Synthesis of 4′-hydroxy-3′-piperidinomethylchalcone derivatives and their cytotoxicity against PC-3 cell lines

Article abstract of DOI:10.1002/ardp.200600072

A new series of mono Mannich bases of 4′-hydroxychalcones 2a-e carrying a variety of aryl groups was synthesized and the in vitro cytotoxic activities of the new compounds were screened against PC-3 cell lines. Bioactivities of 2a-e, which are reported for the first time in this study, were compared against their precursor 4′-hydroxychalcones 1a-e. Compound 2b was found to be the most potent (IC₅₀ = 3.7 μM) among the compounds synthesized. In addition, the compounds 1a-c and 2d showed moderate cytotoxicity. Incorporation of the 3′-piperidinomethyl group in 1b and 1d raised the potency by 1.68 and 2.19 times respectively and, therefore, seemed to be a noteworthy molecular modification. Correlations were noted between cytotoxicity and one or more physiochemical constants of the aryl ring as well as log P values for the compounds 2a-e. The significant improvement of cytotoxicity of 2b, 2d, and 2e against PC-3 cell lines compared with their chalcone precursors suggests that the incorporation of a piperidinomethyl group is a useful molecular modification and further development of these compounds as candidate cytotoxic agents may be warranted.

Full text of DOI:10.1002/ardp.200600072

Arch. Pharm. Chem. Life Sci. 2007, 340, 195–201
H. I. Gul et al.
195
Full Paper
Synthesis of 49-Hydroxy-39-piperidinomethylchalcone
Derivatives and Their Cytotoxicity Against PC-3 Cell Lines
1
1
2
3
4
4
Halise I. Gul , Kadir O. Yerdelen , Mustafa Gul , Umashankar Das , Bulbul Pandit , Pui-Kai Li ,
5
6
Hasan Secen , and Fikrettin Sahin
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk University, Erzurum, Turkey
Department of Physiology, Faculty of Medicine, Ataturk University, Erzurum, Turkey
College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Canada
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University,
Columbus, OH, USA
Department of Chemistry, Faculty of Arts and Sciences, Ataturk University, Erzurum, Turkey
Department of Genetic and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University,
Istanbul, Turkey
2
3
4
5
6
A new series of mono Mannich bases of 49-hydroxychalcones 2a–e carrying a variety of aryl
groups was synthesized and the in vitro cytotoxic activities of the new compounds were screened
against PC-3 cell lines. Bioactivities of 2a–e, which are reported for the first time in this study,
were compared against their precursor 49-hydroxychalcones 1a–e. Compound 2b was found to
be the most potent (IC50 = 3.7 lM) among the compounds synthesized. In addition, the com-
pounds 1a–c and 2d showed moderate cytotoxicity. Incorporation of the 39-piperidinomethyl
group in 1b and 1d raised the potency by 1.68 and 2.19 times respectively and, therefore, seemed
to be a noteworthy molecular modification. Correlations were noted between cytotoxicity and
one or more physiochemical constants of the aryl ring as well as log P values for the compounds
2
a–e. The significant improvement of cytotoxicity of 2b, 2d, and 2e against PC-3 cell lines com-
pared with their chalcone precursors suggests that the incorporation of a piperidinomethyl
group is a useful molecular modification and further development of these compounds as candi-
date cytotoxic agents may be warranted.
Keywords: Chalcone / Cytotoxicity / Mannich base / PC-3 cells / Synthesis /
Received: April 19, 2006; accepted: October 13, 2006
DOI 10.1002/ardp.200600072
Introduction
nucleophiles that are liberated in situ following deamina-
tion [6, 7]. Chalcones are a,b-unsaturated ketones, and
The principal aim of our laboratory is the discovery of they are associated with diverse biological activities.
novel cytotoxic and anticancer alkylating agents. Several Recently, 4-hydroxychalcones have demonstrated their
publications on Mannich ketones as potential cytotoxins effectiveness as cytotoxic [8, 9], antitumor [10, 11] antiox-
have emerged from our research [1–5]. Bioactivity of this idative [10], antibacterial [12], antifungal [13], antileish-
class of compounds has been proposed due to the alkylat- manial [14], and antinociceptive [15] agents. Antimicro-
ing potential of a,b-unsaturated ketones towards cellular bial [16] and mosquito repellent [17] activities of ortho-
aminomethyl phenol derivatives (Mannich phenols) are
also noteworthy.
