CYP enzymes, expressed within live human suspension cells, are superior to widely-used microsomal enzymes in identifying potent CYP1A1/CYP1B1 inhibitors: Identification of quinazolinones as CYP1A1/CYP1B1 inhibitors that efficiently reverse B[a]P toxicity and cisplatin resistance

Article abstract of DOI:10.1016/j.ejps.2019.02.016

Microsomal cytochrome P450 (CYP) enzymes, isolated from recombinant bacterial/insect/yeast cells, are extensively used for drug metabolism studies. However, they may not always portray how a developmental drug would behave in human cells with intact intracellular transport mechanisms. This study emphasizes the usefulness of human HEK293 kidney cells, grown in ‘suspension’ for expression of CYPs, in finding potent CYP1A1/CYP1B1 inhibitors, as possible anticancer agents. With live cell-based assays, quinazolinones 9i/9b were found to be selective CYP1A1/CYP1B1 inhibitors with IC₅₀ values of 30/21 nM, and > 150-fold selectivity over CYP2/3 enzymes, whereas they were far less active using commercially-available CYP1A1/CYP1B1 microsomal enzymes (IC₅₀, >10/1.3–1.7 μM). Compound 9i prevented CYP1A1-mediated benzo[a]pyrene-toxicity in normal fibroblasts whereas 9b completely reversed cisplatin resistance in PC-3/prostate, COR-L23/lung, MIAPaCa-2/pancreatic and LS174T/colon cancer cells, underlining the human-cell-assays’ potential. Our results indicate that the most potent CYP1A1/CYP1B1 inhibitors would not have been identified if one had relied merely on microsomal enzymes.

Full text of DOI:10.1016/j.ejps.2019.02.016

Accepted Manuscript
CYP enzymes, expressed within live human suspension cells, are
superior to widely-used microsomal enzymes in identifying potent
CYP1A1/CYP1B1 inhibitors: Identification of quinazolinones as
CYP1A1/CYP1B1 inhibitors that efficiently reverse B[a]P
toxicity and cisplatin resistance
Vinay R. Sonawane, Mohd Usman Mohd Siddique, Linda
Gatchie, Ibidapo S. Williams, Sandip B. Bharate, Venkatesan
Jayaprakash, Barij N. Sinha, Bhabatosh Chaudhuri
PII:
S0928-0987(19)30069-7
https://doi.org/10.1016/j.ejps.2019.02.016
PHASCI 4846
DOI:
Reference:
To appear in:
European Journal of Pharmaceutical Sciences
Received date:
Revised date:
Accepted date:
20 September 2018
26 December 2018
12 February 2019
Please cite this article as: V.R. Sonawane, M.U.M. Siddique, L. Gatchie, et al., CYP
enzymes, expressed within live human suspension cells, are superior to widely-used
microsomal enzymes in identifying potent CYP1A1/CYP1B1 inhibitors: Identification of
quinazolinones as CYP1A1/CYP1B1 inhibitors that efficiently reverse B[a]P toxicity and
cisplatin resistance, European Journal of Pharmaceutical Sciences, https://doi.org/10.1016/
j.ejps.2019.02.016
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ACCEPTED MANUSCRIPT
CYP enzymes, expressed within live human suspension cells, are superior
to widely-used microsomal enzymes in identifying potent CYP1A1/CYP1B1
inhibitors: Identification of quinazolinones as CYP1A1/CYP1B1 inhibitors
that efficiently reverse B[a]P toxicity and cisplatin resistance
a,#
b,#
a
a
Vinay R. Sonawane , Mohd Usman Mohd Siddique , Linda Gatchie , Ibidapo S Williams ,
c
b
b
a,
Sandip B. Bharate , Venkatesan Jayaprakash , Barij N. Sinha , Bhabatosh Chaudhuri *
a
CYP Design Ltd, The Innovation Centre, 49 Oxford Street, Leicester, LE1 5XY, UK.
b
Department of Pharmaceutical Sciences & Technology, Birla Institute of Technology,
Mesra, Ranchi-835215, India
b
Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India.
#
Contributed equally to the work described
*
Corresponding author:
*Tel: 00-44-7805230121; E-mail: bchaud00@gmail.com (BC)
1

