The combination of quinazoline and chalcone moieties leads to novel potent heterodimeric modulators of breast cancer resistance protein (BCRP/ABCG2)

Article abstract of DOI:10.1016/j.ejmech.2016.03.067

During the last decade it has been found that chalcones and quinazolines are promising inhibitors of ABCG2. The combination of these two scaffolds offers a new class of heterocyclic compounds with potentially high inhibitory activity against ABCG2. For this purpose we investigated 22 different heterodimeric derivatives. In this series only methoxy groups were used as substituents as these had been proven superior for inhibitory activity of chalcones. All compounds were tested for their inhibitory activity, specificity and cytotoxicity. The most potent ABCG2 inhibitor in this series showed an IC₅₀ value of 0.19 μM. It possesses low cytotoxicity (GI₅₀ = 93 μM), the ability to reverse MDR and is nearly selective toward ABCG2. Most compounds containing dimethoxy groups showed slight activity against ABCB1 too. Among these three compounds (17, 19 and 24) showed even higher activity toward ABCB1 than ABCG2. All inhibitors were further screened for their effect on basal ATPase activity. Although the basal ATPase activity was partially stimulated, the compounds were not transported by ABCG2. Thus, quinazoline-chalcones are a new class of effective ABCG2 inhibitors.

Full text of DOI:10.1016/j.ejmech.2016.03.067

Accepted Manuscript
The combination of quinazoline and chalcone moieties leads to novel potent
heterodimeric modulators of Breast Cancer Resistance Protein (BCRP/ABCG2)
Stefanie Kraege, Katja Stefan, Kapil Juvale, Thomas Ross, Thomas Willmes, Michael
Wiese
PII:
S0223-5234(16)30249-5
10.1016/j.ejmech.2016.03.067
EJMECH 8492
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 December 2015
Revised Date: 8 March 2016
Accepted Date: 25 March 2016
Please cite this article as: S. Kraege, K. Stefan, K. Juvale, T. Ross, T. Willmes, M. Wiese, The
combination of quinazoline and chalcone moieties leads to novel potent heterodimeric modulators of
Breast Cancer Resistance Protein (BCRP/ABCG2), European Journal of Medicinal Chemistry (2016),
doi: 10.1016/j.ejmech.2016.03.067.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
The combination of quinazoline and chalcone moieties leads to novel potent
heterodimeric modulators of Breast Cancer Resistance Protein
(BCRP/ABCG2)
‡
‡
Stefanie Kraege, Katja Stefan, Kapil Juvale, Thomas Ross, Thomas Willmes and Michael
*
Wiese
Pharmaceutical Chemistry II, Pharmaceutical Institute, University of Bonn, An der
Immenburg 4, 53121 Bonn, Germany
‡
These authors contributed equally.
Keywords. ABC transporter, ABCG2, Multidrug resistance, Chalcones, Quinazolines,
Quinazoline-chalcones, Pheophorbide A assay, ATPase
1

ACCEPTED MANUSCRIPT
Abstract: During the last decade it has been found that chalcones and quinazolines are
promising inhibitors of ABCG2. The combination of these two scaffolds offers a new class of
heterocyclic compounds with potentially high inhibitory activity against ABCG2. For this
purpose we investigated 22 different heterodimeric derivatives. In this series only methoxy
groups were used as substituents as these had been proven superior for inhibitory activity of
chalcones. All compounds were tested for their inhibitory activity, specificity and
cytotoxicity. The most potent ABCG2 inhibitor in this series showed an IC value of 0.19
5
0
µM. It possesses low cytotoxicity (GI = 93 µM), the ability to reverse MDR and is nearly
5
0
selective toward ABCG2. Most compounds containing dimethoxy groups showed slight
activity against ABCB1 too. Among these three compounds (17, 19 and 24) showed even
higher activity toward ABCB1 than ABCG2. Selected inhibitors were further screened for
their effect on basal ATPase activity. Although the basal ATPase activity was partially
stimulated, the compounds were not transported by ABCG2. Thus, quinazoline-chalcones are
a new class of effective ABCG2 inhibitors.
2

ACCEPTED MANUSCRIPT
Introduction
Chemotherapy is one of the common treatments of cancer, but often fails due to the
emergence of multidrug resistance (MDR). Often MDR is caused by overexpression of efflux
1
transport proteins from the family of ATP-binding cassette (ABC) transporters. Currently 48
members of ABC transporters are known in humans, which act as exporters and are divided
into seven subfamilies. The typical ABC transporter consists of two nucleotide binding
domains (NBDs) and two transmembrane domains (TMDs), containing six transmembrane
helices each, as functional units. Hydrolysis of ATP yields the energy necessary for active
transport and is performed by the NBDs. While most ABC transporters have specific
substrates, some are polyspecific, recognizing a wide variety of structurally unrelated
compounds. Three polyspecific ABC transporters, mostly associated with MDR in cancer, are
2
,3
ABCB1 (P-glycoprotein), ABCC1 (MRP1) and ABCG2 (BCRP).
The most recently described polyspecific ABC transporter ABCG2 was first discovered in
998 in the multidrug resistant breast cancer cell line MCF-7/AdrVp. It has a molecular
1
4
,5
weight of 72 kDa and consists of 655 amino acids. ABCG2 contains only one NBD and one
6
,7,8
TMD and dimers or tetramers are considered to form the functional transporter.
The
protein is the second member of the ABCG subfamily and is present in various organs with
9
barrier functions, and is expressed in many types of cancer as well. It is involved in MDR by
transporting molecules with amphipathic characteristics, which can be neutral, positively or
negatively charged. The structural diversity of substrates ranges from anticancer drugs such as
mitoxantrone over various non-chemotherapy drugs to fluorescence dyes, like pheophorbide
1
0,11
A.
The development of potent, selective and non-toxic inhibitors is one possibility to
overcome MDR. A variety of compounds that can inhibit ABCG2 have been investigated
during the last decade including fumitremorgin C (FTC), a highly potent and selective
1
2,13,14
inhibitor produced by Aspergillus fumigatus, that is also highly neurotoxic.
Based on the
1
5
scaffold of FTC the non-toxic inhibitor Ko143 was developed. Tariquidar, a potent inhibitor
3

ACCEPTED MANUSCRIPT
1
6
of ABCB1, has been shown to also inhibit ABCG2, though with lesser potency. Derivatives
lacking the tetrahydroisoquinoline moiety of tariquidar lost their ABCB1 inhibiting potency
1
7,18,19,20
but retained their ABCG2 inhibition potential.
Naturally occurring flavonoids and
2
1,22,23,24
derivatives have been described as potent inhibitors of ABCG2.
Also the investigation
of their biochemical precursors, the chalcones and their derivatives as selective inhibitors of
ABCG2 showed the possibility to overcome MDR in vitro. Chalcones were found to be potent
ABCG2 inhibitors with IC50 values in the low micromolar range and the most potent
2
5
derivative having an IC50 value of 0.53 µM in the Hoechst 33342 accumulation assay.
Nevertheless chalcones are limited in their maximum inhibitory potential. Quinazolines like
the tyrosine kinase inhibitor gefitinib and structurally related derivatives are even more potent
2
6,27,28
inhibitors of ABCG2 with IC values in the high nanomolar range.
In 2014 Winter et
5
0
2
9
al. developed a series of quinoxaline-substituted chalcones as new inhibitors of ABCG2.
They replaced the ring B of the chalcone by a quinoxaline, a 2-napthyl or a 3,4-
methylenedioxyphenyl residue and substituted ring A with varying methoxy group
substitution patterns. In general the quinoxaline derivatives were found to be most potent and
the presence of at least two methoxy groups on the A-ring is necessary for an effective
inhibition of ABCG2. The most potent compounds possessed IC50 values in the low
micromolar range in the mitoxanthrone efflux assay. Thus, the combination of two
substructures of ABCG2 inhibitors provided more potent compounds. The benefit of
combination of substructures was previously shown on the example of linked homodimers as
modulators of ABCB1. In several cases an increase in the inhibitory potential of the
3
0,31,32
compounds was observed.
Even strong potentiation of inhibitory activity was reported.
For example, the galantamine dimer (Gal-2) inhibited ABCB1 efflux with an IC of 0.8 µM,
5
0
3
3
while the monomer showed no inhibition up to 100 µM.
Based on these results we decided to combine chalcones with quinazolines, in order to
investigate whether there is also a benefit of combining both scaffolds of ABCG2 inhibitors.
4

ACCEPTED MANUSCRIPT
Our decision was based on the available data regarding the binding region of chalcones and
quinazolines. From the available literature data it can be concluded that both types of
inhibitors most probably bind to the NBDs of ABC transporters and not to the common drug
binding site located in the transmembrane domains. In case of quinazolines the possibility of
attacking both binding sites exist as outlined below. For flavonoids, the successor of
chalcones, several docking and 3D QSAR studies demonstrated that they target the nucleotide
3
4,35,36
binding domain of ABCB1.
With regard to ABCG2 no detailed studies are available,
but based on the highly conserved structure of the nucleotide binding domains, it is suggested
3
7
that flavonoids inhibit similarly via interaction with the NBD. Chalcones have not been
investigated that intensively but they were shown to interact with the NBDs of ABCB1 by
3
8,39
Bois et al.
Again nothing definite is known for ABCG2, but due to the conserved NBD
structures, an analogous interaction can be assumed, as for flavonoids. This is corroborated by
the finding that chalcones can interact with the ATP-binding site of tyrosine kinases, the
4
0,41
targets of several quinazoline based TKIs.
Also structurally related
4
2
dihydroxybenzophenones have been shown to bind to the NBD of ABCB1. TKIs with
quinazoline scaffold are known to bind to the ATP-binding site of the receptor tyrosine
4
3,44,45
kinases.
Only a few quinazoline based TKIs have been investigated in detail for
interaction with ABCB1 and ABCG2. While stimulation of ATPase activity was observed in
1
25
submicromolar concentrations, replacement of radiolabelled [ I] IAAP from ABCG2
4
6
membranes required much higher concentrations, or the compounds were not active at all.
Additionally Saito et al. demonstrated in kinetic analysis experiments that Gefitinib binds to
4
7
an ABCG2-ATP complex.
Therefore we designed and synthesized novel heterodimeric modulators based on the
combination of chalcones with quinazolines, in order to investigate whether there is also a
benefit of combining both scaffolds of ABCG2 inhibitors.
5

