7-Substituted-[1,4]dioxano[2,3-g]quinazolines as inhibitors of epidermal growth factor receptor kinase

Article abstract of DOI:10.1002/ardp.200290003

With the aim of developing inhibitors of EGFR tyrosine kinase, the 7-methoxymethyl-[1,4]dioxano[2,3-g]quinazolines (3a-b) and 7-mono- or di-alkylaminomethyl-[1,4]dioxano[2,3-g]quinazolines (4a-i) were prepared and evaluated for the inhibition of EGFR tyrosine kinase and the growth inhibition of human tumor cell lines. Among them, compounds 4d and 4h showed potencies against both EGFR tyrosine and the A431 cell line similar to that of PD153035 with greater aqueous solubilities of their HCI salts.

Full text of DOI:10.1002/ardp.200290003

Arch. Pharm. Pharm. Med. Chem. 2002, 10, 487–494
7-Substituted-[1,4]dioxano[2,3-g]quinazolines 487
Jae Yeol Lee,
7-Substituted-[1,4]dioxano[2,3-g]quinazolines as
Inhibitors of Epidermal Growth Factor Receptor
Kinase
Yong Kyu Park,
Seon Hee Seo,
Beom-Seok Yang,
Hokoon Park,
Yong Sup Lee
With the aim of developing inhibitors of EGFR tyrosine kinase, the 7-methoxymeth-
yl-[1,4]dioxano[2,3-g]quinazolines (3a–b) and 7-mono- or di-alkylaminomethyl-
[1,4]dioxano[2,3-g]quinazolines (4a–i) were prepared and evaluated for the inhibi-
tion of EGFR tyrosine kinase and the growth inhibition of human tumor cell lines.
Among them, compounds 4d and 4h showed potencies against both EGFR tyro-
sine and the A431 cell line similar to that of PD153035 with greater aqueous solubili-
ties of their HCl salts.
Medicinal Chemistry Research
Center, Korea Institute of
Science & Technology,
P.O. Box 131, Cheongryang,
Seoul 130–650, Korea
Keywords:EGFR;Tyrosine kinase;Inhibitor;Water solubility;Dioxane;Quinazoline;
A431; Antitumor
Received: March 12, 2002 [FP683]
fused tricyclic quinazolines have therefore been pre-
pared where various solubilizing groups were appended
Introduction
The epidermal growth factor receptor (EGFR) plays a
central role in growth signaling and regulation of mam-
malian cells [1]. Like other protein kinases, it mainly
transduces signals to a target cell by catalyzing the
transfer of the terminal phosphate of ATP to tyrosine resi-
dues in protein substrates.Overexpression of EGFR has
been implicated in a significant proportion of human tu-
mors and other proliferative diseases [2].EGFR is there-
fore expected to be an attractive target for cancer
chemotherapy. Due to the relative conservation of the
ATP binding site of protein kinases, the development of
selective inhibitors of protein kinases of the EGFR has
been an important concern. As a result of extensive re-
search, the 4-anilinoquinazoline class of compounds
(e.g.PD153035, I) have recently proven to be potent and
selective inhibitors of tyrosine kinase activity of EGFR
via competitive binding at the ATP site of the enzyme [3].
Based on these results, considerable efforts have been
devoted to the synthesis of PD153035 analogues for
studying structure-activity relationships to develop tyro-
sine kinase inhibitory antitumor drugs [4–7].
to amine substituents [9–11]. Recently, we have also re-
ported that 4-anilino-[1,4]dioxano[2,3-g]quinazolines (2)
are effective inhibitors of EGFR as judged by their inhibi-
tory activity against EGFR kinase and the growth of hu-
man tumor cell lines [12].
With a view to the further investigation of structure-activi-
ty relationships (SAR) and improving water solubility in
this series of [1,4]dioxano[2,3-g]quinazoline inhibitors,
we report here the synthesis and biological activity of a
series of [1,4]dioxano[2,3-g]quinazolines substituted
with methoxymethyl (3a–b) or mono- or dialkylami-
nomethyl group (4a–i) at 7-position, based on the our re-
cent report that a [1,4]dioxano[2,3-g]quinazoline ring
would be a new scaffold for EFGR kinase inhibitor [12].
Synthesis
First, compounds of the 7-methoxymethyl-[1,4]diox-
ano[2,3-g]quinazoline series (3a–b) were prepared by
the known procedures as shown in Scheme 1 [8, 12–14].
Commercially available 2-hydroxymethylbenzo[1,4]-
dioxane (5) was protected with NaH and CH3I to afford
2-methoxymethylbenzo[1,4]dioxane (6) in 78 % yield.
Friedel-Crafts acylation of compound 6 using acetyl
chloride gave 7-acylbenzodioxane 7 (70 %) along with
6-acyl isomer (30 %) in 84 % yield. Because 6-acyl iso-
mer could not be separated by column chromatography,
the isomeric mixture was used for the next reactions [13].
Oxidation of the isomeric mixture with NaOCl (48 %), ni-
While PD153035 (1) has an EGFR tyrosine kinase inhib-
itory activity with IC50 = 25 pM (Ki 6 pM) and shows little
effect on the activity of other kinase [3], its poor water sol-
ubility complicated its in vivo evaluation in tumor models
[8].With a view to overcoming the poor aqueous solubili-
ty of the parent compound, pyrido[d]pyrimidines and
Correspondence: Yong Sup Lee, Medicinal Chemistry
Research Center, Korea Institute of Science & Technology,
P.O. Box 131 Cheongryang, Seoul 130–650, Korea.
tration (54 %), and reduction (65 %) provided
a
Phone:
+8229585167,
Fax:
+8229585189,
e-mail:
yslee@kist.re.kr.
