Investigation of quinazolines as inhibitors of breast cancer resistance protein (ABCG2)

Bioorganic & Medicinal Chemistry 21 (2013) 7858–7873

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Investigation of quinazolines as inhibitors of breast cancer resistance

protein (ABCG2)

⇑

Kapil Juvale, Jennifer Gallus, Michael Wiese

Pharmaceutical Institute, University of Bonn, Pharmaceutical Chemistry II, An der Immenburg 4, 53121 Bonn, Germany

a r t i c l e i n f o

a b s t r a c t

Article history:

Chemotherapy is one of the major forms of cancer treatment. Unfortunately, tumors are prone to multi-

drug resistance leading to failure of treatment. Breast cancer resistance protein (BCRP), the second mem-

ber of ABC transporter subfamily G, has been found to play a major role in drug efﬂux and hence

multidrug resistance. Until now, very few potent and selective BCRP inhibitors like Ko143 have been

identiﬁed. In the search for more potent and selective BCRP inhibitors, we synthesized and investigated

a series of differently substituted quinazoline compounds. Several variations at positions 2, 4, 6 and 7 of

the quinazoline scaffold were carried out to develop a structure–activity-relationship analysis for these

compounds. It was found that compounds bearing a phenyl substituent at position 2 of the 4-anilinoqui-

nazoline scaffold were most potent. On the aniline ring at position 4 of the quinazoline moiety substit-

uents like NO₂, CN, CF₃led to very high BCRP inhibition potencies. The most potent compounds were

further investigated for their intrinsic cytotoxicity and their ability to reverse the multidrug resistance.

Compound 20, an anilinoquinazoline bearing a phenyl ring at position 2 and meta-nitro substitution

on the 4-anilino ring, was found to have the highest therapeutic ratio. The most active compounds from

each variation were also investigated for their effect on BCRP expression. It was found that compound 20

has no signiﬁcant effect on BCRP expression, while compound 31 decreased the surface BCRP expression.

The only difference in the two compounds was the presence of a 3,4-dimethoxyphenyl ring in compound

31 instead of phenyl substitution at position 2 of the quinazoline moiety. From the study of all target

compounds, compound 20 was the most prominent compound having inhibitory potency even higher

than Ko143, the most potent BCRP inhibitor known. Compound 20 was also found to be selective towards

BCRP with a very high therapeutic ratio.

Received 6 August 2013

Revised 1 October 2013

Accepted 7 October 2013

Available online 17 October 2013

Keywords:

ATP binding cassette (ABC) transporter

Breastcancerresistanceprotein(BCRP/ABCG2)

Multidrug resistance

Quinazolines

Hoechst 33342 accumulation assay

1. Introduction

helices and one NBD, it is assumed that BCRP forms a homo- or het-

erodimer or some higher oligomers for its functionality.

Occurrence of multidrug resistance (MDR) in tumors is a major

cause of the failure of chemotherapy in the treatment of cancer. In

most cases an overexpression of multispeciﬁc ATP-binding cassette

(ABC) transporter proteins has been observed.¹In humans there

are 48 ABC genes identiﬁed so far, which can be divided into seven

subfamilies from ABC A to ABC G. Among these, the most impor-

tant and best examined multispeciﬁc ABC transporters are P-glyco-

protein (P-gp, ABCB1), multidrug resistance-associated protein 1

(MRP1, ABCC1) and breast cancer resistance protein (BCRP,

ABCG2).²BCRP is the most recently described ABC transporter pro-

tein. It was ﬁrst cloned in 1998 by Doyle et al. and consists of 665

amino acids, with a molecular weight of 72 kDa.³The functional

unit of ABC transporters consists of at least two transmembrane

domains (TMDs) and two nucleotide binding domains (NBDs).⁴

Owing to presence of only one TMD, bearing six transmembrane

One of the strategies to overcome MDR caused by ABC trans-

porters is the direct inhibition of these transporter proteins.

Although inhibition of P-gp in AML patients has not been shown

to result in a beneﬁt for patients,⁵inhibition of BCRP is likely to in-

crease levels of chemotherapeutics within the brain and brain

tumors.⁶In contrast to P-gp, only a few inhibitors of BCRP are

known. Several efforts have been undertaken to identify potent

and selective BCRP inhibitors. Very few potent, selective and non-

toxic BCRP inhibitors like Ko143 have been identiﬁed until now.⁷In

the recent past, a few quinazoline derivatives have been shown to

be substrates as well as inhibitors of ABC transporters. Prazosin, a

quinazoline compound and an

a-adrenergic blocker has been

shown to be transported by BCRP in several studies.^8,9It was also

shown to be transported by P-gp to a lesser extent by Zhou et al.¹⁰

BODIPY-prazosin, a ﬂuorescent derivative, is used widely for trans-

port activity studies on BCRP.¹¹Kodaira et al. have shown the sub-

strate nature of another tyrosine kinase inhibitor (TKI) quinazoline,

erlotinib.¹²

⇑

Corresponding author. Tel.: +49 228 735213; fax: +49 228 737929.

E-mail address: mwiese@uni-bonn.de (M. Wiese).

http://dx.doi.org/10.1016/j.bmc.2013.10.007

K. Juvale et al. / Bioorg. Med. Chem. 21 (2013) 7858–7873

7859

Scheme 1. General scheme for synthesis of target compounds 9–49. Reagents and conditions: (a) DMF, I₂, K₂CO₃70–90 °C, 4–5 h; (b) 150 °C, 10–18 h; (c) POCl₃, reﬂux, 8 h;

(d) substituted aniline, isopropanol, reﬂux, 3–4 h.

total and surface expression of BCRP in EGFR-positive MDCK BCRP

cells by interacting with the PI3K/Akt signaling pathway.¹⁶

In the study done by Pick et al., a few TKI quinazoline com-

pounds were found to be potent inhibitors of BCRP with IC₅₀values

in the sub-micromolar range. But these compounds lack variation

in the substituents present on the quinazoline moiety. Recently

we showed that 2-phenyl substituted 4-anilinoquinazolines are

potent inhibitors of BCRP.¹⁷In the current work we investigated

more quinazolines with the aim of ﬁnding more potent compounds

Scheme 2. General scheme for synthesis of target compounds 50–54. Reagents and

conditions: (a) aniline, isopropanol, reﬂux, 12–24 h.

and possibly identifying the structural requirements for BCRP inhi-

bition. All compounds were additionally investigated for selectivity

over P-gp and MRP1, cytotoxicity and their effect on BCRP

expression.

Recently a few TKI quinazolines were also identiﬁed to be good

inhibitors of BCRP. In the ﬁrst reports by Yanase et al., geﬁtinib

(Iressa)was shownto haveaninhibitoryeffect onBCRP,¹³whilelater

Leggas et al. showed the inhibitory effect of this compound on both

P-gp and BCRP.¹⁴Contradictorily geﬁtinib was found to be a sub-

strate ofBCRPby Agarwal etal., where it was shownthat distribution

of geﬁtinib to the brain was limited through its transport by P-gp and

BCRP.¹⁵We recently conﬁrmed the BCRP inhibitory effect of geﬁti-

nib, where we also showed that geﬁtinib was able to decrease the

2. Result and discussion

2.1. Chemistry

The quinazoline TKIs investigated until now for BCRP inhibition

possess substituents only at positions 4, 6 and 7 of the quinazoline

moiety. Hence, in this work compounds with different substitution,

starting with 2-phenyl substituted derivatives were investigated

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K. Juvale et al. / Bioorg. Med. Chem. 21 (2013) 7858–7873

Table 1

Structures and inhibitory potencies of the compounds 9–49 against BCRP

Compound

R²

R⁶

R⁷

R⁰

Hoechst 33342

IC₅₀SD (

M)^*

Pheophorbide A

l

IC₅₀SD (l

M)^*

9^a

Phenyl

3,4-Dimethoxyphenyl

H

2-Br

3-Br

4-Br

2.57 0.28

3.75 0.53

0.57 0.04

6.83 0.97

1.93 0.28

1.66 0.11

2.25 0.07

1.32 0.10

1.93 0.11

0.19 0.01

5.93 0.10

0.13 0.03

0.97 0.24

2.46 0.14

0.14 0.04

0.25 0.10

1.53 0.13

0.15 0.03

1.99 0.20

0.29 0.03

0.42 0.05

0.64 0.08

0.076 0.019

0.30 0.04

0.19 0.01

0.26 0.07

0.14 0.03

0.47 0.05

1.08 0.25

1.18 0.24

1.13 0.37

0.94 0.19

4.79 0.87

1.73 0.15

2.40 0.79

1.06 0.12

3.80 0.53

0.81 0.06

2.92 0.39

7.38 2.19

n.t.

10^a

11^a

12^a

13^a

14^a

15^a

16^a

17^a

18^a

19^a

20^a

21^a

22^a

23^a

24^a

25^a

26

3-Cl

3-Cl,4-F

2-OCH₃

3-OCH₃

4-OCH₃

3,4-OCH₃

2-NO₂

3-NO₂

4-NO₂

3-NO₂,4-F

3-CN

3-CF₃

4-CF₃

3-OH

H

3-Br

3,4-OCH₃

2-NO₂

3-NO₂

4-NO₂

3-CF₃

3-CN

2-OH

3-OH

4-OH

3-Br

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

4.09 1.60

0.40 0.09

0.61 0.04

1.55 0.29

0.092 0.024

0.32 0.09

0.24 0.05

0.30 0.06

0.29 0.03

0.11 0.01

0.68 0.12

1.48 0.19

2.03 0.55

0.89 0.14

1.55 0.33

3.38 0.76

2.32 0.70

1.98 0.42

0.93 0.06

3.97 0.61

0.91 0.36

2.46 0.50

3.79 0.27

3,4-Dimethoxyphenyl

H

3-CN

3-NO₂

3-CF₃

3,4-OCH₃

3-Br

3-CF₃

3-CN

2-NO₂

3-NO₂

4-NO₂

3,4-OCH₃

H

OCH₃

*

Data are expressed as mean SD (n = 3); n.t. = not tested in pheophorbide A assay.

Data for the compounds 9–25 were taken from our previous study.¹⁷

a

for their activity against BCRP. Different combinations of modiﬁca-

tions at position 2, 4, 6 and 7 of the quinazoline moiety were made.

In general, synthesis of target compounds (9–49) ﬁrst involved

preparation of 4-quinazolinones (1–4). The synthesis of 4-quinaz-

olinones was carried out using a modiﬁed Niementowski reac-

acid and formamide. These 4-quinazolinones were further reacted

with excess of phosphoryl chloride to obtain 4-chloroquinazolines

(5–8). The target compounds (9–49) were synthesized by reacting

different substituted anilines with corresponding 4-chloroquinazo-

lines. Reactions involved in the synthesis of compounds 9–49 are

depicted in Scheme 1. Target compounds 50–54 were synthesized

by reacting double equivalent quantity of substituted anilines with

2,4-dichloro-6,7-dimethoxyquinazoline which was procured from

commercial source. The reaction involved in the synthesis of com-

pounds 50–54 is depicted in Scheme 2. All synthesized compounds

were characterized by ¹H NMR, ¹³C NMR and purity of target

tion.¹⁸For the compounds bearing

a phenyl substituent at

position 2 of quinazoline (1–2), the synthesis was carried out using

anthranilamide and a substituted benzaldehyde in presence of io-

dine and potassium carbonate. On the other hand for compounds

bearing no substitution at position 2 of the quinazoline structure

(3–4), the synthesis was carried out using a substituted anthranilic

K. Juvale et al. / Bioorg. Med. Chem. 21 (2013) 7858–7873

7861

Table 2

Structures and inhibitory potencies of the compounds 50–54 against BCRP

Compound

R⁰

Hoechst 33342

IC₅₀SD (

M)^*

Pheophorbide A

IC₅₀SD (

M)^*

l

50

51

52

53

54

Ko143

3-Br

0.67 0.10

0.80 0.02

0.48 0.04

0.33 0.08

1.74 0.26

0.25 0.02

0.86 0.14

0.61 0.15

0.47 0.03

0.32 0.06

1.52 0.30

0.33 0.05

3-CF₃

3-CN

3-NO₂

3,4-OCH₃

—

Figure 2. Scatterplot of pIC₅₀values for ABCG2 inhibition obtained in the Hoechst

33342 assay against pheophorbide A assay for compounds 27–54. Each point

indicates mean of IC₅₀values obtained in three independent experiments and error

bars indicate standard deviation. The squared correlation coefﬁcient r²= 0.94.

^*Data are expressed as mean SD (n = 3).

Figure 3. Structural features of quinazolines as BCRP inhibitors. +: Showed good

activity, ++: showed very high activity, -: led to decrease in activity.

the current work. The most potent and promising BCRP inhibitors

were further investigated for selectivity by checking their ability to

inhibit P-gp and MRP1 using the calcein AM assay. Intrinsic cyto-

toxicity and efﬁcacy to reverse MDR of selected compounds were

determined in the MTT cytotoxicity assay.

2.2.1. Characterization of the cell lines

Before carrying out functional assays using different cell lines,

we ﬁrst conﬁrmed that each cell line has adequate expression lev-

els of corresponding ABC transporter protein.

The MDCK BCRP cell line has been shown by several research

groups to express high levels of BCRP.^19,20This was conﬁrmed by

western blot analysis of a membrane preparation of MDCK BCRP

cells using the BCRP speciﬁc antibody BXP-21. In the western blot

of MDCK BCRP cells, two bands were observed between 68 and

72 kDa. This is due to different glycosylation states of BCRP. Three

possible putative N-linked glycosylation sites at amino acids 418,

557 and 596 of human BCRP have been reported.^21,22

Figure 1. Characterization of resistant cell lines for the overexpression of corre-

sponding ABC transporter. Membrane protein preparations (30 g) of MDCK BCRP,

A2780 adr (P-gp) and 2008 MRP1 cells were electrophoretically separated and

transferred to nitrocellulose membrane. Western blots were probed with speciﬁc

antibodies against BCRP (BXP-21), P-gp (C219) and MRP1 (QCRL1), respectively.

l

Similarly, the protein expression of P-gp and MRP1 was con-

ﬁrmed by western blotting using anti-Pgp antibody C219 and

anti-MRP1 antibody respectively. Figure 1 shows the western blots

for MDCK BCRP, A2780 adr (P-gp) and 2008 MRP1 cell lines.

compounds was conﬁrmed by elemental analysis. All target com-

pounds are listed in Tables 1 and 2.

