Discovery and characterization of novel CYP1B1 inhibitors based on heterocyclic chalcones: Overcoming cisplatin resistance in CYP1B1-overexpressing lines

Article abstract of DOI:10.1016/j.ejmech.2017.02.016

The structure of alpha-napthoflavone (ANF), a potent inhibitor of CYP1A1 and CYP1B1, mimics the structure of chalcones. Two potent CYP1B1 inhibitors 7k (DMU2105) and 6j (DMU2139) have been identified from two series of synthetic pyridylchalcones. They inh

Full text of DOI:10.1016/j.ejmech.2017.02.016

Accepted Manuscript
Discovery and characterization of novel CYP1B1 inhibitors based on heterocyclic
chalcones: Overcoming cisplatin resistance in CYP1B1-overexpressing lines
Neill J. Horley, Kenneth J.M. Beresford, Tarun Chawla, Glen J.P. McCann, Ketan
C. Ruparelia, Linda Gatchie, Vinay R. Sonawane, Ibidapo S. Williams, Hoon L. Tan,
Prashant Joshi, Sonali S. Bharate, Vikas Kumar, Sandip B. Bharate, Bhabatosh
Chaudhuri
PII:
S0223-5234(17)30081-8
10.1016/j.ejmech.2017.02.016
EJMECH 9212
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 December 2016
Revised Date: 6 February 2017
Accepted Date: 7 February 2017
Please cite this article as: N.J. Horley, K.J.M. Beresford, T. Chawla, G.J.P. McCann, K.C. Ruparelia,
L. Gatchie, V.R. Sonawane, I.S. Williams, H.L. Tan, P. Joshi, S.S. Bharate, V. Kumar, S.B. Bharate, B.
Chaudhuri, Discovery and characterization of novel CYP1B1 inhibitors based on heterocyclic chalcones:
Overcoming cisplatin resistance in CYP1B1-overexpressing lines, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.02.016.
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GRAPHICAL ABSTRACT
Unique features of 7k and
j vs ANF
6
VHigh CYP1B1 selectivity
VHighly active in live cells
VBetter aqueous solubility
VBetter potency for cisplatin
resistance reversal
6
j with CYP1B1
Pyridylchalcones were identified as potent and highly selective CYP1B1 inhibitors. These potent cell-
permeable and water-soluble CYP1B1 inhibitors are likely to have useful roles in the treatment of
cancer, glaucoma, ischemia and obesity.
1

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Discovery and characterization of novel CYP1B1 inhibitors based
on heterocyclic chalcones: Overcoming cisplatin resistance
in CYP1B1-overexpressing lines
a,#
a, #
a, #
a
Neill J. Horley, Kenneth J. M. Beresford, Tarun Chawla, Glen J. P. McCann, Ketan
a
b
a
b
a
C. Ruparelia, Linda Gatchie, Vinay R. Sonawane, Ibidapo S. Williams, Hoon L. Tan,
c
c
c
c,*
Prashant Joshi, Sonali S. Bharate, Vikas Kumar, Sandip B. Bharate, Bhabatosh
a,b,*
Chaudhuri
a
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
Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India.
#
Contributed equally to the work described.
*
*
Corresponding author
Prof. Bhabatosh Chaudhuri, Professor of Cancer Studies,
Leicester School of Pharmacy, De Montfort University, Leicester, LE1 9BH, UK
Fax: Fax: +44(0) 116 257 7287; Tel: 44(0)116 250 7280
*
*
E-mail: E-mail: bchaudhuri@dmu.ac.uk (BC),
Dr. Sandip B. Bharate, Senior Scientist, Indian Institute of Integrative Medicine (CSIR),
Canal Road, Jammu-180001, India. E-mail: sbharate@iiim.ac.in (SBB)
1

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ABSTRACT
The structure of alpha-napthoflavone (ANF), a potent inhibitor of CYP1A1 and CYP1B1,
mimics the structure of chalcones. Two potent CYP1B1 inhibitors 7k (DMU2105) and 6j
(DMU2139) have been identified from two series of synthetic pyridylchalcones. They inhibit
TM
human CYP1B1 enzyme bound to yeast-derived microsomes (Sacchrosomes ) with IC50
values of 10 and 9 nM, respectively, and show a very high level of selectivity towards
CYP1B1 with respect to the IC50 values obtained with CYP1A1, CYP1A2, CYP3A4,
TM
CYP2D6, CYP2C9 and CYP2C19 Sacchrosomes . Both compounds also potently inhibit
CYP1B1 expressed within ‘live’ recombinant yeast and human HEK293 kidney cells with
IC50 values of 63, 65, and 4, 4 nM, respectively. Furthermore, the synthesized
pyridylchalcones possess better solubility and lipophilicity values than ANF. Both
compounds overcome cisplatin–resistance in HEK293 and A2780 cells which results from
CYP1B1 overexpression. These potent cell-permeable and water-soluble CYP1B1 inhibitors
are likely to have useful roles in the treatment of cancer, glaucoma, ischemia and obesity.
KEYWORDS:
Pyridylchalcones; cisplatin-resistance; CYP1B1 inhibitors; live CYP1B1-expressing human
TM
cells; live CYP1B1-expressing yeast cells; Sacchrosomes
2