[1,4]dioxane-fused anthranilic acid (10) as a precursor
© 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/010/0487 $ 17.50+.50/0

488 Lee et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 487–494
Scheme 1a
for cyclization. At this stage, the impurity from 6-acyl iso-
mer could be removed by recrystallization with EtOAc.
The cyclization of pure compound 10 with triazine was
conducted in the presence of piperazine to afford 4-hy-
droxyquinzaoline (11) in 63 % yield, which was easily
converted into 4-chloroquinazoline (12) with POCl3 in
90 % yield. The coupling reaction between 4-chloro-
quinazoline (12) and appropriate anilines was finally
conducted to provide 4-anilino-7-methoxymethyl-
[1,4]dioxano[2,3-g]quinazolines (3a–b) in iso-propanol
under reflux in 63 and 94 % yields. The structures and
yields of these compounds are summarized in
Table 1.The 7-mono or di-alkylaminomethylquinazoline
Scheme 2a

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 487–494
7-Substituted-[1,4]dioxano[2,3-g]quinazolines 489
Table 1. Structures, yields, physical and biological properties of 7-substituted-[1,4]-dioxano[2,3-g]quinazolines.
derivatives (4a–i) were prepared from the selected 7-
methoxymethyl-[1,4]-dioxano[2,3-g]quinazolines (3a–
b) by demethylation of 7-methoxymethyl group of com-
pound (3a–b) with BBr3, mesylation of the resultant free
hydroxy group with MsCl/TEA, and subsequent dis-
placement of mesylate (14a–b) with various amines as
shown in Scheme 2.In order to improve the aqueous sol-
ubility of parent compounds, all compounds 4a–i were
also prepared as hydrochloride salts (>99 %) by treating
the parent compounds with EtOH/AcCl. The structures
and yields of these compounds 4a–i are also summa-
rized in Table 1.
ation of tyrosine kinase substrate 2 by EGFR and the
growth of A431 cell line (uterus cancer), which highly
overexpresses EGFR. To compare these data with that
of standard, the inhibitory activity of the PD153035 un-
der our assay conditions was inserted into the data set.
The IC50 value of PD153035 we obtained is significantly
higher and lower, respectively, than reported by previous
workers [3, 15]. It was reported that the difference is at
least partly attributable to the nature of the enzyme and
substrate as well as the overall assay conditions [16].
In the case of 7-methoxymethylquinazoline compounds
(3a and 3b), both compounds showed better potencies
(GI50 = 0.59 and 0.85 nM, respectively) than the stan-
dard compound, PD153035 (GI50 = 1.03 nM) against the
growth of the A431 cell line, but less inhibitory activities
Results and discussion
The compounds (3a–b) and (4a–i) shown in Table 1
were evaluated for their ability to inhibit the phosphoryl-

490 Lee et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 487–494
Figure 1. PD153035 (1), its cyclic analogue (2), and 7-substituted-[1,4]dioxano[2,3-g]quinazolines (3 and 4).
(3a and 3b; IC50 = 35.5 and 28.2 nM, respectively) than
PD153035 (IC50 = 6.6 nM) against the isolated enzyme.
Among the synthesized compounds, compound 3a,
containing a 3-bromoaniline ring at 4-position, was the
most potent compound against the growth of A 431 cells.
Table 2. Inhibition of the growth of selected cell lines.
In the case of 7-mono- or di-alkylaminomethylquinazo-
line derivatives (4a–i), several points are apparent from
the data shown inTable 1:First, all compounds generally
showed similar activities to PD153035 against the
growth of the A 431 cell line, while the range of enzyme
inhibitory activities (IC50 = 8.9 to >100 nM) against the
isolated enzyme are relatively broad. As expected, sec-
ondly, HCl salts of all compounds showed much greater
aqueous solubilities (>200 mM) than PD153035
(0.056 mM) under those experiments. With regard to
isolated enzyme, compound 4d, containing (4-methyl-
piperazino)methyl group at 7-position, was the most po-
tent compound (IC50 = 8.9 nM) and compound 4h, bear-
ing bis(ethanol)aminomethyl group, showed an activity
as effective (IC50 = 9.5 nM) as compound 4d.In the case
of other compounds, the substitution of 7-mono- or -di-
alkylamino side chain generally resulted in decreasing
inhibitory potency (IC50 = 28.2 to >100 nM) against the
isolated enzyme when compared with their parent com-
pounds (3a and 3b; IC50 = 35.5 and 28.2 nM, respec-
tively).
(GI50 = 1.03 µM) against the A431 cell line with the ex-
ception of compounds 4b and 4h (GI50 = 2.57 and
2.21 µM, respectively), irrespective of enzyme inhibitory
activity.Therefore, it is difficult to interpret the structure-
activity relationship of compounds inTable1 between en-
zyme inhibitory activity and cell growth inhibitory activity.
Among this series, compound 4f, having a 2-(pyrrolidi-
no)ethylamino group at 7-position, was the most potent
compound (GI50 = 0.71 µM).