ACCEPTED MANUSCRIPT
An amino linker at position 4 of the quinazoline moiety was used to couple the quinazolines
with different substituted chalcones. Based on previous results from studies of both individual
compound classes, 22 new derivatives were synthesized. Further modifications, like methoxy
groups at position 6, 7 and the addition of a phenyl ring in position 2 of the quinazoline
moiety, led to increased activity. The compounds were investigated for their inhibitory
activity in MDCK II cells expressing ABCG2 using the pheophorbide A assay. ABCG2
selectivity was analyzed by screening for ABCB1 and ABCC1 inhibition using the calcein
accumulation assay. Additionally, the compounds effect on basal ATPase activity was studied
and enzyme kinetic measurements were performed to characterize the type of interaction with
the substrate pheophorbide A.
Results and Discussion
Chemistry. The synthesis of the target compounds 17-38 is shown in Scheme 1.
First the quinazoline ring system was built by synthesizing quinazolinones. For this purpose
two different methods, depending on the substitution in position 2 of the quinazolines, were
used. For compounds without substitution at position 2 anthranilic acid or 2-amino-4,5-
dimethoxybenzoic acid and excess formamide were reacted to yield compounds 1 and 2. To
obtain compounds 3 and 4 anthranilamide and an accordingly substituted benzaldehyde in
dimethylformamide in presence of iodine and anhydrous potassium carbonate were reacted to
yield the 2-substituted quinazolinones. Then chlorination with phosphoryl chloride produced
the intermediates 5-8. The substituted 4-chloroquinazolines were transformed into 4-
anilinoquinazolines by reaction with 3- or 4-aminoacetophenones to produce the precursors 9-
1
6
1
6. Finally, Claisen-Schmidt condensation of the ketones with different benzaldehydes,
1
5
using LiOH as catalyst, led to the desired quinazoline-chalcones. All synthesized
compounds were characterized by NMR spectroscopy and the purity confirmed by LC-MS.
6

ACCEPTED MANUSCRIPT
Biological Testing. Inhibition of ABCG2. The 22 synthesized quinazoline-chalcones were
examined for their ability to inhibit ABCG2 using the pheophorbide A assay and the MDCK
II BCRP cell line (Table 1). Pheophorbide A is a fluorescent breakdown product of
1
1
chlorophyll and a well-known specific substrate of ABCG2. The assay was performed as
4
8
previously described with minor modifications. Previous results from compounds with
different substituents on ring B of the chalcone showed the necessity of a substituted phenyl
ring. The unsubstituted derivatives were either inactive or showed only very low inhibitory
activity, depending on the substitution pattern on ring A of the chalcone. From the
investigated substituents methoxy substitution, especially 3,4-dimethoxy substitution, yielded
2
5
the maximum inhibitory activity against ABCG2. Compared to the quinazoline and chalcone
building blocks the combination of both moieties generally led to increased activity. For
example for the quinazoline 2-(3,4-dimethoxyphenyl)-4-anilinoquinazoline an IC value of
5
0
2
6
4
.09 µM was reported and for the chalcone (E)-1,3-Bis(3,4-dimethoxyphenyl)prop-2-en-1-
4
9
one an IC of 5.12 µM.
5
0
The first series of compounds (17-26) started with a meta-acryloylphenyl residue on ring A of
the chalcone. All synthesized compounds showed an inhibitory effect against ABCG2 with
IC -values in the range of 0.2 to 2 µM. Comparison of compounds 17 to 20 showed no
5
0
significant differences related to methoxy substitution in positions 6 and 7 of the quinazoline
moiety unsubstituted at position 2. Only the combination of 6,7-OCH and 4’-OCH on the
3
3
chalcone residue leads to increased activity with compound 20 being most potent (IC = 0.32
5
0
µM). In both cases a 3’,4’-dimethoxy substitution on ring B slightly decreased the inhibitory
potency. This is in agreement with previous findings for 4-phenyl substituted quinazolines,
where the additional methoxy groups at the quinazoline moiety had mostly an activity
2
6
decreasing effect.
Different results were obtained for quinazolines substituted at position 2 with a phenyl ring
(21-23). The 3’,4’-dimethoxy substitution was most potent, followed by the meta-substituted
7

ACCEPTED MANUSCRIPT
methoxy derivative and the para-substituted analogue being the least active. If additional
methoxy groups at the 2-phenyl ring were present (24-26) the activity pattern was identical to
the unsubstituted quinazolines 17-20 (4’-methoxy substitution being superior to 3’,4’
disubstitution). In case of the quinazoline-chalcones there is still a positive effect of
substituents at position 2 of the quinazoline moiety, but it is less pronounced as for the 2-
2
6
phenyl substituted quinazolines without chalcone moitey.
The second series of compounds (27-38) contained a para-acryloylphenyl residue on ring A
of the chalcone. As before, compounds unsubstituted at position 2 of the quinazoline moiety,
with or without methoxy groups on position 6 and 7 (27-30), were investigated. In this series
the quinazoline-chalcones were substituted with methoxy groups in position 3’ and 3’,4’ on
ring B. Again the mono-substituted derivatives were more potent than their 3’,4’-disubstituted
counterparts. Comparing the meta-acryloyl with the para-acryloyl residues, the latter showed
a slightly increased activity, compare 17 (IC = 1.30 µM) with 27 (IC = 0.84 µM) and 19
5
0
50
(
IC50 = 1.71 µM) with 29 (IC50 = 1.23 µM). The two derivatives with 6,7-methoxy
substitution and a single methoxy group at the chalcone ring B (20 and 30) possessed the
highest inhibitory activity among the eight derivatives unsubstituted in position 2 of the
quinazoline.
For the quinazoline chalcones 31-33 bearing a 2-phenyl ring the same effect of the
substitution pattern was observed as for the meta-acryloyl derivatives 21-23. Again the 3’,4’-
methoxy derivative 31 showed the highest activity (IC = 0.29 µM), while a 4’-methoxy
5
0
group was detrimental (33: IC = 3.55 µM). As seen for the meta-acryloyl substituted
5
0
compounds, a 3,4-dimethoxy substituted phenyl ring at position 2 of the quinazoline
improved activity further in the series of para-acryloyl substituted derivatives. Compound 35
turned out to be the best in the whole series, with an IC -value of 0.19 µM only. But also the
5
0
3
’-methoxy derivative 36 (IC = 0.36 µM) was nearly as potent as Ko143, the most potent
50
ABCG2 inhibitor known so far. Interestingly the quinazoline-chalcones from both the meta
8

ACCEPTED MANUSCRIPT
and para series without a substituent at ring B showed almost identical IC -values (34: 0.92
5
0
µM vs. 38: 1.09 µM). A graphical summary of the effect of structural features of the
quinazoline-chalcone scaffold for ABCG2 inhibition is given in Figure 1.
Taken together the comparisons of all synthesized quinazoline-chalcones, it can be concluded
that compounds containing a para-acryloylphenyl residue are better inhibitors than those with
a meta-acryloylphenyl residue and that the presence of 2-phenyl substitution at the
quinazoline moiety leads to an increase in ABCG2 inhibition.
To examine the type of inhibition of quinazoline-chalcones enzyme kinetic studies were
performed using the most potent compound 35 and pheophorbide A. The Lineweaver-Burk
plot of the transport velocities for a range of substrate concentrations in absence or presence
of various concentrations of compound 35 reveals that it is a non-competitive inhibitor of
ABCG2 with regard to pheophorbide A, indicating that substrate and inhibitor have different
binding sites (Figure 2).
Calcein accumulation assay to determine ABCB1 and ABCC1 inhibition. All synthesized
compounds were additionally screened for their inhibition of ABCB1 and ABCC1 utilizing
the calcein accumulation assay. The screening was conducted at a fixed concentration of 10
µM using A2780adr (ABCB1) and H69AR (ABCC1) cell lines. Cyclosporine A (10 µM) was
used as standard inhibitor for both ABCB1 and ABCC1, respectively. Figure 3 illustrates the
effects of the 22 quinazoline-chalcones on the accumulation of calcein and exemplifies that
the studied substances possess higher affinity for ABCB1 (Figure 3A) than ABCC1 (Figure
3
B). As could be expected, compounds with dimethoxy-substitution showed relatively high
inhibition of ABCB1. Within the two series, the chalcones with meta-acryloylphenyl residue
17-26) were found to inhibit ABCB1 more than compounds with the para-acryloylphenyl
(
residue (27-38). Concentration-response curves were generated for compounds showing more
than 25 % inhibition at 10 µM. The results are depicted in Table 2 and reveal that three
compounds 17 (IC = 0.42 µM), 19 (IC = 0.86 µM) and 24 (IC = 0.48 µM) have a
5
0
50
50
9

