Syntheses of 4-(indole-3-yl)quinazolines - A new class of epidermal growth factor receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2007.09.018

The epidermal growth factor (EGF) family of membrane receptors has been identified as a key element in the complex signaling network that is utilized by various classes of cell-surface receptors. The synthesis and pharmacological results of 4-(indole-3-yl)quinazolines are described. The synthesized compounds are new high potent EGFR-tyrosine kinase inhibitors with excellent cytotoxic properties at different cell lines. Furthermore the 4-(indole-3-yl)quinazolines show some tendencies to inhibit the HER-2 TK, too. Moreover this substance class has remarkable strong fluorescence properties.

Full text of DOI:10.1016/j.ejmech.2007.09.018

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1478e1488
http://www.elsevier.com/locate/ejmech
Original article
Syntheses of 4-(indole-3-yl)quinazolines e A new class of epidermal
growth factor receptor tyrosine kinase inhibitors
*
Anja Luth , Werner Lowe
¨
¨
Freie Universita¨t Berlin, Institut fu¨r Pharmazie, Ko¨nigin-Luise-Strasse 2þ4, 14195 Berlin, Germany
Received 21 November 2006; received in revised form 28 August 2007; accepted 10 September 2007
Available online 29 September 2007
Abstract
The epidermal growth factor (EGF) family of membrane receptors has been identified as a key element in the complex signaling network that
is utilized by various classes of cellesurface receptors. The synthesis and pharmacological results of 4-(indole-3-yl)quinazolines are described.
The synthesized compounds are new high potent EGFR-tyrosine kinase inhibitors with excellent cytotoxic properties at different cell lines. Fur-
thermore the 4-(indole-3-yl)quinazolines show some tendencies to inhibit the HER-2 TK, too. Moreover this substance class has remarkable
strong fluorescence properties.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Tyrosine kinase inhibitor; EGFR; HER-2; ATP antagonist
1. Introduction
cells, and this increase in receptor levels has been associated
with a poor clinical prognosis.
The epidermal growth factor (EGF) family of membrane
receptors has been well characterized in cancer research. Con-
clusive evidence from studies in the last two decades has dem-
onstrated that dysregulation of the epidermal growth factor
receptor (EGFR, also known as HER-1, ErbB-1) is associated
with the principal characteristics of cancer, including autono-
mous cell growth, invasion and angiogenesis. The EGFR is
a transmembrane glycoprotein with an extracellular ligand-
binding domain and an intracellular domain with tyrosine
kinase activity for signal transduction. Ligand binding acti-
vates the receptor and its signaling pathways leading to the
activation or modulation of cellular processes [1,2]. The recep-
tor is expressed on healthy cells (40e100 EGFR/cell) as well
as on malignant tissues (more than 1 000 000 EGFR/cell) [3].
EGF and transforming growth factor-a (TGF-a) are the most
important stimulatory ligands for EGFR. Overexpression of
EGFR has been demonstrated in a wide variety of malignant
Thus, therapeutic strategies to inhibit EGFR and EGFR-
related pathways have been pursued, including the develop-
ment of ATP-competitive small molecule inhibitors of the
intracellular tyrosine kinase domain of the receptor or inhibi-
tors of downstream effectors of EGFR signaling pathways,
like Gefitinib 1 (IC50 20 nM) and Erlotinib 2 (IC50 1 nM) as
shown in Fig. 1 [4,5]. The 4-anilinoquinazoline derivatives
are both selective and effective inhibitors of the EGFR tyro-
sine kinase.
Most of the EGFR-tyrosine kinase inhibitors have the same
4-anilinoquinazoline skeleton, only the substituents and the
side chains are variable. Therefore, the replacement of the an-
iline structure by an indole nucleus could rigid the resulting
structure by only one single bonding between the indole and
the quinazoline nucleus. On the other hand, hydrogen bonding
between the indoleeNH and the peptide backbone of the EGF
receptor could afford specific conformations, improving the
inhibitory activities of the resulting derivatives.
In an earlier work by the Rhone-Poulenc Rorer group three
4-(indole-3-yl)quinazolines have already been described. Here
it was discovered that 4-(indole-3-yl)-substitution at the
* Corresponding author. Tel.: þ49 30 838 54109; fax: þ49 30 838 53854.
E-mail address: anja.lueth@fu-berlin.de (A. Luth).
¨
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.09.018

A. Lu¨th, W. Lo¨we / European Journal of Medicinal Chemistry 43 (2008) 1478e1488
1479
NH2
NH2+
O
O
N
O
O
NH2
O
O
N
O
125°C
N
O
NH
Cl
HN
Cl
F
OH
N
3
O
O
S
90°C
Cl, DMF
1
N
O
O
O
O
O
O
N
N
N
Cl
4
C
HN
CH
Scheme 1. Syntheses of 4-chloro-6,7-dimethoxyquinazoline 4.
2
The syntheses of the indoles are based on a procedure re-
ported by Hemetsberger-Knittel and co-workers [12]. Treat-
ment of methylazidoacetate 5 with substituted benzaldehydes
gave a,b-unsaturated cinnamic acid esters 6e7. Refluxing
compounds 6e7 in xylene resulted in cyclization of the
cinnamic esters to give the indole-2-carboxylic acid esters
8e10. The intramolecular cyclization of the cinnamic acid es-
ter 6 did not occur regiospecific in position 6 of the benzene
ring but also in position 2. At this point of the synthesis, a mix-
ture of two indole carboxylic esters was isolated. After that,
the hydrolysis of the esters afforded the corresponding indole
carboxylic acids 11e13 which were decarboxylated to give
the desired indoles 16e18. The resulting indole isomers
were easily separated by sc-chromatography.
Fig. 1. The 4-anilinoquinazolines Gefitinib 1 and Erlotinib 2.
quinazoline moiety is not compatible with good activity [6].
For example, the Rhone-Poulenc Rorer ‘‘compound 90’’ was
evaluated for its activity toward EGFR and HER-2 tyrosine
kinases and was described in a review article by Bridges as
follows: ‘‘Compound 90 represents a more fundamental
change in the pharmacophore and claims moderate ErbB-2 ac-
tivity with little or no EGFR activity’’ (Fig. 2) [7]. But these
mentioned compounds possess no halogen substitution pattern.
Therefore the benzene ring of the indole moiety of 4-(indole-
3-yl)quinazolines should be substituted by halogen, preferably
by fluorine and chlorine, which are essential for the activity of
the comparable 4-anilinoquinazolines like Gefitinib.
The syntheses of 4-(indole-3-yl)quinazolines (19e22, 28,
36) are based on a chemoselective and regioselective hetaryla-
tion reaction. Searching for a cross-coupling approach to
connect the quinazoline and indole moieties, an interesting
method was discovered, which afforded a hetarylation reaction
with the help of Grignard compounds. In the key step
2. Results and discussion
2.1. Chemistry
Since an earlier work by the Parke-Davis group with quina-
zoline-based inhibitors of EGFR established that 6,7-dialkoxy
substitution is compatible with good activity, we decided to re-
tain this feature in our initial compounds [8]. Therefore com-
pound 4 was synthesized according to the procedure of Zeneca
Ltd depicted in Scheme 1 [9]. Starting with 6,7-dimethoxyan-
thranilic acid the amino group was condensed with formami-
dine acetate in 2-methoxyethanol, followed by treatment
with thionyl chloride and N,N-dimethylformamide to give
4-chloro-6,7-dimethoxyquinazoline (4). On the other side, it
was necessary to prepare the differently substituted indoles
16e18 as outlined in Scheme 2 [10,11].