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1
. INTRODUCTION
The cytochrome P450 (CYP) enzyme superfamily plays a key role in the phase 1 oxidative
metabolism and detoxification of both endogenous and xenobiotic substances [1]. However,
certain members of the CYP superfamily of enzymes are also involved in the bio-activation
of pro-carcinogens such as polyaromatic hydrocarbons, aromatic amines and nitro-polycyclic
hydrocarbons [2-4]. The CYP1 subfamily of enzymes comprising 1A1, 1B1 and 1A2 fall into
this category [5]. The CYP1A1 and CYP1B1 proteins are mainly expressed in extrahepatic
tissues, while CYP1A2 is a major P450 found in the human liver (i.e. consisting of about
1
3% of the total CYP content in the human liver) [6]. CYP1B1 has been identified as a key
enzyme in the carcinogenic action of 17-β-estradiol (E ) and participates by catalysing
2
hydroxylation at C-4 in E to generate 4-hydroxyestradiol (4-OHE ). Subsequent oxidation
2
2
of 4-OHE to estradiol-3,4-quinone results in a quinone-DNA adduct responsible for
2
estrogen-related carcinogenesis. The other two members of the CYP1 family selectively
metabolise E to give 2-hydroxyestradiol which is not mutagenic [7].
2
CYP expression has been studied in a variety of human tumors. It is now recognized that
many cancers have distinct P450 profiles and overexpress specific P450 protein sub-families.
CYP1B1 is the only CYP1 family member that is significantly and consistently
overexpressed in tumors [8-10]. CYP1B1 has been shown to be active within tumors and is
also capable of metabolizing a structurally diverse range of anticancer drugs [11, 12].
Because of this, and its role in the activation of pro-carcinogens, CYP1B1 is seen as an
important target for anticancer drug development. Selective inhibitors of this enzyme could
have a major impact in cancer prevention. Particularly, as the extent of CYP1B1 expression
appears to be a critical determinant of carcinogenesis in mammary, endometrial and ovarian
tissues [7, 13-16].
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It has been reported that CYP1B1 overexpression in normal mammalian cells leads to
resistance to docetaxel [12], which is reversed in the presence of a CYP1B1 inhibitor [17].
Other chemotherapeutic agents which are subject to CYP1B1-mediated resistance include
cisplatin, tamoxifen and nucleoside analogues [17-22]. Furthermore, it has been reported that
CYP1B1 also plays a role in obesity. Disruption of the CYP1B1 gene in mice suppresses
high-fat diet (HFD)-induced obesity suggesting that CYP1B1 is a potential target for
identifying novel anti-obesity drugs [23]. Recently, it has been found that CYP1B1
contributes to the development of atherosclerosis and hypertension in apolipoprotein E-
deficient mice which are known to rapidly develop severe hypercholesterolemia and
spontaneous atherosclerotic lesions similar to those observed in humans. This would indicate
that potent CYP1B1-specific inhibitors could be used for the treatment of atherosclerosis and
hypertension caused by hypercholesterolemia [24, 25].
Human CYP1B1 shares only 38% amino acid sequence identity with CYP1A1 and 36% with
1
A2 but seems to have an overlapping substrate profile with both, which makes the
development of potent and selective inhibitors a significant challenge [26]. Despite this, a
number of highly selective CYP1B1 inhibitors have been reported, several of these are based
on the structure of resveratrol and its natural analogues. Notable examples include 2,4,2',6'-
tetramethoxy-trans-stilbene (1) and 3,4,2'-trimethoxy-trans-stilbene (2) with IC values for
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CYP1B1 of 2 and 4 nM, respectively [26, 27]. Methoxyflavanoids have shown potent
inhibition of CYP1B1, the naturally occurring flavone acacetin (3) having an IC value of 12
5
0
nM [28]. Methoxyflavones can also act as CYP1A1/CYP1B1 substrates, undergoing O-
demethylation and aromatic hydroxylation reactions. It has been proposed that the
metabolites of these reactions make a significant contribution to their anti-proliferative
activities in cancer cells [29].
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Alpha-naphthoflavone (ANF) (4) is a synthetic flavonoid and a strong CYP1 inhibitor [30].
Recently, it was used as a lead to design a series of trimethoxy-naphthoflavones of which
compound 5 was the most potent and selective inhibitor of CYP1B1 (IC of 0.043 nM) [31].
5
0
A water-soluble naphthoflavone, in this series, also overcame the docetaxel-mediated drug
resistance which was induced by the over-expression of CYP1B1 in MCF-7 breast cancer
cells. Molecular modelling in this and other papers suggest that flavones bind into the active
site of the CYP1 family enzymes with the B-ring orientated towards the heme prosthetic
group. It seems that changing substituents on the B-ring has a profound effect on both
activity and selectivity [28, 31].
Chalcones are precursors in the biosynthesis of flavones and are widely distributed in nature.
They have received considerable attention due to their wide range of biological actions [32-
3
9]; however, there are few reports of them acting as CYP1 inhibitors [30, 40]. Like
methoxyflavones, certain chalcone derivatives are CYP1A1/CYP1B1 substrates, undergoing
O-dealkylation and aromatic hydroxylation reactions. Comparing the chalcone and flavone
structures, we reasoned that chalcones should fit into the narrow active site of CYP1B1 in a
similar fashion to the flavones, with an aromatic ring adjacent to the heme iron [6]. If this
ring was replaced by a group which could interact strongly with the heme iron, inhibition
should result. Pyridine derivatives are known to inhibit CYP450 enzymes via coordination of
the pyridine nitrogen to the heme iron and interactions of ring substituents with lipophilic
regions of the enzyme pocket [41]. It has been reported that the addition of a suitably
positioned pyridyl moiety to flavones improves their CYP450 inhibition by up to two orders
of magnitude [41-43]. Herein, we report the synthesis and biological evaluation of a series of
pyridylchalcones with the aim of finding potent and selective CYP1B1 inhibitors.
Furthermore, it was conjectured that incorporating a pyridine ring into the flexible chalcone
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structure would result in greater water solubility. Derivatives were prepared in which either
the B or A-ring of the chalcone was replaced by a pyridyl group (shown in Figure 1A). The
remaining ring was then altered in order to enhance inhibition and selectivity for CYP1B1.
Molecular modeling was used to rationalize the empirical results obtained.
Figure 1
2
2
. RESULTS AND DISCUSSION
.1. Chemistry. The most common method for the synthesis of chalcones is the Claisen-
Schmidt condensation reaction, in which equimolar quantities of a benzaldehyde and an
acetophenone are reacted together with either a base or acid catalyst [44]. Typically, either
sodium or potassium hydroxide is used with methanol as solvent. Initial attempts to prepare
3
-(3-pyridyl)chalcones
6
using these standard conditions by reacting 3-
pyridinecarboxaldehyde 9a with an acetophenone 8a were unsuccessful (Figure 1B). With 5
equivalents of aqueous sodium hydroxide at room temperature, little of the desired products
were obtained. An alternative approach using aprotic anhydrous conditions (Figure 1B,
Method 1) proved more fruitful. With 1 equivalent of freshly prepared lithium
diisopropylamide (LDA) as base in THF, a series of pyridylchalones (Table 1) were prepared
in yields of 25-88% after purification by flash chromatography. Reducing the amount of
aqueous sodium hydroxide to 2 equivalents, and cooling the reaction to 0 °C for 2 h prior to
warming to room temperature appeared to overcome initial difficulties. This method 2 was
used to prepare further derivatives in yields of 8-61% (Table 1). For the synthesis of
compound 6m, 4-pyridinecarboxaldehyde (9b) was used in place of 3-pyridine
carboxaldehyde (9a). Due to the poor solubility of the polyaromatic acetophenones in
methanol during synthesis of 6n, 6o and 6p, 25% of dichloromethane was added as a co-
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solvent. An unexpected result was obtained in the reaction between 9a and 3-
chloroacetophenone 8 using method 2. Instead of the desired product 6g, the major product of
this reaction was the adduct 10, presumably resulting from conjugate addition of the enolate
of 3-chloroacetophenone to the previously formed chalcone 6g (Figure 1B). A similar result
was obtained with 3-bromoacetophenone. Although some chalcone was produced using the
aprotic conditions (Method 1), it was not until a solvent free approach (Method 3) was
adopted that these halogenated derivatives were prepared in acceptable yields [45]. No
additional products were obtained under these conditions. 1-(3-Pyridyl)chalcones 7a-l were
prepared from the appropriate aldehydes 12 and 3-acetylpyridine 11 using either methods 2
or 3. Yields ranged from 13-76% after purification by flash chromatography (Table 2).
TM
2
.2. Yeast microsomes (Sacchrosomes ) as a screening tool for CYP1 inhibitors. The
human CYP1B1 gene was isolated from a human liver cDNA library that was constructed by
BioCat (Germany). The baker’s yeast, Saccharomyces cerevisiae, was chosen for the
production of human CYPs because the intracellular architecture of the compartments within
yeast cells closely resembles that of human cells. Because of this, yeast cells have been
widely used as a model to study human biochemical pathways and to express heterologous
human proteins [46-48]. The details of CYP1B1 expression in baker’s yeast are provided in
supporting information (Section S2). The presence of the CYP1B1 protein in the CYP1B1
Sacchrosomes was confirmed using Western blotting. The activity of the CYP1B1-
Sacchrosomes (microsomal membranes isolated from recombinant yeast cells) was then
TM
compared with that of CYP1B1-Supersomes
(microsomal membranes isolated from
recombinant insect cells and obtained from Corning-Gentest). The Supersomes, isolated from
insect cells, are recognised as the benchmark for commercially produced recombinant
CYP450s across the world. Results indicated that relative CYP1B1 activity in Sacchrosomes
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is around 3-fold higher than that present in Supersomes (Section S3 of supporting
information). It can be argued that the much higher activity of CYP1B1 seen in
Sacchrosomes, when compared to the insect cell derived CYP1B1-Supersomes, could be due
to the intracellular organization of yeast being akin to that of human cells and that this may
have influenced the increased activity of the CYP1B1-Sacchrosomes. Hence, it became clear
that the yeast-derived human microsomal CYP1B1 enzyme could, in theory, be used for rapid
screening of the potential CYP1B1 inhibitors 6-7 that had been synthesized.
Before embarking on screening the pyridylchalcone structure-based library of compounds, it
was essential that one proved that the active site of the CYP1B1-Sacchrosomes was similar in
conformation to the reported recombinant CYP1B1 enzymes. Therefore, the ability of ANF, a
known inhibitor of CYP1B1, to inhibit CYP1B1-Sacchrosomes was investigated. ANF is not
a specific inhibitor for any of the CYP1 family of enzymes [31]. It inhibits all the CYP1
enzymes (CYP1A1, CYP1A2 and CYP1B1) to a certain degree. Results with CYP1B1-
Sacchrosomes showed that there was >80% inhibition at 1 µM of ANF. Its IC for inhibition
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of yeast-produced CYP1B1 enzyme was found to be 0.008 µM (Section S4 of supporting
information) which was close to the published value of 0.005 µM for ANF observed using
CYP1B1 enzyme isolated from a recombinant human lymphoblastoid cell line [49]. ANF’s
IC for inhibition of CYP1A1-Sacchrosomes is 0.01 µM and of CYP1A2-Sacchrosomes is
5
0
0
.03 µM, which are close to published values obtained with CYP1A1-Supersomes and
CYP1A2-Supersomes, 0.018 µM and 0.012 µM, respectively [50, 51]. This re-affirmed the
validity of using CYP-bearing Sacchrosomes for further CYP inhibition studies.
2
.3. Screening of pyridylchalcones for CYP1 inhibition in yeast microsomes
TM
(
Sacchrosomes ). After confirming the validity of Sacchrosomes, containing active
CYP1B1 enzyme, they were used for screening of the pyridylchalcones 6a-p and 7a-l. The
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compounds were first tested at a single concentration of 5 µM to test their effects on CYP1B1
activity. A percentage inhibition value for each of the compounds in the library was obtained
after testing each compound twice. The compounds with the best percentage inhibition were
chosen for determination (in triplicate) of IC s, in at least three separate experiments; the
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values are presented in Tables 1 and 2.
Table 1 and 2
The graphs obtained for 7k and 6j in CYP1B1-bearing Sacchrosomes using 7-
ethoxyresorufin (7-ER) (5 µM final concentration) as substrate, are depicted in Section S10
of the supporting information. The results indicate that 7k and 6j are indeed potent inhibitors
of CYP1B1 showing IC values of 10 and 9 nM, respectively. Amongst all the compounds
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screened, the compounds bearing a naphthyl ring were found to be the most potent. Among
naphthyl linked compounds those with a pyridin-3-yl on the other side were better CYP1B1
inhibitors than 6m which has a pyridine-4-yl ring.
2
.4. Specificity of the potent CYP1B1 inhibitors 7k and 6j in Sacchrosomes. Since ANF
is a non-specific inhibitor of the CYP1 family of enzymes [6, 51, 52], it was important to
determine if the compounds identified as CYP1B1 inhibitors in the CYP1B1-Sacchrosome
TM
screen, were specific to CYP1B1 (Tables 1 and 2). Hence, Sacchrosomes containing
CYP1A1 and CYP1A2 were used to test the ability of the two compounds to inhibit these
CYP1 enzymes. It was found that the two most potent inhibitors identified, 7k and 6j, were
specific. Compound 7k shows 74 and 120-fold selectivity for CYP1B1 over CYP1A1 and
CYP1A2, whereas 6j shows 88 and 133-fold selectivity over the same enzymes.
Later, the most potent CYP1B1 inhibitors (i.e. IC < 0.1 µM) which showed specificity
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towards CYP1B1 were selected and tested for the inhibition of CYP2D6 and CYP3A4
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Sacchrosomes. The CYP2D6 and CYP3A4 enzymes are involved in the metabolism of the
majority of approved pharmaceuticals. If any of the chalcone based CYP1B1-specific
inhibitors are to be developed for further therapeutic use, it is essential that they do not inhibit
these two enzymes. Medicinal entities which inhibit CYP2D6 or CYP3A4 have the potential
of being involved in drug-drug interactions in patients who are prescribed more than one
drug. The results obtained are presented in Tables 1 and 2. They indicate that, for the CYPs
investigated, the two most potent inhibitors of CYP1B1, 7k and 6j, are CYP1B1-specific.
2
.5. Pyridylchalcones 7k and 6j potently inhibit CYP1B1 expressed within live yeast
cells. The two most potent CYP1B1-specific inhibitors were tested in live yeast cells to
establish if the compounds could penetrate the cell wall of live eukaryotic cells. We chose
recombinant CYP1B1-bearing baker’s yeast S. cerevisiae cells for our studies, this yeast
being a unicellular eukaryote. The majority of this particular yeast’s proteins share a high
degree of homology with the primary sequences of human proteins. It was conjectured that, if
the compounds did penetrate yeast cells to inhibit CYP1B1 bound to the endoplasmic
reticular membranes, they could find therapeutic applications in human cells (which are
multicellular eukaryotes) that overexpress CYP1B1 under certain diseased conditions.
The graphs that allowed the determination of the IC s of 7k and 6j, in live yeast cells using
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7
-ER (with 5 µM final concentration) as substrate, are depicted in Section S11 of the
supporting information. These compounds showed IC values of 64 and 65 nM, respectively.
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The results indicate that the two most potent chalcone-based CYP1B1 inhibitors can enter
live yeast cells to inhibit the endoplasmic reticular membrane-bound CYP1B1. This would
suggest that 7k and 7j could further be assessed for their possible therapeutic potential in
human cells. The two IC values in recombinant yeast cells are almost identical, as was seen
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in the assay using CYP1B1 Sacchrosomes. The IC50 value for a compound is always
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expected to be higher in cellular assays since all chemical compounds have some affinity
towards both extracellular and intracellular membranes. Hence, one can conclude that there
is a correlation between the results obtained from the yeast live cell assays and those obtained
with microsomal CYP1B1 enzyme borne on Sacchrosomes. This highlights the fact that the
CYP1B1-bearing live yeast cells, developed here for these studies, could provide an
inexpensive route towards the screening of potential inhibitors of CYP1B1 which could have
roles in the treatment of cancer, glaucoma, ischemia and obesity.
2
.6. Pyridylchalcones 7k and 6j potently inhibit CYP1B1 enzyme expressed within live
human cells. In the pharmaceutical industry, CYP450 assays are often performed in
hepatocytes grown in suspension, where studies normally cannot last for more than 6 hours
because of the rapid degradation of CYP enzyme activities. Hepatocytes contain a
conglomeration of CYPs and it is often difficult, if not impossible, to precisely determine
IC values of an inhibitor of a specific CYP of interest using convenient fluorescence-based
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assay protocols which depend on the inhibition of specific CYP-mediated conversion of a
non-fluorescent substrate to a fluorescent product. This is also true when using CYP-specific
chemical substrates, with medicinal properties, for IC50 determinations of potential CYP
inhibitors.
Until now, CYP assays have never been attempted in live recombinant human ‘suspension’
cells that over-express specific human CYP genes. We have adapted adherent human kidney
HEK293 cells for growth in suspension. These cells were transfected with an expression
plasmid (pCDM/hCYP1B1; pCDM obtained from CYP Design) encoding the same human
CYP1B1 gene which had been cloned previously in a yeast expression plasmid (details are
provided in section S5 of supporting information). The graphs that allowed determination of
the IC s of 7k and 6j, in live human HEK293 cells, are depicted in section S12 of the
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supporting information. At each concentration of the compound, cells were incubated for 30
min. Two different substrates, 7-ER and 3-cyano-7-ethoxycoumarin (CEC) were used for
determining the inhibitory capacity of the two compounds. IC values were found to be in
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the range of 4-53 nM, for both compounds. As mentioned before, 7-ER is converted to
fluorescent resorufin by CYP1B1 enzyme activity, whereas the same enzyme converts CEC
to 3-cyano-7-hydroxycoumarin. In both biotransformation reactions, CYP1B1 is responsible
for de-ethylation.
The results indicate that two compounds potently inhibit the human CYP1B1 enzyme that
resides on the endoplasmic reticular membranes within human cells. It can be conceived that
the enzyme activity within human HEK293 cells would be closely mimicking the activities in
human tissues which overexpress CYP1B1 protein under certain diseased conditions like
cancer and obesity. Although the tissues involved in these diseases are different, the proteins
present in the endoplasmic reticular membranes, to which CYP1B1 must be bound to
manifest activity, should be the same (if not identical) in all tissue types. Thus the newly
developed assay, based on HEK293 cells grown in suspension, can open up new avenues for
rapid screening of potential CYP1B1 inhibitors which would be directly pertinent to further
pre-clinical studies. In fact, we have shown that 200-300 compounds can be screened without
automation, per week, in such CYP-expressing human cell assays (manuscript in
preparation). The extraordinary CYP1B1-inhibiting potency of 7k and 6j, corroborated in
live human cells, did open up the possibility of further exploring the compounds’ therapeutic
potential in the context of human diseases like cancer.
2
.7. Pyridylchalcones 7k and 6j overcome cisplatin resistance in adherent HEK293 cells
that overexpress CYP1B1. pcDNA3.1/hCYP1B1 (Figure S7, in section S6, of supporting
information), is a plasmid where the hCYP1B1 gene was cloned in the vector pcDNA3.1
1
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(
Invitrogen) with the view of creating stable adherent cell lines. CYP1B1 protein expression
was confirmed through Western blotting (Figure 2A). Mechanically scraped cells, after
transfection, showed CYP1B1 enzyme activity (albeit far lower than in the same number of
suspension cells) using 7-ER as substrate (results not shown).
Figure 2
We also observed that overexpression of CYP1B1 protein, from the pcDNA3.1/hCYP1B1
(Figure 2A), in adherent HEK293 cells results in resistance to cisplatin when compared to
cells that do not express CYP1B1. Resistance was observed by determining EC values (i.e.
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the concentration of cisplatin that provides half-maximal response to cell growth) by
monitoring cell viability in the presence of cisplatin (Figure 2B). We then attempted to find
out if the two potent CYP1B1-specific inhibitors, 7k, 6j, can overcome resistance to cisplatin
observed in CYP1B1 overexpressing HEK293 cells. The results show that the two CYP1B1-
specific inhibitors can indeed overcome CYP1B1-mediated resistance to cisplatin (Figure
2
C). Figure 2C shows that upon co-administration of CYP1B1-specific inhibitors with
cisplatin in HEK293 cells, transfected with pcDNA3.1/hCYP1B1, the cisplatin resistant cells
become re-sensitized to cisplatin. EC50 values of cisplatin in the presence of 7k and 6j
abruptly diminish from the EC value seen using cells which were not treated with CYP1B1
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inhibitors. In the presence of 6j, the EC is reversed back to 8.3 µM from 61 µM (seen in
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CYP1B1-expressing cells without any inhibitor). The EC50 value, in the presence of 6j,
resembles the EC of cisplatin, 8.7 µM, in cells transfected with the empty plasmid which
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has no CYP1B1 gene and therefore cannot express CYP1B1 protein.
In the presence of 7k however, the EC goes down to 1 µM indicating that the cells had
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suffered from toxicity which may have been mediated by CYP1B1 inhibition. Un-transfected
cells (HEK293: pcDNA3.1), when treated with cisplatin and 7k (10 x IC ) did not show any
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perceptible decrease of cisplatin EC (8.5 µM ± 0.9). ANF, the known inhibitor of CYP1
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enzymes reduces the cisplatin EC50 from 61 µM (seen in the presence of CYP1B1
overexpression from the pcDNA3.1 plasmid) only to 40 µM. This is much higher than 8.7
µM (seen with the cell line transfected with the control plasmid) indicating that ANF may not
be effective in reversing resistance to cisplatin.
2
.8. Sensitization of CYP1B1-overproducing ovarian cancer cells, resistant to cisplatin,
by treatment with pyridyl chalcones 7k and 6j. Gene expression studies comparing
cisplatin-sensitive and cisplatin-resistant ovarian cancer cells, A2780 and A2780cis, have
shown, using real time RT-PCR (reverse transcriptase mediated PCR), that CYP1B1 is a
gene, amongst five genes, that is highly elevated in resistant A2780cis cells compared to
sensitive A2780 cells (section S6 of supporting information) [53]. We show that indeed
CYP1B1 protein is selectively overproduced in the cisplatin-resistant A2780cis cells when
compared with the parent A2780 cells (Figure 3A). Western blotting confirms the earlier
proteomic studies [53].
Figure 3
In analogy to results obtained with HEK293 cells (Figure 2B), the ovarian cancer cell line
A2780 when transfected with the plasmid pcDNA3.1/CYP1B1 (Figure 3A) also showed
resistance to cisplatin. Resistant cells could be sensitised once again by the two CYP1B1-
specific inhibitors, 7k and 6j (Figure 3B).
Although we could measure CYP1B1 activity in mechanically scraped A2780cis cells,
unfortunately we have not been able to show that the resistant cells can be re-sensitised to
cisplatin using the two potent CYP1B1-specific inhibitors. The supplier of the cell line
(ECACC) states that the cisplatin-resistant A2780cis cells (catalogue no. 93112517),
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developed by chronic exposure of the parent cisplatin-sensitive A2780 cell line (ECACC
catalogue no. 93112519) to increasing concentrations of cisplatin, has an increased ability to
repair DNA damage as well as has cytogenetic abnormalities. We have wondered whether
these were the reasons for our observations with our potent CYP1B1 inhibitors.
2
.9. Experimental solubility and lipophilicity of ANF (4) and pyridylchalcones 6j and
7
k. ANF has been reported as highly insoluble in water because of its rigid and polyaromatic
(hydrophobic) structure. Furthermore, it has been reported that it precipitates in cell culture
medium at 5 µM concentration [31]. It is well reported fact that the incorporation of
structural flexibility in planar and rigid structures results in improved aqueous solubility. The
pyridylchalcones prepared herein, were expected to possess better solubility and lipophilicity
than ANF, because of its increased flexibility than ANF. In order to find out the comparative
solubility and lipophilicity profile of ANF and pyridyl chalcones, we determined their
experimental solubility (in three biological media viz. PBS, SGF and SIF) and lipophilicity.
Results are presented in Table 3. ANF showed poor solubility in all three media with
solubility values less than 6.85 µg/ml. The predicted Log P/ Log D values were also very
high (4.52 and 4.87, respectively). Pyridylchalcones showed 2-3 fold improvement in
solubility in PBS. However, highly significant improvement (83-fold increase) in the
solubility of 6j in simulated gastric fluid (SGF: pH 1.2) was observed. The improvement in
solubility was corroborated in lipophilicity values, as both pyridylchalcones have improved
Log P/ Log D values when compared to ANF.
Table 3
2
.10. Molecular modelling studies of pyridylchalcones 7k and 6j with a panel of CYP
enzymes. ANF is a well-known pan-inhibitor of CYP1 family isoforms with poor selectivity
<10-fold difference in selectivity for 1B1 over 1A1). Interactions of ANF with CYP1
(
1
5