With regard to cellular assay, most compounds showed
similar potencies (GI50 = 0.71 to 1.21 µM) to PD153035
Some compounds (4a–d) were further evaluated
against human cancer cell lines for their selectivity:

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 487–494
7-Substituted-[1,4]dioxano[2,3-g]quinazolines 491
layer thickness 0.25 mm). Flash column chromatography was
performed with Merck Kiesegel 60 Art 9385 (230–400 mesh).
All solvents used were purified according to standard proce-
dures.
HCT116 (colon cancer) and SNU638 (stomach cancer),
which express low levels of EGFR. As a result, all com-
pounds showed more than 10-fold selectivities for the in-
hibition of growth of A431 over HCT116 and SNU638, as
shown in Table 2. This suggests that the mechanism of
growth inhibition is largely due to the targeting of EGFR.
2-Methoxymethyl-benzo[1,4]dioxane (6)
To a susupension of NaH (5.0 g, 0.20 mol) in 60 mL of anhy-
drous THF was added 2-hydroxymethylbenzo[1,4]dioxane
(28.9 g, 0.17 mol) at 0°C, and then the mixture was stirred at
room temperature for 30 min followed by addition of methyl
iodide (16.3 mL, 0.26 mol).The reaction mixture was stirred at
room temperature for 48 h, quenched with 10 mL of water, and
extracted with ethyl acetate. The combined organic layer was
dried over MgSO4 and concentrated in vacuo to give 6 (24.0 g,
78 %) as an oil: 1H NMR (CDCl3) δ 6.96–6.85 (m, 4H), 4.32–
4.26 (m, 2H), 4.05 (m, 1H), 3.67–3.54 (m, 2H), 3.42 (s, 3H).
With regard to water solubility, the parent compound 3a
was 10-fold more soluble (0.6 mM) than PD153035
(0.06 mM) and compounds 4a–i as HCl salts showed
much greater aqueous solubilities (>200 mM) in those
experiments. Meanwhile, the formation of 4c as HCl salt
resulted in slight loss of activity against the isolated en-
zyme and A431 cells, when compared with 4c as a free
base (IC50’s of 42.7 and 28.2 nM; GI50’s 2.48 and
1.50 µM, respectively – the data for HCl salt compounds
are not shown in Table 1). In spite of poor enzyme inhibi-
tory activities of some compounds, therefore, their high
potency against the growth of cell lines would be ex-
plained by good cellular uptake of compounds.
7-Acetyl-2-methoxymethyl-benzo[1,4]dioxane (7)
A mixture of anhydrous AlCl3 (4.4 g, 33.3 mmol) and N,N-
dimethylacetamide (1.3 mL, 16.7 mmol) was stirred at room
temperature for 30 min. To the mixture was added the above
compound 6 (0.5 g, 3.8 mmol), and the mixture was stirred at
the same temperature for 15 min, followed by addition of acetyl
chloride (0.3 mL, 3.3 mmol). After stirring at room temperature
for 5 h, the reaction mixture was cooled to 0°C, treated with ice-
water, neutralized with saturated NaHCO3 solution, and ex-
tracted with ethyl acetate. The combined organic layer was
dried over MgSO4 and concentrated in vacuo to give 7 (520 mg,
84 %) as an isomeric mixture, which was used for the next reac-
tion without further purification:1H NMR (CDCl3) δ 7.53 (s, 1H),
7.50 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 4.37–4.23 (m,
2H), 4.11 (m, 1H), 3.67–3.57 (m, 2H), 3.42 (s, 3H), 2.51 (s,
3H).
In conclusion, we prepared new 7-methoxymethyl-
[1,4]dioxano[2,3-g]quinazolines (3a–b) and 7-mono-
or di-alkylaminomethyl-[1,4]dioxano[2,3-g]quinazolines
(4a–i) derivatives to probe the effect of 7-substituents on
the inhibition of EGFR tyrosine kinase and the growth in-
hibition of human tumor cell lines.Most compounds were
as potent as PD153035 against the A431 cell line and
some compounds (4a–d) showed good selectivities for
A431 over other tumor cell lines such as HCT116 and
SNU638. HCl salts of most compounds showed much
greater aqueous solubilities (>200 mM) than PD153035.
In particular, compounds 4d and 4h showed similar po-
tencies to PD153035 against both EGFR and the A431
cell line.The above results demonstrate that 7-mono- or
dialkylaminomethylquinazoline derivative would be an
effective inhibitor of EGFR when considered in the light
of the inhibitory activity against EGFR kinase, the growth
of human tumor cell lines, and the aqueous solubility of
its HCl salt.
3-Methoxymethyl-benzo[1,4]dioxane-6-carboxylic acid (8)
A solution of 13 % NaOCl (140 mL; Fluka®) was added to 7
(12.9 g, 57.8 mmol).The reaction mixture was heated at 65°C
for 30 min, cooled to room temperature, and further stirred at
the same temperature for 30 min.The insoluble precipitate was
filtered off and the filtrate was acidified to pH 3–4 with c-HCl to
give a solid. The filtered solid was washed with water and re-
crystallized with water/ethanol (v/v = 2/3) to afford 8 (6.3 g,
48 %) as a white solid: 1H NMR (CD3OD3) δ 7.48 (d, J = 8.5 Hz,
1H), 7.45 (s, 1H), 6.85 (d, J = 8.5 Hz, 1H), 4.35–5.25 (m, 2H),
4.05 (m, 1H), 3.62–3.58 (m, 2H), 3.25 (s, 3H).