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ABSTRACT
Microsomal cytochrome P450 (CYP) enzymes, isolated from recombinant
bacterial/insect/yeast cells, are extensively used for drug metabolism studies. However, they
may not always portray how a developmental drug would behave in human cells with intact
intracellular transport mechanisms. This study emphasizes the usefulness of human HEK293
kidney cells, grown in ‘suspension’ for expression of CYPs, in finding potent
CYP1A1/CYP1B1 inhibitors, as possible anticancer agents. With live cell-based assays,
quinazolinones 9i/9b were found to be selective CYP1A1/CYP1B1 inhibitors with IC50
values of 30/21 nM, and >150-fold selectivity over CYP2/3 enzymes, whereas they were far
less active using commercially-available CYP1A1/CYP1B1 microsomal enzymes (IC50
,
>10/1.3-1.7 µM). Compound 9i prevented CYP1A1-mediated benzo[a]pyrene-toxicity in
normal fibroblasts whereas 9b completely reversed cisplatin resistance in PC-3/prostate,
COR-L23/lung, MIAPaCa-2/pancreatic and LS174T/colon cancer cells, underlining the
human-cell-assays’ potential. Our results indicate that the most potent CYP1A1/CYP1B1
inhibitors would not have been identified if one had relied merely on microsomal enzymes.
KEYWORDS: Microsomal CYP450 enzymes; cisplatin resistance; CYP1A1 inhibitors;
CYP1A1-mediated benzo[a]pyrene toxicity; CYP1B1 inhibitors; hard to metabolize
medications; live CYP-expressing human cells.
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INTRODUCTION
Cytochrome P450 (CYP) enzymes belong to a superfamily of monooxygenase enzymes. The
human CYP family consists of 57 proteins (Lewis, 2004). Around a dozen human CYP
enzymes are responsible for the metabolism of xenobiotics, which include pharmaceutical
drugs, chemicals and other substances that humans imbibe in their daily existence. These
CYPs account for ~70-80% of liver enzymes involved in the metabolism of xenobiotics by
the liver (Fasinu et al., 2012). In general, the liver CYPs enable conversion of hydrophobic
molecules to water-soluble products, to facilitate excretion. Essentially, these CYPs bring
about oxidation of chemicals, usually with the incorporation of polar moieties or removal of
hydrophobic groups so as to render them soluble for excretion (Nebert et al., 2013).
At the pre-clinical stage of drug discovery, CYP enzymes play critical roles in the
evaluation of metabolic profiles of drug-like candidates (Nebert and Russell, 2002). Human
liver CYP enzymes are bound to human liver microsomes (HLMs) which are principally
endoplasmic reticular (i.e. ER) membranes. Human CYPs bound to the ER of baker’s yeast
cells and insect cells or the periplasmic membranes of bacterial cells (since prokaryotic cells
do not contain ER) are commonly used for pre-clinical drug development (Hausjell et al.,
2
018). Although microsomal CYPs isolated from such recombinant cells are accepted as in
vitro tools for research linked to drug metabolism, they are not always able to predict how a
compound would inhibit or be metabolized by a CYP enzyme within live human cells where
intracellular compartments (and proteins contained in these compartments), responsible for
transport of chemicals, are fully intact. Hence, a type of live liver cells (i.e. hepatocytes) are
the most preferred in drug metabolism studies (Basu & Shaik, 2016). Unfortunately,
hepatocytes are inherently inconsistent in their content because they are usually created from
the pooling of a large number of livers derived from individuals who do not share similar
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genetic makeup. Hepatocytes provide reproducible results only after extensive initial
standardization. Therefore, an alternative technology that would eliminate the negative
aspects of hepatocytes and isolated microsomal enzymes but would preserve their positive
features could be useful in facilitating drug discovery research.
This report compares, for the first time, screening of a small chemical library with the help
of (a) CYP-expressing live human kidney (HEK293) cells, grown in ‘suspension’ (it should
be noted that hepatocytes are also grown in ‘suspension’), and (b) microsomal CYP enzymes,
in the identification of inhibitors of CYP1A1 and CYP1B1, two CYP enzymes which have
critical roles in the genesis of cancer. Unlike microsomal enzymes and hepatocytes which
lose all CYP activity within a few hours (i.e. a maximum of 6 h) at 37 ºC (Donato et al.,
2
004), we see that CYP activities within live whole cells can be maintained over 4-5 days.
CYP activities, expressed within live HEK293 ‘suspension’ (HEK293S) cells, also do not
suffer from the drawback of being inherently dependent on a NADPH-regenerating system,
which is absolutely essential for all microsomal assays (Mazur et al., 2009). Our results from
screening of this chemical library indicate that there are stark differences in the inhibition of
cellular CYP1A1 and CYP1B1 enzymes when compared with the inhibition of the same
enzymes isolated as microsomal entities from baker’s yeast (Sacchrosomes™; CYP Design
Ltd), from insect cells (Supersomes™; Corning) or from bacteria (Bactosomes™; Cypex).
The company Invitrogen (Thermo-Fisher) also markets CYP-microsomes (Baculosomes™)
isolated from insect cells; however, they do not sell the human CYP1A1 and CYP1B1
microsomal enzymes.
The CYP1A1 enzyme, belonging to the CYP1 sub-family, mediates the metabolism of
polyaromatic hydrocarbons (PAHs) (Moorthy et al., 2015). PAHs, such as benzo[a]pyrene
(B[a]P) and 7,12-dimethylbenz(a)anthracene (DMBA), are present in cigarette smoke and
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charcoal-broiled foods. PAHs form diol-epoxides and o-quinones which intercalate DNA at
the DNA synthesis (S) phase of the cell division cycle, and thereby cause mutations in DNA
which lead to carcinogenesis. It has been reported that this is one of the main causes of lung
cancer. Hence, it has been inferred that inhibition of CYP1A1 enzyme can prevent lung
cancer (Moorthy et al., 2015; Androutsopoulos et al., 2009). It has also recently been
published that hyperactive CYP1A1 effects intestinal homeostasis in mice which leads to
colon cancer (Schiering et al., 2017).
The CYP1B1 isoform, also of the CYP1 sub-family, mainly mediates hydroxylation of
endobiotics, chemicals endogenous to the human body, such as estradiol and fatty acids.
CYP1B1 is unusual amongst CYP proteins. It is the only CYP which is not present in normal
healthy tissues but is significantly overexpressed in most hormonal cancers that are linked to
the ovary, prostate, uterus, mammary glands, pituitary. Overexpression of CYP1B1 is
unrelated to the genetic origin of these cancers (McFadyen et al., 1999; Murray et al., 1997;
Muskhelishvili et al., 2001; Gajjar et al., 2012; Sutter et al., 1994; Carnell et al., 2004). It has
been shown that in cancer and precancer tissues of the mammary, prostate and ovary,
CYP1B1 mediates regio-specific conversion of estradiol to 4-hydroxy estradiol (4-OHE2)
while CYP1A1 catalyzes the formation of 2-hydroxy estradiol (2-OHE2). Only the 4-OHE2
metabolite, not 2-OHE2, induces oncogenesis (Yager, 2000). In cancer cells, 4-OHE2 is
further oxidized to 2,3-estradiol quinone and 3,4-estradiol quinone, which are initiators of
tumor formation. 2,3-Estradiol quinone forms a stable adduct with DNA, whereas binding of
3
,4-estradiol quinone to DNA results in its depurination that leads to genotoxic mutations
Cavalieri et al., 2002; Cavalieri et al., 1997; Hayes et al., 1996; Rogan et al., 2003; Zhu &
Conney, 1998; Rochat et al., 2001; Newbold & Liehr, 2000; Cavalieri & Rogan, 2004; Liehr
Ricci, 1996). Overexpression of CYP1B1 in the endometrium, mammary and ovarian
(
&
tissues is now considered as a biomarker for hormonal oncogenesis (Liehr & Ricci, 1996;
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Spink et al., 1998; Tait et al., 1990). CYP1B1 overexpression also leads to the inactivation of
(i.e. resistance to) various cancer drugs such as trans-tamoxifen (Crewe et al., 2002),
docetaxel (Chang et al., 2015), cisplatin and nucleoside analogues (Martinez et al., 2008) in
diverse cancer cell types. Hence, it has been proposed that CYP1B1-specifc inhibitors should
be able to reverse resistance to such widely used anticancer agents.
In previous studies (Horley et al., 2017; Mohd Siddique et al., 2016; Mohd Siddique et al.,
2
017; Joshi et al., 2017), we have successfully identified new structural classes of potent
CYP1A1 and CYP1B1 inhibitors using Sacchrosomes™ and live human HEK293 cells.
However, we have never attempted a rigorous head-to-head comparison of the results
obtained from the two dissimilar assay systems although there have been hints that there may
be some differences between a compound’s ability to inhibit CYP1A1/CYP1B1 in
Sacchrosomes-based microsomal assays and live human cells. Here, we show clearly that the
sole use of microsomal CYP enzymes, Sacchrosomes™, Supersomes™ and/or Bactosomes™
would not have identified molecules that are potent CYP1A1 and CYP1B1 inhibitors.
Instead, live ‘suspension’ human kidney cells, which express CYP enzymes intracellularly,
were essential for the revelation of compounds with possible therapeutic potential.
We have earlier reported biphenyl urea and quinazolinone derivatives as highly selective
and potent CYP1B1 inhibitors (Mohd Siddique et al., 2016; Mohd Siddique et al., 2017). In
continuation of these efforts, we have now further explored the quinazoline scaffold to
discover potent inhibitors of both CYP1A1 and CYP1B1, using the live human cell system,
in parallel with the use of microsomal enzymes. We show that presence of bulkier side-chains
on meta and para-positions of the aryl ring at position 2 of quinazolinones establish favorable
H-bonding and hydrophobic interactions with amino acid residues within the active sites of
these enzymes, while keeping the quinazolinone ring close to heme. An additional –NH-CO-
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group, incorporated in the side-chain, improves polar interactions that may, in turn, improve
the potency of this class of drugs. Furthermore, we have profiled the CYP1A1 and CYP1B1
inhibitors, identified in the human cell system, as true inhibitors of the two enzymes. The
CYP1A1 inhibitors rescue CYP1A1-overexpressing normal fibroblast cells from B[a]P-
mediated toxicity while the CYP1B1 inhibitors overcome cisplatin resistance in multiple
cancer cells that overproduce CYP1B1.
RESULTS AND DISCUSSION
Development of CYP-expressing HEK293 Cellular Assays for Screening of CYP
Inhibitors and Drug Metabolism Studies
Adaptation of HEK293 Monolayer Adherent Cells for Growth as ‘Suspension’
Cultures. In the early stages of drug discovery, recombinant proteins expressed transiently in
mammalian cells are considered to be an important alternative to stable gene expression since
chromosomal integration of DNA is time-consuming and costly (Daramola et al., 2013;
Meissner et al., 2001). Transient expression of recombinant proteins in several mammalian
cell lines has been demonstrated successfully (Flied et al., 2015; Han et al., 2007). In this
study, attempts have been made to express major CYP enzymes transiently in human kidney
HEK293 cells, grown in ‘suspension’ (i.e. HEK293S cells), so that metabolically-active,
whole live cells could be used to demonstrate their application in screening of chemical
libraries.
Until recently, CYP assays have never been performed in live recombinant human
‘suspension’ cells that overexpress specific human CYP enzymes of interest. In the past, CYP
enzyme expression has been attempted in adherent cell cultures. Upon harvesting the
cultures, the metabolically-inactive cells yielded quite poor activity with fluorogenic
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substrates (Donato et al., 2004). We have now adapted ‘adherent’ HEK293 cells (Figure 1A)
for growth in ‘suspension’. The cells in ‘suspension’ have been re-named as HEK293S cells
(Figure 1B). Around 30% transfection efficiency (Figures 1C to 1E) was seen when
HEK293S cells were co-transfected with two separate plasmids, one encoding the human
CYP1A1 gene and the other encoding the gene for the enhanced green fluorescent protein
(eGFP).
Establishment of the Live HEK293S Cell CYP-expression System for Screening of
CYP Inhibitors. HEK293S cells, suspended in 100 mL culture medium in polycarbonate
shake flasks, were transfected with expression plasmids containing either the human
CYP1A1 or the human CYP1B1 genes (Supplementary Information, SI; Figure S1 and S2).
The expression of CYP1A1 and CYP1B1 proteins, in HEK293S cells, was confirmed by
Western blotting (Figures 2A and 3A, respectively). The activities of the CYP1A1 and
CYP1B1 enzymes were assessed (Figures 2B and 3B, respectively). The kinetics of CYP1A1
and CYP1B1-mediated reactions, with the fluorogenic compound 7-ethoxyresorufin (7-ER)
as substrate, were found to be linear until 7-ER became limiting. The activities of CYP1A1
and CYP1B1 enzymes, in live cells, were monitored up to 120 h post-transfection (Figures
2
C and 3C, respectively). It should be noted that CYP enzyme activity in microsomal enzyme
assays and in hepatocytes become immeasurably small within 6 h of incubation at 37 ºC
Basu & Shaik, 2016; Donato et al., 2004). Reliability of CYP1A1/CYP1B1 enzyme
(
activities, produced within recombinant HEK293S cells, was corroborated by testing for their
inhibition by the FDA-recommended CYP1A1/CYP1B1 inhibitor, α-naphthoflavone (ANF;
Figures 2D and 3D, respectively).
The CYP enzymes, 1A2, 2C9, 2C19, 2D6 and 3A4, which metabolize >80% of approved
pharmaceutical drugs, were expressed similarly to CYP1A1/CYP1B1, in HEK293S cells.
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The expression of the individual proteins from different plasmids (Supporting Information,
Figures S3 to S7), the kinetics of their enzyme activities, their activities over 120 h of culture
and their ability to be inhibited by FDA-recommended inhibitors were also studied
(Supporting Information, Figures S8 to S12; and Table 1).
The IC50 values obtained in live HEK293S cells (Table 1), grown in ‘suspension’, were
found to be in good agreement with the data reported in the literature for hepatocytes which
normally are also grown in ‘suspension’. This would indicate that the active site 3D geometry
of recombinant enzymes within live HEK293S cells and hepatocytes are similar. However,
our IC50 data obtained in HEK293S cells and that published in hepatocytes have some
obvious inconsistencies with the data that have been described in the literature for human
liver microsomes (HLMs) and recombinant microsomes (see Table 1).
Screening for CYP1 Inhibitors in a Chemical Library of Quinazolinones using CYP1-
expressing HEK293 Cells and Microsomal Enzymes, followed by SAR Studies
Chemistry. Previously, we had described (Mohd Siddique et al., 2017) a simple and
efficient route to constructing quinazoline-4-(3H)-ones (depicted in Scheme 1) via
condensation of 2-amino benzamide with an aldehyde using DMF as a solvent. Sodium
acetate was used as a base while in situ oxidation was performed by iodine. The rare
substituted aldehydes were prepared separately, as depicted in Scheme 1. Various substituted
anilines were treated with chloroacetyl chloride to prepare acetamide 1. The reaction was
carried out in glacial acetic acid at room temperature. The resultant acetamide was then
reacted with 4-hydroxy benzaldehyde (2a), vanillin (2b) or isovanillin (2c) in presence of
potassium carbonate and potassium iodide using acetone as solvent under reflux conditions.
The prepared aldehydes 3, 6 & 8 were condensed with 2-amino benzamide (4) in DMF at 60-
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7
0 °C, to synthesize the quinazolinones 5a-i, 7a-g, 9a-k. The compounds were purified
simply via washing with ethyl acetate and petroleum ether in 40:60 ratio.
TM
TM
Screening for CYP1B1 and CYP1A1 Inhibition in Sacchrosomes , Bactosomes ,
SupersomesTM and Live HEK293S Cells Expressing CYP1A1 or CYP1B1 Enzymes. At
first, all the 27 quinazolinones in the chemical library, that had been synthesized, were
evaluated for inhibition of CYP1A1 and CYP1B1 only in microsomal enzyme assays using
(
i) SacchrosomesTM (ER membrane-bound CYP1A1 or CYP1B1 enzyme, isolated from
recombinant baker’s yeast cells), (ii) SupersomesTM (ER membrane-bound CYP1A1 or
CYP1B1 enzyme isolated from recombinant insect cells), and (iii) BactosomesTM
(periplasmic membrane-bound CYP1A1 or CYP1B1 enzyme isolated from recombinant
bacterial cells). Later, live human HEK293 ‘suspension’ cells (i.e. HEK293S cells), which
contained plasmids bearing the human CYP1A1 gene or the human CYP1B1 gene, were used
for assessing enzyme inhibition by the compounds.
The results obtained from microsomal and live HEK293S-cell-based assays are shown in
Tables 2-4. Some of the compounds displayed IC50 values at low nanomolar concentrations,
in live HEK293S cells. Surprisingly, these IC50 values were at variance to the values obtained
TM
TM
and BactosomesTM.
in the microsomal assays using Sacchrosomes , Supersomes
Moreover, compounds which had minimal activity at 10 µM concentrations in the initial
microsomal assays were found to be quite active in the inhibition of CYP1A1 or CYP1B1
enzymes expressed within live HEK293S cells.
For example, compound 9i showed no measurable CYP1A1 or CYP1B1 inhibitory
capacity, at 10 µM concentration, in microsomal enzyme assays whereas it inhibited the
CYP1A1 enzyme, expressed within HEK293S cells, with an IC50 of 30 nM (Table 4).
Compound 9i was the most potent CYP1A1 inhibitor that was identified in HEK293S cells
1
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and it had >250-fold specificity with respect to CYP1B1, where the IC50 for inhibition was
7
.8 µM in HEK293S cells (Table 4). Similarly, compound 7g was identified as a potent
CYP1A1 inhibitor only in HEK293S cells (IC50, 60 nM; Table 3); in CYP1A1 microsomal
assays, IC50 values ranged from 4.7 to 5.7 µM. Compound 7g was also specific to CYPA1
vis-à-vis CYP1B1 (>75-fold specificity); the CYP1B1 IC50 value in HEK293S cells was 4.6
µM (6.5-7.8 µM in microsomal assays; Table 3).
Parallel experiments with CYP1B1 enzyme showed that compound 9b had the best
potency in inhibiting CYP1B1 expressed within HEK293S cells; the IC50 value was only 21
nM which was much lower than the IC50 values of 1.3-1.7 µM obtained using isolated
TM
TM
TM
CYP1B1-bound microsomes (Sacchrosomes , Bactosomes and Supersomes ; Table 4).
Likewise, compound 5e inhibited CYP1B1 enzyme, expressed within live HEK293S cells,
with an IC50 value of 42 nM whereas, using CYP1B1 microsomal enzymes, the IC50 values
ranged from 4.1 to 5.1 µM (Table 2). However, compound 5e was found to be similar in its
inhibition of CYP1A1 and CYP1B1 in microsomal assays (CYP1A1/CYP1B1 IC50 values
ranged from 4.1 to 6.9 µM). Both compounds 5e and 9b were specific (i.e. >100-fold
specificity) to CYP1B1 when their IC50 values were compared with inhibition of CYP1A1.
CYP1A1 IC50 value, in HEK293S cells, was >20 µM (compared to 6.1-6.9 µM in
microsomes) for compound 5e, and 2.3 µM (compared to >20 µM in microsomes) for
compound 9b (Tables 2 and 4).
It was also noticed that there was no clear difference in CYP1B1 inhibition by compound
7
c when tested in HEK293S cells and in microsomes (Table 3). In contrast, compound 7b
having the -OMe group in position 2 rather than in position 3 of the aromatic ring ‘C’ as in
c) was specific to CYP1B1 (IC50, 32 nM, with >180-fold specificity with respect to
inhibition of CYP1A1 in the HE293S-cell-assays).
(
7
1
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In summary, there appears to be a close correlation between enzyme inhibition in
microsomal assays and within live cells in the synthesized quinazolinones 5a to 5d, 5f, 5i, 7a,
7
c to 7f, 9d to 9h, 9j and 9k (a total of 18 compounds). However, there was a clear difference
in the inhibition of CYP1A1 and CYP1B1 enzymes, within HEK293S cells and those that are
bound to microsomal membranes, by the rest of the 9 compounds in the quinazolinone
library. Comparison of the results obtained in the HEK293S-cell-assays and the microsomal
enzyme assays (in Tables 2-4) undoubtedly suggest that one would have missed the potent
inhibitors of CYP1A1/CYP1B1 enzymes if one had relied merely on the initial screening of
compounds with the microsomal CYP1A1/CYP1B1 enzymes.
The discrepancy between IC50 values determined using whole HEK293 cells and
microsomes for some of the compounds in the chemical library may not be because of
problems of outer cell wall permeabilization. We obtain very low IC50 values with CYP-
expressing whole cells compared to the values obtained with enzymes bound to endoplasmic
reticular (ER) (i.e. microsomal) membranes, the ER membranes being far more permeable
than the outer cell wall. We think that, in whole cells, the specific proteins existing in the ER,
and perhaps the vesicular proteins in the ER to Golgi secretory pathway (i.e. ER to Golgi
transporters) may have a role to play on enzyme activity of CYPs bound to microsomal
membranes. In the presence of tunicamycin (that inhibits glycosylation of ER proteins) and
brefeldin (which blocks ER to Golgi traffic), IC50 values of the compounds that are low using
CYP-expressing HEK293 cells increase perceptibly, resembling those obtained with
microsomal enzymes (unpublished observations). That is why we think that the CYP-
expressing HEK293 cells are more akin to hepatocytes rather than human liver microsomes
(HLMs) which only contain ER proteins but are devoid of Golgi membrane proteins and also
vesicular proteins (unpublished observations).
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The observations with recombinant HEK293S cells would indicate that there exists a key
SAR feature of the quinazolinones for inhibition of CYP1A1 and CYP1B1. As substitution at
R1 position in all the three series are different, the structure-activity relationship write-up
considered only the compounds with similar substitutions among the three series. In
th
compound series 5a-i having oxy-acetamide substitution at 4 position of ring C, Compounds
5
g & 5h having naphthyl substitution and compounds 5d & 5e having phenyl ring with
electron-withdrawing substitutions were found to be active and selective towards CYP1B1
Table 2). Introduction of methoxy group ortho to oxy-acetamide group on ring C, reverses
the selectivity and increase the potency of compound 7g, in comparison with 5g (Table 3 &
). Compounds having electron-donating (7b-7e) as well as electron-withdrawing (7f) groups
(
2
are now active and the one having ortho-methoxy (7b) exhibited potent and selective
inhibition of CYP1B1 with IC50 of 0.032 µM (Table 3). Reversing the position of oxy-
acetamide and methoxy groups provided 9a-k series. This series of compounds provided the
most potent and selective inhibitor of both CYP1A1 and 1B1 isoforms. Compound 9b with
ortho-methoxy substitution displayed improved potency in comparison with 7b, while 9i with
para-fluro exhibited reversal of selectivity with improved potency in comparison with 7f
(Table 4 & 3). In summary, oxy-acetamide at meta-position of ring C having methoxy
substitution ortho to it provided favorable activity profile (9a-k series). Comparison of
compounds carrying substitution on the phenyl ring of oxy-acetamide side chain (9b-j) with
its unsubstituted counterpart (9a) revealed (i) electron-donating group (methoxy) at ortho-
position is favorable for potent and selective inhibition of CYP1B1 and (ii) electron-
withdrawing group (fluro) at para-position is favorable for potent and selective inhibition of
CYP1A1.
Molecular Modelling. CYP enzymes are part of a biological hydroxylation machinery
responsible for Phase I hydroxylation reactions observed in drug metabolism. They confer
1
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hydroxylation of substrate through sequential events involving electron transfer. The
inhibitors designed were supposed to displace the water molecule in CYP enzymes and
disrupt the formation of heme-oxo intermediate between the substrate, heme and CYP
enzyme. Molecular modelling studies at the substrate binding site indicate that both rings of
quinazoline-2-one nucleus display π-π interactions with the Phe 231 residue in F-helix. This
is one of the crucial hydrophobic interactions required for inhibition of CYP1 family
isoforms (Wang et al., 2011; Walsh et al., 2013). The higher potency of 9b over 7b for
CYP1B1 inhibition could be justified due to the presence of the additional π-π interactions
with the Phe-134 residue. Besides this, the chromene nucleus of ANF display strong H-
bonding with the anionic charged Asp 333 residue (distance is 2.76 Å) in I helix. In case of
7
b and 9b, we were able to mimic these interactions through C-2 carbonyl group with the
distance of < 3 Å. The OMe group of the terminal ring was present in close proximity to
heme and the linker chain interacts by favorable van der Waal’s interactions with the residue
lining the binding cavity. These residues include Asp 326, Val 395, Asp 333, Thr 510, Asn
2
28 and Phe 268 (Figure 4A). The phenyl ring attached linker -OCH CONH- offers
2
flexibility in the molecule and thus terminal 2-methoxyphenyl ring can adopt multiple ring
conformations at amide linkages in 7b and 9b. This flexibility of 2-methoxy phenyl ring in
7
b lead to folding of ring conformation in such a way that the 7b linker obtains direct access
to the deep pocket where NH group is in close vicinity to the heme metal atom. However,
surprisingly in case of 9b (with isovanillin attached linker), the amide bond flexibility
allowed the 2-methoxy group of terminal phenyl ring to interact with the heme atom (distance
is 2.33 Å) as shown in Figure 4B. The close and open conformations of the 2-methoxy phenyl
ring at amide linkage are shown in overlaid images of 7b and 9b in substrate binding site of
CYP1B1 (Figure 4C).
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4