ACCEPTED MANUSCRIPT
isoforms 1A1, 1B1 and 1A2 has been reported in co-crystallized structures [6, 51, 52].
Molecular modelling studies indicate that, unlike ANF, the hydrophobic 6-methoxynaphthyl
group (ring A and D) of chalcone 6j co-ordinates with the heme molecule (distance is <5 Aº)
and interacts with hydrophobic residue Phe134. It leads to an alignment of the flexible semi-
polar linker with the peptide bond between Ala330-Gly329 in I helix. Further, the π electron
cloud of the heteroaromatic pyridyl ring (ring B) interacts with the conserved Phe231 residue
of the distorted F helix to complete the slot-like structure as shown in Figure 4b. However, in
the case of 7k, the flexible linker between the two hydrophobic rings (A and B) is in the
reverse position with the heteroatom present in ring A. This leads to the exposure of bulkier
hydrophobic naphthyl group to the deeper catalytic pocket where it interacts with the heme
molecule (distance is <5 Aº). The reverse linker also has close alignment with the peptide
bond of Ala330-Gly329 in I helix at one side of the slot cavity. The π electron cloud of the
heteroaromatic pyridyl ring (ring B) interacts with the conserved Phe231 residue of the
distorted F helix and the conserved Phe134 residue of B-C loop by π-π interactions (Figure
4
c).
The 180 ºC flip in orientation of the A and B rings of 6j with respect to the chromene nucleus
of ANF towards the heme molecule is due to the compact size of the binding cavity where a
phenyl or 6-methoxynaphthyl ring can easily be accommodated while a benzo(h)chromene
nucleus is not. The driving force for the binding of 6j seems to be the additional hydrophobic
interactions of the A ring with the hydrophobic Phe123 residue of the binding cavity.
However, in the case of 7k the semi-polar linker is present in a reverse orientation, and the
size of the naphthyl ring is smaller than the benzo(h)chromene nucleus which allows it to fit
in the binding cavity, thereby enabling the naphthyl group (ring B) to coordinate with the
heme molecule.
1
6

