Solution phase combinatorial synthesis and screening of mini libraries of arylchalcones for antibacterial activity

Article abstract of DOI:10.3797/scipharm.0803-36

The solution-phase combinatorial synthesis of aryl chalcones was studied by synthesizing a 6x4 array mini library. The mini libraries of chalcones were synthesized by condensation of 4-substituted acetophenones and various aryl/heteroaryl carbaldehydes. A

Full text of DOI:10.3797/scipharm.0803-36

Sci Pharm. 2008; 76: 259–267
doi:10.3797/scipharm.0803-36
259
© Österreichische Apotheker-Verlagsgesellschaft m. b. H., Vienna, Austria
Reproduction is permitted for non-commercial purposes.
Solution Phase Combinatorial Synthesis and
Screening of Mini Libraries of
Arylchalcones for Antibacterial Activity
2
Neela M. BHATIA * 1, Kakasaheb MAHADIK
1 Bharati Vidyapeeth College of Pharmacy, Near Chitranagari,
Kolhapur – 416013, Maharashtra, India.
2 Bharati Vidyapeeth University’s Poona College of Pharmacy and Research Centre,
Erandwane, Pune – 411038, Maharashtra, India.
Abstract
The solution-phase combinatorial synthesis of aryl chalcones was studied by
synthesizing a 6x4 array mini library. The mini libraries of chalcones were
synthesized by condensation of 4-substituted acetophenones and various
aryl/heteroaryl carbaldehydes. All the synthesized mini libraries were screened for
antibacterial activity using serial dilution method. The mini-libraries 3{1a,2a–d},
3{1b,2a–d}, 3{1a–f,2a} and 3{1a–f,2c} were found to be most active of the
synthesized mini libraries. The common elements present in the identified pool of
interest were used to individually synthesize four compounds which were
individually subjected to antibacterial activity evaluation. The compound 3-(4-
chlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one exhibited significant activity that
was better than that of the other three compounds synthesized. The anti-bacterial
activity of the identified hit was also found to be better than all mini-libraries,
indicating utility of combinatorial synthesis and the assumption there in for lead
identification.
Keywords
Combinatorial • Chalcones • Antibacterial • Leads
* Corresponding author: Tel.: +91-9823463687.
E-mail: neelabhatia@yahoo.co.uk (N. M. Bhatia).

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N. M. Bhatia and K. Mahadik:
Introduction
Combinatorial chemistry has emerged as a powerful tool for the discovery and
optimization of new leads in the pharmaceutical industry. Because of their ability to
mimic the structure of peptides and their ability to reverse binding to proteins,
functionalized heterocycles are interesting scaffolds for the preparation of diversity-
oriented compound libraries for medicinal and pharmaceutical applications [1–4].
So far, several chalcone containing biologically active compounds have been
synthesized [5–7]. However, there are few reports on solution phase combinatorial
synthesis and combinatorial antibacterial screening, despite the simplicity, time and
material saving involved in the process. Aryl/heteroaryl substituted aldehydes and
4-substituted acetophenones, which are versatile reagents in heterocyclic
synthesis, were used as components of mini-library. The synthetic availability and
interesting structural features of chalcones, a ubiquitous structural unit in many
biologically active compounds, prompted us to carry out the solution phase
combinatorial
synthesis
of
1-(4-substituted phenyl)-3-(aryl)prop-2-en-1-one
derivatives for combinatorial screening leading to identification of structures which
could be novel leads for antibacterial activity optimization.
Results and Discussion
Parallel Solution-Phase Synthesis of 1-(4-substituted phenyl)-3-(aryl)prop-2-
en-1-one mini-libraries
We studied parallel solution-phase coupling of the aldehydes with ketones. For
efficient synthesis of larger combinatorial libraries, we were especially interested in
identifying a solvent/catalyst that would allow the conjugation process with a
minimized load of byproducts. During initial studies, we found that solvents yielding
proton were very useful than basic solvents. We also found sodium hydroxide to be
an appropriate catalyst, providing stringent conditions to the reaction for complete
conversion and easy recovery.

