(E)-3-(3,4,5-Trimethoxyphenyl)-1-(pyridin-4-yl)prop-2-en-1-one, a heterocyclic chalcone is a potent and selective CYP1A1 inhibitor and cancer chemopreventive agent

Article abstract of DOI:10.1016/j.bmcl.2017.11.009

The overexpression of CYP1 family of enzymes is reported to be associated with development of human carcinomas. It has been well reported that CYP1A1 specific inhibitors prevents carcinogenesis. Herein, thirteen pyridine-4-yl series of chalcones were synt

Full text of DOI:10.1016/j.bmcl.2017.11.009

Accepted Manuscript
(E)-3-(3,4,5-Trimethoxyphenyl)-1-(pyridin-4-yl)prop-2-en-1-one, a heterocy-
clic chalcone is a potent and selective CYP1A1 inhibitor and cancer chemopre-
ventive agent
Neill J. Horley, Kenneth J.M. Beresford, Supriya Kaduskar, Prashant Joshi, Glen
J.P. McCann, Ketan C. Ruparelia, Ibidapo S. Williams, Linda Gatchie, Vinay
R. Sonawane, Sandip B. Bharate, Bhabatosh Chaudhuri
PII:
S0960-894X(17)31085-5
https://doi.org/10.1016/j.bmcl.2017.11.009
BMCL 25415
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 October 2017
3 November 2017
5 November 2017
Please cite this article as: Horley, N.J., Beresford, K.J.M., Kaduskar, S., Joshi, P., McCann, G.J.P., Ruparelia, K.C.,
Williams, I.S., Gatchie, L., Sonawane, V.R., Bharate, S.B., Chaudhuri, B., (E)-3-(3,4,5-Trimethoxyphenyl)-1-
(pyridin-4-yl)prop-2-en-1-one, a heterocyclic chalcone is a potent and selective CYP1A1 inhibitor and cancer
chemopreventive agent, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.
2017.11.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
(E)-3-(3,4,5-Trimethoxyphenyl)-1-(pyridin-4-
yl)prop-2-en-1-one, a heterocyclic chalcone is a
potent and selective CYP1A1 inhibitor and
cancer chemopreventive agent
Leave this area blank for abstract info.
Neill J. Horley, Kenneth J. M. Beresford, Supriya Kaduskar, Prashant Joshi, Glen J.P. McCann, Ketan C. Ruparelia,
Ibidapo S. Williams, Linda Gatchie, Vinay R. Sonawane, Sandip B. Bharate,* Bhabatosh Chaudhuri*
16
14
12
10
8
13.8
OMe
MeO
MeO
N
PHE319
B[a]P,
EC50 (µM)
PHE224
6
O
8a
LEU 254
4
PHE258
CYP1A1: IC50 = 0.058 µM
CYP1B1: IC50 = 8.18 µM
CYP1A2: IC50 = 1.12 µM
LEU 314
0.8
2
ASN 255
GLU 256
LEU 312
0
B[a]P
B[a]P + 8a
CYP2D6, 2C9, 2C19, 3A4:IC50=>10 µM
Control
8a
SER 116
B[a]P toxicity in CYP1A1
overexpressing cells
Potent and selective CYP1A1 inhibitor
Fonts or abstract dimensions should not be changed or altered.

1
Bioorganic Medicinal Chemistry Letters
jo u r n a l h o m e p a g e : w w w . e ls e v ie r . c o m
(E)-3-(3,4,5-Trimethoxyphenyl)-1-(pyridin-4-yl)prop-2-en-1-one, a heterocyclic
chalcone is a potent and selective CYP1A1 inhibitor and cancer chemopreventive
agent
Neill J. Horley,a, Kenneth J. M. Beresford,a,  Supriya Kaduskar,a,  Prashant Joshi,c, d Glen J.P. McCann,a Ketan C.
Ruparelia,a Ibidapo S. Williams,a,b Linda Gatchie,a,b Vinay R. Sonawane,a Sandip B. Bharate,c,d,* Bhabatosh
Chaudhuria,b,*
a Leicester School of Pharmacy, De Montfort University Leicester, LE1 7RH, UK.
b CYP Design Ltd, The Innovation Centre, 49 Oxford Street, Leicester, LE1 5XY, UK.
c Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-180001, India.
d Academy of Scientific & Innovative Research (AcSIR), CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-180001, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The overexpression of CYP1 family of enzymes is reported to be associated with
development of human carcinomas. It has been well reported that CYP1A1 specific
inhibitors prevents carcinogenesis. Herein, thirteen pyridine-4-yl series of chalcones
were synthesized and screened for inhibition of CYP1 isoforms 1A1, 1B1 and 1A2 in
SacchrosomesTM and live human HEK293 cells. The structure-activity relationship
analysis indicated that chalcones bearing tri-alkoxy groups (8a and 8k) on non-
heterocyclic ring displayed selective inhibition of CYP1A1 enzyme, with IC50 values of
58 and 65 nM, respectively. The 3,4,5-trimethoxy substituted derivative 8a have shown
>10 fold selectivity towards CYP1A1 with respect to other enzymes of the CYP1 sub-
family and >100-fold selectivity with respect to CYP2 and CYP3 family of enzymes.
The potent and selective CYP1A1 inhibitor 8a displayed antagonism of B[a]P mediated
activation of aromatic hydrocarbon receptor (AhR) in yeast cells, and also protected
human cells from CYP1A1-mediated B[a]P toxicity in human cells. This potent and
selective inhibitor of CYP1A1 enzyme have a potential for development as cancer
chemopreventive agent.
Available online
Keywords:
Chalcones,
CYP1A1,
cancer chemoprevention,
B[a]P,
AhR antagonism
2017 Elsevier Ltd. All rights reserved.
Cancer is considered as one of the most lethal diseases worldwide,
However, their expression increases several fold when normal cells
are exposed to PAHs. This leads to over expression of CYP1
enzymes. For example, exposure of lung and skin tissues to B[a]P
leads to increase in CYP1A1 levels. Similarly, exposure of ovarian,
mammary and uterine cells to PAHs leads to increased expression of
CYP1B1.13-17 The aromatic hydrocarbon receptor, AhR, is a
transcription factor that belongs to the hormone receptor family.