ACCEPTED MANUSCRIPT
Both 6j and 7k are specific to the CYP1B1 enzyme isoform within the CYP1 family. They
inhibit the CYP1B1 isoform with >74 fold selectivity in comparison with the 1A1 and 1A2
isoforms, which could possibly be due to the differences in amino acid residues in the
primary sequences of these enzymes (only 38% and 36% sequence homology of CYP1A1
and CYP1A2, with CYP1B1). To gain a deeper understanding of the experimentally observed
selectivity, the binding sites of CYP1B1, CYP1A1 and CYP1A2 were aligned with respect to
both compounds. It was observed that, in the case of 6j, the 6-methoxynaphthyl group (ring
A) interacts with Phe134 residue by additional hydrophobic π-π interactions. This crucial
interaction was not present in the 1A1 and 1A2 isoforms, due to the 180 ºC flip in orientation
of 6j (see section S9 of supporting information) in the active site of enzyme. However, in the
case of 7k a difference exists in the interacting amino acid residues despite having a similar
interaction pattern. The structural difference in the CYP1 family members and their
interactions with the inhibitors 6j and 7k rationalize the manifold difference in selectivity, as
shown in Figure 4d.
CYP2 and CYP3 family members CYP3A4, CYP2D6, CYP2C9 and CYP2C19 are involved
in the metabolism of most pharmaceuticals and consequently their inhibition is the major
reason for drug-drug interactions. The lower tendency to inhibit CYP2 and CYP3 family
members and specificity towards CYP1 family enzymes indicate that both 7k and 6j would
be free from any possible drug-drug interactions. The observed trend in selectivity toward
CYP1B1 enzyme over CYP3A4, CYP2D6 could be due to the lack of interactions of both 7k
and 6j with the heme iron (distance is >5.0 Aº). Moreover, in CYP1B1 enzyme, I helix is
aligned to the semipolar linker of 6j and 7k and aromatic naphthyl and pyridyl rings were
stabilized by π electron cloud of multiple phenyl alanine residues. These non-bonded
interactions are missing in CYP2D6 and CYP3A4 enzymes. The literature [54-57] on
1
7