N+
O
O
NaN3
20°C
Br
N
N
-
O
O
5
R2
R1
CHO
20°C
R3
R3
H
N
R2
R1
R2
R1
N+
O
O
O
-N
N
O
140°C
8-10
6-7
N
O
O
NaOH/EtOH
20°C
N
R3
R3
H
N
H
N
R2
R1
R2
R1
O
O
N
260°C
OH
11-13
16-18
6, 8, 11, 16 R1 = Cl, R2 = F, R3 = H
9, 12, 17 R1 = H, R2 = Cl, R3 = F
7, 10, 13, 18 R1 = CH3, R2 = F, R3 = H
90
Fig. 2. Rhone-Poulenc Rorer ‘‘compound 90’’.
Scheme 2. Syntheses of indoles 16e18.

1480
A. Lu¨th, W. Lo¨we / European Journal of Medicinal Chemistry 43 (2008) 1478e1488
metalated indolyl-magnesium compounds were reacted with
4-chloroquinazolines to give 4-(indole-3-yl)quinazolines 19e
22, 28, 36 as shown in Scheme 3. To prove that method, firstly
we prepared the unsubstituted compound 19. In this case the
indolyl-magnesium compound was reacted with 4-chloroqui-
nazoline to give the desired 4-(indole-3-yl)quinazoline 19.
After a successful isolation of compound 19 it was proved,
whether indoles with halogen substitution retain its halogen-
pattern permanently under hetarylation conditions. Therefore
compound 20 was synthesized from the above used 4-
chloro-6,7-dimethoxyquinazoline 4 and commercially avail-
able 5-bromoindole.
Because of the successful synthesis of the halo substituted
compound 20 the hetarylation reaction with help of Grignard
compounds is suitable to yield all the desired substituted 4-
(indole-3-yl)quinazolines. The first part of the syntheses of
this new class deals with the preparation of 4-(indole-3-yl)qui-
nazolines 19, 20e22 and 27 with constant quinazoline and
variable indole substitution. Because of the excellent pharma-
cologically profile of 21 indole 16 was used for the following
hetarylations.
The second section of the work deals with the syntheses of
4-(indole-3-yl)quinazolines, showing different quinazoline and
constant indole substitution patterns. In this part at first com-
pound 28 was synthesized, whose quinazoline structure is com-
parable with that of Erlotinib (2). The quinazoline component
27 was completely synthesized before the cross-coupling reac-
tion. Therefore, ethyl-3,4-dihydroxybenzoate was alkylated
with bromoethylmethylether to give compound 23. Subse-
quently the nitro group was introduced in 23 by an electrophilic
substitution at the aromatic benzene ring resulting in substance
24. After reduction of the nitro group to give compound 25 the
following condensation of the anthranilic acid moiety with for-
mamidine acetate yielded in the cyclized quinazolinone 26,
which was then chlorinated in position 4 in the presence of thi-
onyl chloride and N,N-dimethylformamide to give compound
27. In the final step the desired compound 28 was separated
after the above-mentioned cross-coupling reaction with the in-
dole moiety 16 (see Scheme 4).
In case of the Gefitinib-like compound 36 we had to prepare
a special 4-chloroquinazoline with a suitable 6-position for the
substitution of a specific side chain, comparable with that of
Gefitinib (1, Scheme 5). Here the hetarylation was not the final
step during the synthesis. The quinazoline side chain at posi-
tion 6 of 36 was introduced only after cross-coupling reactions.
Firstly, the condensation reaction of the 3,4-dimethoxyanthra-
nilic acid with formamidine acetate gave the quinazolinone
29. Treatment with methane sulphuric acid in the presence of
methionine dealkylates only one methoxy group in the position
6 of 29. The resulting free phenolic function of compound 30
was protected using acetic anhydride in the presence of pyri-
dine to give compound 31. The following halogenation in
position 4 was performed with thionyl chloride and N,N-dime-
thylformamide to yield the 4-chloro-compound 32. Subse-
quently, 5-chloro-6-fluoroindole (16) was metalated using the
Grignard compound methylmagnesium iodide to activate the
3-position of the indole heterocycle for the electrophilic hetar-
ylation reaction with the 4-chloroquinazoline 32. After the suc-
cessful cross-coupling reaction resulting in substance 33 the
prepared 4-(3-chloropropyl)morpholine side chain was intro-
duced in the 6-position of the quinazoline moiety of 34. In
this case, firstly the indoleeNH in compound 33 was protected
with BOC to introduce the side chain regiospecific in the de-
sired position 6 of compound 34. It was difficult to unprotect
the BOC group of 35 because of the inclusion of solvents or in-
organic salts. Therefore that reaction was done with formic acid
and Triton B which were suitable to neutralize the acidic solu-
tion. In this case the voluminous cation could not be included in
the podand-like structure of compound 36.
R5
R5
Mg-I
N
H
N
R4
R3
R4
Mg, I2, CH3I, H3C-O-CH3
5°C
R3
14- 16,18
R2
R1
N
14 R3 = H,R4 = H,R5 = H
15 R3 = Br, R4 = H,R5 = H
16 R3 = Cl, R4 = F,R5 = H
The synthesized 4-(indole-3-yl)quinazolines show remark-
able strong fluorescence in the ultraviolet light. For example
the fluorescence of compound 36 is comparable with that of
quinine-sulphate (Fig. 3).
N
Cl
18 R3 = CH3, R4 = F, R5 = H
60°C
R2
R2
R1
N
N
H2O
2.2. In vitro EGFR inhibition activity
N
N
R1
R3
R4
R3
R4
Compounds 20e22, 28 and 36 were evaluated for their
ability to inhibit EGFR tyrosine kinase by MDS Pharma Ser-
vices, Taiwan [13,14]. The results are reported in Table 1. The
assay was based on the inhibition of phosphorylation of poly-
glutamic acid/tyrosine [poly(glu/tyr, 4:1)] by EGFR tyrosine
kinase and was performed in an IC50 semi-quantitative analy-
sis (five concentrations in duplicate: 10 tubes). The IC50 values
of the EGFR-TK inhibition (131e533 nM) show that com-
pounds 20e22, 28 and 36 are relatively potent inhibitors of
EGFR-TK activity. Because of the bromine induced polarity
we suppose, but we cannot prove it, that the cytotoxicity of
20 is somewhat weaker compared with the more lipophilic
N
H
N
Mg-I
R5
R5
19-22, 28, 36
19 R1 = H, R2 = H, R3 = H, R4 = H, R5 = H
20 R1 = OCH3, R2 = OCH3, R3 = Br, R4 = H, R5 = H
21 R1 = OCH3, R2 = OCH3, R3 = Cl, R4 = F, R5 = H
22 R1 = OCH3, R2 = OCH3, R3 = CH3, R4= F, R5 = H
O
O
28 R1 =
, R2 =
, R3 = Cl, R4 = F, R5 = H
O
O
O
N
36 R1 = O
, R2 = OCH3, R3 = Cl, R4 = F, R5 = H
Scheme 3. Hetarylation reaction between the indole and quinazoline part.

