Synthesis and evaluation of p-N,N-dialkyl substituted chalcones as anti-cancer agents

Article abstract of DOI:10.1007/s00044-013-0469-8

Several new N,N-dialkyl substituted chalcones (chalconoids or benzylideneacetophenones) have been synthesized via the condensation of corresponding N,N-dialkylbenzaldehyde with various aryl methyl ketones. All the chalcones have been synthesized from readily available and cheap starting materials under environmentally benign conditions in very high yields without work up and column chromatographic purification. Synthesized compounds have been tested for their biological activity against pathogenic microorganisms such as Escherichia coli, Bacillus subtilis, and Mycobacterium smegmatis. Anti-cancer activity of these compounds has also been tested against multiple myeloma (RPMI-8226) and human mammary adenocarcinoma (MCF-7) cell lines. The most hydrophilic molecules 23 and 24 showed very good anti-cancer activity against MCF-7 cell lines at low micro-molar concentrations. All the compounds have also been evaluated for their activity against Beta-secretase 1 enzyme. One of the synthesized compounds showed Beta-secretase 1 enzyme inhibition activity at micro-molar concentration.

Full text of DOI:10.1007/s00044-013-0469-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:4610–4614
DOI 10.1007/s00044-013-0469-8
ORIGINAL RESEARCH
Synthesis and evaluation of p-N,N-dialkyl substituted chalcones
as anti-cancer agents
•
Grady Nelson Mohammad A. Alam Tyler Atkinson Shirisha Gurrapu
•
•
•
•
J. Sravan Kumar Chris Bicknese Joseph L. Johnson Michael Williams
•
•
Received: 23 August 2012 / Accepted: 3 January 2013 / Published online: 16 January 2013
Ó Springer Science+Business Media New York 2013
Abstract Several new N,N-dialkyl substituted chalcones
(chalconoids or benzylideneacetophenones) have been
synthesized via the condensation of corresponding N,N-
dialkylbenzaldehyde with various aryl methyl ketones. All
the chalcones have been synthesized from readily available
and cheap starting materials under environmentally benign
conditions in very high yields without work up and column
chromatographic purification. Synthesized compounds
have been tested for their biological activity against path-
ogenic microorganisms such as Escherichia coli, Bacillus
subtilis, and Mycobacterium smegmatis. Anti-cancer
activity of these compounds has also been tested against
multiple myeloma (RPMI-8226) and human mammary
adenocarcinoma (MCF-7) cell lines. The most hydrophilic
molecules 23 and 24 showed very good anti-cancer activity
against MCF-7 cell lines at low micro-molar concentra-
tions. All the compounds have also been evaluated for their
activity against Beta-secretase 1 enzyme. One of the syn-
thesized compounds showed Beta-secretase 1 enzyme
inhibition activity at micro-molar concentration.
Introduction
Chalcones of various classes have been extensively investi-
gated for anti-proteasomal activity (Bazzaro et al., 2011;
Achanta et al., 2006), anti-cancer activity (Kim et al., 2010;
Dimmock et al., 1999; Echeverria et al., 2009; Go et al.,
2005;Zhouetal.,2009),anti-microbialactivity(Ahmadetal.,
2011; Venkatesan and Maruthavanan, 2011; Choudhary
et al., 2011; Liaras et al., 2011; Karamunge et al., 2011), and
several other therapeutic uses (Jianzhang et al., 2011; Fei
et al., 2011; Umair et al., 2011; Ramesh and Babitha, 2009).
Very recently (Chiaradia et al., 2012) reported naphthaline
derived chalcones as a potent inhibitor of Mycobacterium
tuberculosis protein tyrosine phosphatase.
Beta-secretase 1 or beta-site APP cleaving enzyme 1
(BACE1), the main cause of Alzheimer’s disease (AD) is an
aspartic-acid protease encoded by BACE1 gene. AD is a neu-
rodegenerative disease and the most common type of dementia.
AD affects millions of elderly persons worldwide and is a major
global social and financial burden. In the US alone, there are 5.4
million people suffering from AD. These people are cared for
by 14.9 million unpaid caregivers and AD costs 183 billion
annually. Based on mortality data of this decade, death rates
have declined for most major diseases while deaths from AD
have risen 66 % during the same period (Thies and Bleiler,
2012). Owing to the importance to get an effective therapeutic
agent, there are so many groups all over the globe working to
treat AD (Malamas et al., 2010). Chalcones have also been
explored as BACE1 inhibitors (Ma et al., 2011).