ACCEPTED MANUSCRIPT
substantial ABCB1 inhibitory activity being mostly more active than in case of ABCG2.
Therefore, these compounds represent dual inhibitors. Interestingly the dose-response curves
of all compounds levelled off below the maximum fluorescence reached by cyclosporine A,
pointing to the possibility of partial inhibition. Comparing the activities against both
transporters the scatterplot shown in Figure 4 demonstrates that there is no correlation
between the IC50 values for ABCG2 and ABCB1. The most potent ABCG2 inhibitor, 35
shows only a weak inhibition of ABCB1 function, no ABCC1 inhibition and can be classified
as rather selective ABCG2 inhibitor.
Effects of quinazoline-chalcones on ATPase activity of ABCG2. To understand the
interactions between quinazoline-chalcones and ABCG2 in more detail, we examined the
effects of quinazoline-chalcones (1 µM) on the vanadate sensitive ATPase activity. As shown
in Figure 5 most of the compounds lead to an increase of the ATPase activity. Additionally,
compounds 27, 31 and 35 activate the membrane ATPase nearly to the same extent as
5
5
quercetin, a well-known potent activator of ABCG2 ATPase activity. These compounds
contain a para-acryloylphenyl residue linked to ring A and a 3,4-dimethoxy substitution at
ring B of the chalcone. The data obtained suggest that quinazoline-chalcones act as ATPase
activators and can be considered as substrates although the compounds were found to be
inhibitors in the pheophorbide A accumulation assay. On the other hand compounds with two
methoxy groups in positions 6 and 7 of the quinazoline scaffold (19, 20, 29 and 30) show an
inhibiting or no effect on ABCG2 ATPase activity acting like Ko143 as inhibitors.
The effects of compound 30 (ATPase inhibiting compound) and 27, 35 (ATPase activating
compounds) on ATPase activity were further characterized by determining concentration
response curves (Figure 6). We observed that 35 increases ATPase activity up to a
concentration of 1 µM followed by a decrease at higher concentrations. In contrast, compound
3
0 inhibits ATPase activity of ABCG2 up to 1 µM and showed no activating effect at higher
concentrations.
1
0

ACCEPTED MANUSCRIPT
The activation or inhibition of the ATPase is apparently influenced also by the scaffolds of the
quinazoline-chalcones and depends on the concentration of the substances.
Investigation of compound 27 for its accumulation in ABCG2 overexpressing cells via
primary fluorescence. The ability of quinazoline-chalcones to stimulate the ATPase of
ABCG2 in nearly the same way as the standard substrate quercetin, supports the possibility
that the compounds are transported by ABCG2.
Fluorescence microscopy was used to investigate this possibility. Among the ATPase
stimulating derivatives compound 27 showed moderate fluorescence when excited at a
wavelength of 405 nm and was selected for this investigation. Because of the similar
excitation and emission wavelength of GFP (green fluorescent protein), linked to cDNA of
ABCG2 transfected MDCK II cells, fluorescence studies were performed using parental
MCF-7 and MCF-7 MX cells expressing ABCG2. Cells were incubated with compound 27 at
1
0 µM for 2 h, washed and cellular fluorescence was visualized using a Nikon A1 R confocal
laser scanning microscope after 30 min. As it can be seen in Figure 7A and B, compound 27
showed no difference in fluorescence intensity between the wild-type MCF-7 and the ABCG2
overexpressing cell line MCF-7 MX, thus the efflux of the substance could be disproved. Our
findings were confirmed by experiments in combination with the BCRP substrate
pheophorbide A, showing its accumulation in resistant cell line MCF-7 MX in presence of
compound 27 during the incubation time. Hereby, the fluorescence intensity was equal
compared to the parental cell line MCF-7. Figure 7C-E highlight the results that the
quinazoline-chalcones inhibit the transport of pheophorbide A but are not transported
themselves.
Further kinetic studies were performed using fluorescence spectroscopy. Parental MCF-7 and
MCF-7 MX cells were incubated with 27 for varying time intervals. After washing, the
fluorescence of the cells was measured in a fluorimeter. The time-dependent determination of
the intracellular fluorescence concentration for up to 3 h indicated that compound 27 behaved
1
1

ACCEPTED MANUSCRIPT
in both cell lines in the same manner. Within the first minutes the intracellular concentration
increases until reaching a plateau after approximately 60 min (Figure 8).
This is in agreement with the results from the confocal laser scanning microscopy disproving
the assumption of transport of the quinazoline-chalcone by ABCG2. It seems that stimulation
of the ATPase activity is not related to drug transport, e.g. behavior as a substrate.
Cytotoxicity of Quinazoline-chalcones. To eliminate cell specific effects and for
determination of therapeutic ratios, all compounds were screened for their cytotoxic effects in
the same MDCK II BCRP and MDCK II wild type cells. Cells were treated with a fixed
concentration of 10 µM of the compounds. After 72 h incubation cell viability was
determined using MTT colorimetric assay. As shown in Figure 9, most of the compounds
have only low cytotoxic effects at a concentration of 10 µM. Only compounds 23, 28, 31 and
3
2 show high cytotoxicity, but no conclusion could be drawn with respect to their chemical
structure. For the most potent representative of our compound set (35) complete dose
response curves were generated. The result is in agreement with the screening, featuring no
detectable cytotoxicity, with a GI value of about 93 µM (Figure 10). The ratios of IC
50
5
0
values and GI50 values yield the therapeutic ratio (Table 3). Among the investigated
derivatives compound 35 has the highest therapeutic ratio, pointing to its usability for further
in vivo investigations. In comparison to this, the standard inhibitor Ko143 has a 10 fold lower
therapeutic value due to its cytotoxicity.
Furthermore for selected compounds, which showed high inhibitory potencies towards
ABCB1, we determined the cytotoxicity in A2780adr and parenteral A2780 cells additionally
(Table 4). Compared to MDCK II cells all compounds were more toxic in A2780 parental and
adr cells. When comparing the GI50-values of the compounds high intercorrelations become
apparent (Table 5). Thus the compounds behave in all cell lines similar with varying baseline
toxicity. As the cytotoxicities of the compounds in the wild type and the transporter
containing cell lines do not differ significantly, it can be concluded that they are no or only
1
2

ACCEPTED MANUSCRIPT
poor substrates of ABCG2 and ABCB1, in agreement with the results of the fluorescence
measurements described above.
Quinazoline-chalcones potently reverse resistance to SN-38.
Furthermore, we checked the substances for their ability to reverse the multidrug resistance in
ABCG2 overexpressing cells. For better comparison we also investigated the efficacy of
Ko143, as known standard inhibitor of ABCG2. Compound 35, which shows highest ABCG2
inhibition in the pheophorbide A accumulation assay as well as Ko143 were examined at final
concentrations of 0.01 µM and 0.1 µM. Figure 11 depicts the shift in the dose response curves
of the ABCG2 substrate SN-38, the active metabolite of irinotecan, (Figure 11A and B) in
presence of the test compounds. These dose-response curves indicate an exceeding decrease
in cell viability in ABCG2 overexpressing cells than in wild type cells. Compound 35 and
Ko143 are able to partially reverse the ABCG2-mediated transport of SN-38 out of the cells,
even at nanomolar concentrations.
Pharmacophore Search.
5
0
Matsson et al. reported in 2007 a minimum pharmacophore model for ABCG2 substrates. It
was derived from the effect of 123 diverse drugs or drug-like compounds on mitoxantrone
efflux in Saos-2 cells transfected with human ABCG2. The pharmacophore model consists of
two hydrophobic centers and one hydrogen bond acceptor feature. Together with lipophilicity
as second descriptor, the pharmacophore model had a high discriminating power. More
5
1
recently new pharmacophore models were generated by Ding et al. These authors created a
pharmacophore ensemble consisting of three different pharmacophores. For comparative
purposes they merged parts from their pharmacophore models and found an agreement with
the model of Matsson et al. Therefore it was of interest to investigate how well our
compounds would fit to this pharmacophore model. The best compound 35 was used for
1
3

ACCEPTED MANUSCRIPT
pharmacophore search. The three-point pharmacophore was built in MOE, the hydrophobic
and hydrogen bond acceptor points defined and a pharmacophore search performed.
Investigation of the matched conformations showed that basically two different overlays were
found as shown in Figure 12.
In one alignment only the chalcone part was involved as shown in panel A of Figure 12.
Although the fit is reasonable, it does not explain the gain in activity observed when
combining quinazoline and chalcone substructures. The second alignment presented in panel
B involves the 2-phenyl ring of the quinazoline part as hydrophobic center. The aromatic
residue of the aniline linker, present in the quinazoline and chalcone substructure, is mapped
on one of the hydrophobic centers. The acceptor feature is fulfilled by the carbonyl oxygen of
the chalcone substructure. Again the pharmacophore fit cannot explain the observed SAR.
The 2-phenyl ring is preferable, but not necessary for high activity as exemplified by
compounds 18 and 20, that belong to the most active derivatives. Also compounds 18 and 22
that differs only by the pharmacophoric 2-phenyl ring, possess almost identical potencies.
Thus other structural motifs seem to be responsible for the activity differences within our
series of compounds. This could be explained taken into account the assumption of binding of
our derivatives to the NBD of ABCG2. The pharmacophore model of Matsson et al. was
developed from drugs that almost exclusively are assumed to bind to the transmembrane
region of ABCG2, distant from the NBD.
Conclusion
The combination of the quinazoline and chalcone inhibitors, leading to heterodimers yielded
inhibitors with mostly IC50 values in the submicromolar range. Compared to the formal
building blocks, the quinazoline 2-(3,4-dimethoxyphenyl)-4-anilinoquinazoline (IC : 4.09
5
0
µM) and the chalcone (E)-1,3-Bis(3,4-dimethoxyphenyl)prop-2-en-1-one (IC : 5.12 µM),
5
0
1
4