A. Lu¨th, W. Lo¨we / European Journal of Medicinal Chemistry 43 (2008) 1478e1488
1481
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OH
OH
O
O
O
O
HNO3 68%
Br, K2CO3
80°C
, Pd/C 10%
80°C
NH2
NO2
23
24
25
O
O
O
NH2
NH2+
125°C
H
O
O
O
O
F
N
Cl
O
N
O
O
N
O
O
N
Cl
O S
Cl, DMF
90°C
19
60°C
N
NH
N
O
27
26
Cl
F
Cl
O
O
O
N
H
28
Scheme 4. Syntheses of the Erlotinib-like compound 28.
chloro-fluoroindole-substitution pattern of compounds 21
and 36.
Compound 36 showed a very strong growth inhibition
(GI50 w 10ꢀ8 M) against lung cancer cell lines EKVX and
NCI-H322M. The same phenomenon (GI50 w 10ꢀ8 M) was
observed for cell lines of CNS cancer (SNB-75) and renal can-
cer (TK-10). The other mentioned cell lines in Table 2 were
inhibited by compound 36 at concentrations in the range of
10ꢀ6e10ꢀ7 M (GI50). In case of compound 21 the GI50 values
are observed between 10ꢀ6 and 10ꢀ7 M. Surprisingly the GI50
of CNS cancer cell line SNB-75 is weaker (w10ꢀ7 M) than
the comparable result with compound 36 (w10ꢀ8 M). These
results give no remark of a connection between the cytotoxic-
ity and the EGFR-TK inhibition. The mechanism of action
which is responsible for the cytotoxic properties remains yet
unclear.
2.3. In vitro HER-2 inhibition activity
Compounds 21 and 36 were additionally tested for their
HER-2-tyrosine kinase inhibition activity by a two-point mea-
sure (primary screen) by MDS Pharma Services, Taiwan
[15,16]. Compound 21 inhibited the HER-2 tyrosine kinase
at a concentration of 100 nM with a rate of 19% and substance
36 at the same concentration with a result of 3%. These results
indicate that compounds 21 and 36 could be discussed as dual
EGFR-TK and HER-2-TK inhibitors.
2.4. In vitro cytotoxicity assay
3. Conclusions
Compounds 21 and 36 were submitted for in vitro biologi-
cal testing against 60 cancer cell lines provided by the Na-
tional Cancer Institute-Developmental Therapeutics Program
(NCI-DTP). The NCI-DTP screening procedure is described
in detail elsewhere [17e19]. The results, summarized in Table
2, reveal that compounds 21 and 36 exhibited excellent growth
inhibition (GI50 < 10ꢀ6 M) against different cancer cell lines.
Our new synthetic pathways are suitable to guarantee the
desired 4-(indole-3-yl)quinazolines with a halogen substitu-
tion pattern in the molecules.
The synthesized compounds 20e22, 28 and 36 were tested
for their in vitro EGFR-tyrosine kinase inhibition. Furthermore
compounds 21 and 36 were evaluated for HER-2-tyrosine
O
S
O
N
O
O
Cl
NH2
NH2
O
O
O
O
N
O
OH
O
O
N
O
O
O
NH2
O
N
O
,
O
S
N
O
+
Cl, DMF
90°C
, L-Methionin
140°C
100°C
125°C
NH
NH
O
NH
N
HO
31
29
30
32
OH
Cl
O
O
H
N
F
Cl
19
N
N
Cl
O
O
O
O
O
N
O
N
N
O
O
N
K2CO3, 18-crown-6
HCOOH , Triton B
20°C
N
BOC,
60°C
20°C
N
N
N
N
O
Cl
F
Cl
F
Cl
F
Cl
O
N
O
N
O
O
F
N
N
N
H
N
36
35
34
33
H
BOC
BOC
Scheme 5. Syntheses of the Gefitinib-like compound 36.

1482
A. Lu¨th, W. Lo¨we / European Journal of Medicinal Chemistry 43 (2008) 1478e1488
10000
8000
6000
4000
2000
0
relative
intensity
10000
relative
intensity
350 nm
8000
6000
4000
2000
0
250 nm
225 nm
300 nm
260 nm
345 nm
319 nm
400
500
600
400
450
500
wavelength [nm]
wavelength [nm]
36
quinine-sulphate
emission
intensity
intensity
excitation
emission
excitation
260
413.8
4258
225
461.4
2416
300
350
413.6
413.6
4298
7948
250
319
345
470.6
468.6
468.6
7089
1542
1556
Fig. 3. Fluorescence analysis of compound 36 at a concentration of 5 mm and quinine-sulphate at a concentration of 1 mm.
kinase inhibition and cytotoxicity testing. Partly the results
show a relatively potent inhibition of the EGFR tyrosine ki-
nase and for 28 and 36 also a tendency for inhibition of the
HER-2 tyrosine kinase. Remarkable are the outstanding cyto-
toxic qualities at different cancer cell lines with GI50 values
between 10ꢀ6 and 10ꢀ8 M.
The results of the EGFR-TK inhibition and the cytotoxity
give this new substance class a very interesting option in treat-
ment of EGFR-dependent tumors.
4.2. 4-Chloro-6,7-dimethoxyquinazoline (4)
A stirred mixture of 3 (2.00 g, 9.67 mmol), thionyl chloride
(30 mL) and N,N-dimethylformamide (0.6 mL) was heated un-
der reflux for 4 h. The organic layer was evaporated. The res-
idue was washed with diethyl ether and dried to give 1.80 g
(82.8%) of a beige solid. 1H NMR (DMSO-d6): d 8.89
(s, 1H, H-2), 7.47 (s, 1H, H-5), 7.41 (s, 1H, H-8), 4.01 (s, 6H,
OCH3); MS: m/z 223.9 (Mþꢂ).
It is conspicuous that the compounds show strong
fluorescences.
4.3. Methylazidoacetate (5)
The EGF receptor appears more flexible, as assumed until
now. Therefore more structure variations in position 4 of the
quinazolines are possible to yield quite new structures of mod-
ern and innovative EGFR-TK inhibitors.
Sodium azide (44.00 g, 676.81 mmol) and methylbromoa-
cetate (96.90 g, 687.44 mmol) were suspended in N,N-dime-
thylformamide and stirred at ambient temperature for 16 h.
The reaction mixture was given into ice water and was ex-
tracted with diethylether. The organic layer was washed with
water and dried. After evaporation of N,N-dimethylformamide
the crude product was distillated under vacuo to give 64.20 g
4. Experimental section
1
(82.4%) of a colourless oil. H NMR (DMSO-d6): d 3.90 (s,
4.1. 6,7-Dimethoxy-3,4-dihydroquinazoline-4-on (3)
2H, CH2), 3.81 (s, 3H, OCH3); MS: m/z 115.1 (Mþꢂ).
4.4. Methyl-a-azido-3-chloro-4-fluorocinnamate (6)
Under cooling sodium (1.35 g) was solved in methanol
2-Amino-4,5-dimethoxybenzoic acid (6.30 g, 38.14 mmol)
and formamidine acetate (8.50 g, 81.65 mmol) in 2-methoxye-
thanol (100 mL) were heated under reflux for 8 h. After evapo-
ration of 2-methoxyethanol the resulting residue was stirred
with ammonia (10%). The residue was isolated, washed with
water and dried to give 5.90 g (89.5%) of a redbrown solid.
(30 mL).
