Combinatorial approach: Identification of potential antifungals from triazole minilibraries

Article abstract of DOI:10.1007/s00044-009-9283-8

Employing basic principles of solution phase combinatorial chemistry, a solution phase combinatorial synthesis and screening of mini libraries of 1,2,4-triazole derivatives has been carried out. 6 x 6 indexed mini libraries were synthesized comprising of 36 compounds. The libraries were analyzed by liquid chromatography - mass spectrometry - mass spectrometry (LC - MS - MS) analysis. All the synthesized mini libraries were screened for antifungal activity and by deconvolution methodology leads for every fungi used for study were identified. The leads were synthesized individually and screened for activity. The antifungal activity of individually synthesized leads was improved as anticipated, in comparison with that of any of the mini libraries. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-009-9283-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:116–120
DOI 10.1007/s00044-009-9283-8
ORIGINAL RESEARCH
Combinatorial approach: identification of potential antifungals
from triazole minilibraries
•
Manish Sudesh Bhatia Bandu Eknath Zarekar
•
•
•
Prafulla Balkrishna Choudhari Kundan Bhanudas Ingale
Neela Manish Bhatia
Received: 30 March 2009 / Accepted: 3 December 2009 / Published online: 9 January 2010
Ó Springer Science+Business Media, LLC 2010
Abstract Employing basic principles of solution phase
combinatorial chemistry, a solution phase combinatorial
synthesis and screening of mini libraries of 1,2,4-triazole
derivatives has been carried out. 6 9 6 indexed mini
libraries were synthesized comprising of 36 compounds.
The libraries were analyzed by liquid chromatography–
mass spectrometry–mass spectrometry (LC–MS–MS)
analysis. All the synthesized mini libraries were screened
for antifungal activity and by deconvolution methodology
leads for every fungi used for study were identified. The
leads were synthesized individually and screened for
activity. The antifungal activity of individually synthesized
leads was improved as anticipated, in comparison with that
of any of the mini libraries.
and yields much more analogues. The advantage of solu-
tion phase over solid phase combinatorial chemistry is that
there is no need to develop strategy for either attachment of
substrate to the solid support or its subsequent cleavage.
Solution phase techniques are, however, limited to short
reaction sequences that either use reagents in stoichiome-
tric quantities, or use reagents that can be easily separated
from the final products (Patrick, 2003; Terret, 1998).
Various derivatives of 1,2,4-triazole are known to exhibit
pharmacological properties like antibacterial, antifungal
anticancer, anti-viral, anti-inflammatory, CNS depressant
and anticonvulsant activity (Amir and Azam, 2004; Amir
et al., 1999; Banachiewicz et al., 2004, Colanceska-
Ragenovic et al., 2001, Metwally et al., 2007, Mishra et al.,
1991; Parmar et al., 1972; Ravi and Rajkannan, 2004). In
this work, focus is mainly on combinatorial synthesis and
high throughput screening of 1,2,4-triazole derivatives
(shown in Scheme 1 and Table 1) and identification of hits
for antifungal activity by 2D deconvolution analysis. The
synthesized mini libraries were confirmed by the separation
of the components by TLC, and co-spotting of the indi-
vidually synthesized components of library. The synthe-
sized mini libraries were screened for antifungal activity
and deconvolution studies identified hits which were syn-
thesized individually and screened for their antifungal
activity.
