1-(3-Aminomethyl-4-hydroxyphenyl)-3-pyridinyl-2-propen-1-ones: A novel group of tumour-selective cytotoxins

Article abstract of DOI:10.3109/14756366.2012.700927

Two series of 1-(3-aminomethyl-4-hydroxyphenyl)-3-pyridinyl-2-propen-1- ones, designed as novel cytotoxins, were synthesized. The compounds had low CC50 values in the micromolar range against HL-60 promyelocytic leukemic cells and HSC-2, HSC-3 and HSC-4 oral squamous cell carcinomas. The CC50 values of these compounds were higher towards non-malignant HGF (gingival fibroblasts), HPC (pulp cells), and HPLF (periodontal ligament fibroblasts) cells, which reveals the tumour-selectivity of these enones. A representative compound 4c caused cleavage of PARP1 in HSC-2 cells but not in HGF cells, which may be a contributing factor to the tumour-selectivity.

Full text of DOI:10.3109/14756366.2012.700927

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(5): 974–980
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.700927
research arTIcLe
1-(3-Aminomethyl-4-hydroxyphenyl)-3-pyridinyl-2-propen-1-
ones: A novel group of tumour-selective cytotoxins
Sinan Bilginer1, Halise Inci Gul1, Ebru Mete2, Umashankar Das3, Hiroshi Sakagami4, Naoki
Umemura4, and Jonathan Richard Dimmock3
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk University, Erzurum, Turkey, 2Department
of Chemistry, Faculty of Sciences, Ataturk University, Erzurum, Turkey, 3Drug Design and Discovery Research Group,
College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, and 4Division of
Pharmacology, Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan
abstract
Two series of 1-(3-aminomethyl-4-hydroxyphenyl)-3-pyridinyl-2-propen-1-ones, designed as novel cytotoxins, were
synthesized. The compounds had low CC50 values in the micromolar range against HL-60 promyelocytic leukemic
cells and HSC-2, HSC-3 and HSC-4 oral squamous cell carcinomas. The CC50 values of these compounds were higher
towards non-malignant HGF (gingival fibroblasts), HPC (pulp cells), and HPLF (periodontal ligament fibroblasts) cells,
which reveals the tumour-selectivity of these enones. A representative compound 4c caused cleavage of PARP1 in
HSC-2 cells but not in HGF cells, which may be a contributing factor to the tumour-selectivity.
Keywords: Mannich bases, azachalcones, cytotoxicity, molecular modelling, PARP1
Introduction
activities4,5. e cytotoxic activities of this class of com-
pounds has been proposed due to the alkylating poten-
tial of α,β-unsaturated ketones towards cellular
nucleophiles that are liberated in situ following deami-
nation6. 4-Hydroxychalcones have demonstrated their
effectiveness as cytotoxic7,8, antitumour9, antioxidant9
agents. e antimicrobial10 and mosquito repellent11
activities of o-aminomethyl phenol derivatives (Mannich
phenols) are also noteworthy. In view of potential
cytotoxicity associated with 4′-hydroxychalcones and
Mannich ketones, we were interested in preparing a
novel series of Mannich chalcones (2a–f, 4a–e Scheme 1)
in order to explore their effectiveness as potent cytotoxic
agents.
One reason for this pursuit is that conjugated enones
react exclusively or preferentially with thiols in contrast
to amino or hydroxyl groups12. Since the latter two
groups, but not thiols, are found in nucleic acids, the
genotoxic side effects of many anticancer drugs should
be avoided. e interest in chalcones is due not only to
A major problem facing the human population at the
current time is the devastating ravages of cancer. For
example, in USA during 2010 at total of 569,490 individu-
als were expected to die from cancer i.e., more than 1500
people per day while over 1.5 million new cases of can-
cer are predicted to be diagnosed during 20101. Some of
the major problems of current anticancer drug therapy
include their lack of substantially greater toxicity for
cancers compared to normal tissues and the emergence
of multidrug resistance (MDR). Hence tumour-selective
cytotoxins are urgently required whose structures are
sufficiently divergent from contemporary anticancer
medication so that MDR tumours are sensitive to these
compounds.
e principal aim of our laboratory is the discovery of
novel cytotoxic and anticancer alkylating agents. Some
Mannich ketones as potential cytotoxins have emerged
from our research2,3. Chalcones are α,β-unsaturated
ketones, and they are associated with diverse biological
Address for Correspondence: Prof. H. Inci Gul, Department of Pharmaceutical Chemistry, Ataturk University, Erzurum, Turkey.