ACCEPTED MANUSCRIPT
the best heterodimeric compound 35 (IC : 0.19 µM) showed 25 fold higher inhibitory
5
0
potency against ABCG2.
As for quinazolines structural features like a 2-phenyl ring bearing two methoxy groups led to
an increase of inhibitory activity. The majority of the substances showed low inhibition of
ABCB1 and no activity against ABCC1. But three compounds (17, 19 and 24) had higher
potencies for ABCB1 than ABCG2. Compound 24, as an example, was found to be a dual
inhibitor with equal potencies for ABCG2 and ABCB1 (24: IC , ABCG2: 0.60 µM; IC ,
5
0
50
ABCB1: 0.48 µM). Due to the determined high cytotoxicity these compounds are limited in
their therapeutic usage as dual inhibitors.
We further investigated the stimulation of ATPase activity and found most of the quinazoline-
chalcones being ATPase activators, pointing to substrate properties despite their inhibitory
activity in the pheophorbide A assay. Therefore we controlled, if the compounds were
transported by ABCG2. Both experiments, laser scanning microscopy and fluorescence
spectrometry, disproved the assumption of possible transport. For future work it will become
more and more relevant to develop non-toxic compounds to inhibit the efflux function of
ABC transporters and thus to increase the efficacy of chemotherapy in combination with
anticancer drugs. Our compounds showed low cytotoxicity and the most potent ABCG2
inhibitor 35 had a GI value of about 93 µM. This compound was able to reverse MDR for
5
0
the ABCG2 substrate SN-38 in the same concentration range and to the same extent as
Ko143. The behavior of compound 35 in combination with conventional anticancer drugs in
vitro and in vivo will be evaluated in further research. In summary, highly potent, partially
selective and low toxic chalcone derivatives were developed that are able to overcome
multidrug resistance in tumor cells.
1
5

ACCEPTED MANUSCRIPT
EXPERIMENTAL SECTION
Chemistry. Materials. All utilized chemicals were purchased from Sigma-Aldrich, Acros
Organics or Alfa Aesar and were used without further purification. During the synthesis
reaction progress was monitored by thin layer chromatography (TLC) on aluminium sheets
coated with silica gel 60 F254 (Merck). For microwave reactions, a CEM Discover-SP
W/ACTIVENT Microwave was used. The structures of target compounds were approved by
NMR and purity by LC-MS. NMR spectra were recorded on a Bruker Avance 500 MHz
1
13
1
NMR spectrometer at 500 MHz ( H) or 126 MHz ( C) or Bruker Advance 600 MHz ( H,
13
6
00MHz/ C, 151MHz) NMR spectrometer. DMSO-d was used as solvent at 303 K. The
6
chemical shifts are reported as δ values (ppm) using the solvent peak as internal standard.
Coupling constants J are given in Hertz and multiplicities of resonance signals are indicated
as s (singlet), d (doublet), doublet of doublets (dd), triplet of doublets (td), t (triplet), doublet
1
3
of triplets (dt), q (quartet) and m (multiplet). The C signals were assigned with the aid of
distortion less enhancement by polarization transfer (DEPT) and attached proton test (APT).
The purity of final compounds were analyzed by ESI-mass spectra obtained on an LCMS
instrument (Applied Biosystems API 2000 LCMS/MS, HPLC Agilent 1100). The purity of all
target compounds was assigned to be ≥ 95 % by LC-MS.
1
6

ACCEPTED MANUSCRIPT
Preparation of substituted quinazolin-4(3H)-ones.
Two different routes were applied for the synthesis of quinazolin-4(3H)-ones depending on
presence or absence of the phenyl ring at position 2 of the quinazoline moiety. For the
compounds bearing no substituent at position 2 method A was used, while for compounds
with substitution at position 2 method B was employed.
Method A: To an amount of 1 eq. anthranilic acid or 2-amino-4,5-dimethoxybenzoic acid was
added 4 eq. formamide and the reaction mixture was heated for 8-10 hours at 150 °C. Then,
the mixture was cooled to room temperature and diluted with 50 ml water which resulted in
the precipitation of solid. The precipitate was filtered and washed with water.
Quinazolin-4(3H)-one (1). Synthesized from anthranilic acid (0.033 mol, 4.5 g) and
formamide (0.13 mol, 6g). The product was recrystallized from ethanol to yield white crystals
1
(
73 %). H NMR (500 MHz, DMSO-d ) δ 12.20 (s, 1H), 8.13 – 8.10 (m, 1H), 8.07 (s, 1H),
6
1
3
7
.82 – 7.78 (m, 1H), 7.65 (dd, J = 8.2, 0.6 Hz, 1H), 7.53 – 7.49 (m, 1H). C NMR (126 MHz,
DMSO-d ) δ 160.88, 148.88, 145.52, 134.43, 127.32, 126.86, 125.96, 122.77.
6
6
,7-Dimethoxyquinazolin-4(3H)-one (2). Synthesized from 2-amino-4,5-dimethoxybenzoic
acid (5 mmol, 1 g) and formamide (20 mmol, 0.92 g). The product was recrystallized from
1
ethanol to yield brown solid (48 %). H NMR (500 MHz, DMSO-d ) δ 12.02 (s, 1H), 7.96 (s,
6
1
3
1
1
H), 7.43 (s, 1H), 7.12 (s, 1H), 3.89 (s, 3H), 3.86 (s, 3H). C NMR (126 MHz, DMSO-d ) δ
6
60.13, 154.58, 148.68, 144.99, 143.92, 115.73, 108.15, 105.08, 56.03, 55.81.
Method B: To a solution of 20 mmol anthranilamide and 20 mmol substituted benzaldehyde
in DMF (20 ml) were added 25 mmol iodine and 20 mmol anhydrous potassium carbonate.
Then, the mixture was stirred under for 4-5 hours at 90 °C. After the completion of the
reaction, the mixture was poured on to crushed ice resulting in a precipitation of solid. The
1
7

ACCEPTED MANUSCRIPT
precipitate was filtered and washed with 100 ml 20% solution of sodium thiosulfate, to
remove residual iodine, and finally with water.
2
-Phenylquinazoline-4(3H)-one (3). Synthesized from anthranilamide and benzaldehyde.
1
The product was recrystallized from ethanol to yield pale yellow solid (71 %). H NMR (500
MHz, DMSO-d ) δ 12.41 (s, 1H), 8.19 – 8.17 (m, 2H), 8.15 (dd, J = 7.9, 1.6 Hz, 1H), 7.85 –
6
7
1
1
2
3
.80 (m, 1H), 7.75 – 7.71 (m, 1H), 7.58 – 7.56 (m, 1H), 7.56 – 7.52 (m, 2H), 7.51 – 7.49 (m,
1
3
H). C NMR (126 MHz, DMSO-d ) δ 162.55, 152.64, 148.85, 134.64, 133.00, 131.46,
6
28.70 (2C), 127.89 (2C), 127.52, 126.63, 125.98, 121.13.
-(3,4-Dimethoxyphenyl)quinazolin-4(3H)-one (4). Synthesized from anthranilamide and
,4-dimethoxybenzaldehyde. The product was recrystallized from ethanol to yield pale brown
1
solid (44 %). H NMR (500 MHz, DMSO-d ) δ 12.32 (s, 1H), 8.12 (dd, J = 8.1, 1.3 Hz, 1H),
6
7
.87 (dd, J = 8.5, 2.2 Hz, 1H), 7.81 (d, J = 2.2 Hz, 1H), 7.80 – 7.77 (m, 1H), 7.70 (dd, J = 8.1,
.5 Hz, 1H), 7.49 – 7.44 (m, 1H), 7.10 (dd, J = 8.2, 4.0 Hz, 1H), 3.88 (s, 3H), 3.84 (s, 3H).
0
1
3
C NMR (126 MHz, DMSO-d ) δ 162.67, 152.17, 151.72, 149.01, 148.70, 134.58, 127.32,
6
1
26.17, 125.96, 125.05, 121.29, 120.83, 111.55, 110.92, 55.84, 55.83.
General procedure for the Preparation of substituted 4-chloroquinazolines (5-8). The
selected quinazolin-4(3H)-one (10 mmol) was mixed with 10 ml phosphoryl chloride and was
then stirred under reflux for 9 hours. After completion of the reaction the solvent was
evaporated under reduced pressure. Ice-water was added to the residue and the formed
precipitate was neutralized with ammonium hydroxide and was filtered off.
4
-Chloroquinazoline (5). The product was synthesized from compound (1) and recrystallized
1
from ethanol to yield pale yellow solid (50 %). H NMR (500 MHz, DMSO-d ) δ 8.62 (s,
6
1
H), 8.17 – 8.12 (m, 1H), 7.91 – 7.86 (m, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.63 – 7.57 (m, 1H).
1
3
C NMR (126 MHz, DMSO-d ) δ 160.01, 147.16, 144.23, 135.22, 127.85, 126.40, 124.24,
6
1
22.02.
1
8

ACCEPTED MANUSCRIPT
4
-Chloro-6,7-dimethoxyquinazoline (6). The product was synthesized from compound (2)
1
and recrystallized from ethanol to yield pale yellow solid (64 %). H NMR (500 MHz,
1
3
DMSO-d ) δ 8.86 (s, 1H), 7.42 (s, 1H), 7.36 (s, 1H), 4.00 (s, 3H), 3.98 (s, 3H). C NMR (126
6
MHz, DMSO-d ) δ 158.00, 156.88, 152.31, 151.53, 148.72, 118.72, 107.00, 102.37, 56.65,
6
5
6.30.
4
-Chloro-2-phenylquinazoline (7). The product was synthesized from compound (3) and
1
recrystallized from ethanol to yield brown solid (68 %). H NMR (500 MHz, DMSO-d ) δ
6
1
3
8
.19 – 8.12 (m, 3H), 7.90 – 7.85 (m, 2H), 7.67 – 7.62 (m, 1H), 7.61 – 7.55 (m, 3H). C NMR
(
126 MHz, DMSO-d ) δ 161.80, 153.93, 146.17, 135.15, 132.29, 131.09, 128.78, 128.50,
6
1
27.31, 126.21, 125.70, 120.75.
4
-Chloro-2-(3,4-dimethoxyphenyl)quinazoline (8). The product was synthesized from
1
compound (4) and recrystallized from ethanol to yield brown solid (91 %). H NMR (500
MHz, DMSO-d ) δ 8.22 (dt, J = 8.3, 0.9 Hz, 1H), 8.09 (dd, J = 8.5, 2.0 Hz, 1H), 8.06 (d, J =
6
3
3
1
.5 Hz, 2H), 8.00 (d, J = 2.0 Hz, 1H), 7.79 – 7.75 (m, 1H), 7.13 (d, J = 8.5 Hz, 1H), 3.89 (s,
13
H), 3.85 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 161.79, 158.95, 152.05, 151.43, 149.03,
6
35.91, 128.86, 128.60, 128.41, 125.74, 122.11, 121.55, 111.75, 110.97, 55.80, 55.71.
General procedure for the preparation of 4-anilinoquinazolines (9-16). A mixture of
substituted 4-chloroquinazoline (2 mmol) and 3- or 4-aminoacetophenone (2 mmol) was
dissolved in hot 2-propanol (25 ml) and refluxed for 1-2 hours. After completion of the
reaction the formed precipitate was filtered off and washed with 2-propanol (15 ml). The
product was recrystallized from ethanol.
1
-(4-(quinazolin-4-ylamino)phenyl)ethanone (9). The product was synthesized from 4-
chloroquinazoline (5) (1.9 mmol, 312 mg) and 4-aminoacetophenone (1.9 mmol, 256 mg) to
1
yield pale yellow solid (58 %). H NMR (500 MHz, DMSO-d ) δ 11.92 (s, 1H), 9.06 (d, J =
6
8
.3 Hz, 1H), 8.99 (s, 1H), 8.14 – 8.09 (m, 1H), 8.09 – 8.03 (m, 3H), 8.01 – 7.97 (m, 2H), 7.90
13
–
7.85 (m, 1H), 2.60 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 197.05, 160.04, 151.20,
6
1
9