Keywords Solution phase ꢀ Combinatorial synthesis ꢀ
1,2,4-Triazole ꢀ Antifungal activity
Introduction
Combinatorial chemistry is a technique by which large
numbers of structurally distinct molecules can be synthe-
sized in a short time and submitted for pharmacological
assay. This process is thus rapid and interactive fine tuning
similar to conventional lead optimization is much faster
Result and discussion
Esters of acids like benzoic acid, anthranilic acid, salicylic
acid, 4-aminobenzoic acid, phenyl acetic acid were reacted
with hydrazine hydrate (99%) to obtain the respective
hydrazides. These hydrazides were cyclized to form 1,2,4-
triazole using carbon disulphide and potassium hydroxide in
M. S. Bhatia (&) ꢀ B. E. Zarekar ꢀ P. B. Choudhari ꢀ
K. B. Ingale ꢀ N. M. Bhatia
Department of Pharmaceutical Chemistry, Bharati Vidyapeeth
College of Pharmacy, Near Chitranagri, Kolhapur 416013,
Maharashtra, India
e-mail: manish_bhatia13@yahoo.co.in
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Med Chem Res (2011) 20:116–120
117
with selected aromatic aldehydes in acetic acid. The syn-
thesized lead molecules were also screened for their anti-
fungal activity and the screening results are shown in
Table 2. The mini-libraries with best activity against each
fungal strain are highlighted in bold and were employed for
identifying the lead structures. The synthesized lead mole-
cules were characterized by IR, 1H-NMR, and elemental
analysis. In each of the synthesized mini-libraries, the
absence of signals in the region 1,240–1,275 cm-1 in IR
spectral data established the absence of C=S. The IR spectra
of schiff bases showed characteristic absorption bands
between 1,570 and 1,600 cm-1. The 1H-NMR characteristic
signals of compounds were observed at d 8.58–8.78 ppm (s,
1H, N=CH). Moreover, the elemental analysis results were
all in good agreement with the structures proposed for lead
molecule. The hits identified were 5-benzylthio-N-[(4-
methoxyphenyl)methylene]-3-pyridin-4-yl-4H-1,2,4-tria-
zol-4-amine (L1) for A. niger, 3-(2-aminophenyl)-N-[(4-
chlorophenyl)methylene]-5-(benzylthio)-4H-1,2,4-triazol-
4-amine (L2) for A. flavus, 3-(2-aminophenyl)-N-[(4-
methoxyphenyl)methylene]-5-benzylthio-4H-1,2,4-triazol-
4-amine (L3) for P. cryogenus and 3-(2-aminophenyl)-N-
[(4-dimethlyphenyl) methylene]-5-(benzylthio)-4H-1,2,4-
triazol-4-amine (L4) for F. oxysporium. The hits were syn-
thesized individually and complete characterization of the
hits was performed to confirm the synthesis. The IR spectrum
of the compounds showed peaks at 1710 C=O, 1105 C–O–C,
1670 C=N. The peak of NH stretching disappeared and
emergence of C=N peak confirmed formation of imines. The
N
N
i) CS2/KOH,ROH
NH2NH2.H2O
ArCONHNH2
ArCOOR
Ar
SH
N
ii) NH2NH2.H2O
NH2
ROH/KOH
N
C6H5CH2Cl
N
N
N
Ar
N
N
SCH2C6H5
Ar'CHO
Ar
N
SCH2C6H5
CH3COOH
R
NH2
Scheme 1 For synthesis of mini-libraries
the presence of alcohol via formation of potassium-3-
dithiocarbazinate. These 3-substituted-4-amino-5-mer-
capto-4H-1,2,4-triazoles were further reacted with benzyl
chloride in the presence of KOH to form substituted triazoles
which were used as building blocks of the mini-combinato-
rial libraries. The minilibraries were prepared by reacting
substituted triazole and aromatic aldehydes to form pool of
imines with 1,2,4-triazole as basic core and vise a versa to
form a pool of imines with a common aromatic structure and
different 1,2,4-triazoles. The libraries thus formed were
confirmed by TLC, IR, and liquid chromatography–mass
spectrometry–mass spectrometry (LC–MS–MS) data and
screened for antifungal activity. The hits were identified by
2D deconvolution methodology and then synthesized indi-
vidually. Hits identified were synthesized by the condensa-
tion of 4-amino-5-benzylthio-3-subtituted-4H-1,2,4-triazole
Table 1 Combinatorial Libraries
Sr.no.
BM1
BM2
BM3
BM4
BM5
BM6
Ar
R
1
C6H5–
2-NH2C6H5–
2-OHC6H5–
4-NH2–C6H5–
C6H5–CH2–
4-C5H5N–
H
H
H
H
H
H
2
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
3
2-OH
2-OH
2-OH
2-OH
2-OH
2-OH
4
4-N(CH3)2
4-OCH3
3,4,5-OCH3
4-N(CH3)2
4-OCH3
3,4,5-OCH3
4-N(CH3)2
4-OCH3
3,4,5-OCH3
4-N(CH3)2
4-OCH3
3,4,5-OCH3
4-N(CH3)2
4-OCH3
3,4,5-OCH3
4-N(CH3)2
4-OCH3
3,4,5-OCH3
5
6
Sr.no.