2.2. Biological investigation

2.2.2. Hoechst 33342 and pheophorbide A assay to investigate

BCRP inhibition

Target compounds were investigated for BCRP inhibition in

Hoechst 33342 (compounds 9–54) and pheophorbide A assay

(compounds 27–54) using MDCK BCRP cells. The Hoechst 33342

assay data for compounds 9–25 were taken from our earlier preli-

minary work reported elsewhere¹⁷for comparison purposes. Se-

lected compounds from this series have been further studied in

The effect of the target compounds on the accumulation of Hoe-

chst 33342 and pheophorbide A, two substrates of BCRP, was inves-

tigated in BCRP overexpressing MDCK cells. The accumulation of

Hoechst 33342 and pheophorbide A in the non-BCRP expressing

parental MDCK cells was also studied and was considered as

maximum accumulation resulting from the absence of BCRP

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K. Juvale et al. / Bioorg. Med. Chem. 21 (2013) 7858–7873

Figure 4. Inﬂuence of selected potent quinazolines at 10 lM concentration on accumulation of calcein AM in P-gp (A) and MRP1 (B) overexpressing cells. Cyclosporine

A (Cs A, 10 lM) was used as a positive control. Response in absence of any compound was used as negative control. Data are expressed as response in percentage of control

(accumulation of calcein AM in non P-gp or MRP1 expressing sensitive cells) and presented as the mean SD of three independent experiments.

Table 3

Inhibitory potencies of selected quinazolines against P-gp and MRP1 using A2780 adr

and 2008 MRP1 cells in the calcein AM assay

(0.25

more polar substituent (OH) was also a very active BCRP inhibitor

with an IC₅₀value of 0.15 M.

lM). It was interesting to see that compound 26 bearing a

Compound

A2780 adr

IC₅₀SD (

2008 MRP1

IC₅₀SD (

l

M)^*

l

M)^*

The second series of quinazolines consists of 2-(3,4-dimethoxy-

phenyl)-4-anilinoquinazolines (27–37), where the inﬂuence of

additional methoxy groups at the 2-phenyl ring was investigated.

It can be seen from Table 1, that in most cases the 3,4-dimethoxy-

phenyl substitution led to a slight increase in the activity. A similar

effect of substituents present on the 4-anilino ring on BCRP inhibi-

tion was observed for compounds 27–37 as for compounds 9–26.

meta-Substituted compounds (see 28, 31, 33, 34 and 36) were

highly active as compared to ortho- (see 30 and 35) or para- (see

32 and 37) substituted compounds. In this series compound 31

11

18

28

29

31

36

12.38 1.72

5.85 0.79

11.76 1.03

3.49 0.23

9.56 0.81

18.21 2.10

0.92 0.12

n.t.

11.18 0.58

14.09 1.65

7.82 0.62

n.t.

2.70 0.58

Cyclosporine A

*

Data are expressed as mean SD (n = 3). Cyclosporine A was used as standard.

n.t. = not tested, showed response less than 25% of control in screening at 10

concentration (see Fig. 4).

lM

was the most potent compound with an IC₅₀value of 0.076

lM

in the Hoechst 33342 assay and 0.092 M in the pheophorbide A

assay. This compound was found to be most potent amongst all

quinazolines investigated in the current work.

l

expression. The IC₅₀values obtained for the investigated com-

pounds in the Hoechst 33342 and pheophorbide A assays are given

in Tables 1 and 2. The comparison of the pIC₅₀values obtained using

these two different substrates produced a good correlation with a

squared correlation coefﬁcient (r²) of 0.94, which indicates the

non-substrate speciﬁcity of the investigated compounds. The scat-

terplot obtained by plotting pIC₅₀values in the Hoechst 33342 as-

say against the pheophorbide A assay is shown in Figure 2.

The ﬁrst series of quinazolines consists of several 2-phenyl-4-

anilinoquinazolines (9–26). All compounds in this class of quinazo-

lines were found to be active. From Table 1, it is apparent that,

from the anilinoquinazolines 9–26, those bearing a meta-substitu-

ent on the aniline ring are more potent than ortho- or para-substi-

tuted analogs (see compounds 11, 20, 23, 24 and 26). Compounds

bearing substituents at ortho position of the aniline ring were the

least active compounds followed by para-substituted compounds.

Compound 20 was the most potent in the set, with an IC₅₀of

Next, compounds bearing no substituent at position 2 of the

quinazoline moiety were investigated, which include compounds

having no substitution at positions 2, 6 and 7 of the quinazoline

(38–42) and compounds bearing no substitution at position 2,

but having methoxy substituents at position 6 and 7 (43–49). In

these two series of quinazolines, mostly compounds bearing

meta-substituted anilines were chosen based on earlier results. It

was clearly observed that removal of the phenyl substituent at po-

sition 2 of the quinazoline led to a strong decrease in activity.

There was no substantial difference in the activities of compounds

with or without 6,7-dimethoxy substitution. This suggests that, the

presence of methoxy substituents at position 6 and 7 of the quinaz-

oline scaffold was not important for the BCRP inhibition. For com-

parison purposes, the TKI quinazoline PD153035 (43) was included

in the current study and gave results comparable to those reported

by Pick et al.¹⁶

0.13 lM, which is even lower than that of the standard Ko143

K. Juvale et al. / Bioorg. Med. Chem. 21 (2013) 7858–7873

7863

Figure 5. Cytotoxicity of selected compounds was determined in the MTT assay using MDCK BCRP and sensitive MDCK cells. Quinazolines 20, 26, 31 and 36 were investigated

up to 100 M concentration for 72 h. Open circle: MDCK BCRP cells, closed circle: MDCK sensitive cells and closed triangle: dilution solvent (PBS) containing 2% methanol in

l

highest concentration.

Table 4

during testing of these compounds was their poor aqueous solubil-

ity. For testing these compounds, a slightly higher amount of meth-

anol (not more than 5% in highest concentration) was used. These

compounds were also found to be good inhibitors of BCRP with

sub-micromolar IC₅₀values. As it can be seen from Table 2, the

compound with a 3-nitro substituent at the aniline (53) yielded

lower IC₅₀values than other substituents. Despite of good inhibi-

tory activity of these compounds, low solubility could limit their

further use.

After comparing all the activity data of all series of quinazolines,

it can be suggested that the presence of 2-phenyl substitution led

to a strong increase in BCRP inhibition. In all types of quinazolines

investigated, compounds bearing meta-substitution on the 4-anili-

no ring of anilinoquinazoline were the most active. From the activ-

ity data of compounds 38–42 and 43–49, it can be proposed that

substitution at position 6 and 7 of the quinazoline is not needed

for BCRP inhibition, although substitution at position 2 is desirable.

A summary of structural features of the quinazoline scaffold for

BCRP inhibition is given in Figure 3.

Cytotoxicity and therapeutic ratio of selected compounds, geﬁtinib was used as

standard

a

b

Compound

GI₅₀

(l

M)

IC₅₀

(l

M)

Therapeutic ratio

(GI₅₀/IC₅₀

)

20

26

31

36

40

47

Geﬁtinib

67.8

51.1

20.8

4.61

18.3

1.50

1.45

0.13

0.15

520

340

270

33

16

1.85

0.45

0.076

0.14

1.13

0.81

3.19

a

GI₅₀obtained in MTT cytotoxicity assay.

IC₅₀obtained in Hoechst 33342 accumulation assay.

b

The last series of quinazolines investigated for BCRP inhibition

consists of compounds bearing 2,4-bis-anilino substituents at the

quinazoline scaffold. Compounds 50–54 bear the same substituent

at position 2 and 4 of the quinazoline. The major problem occurred

Figure 6. Representative dose–response curves of SN-38 and Hoechst 33342 cytotoxicity. Left: effect of compound 20 on SN-38 cytotoxicity in BCRP cells. Right: effect of

compound 26 on cytotoxicity of Hoechst 33342. Both compounds were investigated at 1 M (open circles) and 5 M (closed circles) concentrations. BCRP cells without

inhibitor (open triangles) showed less sensitivity towards SN-38 or Hoechst 33342, than sensitive cells (closed squares).

l

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K. Juvale et al. / Bioorg. Med. Chem. 21 (2013) 7858–7873

Figure 7. Inﬂuence of different concentrations of selected BCRP inhibitors (20, 31, 40, 47 and geﬁtinib) on the membrane (5D3; shaded bars) and total (GFP; black bars) BCRP

expression levels in MDCK BCRP cells after 72 h incubation. Fluorescence for untreated cells (0 M) was considered as 100% and data was normalized for test compounds.

l

2.2.3. Determination of P-gp and MRP1 inhibition by the calcein

AM assay

The most active compounds from each series of quinazoline

were selected and screened for their P-gp and MRP1 inhibition in

order to determine selectivity towards BCRP inhibition. The initial

are expressed as % response obtained in sensitive cells (positive

control), while the response obtained in absence of any compound

was used as negative control. As can be seen from Figure 4, most of

the compounds showed very low inhibitory activity against P-gp

and MRP1. Only compounds bearing a 3,4-dimethoxy substitution

on the aniline ring of anilinoquinazolines showed more than 50%

inhibition when compared to control. Amongst the most potent

BCRP inhibitors, compounds 20 and 26 showed no effect on both

P-gp and MRP1, while compounds 31 and 36 showed a slight effect

screening was done at a concentration of 10

assay using A2780 adr (for P-gp) and 2008 MRP1 (for MRP1) cell

lines. Cyclosporine A (10 M) was used as a standard for both

P-gp and MRP1 inhibition. The data of the investigated compounds

lM, in the calcein AM

l

K. Juvale et al. / Bioorg. Med. Chem. 21 (2013) 7858–7873

7865

on P-gp and even less on MRP1. The inhibitory effect of compounds

31 and 36 on P-gp could be due to the presence of 3,4-dimethoxy

substitution on the 2-phenyl ring of these quinazolines. Com-

pounds showing response of more than 25% of the control were

further investigated to determine their IC₅₀values and these are gi-

ven in Table 3.

nizes an external epitope of ABCG2. For determining total expres-

sion, ﬂuorescence of the reporter gene GFP was measured. Before

carrying out measurements, cells were incubated with different

concentrations (0.5 lM, 1.0 lM and 1.8 lM) of selected com-

pounds (20, 31, 40, 47) for 72 h. As a positive control, geﬁtinib at

the same concentrations was used. After the incubation period,

measurements were done by ﬂow cytometry to determine bound

antibody (FL2) and GFP (FL1) ﬂuorescence. Expression of the un-

treated cells was taken as 100% and the data for test compounds

was normalized. The results obtained in this study are depicted

in Figure 7.

From Figure 7, it can be seen that geﬁtinib decreased plas-

ma membrane and total BCRP expression in MDCK BCRP cells,

as reported previously. From the test compounds, compound

20 showed no substantial effect on surface BCRP expression

2.2.4. MTT cytotoxicity assay

Quinazoline compounds have been shown to be inhibitors of

tyrosine kinases.^23–26Quinazolines like geﬁtinib and erlotinib are

inhibitors of epidermal growth factor receptor (EGFR) tyrosine ki-

nase, and are marketed for the treatment of cancers with mutated

and overactive EGFR (especially for non-small cell lung cancer).²⁷

Interestingly, from the literature survey it was found that, quinazo-

lines reported as potent TKIs do not bear a 2-phenyl substitution

and most of them possess substitutions at position 6 and 7.^28–30

Also, the most potent BCRP inhibitors found in the current study

(20, 23, 26, 31 and 36) lack the substitution at position 6 and 7,

which has been shown to be important for TKI activity. Despite

these ﬁndings, it was important to check the possible intrinsic

cytotoxicity of the synthesized compounds. The cytotoxicity of se-

lected compounds 20, 26, 31, 36, 40 and 47 was studied in the MTT

cytotoxicity assay. The toxicity studies of these compounds were

performed using MDCK BCRP and sensitive MDCK cells. The con-

centration-response curves obtained for compounds 20, 26, 31,

36 are shown in Figure 5. It can be seen that all four quinazolines

investigated showed a varying amount of cytotoxicity. Especially

compound 36 bearing a meta-OH substituent on the 4-anilino ring

of the quinazoline showed high intrinsic cytotoxicity. The thera-

peutic ratio which is an important parameter for considering com-

pounds for future in vivo experiments was calculated for selected

compounds from the IC₅₀values obtained in the Hoechst assay

and GI₅₀values from MTT assay and is summarized in Table 4.

Compounds bearing no phenyl ring at position 2 of the quinazoline

moiety (40 and 47) have the lowest therapeutic ratio due to their

high toxicity. In contrast, the 2-phenyl-4-anilinoquinazolines 20

and 26 are promising BCRP inhibitors owing to their high thera-

peutic ratio. The increase in the cytotoxicity of compounds 31

and 36 when compared to compounds 20 and 26, could be the re-

sult of the extra 3,4-dimethoxy substituents on the 2-phenyl ring

of the anilinoquinazolines.

even at the highest concentration tested, while showing

a

slight increase in total BCRP expression. In contrast, compound

31 showed slight reduction in surface expression (down to 75%

of control) along with a signiﬁcant increase in the total BCRP

expression. This could be due to translocation of BCRP from

plasma membrane to the cytoplasmic compartment. In case

of compound 40 there was no signiﬁcant change in BCRP

membrane expression but an increase in the total BCRP

expression. Compound 47 led to considerable decrease in the

surface expression (down to 60% of the control), but only very

slight decrease in total BCRP expression. The decrease in sur-

face and total expression of BCRP in presence of compound

47 could be due to its interaction with the PI3K/Akt signaling

pathway in EGFR expressing MDCK cells. This result is in

accordance with results obtained for geﬁtinib, which similarly

to compound 47 lacks substitution at position 2 and has sub-

stituents at position 6 and 7 of the quinazoline moiety.