ACCEPTED MANUSCRIPT
CYP3A4 inhibitors indicates that the presence of pyridyl or imidazolyl ring in the inhibitor
drastically improves potency due to heme ligation by the heteroaromatic N atom. However,
our observations indicate that such heteroatom-heme ligation does not appear to be the
crucial factor for higher potency against CYP1 family enzymes. This could be due to the
binding site of CYP1 family enzymes being compact in nature in contrast to the CYP2 and
CYP3 family enzymes which comprise large and flexible binding sites to accommodate a
wide range of xenobiotics. Based on these observations, it may be concluded that heme
chelation is not a crucial factor for CYP1 family enzyme inhibition by pyridylchalcones.
Figure 4
3
. CONCLUSIONS
Based on a pyridylchalcone structure, a small library of compounds was synthesized with a
view to identifying CYP1B1 inhibitors. A number of promising molecules were detected,
with several being CYP1B1-specific. Two of the most potent molecules, 7k and 6j, were
further evaluated for their inhibitory potential in live yeast and human cell assays. In order to
do this, novel cellular assays were devised. After confirmation of their potencies and
CYP1B1-specificity, the compounds were evaluated to determine if they could overcome
cisplatin resistance mediated by CYP1B1 overproduction, having already established that
CYP1B1 is one of five proteins which is overproduced in cisplatin-resistant cells. It was seen
that the cisplatin resistance mediated by CY1B1 overproduction could be overcome in both
HEK293 cells and an ovarian cancer cell line, A2780. The identified pyridylchalcones
possess better water solubility and lower lipophilicity than ANF, which makes them show
better effect in cell based assays.
These results open up avenues for further pre-clinical evaluation of the anticancer potential of
7
k and 6j in SCID mice-human tumour models. Because of the compounds’ CYP1B1
1
8

ACCEPTED MANUSCRIPT
inhibitory potencies and remarkable specificities they may also find applications in animal
models for ischemia, obesity and glaucoma.
4
4
. EXPERIMENTAL SECTION
.1. General. All solvents and chemicals were used as purchased without further purification.
1
13
The H and C-NMR spectra were recorded on a Bruker Avance AV400 NMR spectrometer
at 30°C. Chemical shifts are reported in δ units (ppm) relative to either TMS or the residual
solvent signal.
IR spectra were recorded as KBr discs on a Perkin-Elmer 298
spectrophotometer. HRMS was performed using a Thermo Scientific LTQ Orbitrap XL at the
EPSRC National Mass Spectrometry Service Centre (Swansea, UK). Melting points
(uncorrected) were determined on a Gallenkamp melting point apparatus in open glass
capillary tubes. TLC was performed on Merck Silica Gel 60f254 coated plates. Plates were
visualised under UV light (254/366nm) and stained with either 2,4-dinitrophenylhydrazine,
iodine or phosphomolybdic acid. Fluka Silica gel 60 (30-45µ) was used for flash
chromatography. The purities of compounds were determined using either elemental
analyses (C, H, N) or HPLC. The former was performed on a CE440 elemental analyser by
Warwick Analytical Services. Results were within ±0.4% of the theoretical values. HPLC
was carried out on a Perkin Elmer 200 series chromatography system using a HAISIL 100
C18 (250 x 4.6mm) column eluting with 60 % acetonitrile/ water (Flow rate = 1.0 mL/ min, λ
=
300 nm.). All samples had purity greater than 95%.
4
.2. General methods for chalcone synthesis
Method 1: A solution of n-butyl lithium (3.13 mL, 1.6 M in hexanes, 5.0 mmol) was added
dropwise to a stirred solution of N,N-diispropylethylamine (0.87 mL, 5.0 mmol) in anhydrous
THF (10 mL) at -78°C under nitrogen. After 30 min a solution of the acetophenone (5.0
mmol) in anhydrous THF (5 mL) was added followed, after 10 min, by a solution of the
1
9

ACCEPTED MANUSCRIPT
aldehyde (5.0 mmol) in THF (5 ml). The reaction was allowed to warm to RT and stirred
overnight. After quenching with water (20 mL) the resulting mixture was neutralized with
1
M hydrochloric acid and extracted with ethyl acetate (3x50 mL). The combined organics
extracts were washed with brine (20 ml), dried (MgSO ) and the solvent removed under
4
vacuum. Products were purified by flash chromatography on silica using a gradient elution
with hexane: ethyl acetate: triethylamine (10 to 80% ethyl acetate, 5% triethylamine).
Method 2: Aqueous sodium hydroxide solution (50% w/v, 0.8 mL, 10 mmol) was added to a
stirred solution of the aectophenone (5.0 mmol) and aldehyde (5.0 mmol) in methanol (20
mL) at 0°C. The resulting mixture was stirred at 0°C for two hours before warming up to rt
overnight. The reaction was quenched with water (20 mL), neutralised with 1M hydrochloric
acid and extracted with ethyl acetate (3x50 mL). The combined organic layers were washed
with saturated brine (20 mL), dried (MgSO ) and the solvent removed under vacuum.
4
Products were purified as in method 1.
Method 3: The aectophenone (6.4mmol) and aldehyde (6.4 mmol) were mixed and then
ground with powdered sodium hydroxide (0.26 g, 6.4mmol) using a pestle and mortar. This
was continued until no further change in consistency and colour was observed. The resulting
mixture was dissolved in a mixture of ethyl acetate and water (200 mL, 3: 1). The organic
layer was partitioned, washed with saturated brine (20 mL), dried (MgSO ) and the solvent
4
removed under vacuum. Products were purified as in method 1.
4
.2.1. (E)-1-(3-Methoxyphenyl)-3-(3-pyridinyl) prop-2-en-1-one (6a, DMU713): Method 1;
1
Light brown solid (25%). mp 83-84°C; H NMR (CDCl ): δ 3.90 (3H, s), 7.15 (1H, dd, J =
3
3
.7, 8.8 Hz), 7.36 (1H, dd, J = 4.7, 8.8 Hz), 7.43 (1H, t, J = 8.4 Hz), 7.54-7.63 (3H, m), 7.79
(1H, d, J = 15.8 Hz), 7.93-7.98 (1H, m), 8.64 (1H, dd, J = 1.9, 4.7 Hz), 8.87 (1H, d, J = 2.7
2
0

ACCEPTED MANUSCRIPT
1
3
Hz). C NMR (CDCl ): δ 55.48, 112.90, 119.61, 120.77, 121.30,123.84, 129.69, 130.65,
3
-1
1
39.08, 140.90, 150.3, 151.11, 159.96, 189.49; IR (KBr) /cm 1663 (C=O); HRMS found
[
M+1]+ 240.1019, C H NO requires [M+1]+ 240.1019; Anal. Calcd C H NO : C, 75.30;
15
13
2
15 13
2
H, 5.48, N, 5.85; Found C, 75.53; H, 5.42; N, 5.67.
4
.2.2. (E)-1-(4-Methoxyphenyl)-3-(3-pyridinyl)prop-2-en-1-one (6b, DMU714)[44, 58]
1
Method 1; Yellow solid (48%). mp 105-106°C; H NMR (CDCl ): δ 3.90 (3H, s), 7.00 (2H,
3
d, J = 8.8 Hz), 7.35 (1H, dd, J = 4.6, 8.3 Hz), 7.61 (1H, d, J = 16.2 Hz), 7.78 (1H, d, J = 16.2
Hz), 7.92-7.96 (1H, m), 8.04 (2H, d, J = 8.8 Hz), 8.62 (1H, dd, J = 4.2, 1.3 Hz), 8.87 (1H, d,
1
3
J = 2.5 Hz); C NMR (CDCl ): δ55.55, 114.00, 123.77, 130.69, 130.90, 134.56, 137.38,
3
-
1
1
2
7
40.08, 149.88, 150.93, 163.72, 188.02; IR (KBr) /cm 1662 (C=O); HRMS found [M+1]+
40.1017, C H NO requires [M+1]+ 240.1019; Anal. Calcd C H NO .0.15H O: C,
1
5
13
2
15 13
2
2
4.46; H, 5.54, N, 5.79; Found C, 74.41; H, 5.43; N, 5.77.
4
.2.3. (E)-1-(2, 4-Dimethoxyphenyl)-3-(3-pyridinyl) prop-2-en-1-one (6c, DMU715)[44,
1
5
3
7
1
1
1
8] Method 1; Bright yellow solid (55%). mp 91-93°C; H NMR (CDCl ): δ 3.88 (3H, s),
3
.92 (3H, s), 6.52 (1H, d, J = 2.3 Hz), 6.58 (1H, dd, J = 2.3, 9.4 Hz), 7.34 (1H, dd, J = 5.3,
.0 Hz), 7.58-7.94 (2H, m), 7.81 (2H, d, J = 9.4 Hz), 7.86-7.94 (1H, m), 8.59 (1H, dd, J =
1
3
.2, 5.4 Hz), 8.84 (1H, d, J = 2.4 Hz); C NMR (CDCl ): δ55.60, 55.78, 98.65, 105.46,
3
21.75, 123.69, 129.07, 131.35, 133.17, 134.64, 137.81, 149.73, 150.51, 160.65, 164.60,
-1
89.59; IR (KBr) /cm 1656 (C=O); HRMS found [M+1]+ 270.1126, C H NO requires
1
6
15
3
[
M+1]+ 270.1125; Anal. Calcd C H NO : C, 71.36; H, 5.61, N 5.20 Found C, 71.03; H,
16 15 3
5
.65 N 5.18.
4
.2.4. (E)-1-(3, 4-Dimethoxyphenyl)-3-(3-pyridyl) prop-2-en-l-one (6d, DMU716).
1
Method 1; Light yellow solid (37%). mp 91-93°C; H NMR (CDCl ): δ 3.99 (6H, s), 6.94
3
2
1