MB1
H
MB2
2-Cl
MB3
MB4
MB5
MB6
R
Ar
1
2-OH
4-N(CH3)2
4-OCH3
3,4,5-OCH3
C6H5N–
C6H5N–
C6H5N–
C6H5N–
C6H5N–
C6H5N–
2
2-NH2C6H5–
2-OHC6H5–
4-NH2C6H5–
C6H5–CH2–
4-C5H5–
2-NH2C6H5–
2-OHC6H5–
4-NH2C6H5–
C6H5–CH2–
4-C5H5–
2-NH2C6H5–
2-OHC6H5–
4-NH2C6H5–
C6H5–CH2–
4-C5H5–
2-NH2C6H5–
2-OHC6H5–
4-NH2C6H5–
C6H5–CH2–
4-C5H5–
2-NH2C6H5–
2-OHC6H5–
4-NH2C6H5–
C6H5–CH2–
4-C5H5–
2-NH2C6H5–
2-OHC6H5–
4-NH2C6H5–
C6H5–CH2–
4-C5H5–
3
4
5
6
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Med Chem Res (2011) 20:116–120
Table 2 Showing MIC of mini
libraries
Library code MIC (lg/ml)
Aspergillus niger Penicillium chrysogenum Aspergillus flavus Fusarium oxysporum
BM1
250
250
250
250
250
62
125
62
250
62
62
125
250
250
250
125
500
125
125
62
BM2
BM3
1000
125
250
1000
125
250
250
62
250
125
125
500
250
250
500
250
62
BM4
BM5
BM6
MB1
125
62
MB2
MB3
500
500
125
250
–
MB4
MB5
500
1000
–
125
250
–
MB6
500
–
Fluconazole
Table 3 Table showing IR and NMR data of lead molecules
Lead
L1
R
Ar
I.R (KBr) cm-1
NMR (DMSO)d ppm
4-OCH3
C6H5N–
2290 (CH), 1647.36 (C=N),
2830.15(O-CH3).
9.19 (s, 1H, CH=N), 8.48 (s, 2H, pyridine), 7–8.3
(m, 9H Ar) 6.73 (s, 2H, pyridine), 3.28 (S, 3H
OCH3), 2.51 (s, 2H, SCH2).
L2
L3
L4
4-Cl
2-NH2C6H5–
2-NH2C6H5–
C6H5CH2
3457 (N–H), 2290 (CH), 1647.36 (C=N),
742.46 (C–Cl).
9.19 (s, 1H, CH=N), 7–8.5 (13H), 2.52 (s, 2H,
SCH2), 2.1 (s, 2H, NH2).
4-OCH3
4-OCH3
3447 (N–H), 1647.36(C=N),
2830.15(O-CH3).
9.12 (s, 1H, CH=N), 6.9–7.8 (m, 13H, Ar), 3.28 (S,
3H OCH3), 2.51 (s, 2H, SCH2), 1.44 (s, 2H, NH2)
3138 (C–H), 2830.15 (O-CH3), 1626
(C=N), 830 (C–N).
9.13 (s, 1H, CH=N), 7.1–8.2 (m, 14H, Ar), 3.30 (s,
3H, OCH3), 2.66 (s, 2H, C6H5CH2), 2.51 (s, 2H,
SCH2).
Table 4 Table showing
antibacterial activity of the lead
molecules
Library code MIC (lg/ml)
Aspergillus niger Penicillium chrysogenum Aspergillus flavus Fusarium oxysporum
L1
31
125
62
125
31
250
125
32
250
62
250
32
–
L2
L3
125
250
–
L4
62
125
–
Fluconazole
–
imine CH=N peak at 8.717 in NMR spectrum confirms the
formation of predicted structures. The IR and NMR spectral
data of lead compounds is given in Table 3. The leads were
then screened for antifungal activity and the results of screen-
ing are given in Table 4. From the results of screening it was
seen that the compounds synthesized as hits exhibited better
activity, than that observed in case of any of the mini libraries.
These results indicate the reliable utility of the 2D deconvo-
lution studies in the inexhaustible drug discovery process.
Conclusion
Synthesis of mini-combinatorial libraries and subsequent
screening carried out was based on extensive study of lit-
erature and text. The appealing content and correlation of
reported and known facts with various hypotheses can be
discussed to satisfactorily justify our attempt of combina-
torially synthesizing and screening for antifungal activity
of 1,2,4-triazole derivatives. All the synthesized hits were
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Med Chem Res (2011) 20:116–120
119
found to be active against fungi. Activities of the hits for
most of the biological activities attempted were better than
those of the mini-combinatorial libraries for that activity.
These results justify our approach of employing solution
phase combinatorial synthesis and screening for this work,
and support the utility of combinatorial methodologies in
saving time and material for obtaining significant research
outcome. Further optimization of the lead structures
employing targeted or focused combinatorial libraries with
similar approach could yield clinically useful antifungal
agents.
refluxed for 10 min in microwave at 455 W, with contin-
uous stirring. The color of the reaction mixture changed to
green, hydrogen sulfide was evolved (lead acetate paper
and odor), and a homogeneous solution resulted. Reaction
mixture was diluted with 100 ml of cold water and acidi-
fied with 1:1 HCl to Congo red. The white precipitate
obtained was filtered, dried and recrystallized using
ethanol.