ACCEPTED MANUSCRIPT
1
2
1
41.30, 139.39, 136.45, 134.47, 130.61, 129.00 (2C), 128.81, 125.23, 124.30, 120.23, 113.95,
6.83.
-(3-(quinazolin-4-ylamino)phenyl)ethanone (10). The product was synthesized from 4-
chloroquinazoline (5) (1.9 mmol, 312 mg) and 3-aminoacetophenone (1.9 mmol, 256 mg) to
1
yield pale yellow solid (49 %) H NMR (500 MHz, DMSO-d ) δ 11.98 (s, 1H), 9.04 (d, J =
6
8
.1 Hz, 1H), 8.95 (s, 1H), 8.32 (t, J = 1.8 Hz, 1H), 8.14 – 8.09 (m, 1H), 8.08 – 8.01 (m, 2H),
1
3
7
.94 – 7.91 (m, 1H), 7.89 – 7.85 (m, 1H), 7.64 (t, J = 7.9 Hz, 1H), 2.61 (s, 3H). C NMR
(
126 MHz, DMSO-d ) δ 197.49, 160.18, 151.17, 138.96, 137.49, 137.35, 136.42, 129.51,
6
1
29.30, 128.79, 126.60, 125.16, 124.34, 119.95, 113.71, 26.95.
1
-(4-((6,7-dimethoxyquinazolin-4-yl)amino)phenyl)ethanone (11). The product was
synthesized from 4-chloro-6,7-dimethoxyquinazoline (6) (1.25 mmol, 280 mg) and 4-
1
aminoacetophenone (1.25 mmol, 168 mg) to yield pale yellow solid (94 %). H NMR (500
MHz, DMSO-d ) δ 11.65 (s, 1H), 8.87 (s, 1H), 8.46 (s, 1H), 8.04 – 8.01 (m, 2H), 7.98 – 7.94
6
1
3
(
m, 2H), 7.41 (s, 1H), 4.03 (s, 3H), 3.98 (s, 3H), 2.59 (s, 3H). C NMR (126 MHz, DMSO-
d6) δ 196.95, 158.11, 156.58, 150.42, 148.66, 141.64, 136.19, 133.99, 128.88 (2C), 124.03
2C), 107.81, 104.37, 99.96, 57.26, 56.59, 26.74
-(3-((6,7-dimethoxyquinazolin-4-yl)amino)phenyl)ethanone (12). The product was
(
1
synthesized from 4-chloro-6,7-dimethoxyquinazoline (6) (2.5 mmol, 561 mg) and 3-
1
aminoacetophenone (2.5 mmol, 338 mg) to yield white solid (94 %). H NMR (500 MHz,
DMSO-d ) δ 11.61 (s, 1H), 8.83 (s, 1H), 8.41 (s, 1H), 8.27 (t, J = 1.8 Hz, 1H), 8.06 – 8.01 (m,
6
1
2
1
2
1
H), 7.91 – 7.87 (m, 1H), 7.62 (t, J = 7.9 Hz, 1H), 7.39 (s, 1H), 4.03 (s, 3H), 3.99 (s, 3H),
13
.61 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 197.57, 158.36, 156.53, 150.41, 148.82,
6
37.65, 137.45, 135.90, 129.41, 129.22, 126.20, 124.09, 107.52, 104.28, 99.96, 57.19, 56.61,
6.95.
-(4-((2-phenylquinazolin-4-yl)amino)phenyl)ethanone (13). The product was synthesized
from 4-chloro-2-phenylquinazoline (7) (9 mmol, 2.2 g) and 4-aminoacetophenone (9 mmol,
2
0

ACCEPTED MANUSCRIPT
1
1
.2 g) to yield yellow solid (88 %). H NMR (500 MHz, DMSO-d ) δ 11.50 (s, 1H), 8.95 (d, J
6
=
8.2 Hz, 1H), 8.45 – 8.41 (m, 2H), 8.31 (d, J = 8.2 Hz, 1H), 8.11 (s, 4H), 8.08 (t, J = 7.7 Hz,
1
H), 7.81 (t, J = 7.5 Hz, 1H), 7.69 (t, J = 7.1 Hz, 1H), 7.63 (t, J = 7.4 Hz, 2H), 2.62 (s, 3H).
1
3
C NMR (126 MHz, DMSO-d ) δ 197.01, 159.05, 157.78, 141.98, 135.81 (2C), 133.84,
6
1
33.00, 129.23 (2C), 129.13 (4C), 128.05, 124.62, 123.41 (2C), 113.31, 26.79. Two
quaternary C atoms are missing.
-(3-((2-phenylquinazolin-4-yl)amino)phenyl)ethanone (14). The product was synthesized
1
from 4-chloro-2-phenylquinazoline (7) (1 mmol, 240 mg) and 3-aminoacetophenone (1 mmol,
1
1
35 mg) to yield yellow solid (88 %). H NMR (500 MHz, DMSO-d ) δ 11.68 (s, 1H), 8.97
6
(d, J = 8.2 Hz, 1H), 8.59 (s, 1H), 8.44 (d, J = 7.3 Hz, 2H), 8.37 (d, J = 8.3 Hz, 1H), 8.15 (dd, J
=
8.0, 1.3 Hz, 1H), 8.08 (t, J = 7.6 Hz, 1H), 7.92 (d, J = 7.8 Hz, 1H), 7.82 (t, J = 7.6 Hz, 1H),
1
3
7
1
1
1
.71 – 7.65 (m, 2H), 7.61 (t, J = 7.6 Hz, 2H), 2.63 (s, 3H). C NMR (126 MHz, DMSO-d ) δ
6
97.58, 159.12 (2C), 157.51, 137.83, 137.40 (2C), 135.90, 133.24, 129.34 (2C), 129.23 (2C),
29.06 (2C), 128.66, 128.14, 126.01, 124.66, 123.61, 113.03, 26.94.
-(4-((2-(3,4-dimethoxyphenyl)quinazolin-4-yl)amino)phenyl)ethanone (15). Synthesized
from 4-chloro-2-(3,4-dimethoxyphenyl)quinazoline (8) (1.2 mmol, 360 mg) and 4-
1
aminoacetophenone (1.2 mmol, 162 mg) to yield yellow solid (86 %). H NMR (500 MHz,
DMSO-d6) δ 11.58 (s, 1H), 8.88 (d, J = 8.3 Hz, 1H), 8.46 (d, J = 8.0 Hz, 1H), 8.14 – 8.11 (m,
2
3
1
1
H), 8.10 – 8.07 (m, 2H), 8.07 – 8.03 (m, 3H), 7.77 (t, J = 7.6 Hz, 1H), 7.20 – 7.17 (m, 1H),
1
3
.88 (s, 3H), 3.87 (s, 3H), 2.61 (s, 3H). C NMR (126 MHz, DMSO) δ 197.00, 158.72,
56.64, 153.55, 148.88, 141.67, 135.96 (2C), 134.12, 128.98, 128.93 (2C), 127.87, 124.67,
23.93, 123.78, 112.78, 112.17, 111.82, 56.04, 55.97, 26.80. Two quaternary C atoms are
missing.
-(3-((2-(3,4-dimethoxyphenyl)quinazolin-4-yl)amino)phenyl)ethanone (16). Synthesized
1
from 4-chloro-2-(3,4-dimethoxyphenyl)quinazoline (8) (0.67 mmol, 200 mg) and 3-
1
aminoacetophenone (0.67 mmol, 91 mg) to yield yellow solid (88 %). H NMR (500 MHz,
2
1