PAHs are ligands of AhR but also act as substrates for CYP1A1 and
CYP1B1 enzymes. Within healthy cells, PAHs bind to the ligand-
binding domain of AhR and activate the AhR signalling pathway.
This results in increased expression of the components of the
machinery (i.e. the CYP1 sub-family of enzymes) that metabolizes
xenobiotics. CYP1A1 and CYP1B1 over expression is specifically
linked to the metabolism, through hydroxylation, of B[a]P and
estradiol, respectively. CYP1A1 metabolizes B[a]P into
benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, whereas CYP1B1
converts the estrogen receptor ligand, estradiol (overproduction of
which is the cause of the majority of breast cancers), to its 4-
hydroxylated derivative. Both products form quinone intermediates
which covalently bind to DNA leading to DNA damage,
chromosomal aberrations and generation of excessive reactive
oxygen species (ROS). It has been reported that (a) CYP1A1-
mediated metabolism of B[a]P leads to lung cancer18-27 and (b)
CYP1B1-mediated metabolism of estradiol leads to breast cancer.12,
2
particularly in developing countries.1, From the past 30 years’
experience, it has been learnt that current cancer treatment regimens,
which include conventional and advanced therapeutics alongside
surgical procedures, are associated with poor patient survival and
high mortality rate. This is because drug toxicity, inflammation and
pain reduce overall patient compliance. Hence, there remain
considerable challenges associated with these therapies. Therefore,
‘chemoprevention’ is gaining traction in the scientific community.3-9
“Carcinogenesis” in dividing cells usually occurs through the three
distinct phases of initiation, promotion, and progression.10 These
sequential cellular events may be triggered by exogenous factors
such as carcinogenic chemicals (for example, polyaromatic
hydrocarbons), radiation, infectious organisms (e.g. viruses), or
endogenous factors such as inherited mutations in the genomic DNA,
upregulated hormone levels and downregulated immune status.11, 12
The CYP1 sub-family consists of the haem containing
monooxygenases 1A1, 1B1 and 1A2, which are involved in the
hydroxylation of a large number of pro-carcinogens, such as
polyaromatic hydrocarbons (PAHs) benzo[a]pyrene [BaP], oxides,
amines, and estrogens to cytotoxic, mutagenic and carcinogenic
chemicals. The CYP1 enzymes are present at basal levels in all
healthy cells and are involved in normal cellular metabolism.
------
*Corresponding author. E-mail: bchaudhuri@dmu.ac.uk (BC), sbharate@iiim.ac.in (SBB)
# NJH, KJMB and SK contributed equally to the work described.

2
Bioorganic Medicinal Chemistry Letters
16, 28, 29
O
O
Hence, both B[a]P and estradiol are considered as pro-
carcinogens which are converted to carcinogenic entities in the
Ar
a
+
Ar-CHO
presence of CYP1A1 and CYP1B1.11,
It has also been
24, 30-34
N
N
8a-8m
6
7
observed, in studies of proteomics that CYP1B1 over expression can
lead to resistance to docetaxel and cisplatin due to extensive
metabolism.15, 35-37 It has also been reported that CYP1A1 mediated
metabolism results in generation of the more active metabolites. This
strategy was successfully employed in prodrug design which bio
activated upon metabolism by CYP1A1 enzyme.38-41
8a: Ar = Ph (3,4,5-tri-OMe)
8b: Ar = Ph (3,4-di-OMe)
8c: Ar =Ph (2-OMe)
8d: Ar = Ph (3-OMe)
8e: Ar = Ph (4-OMe)
8f: Ar = Ph (3,4-methylene-dioxy)
8g: Ar =Naphthalen-1-yl
8h: Ar = Ph (3,5-di-OMe)
8i: Ar = Ph (2,3,4-tri-OMe)
8j: Ar = Ph (3,5-di-OMe, 4-OEt)
8k: Ar = Ph (3,4,5-tri-OEt)
8l: Ar = Ph (3,4-Di-OMe, 5-OEt)
8m:Ar = Ph (3,4-di-OEt, 5-OMe)
Various biochemical and cellular studies suggest that CYP1 enzymes
are expressed only at basal levels in healthy tissues, while the same
tissues when exposed to pro-carcinogens induce high level of
expression of these enzymes. This selective behavior provides an
opportunity for medicinal chemists to prevent CYP1A1 and CYP1B1
mediated conversion of pro-carcinogens to carcinogens which is the
genesis of cancer. Several natural products including resveratrol (1),
quercetin (2), genistein (3) and many more dietary flavonoids
possess cancer chemopreventive activity.38, 42-47 Similarly, the ANF
derivative 4 can overcome the resistance of CYP1B1 over expressing
cells to docetaxel36 and 3-pyridyl chalcone 5 overcome the resistance
of CYP1B1 over expressing cells to cisplatin37 (Figure 1).
Figure 2. Synthesis of pyridine-4-yl chalcones 8a-m. Reagents and
conditions: (a) NaOH/ KOH and solid state grinding using mortar and pestle,
yield: 2-18%.
CYP inhibition activity: All synthesized pyridine-4yl chalcones
were screened for inhibition of CYP1, CYP2 and CYP3 family
enzymes using SacchrosomesTM (Sacchrosomes are endoplasmic
reticular membrane bound CYP-reductase complexes which were
isolated from recombinant baker’s yeast, Saccharomyces cerevisiae,
cells) in EROD assay.52 The results of CYP inhibition in
Sacchrosomes for CYP1 (1A1, 1B1 and 1A2), CYP2 (2D6, 2C9 and
2C19) and CYP3 (3A4) family isoforms are depicted in Table 1.