3-Methoxymethyl-7-nitro-benzo[1,4]dioxane-6-carboxylic acid
(9)
To a solution of 3 (0.8 g, 3.5 mmol) in 2 mL of glacial acetic acid
was added a mixture of H2SO4 (3 mL) and HNO3 (3 mL) at room
temperature during a period of 30 min. After further stirring for
30 min, the reaction mixture was treated with 10 mL of water to
produce a solid.The solid was filtered, washed with water and
ether, and dried in vacuo to afford 9 (0.5 g, 54 %) as a white
solid: 1H NMR (CDCl3) δ 7.41 (s, 1H), 7.34 (m, 1H), 4.52–4.37
(m, 2H), 4.16 (m, 1H), 3.70–3.61 (m, 2H), 3.44 (s, 3H).
Acknowledgement
This work was supported by Korea Institute of Science
and Technology (2E17073).
Experimental part
7-Amino-3-methoxymethyl-benzo[1,4]dioxane-6-carboxylic
acid (10)
Chemistry
To a solution of 9 (1.2 g, 4.53 mmol) in 10 mL of methanol was
added 10 % Pd/C (50 mg), and the reaction mixture was stirred
at room temperature for 24 h. After filtering through Celite 545,
the filtrate was concentrated in vacuo and the residue was re-
crystallized with EtOAC to afford pure 10 (0.7 g, 65 %) as a pale
yellow solid: mp 161–167°C; 1H NMR (CDCl3) δ 7.50 (s, 1H),
7.28 (s, 2H), 6.17 (s, 1H), 4.34–4.24 (m, 2H), 4.12 (m, 1H),
3.67–3.59 (m, 2H), 3.44 (s, 3H).
1H NMR spectra were recorded on a Gemini Varian-300
(300 MHz). Mass spectra (EI) were determined on HP GC
5972 and HP MS 5988A system at 70 eV.HPLC was performed
on a Waters pump model 501, with a UV detector (λ = 254 nm)
model486systemusingaLiChrosorbRP-18(10mmi.d.×25 cm,
Merck).Analytical thin layer chromatography (TLC) was carried
out on precoated silica gel (E. Merck Kiesegel 60F254

492 Lee et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 487–494
{4-[(3Ј-Bromophenyl)amino]-[1,4]dioxano[2,3-g]quinazolin-
yl}methyl methanesulfonate (14a): General procedure for
mesylation of 13a and 13b in Scheme 2
7-Methoxymethyl-[1,4]dioxano[2,3-g]quinazolin-4-ol (11)
A solution of 10 (0.4 g, 1.53 mmol), triazine (0.2 g, 2.30 mmol),
and piperidine (0.015 mL, 0.15 mmol) in 10 mL of methanol was
heated at reflux for 4 h. After cooling to room temperature, the
resultant solid was filtered, washed with methanol, and dried in
vacuo to afford 11 (0.24 g, 63 %) as a white solid: mp 246–
251°C;1H NMR (DMSO-d6) δ 7.92 (s, 1H), 7.46 (s, 1H), 7.09 (s,
1H), 4.46–4.19 (m, 2H), 4.13 (m, 1H), 3.62–3.60 (m, 2H), 3.32
(s, 3H).
To a solution of 13a (200 mg, 0.52 mmol) in 10 mL of CH2Cl2
was added triethylamine (0.72 mL, 5.15 mmol) followed by ad-
dition of methanesulfonyl chloride (0.40 mL, 5.15 mmol) at 0°C.
The reaction mixture was stirred at the same temperature for
1 h and further stirred at room temperature for 4 h. After treat-
ing with saturated with NaHCO3 solution, the reaction mixture
was extracted with CH2Cl2. The combined organic layer was
dried over MgSO4, concentrated, and purified with column
4-Chloro-7-methoxymethyl-[1,4]dioxano[2,3-g]quinazoline
(12)
chromatography (EtOAc/hexane
= 2/1) to provide 14a
(144 mg, 60 %) as a pale yellow solid: 1H NMR (CDCl3) δ 8.42
(s, 1H), 8.10 (s, 1H), 7.91 (s, 1H), 7.70 (m, 1H), 7.28–7.27 (m,
2H), 7.21 (s, 1H), 4.65 (m, 1H), 4.60–4.50 (m, 3H), 4.26 (m,
1H), 3.10 (s, 3H).
To a solution of 11 (0.5 g, 2.0 mmol) in 25 mL of POCl3 was ad-
ded N,N-dimethylaniline (0.64 mL, 5.49 mmol), and the reac-
tion mixture was heated at reflux for 2 h. After excess of POCl3
was distilled off under reduced pressure, the residue was dis-
solved in 30 mL of CH2Cl2 and treated with 50 mL of ice-water.
The separated organic layer was washed with saturated
NaHCO3, dried over MgSO4, and concentrated in vacuo to pro-
vide 12 (0.5 g, 90 %), which was used for the next reaction with-
out further purification:1H NMR (DMSO-d6) δ 8.88 (s, 1H), 7.46
(s, 1H), 7.10 (s, 1H), 4.59–4.55 (m, 2H), 4.26 (m, 1H), 3.67–
3.66 (m, 2H), 3.25 (s, 3H).