ACCEPTED MANUSCRIPT
DMSO-d6) δ 11.48 (s, 1H), 8.82 (d, J = 8.1 Hz, 1H), 8.46 (s, 1H), 8.36 (d, J = 8.1 Hz, 1H),
8
.14 (d, J = 7.8 Hz, 1H), 8.11 (dd, J = 8.5, 2.0 Hz, 1H), 8.07 (t, J = 7.6 Hz, 1H), 8.04 (d, J =
.1 Hz, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.79 (t, J = 7.7 Hz, 1H), 7.69 (t, J = 7.9 Hz, 1H), 7.19
2
1
3
(
d, J = 8.6 Hz, 1H), 3.88 (s, 3H), 3.83 (s, 3H), 2.64 (s, 3H). C NMR (126 MHz, DMSO-d ) δ
6
1
1
97.63, 158.85, 156.67, 153.60, 148.89, 137.69, 137.40, 135.93, 129.16, 129.09, 127.89,
26.29, 124.63, 123.86, 123.80, 112.64, 112.20, 111.72, 56.06, 55.99, 26.97. Two quaternary
C atoms are missing.
Preparation of quinazoline-chalcones. General procedure for synthesis of compound 17-
3
8. A mixture of 1 eq. 4-anilinoquinazoline, 1eq. substituted benzaldehyde and 7 eq. lithium
hydroxide in 5 ml methanol was stirred under microwave irradiation (120W, 100°C) for 30
minutes. After completion of the reaction the solution was acidified with diluted hydrochloric
acid. Then the solvent was evaporated under reduced pressure and the product was
recrystallized from ethanol/water (1:1).
(E)-3-(3,4-dimethoxyphenyl)-1-(3-(quinazolin-4-ylamino)phenyl)prop-2-en-1-one (17).
The product was synthesized from 1-(3-(quinazolin-4-ylamino)phenyl)ethanone (10) and 3,4-
1
dimethoxybenzaldehyde to yield yellow solid (34 %). H NMR (500 MHz, DMSO-d ) δ 11.87
6
(
s, 1H), 9.00 (d, J = 8.1 Hz, 1H), 8.96 (s, 1H), 8.47 (t, J = 1.8 Hz, 1H), 8.15 – 8.12 (m, 2H),
.10 (dd, J = 8.8, 1.1 Hz, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.91 – 7.87 (m, 1H), 7.82 (d, J = 15.5
Hz, 1H), 7.75 (d, J = 15.5 Hz, 1H), 7.70 (t, J = 7.9 Hz, 1H), 7.54 (d, J = 1.9 Hz, 1H), 7.41 (dd,
8
1
3
J = 8.4, 1.9 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 3.86 (s, 3H), 3.82 (s, 3H). C NMR (126 MHz,
DMSO-d ) δ 188.66, 160.11, 151.63, 151.38, 149.22, 145.18, 139.32, 138.60, 137.51, 136.38,
6
1
1
29.35, 129.14, 128.80, 127.56, 126.72, 125.00, 124.63, 124.18, 120.27, 119.67, 113.77,
+
11.80, 111.14, 55.99, 55.80. LC-MS (m/z): positive mode 412 [M+H] . Purity by HPLC-UV
(254 nm)-ESI-MS: 96 %.
(E)-3-(4-methoxyphenyl)-1-(3-(quinazolin-4-ylamino)phenyl)prop-2-en-1-one (18).
2
2

ACCEPTED MANUSCRIPT
The product was synthesized from 1-(3-(quinazolin-4-ylamino)phenyl)ethanone (10) and 4-
1
methoxybenzaldehyde to yield ochre solid (33 %). H NMR (500 MHz, DMSO-d ) δ 11.94 (s,
6
1
–
=
H), 9.02 (d, J = 8.3 Hz, 1H), 8.97 (s, 1H), 8.46 (t, J = 1.8 Hz, 1H), 8.15 – 8.10 (m, 2H), 8.09
8.05 (m, 1H), 8.02 (d, J = 7.7 Hz, 1H), 7.91 – 7.88 (m, 1H), 7.87 – 7.84 (m, 2H), 7.77 (d, J
1
3
3.0 Hz, 2H), 7.69 (t, J = 7.9 Hz, 1H), 7.04 – 6.99 (m, 2H), 3.82 (s, 3H). C NMR (126
MHz, DMSO-d ) δ 188.58, 161.67, 160.18, 151.26, 144.65, 139.04, 138.56, 137.43, 136.43,
6
1
31.02 (2C), 129.38, 129.26, 128.81, 127.36, 126.73, 125.07, 124.81, 120.05, 119.54, 114.60
+
(
2C), 113.72, 55.56. LC-MS (m/z): positive mode 382 [M+H] . Purity by HPLC-UV (254
nm)-ESI-MS: 98 %.
E)-3-(3,4-dimethoxyphenyl)-1-(3-((6,7-dimethoxyquinazolin-4-yl)amino)phenyl) prop-2-
(
en-1-one (19). The product was synthesized from 1-(3-((6,7-dimethoxyquinazolin-4-
yl)amino)phenyl)ethanone (12) and 3,4-dimethoxybenzaldehyde to yield yellow solid (30 %).
1
H NMR (500 MHz, DMSO-d ) δ 9.66 (s, 1H), 8.49 (s, 1H), 8.37 (t, J = 1.9 Hz, 1H), 8.28 –
6
8
.24 (m, 1H), 7.95 – 7.93 (m, 1H), 7.89 (s, 1H), 7.80 (d, J = 15.5 Hz, 1H), 7.74 (d, J = 15.5
Hz, 1H), 7.59 (t, J = 7.9 Hz, 1H), 7.53 (d, J = 2.0 Hz, 1H), 7.40 (dd, J = 8.4, 2.0 Hz, 1H), 7.20
(s, J = 1.9 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 3.98 (s, 3H), 3.94 (s, 3H), 3.85 (s, 3H), 3.82 (s,
1
3
3
1
1
H). C NMR (126 MHz, DMSO-d ) δ 189.23, 156.45, 154.52, 152.89, 151.53, 149.22,
6
49.16, 147.26, 144.83, 140.27, 138.44, 128.92, 127.66, 126.77, 124.03, 123.71, 121.63,
19.96, 111.81, 111.11, 109.09, 107.39, 102.05, 56.40, 55.99, 55.92, 55.79. LC-MS (m/z):
+
positive mode 472 [M+H] . Purity by HPLC-UV (254 nm)-ESI-MS: 99 %.
(E)-1-(3-((6,7-dimethoxyquinazolin-4-yl)amino)phenyl)-3-(4-methoxyphenyl)prop-2-en-
1
-one (20). The product was synthesized from 1-(3-((6,7-dimethoxyquinazolin-4-
yl)amino)phenyl)ethanone (12) and 4-methoxybenzaldehyde to yield pale yellow solid (13
1
%
). H NMR (500 MHz, DMSO-d ) δ 9.65 (s, 1H), 8.49 (s, 1H), 8.37 (s, 1H), 8.25 (dd, J =
6
8
.0, 1.6 Hz, 1H), 7.92 (d, J = 7.7 Hz, 1H), 7.89 – 7.83 (m, 3H), 7.76 (d, J = 1.6 Hz, 2H), 7.58
(t, J = 7.9 Hz, 1H), 7.20 (s, 1H), 7.02 (d, J = 8.8 Hz, 2H), 3.98 (s, 3H), 3.94 (s, 3H), 3.82 (s,
2
3

ACCEPTED MANUSCRIPT
1
3
3
1
1
H). C NMR (126 MHz, DMSO-d ) δ 189.15, 161.58, 156.43, 154.51, 152.89, 149.15,
6
47.25, 144.29, 140.25, 138.39, 130.93 (2C), 128.94, 127.44, 126.76, 123.62, 121.64, 119.82,
14.61 (2C), 109.07, 107.38, 102.03, 56.39, 55.98, 55.54. LC-MS (m/z): positive mode 442
+
[
M+H] . Purity by HPLC-UV (254 nm)-ESI-MS: 99 %.
(E)-3-(3,4-dimethoxyphenyl)-1-(3-((2-phenylquinazolin-4-yl)amino)phenyl)prop-2-en-1-
one (21). The product was synthesized from 1-(3-((2-phenylquinazolin-4-
yl)amino)phenyl)ethanone (14) and 3,4-dimethoxybenzaldehyde to yield yellow solid (34 %).
1
H NMR (500 MHz, DMSO-d ) δ 11.53 (s, 1H), 8.94 (d, J = 7.6 Hz, 1H), 8.77 (s, 1H), 8.44
6
(
d, J = 7.4 Hz, 2H), 8.28 (d, J = 6.6 Hz, 1H), 8.21 – 8.17 (m, 1H), 8.14 – 8.05 (m, 2H), 7.88 –
.81 (m, 2H), 7.78 (d, J = 15.5 Hz, 1H), 7.73 (t, J = 7.9 Hz, 1H), 7.63 (t, J = 7.2 Hz, 1H), 7.56
t, J = 7.6 Hz, 2H), 7.49 (d, J = 1.9 Hz, 1H), 7.37 (dd, J = 8.3, 1.8 Hz, 1H), 7.00 (d, J = 8.4
7
(
1
3
Hz, 1H), 3.81 (s, 3H), 3.78 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 188.76, 159.07,
6
1
57.78, 151.58, 149.18 (2C), 145.05, 138.48, 138.16, 138.11, 135.69, 129.28 (2C), 129.14
(2C), 129.03 (2C), 128.15, 128.02, 127.58, 125.93, 124.46, 124.13 (2C), 123.89, 119.66 (2C),
+
1
13.18, 111.77, 111.07, 55.87, 55.79. LC-MS (m/z): positive mode 488 [M+H] . Purity by
HPLC-UV (254 nm)-ESI-MS: 96 %.
(E)-3-(4-methoxyphenyl)-1-(3-((2-phenylquinazolin-4-yl)amino)phenyl)prop-2-en-1-one
(22).
The
product
was
synthesized
from
1-(3-((2-phenylquinazolin-4-
1
yl)amino)phenyl)ethanone (14) and 4-methoxybenzaldehyde to yield yellow solid (22 %). H
NMR (500 MHz, DMSO-d ) δ 10.06 (s, 1H), 8.82 (t, J = 1.8 Hz, 1H), 8.62 (d, J = 8.2 Hz,
6
1
H), 8.52 – 8.48 (m, 2H), 8.26 (dd, J = 8.1, 1.4 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.90 – 7.88
(m, 2H), 7.82 – 7.78 (m, 4H), 7.68 – 7.66 (m, 1H), 7.66 – 7.62 (m, 1H), 7.48 – 7.44 (m, 3H),
1
3
6
1
1
.99 (d, J = 8.8 Hz, 2H), 3.81 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 189.16, 161.55,
6
59.15, 158.05, 150.70, 144.24, 140.07, 138.45, 138.39, 133.50, 130.88 (2C), 130.43, 129.05,
28.55 (2C), 128.34, 128.09 (2C), 127.44, 126.32, 126.23, 123.76, 123.18, 121.81, 119.82,
2
4