Tel.: +90 442 231 5203. Fax: +90 442 236 0962. E-mail: incigul1967@yahoo.com
(Received 10 April 2012; revised 31 May 2012; accepted 31 May 2012)
974

A novel group of tumour-selective cytotoxins 975
their antineoplastic properties but also to the absence
of their displaying pronounced toxicity in general13. e
thiol-alkylating properties of chalcones will be enhanced
by the presence of a strongly electron-withdrawing
group in ring B such as a nitro substituent which will
increase the electrophilicity of the adjacent olefinic
carbon atom (Figure 1). However nitroaromatics can be
reduced to form nitroso and amino metabolites as well
as reactive nitro and nitroxyl radicals all of which can
induce oxidative stress14. Hence the decision was made
to replace aryl ring B of the chalcones by an electron-
withdrawing pyridinyl ring.
will increase in acidic tumours and hence selective toxicity
to neoplasms may be demonstrated. Various amines were
considered in order to observe whether cytotoxic potencies
are governed by the nature of the basic group.
An additional substituent to be placed in ring A was
a hydroxyl group for two reasons. First, this substituent
may assist in hydrogen bonding at a receptor. Second, it
is a suitable group for analogue development should this
group of compounds prove to be promising lead mol-
ecules. For example, if the logP values are too high, the
hydroxyl group can be converted into a disodium phos-
phate ester [OP(O)(ONa)2].
Further modifications of the chalcone motif were pro-
posed. One objective was to introduce a basic group into
ring A. When this substituent is protonated, it will exert an
electron-withdrawing effect which will reduce the electron
density on the olefinic carbon atom adjacent to the pyridi-
nyl ring. In so doing thiol alkylation will be enhanced and
hence cytotoxic potencies may increase. Nitrogen atoms
directly attached to aryl rings are weak bases and therefore
a methylene spacer between ring A and the basic centre was
proposed as indicated in Figure 1. As the pH falls, so the per-
centage of the molecules in the ionized form will increase.
e Hammett σm value of the dimethylaminomethyl group
[CH2N(CH3)2], for example, is 0.00 but on protonation this
substituent [CH2NH(CH3)2] is now a strongly electron-
withdrawing group with a σm figure of 0.4015. A number of
tumours are more acidic than the corresponding normal
cells16 and in these cases electrophilic attack of cellular thiols
erefore, the objectives of the present study are: (i) to
develop a series of 1-(3-aminomethyl-4-hydroxyphenyl)-
3-pyridinyl-prop-2-en-1-ones in order to find if cytotoxicity
towards neoplasms was demonstrated and greater toxic-
ity to malignant cells than normal cells is achieved; (ii) to
discern any structural features of this series of compounds
which govern cytotoxicity and tumour-selective toxicity;
(iii) to investigate the possible mode of action of repre-
sentative compounds by which cytotoxicity and selective
tumour toxicity is mediated. Such information will be valu-
able in affording guidelines for expanding the project.
Materials and methods
Synthesis of the compounds 1–4
Melting points were determined using an Electrothermal
9100 (IA9100, UK) instrument and are uncorrected1. H
Scheme 1. Structure and synthesis of the compounds 1–4. e reagents used in the synthesis are i = NaOH, ii = HCHO/amine.
Figure 1. Design of the compounds in series 2 and 4.
© 2013 Informa UK, Ltd.

976 S. Bilginer et al.
NMR (400 MHz) and 13C NMR (100 MHz) spectra were
obtained using a Varian Mercury Plus spectrometer (Palo
Alto, USA). Chemical shifts (δ) are reported in ppm.
Mass spectra were undertaken on a HPLC-TOF Waters
Micromass LCT Premier XE (Milford, MA, USA) mass
spectrometer using an electrospray ion source (ESI).