ACCEPTED MANUSCRIPT
(1H, d, J = 7.5 Hz), 7.36 (1H, dd, J = 5.0, 8.0 Hz), 7.63 (1H, d, J = 15.9 Hz), 7.64 (1H, d, J =
2
.0 Hz), 7.68 (1H, dd, J = 2.0, 7.5 Hz), 7.78 (1H, d, J = 15.9 Hz), 7.93-7.96 (1H, m), 8.63
13
(
1H, dd, J = 1.5, 5.0 Hz), 8.87 (1H, d, J = 1.5 Hz); C NMR (CDCl ) δ 56.18, 109.96,
3
1
1
10.81, 122.70, 123.69, 124.50, 130.91, 134.59, 140.10, 149.40, 149.87, 150.86, 153.69,
-1
87.94; IR (KBr) /cm 1655 (C=O); HRMS found [M+1]+ 270.1125, C H NO requires
1
6
15
3
[
M+1]+ 270.1125; Anal. Calcd C H NO : C, 71.36; H, 5.61, N, 5.20; Found C, 71.03; H,
16 15 3
5
.65; N, 5.14.
4
.2.5. (E)-1-(3, 5-Dimethoxyphenyl)-3-(3-pyridyl) prop-2-en-l-one (6e, DMU717)
1
Method 1: Yellow solid (34%). mp 90-91°C; H NMR (CDCl ): δ 3.87 (6H, s), 7.69 (1H, t, J
3
=
2.1 Hz), 7.15 (2H, d, J = 2.1 Hz), 7.37 (1H, dd, J = 4.8, 6.4 Hz), 7.54 (1H, d, J = 16.0 Hz),
7
1
1
2
1
.78 (1H, d, J = 16.0 Hz), 7.92-7.96 (1H, m), 8.63 (1H, dd, J = 1.6, 4.8 Hz), 8.86 (1H, d, J =
13
.6 Hz); C NMR (CDCl ): δ 55.67, 105.30, 106.45, 123.78, 123.92, 130.67, 134.54, 139.73,
3
-1
41.02, 150.06, 151.16, 161.03, 189.47; IR (KBr) /cm 1666 (C=O); HRMS found [M+1]+
70.1124, C H NO requires [M+1]+ 270.1125; Purity >99 % HPLC (Retention time
16
15
3
2.012 min).
4
.2.6. (E)-3-(3-Pyridyl)-1-(2, 3, 4-trimethoxyphenyl)prop-2-en-l-one (6f, DMU718)[58,
1
5
9]Synthetic Method 1; Yellow solid (51%). mp 77-78°C; H NMR (CDCl ): δ 3.92 (3H, s)
3
3
.93 (3H, s), 3.94 (3H, s), 6.67 (1H, d, J = 8.8 Hz), 7.34 (1H, dd, J = 5.2, 7.2 Hz), 7.53 (1H,
d, J = 8.8 Hz), 7.59 (1H, d, J = 16.0 Hz), 7.68 (1H, d, J = 16.0 Hz), 7.70-7.94 (1H, m), 8.61
1
3
(
1H, dd, J = 1.0, 5.2 Hz), 8.83 (1H, d, J = 2.1 Hz); C NMR (CDCl ): δ56.17, 61.07, 62.09,
3
1
1
07.50, 123.74, 126.04, 126.27, 128.46, 131.05, 134.47, 138.73, 142.18, 149.95, 150.77,
-1
53.97, 157.48, 189.98; IR (KBr) /cm 1666 (C=O); HRMS found [M+1]+ 300.1229,
C H NO requires [M+1]+ 300.1230; Purity >99 % % HPLC (Retention time 12.168 min).
17
17
4
2
2

ACCEPTED MANUSCRIPT
4
.2.7. (E)-1-(3-Chlorophenyl)-3-(3-pyridinyl)prop-2-en-1-one (6g, DMU2123) Method 3;
o
1
Pale yellow solid (52%). mp 128-130 C; H NMR (CDCl ): δ 7.37 (1H, dd, J = 5.2, 7.7 Hz),
3
7
.45 (1H, t, J = 6.6 Hz), 7.51-7.60 (2H, m), 7.80 (1H, d, J = 16.0 Hz), 7.89 (1H, d, J = 7.6
1
3
Hz), 7.94-8.02 (2H, m), 8.65 (1H, d, J = 5.2 Hz), 8.86 (1H, s); C NMR (CDCl ): δ 123.19,
3
1
1
23.84, 126.60, 128.62, 130.09, 130.40, 133.04, 135.07, 139.30, 141.77, 150.15, 151.40,
-1
88.44; IR (KBr) /cm 1665 (C=O); HRMS found [M+1]+ 244.0524, C H NOCl requires
14
10
[
M+1]+ 244.0524; Anal. Calcd C H NOCl: C, 69.00; H, 4.14, N, 5.75; Found C, 68.86; H,
14 10
4
.12, N, 5.66.
4
.2.8. (E)-1-(3-Bromophenyl)-3-(3-pyridinyl)prop-2-en-1-one (6h, DMU2127) Method 3;
o
1
Pale yellow solid (39%). mp 126-128 C; H NMR (DMSO-d ): δ 7.51 (1H, dd, J = 7.4, 3.9
6
Hz), 7.56 (1H, t, J = 7.7 Hz), 7.81 (1H, d, J = 15.8 Hz), 7.87-7.92 (1H, m), 8.11 (1H, d, J =
1
5.8 Hz), 8.16-8.19 (1H, m), 8.35-8.42 (2H, m), 8.64 (1H, dd, J = 2.1, 7.4 Hz), 9.06 (1H, d, J
1
3
=
2.8 Hz); C NMR (DMSO-d ) δ122.36, 123.41, 123.88, 127.58, 130.37, 131.04, 135.31,
6
-1
1
35.96, 139.27, 141.36, 150.54, 151.17, 187.65; IR (KBr) /cm 1665 (C=O); HRMS found
[
M]+ 288.0025, C H BrNO requires [M]+ 288.0019; Anal. Calcd C H BrNO: C, 58.36;
14
10
14 10
H, 3.50, N, 4.86; Found C, 58.26; H, 3.44; N, 4.83.
4
.2.9. (E)-1-(2-Naphthyl)-3-(3-pyridyl)prop-2-en-1-one (6i, DMU745) Method 1; Pale
1
brown solid (52%). mp 117-118°C; H NMR (CDCl ): δ 7.38 (1H, dd, J = 4.2, 7.8 Hz), 7.55-
3
7
.63 (2H, m), 7.75 (1H, d, J = 16.0 Hz), 7.86 (1H, d, J = 16.0 Hz), 7.90 (1H, d, J = 8.6 Hz),
.96 (1H, d, J = 9.3 Hz), 7.98-8.02 (2H, m), 8.10 (1H, dd, J = 2.6, 9.3 Hz), 8.53 (1H, s), 8.64
7
1
3
(
1H, dd, J = 1.9, 4.2 Hz), 8.90 (1H, d, J = 2.6 Hz); C NMR (CDCl ) δ 123.80, 123.89,
3
1
1
24.37, 126.94, 127.88, 128.76, 128.76, 129.58, 130.15, 130.77, 132.56, 134.65, 135.12,
-1
35.64, 140.86, 150.05, 151.14, 189.58; IR (KBr) /cm 1651 (C=O); HRMS found [M]+
2
3