{4-[(3Ј-Iodophenyl)amino]-[1,4]dioxano[2,3-g]quinazolin-7-
yl}methyl methanesulfonate (14b):Demethylation and succes-
sive mesylation of 4-[(3Ј-iodophenyl)amino]-7-methoxyme-
thyl-[1,4]dioxano[2,3-g]quinazoline 3b (960 mg, 2.47 mmol) af-
1
forded 14b (756 mg, 59 %) as a pale yellow solid: H NMR
(CDCl3) δ 8.69 (s, 1H), 8.19 (s, 1H), 8.02 (s, 1H), 7.75 (d,
J = 8.3 Hz, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.43 (s, 1H), 7.38 (s,
1H), 7.12 (dd, J = 8.3, 8.7 Hz, 1H), 4.60 (m, 1H), 4.55-4.44 (m,
3H), 4.25 (m, 1H), 3.13 (s, 3H).
4-[(3Ј-Bromophenyl)amino]-7-methoxymethyl-[1,4]di-
oxano[2,3-g]quinazoline (3a): General procedure of coupling
reaction in Scheme 1
7-(4-Morpholino)methyl-4-[(3Ј-bromophenyl)amino]-[1,4]-
dioxano[2,3-g]quinazoline (4a):General procedure of substitu-
tion in Scheme 2
A solution of 12 (40 mg, 0.15 mmol) and 3-bromoaniline
(33 mg, 0.3 mmol) in 2.5 mL of 2-propanol was treated with a
catalytic amount of c-HCl, and the reaction mixture was heated
at reflux for 24 h. After cooling to room temperature. the result-
ant solid was filtered, washed with a small volume of iso-propa-
nol, and dried in vacuo to afford 3a (24 mg, 47 %) as a white sol-
id:mp 268–274°C;1H NMR (DMSO-d6) δ 11.23 (s, 1H), 8.89 (s,
1H), 8.49 (s, 1H), 8.07 (m, 1H), 7.78 (m, 1H), 7.50–7.40 (m,
2H), 7.46 (s, 1H), 4.62–4.58 (m, 2H), 4.30 (m, 1H), 3.68 (d, J =
4.5 Hz, 2H), 3.34 (s, 3H).
To a stirred solution of 14a (64 mg, 0.13 mmol) in 10 mL of THF
was added morpholine (113 mL, 1.3 mmol) and triethylamine
(0.36 mL, 1.3 mmol) at 0°C, and the reaction mixture was heat-
ed at reflux for 3 h.After concentration, the residue was washed
successively with water and ethyl ether and purified by column
chromatography (EtOAc/hexane = 2/1) to afford 4a (42 mg,
68 %) as a white solid: mp 229–230°C; IR (KBr) 3358, 2826,
2362, 1506, 1426, 1270, 1228 cm–1; 1H NMR (CD3OD) δ 8.41
(s, 1H), 8.11 (s, 1H), 7.83 (s, 1H), 7.70 (m, 1H), 7.28–7.27 (m,
2H), 7.17 (s, 1H), 4.52–4.45 (m, 2H), 4.16 (m, 1H), 3.72–3.66
(m, 4H), 2.72–2.55 (m, 6H).
4-[(3Ј-Iodophenyl)amino]-7-methoxymethyl-[1,4]dioxano-
[2,3-g]quinazoline (3b)
7-(Diethanolamino)methyl-4-[(3Ј-bromophenyl)amino]-
[1,4]dioxano[2,3-g]quinazoline (4b)By reaction of 14a with di-
ethanolamine (10 equiv): 40 % (a pale yellow slid); mp
188–191°C; IR (KBr) 2978, 1506, 1428, 1274, 1234 cm–1; 1H
NMR (CD3OD) δ 8.41 (s, 1H), 8.10 (s, 1H), 7.85 (s, 1H), 7.72
(d, J = 8.2 Hz, 1H), 7.28–7.26 (m, 2H), 7.18 (s, 1H), 4.54–4.43
(m, 2H), 4.20 (m, 1H), 3.67–3.59 (m, 4H), 3.30 (m, 1H), 2.92
(m, 1H), 2.89–2.73 (m, 4H).
By reaction of 12 with 3-iodoaniline (2 equiv): 94 % (yellow
1
solid); mp 175–177°C; H NMR (DMSO-d6) δ 10.70 (s, 1H),
9.19 (s, 1H), 8.31 (s, 1H), 8.21 (s, 1H), 7.81 (d, J = 8.2 Hz, 1H),
7.64 (d, J = 8.4 Hz, 1H), 7.30 (s, 1H), 7.27 (dd, J = 8.2, 8.4 Hz,
1H), 4.61–4.58 (m, 2H), 4.28 (m, 1H), 3.68 (d, J = 4.7 Hz, 2H),
3.34 (s, 3H).
7-(Ethanolamino)methyl-4-[(3Ј-bromophenyl)amino]-[1,4]-
dioxano[2,3-g]quinazoline (4c)
4-[(3Ј-Bromophenyl)amino]-7-hydroxymethyl-[1,4]di-
oxano[2,3-g]quinazoline (13a): General procedure of demeth-
ylation of 3a and 3b in Scheme 2
By reaction of 14a with 2-ethanolamine (10 equiv): 42 % (a yel-
low solid); mp 168–172°C; IR (KBr) 2910, 2834, 1602,
1570 cm–1; 1H NMR (CD3OD) δ 8.42 (s, 1H), 8.10 (s, 1H), 7.88
(s, 1H), 7.71 (m, 1H), 7.28–7.27 (m, 2H), 7.20 (s, 1H), 4.52–
4.44 (m, 2H), 4.17 (m, 1H), 3.69 (t, J = 5.2 Hz, 2H), 2.97–2.84
(m, 2H), 2.82 (t, J = 5.2 Hz, 2H).