3. Conclusion

In this study different modiﬁcations in the quinazoline scaffold

were carried out to study the effect of different substituents on

BCRP inhibition. It was observed that quinazoline compounds

bearing a phenyl substitution at position 2 of the quinazoline

moiety were potent inhibitors of BCRP. From the structure–activ-

ity-relationship analysis, it was observed that a meta-substituted

aniline group at position 4 of the quinazoline produced higher

inhibitory effects in comparison to a ortho- or para-substituted ani-

line ring. Substituents like NO₂, CN, CF₃on the aniline ring at posi-

tion 4 of the quinazoline moiety were found to be optimum for the

BCRP inhibition. Compounds 20 and 31, both bearing a NO₂-substi-

tuent were the most active compounds amongst all investigated

quinazolines. After investigating compound 20 and 31 for their

intrinsic cytotoxicity, it was found that compound 20 has twice

the therapeutic ratio of 31, indicating its higher usability in future

in vivo studies. These compounds were also further investigated

for their effect on BCRP expression, where compound 20 showed

no effect on plasma membrane BCRP expression and negligible ef-

fect on total BCRP expression, which is an indication that the inhib-

itory potencies are due to only direct BCRP inhibition. The

qualitative structure–activity-relationship for BCRP inhibition will

be useful to ﬁnd more potent, selective and nontoxic BCRP

inhibitors.

To determine the effectiveness of promising compounds 20 and

26 to reverse MDR in BCRP overexpressing cells, MTT assays were

performed. Test compounds were investigated at ﬁnal concentra-

tions of 1 lM and 5 lM for their ability to reverse resistance to

Hoechst 33342 and SN-38 in MDCK BCRP cells. Figure 6 depicts

the shift in the dose–response curves of SN-38 and Hoechst

33342 in MDCK BCRP cells in presence of compounds 20 and 26,

respectively. This shift in dose–response curves towards lower

concentrations indicates sensitization of MDCK BCRP cells to cyto-

toxicity of SN-38 and Hoechst 33342. Both compounds were able

to signiﬁcantly reverse the resistance even at a concentration as

low as 1

lM and a complete reversal of resistance at 5 lM concen-

tration was observed.

2.2.5. Inﬂuence of selected compounds on BCRP expression

Quinazoline TKI compounds, especially the EGFR inhibitor geﬁ-

tinib, were recently shown to reduce the BCRP expression in EGFR-

positive MDCK BCRP cells via the PI3K/Akt signaling pathway.¹⁶

Hence, it was important to conﬁrm that the inhibitory effect of

the promising potent compounds on BCRP is not an effect of reduc-

tion in BCRP expression. For this purpose we planned to determine

the effect of selected target compounds from each series on the

BCRP expression (surface and total expression) in MDCK BCRP

cells. The plasma membrane (surface) expression of BCRP was

determined using the ABCG2 speciﬁc antibody 5D3, which recog-

4. Experimental section

4.1. Chemistry

All chemicals were procured from Acros Organics (Geel,

Belgium), Alfa Aesar (Karlsruhe, Germany), Sigma–Aldrich

7866

K. Juvale et al. / Bioorg. Med. Chem. 21 (2013) 7858–7873

(Steinheim, Germany) or Merck (Darmstadt, Germany) and used

without further puriﬁcation. During the synthesis, reaction progress

was monitored using analytical thin layer chromatography (TLC) on

silica gel plates (Silica Gel 60 F₂₅₄from Merck). Structures of all com-

pounds were conﬁrmed by NMR and purity of target compounds

was checked by elemental analysis. NMR spectra were recorded in

DMSO-d₆. ¹H NMR spectra were obtained on Bruker Advance 500

(500 MHz); ¹³C NMR, Bruker Advance 500 (126 MHz); chemical

shifts are expressed in d values (ppm) using the solvent peak as

internal standard; multiplicity of resonance peaks is indicated as

singlet (s), doublet (d), triplet (t), quartet (q) and multiplet (m).

The ¹³C signals were assigned with the aid of distortionless

enhancement by polarization transfer (DEPT) and attached proton

test (APT), the J values are in Hertz. Elemental analyses were per-

formed on a Vario EL of Elementar. Found values were all within

0.4% of the theoretical values except when indicated.

crystals (2.48 g, 85%): ¹H NMR (DMSO-d₆) d: 12.20 (s, 1H), 8.11

(ddd, J = 7.9, 1.6, 0.5 Hz, 1H), 8.07 (s, 1H), 7.80 (ddd, J = 8.2, 7.1,

1.6 Hz, 1H), 7.65 (ddd, J = 8.2, 1.0, 0.4 Hz, 1H), 7.51 (ddd, J = 8.1,

7.2, 1.2 Hz, 1H); ¹³C NMR (DMSO-d₆) d: 160.86, 148.84, 145.51,

134.40, 127.28, 126.83, 125.93, 122.75.

4.2.4. 6,7-Dimethoxyquinazolin-4(3H)-one (4)

According to method B, 2-amino-4,5-dimethoxybenzoic acid

(3.94 g, 20 mmol) and formamide (0.9 g, 20 mmol) were reacted

to obtain 4 as pale yellow solid (3.66 g, 89%): ¹H NMR (DMSO-d₆)

d: 12.02 (s, 1H), 7.96 (s, 1H), 7.43 (s, 1H), 7.12 (s, 1H), 3.87 (d,

J = 17.4 Hz, 6H); ¹³C NMR (DMSO-d₆) d: 160.16, 154.60, 148.70,

145.01, 143.94, 115.75, 108.17, 105.10, 56.05, 55.83.

4.3. General procedure for synthesis of substituted 4-

chloroquinazolines

4.2. General procedure for synthesis of substituted quinazolin-

4(3H)-ones

To phosphoryl chloride (30 ml, 0.32 mol), the selected quinazo-

lin-4(3H)-one (10 mmol) was added at 0 °C and stirred for 10 min.

The resulting mixture was then reﬂuxed for 9 h. After removal of

excess solvent, the residue was dissolved in ice-water (50 ml)

and the solution was neutralized with ammonium hydroxide.

The solution was extracted three times with dichloromethane

(50 ml). The organic layer was washed with brine (100 ml), dried

over MgSO₄, and the solvent was removed under reduced pressure.

The formed solid was recrystallized from ethanol. For further use

the structure of the compounds were conﬁrmed by NMR.

Two different routes were applied for the synthesis of quinazo-

lin-4(3H)-ones depending on presence or absence of the phenyl

ring at position 2 of the quinazoline scaffold. For the compounds

bearing a 2-phenyl ring method A was used, while for compounds

with no substitution at position 2 method B was employed.

Method A: To a stirred mixture of anthranilamide (20 mmol) and

a selected substituted benzaldehyde (20 mmol) in 20 ml DMF, io-

dine (3.17 g, 25 mmol) and anhydrous potassium carbonate

(2.76 g, 20 mmol) were added. Then the mixture was heated at

70–90 °C for 4–5 h. The mixture was poured on to crushed ice

and the precipitate formed was ﬁltered. The product was washed

with 100 ml 20% solution of sodiumthiosulfate to remove traces

of iodine, followed by washing with water. The product was recrys-

tallized from ethanol to yield pale yellow to dark yellow crystals.

Method B: A mixture of 2-aminobenzoic acid (anthranilic acid)

or 2-amino-4,5-dimethoxybenzoic acid (20 mmol) and formamide

(20 mmol) was heated at 150 °C for 10–18 h. To the resulting mix-

ture 50 ml water was added, which resulted in precipitation of a lot

of solid. The reaction mixture was stirred for another 30–45 min

and the resulting precipitate was ﬁltered and washed with water.

The product was recrystallized from ethanol to yield white to pale

yellow crystals.

4.3.1. 4-Chloro-2-phenylquinazoline (5)

Synthesized from 2-phenylquinazoline-4(3H)-one (2.22 g,

10 mmol) to obtain 5 as pale yellow solid (2.08 g, 87%): ¹H NMR

(DMSO-d₆) d: 8.41–8.36 (m, 2H), 8.01 (ddd, J = 8.0, 1.43, 0.62 Hz,

1H), 7.78 (ddd, J = 8.2, 1.71, 0.52 Hz, 1H), 7.59 (m, 1H), 7.48 (m,

1H), 7.39–7.31 (m, 3H); ¹³C NMR (DMSO-d₆) d: 164.2, 159.8,

153.5, 137.3, 135.6, 132.3, 130.2, 129.8, 128.1, 127.3, 123.9, 120.8.

4.3.2. 4-Chloro-2-(3,4-dimethoxyphenyl)quinazoline (6)

Synthesized from 2-(3,4-dimethoxyphenyl)quinazolin-4(3H)-

one (2.82 g, 10 mmol) to obtain 6 as yellow solid (2.34 g, 78%):

¹H NMR (DMSO-d₆) d: 8.19–8.15 (m, 1H), 8.12 (d, J = 8.0 Hz, 1H),

7.96–7.89 (m, 1H), 7.89–7.82 (m, 2H), 7.60 (ddd, J = 8.1, 7.3,

1.1 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 3.90 (d, J = 19.6 Hz, 6H); ¹³C

NMR (DMSO-d₆) d: 161.20, 154.77, 153.24, 148.66, 135.55,

127.59, 126.44, 123.40, 123.21, 120.00, 112.16, 111.63, 56.09,

56.06.

4.2.1. 2-Phenylquinazoline-4(3H)-one (1)

According to method A, anthranilamide (2.72 g, 20 mmol) and

benzaldehyde (2.12 g, 20 mmol) were reacted to obtain 1 as pale

yellow solid (3.50 g, 79%): ¹H NMR (DMSO-d₆) d: 11.98 (s, 1H),

8.26 (s, 1H), 7.92–7.84 (m, 2H), 7.61 (s, 1H), 7.52 (s, 1H), 7.41 (s,

1H), 7.36–7.28 (m, 3H); ¹³C NMR (DMSO-d₆) d: 164.23, 157.08,

143.68, 138.92, 131.47, 129.01(2), 127.22, 126.91(2), 125.12,

124.37, 123.06, 119.56.

4.3.3. 4-Chloroquinazoline (7)

Synthesized from quinazolin-4(3H)-one (1.46 g, 10 mmol) to

obtain 7 as pale yellow solid (1.35 g, 82%): ¹H NMR (DMSO-d₆) d:

8.31 (s, 1H), 8.01 (dd, 1H, J = 7.6, 1.2 Hz), 7.79 (t, 1H, J = 8.0 Hz),

7.69 (d, 1H, J = 7.9 Hz), 7.51 (t, 1H, J = 7.2 Hz); ¹³C NMR (DMSO-

d₆) d: 161.07, 146.01, 144.71, 133.85, 126.92, 125.31, 124.05,

118.21.

4.2.2. 2-(3,4-Dimethoxyphenyl)quinazolin-4(3H)-one (2)

According to method A, anthranilamide (2.72 g, 20 mmol) and

3,4-dimethoxybenzaldehyde (3.32 g, 20 mmol) were reacted to ob-

tain 2 as yellow solid (4.85 g, 86%): ¹H NMR (DMSO-d₆) d: 12.37 (s,

1H), 8.14–8.10 (m, 1H), 7.89–7.84 (m, 1H), 7.82–7.80 (m, 1H),

7.79–7.77 (m, 1H), 7.70 (dd, J = 8.2, 0.5 Hz, 1H), 7.46 (ddd, J = 8.1,

7.1, 1.2 Hz, 1H), 7.09 (dd, J = 10.0, 5.7 Hz, 1H), 3.85 (dd, J = 16.6,

7.6 Hz, 6H); ¹³C NMR (DMSO-d₆) d: 162.76, 152.27, 151.72,

149.04, 148.71, 134.57, 127.32, 126.16, 125.97, 125.14, 121.30,

120.84, 111.55, 110.93, 55.85, 55.84.

4.3.4. 4-Chloro-6,7-dimethoxyquinazoline (8)

Synthesized from 6,7-dimethoxyquinazolin-4(3H)-one (2.06 g,

10 mmol) to obtain 8 as yellow solid (1.59 g, 71%); ¹H NMR

(DMSO-d₆) d: 8.51 (s, 1H), 7.45 (s, 1H), 7.23 (s, 1H), 3.91 (s, 3H),

3.88 (s, 3H); ¹³C NMR (DMSO-d₆) d: 159.26, 155.08, 149.32,

148.72, 115.11, 107.00, 105.54, 105.18, 56.31, 56.27, 56.04.

4.4. General procedure for synthesis of 4-anilinoquinazolines

4.2.3. Quinazolin-4(3H)-one (3)

According to method B, anthranilic acid (2.74 g, 20 mmol) and

formamide (0.9 g, 20 mmol) were reacted to obtain 3 as white

4-Chloroquinazolines (1 mmol) were dissolved in reﬂuxing

2-propanol (20 ml) and the substituted aniline (1 mmol) was

added drop wise. The reaction mixture was reﬂuxed for 3–4 h, until

K. Juvale et al. / Bioorg. Med. Chem. 21 (2013) 7858–7873

7867

the reaction was complete as indicated by TLC. The precipitate

4.4.6. N-(3-Chloro-4-ﬂuorophenyl)-2-phenylquinazolin-4-

amine (14)

formed was ﬁltered off and washed with 2-propanol (10 ml) and

then with ether (10 ml). If no precipitate was formed, the solvent

was removed under reduced pressure to yield solid product. The

product was recrystallized from 75% ethanol.

Synthesized from 3-chloro-4-ﬂuoroaniline (145 mg, 1 mmol)

and 4-chloro-2-phenylquinazoline (240 mg, 1 mmol) to obtain 14

as pale yellow solid (255 mg, 73%): ¹H NMR (DMSO-d₆) d: 11.69

(s, 1H), 8.95 (d, J = 8.0 Hz, 1H), 8.38 (d, J = 7.3 Hz, 2H), 8.33 (d,

J = 8.0 Hz, 1H), 8.20 (dd, J = 6.8, 2.4 Hz, 1H), 8.07 (t, J = 7.6 Hz,

1H), 7.89 (ddd, J = 8.9, 4.2, 2.6 Hz, 1H), 7.80 (t, J = 7.7 Hz, 1H),

7.69 (t, J = 7.2 Hz, 1H), 7.61 (dd, J = 13.9, 6.6 Hz, 2H), 7.57 (d,

J = 9.0 Hz, 1H); ¹³C NMR (DMSO-d₆) d: 159.15, 157.54, 156.00,

154.05, 135.90, 134.57, 133.18, 129.19, 129.08, 128.12, 126.34,

124.98, 124.65, 119.30, 119.15, 117.06, 116.89, 112.96; Anal. Calcd

for C₂₀H₁₃ClFN₃: C, 68.67; H, 3.75; N, 12.01. Found: C, 68.52; H,

3.92; N, 11.89.