Results indicate that all compounds are selective CYP1 family
inhibitors. The pyridine-4-yl chalcone 8a displayed >10-fold
selectivity in inhibition of CYP1A1 within CYP1 family isoforms,
with IC50 value of 61 nM. Some of the key structure-activity
relationship features have been deduced from 3 and 4-pyridyl
chalcone class. These include (a) 3-pyridyl substituted CYP1
inhibitors are selective for CYP1B1 isoform, whereas 4-pyridyl
chalcones requires fine tuning of substitutions for necessary π-π and
polar interactions leading to CYP1 inhibition. (b) The 3, 4, 5-alkoxy
substitutions in the non-heterocyclic ring are crucial features for
CYP1A1 specificity. (c) Hydroxyl and halogen substitutions on the
non-heterocyclic ring reduce CYP1 inhibition potency in both 3 and
4-pyridyl heterocyclic chalcones. (d) Alkoxy substitution(s) on the
non-heterocyclic ring is/are critical for nanomolar CYP1 inhibition
potency, in both 3-pyridyl and 4-pyridyl classes of compounds. (e)
The increase in the number of methoxy group seems to favour CYP
inhibition; moreover a larger alkoxy group, like ethoxy, also favours
potency.
Figure 1. Known CYP1 family inhibitors
Using bioisoester approach and conventional drug design, we
recently have carried out successful mimicry of the flavonoid
structure to synthesize biphenyl urea,48 quinazoline,48,
and
49
chalcone37 classes of compounds for reversal of CYP1B1-mediated
chemo-resistance of cisplatin. In continuation of our earlier work in
this area,37, 48-51 herein we describe the design and synthesis of new
pyridine-4yl chalcones as selective CYP1 family inhibitors with the
ability to provide protection of cells against CYP1A1 mediated
B[a]P toxicity.
Literature precedence suggests that most CYP substrates and
inhibitors are devoid of optimal drug-like properties, which is critical
for their development as a drug candidate. Another major reason for
the failure of large numbers of new chemical entities is their lack of
cellular efficacy. Traditionally, CYP screening is performed with
human liver microsomes (HLMs) which harbour the major CYPs
that are important for drug metabolism studies. However, HLMs
suffer from stability issues since CYPs rapidly lose enzymatic
activity, within a few hours, at room temperature and, therefore,
reproducibility of the assay system is difficult to maintain.
Determination of CYP enzyme specificity using HLMs, which
contain so many different CYPs, is also challenging. Instead
recombinant microsomes, bound to individual CYP enzymes,
isolated from bacterial (Bactosomes), insect (Supersomes or
Baculosomes) and yeast (Sacchrosomes) cells, are routinely used.
Unfortunately, results obtained with recombinant microsomal CYP
enzymes often are quite dissimilar to that obtained using HLMs and
even hepatocytes, from which HLMs are derived.
Synthesis: A series of 4-pyridyl chalcones were synthesized using
the classical Claisen-Schmidt condensation. This approach involves
the condensation reaction between equimolar amounts of aromatic
aldehydes (6) and heteroaromatic acetophenones (7) in either acidic
or basic conditions. Initially, aqueous NaOH or aqueous KOH was
used with a miscible organic solvent, methanol or ethanol. However,
this method suffers from poor yield even at high equivalents of base.
The problem was overcome by grinding them physically, without
using any solvent, in which equimolar amounts of aromatic
aldehydes and hetero-aromatic acetophenones were mixed together
by means of physical attrition in presence of 5 equivalents of base,
which was followed by usual aqueous work-up and purification
using silica-gel column chromatography. The synthetic scheme for 4-
pyridyl chalcones is provided in Figure 2. Total thirteen chalcones
8a-m were synthesized and all were characterized by NMR, IR and
MS analysis.

Table 1. The IC50 values for inhibition of CYP SacchrosomesTM by 4-pyridyl chalcones 8a-m.
3
IC50 values for CYP inhibition in SacchrosomesTM (µM)
Entry
1A1
1B1
1A2
2D6
2C9
>20
>20
2C19
>20
3A4
>20
>20
8a
8b
0.061 ± 0.005
0.38 ± 0.02
5.21 ± 0.06
0.17 ± 0.02
1.09 ± 0.05
0.12 ± 0.03
5 ± 0.1
8 ± 0.2
>20
8c
0.57 ± 0.02
0.048 ± 0.003
0.032 ± 0.002
11 ± 0.2
>20
>20
>20
8d
8e
0.59 ± 0.02
6.31 ± 0.06
1.12 ± 0.05
0.51 ± 0.03
0.18 ± 0.01
1.41 ± 0.05
0.23 ± 0.3
0.054 ± 0.002
7.0 ± 0.1
0.020 ± 0.003
12 ± 0.2
12 ± 0.2
15 ± 0.2
12 ± 0.2
18 ± 0.4
16 ± 0.3
11 ± 0.2
12 ± 0.2
14 ± 0.2
17± 0.3
15 ± 0.3
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
8f
2.11 ± 0.08
0.65 ± 0.04
0.041± 0.004
0.032 ± 0.002
0.698 ± 0.04
8.62 ± 0.12
3.81 ± 0.08
2.92 ± 0.04
0.05 ± 0.01
1.5 ± 0.05
8g
0.035 ± 0.004
0.028 ± 0.002
0.104 ± 0.008
2.58 ± 0.08
1.19 ± 0.05
1.32 ± 0.05
0.78 ± 0.01
0.03 ± 0.01
8h
8i
8j
8k
8l
0.092 ± 0.003
0.063 ± 0.002
0.065 ± 0.002
0.01 ± 0.002
8m
ANF
aα-naphthoflavone (ANF) was used as a control in these studies. The IC50 values represent mean and standard deviations from three independent experiments.
Table 2. The IC50 values for inhibition of CYP s expressed in live HEK293 cells by 4-pyridyl chalcones 8a-m.