To a solution of 4-[(3Ј-iodophenyl)amino]-7-methoxymethyl-
[1,4]dioxano[2,3-g]quinazoline (3a; 1 g, 2.5 mmol) in 50 mL of
CH2Cl2 was added 1 M solution of BBr3 in hexane (4.0 mL,
4.0 mmol) under N2 atmosphere at –78°C. The reaction mix-
ture was stirred at the same temperature for 1 h and allowed to
warm to room temperature for 24 h. After treatment with satu-
rated NaHCO3 solution, the reaction mixture was extracted with
ethyl acetate. The combined organic layer was dried over
MgSO4 and concentrated in vacuo to afford 13a (960 mg, 99 %)
as red solid:1H NMR (CD3OD) δ 8.42 (s, 1H), 8.10 (s, 1H), 7.87
(s, 1H), 7.70 (m, 1H), 7.29–7.21 (m, 3H), 4.49 (m, 1H), 4.35
(m, 1H), 4.25 (m, 1H), 3.86 (d, J = 4.8 Hz, 2H), 3.42 (s,
3H).
7-(4-Methyl-1-piperazino)methyl-4-[(3Ј-bromophenyl)ami-
no]-[1,4]dioxano[2,3-g]quinazoline (4d)
By reaction of 14a with 1-methylpiperazine (10 equiv): 80 % (a
pale yellow solid); mp 182–189°C; IR (KBr) 3270, 2940, 2796,
1510, 1424, 1278 cm–1; 1H NMR (CD3OD) δ 8.42 (s, 1H), 8.11
(s, 1H), 7.87 (s, 1H), 7.71 (m, 1H), 7.28–7.27 (m, 2H), 7.20 (s,
1H), 4.51–4.45 (m, 2H), 4.18 (m, 1H), 2.78–2.69 (m, 10H),
2.50 (s, 3H).

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 487–494
7-Substituted-[1,4]dioxano[2,3-g]quinazolines 493
ear response). Conditions of HPLC were as follows: eluant =
CH3CN/H2O (4/1) and flow rate = 1 mL/min. An accurately
weighed amount of compound (5 mg) was supersaturated in
1 mL of distilled water at room temperature and the resultant
supersaturated solution was sonicated for 30 min at the same
temperature. After standing for an additional 30 min at room
temperature, the samples were centrifuged at 14,000 rpm for
5 min.The concentration of compound in the supernatant was
determined by HPLC using the calibration curve determined
[11]. The data of solubility were recorded in Table 1.
7-[(R)-1-Isopropyl-2-ethanolamino]methyl-4-[(3Ј-bromophe-
nyl)amino]-[1,4]dioxano[2,3-g]quinazoline (4e)
By reaction of 14a with (R)-valinol (10 equiv): 84 % (a yellow
solid); mp 170–175°C; 1H NMR (CD3OD) δ 8.41 (s, 1H), 8.10
(s, 1H), 7.86 (s, 1H), 7.71 (m, 1H), 7.28–7.26 (m, 2H), 7.18 (s,
1H), 4.51–4.38 (m, 2H), 4.19 (m, 1H), 3.70–3.62 (m, 2H), 3.48
(m, 1H), 3.01–2.96 (m, 2H), 1.95 (m, 1H), 0.96–0.89 (m, 6H).
7–[(1-pyrrodino)ethylamino]methyl-4-[(3Ј-iodophenyl)ami-
no]-[1,4]dioxano[2,3-g]quinazoline (4f)
By reaction of 14b with 1-(2-aminoethyl)pyrrolidine (10 equiv):
20 % (a yellow solid); mp 195–205°C; 1H NMR (CD3OD) δ 8.42
(s, 1H), 8.22 (s, 1H), 7.87 (s, 1H), 7.75 (d, J = 8.2 Hz, 1H), 7.50
(d, J = 8.0 Hz, 1H), 7.21 (s, 1H), 7.14 (dd, J = 8.0, 8.2 Hz, 1H),
4.53–4.44 (m, 2H), 4.23 (m, 1H), 3.20–2.90 (m, 10H), 2.00–
1.97 (m, 4H).
Pharmacology
Expression of human recombinant EGFR tyrosine kinase do-
main protein
Tyrosine kinase domain in EGFR gene exists in C-terminal cy-
toplasmic domain and the recombinant C-terminal protein was
verified to possess the tyrosine kinase activity which is identical
to the purified natural EGFR protein [11].To express EGFR ty-
rosine kinase domain protein in SF9 insect cells, the expres-
sion vector using pBacPAK8 vector (from Clontech, U.S.A.)
was constructed. The EGFR tyrosine kinase domain (amino
acids 668–1210) was PCR amplified using 5Ј primer (5Ј-
CTCGAGATGCATCATCATCATCATCATCGAA-GGCGCCA-
CATCGTTCG-3Ј) and 3Ј primer (GGTACCTCTAGATCAT-
GCTCCAATAAATTCACTGC-3Ј) and subcloned into XhoI and
KpnI restriction enzyme cloning sites in pBacPAK8 vector.The
amplified gene was sequenced to confirm the absence of mu-
tation.The expressed protein was engineered to have hexahis-
tidine tag in its N-terminal.The baculoviral stock from the vector
was generated using the virus generation kit (from Clontech
U.S.A.). The overexpression of the recombinant protein was
carried in 1 L of suspension culture of the virus infected SF9
cells.The expressed protein was purified using Ni2+ affinity col-
umn (from Novagen, U.S.A.) to over 95 % purity following the
protocol provided by the manufacturer. The purified recom-
binant EGFR tyrosine kinase domain protein has nearly identi-
cal Km value for ATP andVmax compared to those of purified nat-
ural EGFR protein under our assay condition (unpublished ob-
servation).