In view of potential cytotoxicity associated with 49-
Correspondence: Halise Inci Gul, PhD., Associate Professor, Depart-
ment of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk Univer-
sity, 25240 Erzurum, Turkey
E-mail: incigul1967@yahoo.com; incigul@atauni.edu.tr
Fax: +90 442 236-0962
hydroxychalcones and Mannich ketones, we were inter-
ested in preparing a novel series of Mannich chalcones
2a–e, Fig. 1) in order to explore their effectiveness as
potent cytotoxic agents. This combination has also been
(
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H. I. Gul et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 195–201
Table 1. Physical data of compounds 1a–e.
Compounds Mol.
Formula
Mol.
Weight
Yield
(%)
Melting
point (8C)
a)
1
1
1
1
1
a
b
c
d
e
C
15
C
15
C
16
C
16
C
13
H
H
H
H
H
12
O
11ClO
O
O
O
2
224.26
258.71
254.29
238.29
230.29
85
90
86
86
90
174
190
182
193
b)
2
c)
14
14
10
3
2
2
d)
Scheme 1. Formation of cyclohexadienone 4 from 2 on deami-
nation, which generates an additional alkylating site for cellular
sulfahydryl nucleophiles.
e)
S
172
a)
b)
c)
Melting points reported (8C): : 172–173 [8]; : 187–189 [8];
1
:
d)
e)
80–182 [8]; : 190–192 [8]; : 171 [21].
explored by Dimmock et al. to synthesize some Mannich
bases of arylidene cyclohexanones [18].
The rationale to prepare 2a–e included the following
considerations: Firstly, conversion of various acyclic con-
jugated styryl ketones into the corresponding Mannich
bases are often accompanied by increased bioactivity
both in vitro and in vivo [19]. Secondly, deamination of
Mannich chalcones 2 into the corresponding cyclohexa-
dienones 4 (Scheme 1) may be considered as a possibility
to generate a further site for a nucleophilic attack by cel-
lular thiols, and greater bioactivity of Mannich chalcones
can be expected compared with the 49-hydroxychalcones.
Thirdly, selective affinity of a,b-unsaturated ketones
toward thiols rather than hydroxyl and amino groups
present in the nucleic acids may eliminate the problems
of mutagenicity and carcinogenicity, which have been
associated with certain alkylating agents used in cancer
chemotherapy [20].
In summary, the present study aimed at preparing
some new derivatives of Mannich chalcones 2a–e and
evaluating their cytotoxicity against the androgen-inde-
pendent prostrate cancer cell line (PC-3). Biodata gener-
ated for this series of compounds will be helpful to gain
insight whether the incorporation of the 39-piperidino-
methyl group in 49-hydroxychalcones 1a–e leads to
improve the cytotoxicity. In addition, possible correla-
tions between cytotoxicity and various physiochemical
properties of aryl substituents as well as the partition
coefficients of the compounds were investigated. The pre-
sence of such relationships would permit the expansion
of clusters of compounds on a rational basis.
Reaction condition: i: Ar-CHO/NaOH/ethanol; ii: Paraformaldehyde/Piperidine/Ethanol
Ar = a: C ; b: 4-ClC ; c: 4-CH OC ; d: 4-CH ; e: 2-Thienyl
6
H
5
6
H
4
3
6
H
4
3 6 4
C H
Scheme 2. Synthesis of compounds 1a–e and 2a–e.
Here, we report the first-time synthesis of 39-piperidino-
methyl derivatives 2a–e. Mannich chalcones 2 were pre-
pared by reaction of a suitable 49-hydroxychalcone 1 with
paraformaldehyde and piperidine in ethanol. Attempts
to prepare 39, 59-bispiperidinomethyl derivative 3 from 1
or 2 were unsuccessful and only led to the mono deriv-
atives 2 (Scheme 2). The optimum yields of 2 were
obtained using paraformaldehyde, piperidine, and chal-
cone in molar ratios (2:2:1) while use of molar ratios
(1:1:1) extended the reaction time substantially and
resulted in low yields. In a representative case, com-
pound 2c was obtained in 86% yield in 26 hours versus
4
1
4
0% yield in 40 hours using the reactants in 2:2:1 and
:1:1 molar ratios, respectively. To obtain the best yield,
9-hydroxychalcone together was added to a previously
heated reaction mixture of piperidine and paraformalde-
Results and discussion
hyde in ethanol. Refluxing a mixture of piperidine, paraf-
The intermediate 49-hydroxychalcones 1a–e reported in ormaldehyde, and chalcone even in 2:2:1 molar ratio
this communication are known in the literature [8, 21] prolongs the reaction time and results in significant
and were prepared under alkaline condition in 85–90% yield loss. For example, in case of 2c, when all the reac-
yield. Chemical structures of the compounds 1a–e were tants were refluxed together, the reaction time had to be
1
confirmed by H-NMR. Melting points of 1a–e were in extended from 26 to 47 hours with a decline in yield
accordance with the literature reports. The physical data from 86% to 50%. Aminomethylation occurred at the
of compounds 1a–e are presented in Table 1.
ortho position to hydroxyl group as the para position of
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Arch. Pharm. Chem. Life Sci. 2007, 340, 195–201
Cytotoxicity of Chalcone Mannich Bases
197
Table 2. Physical data of Mannich chalcones 2a–e.
