Design, synthesis, and in vitro antitumor activity evaluation of novel 4-pyrrylamino quinazoline derivatives

Article abstract of DOI:10.1111/j.1747-0285.2011.01234.x

Here, we describe the design and synthesis of two series of 4-pyrrylamino quinazolines as new analogs of the epidermal growth factor receptor inhibitor gefitinib. In vitro antitumor activity of these novel compounds against pancreatic (Miapaca2) and prostate (DU145) cancer cell lines was evaluated. Compared with the parental gefitinib, all 18 derivatives show a greatly increased cytotoxicity to cancer cells. In vitro kinase inhibitory activity on epidermal growth factor receptor was also investigated. Among them, compounds GI-6, GII-4, GII-6, GII-8, and GII-9 are more potential receptor tyrosine kinase (RTK) inhibitors. Based on these results, we propose simple structure-activity relationship to provide information for designing and developing more potent antitumor agents.

Full text of DOI:10.1111/j.1747-0285.2011.01234.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01234.x
Chem Biol Drug Des 2011; 78: 932–940
Research Article
Design, Synthesis, and In vitro Antitumor Activity
Evaluation of Novel 4-pyrrylamino Quinazoline
Derivatives
Xiaoqing Wu1,2, Mingdong Li1, Wenhua
of RTKs has shown to be causally associated with the development
and progression of many human cancers (1–3). Consequently, RTKs
are rational targets for molecular targeted cancer therapy. Strate-
gies toward the attenuation and intervention of RTK signaling
include monoclonal antibodies, small-molecule inhibitors, antagonis-
tic ligands, and antisense oligonucleotides.
Tang2, Youguang Zheng1, Jiqin Lian2,
Liang Xu2,* and Min Ji1,*
1School of Chemistry and Chemical Engineering, Southeast
University, Nanjing 210009, Jiangsu, China
2Department of Radiation Oncology, Division of Radiation and
Cancer Biology, University of Michigan Medical School, Ann Arbor,
MI 48109, USA
*Corresponding authors: Min Ji, jimin@seu.edu.cn and
Liang Xu, liangxu@umich.edu
In 2003, Iressa (gefitinib, Figure 1), a selective epidermal growth fac-
tor receptor (EGFR) tyrosine kinase inhibitor, was approved by FDA
for locally advanced or metastatic non-small-cell lung cancer therapy
(4). In 2004, another EGFR small-molecule inhibitor Tarceva (erlotinib)
was launched for same indication (5). Tykerb (lapatinib), a dual tyro-
sine kinase inhibitor of ErbB-2 ⁄ EGFR, was approved by FDA for the
treatment of patients with advanced or metastatic breast cancer in
2007 (6). These three drugs share the same 4-anilinoquinazoline core
structure. Thus, in recent years, 4-anilinoquinazolines have emerged
as a versatile template for the inhibition of diverse RTKs and numer-
ous new derivatives have been designed and synthesized.
Here, we describe the design and synthesis of two
series of 4-pyrrylamino quinazolines as new ana-
logs of the epidermal growth factor receptor
inhibitor gefitinib. In vitro antitumor activity of
these novel compounds against pancreatic (Mia-
paca2) and prostate (DU145) cancer cell lines was
evaluated. Compared with the parental gefitinib,
all 18 derivatives show a greatly increased cyto-
toxicity to cancer cells. In vitro kinase inhibitory
activity on epidermal growth factor receptor was
also investigated. Among them, compounds GI-6,
GII-4, GII-6, GII-8, and GII-9 are more potential
receptor tyrosine kinase (RTK) inhibitors. Based on
these results, we propose simple structure–activ-
ity relationship to provide information for design-
ing and developing more potent antitumor agents.
In this study, using gefitinib as a leading compound, we replaced
the benzene ring with pyrrole ring, designed and synthesized two
series of novel 4-pyrrylamino quinazoline derivatives. Preliminary in
vitro antitumor activity and kinase inhibitory potency of these new
compounds were also evaluated. Our primary objective was to
develop new RTK inhibitors with improved antitumor activity.
Experimental Section
Key words: 4-pyrrylamino quinazoline, antitumor activity, cancer
cells, gefitinib, receptor tyrosine kinase
Synthesis
All reagents were purchased from commercial sources and used with-
out further purification. Melting points were measured in open capil-
laries and are uncorrected. 1H-NMR and 13C-NMR spectra were
recorded in CDCl3 on a Bruker Avance 500 spectrometer (Bruker BioS-
pin GmbH, Rheinstetten, Germany). Chemical shifts (d) are reported in parts
per million (p.p.m.) relative to tetramethylsilane, used as an internal
Received 26 October 2010, revised 12 April 2011 and accepted for pub-
lication 28 August 2011
Molecular targeted therapy is a new approach in oncotherapy
developed in the last 20 years, which selectively targets molecules
related to cancers and possesses higher specificity and lower toxic-
ity and side-effect over traditional cancer therapeutics. Receptor
tyrosine kinases (RTKs) are a subclass of cell-surface growth factor
receptors with an intrinsic, ligand-controlled tyrosine kinase activity
(1) and are main regulators of intracellular communication control-
ling cell proliferation, differentiation, survival, metabolism, and
migration (1,2). RTK activity is resting and tightly controlled in nor-
mal cell. However, gene application and overexpression of RTKs
occur frequently in many human cancers, and abnormal activation
F
O
HN
N
Cl
N
O
O
N
Figure 1: Structure of gefitinib.
932