IC50 values for CYP inhibition in live HEK293 cells (µM)a
Entry
1A1
1B1
1A2
2D6
2C9
2C19
3A4
8a
8b
8c
8d
8e
0.058 ± 0.002
0.42 ± 0.01
0.61 ± 0.03
0.62 ± 0.03
6.61 ± 0.06
1.12 ± 0.04
0.55 ± 0.03
0.22 ± 0.02
1.33 ± 0.04
0.28 ± 0.31
0.092 ± 0.003
0.063 ± 0.002
0.065 ± 0.002
0.38 ± 0.05
8.18 ± 0.22
0.23 ± 0.02
0.06 ± 0.02
0.06 ± 0.02
8.52 ± 0.31
3.52 ± 0.23
0.75 ± 0.04
0.031 ± 0.005
0.022 ± 0.002
0.61 ± 0.04
9.92 ± 0.53
4.76 ± 0.02
3.07 ± 0.15
0.08 ± 0.01
1.12 ± 0.05
0.12 ± 0.03
0.032 ± 0.002
0.020 ± 0.003
12 ± 0.2
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
8f
1.5 ± 0.05
8g
8h
8i
8j
8k
8l
0.028 ± 0.004
0.028 ± 0.003
0.20 ± 0.06
2.21 ± 0.3
1.09 ± 0.05
1.53 ± 0.1
0.82 ± 0.07
0.05 ± 0.01
8m
ANF
a The IC50 values represent mean and standard deviations from three independent experiments
Table 3. The IC50 values for inhibition of CYP s expressed in live yeast cells.
IC50 values for CYP inhibition in live yeast cells (µM) a
Entry
8a
1A1
0.15 ± 0.03
1B1
6.12 ± 0.24
1A2
2A6
>10
2B6
>10
2C8
2E1
>10
2J2
>20
1.82 ± 0.21
8.92 ± 0.64
a The IC50 values represent mean and standard deviations from three independent experiments.
In order to overcome the problems of specificity and reproducibility,
our group has developed a cell based assay for CYP enzyme
inhibition studies.37, 48, 49 It includes transformation of HEK293 cells
with plasmids that encode human CYP genes under the control of a
constitutive promoter. Specifically, adherent human kidney cells are
at first adapted for growth in suspension. The adapted suspension
cells are then transfected with CYP-bearing expression plasmids. The
active CYP1 inhibitors, obtained from screening of the compounds
in Sacchrosomes, were then tested for inhibition of CYP1A1,
CYP1B1, CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4
enzymes which were expressed within live HEK293 cells.
majority of approved drugs. This could be a desirable feature, with
the molecules not having the risk of potential drug-drug interactions,
if one considers developing them as possible chemo-preventives. The
results of CYP inhibition in live cells are depicted in Table 2 and
Table 3.
Further, in order to know whether these compounds are time-
dependent inhibitors of CYP1A1 and CYP1B1 (in both
Sacchrosomes as well as in live cells), representative best
compounds 8a have been pre-incubated with CYP1A1 for 30, 60, 90,
120 minutes.53 In this study different time periods of incubation have
not shown any difference in percentage inhibition of the CYP1A1,
indicating that these compounds are not time-dependent inhibitors.
The inhibition constant (ki) values of compounds 8a for CYP1A1
was found to be 0.28 µM (mean values of 3 independent
experiments) and indicating that they are competitive inhibitors.53
The compounds that inhibited CYP1A1 and CYP1B1 in
Sacchrosomes, were at first assessed in cells which individually
expressed these two enzymes. Inhibitory potential of
CYP1A1/CYP1B1 was determined via de-ethylation of the substrate,
7-ethoxyresorufin, which produces the fluorescent molecule,
resorufin, using a fluorescent plate reader. The compounds 8a, 8k, 8l
and 8m selectively inhibited CYP1A1 isoform, with at least 10-fold
selectivity within CYP1 family isoforms. These compounds do not
show inhibition (IC50 > 10 µM) of enzymes belonging to the CYP2
and CYP3 family which are responsible for the metabolism of the
The best inhibitor 8a was also tested using human CYP enzymes
expressed within live yeast cells.37 The results, obtained in the Table
3, again show that compounds 8a is specific inhibitors of CYP1A1
isoform.

4
Bioorganic Medicinal Chemistry Letters
In order to establish the scientific rationale for the observed efficacy
and selectivity of CYP inhibition by specific chalcones, molecular
modelling studies were carried out with the potent inhibitors using
3D structures of the panel of CYP enzymes used for experimental
studies. Interestingly, X-ray crystallography suggests that various
CYP1 family enzymes share common structural features such as
amino acid sequence similarity, the haem complex which acts as the
O donor and the mechanism for hydroxylation. These aspects are
crucial for determining the xenobiotic metabolic propensity of CYP
enzymes. The X-ray structure of α-naphthoflavone (ANF) co-
crystallised with CYP1A1 displays characteristic canonical helices, β
sheets and two short helixes F’ and G’ which together form the
binding site for the ligand, ANF. The notable structural differences
between 1A1 and 1A2 include a 5-residue extended gap in F-helix,
which controls the size and shape of the binding cavity by providing
the structural flexibility in the amino acid side chains that allows
accommodation of substrate movement and orientation. The ligand
ANF interacts mainly by hydrophobic π-π interactions with multiple
aromatic residues of CYP1A1 and CYP1B1.54-56 The 4-pyridyl
chalcone 8a is specific CYP1A1 inhibitor having excellent
selectivity over CYP2 and CYP3 family enzymes. The 3,4,5-
trimethoxy phenyl ring of 8a was found to interact with the heam
complex, wherein the distance between the ligand and metal ion of
haem was 3.46 Å. The pyridyl ring interacts with Phe-224 and Phe-
258 residues via hydrophobic interactions. The distance between
pyridyl ring of 8a and phenyl ring of Phe-224 and Phe-258 residues
was found to be 3.89 and 4.3 Å. These interactions of 8a with
CYP1A1 isoforms block the formation of reactive haem iron-oxo
intermediate between substrate and enzyme, which is requisite for
hydroxylation (Figure 3).