7-(4-Morpholino)methyl-4-[(3Ј-iodophenyl)amino]-[1,4]-
dioxano[2,3-g]quinazoline (4g)
By reaction of 14b with morpholine (10 equiv): 44 % (a white
solid); mp 238–240°C; IR (KBr) 2966, 2806, 1512, 1424, 1274,
1228 cm–1; 1H NMR (CD3OD) δ 8.41 (s, 1H), 8.25 (s, 1H), 7.86
(s, 1H), 7.76 (d, J = 8.1 Hz, 1H), 7.48 (d, J = 7.9 Hz, 1H), 7.19
(s, 1H), 7.13 (dd, J = 7.9, 8.0 Hz, 1H), 4.53–4.47 (m, 2H), 4.24
(m, 1H), 3.73–3.69 (m, 4H) 2.73–2.56 (m, 6H).
7-(Diethanolamino)methyl-4-[(3Ј-iodophenyl)amino]-[1,4]-
dioxano[2,3-g]quinazoline (4h)
By reaction of 14b with diethanolamine (10 equiv): 66 % (a
white solid); mp 174–175°C; IR (KBr) 2822, 2234, 1568, 1508,
1426, 1274, 1232 cm–1; 1H NMR (CD3OD) δ 8.40 (s, 1H), 8.24
(s, 1H), 7.85 (s, 1H), 7.76 (d, J = 8.2 Hz, 1H), 7.48 (d, J =
8.5 Hz, 1H), 7.19 (s, 1H), 7.13 (dd, J = 8.2, 8.5 Hz, 1H), 4.87–
4.45 (m, 2H), 4.22 (m, 1H), 3.71–3.49 (m, 4H), 2.93–2.90 (m,
2H), 2.80–2.77 (m, 4H).
7-(2-Ethanolamino)methyl-4-[(3Ј-iodophenyl)amino]-[1,4]-
dioxano[2,3-g]quinazoline (4i)
By reaction of 14b with 2-ethanolamine (10 equiv): 77 % (a
white solid); mp 200–201°C; IR (KBr) 3094, 2910, 2838, 1568,
1508, 1424, 1268, 1240 cm–1; 1H NMR (DMSO-d6) δ 8.48 (s,
1H), 8.37 (s, 1H), 8.12 (s, 1H), 7.98 (d, J = 8.2 Hz, 1H), 7.44 (d,
J = 8.1 Hz, 1H), 7.21 (s, 1H), 7.17 (dd, J = 8.1, 8.2 Hz, 1H),
4.54–4.39 (m, 2H), 4.22–4.18 (m, 1H), 3.69 (t, J = 5.3 Hz, 2H),
2.89–2.84 (m, 2H), 2.82 (t, J = 5.2 Hz, 2H).
Tyrosine kinase assays
Total enzymatic inhibition assay was done in total 20 µL reac-
tion mixture containing 10 ng of purified human recombinant
EGFR tyrosine kinase domain protein, 250 µM biotinylated ty-
rosine kinase substrate 2 (purchased from Promega, U.S.A.),
20 µM ATP, 2 µCi p32-gamma-ATP, 5 mM MgCl2, 5 mM DTT,
and 1 µL of appropriate dilution of inhibitor. The reaction was
carried out for 1 h at 30°C.The reaction was terminated by add-
ing 10 µL of 30 % phosphoric acid and spotted in avidin coated
PVDF membrane (purchased from Promega). The membrane
was washed five times with 20 mM Tris-HCl (pH 8.0) contain-
ing 0.2 N NaCl.The radioactivity from each spot was quantitat-
ed using the BAS system (from Kodak).
7-(4-Morpholino)methyl-4-[(3Ј-bromophenyl)amino]-[1,4]-
dioxano[2,3-g]quinazoline hydrochloride (4a HCl): General
procedure of preparation of hydrochloride
To
a
solution of 7-(4-morpholino)methyl-4-[(3Ј-bromophe-
4a (14 mg,
nyl)amino]-[1,4]dioxano[2,3-g]quinazoline
0.03 mmol) in 3 mL of ethanol was added 1 mL of acetyl chlo-
ride at 0°C, and the reaction mixture was stirred at room tem-
perature for 24 h.The reaction mixture was poured into 15 mL of
diethyl ether to give a precipitate.The solid was filtered, washed
with ether and dried in vacuo to provide 4a (15 mg, 99 %) as
HCl salt (a white solid): mp 230–255°C (decomp); IR (KBr)
Cancer cell growth inhibition assay
A431 (uterus cancer), HCT116 (colon cancer), and SNU638
(stomach cancer) human cancer cells were maintained using
RPMI 1640 medium containing 10 % fetal calf serum in 37°C
and 5 % CO2 incubator. A thousand cells were plated in 96 well
plate and incubated overnight. Cells were treated with inhibitor
and left for two days. Cells were fixed with formalin solution
(SIGMA) and washed with tap water. The cells were dried and
stained with 0.1 % sulforhodamine B (SIGMA) for 30 min. The
cells were washed with 1 % acetic acid and the dye was eluted
from cells by adding 0.1 M Tris-HCl (pH 8.0). The absorbance
was measured at 520 nm wavelength using microplate reader
(Molecular Dynamics).The inhibitor concentration, which inhib-
its cell growth by 50 %, was assigned as GI50 value.