A
mixture of 3-chloro-4-fluorobenzaldehyde
(4.65 g, 29.33 mmol) and 5 (6.75 g, 58.65 mmol) in methanol
(10 mL) was dropwise given to the sodium methanolate. The
mixture was stirred at ambient temperature for 3 h. After neu-
tralization with hydrochloric acid (2 N) the reaction mixture
1
mp: 300 ꢁC; H NMR (DMSO-d6): d 12.64 (s, 1H, NH), 7.99
(s, 1H, H-2), 7.44 (s, 1H, H-5), 7.13 (s, 1H, H-8) 3.89 (s, 6H,
OCH3); MS: m/z 206.4 (Mþꢂ). Anal.: (C10H10N2O3) C, H, N.

A. Lu¨th, W. Lo¨we / European Journal of Medicinal Chemistry 43 (2008) 1478e1488
1483
Table 1
Table 2
In vitro cytotoxicity of compounds 21 and 36 in selected cancer cell lines
IC50 of EGFR-tyrosine kinase inhibition of compounds 20e22, 28 and 36 and
HER-2-tyrosine kinase inhibition in % at a concentration of 100 nm of com-
pounds 21 and 36
Cell line
GI50 (M)
21
36
Compound
IC50 of the EGFR-TK
inhibition (nM)
Non-small lung cancer
EKVX
HOP-92
4.10 ꢃ 10ꢀ7
1.75 ꢃ 10ꢀ6
2.55 ꢃ 10ꢀ7
8.97 ꢃ 10ꢀ8
5.04 ꢃ 10ꢀ7
3.80 ꢃ 10ꢀ8
O
O
N
NCI-H322M
N
N
Ovarian cancer
IGROV1
SK-OV-3
Br
131
4.24 ꢃ 10ꢀ7
7.11 ꢃ 10ꢀ7
1.08 ꢃ 10ꢀ7
5.92 ꢃ 10ꢀ7
N
H
20
Renal cancer
A498
ACHN
4.92 ꢃ 10ꢀ7
2.22 ꢃ 10ꢀ7
3.25 ꢃ 10ꢀ7
2.90 ꢃ 10ꢀ6
2.15 ꢃ 10ꢀ7
9.76 ꢃ 10ꢀ8
O
O
N
TK-10
Cl
F
Colon cancer
HT29
209 (HER-2-TK inhibition at a
concentration of 100 nm: 3%)
8.48 ꢃ 10ꢀ7
7.66 ꢃ 10ꢀ7
2.04 ꢃ 10ꢀ7
3.70 ꢃ 10ꢀ6
4.90 ꢃ 10ꢀ7
4.93 ꢃ 10ꢀ8
N
H
Prostate cancer
PC-3
21
CNS cancer
SNB-75
O
O
N
N
CH3
533
472
3
1H, H-2), 7.79 (m, 1H, H-6), 7.22 (t, J ¼ 9.14 Hz, 1H, H-
5), 6.93 (s, 1H, CH aliph), 3.88 (s, 3H, OCH3); MS: m/z
235.3 (Mþꢂ). Anal.: (C11H10FN3O2) C, H, N.
N
H
F
22
O
O
N
4.6. Methyl-5-chloro-6-fluoroindole-2-carboxylate (8)
and methyl-7-chloro-6-fluoroindole-2-carboxylate (9)
N
O
O
Cl
F
N
A mixture of 6 (2.38 g, 10.11 mmol) and xylene (180 mL)
was heated under reflux for 45 min. The solvent was removed.
There were obtained 1.00 g (47.8%) of an almost 1:1 mixture
of 8 and 9 as white crystals which was used in the next step
28
H
N
O
O
N
1
333 (HER-2-TK inhibition at a
concentration of 100 nm: 19%)
without further purification. H NMR: (DMSO-d6) d 12.37
Cl
F
N
O
(s, 1H, NH, 9), 12.22 (s, 1H, NH, 8), 7.91 (d, JHeF ¼ 7.47 Hz,
N
1H, H-4, 8), 7.67 (m, 1H, H-4, 9), 7.35 (d, JHeF ¼ 9.87 Hz,
36
H
3
1H, H-7, 8), 7.29 (s, 1H, H-3, 9), 7.17 (t, J ¼ 9.20 Hz, 1H,
Reference compounds: EGFR-TK inhibition: Tyrphostin 47 e IC50 1.31 mM;
HER-2-TK inhibition: Tyrphostin 47 e IC50 5.70 mM.
H-5, 9), 7.15 (s, 1H, H-3, 8), 3.89 (s, 3H, OCH3), 3.88
(s, 3H, OCH3); MS: m/z 227.0 (Mþꢂ).
was extracted with ethyl acetate. The organic layer was
washed with water and dried. After evaporation of ethyl ace-
tate at a temperature less than 40 ꢁC the resulting residue
was chromatographed on silica gel with ethyl acetate/n-hexane
4.7. Methyl-6-fluoro-5-methylindole-2-carboxylate (10)
This compound was prepared from 7 as previously de-
scribed. The crude yield was 1.00 g (47.8%) of white crystals.
1H NMR (DMSO-d6): d 11.82 (s, 1H, NH), 7.46 (d, JHeF
8.37 Hz, 1H, H-4), 7.03 (d, JHeF ¼ 10.73 Hz, 1H, H-7), 7.01
(s, 1H, H-3), 3.79 (s, 3H, OCH3), 2.20 (s, 1H, CH3); MS:
m/z 207.3 (Mþꢂ).
1
(1:3) to give 4.55 g (60.7%) of white crystals. mp: 58 ꢁC; H
¼
4
NMR (DMSO-d6): d 8.13 (dd, J ¼ 2.16 Hz, JHeF ¼ 7.42 Hz,
3
1H, H-2), 7.90 (m, 1H, H-6), 7.47 (t, J ¼ 9.01 Hz, 1H, H-5),
6.93 (s, 1H, CH aliph), 3.86 (s, 3H, OCH3); MS: m/z 255.1
(Mþꢂ). Anal.: (C10H7ClFN3O2) C, H, N.
4.8. 5-Chloro-6-fluoroindole-2-carboxylic acid (11) and
7-chloro-6-fluoroindole-2-carboxylic acid (12)
4.5. Methyl-a-azido-4-fluoro-3-methylcinnamate (7)
This compound was prepared from 4-fluoro-3-methylben-
zaldehyde and 5 as previously described. The crude yield
A suspension of an almost 1:1 mixture of 8 and 9 (2.04 g,
8.96 mmol) in ethanol (90 mL) and sodium hydroxide solution
(2 N, 45 mL) was stirred at ambient temperature for 2 h. The
alcohol was evaporated and the residue was treated with
1
was 4.74 g (67.0%) of light yellow crystals. mp: 61 ꢁC; H
4
NMR (DMSO-d6): d 7.85 (dd, J ¼ 2.32 Hz, JHeF ¼ 7.69 Hz,

1484
A. Lu¨th, W. Lo¨we / European Journal of Medicinal Chemistry 43 (2008) 1478e1488
hydrochloric acid (2 N). The crystals were isolated, washed
with water and dried. There were obtained 1.8 g (94.1%) of
an almost 1:1 mixture of 11 and 12 as white solid. 1H
NMR: (DMSO-d6): d 13.10 (s, 2H, COOH, 11, 12) 12.18
dried and the ethyl acetate was evaporated. The crude product
was chromatographed over silica gel with ethyl acetate/ethanol
(9:1). There were obtained 0.2 g (45.1%) of a yellow solid.