Solution Phase Combinatorial Synthesis and Screening of Mini Libraries of …
261
The reaction of ketones 1(a–f) with aldehydes 2(a–d) in methanol gave the
corresponding combinatorial mini-libraries. Assignments of the structures of
combinatorial libraries were done on basis of the data obtained by IR analysis. The
absorption bands of α,β-unsaturated carbonyl group were observed at 1720 cm−1.
The various substituent groups on aldehydes and ketones used for the synthesis of
respective mini-libraries exhibited bands at 745–755(C-Cl); 780–785(C-Br); 1050–
1055(C-O); 1350–1375 N-(CH3)2; 1610–1620(NO2); 2800–2900(CH); 3200–
3350(NH).
Analysis of Mini libraries by GC-MS
Gas Chromatography- Mass Spectrometry (GC-MS) was used to establish that
a sizable fraction of the expected compounds are produced in the above synthesis
of ethanol soluble mini libraries. Experiments were designed to determine if the
combination of a 3-(4-methoxyphenyl)prop-2-en-1-one core with a set of ketone
results in the formation of a mixture in which all of the expected compounds are
present above a certain concentration threshold. To investigate the mass-
spectrometric behavior of these molecules, several pure test mixtures of three
compounds were 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 fragmentation was not a factor, we were able to compare
the molecular ion peaks in the mass spectra with the molecular weights expected
for each mini-library, and conclude which compounds had been formed and which
had not. Result of the GC-MS analysis of a mini-library is compiled in Fig. 1. This
indicated that all components might be present in sufficient amount.

262
N. M. Bhatia and K. Mahadik:
Fig. 1. Results of GC-MS analysis of Library 3{1a–f,2a}
Combinatorial screening of the mixtures for Antibacterial activity.
The combinatorial mini-library mixtures were subjected to antibacterial activity
evaluation against four different bacteria strains S. aureus, B. subtilis, E. coli, P.
aeruginosa. 2D deconvolution steps deconvoluted activity pools of interest. In the
family pool 3{1a,2a–d}, 3{1b,2a–d}, 3{1a–f,2a} and 3{1a–f,2c} stood out as most
active mini-libraries.
The common elements present in the identified pool of interest were used to
individually synthesize four compounds which were individually subjected to
antibacterial activity evaluation. The compound 3{1a,2a} exhibited significant
activity that was better than that of the other three compounds synthesized. Activity
of this lead molecule was also better than that of any of the mini-combinatorial
libraries (Table 1).
Thus, synthesis of mixture mini-libraries proved to be a valuable time and
material saving tool in the discovery of new leads for drug discovery projects.
Specifically, preparation and screening of mixtures of 1-(4-substituted phenyl)-3-
(aryl)prop-2-en-1-one derivatives has lead to the discovery of many potent
compounds across a variety of receptor targets.

Solution Phase Combinatorial Synthesis and Screening of Mini Libraries of …
263
Tab. 1. Antibacterial activity data of lead molecules
Comp.
Code
3{1a,2a}
3{1a,2c}
3{1b,2a}
3{1b,2c}
MIC (μg/ml)
S. aureus
31
B. subtilis
E. Coli
16
P. aeruginosa
63
125
63
16
250
250
63
125
125
16
250
250
31
63
The lead 3{1a,2a} identified from the mini-libraries by deconvolutional analysis
after individual synthesis exhibited maximum activity. The lead molecule was an
element of the most active mini-libraries which were synthesized. The results
obtained have potential to tempt for further evaluation and modification by the tools
of combinatorial and computational chemistry for identification of potent and less
toxic molecules.
Experimental
1
The NMR spectra were obtained on a 400 MHz Varian NMR for H, using
DMSO-d6 and TMS as the solvent and internal standard respectively. GC-MS
spectra were recorded on a Shimadzu GC-2010, MS-2P20100 spectrophotometer;
IR spectra, on a Jasco 4100 FTIR spectrophotometer.
Combinatorial Synthesis of 1-(4-substituted phenyl)-3-(aryl)prop-2-en-1-one.
3{1a-f,2a-d} [8–10].
Combinatorial synthesis of mini-library of 1-(4-substituted phenyl)-3-(aryl)prop-
2-en-1-one derivatives was done using the reported procedure of formation of
chalcones. Mini-libraries of 6x4 array were synthesized by reacting aromatic
aldehydes 2{a–d} in stoichiometric amount with the selected ketones 1{a–f} in
methanol. Vice-a-versa the second set was synthesized by condensation of
aldehydes with ketones in stoichiometric amounts in methanol. The reaction
mixture was stirred for 1hr in presence of 10% sodium hydroxide and the mixture
was kept in cold water for 24 hrs. Spectral data of a representative mini-library

264
N. M. Bhatia and K. Mahadik:
3{1a–f,2a} is given: IR (KBr) 745–755(C-Cl); 780–785(C-Br); 1050–1055(C-O);
1350–1375 N-(CH3)2; 1610–1620(NO2); 2800–2900(CH); 3200–3350(NH). 1H NMR
(DMSO) 2.10 (s, 6H, N(CH3)2), 2.50 (s, 3H, CH3), 3.20 (s, 3H, OCH3), 5.34–5.45
(2d, 2H, -CH=CH-), 7.0–8.5 (Br, Ar).
COCH3
O
10% NaOH
Ar
ArCHO
+
R
R
2{a–d}
3{1a–f,2a–d}
1{a–f}
R= OCH3, Cl, CH3, Br, NO2, N(CH3)2.
NH
MeO
OMe
Cl
2a
OMe
N(CH3)2
2b
2c
2d
Fig. 1. Synthesis of compounds
Biological Screening
All the synthesized mini libraries were screened for antibacterial activity [11],
against S. aureus, B. subtilis, E. coli and P. aeruginosa by cup plate agar diffusion
method. Ciprofloxacin was used as standard drug for antibacterial activity. The
activity was measured by serial dilution method to determine the Minimum
Inhibitory Concentrations of each mini-library. In each plate, four cavities of 6 mm
diameter were made with a sterile-borer. Test solutions at the concentrations 1000,
750, 500, 250, 125, 63, 31 and 16 μg/ml were added to the respective cavity

