
a]pyridine), 8.40 (1H, s, N]CH), 11.30 (1H, s, N–NH); MS-FABþ: m/z:
312 [M], 313 [M þ 1]. Anal. Calc. for C16H16N4O3:C, 61.53; H, 5.16; N,
17.94. Found:C, 61.33; H, 5.21; N, 17.76.
5.1.1. Preparation of tetrahydroimidazo[1,2-a]pyridine-2-carboxylic
acid hydrazides (1)
These compounds were prepared according to the previously
reported method, by reacting ethyl tetrahydroimidazo[1,2-a]pyri-
dine-2-carboxylates with hydrazine hydrate.
5.1.2. Preparation of tetrahydroimidazo[1,2-a]pyridine-2-carboxylic
acid arylidenehydrazides derivatives (2a–h)
Equimolar quantities of acid hydrazides (30 mmol) and appro-
priate benzaldehydes in 25 ml of absolute ethanol were refluxed for
3–5 h. The resulting solid was filtered and recrystallized from
ethanol.
5.1.2.5. 5,6,7,8-Tetrahydroimidazo[1,2-a]pyridine-2-carboxylic acid-
(4-nitrobenzylidene)hydrazide (2e). The physical appearance: fine
needle shaped yellow crystals. IR (KBr, cmꢀ1): 3175 (NH), 1633
(C]O),1610 (C]N),1588 (C]C–H),1545 (C]N–Ph),1518,1340 (N–
O), 760 (N]C–H); 1H NMR (250 MHz) (DMSO-d6)
d (ppm): 1.75–
Some characteristics of the synthesized compounds are shown
in Table 1. Analytical and spectral data (IR, 1H NMR, MS-FABþ)
confirmed the structures of the new compounds.
1.95 (4H, br, C6 and C7 protons of tetrahydroimidazo[1,2-a]pyri-
dine), 2.75 (2H, t, C8 protons of tetrahydroimidazo[1,2-a]pyridine),
4.00 (2H, t, C5 protons of tetrahydroimidazo[1,2-a]pyridine), 7.70
(1H, s, C3 protons of tetrahydroimidazo[1,2-a]pyridine), 7.85 and
8.25 (4H, 2 d J ¼ 8.86 Hz and J ¼ 8.84 Hz, 1,4-disubstituted phenyl
protons), 8.60 (1H, s, N]CH), 11.75 (1H, s, N–NH); MS-FABþ: m/z:
314 [M þ 1]. Anal. Calc. for C15H15N5O3:C, 57.50; H, 4.83; N, 22.35.
Found:C, 57.44; H, 4.86; N, 22.38.
5.1.2.1. 5,6,7,8-Tetrahydroimidazo[1,2-a]pyridine-2-carboxylic acid-
(4-methylbenzylidene)hydrazide (2a). The physical appearance:
light brown solid. IR (KBr, cmꢀ1): 3201 (NH), 1635 (C]O), 1600
(C]N), 1534, 1508 (C]N–Ph), 760 (N]C–H); 1H NMR (250 MHz)
(DMSO-d6)
d (ppm): 1.85–1.90 (4H, br, C6 and C7 protons of tetra-
hydroimidazo[1,2-a]pyridine), 2.35 (3H, s, CH3), 2.80 (2H, t, C8
protons of tetrahydroimidazo[1,2-a]pyridine), 4.00 (2H, t, C5
protons of tetrahydroimidazo[1,2-a]pyridine), 7.25 and 7.55 (4H, 2 d
J ¼ 8.01 Hz and J ¼ 8.05 Hz, 1,4-disubstituted phenyl protons), 7.65
(1H, d J ¼ 6.80 Hz, C3 protons of tetrahydroimidazo[1,2-a]pyridine),
8.45 (1H, s, N]CH), 11.35 (1H, s, N–NH); MS-FABþ: m/z: 282 [M],
283 [M þ 1]; Anal. Calc. for C16H18N4O:C, 68.06; H, 6.43; N, 19.84.
Found:C, 68.09; H, 6.41; N, 19.88.
5.1.2.6. 5,6,7,8-Tetrahydroimidazo[1,2-a]pyridine-2-carboxylic acid-
(4-bromobenzylidene)hydrazide (2f). The physical appearance: white
powder. IR (KBr, cmꢀ1): 3192 (NH), 1630 (C]O), 1602 (C]N), 1543
(C]N–Ph), 750 (N]C–H), 510 (C-Br); 1H NMR (250 MHz) (DMSO-d6)
d
(ppm):1.75–1.95(4H, br, C6 and C7 protonsoftetrahydroimidazo[1,2-
a]pyridine), 2.75 (2H, t, C8 protons of tetrahydroimidazo[1,2-a]pyri-
dine), 3.95 (2H, t, C5 protons of tetrahydroimidazo[1,2-a]pyridine),
7.50–7.70 (5H, m, aromatic protons), 8.50 (1H, s, N]CH), 11.50 (1H, s,
N–NH);MS-FABþ: m/z: 346[Mꢀ 1], 347 [M], 348 [Mþ 1], 349 [M þ 2].
Anal. Calc. for C15H15BrN4O:C, 51.89; H, 4.35; N, 16.14. Found:C, 52.01;
H, 4.32; N, 16.02.