1
mp: 91 ꢁC; H NMR (DMSO-d6): d 12.16 (s, 1H, NH), 9.34
(s, 1H, NH, 12), 12.02 (s, 1H, NH, 11), 7.88 (d, JHeF
¼
(s, 1H, H-2), 8.58 (d, 3J ¼ 8.44 Hz, 1H, H-5), 8.34
3
7.48 Hz, 1H, H-4, 11), 7.65 (m, 1H, H-4, 12), 7.33 (d,
JHeF ¼ 9.94 Hz, 1H, H-7, 11), 7.21 (s, 1H, H-3, 12), 7.13
(d, J ¼ 6.90 Hz, 1H, H-40), 8.32 (d, J ¼ 2.88 Hz, 1H, H-20),
3
3
8.09 (t, J ¼ 6.34 Hz, 1H, H-6), 8.05 (d, J ¼ 8.53 Hz, 1H,
3
3
3
(t, J ¼ 9.07 Hz, 1H, H-5, 12), 7.08 (s, 1H, H-3, 11); MS:
H-8), 7.80 (t, J ¼ 8.36 Hz, 1H, H-7), 7.61 (d, J ¼ 8.36 Hz,
m/z 212.9 (Mþꢂ).
1H, H-70), 7.32 (t, J ¼ 8.06 Hz, 1H, H-50), 7.25 (t, 3J ¼
3
7.44 Hz, 1H, H-60); MS: m/z 245.0 (Mþꢂ). Anal.: (C16H11N3)
C, H, N.
4.9. 6-Fluoro-5-methylindole-2-carboxylic acid (13)
This compound was prepared from 10 as previously de-
scribed. The crude yield was 1.72 g (90.4%) as white solid.
1H NMR (DMSO-d6): d (ppm) 12.91 (s, 1H, COOH) 11.75
(s, 1H, NH), 7.50 (d, JHeF ¼ 8.09 Hz, 1H, H-4), 7.08 (d,
JHeF ¼ 10.97 Hz, 1H, H-7), 7.01 (s, 1H, H-3), 2.28 (s, 3H,
CH3); MS: m/z 193.5 (Mþꢂ).
4.13. 4-(5-Bromoindole-3-yl)-6,7-
dimethoxyquinazoline (20)
Under cooling magnesium (0.25 g) and iodine were stirred
with a mixture from methyliodide (1.2 mL) and diethyl ether
(5 mL) for 15 min. Afterwards a solution of 5-bromoindole
(0.50 g, 2.50 mmol) in diethyl ether (15 mL) was given to
the reaction mixture and was stirred for 15 min. Subsequently
4 (0.70 g, 3.11 mmol) was added in portions. The mixture was
heated under reflux for 1 h and then was added to ice water.
After extraction with ethyl acetate the organic layer was dried
and the ethyl acetate was evaporated. The crude product was
chromatographed over silica gel with ethyl acetate/ethanol
(9:1) and was recrystallized from ethyl acetate/n-hexane.
There were obtained 0.15 g (14.2%) of yellow crystals. mp:
256 ꢁC; 1H NMR (DMSO-d6): d 12.08 (s, 1H, NH), 9.09
(s, 1H, H-2), 8.40 (d, 1H, 4J ¼ 2.47 Hz, H-40), 8.39 (d, 1H, J ¼
4.10. 5-Chloro-6-fluoroindole (16) and 7-chloro-6-
fluoroindole (17)
A suspension of 1.92 g (8.99 mmol) of an almost 1:1 mix-
ture of 11 and 12 in diphenyl ether (45 mL) was stirred at
260 ꢁC for 4 h. The reaction mixture was chromatographed
over silica gel with n-hexane and n-hexane/toluene (3:1).
There were obtained 0.70 g (45.9%) of 16 as white and
0.40 g (26.3%) of 17 as light brown crystals. 16: mp: 83 ꢁC;
1H NMR (DMSO-d6): d 11.32 (s, 1H, NH), 7.70 (d, JHeF
¼
3
7.40 Hz, 1H, H-4), 7.41 (t, J ¼ 5.70 Hz, 1H, H-2), 7.38 (d,
JHeF ¼ 10.22 Hz, 1H, H-7), 6.43 (t, J ¼ 5.02 Hz, 1H, H-3);
MS: m/z 169.2 (Mþꢂ). Anal.: (C8H5ClFN) C, H, N.; 17: mp:
42 ꢁC; 1H NMR (DMSO-d6): d 11.61 (s, 1H, NH), 7.53
(m, 1H, H-4), 7.42 (t, J ¼ 5.46 Hz, 1H, H-2) 7.04
(t, 3J ¼ 8.79 Hz, 1H, H-5), 6.54 (t, J ¼ 4.94 Hz, 1H, H-3);
MS: m/z 169.1 (Mþꢂ). Anal.: (C8H5ClFN) C, H, N.
1.31 Hz, H-20), 7.68 (s, 1H, H-5), 7.51 (d, 1H, J ¼ 8.58 Hz,
H-70), 7.38 (s, 1H, H-8), 7.36 (dd, 1H, 3J ¼ 9.19 Hz,
4J ¼ 1.81 Hz, H-60); MS: m/z 383.1 (Mþꢂ). Anal.:
(C18H14BrN3O2) C, H, N.
4.14. 4-(5-Chloro-6-fluoroindole-3-yl)-6,7-
dimethoxyquinazoline (21)
4.11. 6-Fluoro-5-methylindole (18)
This compound was prepared from 16 and 4 as previously
described. There were obtained 0.12 g (12.7%) of yellow crys-
1
This compound was prepared from 13 as previously de-
scribed. The crude yield was 0.9 g (60.7%) as light yellow
crystals. 1H NMR (DMSO-d6): d 11.05 (s, 1H, NH), 7.42
(d, JHeF ¼ 7.68 Hz, 1H, H-4), 7.33 (t, J ¼ 5.48 Hz, 1H, H-2),
7.16 (d, JHeF ¼ 10.88 Hz, 1H, H-7), 6.40 (t, J ¼ 4.13 Hz,
1H, H-3), 2.44 (s, 1H, CH3); MS: m/z 169.1 (Mþꢂ). Anal.:
(C9H8FN) C, H, N.
tals. mp: 245 ꢁC; H NMR (DMSO-d6): d 12.09 (s, 1H, NH),
9.09 (s, 1H, H-2), 8.44 (d, 1H, J ¼ 2.70 Hz, H-20), 8.37 (d, 1H,
JHeF ¼ 7.63 Hz, H-40), 7.68 (s, 1H, H-5), 7.53 (d, 1H, JHeF
¼
9.82 Hz, H-70), 7.39 (s, 1H, H-8), 4.04 (s, 3H, OCH3), 4.01
(s, 3H, OCH3); MS: m/z 357.2 (Mþꢂ). Anal.: (C18H13ClFN3O2)
C, H, N.