Keywords Chalcones ꢀ Anti-cancer ꢀ Anti-microbial ꢀ
Beta-secretase 1 enzyme
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0469-8) contains supplementary
material, which is available to authorized users.
G. Nelson ꢀ M. A. Alam (&) ꢀ T. Atkinson ꢀ S. Gurrapu ꢀ
J. Sravan Kumar ꢀ C. Bicknese ꢀ J. L. Johnson ꢀ M. Williams
Department of Chemistry and Biochemistry, University of
Minnesota Duluth, Duluth, MN 55812, USA
Results and discussions
The ease of synthesis coupled with the wide range of
diverse biological applications of chalcones provides
e-mail: alam@rowan.edu
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Med Chem Res (2013) 22:4610–4614
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tremendous scope to understand the structure activity
relationship and to identify novel chalcones as therapeutic
agents. Some of the important chalcone based therapeutic
agents are listed in Fig. 1 (Nowakowska, 2007; Keedwell
et al., 2004).
N-hydroxyethylpiperazine under the same conditions as
discussed above.
To determine the potency of our synthesized chalcones,
we tested these molecules against recombinant human
BACE1 enzyme. The most hydrophobic chalcone 6
(Fig. 2) showed moderate BACE1 inhibition activity at
50 lM concentration. Due to weak BACE inhibition
activity further studies (IC50) were not pursued.
In this regard, we envisaged the synthesis of piperazinyl,
pyrrolidinyl, piperidinyl, and dibenzyl amino substituted
chalcones and studied their biological properties against
various targets. The synthesis of N,N-dibenzylchalcone
derivatives was started with p-fluorobenzaldehyde. To
obtain N,N-dibenzylbenzaldehyde 2, aniline was treated
with benzyl bromide, K2CO3, and catalytic amount of soap
under refluxing conditions, followed by Vilsmeier–Haack
reaction of the resulting N,N-dibenzylaniline 1 (Scheme 1).
The aldehydes upon condensation with various aryl methyl
ketones in the presence of KOH in ethanol solvent fol-
lowed by treatment with 6 M HCl and filtration provided
the crude chalcones (Gezegen et al., 2010), which were
further purified by recrystallization in methanol.
Synthesized molecules were also tested against Bacillus
subtilis, Escherichia coli, and Candida albicans. For anti-
microbial studies, the colonies were grown in LB broth at
37 1 °C overnight. The microbial suspension was
swabbed on to a LB agar plate. Compound was dissolved in
DMSO to a concentration of 0.1 M. Then 5 lL of the
0.1 M stock solution was added to standard paper disks
(1 cm) to achieve a final concentration of 100 and 50 lg/
disk. The plates were incubated at 37 1 °C for 24 h. The
anti-microbial activity was determined based on the zone
of inhibition around the disk (which was measured in cm).
One of the compounds 9 (Fig. 2) showed weak zone
(1.5 cm) of inhibition against Mycobacterium smegmatis at
10 mM concentration. Hence, minimal inhibitory concen-
trations were not determined for these compounds due to
the apparent lack of significant anti-mycobacterial activity.
Piperidinylbenzaldehyde derivative 12a was readily
synthesized by treating fluorobenzaldehyde 11 with the
piperidine in the presence of K2CO3 at refluxing condition
in water (Grayson and Charles, 2008). This aminoaldehyde
derivative was treated with the corresponding acetophe-
nones to obtain the chalcones (Scheme 2). The products
derived from this aldehyde 12a gave less hydrophobic
chalcones (13–20) than the N,N-dibenzyl derivatives
(3–10) described above in Scheme 1. We also synthesized
amino aldehyde derivative 12b by treating fluorobenzal-
dehyde with pyrrolidine under the same conditions as
described in the synthesis of piperidinylbenzaldehyde 12a.
Chalcones (21, 22, 23, 26, and 27) were obtained by
treating 12b with corresponding acetophenone in excellent
yields (Scheme 2). Finally, hydrophilic chalcones such as
N-hydroxyethylpiperazinyl derivatives (24 and 25)
were synthesized by treating fluorobenzaldehyde with
Anti-cancer activity
Finally, the synthetic chalcones were tested for cytotoxicity
against multiple myeloma and MCF-7 cell lines. For anti-
cancer studies, MCF-7 cells were incubated in 5 % CO2
atmosphere at 37 °C in IMEM medium containing 10 %
Hyclone-III and 1 % Antibiotic (500,000 units pen-strep)
in sterile conditions at 100 % humidity. For the present
studies, sulforhodamine-B (SRB) assay was utilized.