Hz, 1H), 8.67 (d, J = 3.9 Hz, 1H); 13C NMR (CDCl3) δ 11.3,
46.5, 56.9, 116.4, 122.2, 124.4, 125.6, 125.7, 129.3, 129.9,
130.7, 137.1, 141.7, 150.3, 153.7, 164.3, 188.6; Mass spec-
trum: 311.17 (M++1); HRMS (ESI-MS) Calc.: 311.1760 for
C19H22N2O2 [M+H+], Found: 311.1765.
E-1-[4-Hydroxy-3-(pyrrolidin-1ylmethyl)phenyl]-3-(2-
pyridinyl)prop-2-en-1-one (2c)
E-1-(3-(Dimethylaminomethyl-4-hydroxyphenyl)-3-(2-
pyridinyl)prop-2-en-1-one (2a)
Yield: 77%. M.p.125-127°C1.H NMR (CDCl3) δ 1.80–1.96
(m, 4H), 2.50–2.80 (m, 4H), 3.90 (s, 2H), 6.86 (d, J = 8.2
Hz, 1H), 7.26 (dd, J = 7.8, 4.1 Hz, 1H), 7.45 (d, J = 7.8 Hz,
1H), 7.72 (d, J = 7.8 Hz, 1H), 7.74 (d, J = 15.3 Hz, 1H), 7.82
(bs, 1H), 7.99 (d, J = 8.2 Hz, 1H), 8.11(d, J = 15.3 Hz, 1H),
8.67 (d, J = 4.1 Hz, 1H); 13C NMR (CDCl3) δ 23.9, 53.7, 58.7,
116.3, 122.6, 124.4, 125.6, 125.7, 129.3, 129.4, 130.8, 137.1,
141.8, 150.3, 153.7, 163.9, 188.7;
A solution of paraformaldehyde (0.066 g, 2.2 mmol) and
dimethylamine (0.099 g, 2.2 mmol) in acetonitrile (5ml)
was heated under reflux in a microwave oven (80 watts)
at 80°C for 5 minutes. A solution of compound 1 (0.50 g,
2.2 mmol) in acetonitrile (20 ml) was added to the reac-
tion mixture and heating in the oven at 80°C continued.
e reaction was monitored by TLC and after 7.5h, the
solvent was removed in vacuo and the crude product
was chromatographed using a column of silica gel 60
(70–230 mesh) and chloroform: methanol (9:1) as eluant.
Evaporation of the solvent afforded 2a as a yellow oil.
Yield: 15%1.H NMR (CDCl3) δ 2.34 (s, 6H), 3.73 (s, 2H),
6.87 (d, J = 8.7 Hz, 1H), 7.26 (dd, J = 7.8, 4.6 Hz, 1H), 7.45
(d, J = 7.8 Hz, 1H), 7.72 (dt, J = 7.8, 1.8 Hz, 1H), 7.74 (d, J =
15.3 Hz, 1H), 7.81 (d, J = 1.8 Hz, 1H), 8.00 (dd, J = 8.7, 1.8
Hz, 1H), 8.11 (d, J = 15.3 Hz, 1H), 8.66 (d, J = 4.6 Hz, 1H);
13C NMR (CDCl3) δ 44.6, 62.7, 116.4, 122.0, 124.4, 125.6,
125.7, 129.5, 129.9, 130.9, 137.1, 141.8, 150.3, 153.7, 163.8,
188.7; Mass spectrum: 283.14 (M++1); HRMS (ESI-MS)
Calc.: 283.1447 for C17H18N2O2 [M+H+], Found: 283.1445.
e dienones 2b–f and 4a–e were prepared in a similar
manner. A solution of compound 3 was used instead of 1
to synthesize 4a–e. e times of heating in the oven and
the nature of the eluant used in column chromatography
were as follows: 2b: 4h, chloroform: methanol (7:3);
2c: 8h, chloroform: methanol (6:4); 2d: 48h, ethyl
acetate: methanol (8:2); 2e: 49h, chloroform: methanol
(9:1); 2f: 48h, diisopropyl ether: methanol (7:3). 4a:
72h, chloroform: methanol (8:2); 4b: 72h, chloroform:
methanol (8:2); 4c: 40h, chloroform: methanol (95:5); 4d:
84h, ethyl acetate: methanol (9:1); 4e: 40h, chloroform:
methanol (95:5).