ACCEPTED MANUSCRIPT
Specificity against Various CYP Isoforms. The most potent CYP1B1 inhibitors 7b and
b along with few more inhibitors, identified in the human live cell assays using recombinant
9
HEK293S cells, were screened for selectivity against CYPs 1A2, 2C9, 2C19, 2D6 and 3A4.
The results, summarized in Table 5, indicate that none of the compounds show significant
inhibitory activity against CYP 2 and 3 enzymes. Importantly, the compounds 7b, and 9b had
IC50 values greater than 10 µM for inhibition of the CYP2 and CYP3 enzymes. This indicates
that both pairs of compounds are highly selective towards CYP1A1 and CYP1B1. The
rationale for higher selectivity of 7b and 9b for CYP1B1 was also studied by aligning the
binding site of CYP2 and CYP3 family isoforms to CYP1B1. The CYP2 and CYP3 family
enzymes such as 2D6 and 3A4 have large open binding site which is highly flexible so that it
could accommodate a wide range of substrates. Most of the CYP2D6 and CYP3A4 inhibitors
act by ionic charge-charge interactions, which are missing in the case of compounds 7b, and
9
b, as shown in Figures S13 and S14 of Supporting Information.
Validation of the Best CYP1A1/CYP1B1 Inhibitors Identified in CYP1-expressing HEK293
Cells, via Cell Cycle Analyses
Rescue from Benzo[a]pyrene’s Toxicity in CYP1A1-expressing Human Lung
Fibroblast WI-38 Cells, by CYP1A1 Inhibitors. Transformed cancer cell lines have been
used to study cellular responses to carcinogens (Calabrese, 2005). However, these cells do
not reflect the physiological status of normal tissues. Hence, CYP1A1-overexpressing human
lung normal fibroblast cells, WI-38, have been exposed to the pro-carcinogen
benzo[a]pyrene, B[a]P, to address the question whether these cells would metabolize B[a]P
to a carcinogenic product (e.g. benzo[a]pyrene diol epoxide; BPDE) that would form an
adduct with DNA which would block cell growth. As shown in Table 6, the EC50 of B[a]P
1
5