Antagonism of aromatic hydrocarbon receptor (AhR) signalling,
activated by B[a]P, in yeast cells: CYP1A1 expression is induced
by various pro-carcinogens such as 2,3,7, 8-tetrachlorodibenzo-p-
dioxin (TCDD), benzo(a)pyrene (B[a]P) and 7,12-dimethylbenz(a)-
anthracene (DMBA). These pro-carcinogens are mainly present in
cigarette smoke, high-boiling fraction of crude oil, charred meat and
vegetables. A PAH-bound to AhR is responsible for the induction of
the CYP1 genes at the mRNA level. This leads to the over expression
of CYP1 proteins. For example CYP1A1 protein, induced by B[a]P
bound to the ligand-binding domain of AhR, is also responsible for
the hydroxylation of B[a]P to form its metabolite benzo[a]pyrene-
7,8-dihydrodiol-9,10-epoxide, which initiates the process of
carcinogenesis through formation of DNA adducts. It has been
observed that CYP1 inhibitors have the capacity to antagonize B[a]P
mediated activation of AhR.57-60
In order to study the role of heterocyclic pyridyl chalcones in AhR
signalling, yeast cells were created which are able to stably co-
express the proteins AhR and the AHR nuclear translocator (ARNT),
a co-activator of AhR.61 The cells also contained an eGFP reporter
plasmid under the control of a minimal promoter on to which is
embedded a concatemer of 5 XRE sequences. XRE denotes the
xenobiotic response element, which is an enhancer sequence that is
naturally contained in the promoters of the human CYP1A1, CYP1B1
and CYP1A2 genes. A PAH-bound AhR/ARNT transcription factor
complex can bind to XRE elements to induce the transcription of a
reporter, in this case, the enhanced green fluorescent protein (eGFP).
The CYP1 inhibitors, identified using SacchrosomesTM
, were
screened for their ability to compete with the PAH binding site of the
ligand-binding domain of AhR. The 4-pyridyl chalcones 8a, 8i and
8m which were found to be potent CYP1 family inhibitors, strongly
inhibit the activation of AhR signalling by B[a]P, as shown in Figure
4. It would seem the number of alkoxy groups in the aryl ring have
played a role in AhR antagonism. The optimization of B[a]P
concentration for optimal fluorescence production by AhR/ARNT
complex is provided in our previous publications.50
PHE319
120
100
80
60
40
20
0
PHE224
LEU 254
PHE258
LEU 314
ASN 255
LEU 312
GLU 256
SER 116
Figure 3. Interactions of 8a with CYP1A1 (PDB: 4I8V) enzyme isoform.
Control 8a
8c
8d
8h
8i
8k
8l
8m
The Glide Score and Dock score = -8.51. Glide emodel score = -53.85.
1 µM Concentration
Figure 4. Antagonism of the aromatic hydrocarbon receptor (AhR). Potent
CYP1 inhibitors, identified in assays using Sacchrosomes and CYP-
expressing live human cells, were tested for their ability to antagonise AhR
which was activated by B[a]P (0.7 µM), in a yeast-based assay; test
concentration of CYP-inhibitory compounds was 1 µM. The control yeast
cells, co-expressing the AhR-ARNT complex and also containing a reporter
eGFP plasmid, were treated only with B[a]P but had no CYP-inhibitory
compound. The eGFP units for control cells were considered as 100. All
eGFP values for test compounds are relative to the values obtained with
control cells. The units represent the mean and standard deviations of three
independent experiments.
For selectivity studies, ligand bound 10 Å binding site of CYP2 and
CYP3 family isoforms were aligned with the binding site of
CYP1A1 enzyme. It was observed that, in case of CYP2D6 and
CYP3A4, the interactions of 8a with haem complex gets distorted
and π-π interactions with the corresponding phenyl alanine residue
were completely changed. This could be the possible reason for the
observed loss of potency in CYP2 and CYP3 family isoforms. This
loss of interactions with CYP2 and CYP3 may be due to the large
and flexible architecture of CYP2 and CYP3 family isoforms. The
CYP isoforms interaction images are shown in supporting
information section S3.
Restoration of cell growth in CYP1A1-expressing human
HEK293 cells by potent CYP1A1 inhibitors, after cells had been

5
treated with B[a]P: B[a]P is a substrate of CYP1A1. When adherent
human HEK293 cells expressing CYP1A1 from a plasmid, that
encodes the CYP1A1 gene, are treated with B[a]P, CYP1A1 converts
B[a]P to its metabolite benzo[a]pyrene-7,8-dihydrodiol-9,10-
epoxide. Overproduction of the B[a]P metabolite causes excessive
intercalation of DNA, which leads to DNA damage, chromosomal
aberration, generation of excessive ROS and ultimately cell death.58,
used to prevent formation of this carcinogen in breast cells which
overproduce estradiol. More than 80% of breast cancers occur
because of overproduction of estradiol. Hence, inhibitors of CYP1B1
could be used for the prevention of breast cancer.17, 36, 65 Beside this,
CYP1B1 over expression in cells can cause resistance to the
cytotoxic anticancer drugs paclitaxel, docetaxel, doxorubicin,
mitoxantrone, cisplatin and the selective estrogen receptor
modulator, tamoxifen, during treatment of hormonal cancers
especially breast and ovarian.15, 35, 36 Cells resistant to these drugs
overproduce CYP1B1. The relationship between resistance and
overproduction of CYP1B1 is not yet fully understood.
62
Therefore, it can be assumed that CYP1A1 inhibition will protect
cells from B[a]P mediated toxicity. This was successfully
43, 63, 64
demonstrated by several studies.24,
In order to test this
assumption, CYP1A1-expressing adherent HEK293 cells were
treated with B[a]P and CYP1A1-inhibitory compound. The EC50 for
growth inhibition in CYP1A1-expressing cells treated with different
concentrations of B[a]P was 0.8 µM whereas untransformed cells,
that did not express CYP1A1, showed an EC50 of 14 µM (shown in
Figure 5). This confirmed that B[a]P toxicity is mediated by
expression of CYP1A1 enzyme (confirmed by 7-ER based
fluorescent assays; data not shown).
Recently, this type of resistance has been overcome by inhibiting
CYP1B1’s enzymatic activity.36, 37 We have shown previously that
the 3-pyridyl chalcone 5 is capable of reversing cisplatin resistance
in recombinant human kidney HEK293 cells and A2780 ovarian
cancer cells, both of which overexpress CYP1B1.37 Therefore, the
CYP1B1-specific inhibitors, identified in sacchrosomes and
recombinant live human cells, were investigated further for their
ability to sensitise CYP1B1-overexpressing HEK293 cells to
cisplatin’s cytotoxicity.