1
2974, 2596, 1616, 1530, 1446, 1294 cm–1; H NMR (D2O) δ
8.52 (s, 1H), 7.98 (s, 1H), 7.77 (s, 1H), 7.50 (d, J = 8.2 Hz, 1H),
7.45 (d, J = 8.0 Hz, 1H), 7.39 (dd, J = 8.0, 8.2 Hz, 1H), 7.36 (s,
1H), 5.07 (m, 1H), 4.64 (m, 1H), 4.34 (m, 1H), 3.96–3.90 (m,
4H), 3.80–3.70 (m, 2H), 3.60–3.50 (m, 4H).
Determination of water solubility
The water solubility of the compounds was determined as fol-
lows:Stock solutions of compound were made up in DMSO and
used to calibrate the HPLC (peak area in mmol, assuming a lin-

494 Lee et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 487–494
[9] A.M.Thompson, A.J.Bridges, D.W.Fry, A.J.Kraker, W.A.
References
Denny, J. Med. Chem. 1995, 38, 3780–3788.
[1] M. Yamada, T. Ikeuchi, H. Hatanaka, Prog. Neurobiol.
1997, 51, 19–37.
[10] G. W. Rewcastle, B. D. Palmer, A. M. Thompson, A. J.
Bridges, D. J. Fry, A. J. Kraker, W. A. Denny, J. Med. Chem.
1996, 39, 1823–1835.
[2] K. Hickey, D. Grehan, I. M. Reid, S. O’Briain, T. N.Walsh, T.
P. J. Hennessy, Cancer 1994, 74, 1693–1698.
[11] A.M.Thompson, D.K.Murray, W.L.Elliott, D.W.Fry, J.Nel-
son, H. D. H. Showalter, B. J. Roberts, P. W.Vincent, W. A.
Denny, J. Med. Chem. 1997, 40, 3915–3925.
[3] D. W. Fry, A. J. Kraker, A. McMichael, L. A. Ambroso, J. M.
Nelson, W. R. Leopold, R. W. Connors, A. J. Bridges,
Science 1994, 265, 1093–1095.
[12] J.Y. Lee, Y. K. Park, S. H. Seo, I.-S. So, H.-K. Chung, B.-S.
Yang, S. J. Lee, H. Park, Y. S. Lee, Arch. Pharm. Pharm.
Med. Chem. 2001, 334, 357–360.
[4] G.W.Rewcastle, W. A. Denny, A.J. Bridges, H. Zhou, D. R.
Cody, A. McMichael, D. W. Fry, J. Med. Chem. 1995, 38,
3482–3487.
[13] A. G. Suárez, Tetrahedron Lett. 1999, 40, 3523–3526.
[5] W. H. J. Ward, P. N. Cook, A. M. Slater, D. H. Davies, G. A.
Holdgate, L. R. Green, Biochem. Pharmacol. 1994, 48,
659–666.
[14] P. M. Heertjes, B. J. Knape, H. C. A. van Beek, K. van den
Boogaart, J. Chem. Soc. 1957, 3445–3447.
[15] A.Wissner, D. M. Berger, D. H. Boschelli, M. B. Floyd, Jr. L.
M. Greenberger, B. C. Gruber, B. D. Johnson, N. Mamuya,
R. Nilakantan, M. F. Reich, R. Shen, H-R.Tsou, E. Upesla-
cis, Y. F. Wang, B. Wu, F. Ye, N. Zhang, J. Med. Chem.
2000, 43, 3244–3256.
[6] A. Wissner, B. D. Johnson, M. B. Floyd, D. B Kitchen, U.S.
Patent 5,760,041, 1998; CAN 127:190750.
[7] C.M.Discafani, M.L.Carroll, M.B.Floyd Jr., I.J.Hollander,
Z.Husain, B.D.Johnson, D.Kitchen, M.K.May, M.S.Malo,
A. A. Minnick Jr., R. Nilakantan, R. Shen, Y.-F. Wang, A.
Wissner, L. M. Greenberger, Biochem. Pharmacol. 1999,
57, 917–925.
[16] B.D.Smaill, B.D.Palmer, G.W.Rewcastle, W.A.Denny, D.
J.McNamara, E.M.Dobrusin, A.J.Bridges, H.Zhou, H.D.
H.Showalter, R. T. Winters, W. R. Leopold, D. W. Fry, J. M.
Nelson, V. Slintak, W. L. Elliot, B. J. Roberts, P.W.Vincent,
S. J. Patmore, J. Med. Chem. 1999, 42, 1803–1815.
[8] M.W. Kunkel, K. E. Hook, C.T. Howard, S. Przybranowski,
B. J. Roberts, W. L. Elliott, W. R. Leopold, Invest. New
Drugs 1996, 13, 295–302.