ACCEPTED MANUSCRIPT
was significantly lowered (EC50 = 0.8 µM) in the CYP1A1-expressing cell line, WI-
3
8::pcDNA3.1/CYP1A1 (i.e. WI-38 cells stably transfected with the plasmid
pcDNA3.1/CYP1A1), compared to WI-38 cells transfected with the empty plasmid,
pcDNA3.1 (EC50 = 17.2 µM). This would indicate that B[a]P manifests toxicity only in cells
which express CYP1A1. After that, the CYP1A1-overexpressing WI-38 cells were first pre-
treated with the CYP1A1 inhibitors, identified in HEK293S recombinant cell assay, and then
the B[a]P induced toxicity was measured after addition of B[a]P to the cells. As depicted in
Table 7, the potent CYP1A1 inhibitor 9i was able to reverse completely B[a]P’s toxicity in
WI-38::pcDNA3.1/CYP1A1 cells (the EC50 value of B[a]P was back to 17.0 µM). Similarly,
the other CYP1A1 inhibitors 7g and 9c were also able to reverse B[a]P’s toxicity. With the
compounds that were less active in their inhibition of CYP1A1 (e.g. compounds 7c and 7e),
in the HEK293S-cell-assay, less reversal in the toxicity of B[a]P was observed.
The effect of CYP1A1 inhibitors on the cell cycle, in the presence of B[a]P, was
investigated by first pre-treating CYP1A1-expressing WI-38 cells with the most potent
CYP1A1 inhibitors, compounds 7g or 9i, and then exposing the cells to B[a]P. CYP1A1
metabolizes B[a]P to its diol-epoxide (BPDE) and corresponding o-quinone which intercalate
DNA and thereby should block cells primarily at the S phase of the cell division cycle. It has
been suggested that formation of this DNA adduct causes mutations in DNA that ultimately
leads to the onset of cancer (Moorthy et al., 2015; Androutsopoulos et al., 2009).
Upon exposure of CYP1A1-expressing WI-38 normal fibroblast cells (Figure 5A) to an
EC50 concentration of B[a]P for 3 h and then releasing cells into fresh medium for 57 h, the
cells did face a block at the S phase of the cell cycle (Figure 5B). This would be expected if
the CYP1A1 protein (Figure 5E), expressed within WI-38 cells, were to be a functional
enzyme. However, when cells were at first pre-incubated for 20 h with a CYP1A1-specific
1
6