Novel Gefitinib Analogs with Improved Antitumor Activity
standard. Mass spectra (MS) were obtained from Agilent 1100LC ⁄ MS.
sium carbonate (138.1 g, 1.0 mol) in dimethyl formamide (DMF)
(500 mL) was heated at 70 ꢀC for 4 h. The reaction mixture was
cooled to room temperature and then poured slowly into ice water
(3 L) while stirring constantly. The solid formed was filtered off and
washed with cold water. The off-white product was recrystallized
from ethyl acetate (200 mL) to give 113.9 g of 3 in 95% yield. Mp:
111–113 ꢀC; 1H-NMR d: 2.02–2.22 (tt, 2H, -CH2CH2CH2-), 3.65 (t,
2H, -CH2Cl), 3.79 (s, 3H, -OCH3), 3.88 (s, 3H, -OCH3), 4.10 (t, 2H, -
CH2O), 6.84 (d, 1H, HAr), 7.49 (s, 1H, HAr), 7.71 (d, 1H, HAr).
IR spectra were run on FI-IR Spectrometer (Perkin-Elmer, Waltham,
MA, USA). Element analysis was run on Elementa Vario ELIII element
analyzer. All compounds were routinely checked by TLC with silica gel
GF-254 glass plates and viewed under UV light at 254 nm.
Diethyl 2-(2-cyanovinylamino)malonate (1)
To 1.3 L toluene, 62.4 g 50% sodium hydride (1.3 mol) was added
in batches with mechanical agitation and ice water bath. A mixture
of acetonitrile (49.2 g, 1.2 mol), ethyl formate (74.0 g, 1.00 mol),
200 mL toluene, and 2 mL absolute ethanol was then added drop-
wise to the aforementioned solution within 1 h. The reaction mix-
ture was allowed to stir at room temperature for 24 h and then
concentrated under vacuum.
Methyl 5-(3-chloropropoxy)-4-methoxy-2-
nitrobenzoate (4)
Nitric acid (84.5 mL, 66%) was added dropwise at 0–5 ꢀC to a
solution of methyl 3-(3-chloropropoxy)-4-methoxybenzoate (3, 93.0 g,
0.36 mol) in a mixture of acetic acid (300 mL) and acetic anhydride
(100 mL). This mixture was stirred at room temperature for 6 h,
then slowly poured into ice water (2 L), and extracted with ethyl
acetate (4 · 200 mL). The combined organic layer was washed with
saturated sodium bicarbonate (2 · 200 mL) and brine (2 · 100 mL)
and then dried using Na2SO4. The ethyl acetate was evaporated to
give a yellow oil that solidified after standing in a refrigerator for
12 h and was then recrystallized from ethyl acetate ⁄ petroleum
ether to afford the product 4 as light yellow crystals (97.1 g, 89%
The residue was dissolved in 1.5 L ethanol, and glacial acetic acid
was used to adjust the pH value of this solution to 6–7; 50.0 g
sodium acetate and 105 g diethyl 2-aminomalonate (0.5 mol) were
added to the aforementioned solution. This mixture was stirred at
room temperature for 2 days and then concentrated under vacuum.
The residue was dissolved in 2 L water, and sodium bicarbonate
was used to adjust the pH value of the mixture to 8–9. This mix-
ture was extracted with ethyl acetate (5 · 200 mL). The combined
organic layer was washed with saturated sodium bicarbonate solu-
tion (2 · 200 mL) and water (2 · 100 mL) and then dried using
Na2SO4. The ethyl acetate was evaporated to give dark oil that
was then distilled under reduced pressure to collect fraction at
180–183 ꢀC and 2 mmHg as light yellow oil (1).
1
yield). Mp: 54–56 ꢀC; H-NMR d: 2.03–2.24 (tt, 2H, -CH2CH2CH2-),
3.66 (t, 2H, -CH2Cl), 3.78 (s, 3H, -OCH3), 3.89 (s, 3H, -OCH3), 4.12
(t, 2H, -CH2O), 7.82 (s, 1H, HAr), 8.01 (d, 1H, HAr).
Methyl 5-(3-chloropropoxy)-2-amino-4-
methoxybenzoate (5)
Ethyl 3-amino-1H-pyrrole-2-carboxylate
hydrochloride (2)
Powdered iron (50 g, 0.89 mol) was added to acetic acid (500 mL).
The resulting suspension was stirred for 15 min at 50 ꢀC under an
atmosphere of N2, and a solution of methyl 5-(3-chloropropoxy)-4-
methoxy-2-nitrobenzoate (4, 90.0 g, 0.30 mol) in methanol (300 mL)
was added dropwise. The mixture was stirred for another 30 min at
50–60 ꢀC. The catalyst was filtered, and the filtrate was slowly
poured into water (4 L) and extracted with ethyl acetate
(4 · 200 mL). The organic phase was washed with a saturated
solution of sodium carbonate (2 · 100 mL) and brine (2 · 100 mL)
and then dried using Na2SO4. The solvent was removed under
vacuum, and the brown solid residue was recrystallized from ethyl
acetate ⁄ petroleum ether to give the product 5 as light brown
crystals (63.1 g, 77% yield). Mp: 96–98 ꢀC; 1H-NMR d: 1.98–2.20
(tt, 2H, -CH2CH2CH2-), 3.62 (t, 2H, -CH2Cl), 3.76 (s, 3H, -OCH3), 3.85
(s, 3H, -OCH3), 4.07 (t, 2H, -CH2O), 5.10–5.35 (b, 2H,-NH2), 6.09 (d,
1H, HAr), 7.21 (s, 1H, HAr).
To 250 mL absolute ethanol, 2.6 g sodium was added in batches
with ice water bath; 30.0 g diethyl 2-(2-cyanovinylamino) malonate
(1, 0.133 mol) in 50 mL absolute ethanol was then added dropwise
to the aforementioned solution. The mixture was stirred at room
temperature for 24 h and then concentrated under vacuum. The res-
idue was dissolved in 300 mL water, and glacial acetic acid was
used to adjust the pH value of the mixture to 7–8. This mixture
was extracted with ethyl acetate (4 · 200 mL). The combined
organic layer was washed with saturated sodium bicarbonate solu-
tion (2 · 200 mL) and water (2 · 100 mL) and then dried using
Na2SO4. The solvent was evaporated to give 18.1 g light brown oil.
The oil was then dissolved in 100 mL acetone, and dried hydrogen
chloride gas was led in until no more precipitation was generated.
The solid was collected by filtration and washed with acetone.
After air-drying, the crude product was recrystallized from acetone
to afford 20.8 g product 2 as white powder in 82% yield. Mp:
198–200 ꢀC;1H-NMR d: 1.32 (t, 3H), 3.95 (b, 1H), 4.30 (q, 2H), 5.02
(b, 2H), 6.42 (d, 1H), 7.08 (d, 1H). Elem. Anal. Calcd: C, 44.10; H,
5.82; N, 14.70. Found C, 44.19; H, 6.15; N, 14.80.
6-(3-chloropropoxy)-7-methoxyquinazolin-4(3H)-
one (6)
A solution of methyl 5-(3-chloropropoxy)-2-amino-4-methoxybenzoate
(5, 98.2 g, 0.36 mol) and formamidine acetate (52.6 g, 0.51 mol) in
ethanol (800 mL) was heated at reflux for 6 h with overhead stir-
ring. The mixture was allowed to stand in the refrigerator overnight.
The precipitate was then collected by filtration, washed with etha-
nol, and air-dried to give 6 as white powder (88.7 g, 92% yield).
Methyl 3-(3-chloropropoxy)-4-methoxybenzoate
(3)
A
mixture of methyl 3-hydroxy-4-methoxybenzoate (84.6 g,
1
0.47 mol), 1-bromo-3-chloropropane (101.6 g, 0.65 mol), and potas-
Mp: 218–219 ꢀC; H-NMR d: 2.10–2.31 (tt, 2H, -CH2CH2CH2-), 3.72
Chem Biol Drug Des 2011; 78: 932–940
933