Compd.
Mol. Formula
Mol.
Mol Ratios (mmol)
Reaction
Crystallization Yield
Melting
Weight
[paraformaldehyde: time (h)
piperidine : chalcone]
solvent
(%)
point (8C)
2
2
2
2
2
a
b
c
d
e
C
21
C
21
C
22
C
22
C
19
H
H
H
H
H
23NO
22Cl NO
2
321.43
355.87
351.45
335.45
327.45
8.8 : 8.8 : 4.4
7.7 : 7.7 : 3.8
7.8 : 7.8 : 3.9
8.4 : 8.4 : 4.2
8.6 : 8.6 : 4.3
55
47
26
35
39
Ethanol
Ethanol
Ethanol
Ethanol
Methanol
31
66
86
54
52
128
137
117
132
132
2
25NO
25NO
21NO
3
2
2
S
Figure 2. Carbon numbering of compounds 2a–d and 2e.
–
1
1
595 cm . Carbon numbering of 2a–d and 2e is shown
in Fig. 2.
For compounds 2a–e, strong K bands were observed in
the UV spectra at 323–347 nm, which result from the
Figure 1. Pharmacophoric combination rationale of Mannich
chalcones 2.
the phenol motif in chalcone 1 is substituted (Fig. 1). electronic transition of the styryl ketone chromophore
Occasionally, reaction also happens at the para position pfip*. These bands are as important as the olefinic pro-
in case this position is unsubstituted. The molar ratios of ton signals in the NMR spectra to follow adduct reactions
reactants (paraformaldehyde, piperidine, and chalcone) carried out to determine the alkylating antineoplastic
along with the physical data of the compounds 2a-e are activity of the a,b-unsaturated ketones, and also to deter-
presented in the Table 2.
mine adduct-elimination ratios. Maximum absorption
1
13
The H-NMR, C-NMR, UV, and IR spectra of the com- determined at 323 nm wave length in the UV spectrum
pounds 2a–e were in accordance with their chemical of 2a confirms the structure of the conjugated a,b-unsa-
structures (Tables 3 and 4). Elemental analyses results (C, turated ketone. Low intensity O-H stretching of the phe-
H, N) were within 0.4% of the calculated values. The pre- nolic group of Mannich chalcones was observed at 2950–
sence of a 39-piperidinomethyl group was very much evi- 2926 cm . This lowering of the intensity may be due to
dent from the spectral data. For example, in case of 2a, the intermolecular hydrogen bonding of hydroxyl
the piperidine protons were observed as multiplets of six groups. A positive ferric chloride test confirmed the pre-
–
1
protons and four protons at d 1.80–1.30 ppm and 3.20–
sence of phenolic group in the compounds of series 2.
Cytotoxic activities of 49-hydroxychalcones 1a–e and
1
2.00 ppm, respectively in H-NMR spectra. Whereas the
benzylic methylene protons appeared at d 3.75 ppm, the their corresponding 39-piperidinomethyl 2a–e deriv-
olefinic protons of chalcone 2a were observed as doublets atives tested against androgene-independent prostate
(
J = 15.8 Hz) at d 7.78–7.55 ppm. On the basis of the J cancer cell line (PC-3) are shown in Table 5. In series 1,
value, the structures were characterized as E isomers. In cytotoxicity occurs in the order 1a F 1b A 1c A 1d A 1e.
1
3
C-NMR, chemical shifts of carbons of the piperidino- Compounds 1a, 1b, and 1c were moderately active and
methyl group have been observed at d 54.00 ppm. Carbon exhibited 4–5 times lower potency than 5-fluorouracil.
peaks of the olefinic group in 2a have been observed at d Replacement of the phenyl ring by its thiophene bio-iso-
1
43.80 and d 122.10 ppm. In addition, strong absorption ster in compound 1e lowered the potency unexpectedly.
–
1
observed at 1653 cm in the IR spectra of 2a support a,b- Apparently, no correlation exist between cytotoxicity
unsaturated ketone structure [22]. Due to a similar rea- and various physio-chemical properties such as electro-
son, C=C stretching frequency shifts from 1670–1640 to nic (r) [23], hydrophobicity (p) [23], and molar refractivity
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H. I. Gul et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 195–201
1
13
Table 3. H-NMR and C-NMR data of Mannich chalcones 2a–e.