Various CYP1A1 specific inhibitors, 8a, 8k, 8l and 8m were tested
to see if they would rescue CYP1A1-expressing HEK293 cells from
CYP1A1-mediated toxicity of B[a]P. The CYP1A1-expressing cells,
when treated with these inhibitors at 2 x IC50 concentrations, in the
presence of B[a]P, showed EC50 values of around 14 µM once again.
This would indicate that CYP1A1’s metabolic activity on B[a]P was
completely nullified by the CYP1A1 inhibitors identified using
CYP1A1-Sacchrosomes (i.e. CYP1A1-bound microsomal enzymes
isolated from yeast). One can therefore conclude that the identified
CYP1A1 inhibitors 8a, 8k, 8l and 8m have a protective influence on
cells. They are able to evade completely B[a]P’s toxicity manifested
in the presence of CYP1A1. The results are shown in Figure 5.
However, the well-known CYP1 inhibitor, α-naphthoflavone (ANF),
could only partially protect the cells at 2 x IC50 concentration.
Introduction of a plasmid bearing the CYP1B1 gene in to HEK293
cells reduces the cytotoxicity of cisplatin by increasing the EC50 from
10.5 (non-transfected cells) to 65 µM (transfected cells; shown in
Figure 6). In the presence of the CYP1B1 inhibitors 8c, 8d, 8h, and
8i, the EC50 value of cisplatin is restored to ~10 μM. This indicated
complete restoration of cisplatin’s cytotoxicity in cells that
overexpress CYP1B1 (Figure 6). The results also show that that the
identified inhibitors CYP1B1 inhibitors 8c, 8d, 8h, and 8i were
superior to ANF in their ability to overcome resistance to cisplatin.
80
EC50 of cisplatin
Cell Line
70
(µM)
HEK293:: –
10.51 ± 1.31
7.82 ± 0.62
65.01 ± 7.01
60
50
40
30
20
10
0
Cell Line
EC50 of B[a]P (µM)
HEK293::pcDNA3.1
HEK293::pcDNA3.1/CYP1B1
HEK293:: –
14.11 ± 1.21
HEK293::pcDNA3.1
12.82 ± 0.93
0.80 ± 0.14
HEK293::pcDNA3.1/CYP1A1
16
14
12
10
8
Control
8c
8d
8h
8i
ANF
Test compounds at 2X IC50 for CYP1B1 inhibition in live HEK293 cells
6
Figure 6. Overcoming cisplatin resistance by CYP1B1 inhibitors. A range of
concentrations of cisplatin (0.05 µM – 100 µM) were used, in the presence of
2 x IC50 values (as was determined in the human cell assay where cells were
grown in suspension). The cells used were adherent HEK293 cells transfected
with pcDNA3.1/hCYP1B1 (the plasmid which encodes the human CYP1B1
gene). The control cells were treated with a range of concentrations of
cisplatin but did not contain any inhibitor. All values, presented in µM
concentrations, represent the mean and standard deviations of three
independent experiments.
4
2
0
Control
8a
8k
8l
8m
ANF
Test compounds at 2X IC50 for CYP1A1 inhibition in live HEK293 cells
Figure 5. Protection from CYP1A1 mediated B[a]P toxicity by CYP1A1
inhibitors. A range of concentrations of B[a]P (0.05 µM – 100 µM) were
used, in the presence of 2 x IC50 values (as was determined in the human cell
assay where cells were grown in suspension). Adherent HEK293 cells were
transfected with pcDNA3.1/hCYP1A1 (the plasmid which encodes the
human CYP1A1 gene). The control cells were treated with a range of
concentration of B[a]P only and did not contain any inhibitor. All values,
presented in µM concentrations, represent the mean and standard deviations
of three independent experiments.
In conclusion, CYP1A1 structure-guided information has allowed us
to develop SAR for heterocyclic chalcones as CYP1 family
inhibitors. CYP1A1 inhibitory potential of the most potent
compounds were confirmed by their potential to protect against
B[a]P’s cellular toxicity which is mediated by CYP1A1. The pyridyl
chalcones 8a (IC50 for CYP1A1 inhibition = 61 nM) and 8i (IC50 for
CYP1B1 inhibition = 22 nM) displayed B[a]P activated AhR
antagonism. Further cellular studies could lead to establishing the
potential of some of these compounds as chemo-preventives and
novel modulators of chemo-resistance.
CYP1B1 inhibitors can overcome cisplatin resistance seen in
adherent HEK293 cells that overexpress CYP1B1: It is known
that CYP1B1 has a positive role in oestrogen metabolism in breast
cancer. It metabolises estradiol to form the carcinogenic molecule 4-
hydroxy estradiol. Therefore, CYP1B1-specific inhibitors could be

6
Bioorganic Medicinal Chemistry Letters
24. Wen, X.; Walle, U. K.; Walle, T. Carcinogenesis 2005, 26, 803-
Supporting information available. Experimental procedures,
spectral data and spectral data scans. This material is available free
of charge via the internet at http://sciencedirect.com.
809.
25. Yager, J. D. J. Natl. Cancer Inst. Monographs 2000, 27, 67-73.
26. Newbold, R. R.; Liehr, J. G. Cancer Res. 2000, 60, 235 -237.
ACKNOWLEDGEMENTS
27. Cavalieri, E. L.; Stack, D. E.; Devanesan, P. D.; Todorovic, R.;
Dwivedy, I.; Higginbotham, S. M.; Johansson, S. L.; Patil, K.
D.; Gross, M. L.; Gooden, J. K.; Ramanathan, R.; Cerny, R. L.;
Rogan, E. G. Proc. Natl. Acad. Sci. USA 1997, 94, 10937-
10942.
BC would like to thank the UK Higher Education Innovation Fund
(HEIF) and a British Council-UKIERI grant for support. PJ
acknowledges CSIR for an award of Senior Research Fellowship.