Solution Phase Combinatorial Synthesis and Screening of Mini Libraries of …
265
aseptically and labeled accordingly. Standard was added in concentration of
100µg/ml. The plates were kept undisturbed for at least 2 hrs at room temperature
to allow diffusion of the solution properly in the nutrient agar medium. After
incubation of the plates at
37 ± 1°C for 24 hours. The minimum inhibitory
concentration (MIC) values of libraries are given in (Table 2).
Tab. 2. Antimicrobial screening data of mini-libraries.
MIC (μg/ml)
Library
S.
B.
E.
P.
Code
aureus subtilis coli
areuginosa
31
3{1a,2a–d} 63
3{1b,2a–d} 63
3{1c,2a–d} 500
3{1d,2a–d} 250
3{1e,2a–d} 500
125
31
31
63
125
250
750
750
250
63
750
1000
1000
250
31
500
1000
1000
500
63
3{1f,2a–d}
3{1a–f,2a}
3{1a–f,2b}
3{1a–f,2c}
3{1a–f,2d}
Cipro.
1000
63
250
125
250
16
125
63
750
31
1000
125
250
16
750
31
250
31
Acknowledgement
The authors are thankful to Dr. H. N. More, Principal, Bharati Vidyapeeth
College of Pharmacy, Kolhapur, for providing facilities to carry out the research
work.

266
N. M. Bhatia and K. Mahadik:
References
[1]
[2]
[3]
Nicolaou KC, Hanko R, Hartwig W, editors.
Solid-Phase Synthesis of Natural Products and Natural Product-like
Libraries. In Handbook of Combinatorial Chemistry.
Drugs, Catalysts, Materials, Vol. 2, Wiley-VCH Verlag GmbH: Weinheim,
2002: 611–642.
[doi:10.1002/3527603034.ch21]
Dolle RE, editor.
Solid-Phase Synthesis of Heterocyclic Systems.
In Handbook of Combinatorial Chemistry.
Drugs, Catalysts, Materials, Vol. 2, Wiley-VCH Verlag GmbH: Weinheim,
2002: 643–684.
[doi:10.1002/3527603034.ch22]
Pernerstorfer J.
Molecular Design and Combinatorial Compound Libraries.
In Handbook of Combinatorial Chemistry. Drugs, Catalysts, Materials, Vol. 2,
Wiley-VCH Verlag GmbH: Weinheim, 2002: 725–742.
[doi:10.1002/3527603034.ch25]
[4]
[5]
[6]
[7]
Dörwald FZ.
Organic Synthesis on Solid Phase, 2nd ed. Wiley-VCH Verlag GmbH:
Weinheim, 2002: 1–504.
[doi:10.1002/3527600884]
Famita AO.
Synthesis of poly functionally substituted hetroaromatic compounds via
benztriazole chalcones with antimicrobial and antifungal activities.
J Hetrocycl Chem. 2004; 41: 327–333.
Khan MS.
Synthesis anti inflammatroy and antibacterial activity of some new flavonoidal
derivatives.
Indian J Chem. 2003; 42B: 1970–1975.
Solankee A, Patel J.
Synthesis of chalcones, pyrazolines, aminopyrimidines and pyrimidinethiones
as antibacterial agents.
Indian J Chem. 2004; 43B: 1580–1584.
[8]
[9]
Dhar DN.
Chemistry of Chalcones and Related Compounds.
John Wiley and Sons, New York,1981: 1–15.
Tiwari SS, Singh A.
Chalkones as bactericidal compounds.
J Indian Chem Soc. 1961; 38: 931–932.

Solution Phase Combinatorial Synthesis and Screening of Mini Libraries of …
267
[10] Famita AO.
Synthesis of poly functionally substituted hetroaromatic compounds via
benztriazole chalcones with antimicrobial and antifungal activities.
J Hetrocycl Chem. 2004; 41: 327–333.
[11] Indian Pharmacopoeia.
Controller of Publications, 1996; Vol-II: A-100
Received March 31st, 2008
Accepted (after revision) May 26th, 2008
Available online at www.scipharm.at May 28th, 2008