4.4.1. N,2-Diphenylquinazolin-4-amine (9)

Synthesized from aniline (93 mg, 1 mmol) and 4-chloro-2-phe-

nylquinazoline (240 mg, 1 mmol) to obtain 9 as pale yellow solid

(208 mg, 70%): ¹H NMR (DMSO-d₆) d: 11.49 (s, 1H), 8.90 (d,

J = 8.3 Hz, 1H), 8.39–8.34 (m, 2H), 8.31 (d, J = 8.4 Hz, 1H), 8.07 (t,

J = 7.3 Hz, 1H), 7.88–7.84 (m, 2H), 7.80 (t, J = 7.4 Hz, 1H), 7.68 (t,

J = 7.3 Hz, 1H), 7.62 (t, J = 7.5 Hz, 2H), 7.56–7.50 (m, 2H), 7.34 (t,

J = 7.4 Hz, 1H); ¹³C NMR (DMSO-d₆) d: 159.10, 157.52, 137.23,

135.81, 133.18, 129.23, 129.08, 128.84, 128.07, 126.38, 124.60,

124.52, 112.96; Anal. Calcd for C₂₀H₁₅N₃: C, 80.78; H, 5.08; N,

14.13. Found: C, 80.45; H, 5.23; N, 13.87.

4.4.7. N-(2-Methoxyphenyl)-2-phenylquinazolin-4-amine (15)

Synthesized from 2-methoxyaniline (123 mg, 1 mmol) and

4-chloro-2-phenylquinazoline (240 mg, 1 mmol) to obtain 15 as

pale yellow solid (222 mg, 68%): ¹H NMR (DMSO-d₆) d: 11.39 (s,

1H), 8.82 (d, J = 7.9 Hz, 1H), 8.42 (d, J = 8.2 Hz, 1H), 8.27 (d,

J = 7.6 Hz, 2H), 8.08 (t, J = 7.7 Hz, 1H), 7.81 (t, J = 7.6 Hz, 1H), 7.65

(t, J = 7.3 Hz, 1H), 7.61–7.51 (m, 3H), 7.43 (t, J = 7.6 Hz, 1H), 7.25

(d, J = 8.2 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 3.81 (s, 3H); ¹³C NMR

(DMSO-d₆) d: 159.81, 157.21, 154.04, 150.05, 145.43, 135.93,

133.28, 129.17, 128.98, 128.93, 128.22, 127.83, 125.15, 124.47,

120.45, 112.47, 112.33, 55.92; Anal. Calcd for C₂₁H₁₇N₃O: C,

77.04; H, 5.23; N, 12.84. Found: C, 77.34; H, 5.14; N, 12.98.

4.4.2. N-(2-Bromophenyl)-2-phenylquinazolin-4-amine (10)

Synthesized from 2-bromoaniline (172 mg, 1 mmol) and

4-chloro-2-phenylquinazoline (240 mg, 1 mmol) to obtain 10 as

pale yellow solid (304 mg, 81%): ¹H NMR (DMSO-d₆) d: 11.49 (s,

1H), 8.90 (d, J = 8.3 Hz, 1H), 8.39–8.34 (m, 2H), 8.31 (d, J = 8.4 Hz,

1H), 8.07 (t, J = 7.3 Hz, 1H), 7.88–7.84 (m, 2H), 7.80 (t, J = 7.4 Hz,

1H), 7.68 (t, J = 7.3 Hz, 1H), 7.62 (t, J = 7.5 Hz, 2H), 7.56–7.50 (m,

2H), 7.34 (t, J = 7.4 Hz, 1H); ¹³C NMR (DMSO-d₆) d: 159.10,

157.52, 137.23, 135.81, 133.18, 129.23, 129.08, 128.84, 128.07,

126.38, 124.60, 124.52, 112.96; Anal. Calcd for C₂₀H₁₄BrN₃: C,

63.84; H, 3.75; N, 11.17. Found: C, 63.54; H, 4.15; N, 10.75.

4.4.8. N-(3-Methoxyphenyl)-2-phenylquinazolin-4-amine (16)

Synthesized from 3-methoxyaniline (123 mg, 1 mmol) and

4-chloro-2-phenylquinazoline (240 mg, 1 mmol) to obtain 16 as

pale yellow solid (252 mg, 77%): ¹H NMR (DMSO-d₆) d: 11.06 (s,

1H), 8.85 (d, J = 8.2 Hz, 1H), 8.42 (d, J = 7.0 Hz, 2H), 8.23 (d,

J = 8.2 Hz, 1H), 8.02 (t, J = 7.5 Hz, 1H), 7.76 (t, J = 7.4 Hz, 1H), 7.64

(d, J = 7.1 Hz, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.50 (dd, J = 8.0, 1.0 Hz,

1H), 7.41 (t, J = 8.1 Hz, 1H), 6.87 (dd, J = 8.2, 2.0 Hz, 1H), 6.79 (dd,

J = 6.0, 2.8 Hz, 1H), 3.82 (s, 3H); ¹³C NMR (DMSO-d₆) d: 160.11,

159.54, 158.80, 157.87, 154.93, 138.99, 135.23, 132.55, 130.57,

129.51, 128.93, 127.60, 124.28, 116.01, 113.65, 113.28, 111.61,

109.34, 107.39, 55.39; Anal. Calcd for C₂₁H₁₇N₃O: C, 77.04; H,

5.23; N, 12.84. Found: C, 76.82; H, 5.06; N, 12.78.

4.4.3. N-(3-Bromophenyl)-2-phenylquinazolin-4-amine (11)

Synthesized from 3-bromoaniline (172 mg, 1 mmol) and

4-chloro-2-phenylquinazoline (240 mg, 1 mmol) to obtain 11 as

pale yellow solid (293 mg, 78%): ¹H NMR (DMSO-d₆) d: 11.59 (s,

1H), 8.94 (d, J = 8.1 Hz, 1H), 8.44–8.38 (m, 2H), 8.33 (d, J = 8.3 Hz,

1H), 8.25 (s, 1H), 8.07 (t, J = 7.6 Hz, 1H), 7.94–7.86 (m, 1H), 7.81

(t, J = 7.5 Hz, 1H), 7.69 (t, J = 7.3 Hz, 1H), 7.62 (t, J = 7.5 Hz, 2H),

7.54–7.44 (m, 2H); ¹³C NMR (DMSO-d₆) d: 159.09, 157.50, 149.08,

139.01, 135.88, 133.18, 130.70, 129.21, 129.07, 128.64, 128.10,

126.92, 124.63, 123.07, 121.21, 113.06; Anal. Calcd for C₂₀H₁₄BrN₃:

C, 63.84; H, 3.75; N, 11.17. Found: C, 63.61; H, 4.05; N, 11.27.

4.4.4. N-(4-Bromophenyl)-2-phenylquinazolin-4-amine (12)

Synthesized from 4-bromoaniline (172 mg, 1 mmol) and

4-chloro-2-phenylquinazoline (240 mg, 1 mmol) to obtain 12 as

pale yellow solid (309 mg, 82%): ¹H NMR (DMSO-d₆) d: 11.57 (s,

1H), 8.93 (d, J = 8.2 Hz, 1H), 8.41–8.37 (m, 2H), 8.33 (d, J = 8.1 Hz,

1H), 8.07 (t, J = 7.4 Hz, 1H), 7.89–7.84 (m, 2H), 7.81 (t, J = 7.5 Hz,

1H), 7.74–7.70 (m, 2H), 7.68 (d, J = 7.3 Hz, 1H), 7.63 (t, J = 7.5 Hz,

2H); ¹³C NMR (DMSO-d₆) d: 159.03, 157.58, 150.03, 136.75,

135.86, 133.16, 131.69, 129.28, 129.12, 128.10, 126.38, 124.65,

118.42, 113.06; Anal. Calcd for C₂₀H₁₄BrN₃: C, 63.84; H, 3.75; N,

11.17. Found: C, 63.67; H, 4.01; N, 10.94.

4.4.9. N-(4-Methoxyphenyl)-2-phenylquinazolin-4-amine (17)

Synthesized from 4-methoxyaniline (123 mg, 1 mmol) and

4-chloro-2-phenylquinazoline (240 mg, 1 mmol) to obtain 17 as

pale yellow solid (203 mg, 62%): ¹H NMR (DMSO-d₆) d: 11.55 (s,

1H), 8.89 (d, J = 8.2 Hz, 1H), 8.43–8.26 (m, 3H), 8.06 (t, J = 7.4 Hz,

1H), 7.84–7.73 (m, 3H), 7.69 (t, J = 7.3 Hz, 1H), 7.62 (t, J = 7.6 Hz,

2H), 7.12–7.05 (m, 2H), 3.82 (s, 3H); ¹³C NMR (DMSO-d₆) d: 158.82,

157.65, 157.35, 135.78, 133.29, 129.82, 129.26, 129.08, 128.06,

125.98, 124.57, 114.01, 112.80, 55.50; Anal. Calcd for C₂₁H₁₇N₃O:

C, 77.04; H, 5.23; N, 12.84. Found: C, 76.91; H, 5.17; N, 12.81.

4.4.10. N-(3,4-Dimethoxyphenyl)-2-phenylquinazolin-4-amine

(18)

4.4.5. N-(3-Chlorophenyl)-2-phenylquinazolin-4-amine (13)

Synthesized from 3-chloroaniline (127 mg, 1 mmol) and

4-chloro-2-phenylquinazoline (240 mg, 1 mmol) to obtain 13 as

pale yellow solid (229 mg, 69%): ¹H NMR (DMSO-d₆) d: 11.56 (s,

1H), 8.93 (d, J = 8.1 Hz, 1H), 8.46–8.36 (m, 2H), 8.32 (d, J = 8.1 Hz,

1H), 8.11–8.02 (m, 2H), 7.87 (dd, J = 8.1, 1.2 Hz, 1H), 7.81 (t,

J = 7.6 Hz, 1H), 7.69 (t, J = 7.3 Hz, 1H), 7.63 (t, J = 7.5 Hz, 2H), 7.55

(t, J = 8.1 Hz, 1H), 7.38 (dd, J = 7.9, 1.3 Hz, 1H); ¹³C NMR (DMSO-

d₆) d: 159.12, 157.58, 138.94, 136.21, 135.85, 133.13, 132.94,

130.42, 129.17, 129.07, 128.08, 125.73, 124.61, 124.01, 122.70,

113.08; Anal. Calcd for C₂₀H₁₄ClN₃: C, 72.40; H, 4.25; N, 12.66.

Found: C, 72.29; H, 4.75; N, 12.37.

Synthesized from 3,4-dimethoxyaniline (153 mg, 1 mmol) and

4-chloro-2-phenylquinazoline (240 mg, 1 mmol) to obtain 18 as

pale yellow solid (282 mg, 79%): ¹H NMR (DMSO-d₆) d: 11.10

(s, 1H), 8.81 (d, J = 7.9 Hz, 1H), 8.44–8.37 (m, 2H), 8.21 (d,

J = 7.2 Hz, 1H), 8.02 (t, J = 7.6 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H),

7.64 (qd, J = 14.5, 7.0 Hz, 4H), 7.40 (dd, J = 8.6, 2.3 Hz, 1H), 7.08

(d, J = 8.7 Hz, 1H), 3.81 (d, J = 5.0 Hz, 6H); ¹³C NMR (DMSO-d₆)

d: 158.51, 157.72, 148.49, 146.98, 135.27, 132.77, 130.71,

128.97, 127.70, 124.21, 116.09, 113.11, 111.72, 108.78, 55.88,

55.75; Anal. Calcd for C₂₂H₁₉N₃O₂: C, 73.93; H, 5.36; N, 11.76.

Found: C, 73.74; H, 5.50; N, 11.82.

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4.4.11. N-(2-Nitrophenyl)-2-phenylquinazolin-4-amine (19)

Synthesized from 2-nitroaniline (138 mg, 1 mmol) and

4-chloro-2-phenylquinazoline (240 mg, 1 mmol) to obtain 19 as

pale yellow solid (284 mg, 83%): ¹H NMR (DMSO-d₆) d: 11.82 (s,

1H), 8.54–8.47 (m, 1H), 7.94 (ddd, J = 5.2, 4.3, 1.4 Hz, 2H), 7.59–

7.51 (m, 2H), 7.44–7.30 (m, 5H), 7.03–6.97 (m, 2H), 6.60 (ddd,

J = 8.4, 6.8, 1.3 Hz, 2H); ¹³C NMR (DMSO-d₆) d: 165.96, 159.13,

151.40, 146.31, 137.68, 135.80, 134.26, 130.85, 130.47, 128.70,

128.11, 127.75, 127.09, 125.48, 123.44, 119.30, 115.59, 115.01;

Anal. Calcd for C₂₀H₁₄N₄O₂: C, 70.17; H, 4.12; N, 16.37. Found: C,

70.01; H, 3.98; N, 16.41.

as pale yellow solid (252 mg, 69%): ¹H NMR (DMSO-d₆) d: 11.66

(s, 1H), 8.96 (d, J = 8.3 Hz, 1H), 8.48 (s, 1H), 8.41 (dd, J = 14.1,

6.9 Hz, 2H), 8.32 (d, J = 8.0 Hz, 1H), 8.16 (d, J = 8.3 Hz, 1H),

8.08 (t, J = 7.5 Hz, 1H), 7.82 (t, J = 7.7 Hz, 1H), 7.76 (t, J = 7.9 Hz,

1H), 7.68 (dd, J = 14.3, 7.2 Hz, 2H), 7.60 (t, J = 7.6 Hz, 2H); ¹³C

NMR (DMSO-d₆) d: 159.14, 157.61, 138.40, 135.84, 133.10,

130.02, 129.52, 129.27, 129.12, 128.99, 128.08, 127.70, 125.33,

124.59, 123.16, 122.18, 120.80, 113.14; Anal. Calcd for

C₂₁H₁₄F₃N₃: C, 69.04; H, 3.86; N, 11.50. Found: C, 68.74; H,

4.02; N, 11.70.