28. Liehr, J. G.; Ricci, M. J. Proc. Natl. Acad. Sci. USA 1996, 93,
References
3294-3296.
1. Kanavos, P. Ann. Oncol. 2006, 17, 15-23.
29. Tait, L.; Soule, H. D.; Russo, J. Cancer Res. 1990, 50, 6087-
2. Parkin, D. M.; Bray, F.; Ferlay, J.; Pisani, P. Cancer J. Clin.
2005, 55, 74-108.
6094.
30. Wen, X.; Walle, T. Carcinogenesis 2005, 26, 1774-1781.
3. Bishayee, A. Cancer Prev. Res. 2009, 2, 409–418.
31. Uno, S., Dalton, T. P., Dragin, N., Curran, C. P., Derkenne, S.,
Miller, M. L., Shertzer, H. G., Gonzalez, F.J., Nebert, D. W. .
Mol Pharmacol. 2006, 69, 1103-1114.
4. Aziz, M. H.; Kumar, R.; Ahmad, N. Int. J. Oncol. 2003, 23, 17-
28.
5. Jang, M.; Cai, L.; Udeani, G. O. Science 1997, 275, 218-220.
32. Kasala, E. R.; Bodduluru, L. N.; Barua, C. C.; Gogoi, R.
6. Messing, E.; Gee, J. R.; Saltzstein, D. R.; Kim, K.;
diSant'Agnese, A.; Kolesar, J.; Harris, L.; Faerber, A.;
Havighurst, T.; Young, J. M.; Efros, M.; Getzenberg, R. H.;
Wheeler, M. A.; Tangrea, J.; Parnes, H.; House, M.; Busby, J.
E.; Hohl, R.; Bailey, H. Cancer Prev. Res. 2012, 5, 621–630.
Biomed Pharmacother 2016, 82, 568-577.
33. Takemura, H.; Itoh, T.; Yamamoto, K.; Sakakibara, H.; Shimoi,
K. Bioorg. Med. Chem. 2010, 18, 6310-6315.
34. Chen, Z. H.; Hurh, Y. J.; Na, H. K.; Kim, J. H.; Chun, Y. J.;
Kim, D. H.; Kang, K. S.; Cho, M. H.; Surh, Y. J.
Carcinogenesis 2004, 25, 2005–2013.
7. Aqil, F.; Jeyabalan, J.; Munagala, R.; Ravoori, S.; Vadhanam,
M. V.; Schultz, D. J.; Gupta, R. C. Int J Mol Sci 2017, 18, pii:
E425.
35. Rochat, B.; Morsman, J. M.; Murray, G. I.; Figg, W. D.;
McLeod, H. L. J. Pharmacol. Exp. Ther. 2001, 296, 537–541.
8. Santes-Palacios, R.; Ornelas-Ayala, D.; Cabanas, N.;
Marroquin-Perez, A.; Hernandez-Magana, A.; Del Rosario
Olguin-Reyes, S.; Camacho-Carranza, R.; Espinosa-Aguirre, J.
J. Biomed Res Int 2016, 2016, 5341081.
36. Cui, J.; Meng, Q.; Zhang, X.; Cui, Q.; Zhou, W.; Li, S. J. Med.
Chem. 2015, 58, 3534-3547.
37. Horley, N. J.; Beresford, K. J. M.; Chawla, T.; McCann, G. J.
P.; Ruparelia, K. C.; Gatchie, L.; Sonawane, V. R.; Williams, I.
S.; Tan, H. L.; Joshi, P.; Bharate, S. B.; Kumar, V.; Bharate, S.
B.; Chaudhuri, B. Eur. J. Med. Chem. 2017, 129, 159-174.
9. Badal, S.; Delgoda, R. J. Appl. Toxicol. 2014, 34, 743-753.
10. Fimognari, C.; Hrelia, P. Mutat. Res. 2007, 635, 90-104.
11. Cavalieri, E. L.; Devanesan, P.; Bosland, M. C.; Badawi, A. F.;
38. Androutsopoulos, V.; Arroo, R. R.; Hall, J. F.; Surichan, S.;
Rogan, E. G. Carcinogenesis 2002, 23, 329–333.
Potter, G. A. Breast Cancer Res. 2008, 10, R39.
12. Hayes, C. L.; Spink, D. C.; Spink, B. C.; Cao, J. Q.; Walker, N.
J.; Sutter, T. R. Proc. Natl. Acad. Sci. USA 1996, 93, 9776-
9781.
39. Androutsopoulos, V.; Wilsher, N.; Arroo, R. R.; Potter, G. A.
Cancer Lett. 2009, 274, 56-60.
40. Surichan, S.; Androutsopoulos, V. P.; Sifakis, S.; Koutala, E.;
Tsatsakis, A.; Arroo, R. R.; Boarder, M. R. Food Chem.
Toxicol. 2012, 50, 3320-3328.
13. Spyrou, I.; Sifakis, S.; Ploumidis, A.; Papalampros, A. E.;
Felekouras, E.; Tsatsakis, A. M.; Spandidos, D. A.;
Androutsopoulos, V. P. Tumour Biol. 2014, 35, 9549-9556.
41. Sutherland, M.; Gill, J. H.; Loadman, P. M.; Laye, J. P.;
Sheldrake, H. M.; Illingworth, N. A.; Alandas, M. N.; Cooper,
P. A.; Searceycey, M.; Pors, K.; Shnyder, S. D.; Patterson, L. H.
Mol. Cancer Ther. 2013, 12, 27-37.
14. Androutsopoulos, V. P.; Spyrou, I.; Ploumidis, A.;
Papalampros, A. E.; Kyriakakis, M.; Delakas, D.; Spandidos, D.
A.; Tsatsakis, A. M. PLoS One 2013, 8, e82487.
15. McFadyen, M.; McLeod, H.; Jackson, F.; W., M.; J., D.; G., M.
42. Messing, E.; Gee, J. R.; Saltzstein, D. R.; Kim, K.;
diSant'Agnese, A.; Kolesar, J.; Harris, L.; Faerber, A.;
Havighurst, T.; Young, J. M.; Efros, M.; Getzenberg, R. H.;
Wheeler, M. A.; Tangrea, J.; Parnes, H.; House, M.; Busby, J.