1
13
Compound
H-NMR (d)
C-NMR (d)
2
2
2
2
a
b
c
1.80–1.30 (m, 6H, 26C18-H, 26C19-H, 26C20-H), 3.20–2.00
188.8 (C9), 163.7 (C13), 143.8 (C7), 135.4 (C1), 130.5
(C11), 130.4 (C15), 129.8 (C10), 129.6 (C3 and C5),
129.1 (C4), 128.6 (C2 and C6), 122.1 (C8), 121.9
(
m, 4H, 26C17-H, 26C21-H), 3.75 (s, 2H, 26C16-H), 6.86 (d, 1H,
3
C14-H, J14,15= 8.4 Hz), 7.43–7.36 (m, 3H, C3-H, C4-H, C5-H), 7.55
3
(B part of AB system, d, 1H, C8-H, J7,8= 15.8 Hz), 7.63 (dd, 1H, C2-H (C12), 116.2 (C14), 62.1 (C16), 54.0 (C17 and C21),
3 3 4 4
and C6-H, J2,3= J5,6= 7.4 Hz, J2,4= J4,6= 2.2 Hz ), 7.76 (brs, 1H, C11-H, 26.0 (C18, C20), 24.0 (C19).
overlapped with C7-H signal), 7.78 (A part of AB system, d, 1H, C7-
3
3
4
H, J7,8= 15.8 Hz), 7.91 (dd, 1H, C15-H, J14,15= 8.4 Hz, J11,15= 2.2 Hz ) ,
0.22 (brs, 1H, Ar-OH).
1.70–1.20 (m, 6H, 26C18-H, 26C19-H, 26C20-H), 3.20–2.00
m, 4H, 26C17-H, 2x C21-H), 3.74 (s, 2H, 26C16-H), 6.84 (d, 1H,
1
188.3 (C9), 163.8 (C13), 142.2 (C7), 136.2 (C1), 134.0
(C4), 130.4 (C11), 129.8 (C15), 129.7 (C3 and C5),
129.5 (C10), 129.4 (C2 and C6), 122.5 (C8), 121.9
(C12), 116.2 (C14), 62.0 (C16), 54.0 (C17 and C21),
26.0 (C18, C20), 24.0 (C19).
(
3
C14-H, J14,15= 8.4 Hz), 7.35 (B part of AB system, d, 2H, C3-H and
3
3
C5-H, J2,3= J5,6= 8.4 Hz ), 7.50 (B part of AB system, d, 1H, C8-H,
3
J
J
7,8= 15.6 Hz), 7.54 (A part of AB system, d, 2H, C2-H and C6-H,
3
3
3
2,3= J5,6= 8.4 Hz), 7.70 (A part of AB system, d, 1H, C7-H, J7,8= 15.6
3
4
Hz), 7.74 (brs, 1H, C11-H), 7.88 (dd, 1H, C15-H, J14,15= 8.4 Hz, J11,15
1.8 Hz ) , 11.64 (brs, 1H, Ar-OH).
1.80–1.30 (m, 6H, 26C18-H, 26C19-H, 26C20-H), 3.20–2.00
m, 4H, 26C17-H, 26C21-H), 3.76 (s, 2H, 26C16-H), 3,85 (s, 3H,
=
188.8 (C9), 163.4 (C13), 161.6 (C4), 143.6 (C7), 130.3
(C2 and C6), 130.2 (C11), 129.9 (C10), 129.8 (C15),
(
3
OCH3), 6.86 (d, 1H, C14-H, J14,15= 8.4 Hz), 6.93 (B part of AB system, 128.2 (C1), 121.8 (C12), 119.8 (C8), 116.1 (C14),
d, 2H, C3-H and C5-H, J2,3= J5,6= 8.8 Hz ), 7.43 (B part of AB system, 114.6 (C3 and C5), 62.1 (C16), 55.6 (C22), 54.1 (C17
d, 1H, C8-H, J7,8= 15.6 Hz), 7.60 (A part of AB system, d, 2H, C2-H
and C6-H, J2,3= J5,6= 8.8 Hz), 7.75 (d, 1H, C11-H, J11,15= 2.0), 7.76
3
3
3
and C21), 26.0 (C18 and C20), 24.1 (C19).
3
3
4
3
(
A part of AB system, d, 1H, C7-H, J7,8= 15.6 Hz), 7.90 (dd, 1H, C15-H,
3
4
J
14,15= 8.4 Hz, J11,15= 2.0 Hz ) , 9.86 (brs, 1H, Ar-OH).
d
1.80–1.20 (m, 6H, 26C18-H, 26C19-H, 26C20-H), 2.39 (s, 3H,
Ar-CH3), 3.20–2.00 (m, 4H, 26C17-H, 26C21-H), 3.76 (s, 2H,
188.7 (C9), 163.5 (C13), 143.9 (C7), 140.9 (C4), 132.7
(C1), 130.3 (C11), 129.9 (C2 and C6), 129.8 (C10),
128.6 (C3 and C5), 121.8 (C12), 121.1 (C8), 116.2
(C14), 62.1 (C16), 54.1 (C17 and C21), 26.0 (C18 and
C20), 24.2 (C19), 21.8 (C22).