4.4.17. 2-Phenyl-N-(4-(triﬂuoromethyl)phenyl)quinazolin-4-

amine (25)

4.4.12. N-(3-Nitrophenyl)-2-phenylquinazolin-4-amine (20)

Synthesized from 3-nitroaniline (138 mg, 1 mmol) and

4-chloro-2-phenylquinazoline (240 mg, 1 mmol) to obtain 20 as

pale yellow solid (304 mg, 89%): ¹H NMR (DMSO-d₆) d: 11.43

(s, 1H), 9.12 (s, 1H), 8.90 (d, J = 8.3 Hz, 1H), 8.46 (d, J = 7.2 Hz,

2H), 8.35 (dd, J = 8.1, 1.3 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.12

(dd, J = 8.2, 1.7 Hz, 1H), 8.07 (t, J = 7.8 Hz, 1H), 7.86–7.77 (m,

2H), 7.67 (t, J = 7.2 Hz, 1H), 7.61 (t, J = 7.4 Hz, 2H); ¹³C NMR

(DMSO-d₆) d: 158.96, 157.93, 147.97, 146.81, 139.17, 135.60,

132.74, 130.14, 129.49, 129.03, 127.90, 124.34, 119.88, 117.99,

113.34; Anal. Calcd for C₂₀H₁₄N₄O₂: C, 70.17; H, 4.12; N, 16.37.

Found: C, 69.87; H, 4.32; N, 16.73.

Synthesized from 4-(triﬂuoromethyl)aniline (161 mg, 1 mmol)

and 4-chloro-2-phenylquinazoline (240 mg, 1 mmol) to obtain 25

as pale yellow solid (267 mg, 73%): ¹H NMR (DMSO-d₆) d: 11.59

(s, 1H), 8.97 (d, J = 8.2 Hz, 1H), 8.46–8.39 (m, 2H), 8.32 (d,

J = 8.3 Hz, 1H), 8.17 (t, J = 9.6 Hz, 2H), 8.08 (t, J = 7.4 Hz, 1H),

7.93–7.86 (m, 2H), 7.81 (dd, J = 17.0, 9.6 Hz, 1H), 7.73–7.59 (m,

3H): ¹³C NMR (DMSO-d₆) d: 159.16, 157.80, 141.35, 135.82,

132.97, 129.24, 129.10, 128.04, 125.97, 125.94, 125.47, 124.65,

124.17, 123.30, 113.23; Anal. Calcd for C₂₁H₁₄F₃N₃: C, 69.04; H,

3.86; N, 11.50. Found: C, 69.15; H, 3.93; N, 11.45.

4.4.18. 3-((2-Phenylquinazolin-4-yl)amino)phenol (26)

Synthesized from 3-aminophenol (109 mg, 1 mmol) and

4-chloro-2-phenylquinazoline (240 mg, 1 mmol) to obtain 26 as

pale yellow solid (211 mg, 59%): ¹H NMR (DMSO-d₆) d: 10.51 (s,

1H), 8.32 (s, 1H), 8.18 (dd, J = 7.4, 1.6 Hz, 1H), 7.75 (dd, J = 7.7,

1.8 Hz, 1H), 7.67 (td, J = 7.3, 1.7 Hz, 1H), 7.49 (td, J = 7.8, 1.3 Hz,

1H), 7.39–7.25 (m, 5H), 6.88–6.81 (m, 3H), 6.79 (dt, J = 7.5,

1.8 Hz, 1H); ¹³C NMR (DMSO-d₆) d: 161.59, 156.34, 154.82,

147.33, 142.26, 137.06, 131.26, 131.05, 130.57, 128.98, 126.24,

124.22, 122.59, 120.61, 119.35, 116.25, 115.03, 111.50; Anal. Calcd

for C₂₀H₁₅N₃O: C, 76.66; H, 4.82; N, 13.41. Found: C, 76.59; H, 5.03;

N, 13.55.

4.4.13. N-(4-Nitrophenyl)-2-phenylquinazolin-4-amine (21)

Synthesized from 4-nitroaniline (138 mg, 1 mmol) and

4-chloro-2-phenylquinazoline (240 mg, 1 mmol) to obtain 21 as

pale yellow solid (274 mg, 80%): ¹H NMR (DMSO-d₆) d: 11.21 (s,

1H), 8.84 (d, J = 8.1 Hz, 1H), 8.51–8.35 (m, 4H), 8.28 (d, J = 8.7 Hz,

2H), 8.17 (d, J = 7.8 Hz, 1H), 8.05 (t, J = 7.8 Hz, 1H), 7.79 (t,

J = 7.4 Hz, 1H), 7.62 (d, J = 7.5 Hz, 3H); ¹³C NMR (DMSO-d₆) d:

158.89, 157.14, 148.13, 146.57, 140.35, 135.73, 132.16, 132.05,

130.85, 129.22, 128.78, 127.22, 126.19, 124.32, 120.78, 119.47,

112.39; Anal. Calcd for C₂₀H₁₄N₄O₂: C, 70.17; H, 4.12; N, 16.37.

Found: C, 70.25; H, 4.23; N, 16.43.

4.4.14. N-(4-Fluoro-3-nitrophenyl)-2-phenylquinazolin-4-

amine (22)

4.4.19. 2-(3,4-Dimethoxyphenyl)-N-phenylquinazolin-4-amine

(27)

Synthesized from 4-ﬂuoro-3-nitroaniline (156 mg, 1 mmol) and

4-chloro-2-phenylquinazoline (240 mg, 1 mmol) to obtain 22 as

pale yellow solid (234 mg, 65%): ¹H NMR (DMSO-d₆) d: 11.39 (s,

1H), 9.01 (d, J = 4.6 Hz, 1H), 8.85 (d, J = 8.1 Hz, 1H), 8.45 (d,

J = 7.4 Hz, 2H), 8.37–8.27 (m, 1H), 8.20 (d, J = 7.3 Hz, 1H), 8.06 (t,

J = 7.7 Hz, 1H), 7.86–7.70 (m, 2H), 7.64 (dt, J = 15.0, 7.3 Hz, 3H);

¹³C NMR (DMSO-d₆) d: 158.80, 157.98, 150.68, 136.45, 135.46,

133.67, 132.60, 130.94, 128.98, 127.81, 124.21, 120.50, 118.91,

118.74, 113.26; Anal. Calcd for C₂₀H₁₃FN₄O₂: C, 66.66; H, 3.64; N,

15.55. Found: C, 66.61; H, 3.58; N, 15.60.

Synthesized from aniline (93 mg, 1 mmol) and 4-chloro-2-(3,4-

dimethoxyphenyl)quinazoline (300 mg, 1 mmol) to obtain 27 as

yellow solid (222 mg, 62%): ¹H NMR (DMSO-d₆) d: 11.23 (s, 1H),

7.97 (dd, J = 7.7, 16 Hz, 1H), 7.70 (dd, J = 7.5, 1.3 Hz, 1H), 7.68 (td,

J = 7.2, 1.6 Hz, 1H), 7.47 (td, J = 7.7, 1.3 Hz, 1H), 7.30–7.22 (m,

2H), 7.09 (t, J = 7.8 Hz, 2H), 6.87 (d, J = 7.2 Hz, 1H), 6.75–6.67 (m,

3H), 3.80 (d, J = 11.4 Hz, 6H); ¹³C NMR (DMSO-d₆) d: 161.82,

159.14, 152.81, 150.83, 148.62, 140.60, 135.32, 131.96, 131.05,

129.18, 126.22, 124.90, 123.72, 123.18, 119.55, 114.07, 113.26,

107.91, 56.12, 55.95; Anal. Calcd for C₂₂H₁₉N₃O₂: C, 73.93; H,

5.36; N, 11.76. Found: C, 73.84; H, 5.53; N, 11.81.

4.4.15. 3-((2-Phenylquinazolin-4-yl)amino)benzonitrile (23)

Synthesized from 3-aminobenzonitrile (118 mg, 1 mmol) and

4-chloro-2-phenylquinazoline (240 mg, 1 mmol) to obtain 23 as

pale yellow solid (293 mg, 91%): ¹H NMR (DMSO-d₆) d: 11.73 (s,

1H), 8.97 (d, J = 8.3 Hz, 1H), 8.43–8.36 (m, 3H), 8.32 (d, J = 8.3 Hz,

1H), 8.27–8.19 (m, 1H), 8.08 (t, J = 7.4 Hz, 1H), 7.88–7.66 (m, 4H),

7.61 (dd, J = 10.4, 4.7 Hz, 2H); ¹³C NMR (DMSO-d₆) d: 159.22,

157.65, 138.46, 135.93, 133.12, 130.23, 129.39, 129.16, 129.07,

128.98, 128.13, 127.52, 124.66, 118.57, 113.08, 111.60; Anal. Calcd

for C₂₁H₁₄N₄: C, 78.24; H, 4.38; N, 17.38. Found: C, 78.12; H, 4.46;

N, 16.98.

4.4.20. N-(3-Bromophenyl)-2-(3,4-

dimethoxyphenyl)quinazolin-4-amine (28)

Synthesized from 3-bromoaniline (172 mg, 1 mmol) and

4-chloro-2-(3,4-dimethoxyphenyl)quinazoline (300 mg, 1 mmol)

to obtain 28 as yellow solid (375 mg, 86%): ¹H NMR (DMSO-

d₆) d: 11.54 (s, 1H), 8.86 (d, J = 8.2 Hz, 1H), 8.43 (d, J = 8.2 Hz,

1H), 8.17 (t, J = 1.8 Hz, 1H), 8.12 (dd, J = 8.6, 2.2 Hz, 1H), 8.10–

8.00 (m, 2H), 7.87 (ddd, J = 7.8, 1.9, 1.2 Hz, 1H), 7.77 (dd,

J = 11.4, 4.1 Hz, 1H), 7.56–7.43 (m, 2H), 7.18 (d, J = 8.7 Hz, 1H),

3.88 (d, J = 3.0 Hz, 6H); ¹³C NMR (DMSO-d₆) d: 158.83, 156.74,

153.55, 148.92, 138.89, 135.93, 130.66, 128.88, 127.86, 127.07,

124.58, 123.59, 123.42, 121.21, 112.66, 112.08, 111.74, 56.07,

56.02; Anal. Calcd for C₂₂H₁₈BrN₃O₂: C, 60.56; H, 4.16; N, 9.63.

Found: C, 60.71; H, 3.89; N, 9.47.

4.4.16. 2-Phenyl-N-(3-(triﬂuoromethyl)phenyl)quinazolin-4-

amine (24)

Synthesized from 3-(triﬂuoromethyl)aniline (161 mg, 1 mmol)

and 4-chloro-2-phenylquinazoline (240 mg, 1 mmol) to obtain 24

K. Juvale et al. / Bioorg. Med. Chem. 21 (2013) 7858–7873

7869

4.4.21. N,2-Bis(3,4-dimethoxyphenyl)quinazolin-4-amine (29)

Synthesized from 3,4-dimethoxyaniline (153 mg, 1 mmol) and

4-chloro-2-(3,4-dimethoxyphenyl)quinazoline (300 mg, 1 mmol)

to obtain 29 as yellow solid (221 mg, 53%): ¹H NMR (DMSO-d₆)

d: 11.43 (s, 1H), 8.81 (d, J = 8.1 Hz, 1H), 8.45 (d, J = 8.2 Hz, 1H),

8.15–8.08 (m, 2H), 8.07–8.01 (m, 1H), 7.77 (t, J = 7.5 Hz, 1H), 7.48

(d, J = 2.4 Hz, 1H), 7.38 (dd, J = 8.6, 2.4 Hz, 1H), 7.20 (d, J = 8.4 Hz,

1H), 7.09 (d, J = 8.7 Hz, 1H), 3.87 (d, J = 4.3 Hz, 6H), 3.80 (d,

J = 11.7 Hz, 6H); ¹³C NMR (DMSO-d₆) d: 158.47, 156.48, 153.61,

148.88, 148.57, 147.49, 135.78, 130.03, 127.81, 124.43, 123.70,

117.02, 112.51, 112.31, 111.78, 111.59, 109.47, 56.06, 56.03,

55.91, 55.89; Anal. Calcd for C₂₄H₂₃N₃O₄: C, 69.05; H, 5.55; N,

10.07. Found: C, 69.42; H, 5.68; N, 10.14.

111.70, 56.07, 55.97; Anal. Calcd for C₂₃H₁₈F₃N₃O₂: C, 64.94; H,

4.26; N, 9.88. Found: C, 64.72; H, 4.51; N, 9.69.

4.4.26. 3-((2-(3,4-Dimethoxyphenyl)quinazolin-4-yl)amino)-

benzonitrile (34)

Synthesized from 3-aminobenzonitrile (118 mg, 1 mmol) and

4-chloro-2-(3,4-dimethoxyphenyl)quinazoline (300 mg, 1 mmol)

to obtain 34 as yellow solid (340 mg, 89%): ¹H NMR (DMSO-d₆)

d: 11.07 (s, 1H), 8.32 (dd, J = 7.1, 1.7 Hz, 1H), 7.75 (dd, J = 7.3,

1.5 Hz, 1H), 7.69 (td, J = 7.7, 1.6 Hz, 1H), 7.54 (td, J = 7.7, 1.3 Hz,

1H), 7.31 (t, J = 7.0 Hz, 1H), 7.29–7.24 (m, 2H), 7.23–7.16 (m, 2H),

7.01 (dt, J = 7.6, 1.5 Hz, 1H), 6.89 (d, J = 7.4 Hz, 1H), 3.77 (d,

J = 2.4 Hz, 6H); ¹³C NMR (DMSO-d₆) d: 159.99, 155.94, 152.18,

150.83, 147.62, 140.37, 133.29, 131.10, 131.00, 129.77, 127.38,

125.93, 125.11, 124.72, 122.81, 122.86, 118.39, 113.27, 112.31,

111.02, 108.65, 56.33, 55.61; Anal. Calcd for C₂₃H₁₈N₄O₂: C,

72.24; H, 4.74; N, 14.65. Found: C, 71.88; H, 4.83; N, 14.71.