4.15. 4-(6-Fluoro-5-methylindole-3-yl)-6,7-
dimethoxyquinazoline (22)
4.12. 4-(Indole-3-yl)quinazoline (19)
Under cooling magnesium (0.25 g) and iodine were stirred
with a mixture from methyliodide (1.2 mL) and diethyl ether
(5 mL) for 15 min. Afterwards a solution of indole (0.25 g,
2.13 mmol) in diethyl ether (15 mL) was given to the reaction
mixture and was stirred for 15 min. Subsequently 4-chloroqui-
nazoline (0.30 g, 1.82 mmol) was added in portions. The mix-
ture was heated under reflux for 1 h and then was added to ice
water. After extraction with ethyl acetate the organic layer was
This compound was prepared from 18 and 4 as previously
described. There were obtained 0.24 g (22.8%) of yellow crys-
1
tals. mp: 251 ꢁC; H NMR (DMSO-d6): d 11.86 (s, 1H, NH),
9.12 (s, 1H, H-2), 8.31 (d, 1H, J ¼ 2.62 Hz, H-20), 8.10 (d, 1H,
JHeF ¼ 7.89 Hz, H-40), 7.73 (s, 1H, H-5), 7.43 (s, 1H, H-8),
7.32 (d, 1H, JHeF ¼ 10.45 Hz, H-70), 4.06 (s, 3H, OCH3),
3.98 (s, 3H, OCH3), 2.40 (s, 3H, CH3); MS: m/z 337.0
(Mþꢂ). Anal.: (C19H16FN3O2) C, H, N.

A. Lu¨th, W. Lo¨we / European Journal of Medicinal Chemistry 43 (2008) 1478e1488
1485
4.16. Ethyl-3,4-di-(2-methoxyethoxy)benzoate (23)
H-300), 3.71 (m, 6H, H-50, H-500); MS: m/z 294.2 (Mþꢂ).
Anal.: (C14H18N2O5) C, H, N.
Ethyl-3,4-dihydroxybenzoate (7.29 g, 40.02 mmol), bro-
moethylmethylether (11.3 mL) and potassium carbonate anhy-
drous (16.60 g) in acetonitrile (100 mL) were heated under
reflux for 20 h. After filtration the solvent was evaporated
and the crude product was chromatographed over silica gel
with n-hexane/ethyl acetate (3:2). There were obtained
4.20. 4-Chloro-6,7-di-(2-methoxyethoxy)quinazoline (27)
A stirred mixture of 26 (2.00 g, 6.80 mmol), thionyl chlo-
ride (30 mL) and N,N-dimethylformamide (0.6 mL) was
heated at reflux for 4 h. The liquid layer was evaporated.
The residue was washed with diethyl ether and dried to give
1.10 g (51.8%) of a beige solid. 1H NMR (DMSO-d6):
d 8.88 (s, 1H, H-2), 7.50 (s, 1H, H-5), 7.46 (s, 1H, H-8),
4.36 (m, 4H, H-20, H-200), 3.76 (m, 4H, H-30, H-300), 3.35
(m, 6H, H-50, H-500); MS: m/z 312.2 (Mþꢂ).
1
10.00 g (83.6%) as colourless wax-like mass. mp: 53 ꢁC; H
NMR (DMSO-d6): d 7.57 (dd, 4J ¼ 1.96 Hz, 3J ¼ 8.46 Hz,
1H, H-6), 7.47 (d, 4J ¼ 1.95 Hz, 1H, H-2), 7.09 (d,
3J ¼ 8.50 Hz, 1H, H-5), 4.25 (q, 2H, CH2), 4.17 (t, 4H, H-20,
H-200), 4.13 (t, 4H, H-30, H-300), 3.66 (m, 6H, H-50, H-500),
1.28 (t, 3H, CH3); MS: m/z 298.5 (Mþꢂ). Anal.: (C15H22O6)
C, H, N.
4.21. 4-(5-Chloro-6-fluoro-indole-3-yl)-6,7-di-(2-
methoxyethoxy)quinazoline (28)
4.17. Ethyl-4,5-di-(2-methoxyethoxy)-2-
nitrobenzoate (24)
Under cooling magnesium (0.25 g) and iodine were stirred
with a mixture from methyliodide (1.2 mL) and diethyl ether
(5 mL) for 15 min. Afterwards a solution of 16 (0.50 g,
2.99 mmol) in diethyl ether (15 mL) was given to the reaction
mixture and was stirred for 15 min. Subsequently 27 (0.70 g,
3.11 mmol) was added in portions. The mixture was heated un-
der reflux for 1 h and then was added to ice water. After extrac-
tion with ethyl acetate the organic layer was dried and the ethyl
acetate was evaporated. The crude product was chromato-
graphed over silica gel with ethyl acetate/ethanol (9:1) and
was recrystallisated from ethyl acetate/n-hexane. There were
Compound 23 (6.15 g, 20.62 mmol) was added to cooled
nitric acid (65%, 75 mL) in portions and stirred under cooling
for 2 h. The reaction mixture was put into water and extracted
with dichloromethane. After evaporation of the solvent the
crude product was chromatographed over silica gel with
n-hexane/ethyl acetate (3:2). There were obtained 6.90 g
1
(97.4%) of a brown oil. H NMR (DMSO-d6): d 7.67 (s, 1H,
H-3), 7.34 (s, 1H, H-6), 4.27 (m, 10H, CH2, H-20, H-200,
H-30, H-300), 3.68 (m, 6H, H-50, H-500), 1.26 (t, 3H, CH3);
MS: m/z 343.0 (Mþꢂ). Anal.: (C15H21NO8) C, H, N.
1
obtained 0.10 g (9.8%) of yellow crystals. mp: 197 ꢁC; H
NMR (DMSO-d6): d 12.11 (s, 1H, NH), 9.09 (s, 1H, H-2),
8.41 (d, 1H, J ¼ 3.01 Hz, H-20), 8.38 (d, 1H, JHeF ¼ 7.64 Hz,
H-40), 7.73 (s, 1H, H-5), 7.55 (d, 1H, JHeF ¼ 9.93 Hz, H-70),
7.41 (s, 1H, H-8), 4.36 (t, 4H, H-200, H-2000), 4.32 (t, 4H, H-300,
H-3000), 3.76 (m, 6H, H-500, H-5000); MS: m/z 445.1 (Mþꢂ).
Anal.: (C22H21ClFN3O4) C, H, N.
4.18. Ethyl-2-amino-4,5-di-(2-methoxyethoxy)
benzoate (25)
Compound 24 (7.30 g, 21.26 mmol), palladium/carbon
(10%, 2.8 g) and cylohexene (50 mL) in ethanol (500 mL)
were heated under reflux for 9 h. After filtration and evapora-
tion of the solvent the crude product was chromatographed
over silica gel with n-hexane/ethyl acetate (3:2). There were
4.22. 6,7-Dimethoxy-3,4-dihydroquinazoline-4-on (29)
2-Amino-4,5-dimethoxybenzoic acid (6.30 g, 38.14 mmol)
and formamidine acetate (8.50 g, 81.65 mmol) in 100 mL
2-methoxyethanol were heated at reflux for 8 h. After evapora-
tion of 2-methoxyethanol the resulting residue was stirred
with ammonia (10%). The residue was isolated, washed with
water and dried to give 5.90 g (89.5%) of a redbrown solid.
mp: 300 ꢁC; 1H NMR (DMSO-d6): d 12.64 (s, 1H, NH),
7.99 (s, 1H, H-2), 7.44 (s, 1H, H-5), 7.13 (s, 1H, H-8) 3.89
(s, 6H, OCH3); MS: m/z 206.4 (Mþꢂ). Anal.: (C10H10N2O3)
C, H, N.
1
obtained 4.40 g (66.0%) of a brown oil. H NMR (DMSO-
d6): d 7.20 (s, 1H, H-3), 6.43 (s, 2H, NH2), 6.35 (s, 1H, H-6),
4.20 (q, 2H, CH2), 4.05 (t, 2H, H-20), 3.93 (t, 2H, H-200),
3.67 (t, 2H, H-30), 3.58 (t, 2H, H-300), 3.40 (m, 3H, H-50),
3.35 (m, 3H, H-500), 1.27 (t, 3H, CH3); MS: m/z 313.0
(Mþꢂ). Anal.: (C15H23NO6) C, H, N.