3
2
6C16-H), 6.86 (d, 1H, C14-H, J14,15= 8.4 Hz), 7.21 (B part of AB
3
3
system, d, 2H, C3-H and C5-H, J2,3= J5,6= 8.3 Hz ), 7.50 (B part of
3
AB system, d, 1H, C8-H, J7,8= 15.8 Hz), 7.54 (A part of AB system,
d, 2H, C2-H and C6-H, J2,3= J5,6= 8.3 Hz), 7.76 (brs, 1H, C11-H,
overlapped with C7-H signal), 7.77 (A part of AB system, d, 1H,
C7-H, J7,8= 15.8 Hz), 7.90 (dd, 1H, C15-H, J14,15= 8.4 Hz, J11,15= 2.2
Hz), 10.40 (brs, 1H, Ar-OH).
3
3
3
3
4
2e
1.80–1.20 (m, 6H, 26C16-H, 26C17-H, 26C18-H), 3.20–2.00
188.1 (C7), 163.7 (C11), 140.9 (C1), 136.3 (C5), 131.8
(C9), 130.3 (C4), 129.8 (C13), 129.5 (C8), 128.5 (C1;
(
m, 4H, 26C15-H, 26C19-H), 3.75 (s, 2H, 26C14-H), 6.85 (d, 1H,
3
3
3
C12-H, J13,14= 8.4 Hz), 7.07 (dd, 1H, C3-H, J3,4= 3.7 Hz, J4,5= 5.1 Hz, C2, C3), 121.9 (C10), 120.9 (C6), 116.2 (C12), 62.1
7
C6-H, J6,7= 15.0 Hz), 7.38 (d, 1H, C4-H, J4,5= 5.1 Hz), 7.73 (d, 1H,
C9-H, J10,14= 2.0 Hz), 7.88 (dd, 1H, C13-H, J13,14= 8.4 Hz, J10,14= 2.0
3
.32 (d, 1H, C2-H, J3,4= 3.7 Hz), 7.33 (B part of AB system, d, 1H,
(C14), 54.0 (C15 and C19), 26.0 (C16 and C18), 24.0
(C17).
3
3
4
3
4
3
Hz, 7.91 (A part of AB system, d, 1 H, C5-H, J6,7= 15.0 Hz), 10.67
(brs, 1H, Ar-OH).
(MR) [23] of aryl substituents as well as the partition coef- 1.39 times, respectively, when compared with the precur-
ficient (log P) of compounds 1a–e. Calculation of the par- sor 49-hydroxychalcones. Although not reaching the sig-
tition coefficient was carried out using Chem. Office nificance level, the IC50 figures of 2a–d were negatively
ultra 7.0 software. Hence, the future molecular modifica- correlated with sigma (r) constants (r = –0.943, p =
tion should include the preparation of 3-chloro analo- 0.057), pi (p) constants (r = –0.924, p = 0.076). These
gues following the decision-tree approach [24]. The bio- results suggested that the potency increases (lower IC50
)
data generated from 3-chloro analogues would guide the with the increase of hydrophobicity and electron–with-
future modifications of 49-hydroxychalcones. Incorpora- drawing properties of the aryl substituents. Thus, future
tion of the 39-piperidinomethyl group in 49-hydroxychal- molecular modifications should place strongly hydro-
cones led to the formation of 2a–e. Compound 2b was phobic and electron-attracting substituent in the aryli-
the most potent compound (IC50 = 3.7 lM) among all the dene aryl ring such as 3,4-dichloro (r = 0.60, p = 1.42), 4-
compounds in series 1 and 2 and was 2.5 times less potent trifluoromethyl (r = 0.54, p = 0.88), 3-trifluoromethyl (r =
than 5-fluorouracil. Compound 2d showed moderate 0.43, p = 0.88), 3-chloro (r = 0.37, p = 0.71), and 4-bromo
activity, whereas 2a, 2c, and 2e were weakly active. The (r = 0.23, p = 0.86) groups. A significant correlation (r = –
cytotoxic potency of 2b, 2d, and 2e rose by 1.68, 2.19, and 0.95, p = 0.05) was noted between IC50 figures and log P
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Arch. Pharm. Chem. Life Sci. 2007, 340, 195–201
Cytotoxicity of Chalcone Mannich Bases
199
Table 4. UV and IR data of Mannich chalcones 2a–e.