Step IV: general procedure for preparation of 3-
substituted-4-amino-5-benzylthio-(4H)-1,2,4-triazoles
A mixture of suitable 3-substituted triazole-5-thiol (1.48 9
10-3 mol, 0.4 g) and benzyl chloride (1.48 9 10-3 mol,
0.4 g) in ethanolic alkali (0.08 g KOH in 20 ml aqueous
ethanol) was refluxed in microwave. On cooling of the reac-
tion mixture a crude precipitate was obtained, which was
recrystallized from appropriate solvents.
Experimental
Melting points of synthesized compounds were determined
by an open capillary method and are uncorrected. Analyt-
ical TLC was performed using silica gel-G as stationary
phase. The IR (KBr) spectra were recorded on a Jasco-
FTIR 4100 instrument. The 1H NMR spectra of the com-
pounds were recorded on 400 MHz Varian NMR and
DMSO-d6 was used as solvent. Microwave synthesizer of
Catalyst systems, cata-4RI, was used for microwave
assisted synthesis. The LC–MS–MS spectrometer used for
library analysis was of Varian Inc, USA, Model 500 MS IT
with 410 Prostar Binary LC.
Step V: procedure for preparation of combinatorial
libraries
To a solution of 3-substituted-4-amino-5-benzylthio-(4H)-
1,2,4-triazole 1 g (0.1 mol) in glacial acetic acid add dif-
ferent aldehydes (0.01 mol each). The mixture was
refluxed for 15 min at 700 W in microwave. The mixture
was poured into a beaker containing 100 ml ice-water,
precipitate formed was filtered to obtain dry library. The
pool of imines with aromatic region and different triazoles
was prepared with same procedure.
Step-I: general procedure for preparation of hydrazide
Placed a solution of acid ester 2 g (0.0232 mol) in ethanol
(15 ml) in a flask, to this solution add hydrazine hydrate
(7 ml, 99%). The reaction mixture was heated under reflux
in microwave at 455 W for 12 min. The reaction mixture
was poured on ice cold water; hydrazide which separated
out was filtered, product was washed with two 10 ml
portions of ethanol, dried and re-crystallized from ethanol.
Analysis of mini libraries by LC–MS–MS
LC–MS–MS was used to establish that a sizable fraction of
the expected compounds are produced in the above syn-
thesis of mini libraries. Experiments were designed to
determine if the combination of a 3-substituted-4-amino-5-
benzylthio-(4H)-1,2,4-triazoles core with a set of aldehydes
results in the formation of a mixture in which all of the
expected imino compounds are present above a certain
concentration threshold. Observation of a library of six
compounds was done on HPLC and was found to be sat-
isfactory. Figure 1 shows the resolved components on the
binary LC showing six different components. To investi-
gate the mass-spectrometric behavior of these molecules,
several pure imino derivatives of 3-substituted-4-amino-5-
benzylthio-(4H)-1,2,4-triazoles prepared by condensing the
core molecule with one aldehyde at a time was analyzed.
Taken together, the molecular ion peaks obtained from
these measurements are a set of data directly correlated to
the diversity of a given molecular library. Since fragmen-
tation was not a factor, we were able to compare the
Step II: General procedure for preparation of potassium
3-aroylthiocarbazates
To a mixture of 8.4 g (0.15 mol) of potassium hydroxide,
200 ml of absolute ethanol and 0.1 mol of the aroylhyd-
razide, 11.4 g (0.15 mol) of carbon disulfide was added
and the resulting mixture was stirred for 24 h. It was then
diluted with 200 ml of dry ether, and the precipitated solid
was filtered, washed with 2 portions of 50 ml of ether and
vacuum dried.
Step III: general procedure for preparation of 3-
substituted-4-amino-5-mercapto-(4H)-1,2,4-triazoles
A mixture of potassium dithiocarbazate (15 g, 0.1 mol),
hydrazine hydrate 99% (5 ml, 0.1 mol) and 2 ml water was
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Med Chem Res (2011) 20:116–120
method14. The tested compounds were dissolved in dis-
tilled water to get a solution of 1000, 500, 250, 125, 62,
31.5 lg/ml. Distilled water was used as control. Com-
mercial antifungal fuconazole (100 lg/ml) was also tested
under similar conditions for comparison. These pools were
deconvoluted by 2D, and leads were identified based on the
activity data. The identified leads were synthesized and
screened individually for activity. The results of antifungal
activity testing of mini-libraries are given in Table 2.
Acknowledgment The authors are thankful to the Principal, Bharati
Vidyapeeth College of Pharmacy, Kolhapur for providing necessary
facilities for this work.
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