ACCEPTED MANUSCRIPT
inhibitor, for example, compounds 7g or 9i at their IC50 concentrations (0.06 µM and 0.03
µM, respectively), and then treated with an EC50 concentration of B[a]P for 3 h, followed by
release into fresh medium, the cells did not anymore undergo an S phase block (Figures 5C
and 5D). In fact, the cells re-entered the cell cycle as if the cells had not undergone any
treatment with B[a]P. This implied that the CYP1A1-mediated metabolism of B[a]P must
have been prevented by compounds 7g and 9i, demonstrating that, in the cellular context,
they were indeed true inhibitors of the CYP1A1 enzyme.
In summary, we have identified two potent inhibitors of CYP1A1 (compounds 7g and 9i),
using recombinant HEK293S cells, which have IC50 values of 60 nM and 30 nM,
respectively. This would not have been possible if we had relied only on recombinant
microsomal CYP1A1 enzymes.
Reversal of Cisplatin Resistance by CYP1B1 Inhibitors. Since it has been reported that
inactivating CYP1B1 polymorphisms modify the risk towards the onset of prostate (Gajjar et
al., 2012; Li et al., 2015), lung (Lao et al., 2014) pancreatic (Vrana et al., 2010) and colon
cancers (Mei et al., 2012), stable CYP1B1-expressing human cancer cells PC-3/prostate (p53-
null), COR-L23/lung (p53-mutant), MIAPaCa-2/pancreatic (p53-mutant), LS174T/colon
(p53-positive but Bax-null) cells were constructed to investigate if the CYP1B1 inhibitors,
identified in the HEK293S-cell-assay, were able to overcome cisplatin resistance in these
cells (Mohd Siddique et al., 2016; Mohd Siddique et al., 2017). The four cell lines
overexpressing CYP1B1 protein were constructed via stable transfection of the plasmid
pcDNA3.1/CYP1B1 (Mohd Siddique et al., 2016; Mohd Siddique et al., 2017). The EC50
values of cisplatin, in each cell line, were determined (Table 8). The cisplatin EC50 values
increased dramatically, from 3-4.5 µM to 50-90 µM, when CYP1B1 was stably expressed in
these four-different cancer cell types (Figure 6). When the CYP1B1-overexpressing stable
1
7

ACCEPTED MANUSCRIPT
cells were pre-treated for 24 h with one of the CYP1B1 inhibitors, identified in the
HEK293S-cell-assay, the resistance to cisplatin was overcome (Table 9). For example, for
compound 9b which is a potent inhibitor of CYP1B1 enzyme, the EC50 value of cisplatin
reversed back to 2.7 µM from 50 µM in PC-3 cells transfected with CYP1B1 plasmid (Table
9
).
Expression of the CYP1B1 protein in the four cell lines PC-3, COR-L23, MIAPaCa-2 and
LS174T, stably transfected with the plasmid pcDNA3.1/CYP1B1 (Mohd Siddique et al.,
2
016; Mohd Siddique et al., 2017), were confirmed by Western blotting (Figure 6).
The EC50 values of cisplatin in the four CYP1B1 overexpressing cell lines, in the presence
of the most potent CYP1B1 inhibitor, compound 9b, is depicted graphically in Figure 7. Cells
were pre-treated with compound 9b, for 24 h, before treatment with cisplatin for 3 h followed
by release of cells into fresh medium for 33 h. The EC50 graphs for compound 9b, after
testing it in the four cancer cell lines PC-3, COR-L23, MIAPaCa-2 and LS174T, indicated
that there is a dramatic decrease of cisplatin’s EC50 values when cells were pre-incubated
with the CYP1B1-specific inhibitor compound 9b.
A consequence of treating PC-3, COR-L23, MIAPaca-2 or LS174T cells with cisplatin
would be that they would become arrested at the G
987). As expected, MIAPaca-2::pcDNA3.1/CYP1B1 pancreatic cells, stably transfected
with the plasmid encoding the human CYP1B1 gene, indeed did arrest at G /M (Figure 8A).
This G /M block is seen as a protective mechanism that enables cells to pause and repair
2
/M phase of the cell cycle (Eastman,
1
2
2
cisplatin-damaged DNA before crossing the threshold of mitosis (Aguda, 1999).
After this observation, MIAPaCa-2::pcDNA3.1/CYP1B1 pancreatic cells were pre-
incubated (at 2x IC50 concentrations) with the four CYP1B1-specific inhibitors, compounds
1
8

ACCEPTED MANUSCRIPT
5
e, 7b, 7c and 9b, for 24 h, before cells were treated with EC50 concentrations of cisplatin,
which had been determined in the presence of 2x IC50 concentrations of inhibitors (Table 9),
for 3 h. The results clearly show that, after release of CYP1B1-inhibited cells into fresh
medium for 33 h, the cells no more suffered a G
2
/M block but progressed further into the cell
peaks (in
cycle and underwent between 17% to 35% of apoptosis as indicated by the sub-G
1
Figures 8B to 8E). This established that the CYP1B1-expressing cells were being re-
sensitized to cisplatin in the presence of the potent CYP1B1 inhibitors identified in the live
HEK293S-cell-assay.
Compound 9b was the most potent CYP1B1 inhibitor identified in the HEK293S-cell-
assay and had shown the highest levels of apoptosis in the experiment with MIAPaCa-
2
::pcDNA3.1/CYP1B1 cells which were treated first with compound 9b and then with
cisplatin (compare Figures 8B to 8D with Figure 8E). Hence, it was further tested in the three
CYP1B1-expressing human cancer lines that were derived from the prostate (PC-3), lung
(
COR-L23) and colon (LS174T). The results again show that pre-incubation of cells with a
CYP1B1-specific inhibitor, before exposing the recombinant CYP1B1-expressing cells (PC-
-1B1, COR-L23-1B1, LS174T-1B1) to cisplatin (CP), re-sensitizes the cells to cisplatin and
induces a fair amount of apoptosis (i.e. 26-46%) as seen by the percentages of the sub-G
3
1
peaks (in Figures 9A to 9C). Cells were again pre-incubated with the CYP1B1-specific
inhibitor 9b (at 2 x IC50 concentration) for 24h. Like with MIAPaCa-1B1::
pcDNA3.1/CYP1B1 cells, the human CYP1B1 gene-bearing derivatives of the three cell
lines PC-3-1B1, COR-L23-1B1 and LS174T-1B1 had shown mainly a G /M block (results
2
not shown) on exposure to EC50 concentrations of cisplatin alone (i.e. 50, 63 and 90 µM,
respectively; Table 8).
1
9

ACCEPTED MANUSCRIPT
In summary, we have identified potent and selective inhibitors of CYP1B1, compounds
5
e, 7b, 7c and 9b from a small chemical library of 27 compounds. Compound 9b was the
most potent, with an IC50 value of 21 nM, in live human HEK293S cells, which had been
adapted to grow in ‘suspension’. It displays highly selective inhibition of CYP1B1. Its
CYP1B1 inhibitory activity was further validated by investigating its ability to overcome
cisplatin resistance in four different cancer cell lines PC-3, COR-L23, MIAPaCa-2 and
LS174T. The ability of compound 9b to effectively overcome resistance to cisplatin, in four
different cancer cell lines which overexpress CYP1B1, warrants its further investigation in
preclinical studies.
CONCLUSIONS
This study has shown that the human HEK293S-cell-assay system is unique, and that it is
more reliable than the microsomal assays for the identification of CYP1 inhibitors. The
human cell system may find wider use in pre-clinical studies that targets (i) the identification
of the true inhibitors of CYP enzymes, which occur in vivo, with the aim of understanding,
clarifying and predicting possible drug-drug interactions of a potential clinical entity, (ii) the
identification and characterization of metabolites that are formed from CYP-mediated
reactions in vivo, which are essential for embarking on clinical trials and (iii) the compounds
that are difficult, at the moment impossible, to metabolize by hepatocytes which lose all CYP
activity within 6 h of incubation at 37 ºC (Basu & Shaik, 2016).
This novel live cell technology has also the potential to define accurately the metabolic
profiles of a large number of drug candidates, rapidly and reproducibly. It could be developed
further to create tailor-made multiple CYP-expressing systems that are more akin to
hepatocytes. Furthermore, with time and resources, ‘stable’ CYP-expressing HEK293S cells
2
0