Table 6. Correlations (r) and significance levels (p) between
cytotoxicity of the compounds against PC-3 cells, and Hammett
(r) values of the aryl substituents and log P values.
UV
IR
CHCl3
maks
Com-
k
OH
C=O
C=C
1a-e
1a-d
2a-e
2a-d
pound
(log e) nm
stretching stretching stretching
–
1
– 1
– 1
(
cm
)
(cm
)
(cm )
IC50 and log P IC50 and r IC50 and log P IC50 and r
2
2
2
2
2
a
b
c
243 (4.02),
2943
2936
2926
2926
2950
1653
1656
1653
1649
1648
1595
1587
1560
1586
1597
r
p
0.33
0.67
–0.44
0.56
–0.95
0.05
–0.78
0.22
3
23 (4.43)
242 (4.04),
27 (4.46)
244 (4.23),
44 (4.51)
243 (4.38),
30 (4.50)
245 (4.00),
47 (4.50)
3
3
This study was supported by State Planning Organization of
Turkey (DPT Project number 2003K05) and Ataturk University
Research Fund (Project number: 2001/135). The authors thank
Ebru Mete and Baris Anil for technical assistance in elemental
analyses of the compounds and NMR spectra.
d
e
3
3
Table 5. Cytotoxic potencies of 1a–e and 2a–e against PC-3
cell line and log P values.
Compound
Cytotoxicity
Log P
Experimental
(
IC50, lM)
1
1
1
1
1
2
2
2
2
2
5
a
b
c
d
e
a
b
c
6.19
6.22
7.10
14.83
31.21
10.12
3.7
14.41
6.77
22.40
1.5
3.49
4.21
3.41
4.00
3.14
4.47
5.18
4.39
4.97
4.12
–
Materials
All chemicals were purchased from Aldrich Chemical Co.
(
Munich, Germany). Melting points were determined on a Tho-
mas Hoover Capillary Apparatus (Thomas Hoover, Philadelphia,
PA, USA) and were uncorrected. UV spectra were recorded in
CHCl by a Thermo Electron Hekios (a) (UVA 114903, England).
3
1
13
H-NMR (400 MHz) and C-NMR (100 MHz) spectra were obtained
on Varian 400 (100) NMR spectrometer (Varian Inc., Palo Alto,
CA, USA (chemical shift in d, ppm). Elemental analyses were per-
formed on LECO C, H, N, S – 932 (Leco, St. Joseph, MI, USA). The
rections were monitored using silicagel HF254-366 TLC plates (E.
Merck, Darmstadt, Germany).
d
e
-Fluorouracil
values of the compounds 2a–e. This biodata suggested
that the potency increases as the lipophilicity of the com- Synthesis of compounds
pound increases. Therefore, future modifications 2b Synthesis of 49-hydroxychalcone derivatives 1a–e
should include the following considerations: Firstly, (Scheme 2)
These compounds were prepared by following the procedure
described in [8]: An aqueous solution of sodium hydroxide
introduction of one or more methylene groups between
the 39-carbon and the piperidinyl group in addition to
(
10% w/v, 10 mL) was added to a solution of the appropriate aryl
the existing methylene group and, secondly, replace-
ment of the piperidinyl group by an azepinyl group.
In conclusion, this study reports the synthesis and
cytotoxicities of two series of compounds 1 and 2 against
PC-3 cell lines. Significant improvement of cytotoxic
potencies of 2b, 2d, and 2e compared to the precursor 49-
hydroxychalcones suggested that incorporation of a 39-
piperidinomethyl group is a useful molecular modifica-
tion; compound 2b was established as a lead molecule.
aldehyde (0.02 mol) and 4-hydroxyacetophenone (0.02 mol) in
ethanol (6 mL). The reaction mixture was stirred at room tem-
perature for 12 h, poured into water (100 mL) and neutralized
with hydrochloric acid (10% w/v) to provide a yellow solid or yel-
low oil, which solidified on cooling. The compounds were crys-
tallized from a suitable solvent to obtain analytically pure sam-
ples. The structure and purity of the compounds were confirmed
1
by H-NMR and melting points described in literature if avail-
able.
Correlations were noted between cytotoxicities and one Synthesis of 49-hydroxy-39-piperidinomethylchalcone
or more physiochemical constants of the aryl ring as well derivatives 2a–e (Scheme 2)
A solution of suitable chalcone in ethanol (30 mL) was added to a
as the partition coefficient of the molecules (Table 6).
previously heated mixture of paraformaldehyde and piperidine
These observations provided guidelines for future modifi-
in ethanol (20 mL) for 30 min. The reaction mixture was
cations of these novel compounds as candidate cytotoxic
refluxed until the disappearance of the starting material. The
agents.
completion of reaction was monitored by TLC; it took 26–55 h.