ACCEPTED MANUSCRIPT
2
60.1072, C H NO requires [M]+ 260.1070; Purity >99 % HPLC (Retention time 11.757
18
13
min).
4
.2.10. (E)-1-(6-Methoxy-2-naphthyl)-3-(3-pyridyl)prop-2-en-1-one (6j, DMU2139)
1
Method 2: Pale yellow solid (8%). mp 133-134°C; H NMR (CDCl ): δ 3.96 (3H, s), 7.18
3
(1H, d, J = 2.9 Hz), 7.23 (1H, dd, J = 3.3, 8.8 Hz), 7.28 (1H, dd, J = 7.3, 4.4 Hz), 7.75 (1H, d,
J = 15.0 Hz), 7.80-7.92 (3H, m), 7.97-8.02 (1H, m), 8.09 (1H, dd, J = 8.4, 2.2 Hz), 8.48 (1H,
13
s), 8.64 (1H, dd, J = 4.3, 2.2 Hz), 8.90 (1H, d, J = 2.6 Hz); C NMR (CDCl ): δ 55.47,
3
1
1
05.89, 119.88, 123.78, 123.89, 125.12, 127.45, 127.88, 130.09, 130.89, 131.18, 133.14,
-1
34.62, 137.40, 140.42, 149.99, 151.03, 159.96, 189.13; IR (KBr) /cm 1643 (C=O); HRMS
found [M]+ 290.1175, C H NO requires [M]+ 290.1176; Purity >99 % HPLC (Retention
1
9
15
2
time 15.732 min).
4
.2.11. (E)-1-(1-Naphthyl)-3-(3-pyridyl)prop-2-en-1-one (6k, DMU746) Method 1;
1
Brown solid (88%). mp 123-124°C; H NMR (CDCl ): δ 7.34 (1H, dd, J = 8.2, 4.6 Hz), 7.38
3
(1H, d, J = 16.0 Hz), 7.52-7.64 (4H, m), 7.80 (1H, dd, J = 7.3, 1.0 Hz), 7.88-7.94 (2H, m),
8
.02 (1H, d, J = 4.3 Hz), 8.36 (1H, dd, J = 8.2, 1.4 Hz), 8.62 (1H, dd, J = 1.3, 4.1 Hz), 8.78
1
3
(
1H, d, J = 1.3 Hz); C NMR (CDCl ): δ 123.83, 124.50, 125.56, 126.63, 127.47, 128.55,
3
-1
1
28.74, 130.48, 132.12, 134.53, 136.49, 150.07, 151.24, 194.76; IR (KBr) /cm 1662 (C=O);
HRMS found [M]+ 260.1070, C H NO requires [M]+ 260.1070; Purity 99 % HPLC
1
8
13
(Retention time 10.333 min).
4
.2.12. (E)-1-(4-Biphenyl)-3-(3-pyridyl)prop-2-en-1-one (6l, DMU2134) Method 2;
1
Yellow solid (27%). m p 140-141°C; H NMR (DMSO-d ): δ 7.40-7.56 (4H, m), 7.76-7.83
6
(3H, m), 7.89 (2H, d, J = 9.2 Hz), 8.14 (1H, d, J = 14.6 Hz), 8.27 (2H, d, J = 9.2 Hz), 8.36-
13
8
.40 (1H, m), 8.62 (1H, dd, J = 1.0, 4.4 Hz), 9.04 (1H, d, J = 1.9 Hz); C NMR (DMSO-d )
6
2
4

ACCEPTED MANUSCRIPT
δ188.40, 151.01, 150.37, 144.72, 140.52, 138.82, 136.06, 135.17, 130.51, 128.46, 123.95,
-1
1
23.86. IR (KBr) /cm 1659 (C=O); HRMS found [M]+ 286.1224, C H ON requires [M]+
20 16
2
86.1226; Purity 96 % HPLC (Retention time 14.333 min).
4
.2.13. (E)-1-(2-Naphthyl)-3-(4-pyridyl)prop-2-en-1-one (6m, DMU780) Method 3;
1
Yellow solid (39%). mp 126-127°C; H NMR (CDCl ): δ 7.51 (2H, d, J = 6.1 Hz), 7.54-7.68
3
(
(
(
2H, m), 7.75 (1H, d, J = 15.3 Hz), 7.83 (1H, d, J = 15.3 Hz), 7.90 (1H, d, J = 7.6 Hz), 7.96
1H, d, J = 8.3 Hz), 8.00 (1H, d, J = 8.3 Hz), 8.09 (1H, dd, J = 1.0, 9.7 Hz), 8.54 (1H, s), 8.72
1
3
1H, d, J = 6.1 Hz); C NMR (CDCl ): δ 122.07, 124.30, 126.09, 127.01, 127.91, 128.77,
3
1
28.85, 129.60, 130.32, 132.53, 134.90, 135.72, 141.47, 142.17, 150.69, 189.54; IR (KBr)
-1
/
cm 1663 (C=O); HRMS found [M]+ 260.1071, C H NO requires [M]+ 260.1070; Purity
18 13
9
7 % HPLC (Retention time 9.835 min).
4
.2.14. (E)-1-(3-Phenanthrenyl)-3-(3-pyridinyl) prop-2-en-1-one (6n, DMU2136)
1
Method 2; Bright yellow solid (61%). mp 113-114°C; H NMR (CDCl ): δ 7.38 (1H, dd, J =
3
5
.8, 7.1 Hz), 7.61-8.00 (9H, m), 8.19 (1H, dd, J = 5.8, 1.3 Hz), 8.67 (1H, d, J = 7.1 Hz), 8.91
1
3
(
1H, d, J = 1.2 Hz), 9.35 (1H, s); C NMR (CDCl ): δ 122.78, 123.81, 124.06, 124.14,
3
1
1
25.62, 126.30, 127.30, 127.37, 128.89, 129.08, 129.87, 129.93, 130.61, 130.78, 132.23,
-1
34.68, 135.05, 135.44, 140.99, 147.13, 151.07, 150.17, 189.74; IR (KBr) /cm 1655 (C=O);
HRMS found [M]+ 310.1230, C H NO requires [M]+ 310.1226; Purity 96 % HPLC
2
2
15
(Retention time 17.154 min).
4
.2.15. (E)-1-(9-Phenanthrenyl)-3-(3-pyridinyl)prop-2-en-1-one (6o, DMU2137)
1
Synthetic Method 2; Yellow solid (61%). mp 120-121°C; H NMR (CDCl ): δ 7.15 (1H, dd,
3
J = 4.8, 9.1 Hz), 7.42 (1H, d, J = 15.2 Hz), 7.62-7.79 (5H, m), 7.90-7.99 (2H, m), 8.05 (1H,
s), 8.34 (1H, dd, J = 1.0, 8.6 Hz), 8.63 (1H, dd, J = 4.8, 1.1 Hz), 8.72 (1H, d, J = 8.6 Hz),
2
5

ACCEPTED MANUSCRIPT
1
3
8
1
1
.77 (1H, d, J = 8.6 Hz), 8.80 (1H, d, J = 1.5 Hz); C NMR (CDCl ): δ 122.79, 123.05,
3
23.82, 126.45, 127.25, 127.35, 127.45, 128.68, 128.74, 128.82, 129.13, 129.69, 130.10,
-1
30.43, 130.82, 131.64, 134.54, 135.57, 142.04, 150.17, 151.35, 194.94; IR (KBr) /cm 1651
(
C=O); HRMS found [M]+ 310.1228, C H NO requires [M]+ 310.1226; Purity >99 %
22 15
HPLC (Retention time 17.366 min).
4
.2.16. (E)-1-(1-Pyrenyl)-3-(3-pyridyl)prop-2-en-1-one (6p, DMU2140) Method 2; Bright
1
yellow solid (54%). mp 113-114°C; H NMR (CDCl ): δ 7.33 (1H, dd, J = 4.8, 8.0 Hz), 7.53
3
(1H, d, J = 16.1 Hz), 7.65 (1H, d, J = 16.1 Hz), 7.89-7.93 (1H, m), 8.03-8.11 (2H, m), 8.15-
8
.28 (6H, m), 8.61 (1H, dd, J = 1.6, 4.8 Hz), 8.64 (1H, d, J = 8.8 Hz), 8.89 (1H, d, J = 1.6
1
3
Hz); C NMR (CDCl ): δ 123.82, 124.09, 124.39, 124.58, 124.97, 126.14, 126.32, 126.34,
3
1
26.50, 127.18, 129.07, 129.40, 129.58, 130.55, 131.15, 133.59, 134.51, 141.76, 150.13,
-1
1
51.25, 194.96; IR (KBr) /cm 1620 (C=O); HRMS found [M]+ 334.1227, C H NO
24 15
requires [M]+ 334.1226; Purity >99 % HPLC (Retention time 19.736 min).
4
.2.17. (E)-3-(3-Hydroxyphenyl)1-(3-pyridyl)prop-2-en-1-one (7a, DMU767) Method 3;
1
Yellow solid (17%). mp 192-193°C; H NMR (DMSO-d ): δ 6.92 (1H, d, J = 8.0 Hz), 7.24-
6
7
.35 (3H, m), 7.62 (1H, dd, J = 7.6, 5.6 Hz), 7.68 (1H, d, J = 16.0 Hz), 7.84 (1H, d, J = 16.0
Hz), 8.44-8.48 (1H, m), 8.81 (1H, dd, J = 1.0, 5.6 Hz), 9.29 (1H, d, J = 1.2 Hz), 9.78 (1H,
1
3
brs); C NMR (DMSO-d ) δ 115.39,118.15, 120.08, 121.66, 123.93, 129.90, 132.81, 135.70,
6
-1
1
35.97, 145.05, 149.55, 153.35, 157.72, 188.57; IR (KBr) /cm 1662 (C=O); HRMS found
+
+
[
M+1] 226.0863, C H O N requires [M+1] 226.0863; HPLC purity 98 % (Retention time
14 11 2
3
.947 min).
4
.2.18. (E)-3-(3,4-Methylenedioxyphenyl)-1-(3-pyridyl)prop-2-en-1-one (7b, DMU769)
1
Method 2; Yellow solid (76%). mp 144-145°C; H NMR (DMSO-d ): δ 6.11 (2H, s), 7.01
6
2
6