ACCEPTED MANUSCRIPT
could be obtained where CYP genes of clinical interest would be integrated into human cells’
chromosomal loci. Currently each transfection, as described, allows the assaying of 250-300
compounds for inhibition of a CYP enzyme.
MATERIALS AND METHODS
General Information. All the chemicals were purchased from Spectro-Chem, Hi-media,
Acros Organics or Sigma Aldrich. Solvents were purchased from Rankem and used as it was
or otherwise specified accordingly. All the reactions were carried out under open air and in
dried flask. Precoated silica gel aluminum plates (Merck) were used for reaction monitoring
and visualized under UV light. Column chromatography was done using Merck silica gel (60-
1
3
1
1
00 mesh). Carbon ( C) and Proton ( H) nuclear magnetic resonance (NMR) spectra were
recorded on JEOL 400 MHz or Bruker 300 MHz instruments using dimethyl sulfoxide
1
3
(
DMSO-D6) solvent and tetramethylsilane as an internal standard ( C NMR DMSO: δ 39.5,
1
TMS: δ 0.00 ppm and H NMR: DMSO δ 2.5 ppm).
Procedure for Synthesis of Intermediates 3, 6 and 8: Various substituted anilines were
dissolved in glacial acetic acid at 0 °C, to this was added over 30 min choloroacetyl chloride
(2 equivalents). The reaction mixture was brought to room temperature and stirred overnight.
Saturated sodium bicarbonate solution was added till complete neutralization. The resulting
precipitate was filtered off and washed with n-hexane and dried. The resultant product was
used further without any purification. Either of 4-hydroxy benzaldehyde (2a), vanillin (2b) or
isovanillin (2c) (1 equivalent) was dissolved in acetone and potassium carbonate (2
equivalents) was added. Then corresponding substituted acetamide (1) was mixed to the
stirring solution. Finally, potassium iodide (1.5 equivalent) was added. The reaction mixture
was refluxed for 8-10 hrs. After completion of reaction, the resultant mixture was
2
1

ACCEPTED MANUSCRIPT
concentrated and treated with water and extracted with ethyl acetate (3 x 20 ml). The organic
layers were combined and treated with brine and dried over sodium sulfate and concentrated.
The crude mixture was purified over silica gel (60-120) using petroleum ether: ethyl acetate
(9:1).
Procedure for the Synthesis of 5a-i, 7a-g and 9a-k. To the stirring solution of 2-
aminobenzamide (4.1 equivalents) in dimethylformamide, sodium acetate (2 equivalents) was
added. After stirring for 10 minutes the rare substituted benzaldehydes (3, 6 and 8, 1
equivalent) and iodine (2 equivalents) were added. The reaction mixture was heated at 70-
8
0°C for 20-24 hrs. Then reaction mixture was poured on to crushed. The resulting mixture
was treated with sodium thiosulphate (10% w/v in water) to reduce the remaining iodine. The
precipitate was filtered off and washed with n-Hexane: Ethyl acetate (50:50).
N-cyclohexyl-2-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy) acetamide (5a):
1
White powder, 1.214g (yield 87.5%); M.P.: 264-267°C; H-NMR (400 MHz, DMSO-d
6
) δ
1
.20 (d, J = 10.4 Hz, 4H, CH
2
) 1.52 (d, J = 11.5 Hz, 1H, CH), 1.64-1.69 (m, 4H, CH
2
), 2.46
1
3
(
s, 3H, CH
2
), 4.52 (s, 2H, OCH
2
), 8.11-7.04 (m, 9H, ArH), 12.36 (s, 1H, CO-NH); C-NMR
(101 MHz, DMSO-d
6
) δ 25.48, 25.68, 32.79, 48.13, 67.45, 115.16, 116.44, 121.25, 126.37,
1
27.53, 129.98, 131.10, 134.90, 149.49, 160.83, 166.66. HRMS (ESI): m/z [M+H]+
calculated 378.1818, found 378.1819
2
-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)-N-phenylacetamide (5b): White
powder, 1.281g (yield 93.98%); M.P.: 252-253°C; Mass (M+1) Calculated 372.13, Found
1
3
1
1
1
72.2; H NMR (400 MHz, DMSO-d6) δ 4.81 (s, 2H, OCH
2
), 8.18-7.07(m, 13H, Ar-H),
) δ 67.62,
1
3
0.15 (s, 1H, CO-NH), 12.43 (s, 1H, CO-NH). C-NMR (101 MHz, DMSO-D
6
14.92, 117.64, 120.20, 124.21, 126.37, 128.80, 129.27, 129.98, 133.78, 134.62, 135.12,
38.91, 148.54, 152.35, 158.52, 160.94, 164.23, 166.97
2
2

ACCEPTED MANUSCRIPT
2
-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)-N-p-tolylacetamide (5c): White
powder, 1.401 g (Yield 98%); M.P.: 248-250°C; Mass (M+1) Calculated 386.15, Found
1
3
7
86.1; H NMR (400 MHz, DMSO-d
6
) δ 2.24 (s, 3H, Ar-CH
3
), 4.79 (s, 2H, OCH ), 8.18-
2
1
3
.13(m, 12H, Ar-H), 10.08 (s, 1H, CO-NH), 12.42 (brs., 1H, CO-NH); C-NMR (101 MHz,
DMSO-D6) δ 20.89, 67.52, 115.14, 120.15, 125.87, 126.27, 127.74, 129.57, 129.57, 129.87,
1
3
33.16, 135.06, 136.24, 149.33, 162.72, 166.34; HRMS (ESI): m/z [M+H]+ calculated
86.1505, found 386.1501
N-(3-chlorophenyl)-2-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)
acetamide
(5d): White powder, 1.317g (yield 92.13%); M.P.: 252-254°C; Mass (M+1) Calculated
1
4
1
04.1, Found 404.2; H NMR (400 MHz, DMSO-d
6
) δ 4.83 (s, 2H, OCH ), 8.18-7.14 (m,
2
1
3
2H, Ar-H), 10.35 (s, 1H, CO-NH),12.43 (br. s, 1H, CO-NH); C-NMR (101 MHz, DMSO-
D6) δ 67.47, 112.71, 115.19, 115.29, 118.55, 119.63, 121.17, 123.95, 126.02, 126.30, 126.69,
1
1
27.77, 127.85, 129.93, 130.96, 133.54, 135.04, 140.25, 149.35, 152.27, 160.79, 162.77,
+
67.09. HRMS (ESI): m/z [M+H] calculated 406.0958, found 406.0951
N-(4-nitrophenyl)-2-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy) acetamide (5e):
Yellowish orange powder, 0.674g (yield 87.96%); M.P.: 151-155°C; Mass (M+1) Calculated
1
4
1
6
17.11, Found 417.2; H NMR (400 MHz, DMSO-d
6
) δ 4.95 (s, 2H, OCH
2
), 8.18-7.17 (m,
) δ
1
3
2H, Ar-H), 9.86 (s, 1H, CO-NH), 10.91 (s, 1H, CO-NH); C-NMR (101 MHz, DMSO-d
6
7.44, 115.69, 119.35, 125.73, 130.56, 132.55, 143.29, 144.82, 163.23, 167.39, 191.95.
+
HRMS (ESI): m/z [M+H] calculated 417.1199, found 417.1198.
N-(4-ethoxyphenyl)-2-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)
acetamide
(5f): White powder, 0.707g (yield 92.66%); M.P.: 102-104°C; Mass (M+1) Calculated
1
4
14.15, Found 414.3; H NMR (400 MHz, DMSO-d
6
) δ 1.29 (t, J = 6.85 Hz, 3H, CH
3
), 3.96
(
q, J = 6.85 Hz, 2H, OCH
2
), 4.79 (s, 2H, OCH ), 7.88-6.86 (m, 12H, Ar-H), 9.86 (s, 1H, CO-
2
2
3

ACCEPTED MANUSCRIPT
1
3
NH), 10.01 (s, 1H, CO-NH); C-NMR (101 MHz, DMSO-d
6
) δ 14.7, 63.11, 114.40, 115.22,
1
21.34, 130.11, 131.28, 131.78, 162.80, 165.38, 191.38. HRMS (ESI): m/z [M+H]+
calculated 416.161, found 416.1605.
N-(naphthalen-2-yl)-2-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)
acetamide
(5g): Light brown powder, 1.22g (yield 83.5%); M.P.: 190-192 °C; Mass (M+1) Calculated
1
4
1
22.15, Found 422.2; H NMR (400 MHz, DMSO-d
6
) δ 4.89 (s, 2H, OCH ), 8.26 - 7.06 (m,
2
5H, Ar-H), 10.37 (s, 1H, CO-NH), 12.44 (br. s., 1H, CO-NH); 13C-NMR (101 MHz,
) δ 67.62, 115.26, 116.42, 120.75, 121.24, 125.38, 126.00, 126.37, 126.76, 127.04,
27.89, 128.97, 129.99, 130.48, 133.84, 135.13, 136.47, 149.43, 152.46, 160.82, 162.83,
DMSO-d
6
1
1
+
66.69. HRMS (ESI): m/z [M+H] calculated 422.1505, found 422.1499
N-(naphthalen-1-yl)-2-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)
acetamide
(5h): Cream powder, 0.913g (yield 59%); M.P.: 199-201°C; Mass (M+1) Calculated 422.15,
1
Found 422.2; H NMR (400 MHz, DMSO- d
6
) δ 5.13 (s, 2H, OCH
2
), 7.25 -8.00 (m, 13H, Ar-
) δ 67.62,
15.79, 122.92, 123.32, 126.09, 126.50, 126.67, 128.66, 130.65, 132.32, 133.27, 134.23,
63.34, 167.33, 191.95.
1
3
H), 9.89 (s, 1H, CO-NH), 10.23 (s, 1H, CO-NH); C-NMR (101 MHz, DMSO- d
6
1
1
2
-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)-N-phenethyl
acetamide
(5i):
White powder, 1.319g (yield 91%); M.P.: 252-253°C; Mass (M+1) Calculated 400.16,
1
Found 400.2; H-NMR (400 MHz, DMSO- d
6
) δ 2.46 (q, J = 1.8 Hz, 2H, CH
), 7.04-8.21 (m, 13H, Ar-H), 12.40 (s, 1H, CO-NH);
) δ 35.62, 40.90, 67.47, 115.26, 121.21, 125.93, 126.38,
2
), 2.72 (t, J =
7
.4 Hz, 2H, CH
2
), 4.54 (s, 2H, OCH
2
13C-NMR (101 MHz, DMSO-d
6
1
26.67, 127.72, 128.89, 129.17, 129.97, 135.13, 139.79, 152.40, 160.81, 167.71. HRMS
+
(
ESI): m/z [M+H] calculated 400. 1661, found 400.1655
2
4