E.; Hohl, R.; Bailey, H. Cancer Prev. Res. 2012, 5, 621-630.
Biochem. Pharmacol. 2001, 62, 207–212.
16. Spink, D. C.; Spink, B. C.; Cao, J. Q.; De Pasqualle, J. A.;
Pentecost, B. T.; Fasco, M. J.; Li, Y.; Sutter, T. R.
Carcinogenesis 1998, 19, 291-298.
17. Murray, G. I.; Taylor, M. C.; McFadyen, M. C.; McKay, J. A.;
Greenlee, W. F.; Burke, M. D.; Melvin, W. T. Cancer Res.
1997, 57, 3026-3031.
43. Androutsopoulos, V. P.; Tsatsakis, A. M. Toxicol. Lett. 2014,
230, 304-313.
44. Androutsopoulos, V. P.; Spandidos, D. A. J. Nutr. Biochem.
2013, 24, 496-504.
18. Brockdorff, B. L.; Skouv, J.; Reiter, B. E.; Lykkesfeldt, A. E.
Int. J. Cancer 2000, 88, 902-906.
45. Androutsopoulos, V. P.; Papakyriakou, A.; Vourloumis, D.;
19. Carnell DM, S. R., Daley FM, et al. Int. J. Radiat. Oncol. Biol.
Phys. 2004, 58, 500-509.
Spandidos, D. A. Bioorg. Med. Chem. 2011, 19, 2842–2849.
46. De Filippis, B.; Ammazzalorso, A.; Fantacuzzi, M.; Giampietro,
L.; Maccallini, C.; Amoroso, R. ChemMedChem 2017, 12, 558-
570.
20. Gimenez-Conti, I. B.; Slaga, T. J. J. Cell Biochem. 1993, 17,
83-90.
21. Letchoumy, P. V.; Chandra Mohan, K. V.; Kumaraguruparan,
47. Zhou, Y.; Zheng, J.; Li, Y.; Xu, D. P.; Li, S.; Chen, Y. M.; Li,
R.; Hara, Y.; Nagini, S. Oncol. Res. 2006, 16, 167-178.
H. B. Nutrients 2016, 8.
22. Sutter, T. R.; Tang, Y. M.; Hayes, C. L.; Wo, Y. Y. P.; Jabd, E.
W.; Li, X.; Yin, H.; Cody, C. W.; Greenlee, W. F. J. Biol.
Chem. 1994, 269, 13092-13099.
48. Mohd Siddique, M. U.; McCann, G. J. P.; Sonawane, V.;
Horley, N.; Williams, I. S.; Joshi, P.; Bharate, S. B.;
Jayaprakash, V.; Sinha, B. N.; Chaudhuri, B. Org. Biomol.
Chem. 2016, 14, 8931-8936.
23. Whitlock, J. P. Ann. Rev. Pharmacol. Toxicol. 1999, 39, 103–
125.
49. Mohd Siddique, M. U.; McCann, G. J. P.; Sonawane, V.;
Horley, N.; Gatchie, L.; Joshi, P.; Bharate, S. B.; Jayaprakash,

7
V.; Sinha, B. N.; Chaudhuri, B. Eur. J. Med. Chem. 2017, 130,
320-327.
50. Williams, I. S.; Joshi, P.; Gatchie, L.; Sharma, M.; Satti, N. K.;
Vishwakarma, R. A.; Chaudhuri, B.; Bharate, S. B. Bioorg.
Med. Chem. Lett. 2017, 27, 3683-3687.
51. Joshi, P.; McCann, G. J. P.; Sonawane, V. R.; Vishwakarma, R.
A.; Chaudhuri, B.; Bharate, S. B. J. Chem. Inf. Model. 2017, 57,
1309–1320.
52. Pohl, R. J.; Fouts, J. R. Anal. Biochem. 1980, 107, 150-155.
53. Stresser,
D.
M.
https://www.bdbiosciences.com/documents/webinar_2009_04_
cyp_inhibition.pdf.
54. Walsh, A. A.; Szklarz, G. D.; Emily E. Scott, E. E. J. Biol.
Chem. 2013, 288, 12932-12943.
55. Wang, A.; Savas, U.; Stout, C. D.; Johnson, E. F. J. Biol. Chem.
2011, 286, 5736–5743.
56. Sansen, S.; Yano, J. K.; Reynald, R. L.; Schoch, G. A.; Griffin,
K. J.; Stout, C. D.; Johnson, E. F. J. Biol. Chem. 2007, 282,
14348-14355.
57. Puga, A.; Ma, C.; Marlowe, J. L. Biochem. Pharmacol. 2009,
77, 713-722.
58. Cavalieri, E. L.; Rogan, E. G. Ann. N. Y. Acad. Sci. 2004, 1028,
247–257.
59. Hankinson, O. Annu. Rev. Pharmacol.Toxicol. 1995, 35, 307-
340.
60. Nebert, D. W.; Dalton, T. P.; Okey, A. B.; Gonzalez, F. J. J.
Biol. Chem. 2004, 279, 23847-23850.
61. Miller, C. A. Toxicol. Appl. Pharmacol. 1999, 160, 297-303.
62. Lee, H. S.; Jin, C.; Park, J.; Kim, D. H. Biochem. Mol. Biol. Int.
1994, 34, 483-491.
63. Wu, Z.; Uchi, H.; Morino-Koga, S.; Nakamura-Satomura, A.;
Kita, K.; Shi, W.; Furue, M. Exp. Dermatol. 2014, 23, 260-265.
64. Prince, M.; Campbell, C. T.; Robertson, T. A.; Wells, A. J.;
Kleiner, H. E. Carcinogenesis 2006, 27, 1204-1213.
65. Itoh, T.; Takemura, H.; Shimoi, K.; Yamamoto, K. J. Chem. Inf.
model. 2010, 50, 1173-1178.