ACCEPTED MANUSCRIPT
(1H, d, J = 8.5 Hz), 7.36 (1H, dd, J = 1.1, 8.5 Hz), 7.62 (1H, dd, J = 8.5, 4.8 Hz), 7.67 (1H, d
J = 1.1 Hz), 7.73 (1H, d, J = 15.4 Hz), 7.82 (1H, d, J = 15.4 Hz), 8.44-8.48 (1H, m), 8.82
1
3
(
1H, dd, J = 4.8, 1.6 Hz), 9.31 (1H, d, J = 1.1 Hz); C NMR (DMSO-d ) δ 101.70, 107.02,
6
1
1
08.55, 119.67, 123.91, 126.39, 128.95, 133.94, 135.87, 144.83, 146.63, 148.11, 149.55,
-1
+
53.15, 188.19; IR (KBr) /cm 1662 (C=O); HRMS found [M+1] 254.0812, C H O N
1
5
12
3
+
requires [M+1] 254.0823; HPLC purity 97 % (Retention time 5.681 min).
4
.2.19. (E)-3-(3-Methoxyphenyl)-1-(3-pyridyl)prop-2-en-1-one (7c, DMU789) Method 2;
1
Light brown solid (33%). mp 78-79°C; H NMR (CDCl ): δ 3.87 (3H, s), 6.99 (1H, dd, J =
3
8
.4, 2.4 Hz), 7.14-7.19 (1H, m), 7.25 (1H, d, J = 6.6 Hz), 7.36 (1H, t, J = 6.6 Hz), 7.44-7.50
(2H, m), 7.80 (1H, d, J = 15.5 Hz), 8.26-8.31 (2H, m), 8.81 (1H, dd, J = 1.2, 5.4 Hz), 9.23
1
3
(
1H, d, J = 1.1 Hz); C NMR (CDCl ): δ 55.39, 113.57, 116.18, 121.28, 121.74, 123.69,
3
-1
1
30.08, 133.51, 135.83, 145.96, 149.79, 153.21, 160.04, 189.18; IR (KBr) /cm 1662 (C=O);
+
+
HRMS found [M+1] 240.1017, C H O N requires [M+1] 240.1019; Purity >99 % HPLC
15
13
2
(Retention time 6.641 min).
4
.2.20. (E)-3-(2-Methoxyphenyl)-1-(3-pyridyl)prop-2-en-1-one (7d, DMU790) Method 2;
1
Yellow solid (50%). mp 95-96°C; H NMR (DMSO-d ): δ 3.90 (3H, s), 7.04 (1H, t, J = 8.1
6
Hz), 7.13 (1H, d, J = 7.6 Hz), 7.44-7.51 (1H, m), 7.60 (1H, dd, J = 4.9, 7.6 Hz), 7.88 (1H, d,
J = 16.3 Hz), 8.00 (1H, dd, J = 1.6, 7.6 Hz), 8.10 (1H, d, J = 16.3 Hz), 8.41-8.47 (1H, m),
1
3
8
1
1
.82 (1H, dd, J = 1.6, 4.9 Hz), 9.28 (1H, d, J = 2.2 Hz); C NMR (DMSO-d ) δ 55.74,
6
11.83, 120.72, 121.52, 122.63, 123.95, 128.61, 132.70, 134.94, 135.90, 149.48, 153.19,
-1
+
58.39, 188.60; IR (KBr) /cm 1663 (C=O); HRMS found [M+1] 240.1019, C H O N
1
5
13
2
+
requires [M+1] 240.1019; Purity >99 % HPLC (Retention time 7.154 min).
2
7

ACCEPTED MANUSCRIPT
4
.2.21.
(E)-3-(3-Hydroxy-4-methoxyphenyl)-1-(3-pyridyl)prop-2-en-l-one
(7e,
1
DMU2103) Method 3; Bright yellow solid (13%). mp 157-158°C; H NMR (DMSO-d ): δ
6
3
7
1
1
.85 (3H, s), 7.12 (1H, d, J = 7.4 Hz), 7.30-7.35 (2H, m), 7.60 (1H, dd, J = 5.1, 7.4 Hz), 7.63-
.74 (2H, m), 8.41-8.46 (1H, m), 8.81 (1H, dd, J = 5.1, 0.9 Hz), 9.23 (1H, s), 9.28 (1H, d, J =
13
.1 Hz); C NMR (DMSO-d ) δ 55.68, 111.90, 114.95, 119.17, 122.50, 123.90, 127.14,
6
-1
34.94, 135.85, 145.35, 146.63, 149.44, 150.61, 153.04, 188.29; (C=O); IR (KBr) /cm 1658
+
+
(
C=O); HRMS found [M+1] 256.0970, C H O N requires [M+1] 256.0968; Purity >99 %
15 13 3
HPLC (Retention time 4.950 min).
4
.2.22. (E)-3-(3, 4-Dimethoxyphenyl)-1-(3-pyridyl) prop-2-en-1-one (7f, DMU774)
1
Method 3; Yellow solid (15%). mp 88-89°C; H NMR (DMSO-d ): δ 3.81 (3H, s), 3.85 (3H,
6
s), 7.04 (1H, d, J = 9.3 Hz), 7.40 (1H, dd, J = 9.8, 1.6 Hz), 7.56 (1H, d, J = 1.6 Hz), 7.59 (1H,
dd, J = 4.7, 8.8 Hz), 7.74 (1H, d, J = 15.5 Hz), 7.84 (1H, d, J = 15.5 Hz), 8.41-8.46 (1H, m),
1
3
8
1
1
.81 (1H, dd, J = 1.0, 4.7 Hz), 9.14 (1H, d, J = 1.5 Hz); C NMR (DMSO-d ) δ 56.00,
6
10.78, 111.55, 119.31, 123.89, 124.43, 127.14, 133.05, 135.82, 145.31, 149.03, 149.60,
-1
+
51.51, 151.56, 153.13, 188.20; IR (KBr) /cm 1658 (C=O); HRMS found [M+1] 270.1126,
+
C H O N requires [M+1] 270.1125; Purity 99 % HPLC (Retention time 5.852 min).
16
15
3
4
.2.23.
(E)-1-(3-Pyridyl)-3-(3,
4,
5-trimethoxyphenyl)prop-2-en-1-one
(7g,
1
DMU2114)[60] Method 2; Bright yellow solid (37%). mp 123-124°C; H NMR (DMSO-d ):
6
δ 7.24 (3H, s), 7.62 (1H, dd, J = 7.2, 4.8 Hz), 7.75 (1H, d, J = 16.1 Hz), 7.90 (1H, d, J = 16.1
1
3
Hz), 8.43-8.48m (1H, m), 8.83 (1H, dd, J = 4.8, 1.6 Hz), 9.36 (1H, d, J = 1.2 Hz); C NMR
(
DMSO-d ) δ 60.13, 106.73, 120.93, 123.93, 129.94, 132.88, 135.91, 140.00, 145.27,
6
-1
+
1
49.64, 153.08, 153.23, 188.36; IR (KBr) /cm 1662 (C=O); HRMS found [M+1] 300.1231,
+
C H O N requires [M+1] 300.1230; HPLC purity >99 % (Retention time 5.403 min).
17
17
4
2
8