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200
H. I. Gul et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 195–201
Ethanol was removed under vacuum and the viscous residue
was dissolved in cyclohexane. The solution was kept at room
temperature for 24 h and further cooling at –48C lead to yellow
solids. Compounds 2a and 2e were crystallized directly from the
ethanolic solution of the reaction mixture after overnight cool-
ing at –48C. The products obtained were filtered and crystallized
from a suitable solvent. H-NMR and C-NMR data of the com-
pounds 2a–e are presented in Table 3, while UV and IR data
were presented in Table 4.
of each plate. A background value of 0.41 representing the absor-
bance of media and MTS/PMS solution alone was subtracted
from each average. Percent inhibition of cell proliferation was
calculated for each compound on each plate as follows:
½
fControl absorbanceꢀ0:41g ꢀ fSample absorbanceꢀ0:41gꢁ6100
%I ¼
½
fControl absorbance ꢀ 0:41gꢁ
1
13
Compounds that inhibited 50% or higher of cell viability at
50 lM concentration were then used for IC50 determinations
with the starting concentration of the compounds being
1
50 lM. Threefold serial dilutions were performed and the con-
Determination of the cytotoxicity of the compounds
against androgen-independent prostate cancer cells
centration of the last well was 8 nM. Compounds with percent
inhibition between 30–50% had a starting concentration of
200 lM. Following incubation with the drug for 72 h MTS assay
was performed using the CellTiter 96m AQueous kit. IC50 values
were calculated by using the SOFT max Pro plate reader pro-
gram. The control used was 5-FU with IC50 value of 1.5 lM. Each
compound was tested at least three times.
(PC-3)
Materials
PC-3 cells were obtained from the American Type culture collec-
tion (Manassas, VA, U.S.A). RPMI 1640 medium, PBS, FBS, gluta-
mine, and gentamicin reagent solution were purchased from
Gibco (Grand Island, NY, USA). Trypsin-EDTA solution (10x) and
trypan blue solution were purchased from Sigma Chemical
Statistical analyses
(
St. Louis, MO, USA). Cell Titre 96 Aqueous Non-Radioactive Cell
Proliferation kit was purchased from Promega Corporation
Madison, WI, USA). Cell culture flasks, plates, etc. were pur-
Correlations between cytotoxicity of the compounds against PC-
3 cell line and various physiochemical properties such as Ham-
mett (r), Hansch (p), molar refractivity (MR) of the aryl substitu-
ents and partition coefficient (log P) values of the compounds
were generated using the statistical package SPSS (Ver. 14.0) for
(
chased from Corning Inc (USA). Marathon 3200 centrifuge was
purchased from Fisher Scientific (Pittsburg, PA, USA). HERA cell
incubator and HERA safe cell culture safety hood were pur-
chased from Kendro Laboratory products (Newton, CT, USA). An
IV900 series inverted microscope was purchased from Micro-
scoptics, Inc. (Holy, MI, USA). SpectraMax 384 Plus spectrophot-
ometer was purchased from Molecular devices (Sunnyvale, CA,
USA.)
Windows. Values of the correlations between cytotoxicity (IC50
)
and log P for compounds 1a–e and 2a–e, and IC50 and Hammett
(r) for compounds 1a-d and 2a–d, are shown in the Table 6,
others are mentioned in the text where it is appropriate.
References
Cell maintenance
PC-3 cells were maintained in T-75 flasks at 378C and 5% CO
with RPMI-1640 medium supplemented with 10% FBS, 2 mM glu-
tamine, and 0.02% gentamicin solution (10 mg/mL).
2
[
[
[
[
[
1] H. I. Gul, J. Vepsalainen, M. Gul, E. Erciyas, O. Hanninen,
Pharm. Acta Helv. 2000, 74, 393–398.
2] H. I. Gul, M. Gul, E. Erciyas, Arzneimittelforschung 2002,
5
2, 628–635.
Determination of the cytotoxic activity of the compounds
3] H. I. Gul, M. Gul, O. Hꢀnninen, Arzneimittelforschung
2002, 52, 840–843.
[
25]
Cells were plated into 96-well plates at a cell density of 1000 cells
4] H. I. Gul, M. Gul, J. Vepsalainen, E. Erciyas, O. Hanninen,
per well in 100 lL of medium. The cells were incubated at 378C
Biol. Pharm. Bull. 2003, 26, 631–637.
and 5% CO
face. Compounds were weighed out and dissolved in DMSO to
0 mM concentration. Dilutions were made in medium to a final
2
for 24 h to allow the cells to attach to the plate sur-
5] M. Gul, H. I. Gul, U. Das, O. Hanninen, Arzneimittel-
forschung 2005, 55, 332–337.