4.4.22. 2-(3,4-Dimethoxyphenyl)-N-(2-nitrophenyl)quinazolin-

4-amine (30)

Synthesized from 2-nitroaniline (138 mg, 1 mmol) and

4-chloro-2-(3,4-dimethoxyphenyl)quinazoline (300 mg, 1 mmol)

to obtain 30 as yellow solid (229 mg, 57%): ¹H NMR (DMSO-d₆)

d: 11.15 (s, 1H), 8.14–8.09 (m, 1H), 8.06 (dd, J = 14.6, 2.0 Hz, 1H),

7.98–7.92 (m, 1H), 7.91–7.88 (m, 1H), 7.57 (ddd, J = 8.1, 6.0,

2.2 Hz, 1H), 7.38 (ddd, J = 10.0, 6.1, 2.7 Hz, 2H), 7.11 (d, J = 8.5 Hz,

1H), 7.00 (dd, J = 8.6, 1.3 Hz, 1H), 6.63–6.53 (m, 1H), 5.76–5.67

(m, 1H), 3.88 (s, 3H), 3.85 (s, 3H); ¹³C NMR (DMSO-d₆) d: 165.69,

158.99, 151.49, 148.80, 146.31, 135.80, 134.14, 130.30, 127.53,

126.58, 125.48, 123.41, 121.63, 119.30, 115.59, 114.73, 111.60,

111.13, 55.76, 55.63; Anal. Calcd for C₂₂H₁₈N₄O₄: C, 65.66; H,

4.51; N, 13.92. Found: C, 65.39; H, 4.72; N, 14.11.

4.4.27. 2-((2-(3,4-Dimethoxyphenyl)quinazolin-4-yl)amino)-

phenol (35)

Synthesized from 2-aminophenol (109 mg, 1 mmol) and

4-chloro-2-(3,4-dimethoxyphenyl)quinazoline (300 mg, 1 mmol)

to obtain 35 as pale yellow solid (213 mg, 57%): ¹H NMR (DMSO-

d₆) d: 10.15 (s, 1H), 8.34 (s, 1H), 7.94 (dd, J = 6.9, 1.8 Hz, 1H),

7.73 (dd, J = 7.2, 1.3 Hz, 1H), 7.69–7.63 (m, 1H), 7.39 (t, J = 7.2 Hz,

1H), 6.90 (d, J = 7.1 Hz, 2H), 6.88 (d, J = 7.6 Hz, 1H), 6.70–6.56 (m,

4H), 3.59 (d, J = 3.1 Hz, 6H); ¹³C NMR (DMSO-d₆) d: 161.54,

156.08, 152.65, 150.87, 148.01, 145.92, 132.74, 131.90, 130.49,

126.22, 125.34, 123.88, 123.10, 121.98, 121.00, 113.77, 111.24,

107.38, 56.29, 55.90; Anal. Calcd for C₂₂H₁₉N₃O₃: C, 70.76; H,

5.13; N, 11.25. Found: C, 70.61; H, 5.34; N, 11.43.

4.4.23. 2-(3,4-Dimethoxyphenyl)-N-(3-nitrophenyl)quinazolin-

4-amine (31)

Synthesized from 3-nitroaniline (138 mg, 1 mmol) and

4-chloro-2-(3,4-dimethoxyphenyl)quinazoline (300 mg, 1 mmol)

to obtain 31 as yellow solid (290 mg, 72%): ¹H NMR (DMSO-d₆)

d: 11.60 (s, 1H), 8.93 (s, 1H), 8.86 (d, J = 8.2 Hz, 1H), 8.47–8.29

(m, 2H), 8.11 (dt, J = 31.4, 9.8 Hz, 3H), 7.80 (q, J = 7.8 Hz, 2H),

7.17 (d, J = 8.6 Hz, 1H), 3.86 (d, J = 15.9 Hz, 6H); ¹³C NMR (DMSO-

d₆) d: 158.86, 157.03, 148.92, 147.91, 138.78, 135.86, 130.12,

127.80, 124.48, 123.62, 120.35, 118.53, 112.84, 112.01, 111.74,

56.04, 55.88; Anal. Calcd for C₂₂H₁₈N₄O₄: C, 65.66; H, 4.51; N,

13.92. Found: C, 65.48; H, 4.62; N, 13.69.

4.4.28. 3-((2-(3,4-Dimethoxyphenyl)quinazolin-4-yl)amino)-

phenol (36)

Synthesized from 3-aminophenol (109 mg, 1 mmol) and

4-chloro-2-(3,4-dimethoxyphenyl)quinazoline (300 mg, 1 mmol)

to obtain 36 as pale yellow solid (224 mg, 60%): ¹H NMR (DMSO-

d₆) d: 11.32 (s, 1H), 9.75 (s, 1H), 8.81 (d, J = 8.3 Hz, 1H), 8.41 (d,

J = 8.2 Hz, 1H), 8.16 (dd, J = 8.6, 2.2 Hz, 1H), 8.08 (d, J = 2.2 Hz,

1H), 8.07–8.01 (m, 1H), 7.77 (t, J = 7.3 Hz, 1H), 7.35–7.23 (m, 3H),

7.20 (d, J = 8.7 Hz, 1H), 6.76 (ddd, J = 8.0, 2.3, 1.1 Hz, 1H), 3.87 (d,

J = 2.8 Hz, 6H); ¹³C NMR (DMSO-d₆) d: 158.65, 157.79, 156.52,

153.54, 148.89, 138.02, 135.82, 129.37, 127.80, 124.53, 123.55,

115.26, 113.62, 112.58, 112.23, 111.75, 56.07, 55.85; Anal. Calcd

for C₂₂H₁₉N₃O₃: C, 70.76; H, 5.13; N, 11.25. Found: C, 70.56; H,

5.28; N, 11.35.

4.4.24. 2-(3,4-Dimethoxyphenyl)-N-(4-nitrophenyl)quinazolin-

4-amine (32)

Synthesized from 4-nitroaniline (138 mg, 1 mmol) and

4-chloro-2-(3,4-dimethoxyphenyl)quinazoline (300 mg, 1 mmol)

to obtain 32 as yellow solid (273 mg, 68%): ¹H NMR (DMSO-d₆)

d: 11.54 (s, 1H), 8.86 (d, J = 8.2 Hz, 1H), 8.45–8.31 (m, 3H),

8.29–8.17 (m, 2H), 8.12 (dd, J = 6.8, 2.1 Hz, 2H), 8.06 (t, J = 7.7 Hz,

1H), 7.78 (t, J = 7.7 Hz, 1H), 7.20 (d, J = 9.2 Hz, 1H), 3.89 (d,

J = 12.0 Hz, 6H); ¹³C NMR (DMSO-d₆) d: 158.70, 157.07, 153.30,

148.90, 144.03, 135.85, 127.74, 124.53, 124.46, 123.90, 123.57,

113.09, 112.00, 111.87, 56.00, 55.92; Anal. Calcd for C₂₂H₁₈N₄O₄:

C, 65.66; H, 4.51; N, 13.92. Found: C, 65.71; H, 4.32; N, 13.75.

4.4.29. 4-((2-(3,4-Dimethoxyphenyl)quinazolin-4-yl)amino)-

phenol (37)

Synthesized from 4-aminophenol (109 mg, 1 mmol) and

4-chloro-2-(3,4-dimethoxyphenyl)quinazoline (300 mg, 1 mmol)

to obtain 37 as pale yellow solid (213 mg, 57%): ¹H NMR (DMSO-

d₆) d: 11.37 (s, 1H), 9.72 (s, 1H), 8.76 (d, J = 8.2 Hz, 1H), 8.38 (d,

J = 8.3 Hz, 1H), 8.11–7.97 (m, 3H), 7.75 (t, J = 7.4 Hz, 1H), 7.59 (d,

J = 8.8 Hz, 2H), 7.20 (d, J = 8.6 Hz, 1H), 6.91 (d, J = 8.8 Hz, 2H),

3.87 (d, J = 4.3 Hz, 6H); ¹³C NMR (DMSO-d₆) d: 158.35, 156.39,

156.17, 153.53, 148.84, 135.71, 128.16, 127.76, 126.23, 124.38,

123.48, 115.21, 112.44, 112.19, 111.79, 56.05, 55.92; Anal. Calcd

for C₂₂H₁₉N₃O₃: C, 70.76; H, 5.13; N, 11.25. Found: C, 70.82; H,

5.21; N, 11.49.

4.4.25. 2-(3,4-Dimethoxyphenyl)-N-(3-(triﬂuoromethyl)phenyl)-

quinazolin-4-amine (33)

Synthesized from 3-(triﬂuoromethyl)aniline (161 mg, 1 mmol)

and

4-chloro-2-(3,4-dimethoxyphenyl)quinazoline

(300 mg,

1 mmol) to obtain 33 as yellow solid (315 mg, 74%): ¹H NMR

(DMSO-d₆) d: 11.62 (s, 1H), 8.87 (d, J = 8.2 Hz, 1H), 8.43 (d,

J = 7.9 Hz, 1H), 8.31 (s, 1H), 8.18 (d, J = 8.2 Hz, 1H), 8.10–8.04 (m,

3H), 7.82–7.74 (m, 2H), 7.68 (d, J = 7.7 Hz, 1H), 7.16 (d, J = 8.3 Hz,

1H), 3.87 (d, J = 9.8 Hz, 6H); ¹³C NMR (DMSO-d₆) d: 158.99,

156.90, 153.56, 148.95, 138.23, 135.97, 130.02, 129.64, 129.39,

128.25, 127.89, 124.56, 123.65, 122.56, 120.99, 112.76, 112.19,

4.4.30. N-(3-Bromophenyl)quinazolin-4-amine (38)

Synthesized from 3-bromoaniline (172 mg, 1 mmol) and

4-chloroquinazoline (164 mg, 1 mmol) to obtain 38 as white solid

(204 mg, 68%): ¹H NMR (DMSO-d₆) d: 11.89 (s, 1H), 9.03 (dd, J = 8.4,

0.7 Hz, 1H), 8.98 (s, 1H), 8.11 (ddd, J = 8.4, 7.1, 1.2 Hz, 1H), 8.07 (t,

7870

K. Juvale et al. / Bioorg. Med. Chem. 21 (2013) 7858–7873

J = 1.9 Hz, 1H), 8.02 (dd, J = 8.4, 0.8 Hz, 1H), 7.86 (ddd, J = 8.3, 7.1,

1.2 Hz, 1H), 7.80 (ddd, J = 8.0, 2.0, 1.0 Hz, 1H), 7.52 (ddd, J = 8.0,

1.9, 1.0 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H); ¹³C NMR (DMSO-d₆) d:

160.08, 151.21, 139.09, 138.52, 136.41, 130.74, 129.32, 128.77,

127.40, 125.17, 123.82, 121.26, 120.05, 113.74; Anal. Calcd for C_14-

H₁₀BrN₃: C, 56.02; H, 3.36; N, 14.00. Found: C, 55.88; H, 3.50; N,

13.75.

56.61; Anal. Calcd for C₁₆H₁₄BrN₃O₂: C, 53.35; H, 3.92; N, 11.67.

Found: C, 53.07; H, 4.16; N, 11.49.

4.4.36. 6,7-Dimethoxy-N-(3-

(triﬂuoromethyl)phenyl)quinazolin-4-amine (44)

Synthesized from 3-(triﬂuoromethyl)aniline (161 mg, 1 mmol)

and 4-chloro-6,7-dimethoxyquinazoline (224 mg, 1 mmol) to ob-

tain 44 as pale yellow solid (314 mg, 90%): ¹H NMR (DMSO-d₆) d:

11.74 (s, 1H), 8.87 (s, 1H), 8.46 (s, 1H), 8.17 (s, 1H), 8.11 (d,

J = 8.1 Hz, 1H), 7.70 (t, J = 7.9 Hz, 1H), 7.66–7.61 (m, 1H), 7.39 (s,

1H), 4.01 (d, J = 23.6 Hz, 6H); ¹³C NMR (DMSO-d₆) d: 158.35,

156.59, 150.43, 138.15, 136.11, 129.94, 129.60, 129.34, 128.55,

122.48, 121.16, 121.13, 107.65, 104.39, 100.00, 57.25, 56.60; Anal.

Calcd for C₁₇H₁₄F₃N₃O₂: C, 58.45; H, 4.04; N, 12.03. Found: C,

58.39; H, 4.33; N, 12.19.

4.4.31. 3-(Quinazolin-4-ylamino)benzonitrile (39)

Synthesized from 3-aminobenzonitrile (118 mg, 1 mmol) and

4-chloroquinazoline (164 mg, 1 mmol) to obtain 39 as white solid

(175 mg, 71%): ¹H NMR (DMSO-d₆) d: 12.07 (s, 1H), 9.07 (d,

J = 7.8 Hz, 1H), 9.00 (s, 1H), 8.31 (t, J = 1.8 Hz, 1H), 8.16–8.09 (m,

2H), 8.03 (dd, J = 8.4, 0.8 Hz, 1H), 7.88 (ddd, J = 8.3, 7.1, 1.1 Hz,

1H), 7.78 (dt, J = 7.7, 1.3 Hz, 1H), 7.70 (t, J = 8.0 Hz, 1H); ¹³C NMR

(DMSO-d₆) d: 160.19, 151.30, 139.32, 137.94, 136.52, 130.28,

130.03, 129.58, 128.85, 128.08, 125.20, 120.23, 118.47, 113.78,

111.65; Anal. Calcd for C₁₅H₁₀N₄: C, 73.16; H, 4.09; N, 22.75.

Found: C, 73.30; H, 4.28; N, 22.51.

4.4.37. 3-((6,7-Dimethoxyquinazolin-4-yl)amino)benzonitrile

(45)

Synthesized from 3-aminobenzonitrile (118 mg, 1 mmol) and

4-chloro-6,7-dimethoxyquinazoline (224 mg, 1 mmol) to obtain

45 as pale yellow solid (254 mg, 83%): ¹H NMR (DMSO-d₆) d:

11.39 (s, 1H), 8.87 (s, 1H), 8.29 (s, 1H), 8.27 (t, J = 1.8 Hz, 1H),

8.07 (ddd, J = 8.1, 2.2, 1.2 Hz, 1H), 7.75 (dt, J = 7.7, 1.3 Hz, 1H),

7.69 (dd, J = 11.8, 4.1 Hz, 1H), 7.33 (s, 1H), 4.01 (d, J = 10.9 Hz,

6H); ¹³C NMR (DMSO-d₆) d: 158.22, 157.87, 156.63, 150.48,

138.33, 130.29, 129.50, 129.22, 127.76, 118.54, 111.68, 107.70,

103.89, 57.09, 56.65; Anal. Calcd for C₁₇H₁₄N₄O₂: C, 66.66; H,

4.61; N, 18.29. Found: C, 66.40; H, 4.93; N, 18.40.