4.19. 6,7-Di-(2-methoxyethoxy)-3,4-dihydroquinazoline-
4-on (26)
Compound 25 (6.30 g, 21.41 mmol) and formamidine ace-
tate (8.5 g, 81.65 mmol) in 2-methoxyethanol (100 mL) were
heated under reflux for 8 h. After evaporation of the solvent
the crude product was chromatographed over silica gel with
dichloromethane/methanol (9:1). There were obtained 4.00 g
4.23. 6-Hydroxy-7-methoxy-3,4-dihydroquinazoline-4-
on (30)
Compound 29 (1.50 g, 7.27 mmol) was added portionwise
to stirred methanesulphonic acid (10 mL). L-Methionine
(1.25 g) was added and the resultant mixture was stirred and
heated under reflux for 6 h. The mixture was cooled to ambi-
ent temperature and poured onto a mixture of ice and water.
1
(63.5%) of a white solid. mp: 182 ꢁC; H NMR (DMSO-d6):
d 12.07 (s, 1H, NH), 7.98 (s, 1H, H-2), 7.46 (s, 1H, H-5),
7.16 (s, 1H, H-8), 4.25 (t, 4H, H-20, H-200), 4.19 (t, 4H, H-30,

1486
A. Lu¨th, W. Lo¨we / European Journal of Medicinal Chemistry 43 (2008) 1478e1488
The mixture was neutralized by the addition of an aqueous so-
dium hydroxide solution (40%). The precipitate was isolated,
washed with water and dried. There were obtained 0.40 g
4.27. tert-Butyl-3-(6-acetoxy-7-methoxyquinazoline-4-
yl)-5-chloro-6-fluoroindole-1-carboxy-late (34)
1
(28.6%) of a white solid. mp: 293 ꢁC; H NMR (DMSO-d6):
A mixture of 33 (0.21 g, 0.83 mmol), di-tert-butylpyrocar-
bonate and 4-dimethylamino-pyridine in acetonitrile (70 mL)
was stirred at ambient temperature for 24 h. The solvent was
evaporated. were obtained 0.20 g (49.4%) of yellow crystals.
1H NMR (DMSO-d6): d 9.30 (s, 1H, H-2), 8.32 (s, 1H, H-
20), 8.21 (d, 1H, JHeF ¼ 7.52 Hz, H-40), 8.12 (s, 1H, H-5),
8.05 (d, 1H, JHeF ¼ 10.27 Hz, H-70), 7.62 (s, 1H, H-8), 4.03
(s, 3H, OCH3), 2.33 (s, 3H, CH3COOR), 1.68 (s, 9H); MS:
m/z 484.8 (Mþꢂ).
d 11.92 (s, 1H, NH), 9.80 (s, 1H, OH), 7.90 (s, 1H, H-2),
7.38 (s, 1H, H-5), 7.09 (s, 1H, H-8) 3.90 (s, 3H, OCH3);
MS: m/z 192.2 (Mþꢂ). Anal.: (C9H8N2O3) C, H, N.
4.24. 6-Acetoxy-7-methoxy-3,4-dihydroquinazoline-4-
on (31)
A mixture of 30 (6.75 g, 35.13 mmol), acetic anhydride
(55 mL) and pyridine (6.7 mL) was stirred and heated to
100 ꢁC for 2 h. The mixture was poured onto a mixture of
ice and water. The precipitate was isolated, washed with water
and dried. The crude product was chromatographed over silica
gel with dichloromethane/methanol (9:1). There were obtained
3.40 g (41.3%) of a white solid. mp: 293 ꢁC; 1H NMR
(DMSO-d6): d 12.19 (s, 1H, NH), 8.08 (s, 1H, H-2), 7.75 (s,
1H, H-5), 7.28 (s, 1H, H-8) 3.91 (s, 3H, OCH3), 2.30 (s, 3H,
COOR); MS: m/z 234.1 (Mþꢂ). Anal.: (C11H10N2O4) C, H, N.
4.28. tert-Butyl-5-chloro-6-fluoro-3-[7-methoxy-6-
(3-morpholin-4-ylpropoxy)-quinazoline-4-yl]-indole-1-
carboxylate (35)
A mixture of 34 (0.20 g, 0.41 mmol), potassium carbonate
anhydrous, 18-crown-6 and 4-(3-chloropropyl)morpholin
(5 mL) in acetonitrile (50 mL) was heated at 60 ꢁC for 4 h. Af-
ter filtration the solvent was evaporated and the crude product
was chromatographed over silica gel with ethyl acetate/ethanol
(9:1). There were obtained 0.22 g (95.1%) of light yellow
1
crystals. mp: 156 ꢁC; H NMR (DMSO-d6): d 9.17 (s, 1H,
4.25. 4-Chloro-6-acetoxy-7-methoxyquinazoline (32)
H-2), 8.46 (s, 1H, H-20), 8.26 (d, 1H, JHeF ¼ 7.52 Hz, H-40),
8.10 (d, 1H, JHeF ¼ 10.26 Hz, H-70), 7.61 (s, 1H, H-5), 7.45
(s, 1H, H-8), 4.16 (t, 2H, H-10), 4.03 (s, 3H, OCH3), 3.54 (t,
4H, H-6), 2.42 (t, 2H, H-30), 2.33 (t, 4H, H-3, H-5), 1.96
(m, 2H, H-20), 1.67 (s, 9H, C(CH3)3); MS: m/z 570.9 (Mþꢂ).
Anal.: (C29H32ClFN4O5) C, H, N.
A stirred mixture of 31 (2.00 g, 8.54 mmol), thionyl chlo-
ride (30 mL) and N,N-dimethylformamide (0.6 mL) was
heated at reflux for 4 h. The liquid layer was evaporated.
The residue was washed with diethyl ether and dried to give
1.70 g (78.8%) of a beige solid. 1H NMR (DMSO-d6):
d 9.02 (s, 1H, H-2), 8.03 (s, 1H, H-5), 7.65 (s, 1H, H-8),
4.03 (s, 3H, OCH3), 2.36 (s, 3H, CH3COOR); MS: m/z
251.1 (Mþꢂ).