100 lL of 0.5 % SRB (in 1 % acetic acid) was added in
each well and incubated at 37 °C for 45 min. SRB solution
Fig. 1 Biologically active
naturally occurring and
synthetic chalcones
OH
OH
HO
HO
OMe
OMe
O
O
licochalcone C
licochalcone A
OH
O
O
O
N
CO2H
AGN103198
OH
O
crotaramosmin
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Med Chem Res (2013) 22:4610–4614
CHO
O
NH2
NBn2
O
BnBr, K2CO3,
soap, H2O
POCl3, DMF
70 oC, 8 h,
R1
Ar
12 h, reflux,
quatitative
KOH, EtOH, rt,
overnight
90%
Bn2N
R2
NBn2
R3
1
2
Chalcones Substituents
3
R1=R2=R3=H
4
5
6
7
8
R1=R3=H, R2= CH3
R1=R3=H, R2= OCH3
R1=R2=R3=OCH3
R1=R3=H, R2= Cl
R1=R3=H, R2= Br
O
Ar =
9
S
10
Ar =
Scheme 1 Synthesis of N,N-dibenzyl chalcone derivatives
X
O
O
O
O
R1
H
N
H
Ar
H
N
N
R2
KOH, EtOH, rt,
overnight
K2CO3, H2O, 80oC,
F
X
X
R3
overnight
12
11
Compounds
Compounds
Substituents
Substituents
X
X
0
"
21
22
23
24
25
R1=R3=H, R2= CH3
R1=R2=R3=OCH3
R1=R3=H, R2= Cl
13
14
15
16
17
18
CH2
R1=R2=R3=H
"
"
"
"
"
R1=R3=H, R2= CH3
R1=R3=H, R2= OCH3
R1=R2=R3=OCH3
R1=R3=H, R2= Cl
R1=R3=H, R2= Br
"
NCH2CH2OH R1=R3=H, R2= Cl
"
R1=R3=H, R2= Br
O
26
27
Ar
O
19
20
"
"
S
"
S
Scheme 2 Synthesis of pyrrolidine, piperidine, and piperazine Chalcone derivatives
Fig. 2 Active molecules
O
O
OMe
OMe
O
Bn
N
Bn
N
9
6
Bn
OMe
Bn
O
N
Br
N
25
HO
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Med Chem Res (2013) 22:4610–4614
4613
was removed and the wells were washed 5 times with 1 %
acetic acid solution and dried. The cells were dissolved in
400 lL of 10 mM Tris base (pH 10) and absorbance was
recorded. The most hydrophilic (hydroxyethylpiperazine)
chalcones 24 and 25 showed significant cytotoxicity at
50 lM (IC50) concentration.
purification. To a stirred solution of piperazinyl benzalde-
hyde 1 (10 mmol) in 10 mL (EtOH), 2 mmol KOH and
p-chloroacetophenone (1.2 mmol) were added. The reac-
tion mixture was stirred overnight and quenched with HCl.
The resulting solid was filtered and washed several times
with hexane to obtain the chalcone, which was further
purified by recrystallization with diethyl ether (78 %
yield). Yellow solid, mp. 105–106 °C; IR (KBr pellet)
BACE1 activity assays
m 1652, 1584, 1517, 1220, 1009, 812 cm-1 1H NMR
;
BACE1 activity assays were performed using a FRET
peptide substrate Abz-YIWDEIDLMVLD-DNP synthe-
sized by Genscript, Inc ([99 % purity). In kinetic assays,
the peptide with that amino acid sequence was initially
shown by (Turner et al., 2001) to have a higher affinity for
BACE1 as well as a larger second order rate constant.
Recombinant BACE1 was obtained as described previously
(Mallender et al., 2001). Final concentrations of substrate
and enzyme used for the assay were 25 lM and 0.03 lg/
lL, respectively. Assays were performed in Corning half
area 96-well plates and read using a Molecular Devices M5
Multifunction Platereader with excitation and emission
wavelengths of 320 nm and 420 nm, respectively. All
assays were performed in 50 mM sodium acetate, pH 4.5,
with 0.25 mg/ml BSA at 23 °C. The final DMSO concen-
tration for all assays was kept at or below 5 %. BACE1 was
incubated with each compound in buffer at 23 °C for
30 min prior to the initiation of the assays by the addition
of substrate. The reported percent inhibition values are the
average of six independent measurements SEM relative
to the uninhibited assays.