4.4.32. N-(3-Nitrophenyl)quinazolin-4-amine (40)

Synthesized from 3-nitroaniline (138 mg, 1 mmol) and 4-chlo-

roquinazoline (164 mg, 1 mmol) to obtain 40 as pale yellow solid

(210 mg, 79%): ¹H NMR (DMSO-d₆) d: 12.08 (s, 1H), 9.07 (dd,

J = 8.4, 0.7 Hz, 1H), 9.03 (s, 1H), 8.77 (t, J = 2.1 Hz, 1H), 8.31 (ddd,

J = 8.1, 2.1, 0.9 Hz, 1H), 8.18–8.11 (m, 2H), 8.04 (dd, J = 8.4,

0.8 Hz, 1H), 7.89 (ddd, J = 8.3, 7.1, 1.2 Hz, 1H), 7.78 (t, J = 8.2 Hz,

1H); ¹³C NMR (DMSO-d₆) d: 160.20, 151.35, 147.92, 139.51,

138.31, 136.50, 130.86, 130.22, 128.85, 125.19, 120.93, 120.37,

119.21, 113.90; Anal. Calcd for C₁₄H₁₀N₄O₂: C, 63.15; H, 3.79; N,

21.04. Found: C, 62.83; H, 3.91; N, 21.02.

4.4.38. 6,7-Dimethoxy-N-(2-nitrophenyl)quinazolin-4-amine

(46)

Synthesized from 2-nitroaniline (138 mg, 1 mmol) and

4-chloro-6,7-dimethoxyquinazoline (224 mg, 1 mmol) to obtain

46 as pale yellow solid (221 mg, 68%): ¹H NMR (DMSO-d₆) d:

8.59 (s, 1H), 7.94 (dd, J = 8.7, 1.5 Hz, 1H), 7.41–7.32 (m, 4H), 7.28

(d, J = 8.9 Hz, 2H), 7.00 (dd, J = 8.5, 1.3 Hz, 1H), 6.60 (ddd, J = 8.2,

6.8, 1.3 Hz, 1H), 3.92 (d, J = 16.1 Hz, 6H); ¹³C NMR (DMSO-d₆) d:

164.58, 155.62, 149.97, 148.50, 146.65, 136.14, 130.82, 125.82,

119.64, 115.93, 110.57, 107.22, 101.24, 69.89, 56.46, 56.26; Anal.

Calcd for C₁₆H₁₄N₄O₄: C, 58.89; H, 4.32; N, 17.17. Found: C,

58.62; H, 4.52; N, 17.05.

4.4.33. N-(3-(Triﬂuoromethyl)phenyl)quinazolin-4-amine (41)

Synthesized from 3-(triﬂuoromethyl)aniline (161 mg, 1 mmol)

and 4-chloroquinazoline (164 mg, 1 mmol) to obtain 41 as pale

yellow solid (246 mg, 85%): ¹H NMR (DMSO-d₆) d: 10.00 (s, 1H),

8.67 (s, 1H), 8.57 (d, J = 8.0 Hz, 1H), 8.34 (s, 1H), 8.25 (d,

J = 8.2 Hz, 1H), 7.88 (ddd, J = 8.2, 6.9, 1.2 Hz, 1H), 7.82 (d,

J = 7.5 Hz, 1H), 7.71–7.60 (m, 2H), 7.45 (d, J = 7.7 Hz, 1H); ¹³C

NMR (DMSO-d₆) d: 157.73, 154.33, 149.86, 140.34, 133.37,

129.75, 129.52, 129.27, 128.05, 126.64, 125.63, 123.09, 119.80,

119.77, 118.17, 118.13, 115.30; Anal. Calcd for C₁₅H₁₀F₃N₃: C,

62.28; H, 3.48; N, 14.53. Found: C, 62.00; H, 3.61; N, 14.38.

4.4.39. 6,7-Dimethoxy-N-(3-nitrophenyl)quinazolin-4-amine

(47)

4.4.34. N-(3,4-Dimethoxyphenyl)quinazolin-4-amine (42)

Synthesized from 3,4-dimethoxyaniline (153 mg, 1 mmol) and

4-chloroquinazoline (164 mg, 1 mmol) to obtain 42 as yellow solid

(256 mg, 91%): ¹H NMR (DMSO-d₆) d: 11.70 (s, 1H), 8.95 (d,

J = 8.3 Hz, 1H), 8.88 (s, 1H), 8.12–8.04 (m, 1H), 7.98 (d, J = 8.4 Hz,

1H), 7.83 (dd, J = 11.4, 4.2 Hz, 1H), 7.39 (d, J = 2.4 Hz, 1H), 7.30

(dd, J = 8.6, 2.4 Hz, 1H), 7.05 (d, J = 8.7 Hz, 1H), 3.78 (d,

J = 13.4 Hz, 6H); ¹³C NMR (DMSO-d₆) d: 159.71, 148.66, 147.72,

138.50, 136.20, 129.68, 128.63, 124.98, 119.67, 117.34, 113.54,

111.73, 109.68, 55.90, 55.86; Anal. Calcd for C₁₆H₁₅N₃O₂: C,

68.31; H, 5.37; N, 14.94. Found: C, 68.52; H, 5.20; N, 15.27.

Synthesized from 3-nitroaniline (138 mg, 1 mmol) and 4-

chloro-6,7-dimethoxyquinazoline (224 mg, 1 mmol) to obtain 47

as pale yellow solid (307 mg, 94%): ¹H NMR (DMSO-d₆) d: 11.13

(s, 1H), 8.87 (s, 1H), 8.70 (t, J = 2.2 Hz, 1H), 8.27 (ddd, J = 8.1, 2.1,

0.9 Hz, 1H), 8.19 (s, 1H), 8.11 (dd, J = 8.2, 2.2 Hz, 1H), 7.77 (t,

J = 8.2 Hz, 1H), 7.31 (s, 1H), 4.01 (d, J = 12.2 Hz, 6H); ¹³C NMR

(DMSO-d₆) d: 163.54, 155.60, 154.38, 151.12, 149.93, 149.06,

145.63, 129.82, 124.91, 118.21, 116.03, 110.61, 106.92, 104.21,

56.42, 56.21; Anal. Calcd for C₁₆H₁₄N₄O₄: C, 58.89; H, 4.32; N,

17.17. Found: C, 59.00; H, 4.51; N, 17.26.

4.4.40. 6,7-Dimethoxy-N-(4-nitrophenyl)quinazolin-4-amine

(48)

4.4.35. N-(3-Bromophenyl)-6,7-dimethoxyquinazolin-4-amine

(43)

Synthesized from 4-nitroaniline (138 mg, 1 mmol) and

4-chloro-6,7-dimethoxyquinazoline (224 mg, 1 mmol) to obtain

48 as pale yellow solid (271 mg, 83%): ¹H NMR (DMSO-d₆) d:

10.94 (s, 1H), 8.58 (s, 1H), 8.06 (d, J = 7.5 Hz, 2H), 7.68 (s, 1H),

7.30 (s, 1H), 7.09 (d, J = 7.5 Hz, 2H), 3.72 (s, 3H), 3.59 (s, 3H); ¹³C

NMR (DMSO-d₆) d: 158.72, 153.70, 152.39, 148.90, 147.76,

146.88, 141.27, 126.05, 119.22, 111.20, 107.53, 103.70, 56.23,

55.99; Anal. Calcd for C₁₆H₁₄N₄O₄: C, 58.89; H, 4.32; N, 17.17.

Found: C, 59.09; H, 4.41; N, 17.00.

Synthesized from 3-bromoaniline (172 mg, 1 mmol) and

4-chloro-6,7-dimethoxyquinazoline (224 mg, 1 mmol) to obtain

43 as pale yellow solid (302 mg, 84%): ¹H NMR (DMSO-d₆) d:

11.32 (s, 1H), 8.86 (s, 1H), 8.30 (s, 1H), 8.02 (t, J = 1.9 Hz, 1H),

7.77 (ddd, J = 7.9, 2.0, 1.1 Hz, 1H), 7.49 (ddd, J = 8.0, 1.9, 1.1 Hz,

1H), 7.44 (t, J = 8.0 Hz, 1H), 7.34 (s, 1H), 4.01 (d, J = 12.1 Hz, 6H);

¹³C NMR (DMSO-d₆) d: 158.18, 156.52, 150.40, 138.91, 130.72,

128.79, 127.10, 123.50, 121.28, 107.62, 104.00, 100.38, 57.07,

K. Juvale et al. / Bioorg. Med. Chem. 21 (2013) 7858–7873

7871

4.4.41. N-(3,4-Dimethoxyphenyl)-6,7-dimethoxyquinazolin-4-

amine (49)

Synthesized from 3,4-dimethoxyaniline (153 mg, 1 mmol) and

4.5.4. 6,7-Dimethoxy-N²,N⁴-bis(3-nitrophenyl)quinazoline-2,4-

diamine (53)

Synthesized from 3-nitroaniline (345 mg, 2.5 mmol) and 2,4-di-

chloro-6,7-dimethoxyquinazoline (259 mg, 1 mmol) to obtain 53

as a pale yellow solid (310 mg, 67%): ¹H NMR (DMSO-d₆) d:

12.08 (s, 1H), 8.93 (s, 1H), 7.85–7.78 (m, 4H), 7.34–7.28 (m, 2H),

7.10 (ddd, J = 7.7, 4.9, 3.1 Hz, 3H), 6.57 (s, 1H), 3.81 (d, J = 4.9 Hz,

6H); ¹³C NMR (DMSO-d₆) d: 159.07, 157.48, 155.40, 152.32,

149.02, 148.67, 145.05, 144.69, 143.91, 130.67, 128.38, 124.27,

123.11, 115.24, 114.30, 112.74, 110.61, 105.03, 103.22, 56.31,

55.82; Anal. Calcd for C₂₂H₁₈N₆O₆: C, 57.14; H, 3.92; N, 18.17.

Found: C, 56.80; H, 4.11; N, 18.24.

4-chloro-6,7-dimethoxyquinazoline (224 mg, 1 mmol) to obtain

49 as pale yellow solid (259 mg, 76%): ¹H NMR (DMSO-d₆) d:

11.08 (s, 1H), 8.75 (s, 1H), 8.18 (s, 1H), 7.29 (dd, J = 8.7, 5.1 Hz,

2H), 7.19 (dd, J = 8.6, 2.4 Hz, 1H), 7.05 (d, J = 8.7 Hz, 1H), 3.99 (d,

J = 5.0 Hz, 6H), 3.79 (d, J = 10.7 Hz, 6H); ¹³C NMR (DMSO-d₆) d:

156.26, 150.24, 148.75, 147.48, 129.93, 117.35, 111.82, 109.74,

107.25, 103.85, 56.93, 56.56, 55.89; Anal. Calcd for C₁₈H₁₉N₃O₄:

C, 63.33; H, 5.61; N, 12.31. Found: C, 63.20; H, 5.72; N, 12.29.

4.5. General procedure for synthesis of N²,N⁴-disubstituted

quinazolines

4.5.5. N²,N⁴-Bis(3,4-dimethoxyphenyl)-6,7-

dimethoxyquinazoline-2,4-diamine (54)

2,4-Dichloro-6,7-dimethoxyquinazoline (1 mmol) was dis-

solved in reﬂuxing 2-propanol (20 ml) and the substituted aniline

(2.5 mmol) was added drop wise. The reaction mixture was re-

ﬂuxed for 12–24 h, until the reaction was complete as indicated

by TLC. The precipitate formed was ﬁltered off and washed with

2-propanol (10 ml) and ether (10 ml). The product was recrystal-

lized from 75% ethanol.

Synthesized from 3,4-dimethoxyaniline (382 mg, 2.5 mmol)

and 2,4-dichloro-6,7-dimethoxyquinazoline (259 mg, 1 mmol) to

obtain 54 as a pale yellow solid (290 mg, 59%): ¹H NMR (CDCl3)

d: 12.82 (s, 1H), 9.72 (s, 1H), 9.10 (s, 1H), 7.66 (s, 1H), 7.53 (d,

J = 2.2 Hz, 1H), 7.46 (dd, J = 8.6, 2.3 Hz, 1H), 6.82 (dd, J = 8.6,

2.4 Hz, 1H), 6.56 (d, J = 2.4 Hz, 1H), 6.40 (dd, J = 8.7, 1.9 Hz, 2H),

6.29 (s, 1H), 4.07 (s, 3H), 3.82 (d, J = 16.9 Hz, 6H), 3.65 (d,

J = 4.5 Hz, 6H), 3.58 (s, 3H); ¹³C NMR (CDCl₃) d: 157.08, 156.12,

150.47, 148.94, 148.81, 147.36, 146.89, 146.85, 134.75, 130.54,

129.15, 115.07, 114.97, 110.72, 110.61, 107.19, 104.20, 102.60,

97.71, 56.81, 56.13, 56.05, 56.03, 56.00, 55.92; Anal. Calcd for

4.5.1. N²,N⁴-Bis(3-bromophenyl)-6,7-dimethoxyquinazoline-

2,4-diamine (50)

Synthesized from 3-bromoaniline (430 mg, 2.5 mmol) and 2,4-

dichloro-6,7-dimethoxyquinazoline (259 mg, 1 mmol) to obtain

50 as pale yellow solid (307 mg, 58%): ¹H NMR (DMSO-d₆) d:

12.05 (s, 1H), 10.17 (s, 1H), 7.64 (s, 1H), 7.38 (s, 1H), 7.30 (d,

J = 1.3 Hz, 1H), 7.19 (ddt, J = 9.8, 4.4, 2.7 Hz, 5H), 6.88–6.73 (m,

1H), 6.65 (dt, J = 7.1, 1.5 Hz, 1H), 3.65 (s, 3H), 3.58 (s, 3H); ¹³C

NMR (DMSO-d₆) d: 156.97, 155.67, 154.79, 150.42, 145.92,

145.53, 145.00, 130.05, 129.68, 125.54, 125.34, 124.03, 124.03,

122.90, 122.68, 120.78, 120.77, 111.61, 105.76, 101.76, 57.01,

55.39; Anal. Calcd for C₂₂H₁₈Br₂N₄O₂: C, 49.84; H, 3.42; N, 10.57.