4.29. 4-(5-Chloro-6-fluoroindole-3-yl)-7-methoxy-6-(3-
morpholin-4-ylpropoxy)quinazoline (36)
Compound 35 (0.22 g, 0.39 mmol) in formic acid (30 mL)
was stirred at ambient temperature for 24 h. The reaction mix-
ture was poured onto a mixture of Triton B, ice and water with
a pH > 8. After extraction with diethyl ether and ethyl acetate
the organic layer was dried over molecular sieve for 2 d. After
evaporation the crude product was chromatographed over sil-
ica gel with diethyl ether/ethyl acetate/methanol (3:1:1) and
was recrystallisated from ethyl acetate/n-hexane to give
0.04 g (20.5%) of yellow needles. mp: 186 ꢁC; 1H NMR
(DMSO-d6): d 12.10 (s, 1H, NH), 9.09 (s, 1H, H-2), 8.37 (d,
J ¼ 2.74 Hz, 1H, H-20), 8.33 (d, 1H, JHeF ¼ 7.59 Hz, H-40),
7.65 (s, 1H, H-5), 7.54 (d, 1H, JHeF ¼ 10.00 Hz, H-70), 7.39
(s, 1H, H-8), 4.19 (t, 2H, H-10), 4.01 (s, 3H, OCH3), 3.54 (t,
4H, H2, H-6), 2.43 (t, 2H, H-30), 2.34 (t, 4H,þH-3, H-5),
4.26. 4-(5-Chloro-6-fluor-indol-3-yl)-6-acetoxy-7-
methoxyquinazoline (33)
Under cooling magnesium (0.25 g) and iodine were stirred
with a mixture from methyliodide (1.2 mL) and diethyl ether
(5 mL) for 15 min. Afterwards a solution of 16 (0.50 g,
2.99 mmol) in diethyl ether (15 mL) was given to the reaction
mixture and was stirred for 15 min. Subsequently 32 (0.70 g,
2.77 mmol) was added in portions. The mixture was heated
under reflux for 1 h and then was added to ice water. After ex-
traction with ethyl acetate the organic layer was dried and the
ethyl acetate was evaporated. The crude product was chroma-
tographed over silica gel with ethyl acetate/ethanol (9:1) and
was recrystallisated from ethyl acetate/n-hexane. There were
1.96 (m, 2H, H-20); MS: m/z 470.7 (M ). Anal.:
ꢂ
(C24H24ClFN4O3) C, H, N.
1
obtained 0.23 g (21.7%) of yellow crystals. mp: 190 ꢁC; H
NMR (DMSO-d6): d 12.28 (s, 1H, NH), 9.24 (s, 1H, H-2),
8.44 (d, 1H, JHeF ¼ 7.62 Hz, H-40), 8.34 (d, 1H,
4.30. Analyses indicated by the symbols of the elements
or functions were within ꢄ0.4% of the theoretical values
J ¼ 2.88 Hz, H-20), 8.23 (s, 1H, H-5), 7.56 (d, 1H, JHeF
¼
10.00 Hz, H-70), 7.55 (s, 1H, H-8), 4.02 (s, 3H, OCH3), 2.34
(s, 3H, CH3COOR); MS: m/z 343.2 (Mþꢂ). Anal.:
(C19H13ClFN3O3) C, H, N.
4.30.1. In vitro EGFR-tyrosine kinase activity
The EGFR-tyrosine kinase inhibitory activity assay was
carried out by MDS Pharma Services, Taiwan (R.O.C.).

A. Lu¨th, W. Lo¨we / European Journal of Medicinal Chemistry 43 (2008) 1478e1488
1487
10% TCA) and incubated for 60 min at 4 ꢁC. The supernatant
is discarded, and the plates are washed five times with tap wa-
ter and air dried. Sulforhodamine B (SRB) solution (100 mL)
at 0.4% (w/v) in 1% acetic acid is added to each well, and
the plates are incubated for 10 min at room temperature. After
staining, unbound dye is removed by washing five times with
1% acetic acid and the plates are air dried. Bound stain is sub-
sequently solubilized with 10 mM trizma base, and the absor-
bance is read on an automatic plate reader at a wavelength of
515 nm.
Enzyme source
Substrate
Human A31 cells
10 mg/mL polyglutamic
acid/tyrosine [poly(glu/tyr, 4:1)]
1% dimethylsulfoxide
None
Vehicle
Pre-incubation time/temp.
Incubation time/temp.
Incubation buffer
60 min/25 ꢁC
50 mM Hepes, 20 mM MgCl2,
0.2 mM Na3VO4, pH 7.4
ELISA quantitation of poly(glu:p-tyr)
ꢅ50% of maximal
Quantitation method
Significance criteria
stimulation or inhibition
For suspension cells, the methodology is the same except
that the assay is terminated by fixing settled cells at the bottom
of the wells by gently adding 50 mL of 80% TCA (final con-
centration 16% TCA). Using the seven absorbance measure-
ments [time zero (Tz), control growth (C ), and test growth
in the presence of drug at five concentration levels (Ti) ], the
percentage growth is calculated at each of the drug concentra-
tion levels.
The epidermal growth factor was purchased from Sigma
(prod. number: E2645). The enzyme assay was performed in
wells coated with poly(glu/tyr) substrate. Incubation buffer
and test compound were added into the wells. Finally, enzyme
was added and then ATP to start the reaction. For the calculation
of the inhibitory activities, each assay contained two wells for
determination of the maximum phosphorylation rate (p-tyrmax
:
in the absence of inhibitor) and two wells for determination of
the minimum phosphorylation rate (p-tyrmin: in the absence of
enzyme), as well. Quantitation of phosphortyrosine (p-tyr)
was determinated via ELISA [13,14,20e23].
Percentage growth is calculated as [(Ti ꢀ Tz)/C ꢀ Tz)] ꢃ 100
for concentrations for which Ti > Tz and [(Ti ꢀ Tz)/Tz)] ꢃ 100
for concentrations for which Ti < Tz. Three dose response pa-
rameters are calculated for each experimental agent. Growth
inhibition (TGI) is calculated from Ti ¼ Tz. The LC50 concen-
tration of drug resulting in a 50% reduction in the measured
protein at the end of the drug treatment is calculated from
[(Ti ꢀ Tz)/Tz)] ꢃ 100 ¼ ꢀ50.
4.31. Evaluation for cytotoxic activity against a panel of
60 human cancer cell lines
Evaluation of anticancer activity was performed at the Na-
tional Cancer Institute (Bethesda, MD, USA). The compounds
were tested in an in vitro 60-cell line anticancer assay over a 5
log dose range. The cell lines are derived from nine human
cancer cell types: leukaemia, NSCLC, colon, CNS, melanoma,
ovarian, renal, prostate and breast. The tumor cell lines are
grown in RPMI 1640 medium containing 5% fetal bovine se-
rum and 2 mL L-glutamine. For a typical screening experi-
ment, cells are inoculated into 96 well microtiter plates in
100 mM at plating densities ranging from 5000 to 40 000
cells/well depending on the doubling time of individual cell
lines. After cell inoculation, the microtiter plates are incubated
at 37 ꢁC, 5% CO2, 95% air and 100% relative humidity for
24 h prior to addition of experimental drugs.
Values are calculated for each of these three parameters if
the level of activity is reached; however, if the effect is not
reached or exceeded, the value for that parameter is expressed
as greater or less than the maximum or minimum concentra-
tion tested [19,24].
Acknowledgment
The authors thank the National Cancer Institute in Bethesda
(USA) for testing the anticancer activities at 60 human cancer
cell lines.
References
After 24 h, two plates of each cell line are fixed in situ with
TCA to represent a measurement of the cell population for
each cell line at the time of drug addition (Tz). Experimental
drugs are solubilized in DMSO at 400-fold the desired final
maximum test concentration. At the time of drug addition,
an aliquot of concentrate is thawed and diluted to twice the de-
sired final maximum test concentration with complete medium
containing 50 mg/mL gentamicin. Additionally four, 10-fold or
1/2 log serial dilutions are made to provide a total of five drug
concentrations plus control. Aliquots of 100 mL of these differ-
ent drug dilutions are added to appropriate microtiter wells al-
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final drug concentration. Following drug addition, the plates
are incubated for an additional 48 h at the above-mentioned
conditions. For adherent cells, the assay is terminated by the
addition of cold TCA. Cells are fixed in situ by the gentle ad-
dition of 50 mL of cold 50% (w/v) TCA (final concentration,
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