5.1.2.2. 5,6,7,8-Tetrahydroimidazo[1,2-a]pyridine-2-carboxylic acid-
(4-dimethylaminobenzylidene)-hydrazide
(2b). The
physical
appearance: yellow solid. IR (KBr, cmꢀ1): 3389, 3158 (NH), 1640
(C]O), 1596 (C]N), 1524 (C]C–H), 1485 (C]N–Ph), 750 (N]C–
5.1.2.7. 5,6,7,8-Tetrahydroimidazo[1,2-a]pyridine-2-carboxylic acid-
(4-chlorobenzylidene)hydrazide (2g). The physical appearance:
H); 1H NMR (250 MHz) (DMSO-d6)
d
(ppm): 1.80–1.90 (4H, br, C6

¨
A. Ozdemir et al. / European Journal of Medicinal Chemistry 45 (2010) 2080–2084
2084
needle shaped white crystals. IR (KBr, cmꢀ1): 3199 (NH), 1634
the stock solutions at an initial concentration of 2 mg/mL. Serial
dilution series were prepared in 100 l MHB with an equal amount
(C]O), 1605 (C]N), 1539 (C]N–Ph), 750 (N]C–H); 1H NMR
m
(250 MHz) (DMSO-d6)
d
(ppm): 1.80–2.00 (4H, br, C6 and C7 protons
of the test samples. The last row was filled only with water as
growth control for the microorganism. Overnight grown microor-
ganism suspensions were first diluted in double strength MHB and
standardized to 108 CFU/mL (using McFarland No: 0.5) under sterile
conditions. Then each microorganism suspension was pipetted into
each well and incubated at 37 ꢁC for 24 h. Ketoconazole was used as
a standard antifungal agent against Candida spp. Sterile distilled
water and medium served as a positive growth control. The first
well without turbidity was assigned as the minimum inhibitory
concentration (MIC, in mg/mL). Average results of separately per-
formed three experiments as given in Table 2.
of tetrahydroimidazo[1,2-a]pyridine), 2.80 (2H, t, C8 protons of
tetrahydroimidazo[1,2-a]pyridine), 4.00 (2H, t, C5 protons of tet-
rahydroimidazo[1,2-a]pyridine), 7.50 and 7.65 (4H, 2 d J ¼ 8.52 Hz
and J ¼ 8.48 Hz, 1,4-disubstituted phenyl protons), 7.71 (1H, s, C3
protons of tetrahydroimidazo[1,2-a]pyridine), 8.50 (1H, s, N]CH),
11.55 (1H, s, N–NH); MS-FABþ: m/z: 303 [M þ 1], 304 [M þ 2], 305
[M þ 3]. Anal. Calc. for C15H15ClN4O: C, 59.51; H, 4.99; N, 18.50.
Found:C, 59.43; H, 5.00; N, 18.35.
5.1.2.8. 5,6,7,8-Tetrahydroimidazo[1,2-a]pyridine-2-carboxylic acid-
(4-cyanobenzylidene)hydrazide (2h). The physical appearance: pale
yellow crystals. IR (KBr, cmꢀ1): 3189 (NH), 2220 (C^N), 1633 (C]O),
1602 (C]N), 1544 (C]N–Ph), 760 (N]C–H); 1H NMR
5.2.5. Assay for in vitro cytotoxicity
The compounds were tested for their in vitro cytotoxicity against
a panel of four human cancer cell lines [SK-MEL: malignant, mela-
noma, KB: epidermal carcinoma, BT-549: breast ductal carcinoma,
SK-OV-3: ovary carcinoma] and two noncancerous cell lines [VERO:
African green monkey kidney fibroblast and LLC-PK11: pig kidney
epithelial cells] [14]. All the cell lines were from the American Type
Culture Collection (Manassas, VA). Cells (25.000 cells/well) were
seeded to the wells of 96-well plate and incubated for 24 h. Samples
at different concentration were added followed by an incubation for
48 h. The number of viable cells was determined by Neutral Red assay
according to a modification of the procedure of Borenfreund et al.
[16]. Briefly, the cells were washed with saline and incubated for 3 h
with a solution of NeutralRed. The cells werewashed again to remove
extracellular dye. A solution of acidified ethanol was added to liberate
the incorporated dye fromviable cells and the absorbance was read at
450 nm. Percent cell viability was calculated in comparison to solvent
control. Doxorubicin was used as the drug control.
(250 MHz) (DMSO-d6)
d (ppm): 1.85–2.05 (4H, br, C6 and C7 protons
of tetrahydroimidazo[1,2-a]pyridine), 2.82 (2H, t, C8 protons of te-
trahydroimidazo[1,2-a]pyridine), 4.05 (2H, t, C5 protons of tet-
rahydroimidazo[1,2-a]pyridine), 7.75 (1H, s, C3 protons of
tetrahydroimidazo[1,2-a]pyridine), 7.85 and 7.95 (4H, 2 d J ¼ 8.43 Hz
and J ¼ 8.38 Hz,1,4-disubstituted phenyl protons), 8.65 (1H, s, N]CH),
11.75 (1H, s, N–NH); MS-FABþ: m/z: 294 [M þ 1]. Anal. Calc. for
C16H15N5O:C,65.52;H, 5.15;N,23.88. Found:C, 65.33;H, 5.08;N,23.82.
5.2. Bioassays
5.2.1. Antifungal assays and microorganisms
Microorganisms were obtained from ATCC, NRRL and clinical
isolates (Faculty of Medicine, Eskisehir Osmangazi University,
Turkey) and were stored in 15% glycerol containing micro-test
tubes at ꢀ86 ꢁC (strain numbers of microorganisms were given in
Table 2). All Candida strains were inoculated on Sabouraud Dextrose
Agar (SDA) prior the experiments at 37 ꢁC. After sufficient growth
Candida spp. were then transferred to Mueller Hinton Broth (MHB)
for further incubation at the same conditions for another 24 h.
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The activity of the compounds (1a–e, 2a–e) were first screened
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