1
concentration of 50 and 10 lM and a final volume of 200 lL.
With compounds having solubility problems at higher concen-
trations, lower starting concentration of 25 lM was used. The
concentrations of DMSO did not exceed 0.2% of the total volume
in each well. Triplicates per compound per plate were perform-
ed and as well as a no-drug control, which received 100 lL of
medium with DMSO only. The compounds were incubated with
[6] P. N. Gordon, J. D. Johnston, A. R. English, in Antimicro-
bial Agents and Chemotherapy (Ed.: G. L. Hobby), American
Society for Microbiology, Bethesda, 1965, pp. 165–167.
[7] J. R. Dimmock, K. M. Advikolanu, H. E. Scott, M. J. Duffy,
et al., J. Pharm. Sci. 1992, 81, 1147–1152.
[8] J. R. Dimmock, N. M. Kandepu, M. Hetherington, J. W.
Quail, et al., J. Med. Chem. 1998, 41, 1014–1026.
2
the cells for 72 h at 378C and 5% CO . Promega Cell Titer 96 Aque-
ous Non-Radioactive Cell Proliferation assay kit was used to
determine the viability of cells. For four plates 8 mL of MTS solu-
tion was combined with 400 lL of PMS solution. MTS/PMS solu-
tion (20 lL) was added to each well and the 96-well plates were
[9] O. Sabzevari, G. Galati, M. Y. Moridani, A. Siraki, P. J.
O'Brien, Chem. Biol. Interact. 2004, 148, 57–67.
[
[
10] C. A. Calliste, J. C. L. Bail, P. Trouillas, C. Pouget, et al.,
Anticancer Res. 2001, 21, 3949–3956.
2
incubated for 3-5 h at 378C and 5% CO . Absorbance was deter-
11] S. Iwata, T. Nishino, N. Nagata, Y. Satomi, et al., Biol.
Pharm. Bull. 1995, 18, 1710–1713.
mined at 490 nM using a Molecular devices Spectra Max 384
Plus spectrophotometer with SOFTmax Pro software. The absor-
bance data were copied into Microsoft Excel and the replicates
of each compound were averaged, as were values for the control
[12] K. Bowden, A. Dalpozzo, C. K. Duah, J. Chem. Res. S 1990,
12, 377.
i
2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2007, 340, 195–201
Cytotoxicity of Chalcone Mannich Bases
201
[
[
[
13] P. Boeck, P. C. Leal, R. A. Yunes, V. C. Filho, et al., Arch.
[19] J. R. Dimmock, P. Kumar, Curr. Med. Chem. 1997, 4, 1–22.
Pharm. (Weinheim) 2005, 338, 87–95.
[
[
[
20] J. A. Benvenuto, T. H. Connor, D. K. Monteith, J. L. Laid-
14] S. F. Nielsen, S. B. Christensen, G. Cruciani, A. Kharazmi,
law, et al., J. Pharm. Sci. 1993, 82, 988–991.
T. Liljefors, J. Med. Chem. 1998, 41, 4819–4832.
21] F. Sahin, C. Safak, O. Yegen, A. A. Bilgin, FABAD 1984, 9,
15] F. de Campos-Buzzi, J. Pereira de Campos, P. Pozza
124–132.
Tonini, R. Correa, et al., Arch. Pharm. (Weinheim) 2006,
22] R. M. Silverstein, G. C. Bassler, T. C. Morrill, Spectrometric
3
39, 361–365.
rd
identification of organic compounds, 3 Ed., John Wiley &
Sons, Inc., New York, 1974.
[
16] Z. V. Molodykh, L. A. Kudryavtseva, R. A. Shagidullina, L.
V. Shtanova, et al., Farm. Zh. 1987, 21, 182–186.
[23] C. Hansch, A. J. Leo, Substituent constants for correlation
analysis in Chemistry and Biology, John Wiley & Sons, New
York, 1979, pp. 49–50.
[
17] L. G. Kovalenko, O. V. Viktorov-Nabokov, E. M. Ruban, E.
M. Skrynik, Z. A. Denisova, Med. Parazitol. Mosk. 1983, 52,
46–48.
[24] J. G. Topliss, J. Med. Chem. 1972, 15, 1006–1011.
[
18] J. R. Dimmock, P. Kumar, A. J. Nazarali, N. L. Motagana-
[25] A. H. Cory, T. C. Owen, J. A. Barltrop, J. G. Cory, Cancer
Commun. 1991, 3, 207–212.
halli, et al., Eur. J. Med. Chem. 2000, 35, 967–977.
i
2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com