Mass spectrum: 309.16 (M++1); HRMS (ESI-MS) Calc.:
309.1603 for C19H20N2O2 [M+H+], Found: 309.1602.
E-1-[4-Hydroxy-3-(piperidin-1-ylmethyl)phenyl]-3-(2-
pyridinyl)prop-2-en-1-one (2d)
Yield: 47%. M.p. (diethylether-methanol) 139°C1.H NMR
(CDCl3) δ1.58–1.80 (m, 6H), 2.00–3.00 (m, 4H), 3.75 (s, 2H),
6.85 (d, J = 8.2 Hz, 1H), 7.26 (dd, J = 7.8, 4.6 Hz, 1H), 7.45 (d, J
= 7.8 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.74 (d, J = 15.1 Hz, 1H),
7.81 (d, J = 1.8 Hz, 1H), 7.99 (dd, J = 8.2, 1.8 Hz, 1H), 8.11 (d,
J = 15.1 Hz, 1H), 8.67 (d, J = 4.6 Hz, 1H); 13C NMR (CDCl3) δ
24.0, 26.0, 54.0, 62.0, 116.4, 121.8, 124.4, 125.6, 125.7, 129.4,
130.1, 130.7, 137.1, 141.8, 150.3, 153.7, 163.9, 188.6; Mass
spectrum: 323.17 (M++1); HRMS (ESI-MS) Calc.: 323.1760
for C20H22N2O2 [M+H+], Found: 323.1767.
E-1-[4-Hydroxy-3-(4-methylpiperazin-1-ylmethyl)
phenyl]-3-(2-pyridinyl)prop-2-en-1-one (2e)
Yield: 62%. M.p. 119–122°C1.H NMR (CDCl3) δ 2.30 (s,
3H), 2.34–3.00 (m, 8H), 3.80 (s, 2H), 6.87 (d, J = 8.4 Hz,
1H), 7.28 (dd, J = 7.7, 5.0 Hz, 1H), 7.45 (d, J = 7.7 Hz, 1H),
7.72 (d, J = 7.7 Hz, 1H), 7.73 (d, J = 15.2 Hz, 1H), 7.83 (d,
J = 2.0 Hz, 1H), 8.00 (dd, J = 8.4, 2.0 Hz, 1H), 8.11 (d, J =
15.2 Hz, 1H), 8.67 (d, J = 5.0 Hz, 1H); 13C NMR (CDCl3)
δ 46.1, 52.7, 55.0, 61.3, 116.5, 121.3, 124.5, 125.6, 125.7,
129.7, 130.3, 130.9, 137.1, 141.9, 150.3, 153.6, 163.3, 188.6;
Mass spectrum: 338.18 (M++1); HRMS (ESI-MS) Calc.:
338.1869 for C20H23N3O2 [M+H+], Found: 338.1858.
Variations to the general procedure are as follows.
Paraformaldehyde and the appropriate amine were
heated under reflux at 80°C for 0.5h in the synthesis of
2e,f and 4b–e and stirred at room temperature for 0.5h
in the case of 4a. In the syntheses of 4d, a solution of 3
in ethanol (not acetonitrile) was added to paraformalde-
hyde and piperidine dissolved in acetonitrile. e solvent
used in the preparation of 2e, 4c and 4e was ethanol and
not acetonitrile.
E-1-(4-Hydroxy-3-(morpholin-4-ylmethyl)phenyl)-3-(2-
pyridinyl)prop-2-en-1-one (2f)
Yield: 49%. M.p. (diethylether-methanol) 138-139°C1.H
NMR (CDCl3) δ 2.40–2.70 (m, 4H), 3.70–3.78 (m, 4H), 3.79
(s, 2H), 6.88 (d, J = 8.4 Hz, 1H), 7.27 (dd, J = 7.6, 4.6 Hz,
1H), 7.45 (d, J = 7.6 Hz, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.74
(d, J = 15.1 Hz, 1H), 7.83 (d, J = 1.6 Hz, 1H), 8.01 (dd, J =
8.4, 1.6 Hz, 1H), 8.11 (d, J = 15.1 Hz, 1H), 8.67 (d, J = 4.6
Hz, 1H); 13C NMR (CDCl3) δ 53.1, 61.8, 66.9, 116.5, 120.9,
124.5, 125.5, 125.7, 129.9, 130.4, 131.0, 137.1, 142.0, 150.3,
153.6, 163.0, 188.6; Mass spectrum: 325.15 (M++1); HRMS
(ESI-MS) Calc.: 325.1552 for C19H20N2O3 [M+H+], Found:
325.1543.