(500 MHz, CDCl3): d 7.97 (d, J = 8.5 Hz, 2H), 7.79
(d, J = 15.5 Hz, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.48
(d, J = 8.5 Hz, 2H), 7.34 (d, J = 15.5 Hz, 1H), 6.92 (d,
J = 8.5 Hz, 2H), 3.69 (t, J = 5.0 Hz, 2H), 3,36
(t, J = 5.0 Hz, 4H), 2.63–2.71 (m, 7H); 13C NMR
(125 MHz, CDCl3): d 189.5, 153.0, 145.9, 139.0, 137.3,
130.5, 130.0, 129.0, 125.4, 118.0, 115.0, 59.5, 58.0, 52.8,
48.1; CHN Found: C: 68.15, H: 6.05, N: 7.30; Calculated:
C: 68.01, H: 6.25, N: 7.55.
For detailed synthetic procedure and spectral data of all
the chalcones see supporting information.
Acknowledgments We thank Dr. Venkatram Mereddy (University
of Minnesota Duluth) for providing facilities and valuable suggestions.
References
Achanta G, Modzelewska A, Feng L, Khan SR, Huang P (2006) A
boronic-CHALCONE derivative exhibits potent anticancer
activity through inhibition of the proteasome. Mol Pharmacol
70:426–433
Ahmad RM, Sastry GV, Nasreen B, Anwer S, Kumaraswamy G
(2011) Antioxidant and antibacterial activities of some novel
chalcone derivatives and their synthesis by conventional and
microwave irradiation methods. J Chem Phar Res 3:710–717
Bazzaro M, Anchoori RK, Mudiam MKR, Issaenko O, Kumar S,
Karanam B, Lin Z, Vogel RI, Gavioli R, Destro F, Ferretti V,
Roden RBS, Khan SR (2011) a, b-Unsaturated carbonyl system
of chalcone-based derivatives is responsible for broad inhibition
of proteasomal activity and preferential killing of human
papilloma virus (HPV) positive cervical cancer cells. J Med
Chem 54:449–456
Chiaradia LD, Martins PGA, Cordeiro MNS, Guido RVC, Ecco G,
Andricopulo AD, Yunes RA, Vernal J, Nunes JR, Terenzi H
(2012) Synthesis, biological evaluation, and molecular modeling
of chalcone derivatives as potent inhibitors of mycobacterium
tuberculosis protein tyrosine phosphatases (PtpA and PtpB).
J Med Chem 55:390–402
Conclusions
In conclusion, we have synthesized piperidinyl, piperazi-
nyl, and some other amino-based chalcones as potential
therapeutic agents. Different class of compounds showed
different activity i.e., the most hydrophic chalcone: BACE
inhibition, hydrophilic chalcone: cytotoxicity against
MCF-7 cell lines and furan derived chalcone: anti-
mycobacterial activity. So, the current study offers pre-
liminary pointers to further modify, design, and investigate
the structure of these molecules to expand the utility of
these molecules as potential therapeutic agents.
Choudhary AN, Juyal V (2011) Synthesis of chalcone and their
derivatives as antimicrobial agents. Int J Pharm Pharmaceut Sci
3:125–128
Synthetic procedure (synthesis of 25)
Dimmock JR, Elias DW, Beazely MA, Kandepu NM (1999)
Bioactivities of chalcones. Curr Med Chem 6:1125–1149
Echeverria C, Santibanez JS, Donoso-Tauda O, Escobar CA,
Ramirez-Tagle R (2009) Structural antitumoral activity relation-
ships of synthetic chalcones. IJMS 10:221–231
Fei Z, Qing-Jie Z, Da-Zhi Z, Yong-Sheng J, Wei Z (2011) Synthesis
and protein tyrosine phosphatase 1B-inhibitory activity of
chalcones. Asian J Chem 23:5339–5342
To a stirred solution of fluorobenzaldehyde (10 mmol) in
10 mL H2O, N-hydroxyethylpiperazine (15 mmol) and
potassium carbonate (20 mmol) were added. The reaction
mixture was refluxed for 24 h and cooled. The resulting
solid was filtered to obtain the crude aldehyde 9.5 mmol
(95 % yield), which was utilized for the next step without
123

4614
Med Chem Res (2013) 22:4610–4614
Gezegen H, Dingil A, Ceylan M (2010) Three-step synthesis of 2,4-
diaryl-5,6,7,8-tetrahydroquinoline derivatives.