ACCEPTED MANUSCRIPT
(N-butyl-2-(2-methoxy-4-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy) acetamide)
(7a): Yellowish powder, 0.607g (yield 88.5%); M.P.: 185-186°C; (M+1) Calculated 381.17,
1
Found 382.1; H NMR (400 MHz, DMSO- d
6
) δ 0.85 (3 H, t, J= 7.24 Hz, CH
3
), 1.25 (2 H, m,
), 4.55 (2 H, s,
CH
2
), 1.39 (2 H, m, CH
2
), 3.11 (2 H, q, J=6.58 Hz, CH ), 3.89 (3 H, s, OCH
2
3
OCH
2
), 7.01-8.07 (m, 7H, Ar-H), 8.38 (1 H, s, CO-NH), 12.57 (1 H, br. s, CO-NH). HRMS
+
(
ESI): m/z [M+H] calculated 382.1767, found 382.1759
2
-(2-methoxy-4-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)-N-(2- methoxy phenyl)
acetamide (7b). White powder, 0.268g (yield 84.54%); (M+1) Calculated 532.16, Found
1
4
3
32.08; M.P.: 253-255°C; H NMR (400 MHz, DMSO-D6) δ 3.864 (s, 3H, OCH
3
), 3.93 (s,
H, OCH
3
), 4.803 (s, 2H, OCH
2
), 6.883-8.353 (m, 11H, Ar-H) 9.286 (s, 1H, CO-NH), 12.585
1
3
(
s, 1H, CO-NH); C-NMR (101 MHz, DMSO-D6) δ 56.48, 68.36, 111.55, 113.83, 120.39,
1
21.05, 121.71, 124.96, 126.38, 127.10, 134.53, 143.42, 149.13, 149.23, 166.40. HRMS
+
(
ESI): m/z [M+H] calculated 432.1559, found 432.1559
2
-(2-methoxy-4-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)-N-(3-methoxyphenyl)
1
acetamide (7c): White powder, 0.263g (yield 82.96%); H NMR (400 MHz, DMSO- d
6
) δ
), 6.85-8.34 (m, 11H, Ar-H), 9.994
s, 1H, -CO-NH), 12.40 (s, 1H, -CO-NH); C-NMR (101 MHz, DMSO- d ) δ 55.80, 56.38,
8.82, 112.37, 113.77, 114.43, 121.70, 122.63, 125.21, 126.39, 126.75, 131.81, 135.13,
47.79, 152.43, 156.11, 166.44.
3
.71(s, 3H, OCH
3
), 3.86 (s, 3H, OCH
3
), 4.75 (s, 2H, OCH
2
1
3
(
6
6
1
2
-(2-methoxy-4-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)-N-(4-methoxyphenyl)
acetamide (7d): White powder, 1.234 g (yield 77. 91%); M.P.: 161-163°C; Mass (M+1):
1
Calculated 432.15, Found 432.2; H NMR (400 MHz, DMSO- d
6
) δ 3.70 (s, 3H, OCH ), 3.86
3
(
s, 3H, OCH
3
),4.80 (s, 2H, OCH ), 7.76-6.88 (m, 11H, Ar-H), 9.83 (s, 1H, -CO-NH), 10.04
2
(
s, 1H, CO-NH); 13C-NMR (101 MHz, DMSO- d
6
) δ 55.64, 56.11, 68.21, 110.59, 113.28,
2
5

ACCEPTED MANUSCRIPT
1
14.39, 121.49, 126.07, 130.75, 131.91, 149.80, 153.29, 156.00, 165.78, 191.88. HRMS
+
(
ESI): m/z [M+H] calculated 432.1559, found 432.1553
N-(4-ethoxyphenyl)-2-(2-methoxy-4-(4-oxo-3,4-dihydroquinazolin-2-yl) phenoxy)
acetamide (7e): White powder, 1.414g (yield 86.43%); M.P.: 150-152°C; Mass (M+1)
1
Calculated 446.17 Found 446.2; H NMR (400 MHz, DMSO- d
6
) δ 1.300 (t, J = 7.2Hz, 3H,
),4.792(s, 2H, OCH ), 7.530-6.870 (m,
1H, Ar-H), 9.830 (s, 1H, CO-NH), 10.015 (s, 1H, CO-NH); C-NMR (101 MHz, DMSO-
CH
3
), 3.856 (s, 3H, OCH
3
), 3.983-3.931 (q, 2H, OCH
2
2
1
3
1
d
1
6
) δ 15.12, 56.1, 63.56, 68.21, 110.58, 113.28, 114.91, 121.46, 126.08, 130.75, 131.81,
+
49.80, 153.30, 155.25, 165.76, 191.88. HRMS (ESI): m/z [M+H] calculated 446.1716,
found 446.1711
N-(4-fluorophenyl)-2-(2-methoxy-4-(4-oxo-3,
4-dihydroquinazolin-2-yl)phenoxy)
acetamide (7f): Yellowish powder, 1.412g (Yield 91.6%); M.P.: 135-138°C; (M+1)
1
Calculated 420.13, Found 420.2; H NMR (400 MHz, DMSO- d
6
) δ 3.827 (s, 3H, OCH ),
3
4
.827 (s, 2H, OCH
2
), 7.626-7.09 (m, 10H, Ar-H), 9.831, (s, 1H, CO-NH),10.241 (s, 1H, CO-
NH); 13C-NMR (101 MHz, DMSO- d
) δ 56.11, 68.10, 110.61, 113.30, 115.75, 115.97,
6
1
21.69, 121.77, 126.06, 130.79, 135.22, 149.79, 153.23, 157.50, 159.89, 166.25, 191.89.
+
HRMS (ESI): m/z [M+H] calculated 420.136, found 420.1265
2
-(2-methoxy-4-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)-N-(naphthalen-2-yl)
acetamide (7g): White powder, 1.31g (yield 82.28%); M.P.: 232-235°C; Mass (M+1)
1
Calculated 452.16 Found 452.2; H NMR (400 MHz, DMSO- d
6
) δ 3.91 (s, 3H, OCH ), 4.84
3
(
s, 2H, OCH
2
), 7.09-8.27 (m, 14H, Ar-H), 10.34 (s, 1H, CO-NH), 12.46 (s, 1H, CO-NH);
13C-NMR (101 MHz, DMSO- d
) δ 56.36, 68.41, 111.78, 113.76, 116.19, 120.54, 121.22,
6
1
1
21.47, 125.37, 126.30, 127.07, 127.88, 129.05, 130.46, 133.86, 135.16, 136.60, 149.32,
50.62, 152.30, 162.80, 167.01.
2
6