Found: C, 49.79; H, 3.60; N, 10.39.

C₂₆H₂₈N₄O₆: C, 63.40; H, 5.73; N, 11.38. Found: C, 63.21; H, 5.99;

N, 11.50.

4.6. Biology

4.6.1. Materials for biological studies

Ko143 was purchased from Tocris Bioscience (Bristol, United

Kingdom). All other chemicals were purchased from Sigma–Aldrich

Chemicals (Taufkirchen, Germany) unless otherwise speciﬁed. For

the investigation of the synthesized compounds in different cell

based assays 10 mM stock solutions of the test compounds were

prepared in DMSO. Different dilutions of compounds were pre-

pared in sterile ﬁltered Krebs–HEPES buffer (KHB). For some com-

pounds with low solubility methanol was used for preparing 1 mM

concentrations. The amount of methanol was chosen so that it did

not exceed 5% in the highest concentration used in the assay. The

highest concentration of DMSO present in dilutions used for the as-

says was not more than 0.5%.

4.5.2. 6,7-Dimethoxy-N²,N⁴-bis(3-(triﬂuoromethyl)phenyl)-

quinazoline-2,4-diamine (51)

Synthesized

from

3-(triﬂuoromethyl)aniline

(403 mg,

2.5 mmol) and 2,4-dichloro-6,7-dimethoxyquinazoline (259 mg,

1 mmol) to obtain 51 as a pale yellow solid (274 mg, 54%): ¹H

NMR (DMSO-d₆) d: 11.95 (s, 1H), 8.73 (s, 1H), 7.46 (s, 1H), 7.32

(s, 1H), 7.27 (d, J = 1.4 Hz, 1H), 7.12–6.97 (m, 3H), 6.82 (dd,

J = 3.2, 2.5 Hz, 2H), 6.71–6.66 (m, 1H), 6.54 (ddd, J = 5.9, 3.3,

1.7 Hz, 1H), 3.54 (d, J = 6.0 Hz, 6H); ¹³C NMR (DMSO-d₆) d:

158.95, 156.07, 154.32, 149.26, 144.98, 143.04, 141.70, 134.50,

130.31, 128.82, 126.39, 126.07, 124.83, 123.92, 119.25, 118.78,

115.07, 110.54, 106.29, 103.88, 55.72, 54.89; Anal. Calcd for C_24-

H₁₈F₆N₄O₂: C, 56.70; H, 3.57; N, 11.02. Found: C, 56.57; H, 3.70;

N, 11.29.

4.6.2. Cell culture

The cell lines MDCK wt and MDCK BCRP were received as a gen-

erous gift from Dr. A. Schinkel (The Netherlands Cancer Institute,

Amsterdam, The Netherlands). MDCK BCRP cells were generated

by transfection of the canine kidney epithelial cell line MDCKII

with the human wild-type cDNA C-terminally linked to the cDNA

of the green ﬂuorescent protein (GFP). These cells were cultured

in Dulbecco’s modiﬁed eagle medium (DMEM) with 10% fetal bo-

4.5.3. 3,3⁰-((6,7-Dimethoxyquinazoline-2,4-diyl)bis(azanediyl))-

dibenzonitrile (52)

vine serum (FBS), 50

lg/ml streptomycin, 50 U/ml penicillin G

Synthesized from 3-aminobenzonitrile (295 mg, 2.5 mmol) and

2,4-dichloro-6,7-dimethoxyquinazoline (259 mg, 1 mmol) to ob-

tain 52 as a pale yellow solid (308 mg, 73%): ¹H NMR (DMSO-d₆)

d: 12.04 (s, 1H), 9.01 (s, 1H), 7.48 (s, 1H), 7.32 (s, 1H), 7.28 (ddd,

J = 6.9, 6.5, 4.3 Hz, 3H), 7.09 (t, J = 1.7 Hz, 1H), 7.01 (ddd, J = 7.3,

2.5, 1.3 Hz, 2H), 6.71 (dt, J = 7.1, 1.3 Hz, 1H), 6.55 (dt, J = 7.3,

1.7 Hz, 1H), 3.68 (d, J = 5.9 Hz, 6H); ¹³C NMR (DMSO-d₆) d:

158.38, 156.40, 153.12, 150.61, 147.82, 141.30, 141.21, 133.50,

131.17, 130.06, 127.99, 125.31, 123.08, 121.47, 118.62, 116.57,

115.05, 110.33, 106.19, 101.22, 56.07, 55.31; Anal. Calcd for

and 2 mM -glutamine. Human ovarian carcinoma cell lines

L

A2780 and its corresponding MDR1 overexpressing doxorubicin

resistant A2780adr cell line were purchased from ECACC (Nos.

93112519 and 93112520). The cell lines were grown in RPMI-

1640 medium supplemented with 10% FBS, 50

50 U/ml penicillin G, and 2 mM -glutamine. To maintain the P-gp

overexpression in the A2780adr cell line, intermittently cells were

incubated with 1 M doxorubicin for one passage. The human

lg/ml streptomycin,

L

l

ovarian cancer cell line 2008 stably expressing MRP1 generously

received from Prof. Dr. Piet Borst (The Netherlands Cancer Insti-

tute, Amsterdam, The Netherlands) was used for MRP1 testing.

The cell line 2008 MRP1 was grown in the same medium as

C₂₄H₁₈N₆O₂: C, 68.24; H, 4.29; N, 19.89. Found: C, 68.33; H, 4.10;

N, 20.05.

7872

K. Juvale et al. / Bioorg. Med. Chem. 21 (2013) 7858–7873

described for A2780 cells with addition of 400

(Geneticin) for maintaining ABCC1 overexpression.

All cells were maintained in a 5% CO₂humidiﬁed atmosphere at

37 °C. After conﬂuence of 80–90%, subculturing was performed

with 0.05% trypsin and 0.02% EDTA. For performing different cell

based assays, cell counting was performed using a CASY1 model

l

g/ml G-418

to 109 min) were calculated for each concentration and from these

data, concentration response curves were generated by nonlinear

regression using the four-parameter logistic equation with variable

Hill slope (GraphPad Prism v. 5.0, San Diego, CA, USA).

4.6.5. Pheophorbide A assay

TT cell counter with 150

l

m capillary.

Newly investigated compounds 27–54 were additionally tested

for BCRP inhibition in the pheophorbide A assay as described earlier

using MDCK wt and MDCK BCRP cells.^16,34To perform the pheophor-

bide A assay, cells were prepared as described for the Hoechst 33342

assay. Approximately 45,000 cells per well were seeded into U-

shaped clear 96 well plates (Greiner, Frickenhausen, Germany) in a

volume of 160 ll. To this 20 ll of various test compounds in differ-

ent concentrations were added. The prepared 96 well plate was kept

under 5% CO₂and 37 °C for 30 min. After this preincubation period,

20 ll of a 5 lM pheophorbide A solution (protected from light) was

added to each well and plates were further incubated for 120 min.

Fluorescence was measured by ﬂow cytometry (FACScalibur, Becton

Dickinson Biosciences, Heidelberg, Germany). For the measurement,

pheophorbide A was excited at an excitation wavelength of 488 nm

and emission was detected in the FL3 channel (P670 nm). Concen-

tration response curves were generated by nonlinear regression

using the four-parameter logistic equation with variable Hill slope.

4.6.3. Membrane preparation, protein content examination by

western blot analysis

As ABC transporter proteins are expressed in cell membrane, cells

have to be harvested and shredded to get only the membranes with

protein. First, medium was removed from the cells and cells were

treated with 2.4 ml homogenization buffer (20 mM HEPES, 10 mM

Na₂–EDTA) per ﬂask. Then, cells were harvested by a cell scraper

and transferred to a 50 ml tube for further centrifugation. Cells were

then shredded and homogenized by a Polytron-homogenisator for

12 s on ice, followed by a ﬁrst centrifugation (800g, 10 min, 4 °C)

and the second centrifugation of the supernatant at (40,000g,

30 min, 2 °C). After the second centrifugation, a pellet was formed

consisting of protein containing membranes. To the pellet, storage

buffer (20 mM HEPES, 0.1 mM Na₂–EDTA) was added, the cells were

resuspended and again centrifuged at 40,000g, 30 min, 2 °C. The

supernatant was discarded and the procedure was repeated in order

to wash the membrane pellet. In the end, 0.4 ml storage buffer was

added to the pellet and it was stored at ꢀ80 °C until use.

4.6.6. Calcein AM assay

For determining the selectivity of synthesized compounds to-

wards BCRP, these were further tested for their P-gp and MRP1

inhibition in the calcein AM assay as described earlier.^33–35For this

purpose A2780adr cells for P-gp and 2008 MRP1 cells for MRP1

were used. After conﬂuence of 80–90%, subculturing was per-

formed with 0.05% trypsin and 0.02% EDTA. Cells were prepared

as described above and washed three times with KHB buffer and

then seeded into colorless 96 well plates (Greiner, Frickenhausen,

Germany) at a density of approximately 30,000 cells in a volume

Before performing western blot analysis, the protein content in

the membrane preparation was determined by the amido black

protein assay. 30 lg of membrane protein was loaded onto each

lane of SDS–polyacrylamide gel (7.5% for P-gp and MRP1, 10% for

BCRP), subjected to electrophoresis and then electro-transferred

onto nitrocellulose membranes. For determining MRP1-expression

in MRP1 2008 cells, blots were probed with the monoclonal anti-

MRP1-antibody [QCRL1] (Merck, Darmstadt, Germany), while for

MDCK BCRP cells the anti-BCRP antibody [BXP-21] (Abcam, Cam-

bridge, UK) and for A2780 P-gp expressing cells the anti-Pgp anti-

body C219 (Covance, Munich, Germany) were used. As a secondary

antibody, goat anti-Mouse IgG HRP conjugate (H+L)—antibody

(Merck Millipore, Darmstadt, Germany) was used. Results obtained

by western blot analysis for each cell line are shown in Figure 1.

of 90

resulting in a ﬁnal volume of 100

plates were preincubated for 30 min. After the preincubation per-

iod, 33 l of a 1.25 M calcein AM solution, which was protected

ll per well. Then, 10

l

l of the test compounds were added,

l

l per well. The prepared 96 well

l

from light, was added to each well. The ﬂuorescence was measured

immediately in constant time intervals (60 s) up to 90 min using an

excitation wavelength of 485 nm and an emission wavelength of

520 nm with a BMG POLARstar microplate reader tempered at

37 °C. For calculation of inhibitory effects, the ﬁrst linear part of

the ﬂuorescence time curves was used. From the slopes concentra-

tion response curves were generated by nonlinear regression using

the four-parameter logistic equation with variable Hill slope.

4.6.4. Hoechst 33342 accumulation assay

To investigate the inhibitory effect of the synthesized com-

pounds on BCRP, the Hoechst 33342 accumulation assay was per-

formed as described earlier with small modiﬁcations.^9,31–33After

reaching a conﬂuence of 80–90%, cells were harvested by gentle

trypsination (0.05% trypsin/0.02% EDTA) and then transferred to a

50 ml tube followed by centrifugation (266g, 4 °C, 4 min). The cell

pellet obtained was resuspended in fresh culture medium and the

cell density was determined using a CASY1 model TT cell counter

device (Schaerfe System GmbH, Reutlingen, Germany). Followed

by another centrifugation, cells were washed three times with

KHB and seeded into black 96 well plates (Greiner, Frickenhausen,

Germany) at a density of approximately 20,000 cells per well in a

4.6.7. MTT cytotoxicity assay

Intrinsic cytotoxicity of the selected active compounds (20, 26,

31, 36, 40 and 47) was determined in MDCK BCRP and MDCK sen-

sitive cells using the MTT cytotoxicity assay. The assay was per-

formed as described earlier with minor modiﬁcations.^33,35Cells

were seeded into 96-well tissue culture plates (Sarsteddt, Newton,

USA) at a density of 2500 cells per well in a volume of 180

kept under 5% CO₂at 37 °C for 6 h. Attachment of cells was con-

trolled under the microscope and 20 l of the test compounds were

added to achieve the required ﬁnal concentration in a volume of

200 l. Control experiments were performed with medium con-

ll and

volume of 90

centrations were added to a total volume of 100

96 well plate was kept under 5% CO₂and 37 °C for 30 min. After

this preincubation period, 20 l of a 6 M Hoechst 33342 solution

ll. 10

ll of various test compounds in different con-

ll. The prepared

l

(protected from light) was added to each well. Fluorescence was

measured immediately in constant intervals (60 s) up to 120 min

at an excitation wavelength of 355 nm and an emission wave-

length of 460 nm using a 37 °C tempered BMG POLARstar micro-

plate reader (BMG Labtech, Offenburg, Germany). For the analysis

of the data obtained from the assay, ﬁrst ﬂuorescence of KHB

was subtracted from the ﬂuorescence reading obtained from MDCK

cells. Average of ﬂuorescence values in the steady state (from 100

taining 10% (v/v) of DMSO (positive control) and only PBS (negative

control). After an incubation period of 72 h, the MTT reagent was

added (20

ther incubated for 1 h and then the assay was terminated by

removing supernatants and lysing the cells with 100 l DMSO

ll of a 5 mg/ml solution) to each well. Plates were fur-

l

per well. Viability of cells was determined spectrophotometrically

by measuring absorbance at 544 nm and background correction at

710 nm using a BMG POLARstar microplate reader.

K. Juvale et al. / Bioorg. Med. Chem. 21 (2013) 7858–7873

7873

Similar experiments were performed to determine the MDR

reversal ability of selected compounds (20, 26) at a ﬁnal concentra-

tion of 1 M and 5 M. In this case the effect of these compounds

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BCRP expression

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Acknowledgements

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We would like to thank Dr. A. H. Schinkel (The Netherlands Can-

cer Institute, Amsterdam, The Netherlands) for kindly providing

the MDCK BCRP cell line. We also thank Dr. P. Borst (The Nether-

lands Cancer Institute, Amsterdam, The Netherlands) for providing

the 2008 MRP1 cell line. K.J. thanks DAAD (Germany) for providing

doctoral fellowship.

Article Doi

Investigation of quinazolines as inhibitors of breast cancer resistance protein (ABCG2)

DOI: 10.1016/j.bmc.2013.10.007

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Full text of DOI:10.1016/j.bmc.2013.10.007

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