ACCEPTED MANUSCRIPT
+
1
14.57 (2C), 114.16, 55.54. LC-MS (m/z): positive mode 458 [M+H] . Purity by HPLC-UV
254 nm)-ESI-MS: 95 %.
E)-3-(3-methoxyphenyl)-1-(3-((2-phenylquinazolin-4-yl)amino)phenyl)prop-2-en-1-one
(
(
(23).
The
product
was
synthesized
from
1-(3-((2-phenylquinazolin-4-
1
yl)amino)phenyl)ethanone (14) and 3-methoxybenzaldehyde to yield ochre solid (48 %). H
NMR (500 MHz, DMSO-d ) δ 11.62 (s, 1H), 8.97 (d, J = 8.1 Hz, 1H), 8.78 (s, 1H), 8.44 (d, J
6
=
7.3 Hz, 2H), 8.32 (d, J = 7.6 Hz, 1H), 8.20 (dd, J = 7.91, 1.3 Hz, H), 8.13 (d, J = 7.8 Hz,
H), 8.08 (t, J = 7.7 Hz, 1H), 7.96 (d, J = 15.6 Hz, 1H), 7.85 – 7.76 (m, 2H), 7.73 (t, J = 7.9
Hz, 1H), 7.63 (t, J = 7.3 Hz, 1H), 7.56 (t, J = 7.6 Hz, 2H), 7.45 – 7.42 (m, 1H), 7.39 (d, J =
1
1
3
7
.6 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.02 (dd, J = 8.1, 1.8 Hz, 1H), 3.78 (s, 3H). C NMR
(
126 MHz, DMSO-d ) δ 189.33, 159.77, 159.11, 158.02, 150.68, 144.23, 140.09, 138.33,
6
1
1
38.11, 136.18, 133.49, 130.41, 130.04, 129.09, 128.53 (2C), 128.32, 128.06, 126.56, 126.22,
23.94, 123.16, 122.60, 121.86, 121.67, 116.87, 114.56, 114.13, 113.60, 55.38. LC-MS (m/z):
+
positive mode 458 [M+H] . Purity by HPLC-UV (254 nm)-ESI-MS: 99 %.
(
E)-3-(3,4-dimethoxyphenyl)-1-(3-((2-(3,4-dimethoxyphenyl)quinazolin-4-yl)
amino)phenyl)prop-2-en-1-one (24). The product was synthesized from 1-(3-((2-(3,4-
dimethoxyphenyl)quinazolin-4-yl)amino)phenyl)ethanone (16) and 3,4-
dimethoxybenzaldehyde to yield pale yellow solid (31 %). H NMR (500 MHz, DMSO-d ) δ
1
6
1
1.54 (s, 1H), 8.88 (d, J = 8.3 Hz, 1H), 8.69 (s, 1H), 8.42 – 8.33 (m, 1H), 8.17 – 8.11 (m, 3H),
.08 – 8.04 (m, 2H), 7.84 (d, J = 15.5 Hz, 1H), 7.81 – 7.77 (m, 2H), 7.76 – 7.71 (m, 1H), 7.48
8
(d, J = 1.9 Hz, 1H), 7.36 (dd, J = 8.4, 1.9 Hz, 1H), 7.09 (d, J = 8.7 Hz, 1H), 7.00 (d, J = 8.4
1
3
Hz, 1H), 3.81 (s, 6H), 3.80 (s, 3H), 3.79 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 188.70,
6
1
1
1
58.88, 156.82, 153.47, 151.61 (2C), 149.17, 148.88, 145.09, 138.42 (2C), 135.87 (2C),
29.20, 128.69, 127.84, 127.55, 126.18, 124.69, 124.35, 124.13, 119.62 (2C), 112.76, 111.71,
12.15, 111.75, 111.10, 55.94 (2C), 55.90, 55.79. One quaternary C atom is missing. LC-MS
+
(
m/z): positive mode 548 [M+H] . Purity by HPLC-UV (254 nm)-ESI-MS: 95 %.
2
5

ACCEPTED MANUSCRIPT
(E)-1-(3-((2-(3,4-dimethoxyphenyl)quinazolin-4-yl)amino)phenyl)-3-(4-methoxy
phenyl)prop-2-en-1-one (25). The product was synthesized from 1-(3-((2-(3,4-
dimethoxyphenyl)quinazolin-4-yl)amino)phenyl)ethanone (16) and 4-methoxybenzaldehyde
1
to yield yellow solid (18 %). H NMR (500 MHz, DMSO-d ) δ 10.03 (s, 1H), 8.78 (t, J = 1.9
6
Hz, 1H), 8.58 (d, J = 8.4 Hz, 1H), 8.30 – 8.25 (m, 1H), 8.10 (dd, J = 8.4, 2.0 Hz, 1H), 8.04 (d,
J = 1.9 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.94 (d, J = 15.6 Hz, 1H), 7.86 (d, J = 3.6 Hz, 2H),
7
7
1
1
1
1
.78 (d, J = 15.6 Hz, 1H), 7.66 (t, J = 7.9 Hz, 1H), 7.62 – 7.58 (m, 1H), 7.45 – 7.43 (m, 1H),
.40 (d, J = 7.7 Hz, 1H), 7.34 (t, J = 7.9 Hz, 1H), 7.04 – 7.00 (m, 1H), 6.98 (d, J = 8.6 Hz,
1
3
H), 3.81 (s, 3H), 3.78 (s, 3H), 3.76 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 189.36,
6
59.79, 158.91, 157.83, 151.04, 150.81, 148.63, 144.28, 140.14, 138.09, 136.16, 133.41,
30.94, 130.05, 128.99, 128.12, 126.75, 125.75, 123.92, 123.14, 122.58, 121.93, 121.70,
21.28, 116.88, 113.89, 113.64, 111.46, 111.07, 55.62, 55.40, 55.36. LC-MS (m/z): positive
+
mode 518 [M+H] . Purity by HPLC-UV (254 nm)-ESI-MS: 97 %.
(E)-1-(3-((2-(3,4-dimethoxyphenyl)quinazolin-4-yl)amino)phenyl)-3-(3-
methoxyphenyl)prop-2-en-1-one (26). The product was synthesized from 1-(3-((2-(3,4-
dimethoxyphenyl)quinazolin-4-yl)amino)phenyl)ethanone (16) and 3-methoxybenzaldehyde
1
to yield pale yellow (20 %). H NMR (500 MHz, DMSO-d ) δ 11.57 (s, 1H), 8.86 (d, J = 8.1
6
Hz, 1H), 8.68 (s, 1H), 8.37 (d, J = 8.4 Hz, 1H), 8.15 (d, J = 7.9 Hz, 2H), 8.12 (dd, J = 8.5, 2.1
Hz, 1H), 8.07 (t, J = 7.8 Hz, 1H), 8.03 (d, J = 2.1 Hz, 1H), 7.95 (d, J = 15.6 Hz, 1H), 7.81 (d,
J = 7.6 Hz, 1H), 7.78 (d, J = 15.6 Hz, 1H), 7.74 (t, J = 7.9 Hz, 1H), 7.43 (d, J = 2.0 Hz, 1H),
7
1
1
1
1
.39 (d, J = 7.7 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.11 (d, J = 8.7 Hz, 1H), 7.02 (dd, J = 8.1,
1
3
.8 Hz, 1H), 3.80 (s, 6H), 3.79 (s, 3H). C NMR (126 MHz, DMSO) δ 188.90, 159.80,
58.91, 156.93, 156.91, 153.47, 148.91, 144.62, 138.12, 137.94, 136.08, 135.95, 130.07,
29.35, 128.91, 127.89, 127.82, 124.48, 124.35, 124.28, 123.51, 122.30, 121.78, 116.95,
13.72, 112.74, 112.01, 111.76, 55.98, 55.84, 55.46. One quaternary C atom is missing. LC-
+
MS (m/z): positive mode 518 [M+H] . Purity by HPLC-UV (254 nm)-ESI-MS: 97 %.
2
6