E-1-(3-(Diethylaminomethyl-4-hydroxyphenyl)-3-(2-
pyridinyl)prop-2-en-1-one (2b)
Yield: 47%. Yellow oil1.H NMR (CDCl3) δ 1.11 (t, J = 7.1 Hz,
6H), 2.65 (q, J = 7.1 Hz, 4H), 3.85 (s, 2H), 6.84 (d, J = 8.6 Hz,
1H), 7.27 (dd, J = 7.2, 3.9 Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H),
7.72 (d, J = 7.2 Hz, 1H), 7.73 (d, J = 15.4 Hz, 1H), 7.82 (d, J
= 1.8 Hz, 1H), 7.99 (dd, J = 8.6, 1.8 Hz, 1H), 8.11 (d, J = 15.4
Journal of Enzyme Inhibition and Medicinal Chemistry

A novel group of tumour-selective cytotoxins 977
amines and formaldehyde producing the correspond-
ing Mannich bases 2a–f and 4a–e1.H NMR spectroscopy
revealed that aminomethylation occurred in the ortho
position to the aryl hydroxy group and the olefinic double
bond assumed the E configuration. Molecular modelling
of the compounds in series 1–4 was undertaken in order
to determine the atomic charges on the olefinic carbon
atom adjacent to the pyridinyl ring, the θ1 and θ2 torsion
angles (as indicated in Supplementary Figure S1) and
logP values. ese results are portrayed in Table 2.
e cytotoxicity data are presented in Table 1. A rep-
resentative compound 4c caused cleavage of poly(ADP-
ribose)polymerase 1 (PARP1) in HSC-2 but not HGF
cells, and it is presented (Supplementary Figure S2).
e first question to be addressed is whether the
azachalcones 1–4 have antineoplastic properties. e
results portrayed in Table 1 reveal that in general the
CC50 values of the compounds are in the low micro-
molar range towards HL-60, HSC-2, HSC-3 and HSC-4
cells. e potencies of 1-4 towards the tumour cell lines
were compared with an alkylating agent, melphalan.
Compounds 1, 2a–c, 3, 4c, e were more potent than mel-
phalan towards HSC-3 cells while 1, 2b, c, e, f, 3, 4c–e had
lower CC50 values in the HSC-4 bioassay. Furthermore,
1, 3 (HSC-2 screen), 2f, 4a, b, d (HSC-3 test) and 2a, 4a,
b (HSC-4 assay) were equipotent with melphalan. e
unsaturated ketones 1, 2e and 3 had the lowest average
CC50 figures and are clearly lead molecules. In addition
to these compounds, 2b, c, 4c, and e also had lower aver-
age CC50 values than melphalan. us, many of the com-
pounds in series 1–4 possess noteworthy antineoplastic
potency.
e second aspect of these compounds to consider
is whether they are tumour-specific cytotoxins since
tumours are surrounded by different types of normal
cells. Selectivity index (SI) figures were generated which
are the quotients of the average CC50 values of the non-
malignant cells and the CC50 figure of a compound
towards a specific cell line. e results in Table 1 reveal
that SI values of greater than 1 were obtained and thus
series 1–4 are tumour-specific antineoplastic agents. An
average figure of four was arbitrarily chosen as evidence
of significant selective toxicity to neoplasms and was
displayed by the compounds in series 1–4 except 2a, d–f.
e greatest selectivity, which is 6.24, was noted with 4c
(Table 1). e neoplasms differ in their sensitivity to the
compounds, which is possibly caused by their having
somewhat distinctive modes of action in each cell line.