ACCEPTED MANUSCRIPT
2
-(2-methoxy-5-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)-N-phenyl acetamide
(9a): White powder, 1.314g (yield 89.14%); M.P.: 227-229°C; (M-1) Calculated 400.40,
1
Found 400.3; H-NMR (400 MHz, DMSO- d
6
) δ 3.86 (s, 3H, OCH
3
) 4.78 (s, 2H, OCH ),
2
1
3
7
.04-8.33 (m, 12H, Ar-H), 10.08 (s, 1H, CO-NH), 12.46 (d, J = 52.8 Hz, 1H, CO-NH); C-
NMR (101 MHz, DMSO- d ) δ 56.38, 68.87, 112.38, 113.76, 120.07, 124.25, 126.60, 127.77,
6
1
29.36, 134.67, 138.89, 143.42, 147.68, 161.30, 162.74, 167.12. HRMS (ESI): m/z [M+H]+
calculated 402.1454, found 402.1448
2
-(2-methoxy-5-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)-N-(2-methoxy phenyl)
acetamide (9b): White powder, 0.252g (yield 79.49%); M.P. 254.5-257°C; Mass (M+1)
1
Calculated 432.15 found 432.15; H NMR (400 MHz, DMSO- d
6
) δ 3.85 (s, 6H, 2 x OCH ),
3
4
.818 (s, 2H, OCH
2
), 6.904-8.338 (m, 11H, Ar-H), 9.275 (s, 1H, CO-NH), 12.392 (s, 1H,
CO-NH); C-NMR (101 MHz, DMSO- d ) δ 56.49, 56.60, 68.60, 111.81, 112.47, 113.53,
21.06, 123.02, 124.92, 125.24, 126.37, 127.09, 127.84, 135.23, 147.00, 148.95, 152.02,
1
3
6
1
1
+
62.85, 166.74. HRMS (ESI): m/z [M+H] calculated 432.1559, found 432.1551
2
-(2-methoxy-5-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)-N-(3-methoxy phenyl)
acetamide (9c): Light brown powder, 0.587g (yield 92.7%); M.P.: 114-117°C; Mass (M+1)
1
Calculated 432.15, found 432.10; H NMR (400 MHz, DMSO- d
6
) δ 3.710 (s, 3H, OCH ),
3
3
.904 (s, 3H, OCH
3
), 4.764 (s, 2H, OCH ), 6.639 -7.606 (m, 10H, Ar-H), 9.804 (s, 1H, CO-
2
NH), 10.108 (s, 1H, CO-NH); 13C-NMR (101 MHz, DMSO- d
) δ 55.52, 56.54, 68.27,
6
1
1
05.70, 109.62, 111.68, 112.16, 127.68, 130.18, 140.08, 148.50, 155.20, 160.06, 166.75,
+
91.83. HRMS (ESI): m/z [M+H] calculated 432.1559, found 432.1552
2
-(2-methoxy-5-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)-N-(4-methoxyphenyl)
acetamide (9d): White powder, 1.387g (yield 87%); M.P.: 239-240°C; Mass (M+1)
1
Calculated 432.15, Found 432.2; H-NMR (400 MHz, DMSO- d
6
) 3.68 (s, 3H, OCH ), 3.88
3
2
7

ACCEPTED MANUSCRIPT
(
s, 3H, OCH
3
), 4.71 (s, 2H, OCH ), 6.86-7.57 (m, 11H, Ar-H), 9.78 (s, 1H, CO-NH), δ 9.97
2
+
(
s, 1H, CO-NH). HRMS (ESI): m/z [M+H] calculated 432.1559, found 432.1554
N-(4-ethoxyphenyl)-2-(2-methoxy-5-(4-oxo-3,4-dihydroquinazolin-2-yl)-phenoxy)
acetamide (9e): White powder, 1.469g (yield 92.73%); M.P.: 251-253°C; Mass (M+1)
1
Found 448.1; H NMR (400 MHz, DMSO- d
6
) δ 2.72 (t, J = 7.09 Hz, 3H, OCH
), 4.58 (s, 2H, OCH ), 6.98-7.49 (m, 11H, Ar-H), 8.03(s,
H, CO-NH) 9.84 (s, 1H, CO-NH); C-NMR (101 MHz, DMSO- d ) δ 14.68, 55.78, 63.08,
3
), 3.33 - 3.39
(
m, 2H, OCH
2
),3.83 (s, 3H, OCH
3
2
1
3
1
6
1
6
8.33, 111.75, 113.28, 114.40, 121.16, 126.04, 127.26, 131.35, 134.40, 147.32, 151.73,
54.78, 162.43, 166.00. HRMS (ESI): m/z calculated 446.1716, found 446.1712 [M+H]+ ,
2
-(2-methoxy-4-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)-N-o-tolyl
acetamide
(9f): Cream powder, 0.507g (yield 82.97%); M.P.: 124-127 °C; Mass (M+1) Calculated
1
4
16.16, found 416.10; H NMR (400 MHz, DMSO- d
6
) δ 2.204 (s, 3H, CH
), 7.08-7.55 (m, 11H, Ar-H), 9.392 (s, 1H, CO-NH), 9.851 (s, 1H,
CO-NH); C-NMR (101 MHz, DMSO- d ) δ 18.07, 39.38, 39.59, 39.80, 40.01, 40.22, 40.43,
0.63, 56.21, 68.03, 110.54, 113.65, 124.57, 125.82, 126.18, 126.68, 130.93, 135.86, 149.85,
53.05, 166.18, 192.01.
3
), 3.87 (s, 3H,
OCH
3
), 4.872 (s, 2H, OCH
2
1
3
6
4
1
N-(2-chlorophenyl)-2-(2-methoxy-5-(4-oxo-3,4-dihydroquinazolin -2-yl)-phenoxy)
acetamide (9g): Yellowish powder, 1.296g (yield 81.05%); M.P.: 227-229°C; (M+1)
1
Calculated 436.10, Found 436.10; H NMR (400 MHz, DMSO- d
6
) δ 3.867 (s, 3H, OCH ),
3
4
.876 (s, 2H, OCH
2
), 7.143-8.035 (m, 11H, Ar-H), 9.564 (s, 1H, CO-NH), 12.406 (s, 1H,
CO-NH); C-NMR (101 MHz, DMSO- d ) δ 56.46, 68.47, 112.40, 113.58, 122.94, 124.32,
25.22, 126.39, 127.80, 128.34, 130.03, 134.57, 135.17, 147.11, 149.37, 152.28, 162.86,
1
3
6
1
1
+
67.32. HRMS (ESI): m/z [M+H] calculated 436.1064, found 436.1057
2
8

ACCEPTED MANUSCRIPT
N-(4-chlorophenyl)-2-(2-methoxy-5-(4-oxo-3,4-dihydroquinazolin-2-yl) phenoxy)
acetamide (9h): Light brown powder, 0.662g (yield 82.86%); M.P.: 229°C; (M+1)
1
Calculated 436.10, Found 436.10; H NMR (400 MHz, DMSO-d
6
) δ 3.879 (s, 3H, OCH );
3
4
.820 (s, 2H, OCH
NH); 13C-NMR (101 MHz, DMSO- d
26.31, 127.70, 127.78, 129.18, 137.79, 147.64, 167.15. HRMS (ESI): m/z [M+H]+
2
), 8.109-7.71(m, 11H, Ar-H), 10.32 (s, 1H, CO-NH), 12.71 (s, 1H, CO-
6
) δ 56.33, 68.76, 112.37, 113.68, 121.62, 125.14,
1
calculated 436.1064, found 436.1061
N-(4-fluorophenyl)-2-(2-methoxy-5-(4-oxo-3,4-dihydroquinazolin-2-yl)
phenoxy)
1
acetamide (9i): White powder, 1.41g (yield 91.5%); M.P.: 124-126°C; H NMR (400 MHz,
DMSO- d
6
) δ 3.898 (s, 3H, OCH
3
); 4.792 (s, 2H, OCH
2
), 7.810-7.114(m, 10H, Ar-H),9.779
) δ 39.39, 39.59,
1
3
(s, 1H, CO-NH),10.364 (s, 1H, CO-NH), C-NMR (101 MHz, DMSO- d
6
3
1
9.80, 40.01, 40.22, 40.43, 40.63, 56.44, 68.22, 111.83, 112.33, 118.61, 119.49, 123.93,
27.68, 131.09, 133.95, 140.53, 148.41, 154.80, 167.00, 191.83. HRMS (ESI): m/z [M+H]+
calculated 420.136,found 419.9925
2
-(2-methoxy-5-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)-N-(4-nitro
phenyl)
acetamide (9j): Yellowish powder, 0.279g (yield 85%); M.P.: 269-272°C; Mass (M+1)
1
Calculated 447.13, Found 447.10; H-NMR (400 MHz, DMSO- d
6
) δ 3.86 (s, 3H, OCH ),
3
4
.86 (s, 2H, OCH
2
), 7.14- 8.33 (m, 11H, Ar-H ), 10.75 (s, 1H, -CO-NH), 12.52 (s, 1H, -CO-
NH); 13C-NMR (101 MHz, DMSO- d
) δ 56.62, 68.68, 112.45, 113.74, 119.73, 122.86,
6
1
24.92, 125.59, 134.67, 143.02, 143.41, 145.28, 147.65, 152.51, 168.03. HRMS (ESI): m/z
+
[
M+H] calculated 447.1305, found 447.1275
N-butyl-2-(2-methoxy-5-(4-oxo-3,4-dihydroquinazolin-2-yl)phenoxy)
acetamide
(9k): Cream colored powder, 0.236 g (yield 84.61%); M.P.: 194-197°C; Mass (M+1)
1
Calculated 382.17, Found 382; H NMR (400 MHz, DMSO- d
6
) δ 0.759 (t, 3H, J= 6.8Hz, -
2
9