47:1017–1024
Go ML, Wu X, Liu XL (2005) Chalcones: an update on cytotoxic and
chemoprotective properties. Curr Med Chem 12:483–499
Grayson BL, Charles Z M (2008) PCT Int Appl 2008152471
Jianzhang W, Jianling L, Yuepiao C, Yong P, Faqing Y, Yali Z,
Yunjie Z, Shulin Y, Xiaokun L, Guang L (2011) Evaluation and
discovery of novel synthetic chalcone derivatives as anti-
inflammatory agents. J Med Chem 54:8110–8123
Malamas MS, Erdei J, Gunawan I, Turner J, Hu Y, Wagner E, Fan K,
Chopra R, Olland A, Bard J, Jacobsen S, Magolda RL, Pangalos
M, Robichaud AJ (2010) Design and synthesis of 5,50-disubsti-
tuted aminohydantoins as potent and selective human b-secretase
(BACE1) inhibitors. J Med Chem 53:1146–1158
Mallender WD, Yager D, Onstead L, Nichols MR, Eckman C,
Sambamurti K, Kopcho LM, Marcinkeviciene J, Copeland RA,
Rosenberry TL (2001) Characterization of recombinant, soluble
b-secretase from an insect cell expression system. Mol Pharma-
col 59:619–626
J Het Chem
Karamunge KG, Sayyed MA, Vibhute AY, Vibhute YB (2011)
Synthesis of some new chalcones, pyrazolines and acetyl
pyrazolines derived from piperonal and halogenohydroxy ace-
tophenones as antimicrobial agents. J Indian Chem Soc 88:
443–450
Nowakowska Z (2007) A review of anti-infective and anti-inflam-
matory Chalcones. Eur J Med Chem 42:125–137
Ramesh B, Babitha S (2009) Synthesis and anti-inflammatory activity
of some new pyrimidine derivatives. Research J Pharm and Tech
2:830–832
Keedwell RG, Zhao Y, Hammond LA, Qin S, Tsang KY, Reitmair A,
Molina Y, Okawa Y, Atangan LI, Shurland DL, Wen K, Wallace
DM, Bird R, Chandraratna RA, Brown G (2004) A retinoid-
related molecule that does not bind to classical retinoid receptors
potently induces apoptosis in human prostate cancer cells
through rapid caspase activation. Cancer Res 64:3302–3312
Kim TH, Seo WD, Ryu HW, Seo HR, Jin YB, Lee M, Ji YH, Park
KH, Lee YS (2010) Anti-tumor effects by a synthetic chalcone
compound is mediated by c-Myc-mediated reactive oxygen
species production. Chem Biol Interact 188:111–118
Liaras K, Geronikaki A, Glamoclija J, Ciric A, Sokovic M (2011)
Thiazole-based chalcones as potent antimicrobial agents, synthe-
sis and biological evaluation. Bioorg Med Chem 19:3135–3140
Ma L, Yang Z, Li C, Zhu Z, Shen X, Hu L (2011) Design, synthesis
and SAR study of hydroxychalcone inhibitors of human
b-secretase (BACE1). J Enzy Inh and Med Chem 26:643–648
Thies W, Bleiler L (2012) Alzheimer’s disease facts and figures.
Alzheimer’s and Dementia 8:131–168
Turner RT, Koelsch G, Hong L, Castanheira P, Ermoleiff J, Ghosh
AK, Tang
J (2001) Subsite specificity of memapsin 2
(b-secretase): implications for inhibitor design. Biochemistry 40:
10001–10006
Umair A, Kaskhedikar SG, Zafar A, Shahzad A (2011) Synthesis and
screening of substituted chalcones as lipoxygenase inhibitors.
Asian J Chem 23:4993–4996
Venkatesan P, Maruthavanan T (2011) Piperidine-mediated synthesis
of thiazolyl chalcones and their derivatives as potent antimicro-
bial agents. J Het Chem 48:1181–1186
Zhou J, Geng G, Batist G, Wu JH (2009) Bioorg Med Chem Lett
19:1183–1186
123