Lead compounds should possess both marked cyto-
toxic potencies and selective toxicity for tumours. In
order to identify such molecules, a potency selectivity
expression (PSE) was devised which is the product of
the reciprocal of the average CC50 values towards HL-60,
HSC-2-, HSC-3 and HSC-4 cells (a measure of potency)
and the average SI figures towards these cell lines (a
determination of tumour-selectivity) expressed as a per-
centage. e PSE data are given in Table 1. Seven com-
pounds have PSE figures greater than 30 namely 1, 2b,
Biological activity
Cytotoxicity evaluation
e compounds in series 1–4 were assayed towards
HSC-2, HSC-3, HSC-4, HL-60, HGF, HPC and HPLF cells
based on a literature procedure17 with some minor modi-
fications. In brief, cells were cultured in DMEM supple-
mented with 10% foetal bovine serum (FBS) except
the HL-60 cells were cultured in RPMI 1640 medium
supplemented with 10% FBS. Varying concentrations of
the compound in dimethylsulfoxide were added to the
medium and incubated at 37°C for 48h. e viable cell
numbers were determined by the MTT method except
the viability of HL-60 cells was obtained by cell count-
ing with a hemocytometer after staining with 0.15%
trypan blue. e CC50 values were determined from dose-
response curves.
PARP1 cleavage
e antibody against cleavage PARP1 was purchased from
cellSignalingTechnology(Beverly,MA,USA)whiletheanti-
actinantibodywasobtainedfromSantaCruzBiotechnology
(Santa Cruz, CA, USA). e HSC-2 and HGF cells were
cultured in six well plates for 24h and then incubated with
4c for 24h. e cells were scraped and collected in lysis buf-
fer (20mM HEPES pH 7.4, 1% Triton-X 100, 150mM NaCl,
1.5mM MgCl2, 12.5mM β-glycerophosphate, 2mM EGTA,
10mM NaF, 2mM DTT, 1mM Na3PO4, 1mM PMSF plus
1 × protease inhibitor). Equal amounts of proteins were
transferred onto a polyvinyldene difluoride membrane
saturated with 5% non-fat dry milk. Membranes were incu-
bated with primary antibody and then with horseradish
peroxidase-conjugated secondary antibody of 1h at room
temperature. Membranes were washed with Tris-buffered
saline with 0.5% Tween 20 and developed. Secondary anti-
bodies were horseradish peroxidase-conjugated goat anti-
mouse or antirabbit, and reacted with a chemiluminescent
substrate system and exposed to X-ray films. A represen-
tative compound 4c, was examined for its ability to cause
PARP1 cleavage, since it had the highest average SI value
towards the four malignant cells among the compounds in
series 1–4.
Molecular modelling
e models of the compounds in series 1–4
(Supplementary Figure S1) were built using ChemBio
3D ultra 12.0 and exported to Spartan 2008 for energy
minimization using the AM 1 semi-empirical quantum
mechanical chemistry method for determining atomic
charges and torsion angles. e logP values were cal-
culated using ChemBio3D ultra 12.0. e results were
shown at Table 2.
results and Discussion
Condensation between 4-hydroxyacetophenone and the
appropriate formylpyridine afforded the unsaturated
ketones 1 and 3. ese compounds reacted with various
© 2013 Informa UK, Ltd.

978 S. Bilginer et al.
Table 2. Determination of some atomic charges and torsion
angles of 1–4 and their logP values.
Compound
q (esu)†
LogP
θ1 (°)‡
θ2 (°)‡
1
−0.316
−0.321
−0.337
−0.291
−0.216
−0.298
−0.211
−0.071
−0.086
−0.046
−0.037
0.012
−21.35
−21.12
−20.42
19.49
34.60
34.45
34.86
−35.09
29.04
34.42
−32.67
15.57
21.20
−12.77
2.54
2.08
2.24
2.90
2.54
2.96
1.98
1.83
1.86
1.80
2.48
2.11
2.53
1.55
2a
2b
2c
2d
2e
2f
28.48
−21.26
−28.90
−24.74
−29.16
24.05
3
4a
4b
4c
4d
4e
25.19
29.74
−1.03
−0.055
−25.12
15.34
†e q values are the charges on the β carbon atom as indicated in
Supplementary Figure S1.