ACCEPTED MANUSCRIPT
(E)-3-(3,4-dimethoxyphenyl)-1-(4-(quinazolin-4-ylamino)phenyl)prop-2-en-1-one (27).
Synthesized
from
1-(4-(quinazolin-4-ylamino)phenyl)ethanone
(9)
and
3,4-
1
dimethoxybenzaldehyde to obtain 27 as yellow solid (23 %). H NMR (500 MHz, DMSO-d )
6
δ 10.06 (s, 1H), 8.72 (s, J = 8.0 Hz, 1H), 8.62 (d, J = 8.3 Hz, 1H), 8.22 (d, J = 8.9 Hz, 2H),
8
7
3
1
1
.18 (d, J = 8.7 Hz, 2H), 7.90 (t, J = 7.5 Hz, 1H), 7.88 – 7.82 (m, 2H), 7.73 – 7.66 (m, 2H),
.54 (d, J = 1.8 Hz, 1H), 7.38 (dd, J = 8.3, 1.7 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 3.87 (s, 3H),
13
.82 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 187.61, 157.62, 154.34, 151.36, 150.01,
6
49.21, 143.96, 143.92, 133.45, 132.70, 129.51 (2C), 128.10, 127.82, 126.71, 123.93, 123.22,
21.03 (2C), 119.81, 115.50, 111.78, 110.94, 55.92, 55.76. LC-MS (m/z): positive mode 412
+
[
M+H] . Purity by HPLC-UV (254 nm)-ESI-MS: 95 %.
(E)-3-(3-methoxyphenyl)-1-(4-(quinazolin-4-ylamino)phenyl)prop-2-en-1-one
(28).
Synthesized
from
1-(4-(quinazolin-4-ylamino)phenyl)ethanone
(9)
and
3-
1
methoxybenzaldehyde to obtain 28 as mustard solid (44 %). H NMR (500 MHz, DMSO-d )
6
δ 11.05 (s, 1H), 8.90 (s, 1H), 8.84 (d, J = 8.1 Hz, 1H), 8.27 (d, J = 8.8 Hz, 2H), 8.11 (d, J =
8
.7 Hz, 2H), 8.06 – 8.02 (m, 1H), 7.98 (d, J = 15.6 Hz, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.84 –
.79 (m, 1H), 7.73 (d, J = 15.6 Hz, 1H), 7.50 – 7.48 (m, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.37 (t,
7
1
3
J = 7.9 Hz, 1H), 7.03 (dd, J = 8.1, 1.8 Hz, 1H), 3.84 (s, 3H). C NMR (126 MHz, DMSO-d )
6
δ 187.98, 159.83 (2C), 158.96, 152.63, 143.82, 142.62, 136.30, 135.19, 133.94, 130.08,
1
5
29.57 (2C), 127.94, 124.28, 123.72, 122.87, 122.45 (2C), 121.81, 116.81, 114.66, 113.54,
+
5.48. LC-MS (m/z): positive mode 382 [M+H] . Purity by HPLC-UV (254 nm)-ESI-MS: 97
%.
(E)-3-(3,4-dimethoxyphenyl)-1-(4-((6,7-dimethoxyquinazolin-4-yl)amino)phenyl) prop-2-
en-1-one (29). Synthesized from 1-(4-((6,7-dimethoxyquinazolin-4-
yl)amino)phenyl)ethanone (11) and 3,4-dimethoxybenzaldehyde to obtain 29 as mustard solid
1
(
29 %). H NMR (500 MHz, DMSO-d ) δ 11.54 (s, 1H), 8.89 (s, 1H), 8.42 (d, J = 2.0 Hz,
6
1
H), 8.26 (d, J = 8.6 Hz, 2H), 8.04 – 8.00 (m, 2H), 7.86 (d, J = 15.5 Hz, 1H), 7.71 (d, J = 15.5
2
7

ACCEPTED MANUSCRIPT
Hz, 1H), 7.54 (d, J = 1.9 Hz, 1H), 7.41 – 7.37 (m, 2H), 7.02 (d, J = 8.4 Hz, 1H), 4.04 (s, 3H),
1
3
3
1
1
5
.99 (s, 3H), 3.87 (s, 3H), 3.82 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 187.89, 158.10,
6
56.62, 151.49, 150.47, 149.20, 144.50, 141.58, 136.58, 135.00, 129.27 (2C), 127.69, 124.09,
24.00 (2C), 119.66, 111.76, 111.02, 107.92, 104.22, 100.27, 100.25, 57.21, 56.63, 55.94,
+
5.78. LC-MS (m/z): positive mode 472 [M+H] . Purity by HPLC-UV (254 nm)-ESI-MS: 96
%.
(E)-1-(4-((6,7-dimethoxyquinazolin-4-yl)amino)phenyl)-3-(3-methoxy phenyl) prop-2-en-
1
-one (30). Synthesized from 1-(4-((6,7-dimethoxyquinazolin-4-yl)amino)phenyl)ethanone
1
(
11) and 3-methoxybenzaldehyde to obtain 30 as yellow solid (44 %). H NMR (500 MHz,
DMSO-d ) δ 9.73 (s, 1H), 8.58 (s, 1H), 8.23 (d, J = 8.8 Hz, 2H), 8.11 (d, J = 8.8 Hz, 2H), 7.97
6
(d, J = 15.6 Hz, 1H), 7.88 (s, 1H), 7.71 (d, J = 15.6 Hz, 1H), 7.48 (d, J = 2.1 Hz, 1H), 7.44 (d,
J = 7.8 Hz, 1H), 7.37 (t, J = 7.9 Hz, 1H), 7.23 (s, 1H), 7.02 (dd, J = 7.9, 2.1 Hz, 1H), 3.99 (s,
1
3
3
1
1
H), 3.94 (s, 3H), 3.84 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 187.63, 159.82, 155.99,
6
54.70, 152.68, 149.31, 147.53, 144.62, 143.31, 136.40, 131.94, 130.04, 129.69 (2C), 122.52,
21.68, 120.76 (2C), 116.64, 113.50, 109.42, 107.40, 102.00, 56.43, 56.01, 55.45. LC-MS
+
(
m/z): positive mode 442 [M+H] . Purity by HPLC-UV (254 nm)-ESI-MS: 97 %.
(E)-3-(3,4-dimethoxyphenyl)-1-(4-((2-phenylquinazolin-4-yl)amino)phenyl)prop-2-en-1-
one (31). Synthesized from 1-(4-((2-phenylquinazolin-4-yl)amino)phenyl)ethanone (13) and
1
3
,4-dimethoxybenzaldehyde to obtain 31 as yellow solid (16 %). H NMR (500 MHz,
DMSO-d ) δ 10.15 (s, 1H), 8.64 (d, J = 8.4 Hz, 1H), 8.50 (dd, 2H), 8.32 (d, J = 8.8 Hz, 2H),
6
8
7
3
1
1
.26 (d, J = 8.8 Hz, 2H), 7.94 – 7.88 (m, 3H), 7.73 (d, J = 15.4 Hz, 1H), 7.69 – 7.64 (m, 1H),
.59 – 7.50 (m, 4H), 7.41 (dd, J = 8.3, 1.8 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 3.88 (s, 3H),
13
.82 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 187.55, 159.10, 157.89, 151.37, 150.73,
6
49.22, 144.10, 144.02, 138.23, 133.70, 132.66, 130.61, 129.65 (2C), 128.71 (2C), 128.34,
28.11 (2C), 127.86, 126.41, 123.96, 123.31, 120.97 (2C), 119.76, 114.32, 111.78, 111.09,
2
8

ACCEPTED MANUSCRIPT
+
5
5.97, 55.77. LC-MS (m/z): positive mode 488 [M+H] . Purity by HPLC-UV (254 nm)-ESI-
MS: 96 %.
(E)-3-(3-methoxyphenyl)-1-(4-((2-phenylquinazolin-4-yl)amino)phenyl)prop-2-en-1-one
(32). Synthesized 1-(4-((2-phenylquinazolin-4-yl)amino)phenyl)ethanone (13) and 3-
1
methoxybenzaldehyde to obtain 32 as yellow solid (44 %). H NMR (600 MHz, DMSO-d ) δ
6
1
2
1
1
0.15 (s, 1H), 8.64 (d, J = 8.2 Hz, 1H), 8.52 – 8.49 (m, 2H), 8.33 (d, 2H), 8.28 (d, J = 8.8 Hz,
H), 8.04 (d, J = 15.6 Hz, 1H), 7.93 – 7.90 (m, 2H), 7.74 (d, J = 15.5 Hz, 1H), 7.68 – 7.64 (m,
H), 7.58 – 7.54 (m, 2H), 7.54 – 7.52 (m, 1H), 7.51 (d, J = 1.8 Hz, 1H), 7.46 (d, J = 7.6 Hz,
1
3
H), 7.38 (t, J = 7.9 Hz, 1H), 7.03 (dd, J = 8.1, 2.0 Hz, 1H), 3.84 (s, 3H). C NMR (151
MHz, DMSO-d ) δ 187.63, 159.80, 159.06, 157.84, 150.78, 144.35, 143.45, 138.23, 136.38,
6
1
1
33.67, 132.25, 130.57, 130.02, 129.80 (2C), 128.68 (2C), 128.37, 128.07 (2C), 126.39,
23.28, 122.45, 121.75, 120.90 (2C), 116.63, 114.31, 113.57, 55.45. LC-MS (m/z): positive
+
mode 458 [M+H] . Purity by HPLC-UV (254 nm)-ESI-MS: 97 %.
(E)-3-(4-methoxyphenyl)-1-(4-((2-phenylquinazolin-4-yl)amino)phenyl)prop-2-en-1-one
(33). Synthesized 1-(4-((2-phenylquinazolin-4-yl)amino)phenyl)ethanone (13) and 4-
1
methoxybenzaldehyde to obtain 33 as yellow solid (41 %). H NMR (500 MHz, DMSO-d ) δ
6
1
0.13 (s, 1H), 8.64 (d, J = 8.4 Hz, 1H), 8.53 – 8.48 (m, 2H), 8.32 – 8.28 (m, 2H), 8.28 – 8.25
(m, 2H), 7.92 – 7.91 (m, 2H), 7.90 – 7.85 (m, 3H), 7.74 (d, J = 15.5 Hz, 1H), 7.68 – 7.64 (m,
1
3
1
H), 7.58 – 7.49 (m, 3H), 7.06 – 7.03 (m, 2H), 3.83 (s, 3H). C NMR (126 MHz, DMSO-d )
6
δ 187.53, 161.41, 159.08, 157.85, 150.78, 144.09, 143.41, 138.26, 133.66, 132.60, 130.83
(2C), 130.57, 129.60 (2C), 128.68 (2C), 128.38, 128.08 (2C), 127.64, 126.38, 123.27, 120.93
+
(
2C), 119.71, 114.53 (2C), 114.31, 55.51. LC-MS (m/z): positive mode 458 [M+H] . Purity
by HPLC-UV (254 nm)-ESI-MS: 99 %.
E)-3-phenyl-1-(4-((2-phenylquinazolin-4-yl)amino)phenyl)prop-2-en-1-one (34).
(
Synthesized 1-(4-((2-phenylquinazolin-4-yl)amino)phenyl)ethanone (13) and benzaldehyde to
1
obtain 34 as yellow solid (43 %). H NMR (500 MHz, DMSO-d ) δ 11.51 (s, 1H), 8.96 (d, J =
6
2
9