‡e θ1 and θ2 figures are the torsion angles between the aryl and
heterocyclic rings and the adjacent unsaturated groups are
indicated in Supplementary Figure S1.
c, e, 3, 4c, e and serve as lead molecules for analogue
development.
Investigations were also undertaken with a view to
determine structural features which influence potency.
In the first place, evaluations were conducted to find
the optimal amino group(s) in 2a–f and 4a–e. In order
to make these comparisons, a scoring system was
employed whereby the most potent compound in series
2 was assigned a rating of 6, the compound with the next
highest potency awarded 5 points and so forth. Standard
deviations were taken into consideration, the scores for
equipotent compounds were divided equally and a total
of 21 points (Σ 6, 5, 4, 3, 2, 1) were awarded for 2a–f in each
bioassay. In the case of 4a–e, 15 points (Σ 5, 4, 3, 2, 1) were
allocated. Using this approach, the order of potencies (the
scores are in parentheses) for series 2 were as follows,
namely 2e (22.5) >2c (18.5) >2b (14.5) >2f (13.5) >2a (11)
>2d (4). For series 4, the order of potencies were 4c, e (17)
>4d (13.5) >4a (8) >4b (4.5). us in both series 2 and 4,
the 4-methyl-1-piperazinyl and 1-pyrolidinyl groups are
present in the most potent compounds. is can be a use-
ful observation to consider analogue development.
A further issue requiring resolution was whether
greater cytotoxic potency was going to be found in the
2-pyridinyl or 3-pyridinyl series of compounds having
the same amino group. e 2-pyridinyl compounds had
greater potencies than the analogues in series 4 in half
(10/20) of the comparisons made. In six cases, equipoten-
cies were observed while 4d was invariably more potent
than 2d (4/20). It is of interest to note that 1 had lower
CC50 values than 3 in the HL-60 and HSC-3 tests, while
equipotency was observed in the other two bioassays. In
general, therefore lower CC50 values were found in the
2-pyridinyl series 2 than with the structural isomers 4.
e average selectivity index figures reveal that each
of the compounds in series 4 had a higher value than
the analogues in series 2, which have the same amino
Journal of Enzyme Inhibition and Medicinal Chemistry

A novel group of tumour-selective cytotoxins 979
substituent. e average SI figure of 4a–e was 5.31 in con-
trast to 3.90 for 2a–e. In addition, the average SI values
of 1 and 3 were 4.35 and 5.28, respectively. Hence, the
conclusion to be drawn is that in terms of demonstrating
greater toxicity to neoplasms than normal cells, the pres-
ence of a 3-pyridinyl ring is preferable to the 2-pyridinyl
isostere. e three compounds with the highest average
SI figures are 3, 4c, d and are therefore lead molecules.
One may also note that the SI values generated are depen-
dent on the neoplastic cell line. For example, as indicated
in Table 1, the average SI figures of the compounds in
series 1–4 in 5.95 in the HL-60 screen which is more than
double the value observed in the HSC-2 bioassay.
In regard to the PSE figures, the data in Table 1 reveals
that the average PSE values for 2a–e and 4a–e were simi-
lar being 24.9 and 28.0, respectively. e PSE data for 1
and 3 were also virtually identical. is observation con-
firms the earlier conclusions that the presence of a 2-pyr-
idinyl group enhances potency, while the 3-pyridinyl
analogues display greater toxicity to tumourous tissues
than non-malignant cells.
Molecular modelling of the compounds in series
1–4 was undertaken with a view to discern correlations
between various physicochemical characteristics of the
molecules and the bioactivity observed with special
reference to the general observations that potencies
were greater in 2a–e than 4a–e, whereas the SI values
were higher in series 4. us the atomic charges on the
β-carbon atom were obtained. In addition, the torsion
angles θ1 and θ2 between the aryl and pyridinyl rings with
the adjacent unsaturated groups as illustrated in supple-
mentary Figure S1 were generated. Furthermore the logP
values of the compounds in series 1–4 were determined.
ese results are presented in Table 2.
Cellular thiols would be expected to react with α,β-
unsaturated ketones at the β-carbon atom6. Hence if the
rate and extent of thiol alkylation is an important contrib-
utor to cytotoxicity, the charge densities on the β-olefinic
carbon atom will influence potency considerably. e
average atomic charges of 2a–e and 4a–e were −0.293
and −0.047, respectively. In terms of potency, this result
is counterintuitive as one would have predicted that CC50
values would diminish as the fractional negative charge
on the β-carbon atoms was lowered thereby enhancing
nucleophilic attack by thiols. However, selective toxicity,
which is greater in series 4 than 2, may be associated with
the smaller negative charge on the β-carbon atom.
Several studies have revealed that the torsion angles
between an aryl or heterocyclic ring and an adjacent
unsaturated group are correlated with biological
potencies18. Hence the decision was made to determine
the θ1 and θ2 torsion angles which are indicated in
supplementary Figure S1. One may note that in most
cases the tilt of the aryl and pyridinyl rings in 1–4 are in
opposite directions, i.e., the θ1 and θ2 values are in either
clockwise (+) or anticlockwise (−) orientations. Insertion
of aminomethyl groups into the meta position of the aryl
rings in 1 and 3 leading to the meta position series 2 and
4 had a minimal effect on the θ1 values. Examination of
the θ2 figures shows that the point of attachment of the
pyridinyl ring to the β-carbon atom had a marked effect
on the θ2 torsion angles. e average θ2 values of 2a–e
and 4a–e were 33.4° and 10.6°, respectively, which may
contribute to the variation in potency and selectivity
between series 2 and 4. us in the future, replacement
of the heterocyclic ring by an aryl ring possessing
substituents in one or two ortho positions (creating
high θ2 values) as well as placing the same groups in
the meta and para locations (low θ2 figures) should be
undertaken to explore this hypothesis.
e logP values of the compounds in series 1–4 are
presented in Table 2. ey are in the range of 1.5–3.0 and
thus within one of the guidelines for compounds which
are well absorbed after oral dosing19. e logP values
were higher in 2a–e than the analogues in series 4 which
had the same basic group. In developing this project,
compounds with a wider range of hydrophobicity should
be prepared and subjected to bioevaluation. e results
will shed some light on whether an increase in logP val-
ues will enhance cytotoxic potencies, while reduction in
hydrophobicity will lead to compounds with increased
selective toxicity to tumours.
Many cytotoxins exert their bioactivity by inducing
apoptosis20, but single-stranded DNA breaks can be
repaired by poly(ADP-ribose)polymerase 1 (PARP1)21.
e compounds, which cleave PARP1 (and hence reduce
the extent of repair of DNA damage) to a greater extent
in neoplasms compared with normal cells, may be use-
ful agents in cancer chemotherapy. A representative
compound 4c, which had the highest average SI value
towards the four malignant cells among the compounds
in series 1–4, was examined for its ability to cause PARP1
cleavage in HSC-2 and HGF cells. Supplementary Figure
S2 reveals that some cleavage of PARP1 occurs only in
HSC-2 cells not in HGF cells after 24h at 25 µM. Hence,
a possible factor contributing to the tumour-selectivity of
4c (and maybe to other compounds in series 1–4) is the
ability to inhibit DNA repair preferentially in malignant
cells.
conclusions
is study revealed that various 1-(3-aminomethyl-
4-hydroxyphenyl)-3-pyridinyl-2-propen-1-ones possess
not only noteworthy potencies towards several neo-
plasms but demonstrate a greater toxicity to tumours than
various non-malignant cells. In general, the compounds
in series 2 are more potent than the analogues in series
4. On the other hand, greater SI values are found among
4a–e. Using HSC-2 cells, a representative compound 4c
cleaved PARP1 but at the same concentration this effect
was not noted in HGF cells. Various physicochemical
parameters obtained from molecular modelling enabled
some guidelines for amplification of the project to be
made.
© 2013 Informa UK, Ltd.

980 S. Bilginer et al.
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acknowledgments
is study was supported by the Research Foundation of
Ataturk University (Project Number: 2009/317, 2010/166)
Erzurum, Turkey.
Declaration of interest
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Journal of Enzyme Inhibition and Medicinal Chemistry