Wu et al.
(t, 2H, -CH2Cl), 3.83 (s, 3H, -OCH3), 4.02 (t, 2H, -CH2O), 6.98 (d, 1H,
HAr), 7.89 (s, 1H, HAr), 8.02 (d, 1H, HAr), 9.03–9.42 (b, 1H, -NH-).
Ethyl 3-(7-methoxy-6-(3-(4-methylpiperazin-1-
yl)propoxy)quinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (GI-2)
As in the procedure described for GI-1, compound GI-2 was pre-
pared as white powder (GI-2, 0.65 g, 71% yield). Mp: 175–178 ꢀC;
1H-NMR d: 1.43 (t, 3H), 1.58 (m, 2H), 2.15 (t, 2H), 2.34 (s, 3H),
2.62–2.69 (m, 8H), 4.01 (s, 3H), 4.27 (t, 2H), 4.41 (q, 2H), 6.69 (t,
1H), 7.10 (s, 1H), 7.24 (s, 1H), 7.47 (d, 1Hr), 8.58 (b, 1H), 8.71 (d,
1H), 9.54 (b, 1H); 13C-NMR d: 14.73, 26.53, 46.01, 53.21, 55.02,
55.20, 56.11, 60.11, 67.89, 101.33, 103.73, 107.95, 108.42, 109.36,
122.35, 147.20, 149.03, 153.87, 155.18, 155.29, 161.78; IR ( ⁄ cm):
2952, 2936, 2873, 2776, 1659, 1624, 1604, 1585, 1559, 1513, 1471,
1437, 1413, 1398, 1316, 1240, 1212, 1146, 1067, 1044, 987, 926,
844, 777, 617; m ⁄ z: 469.2 ([M+H]+, 100%). Elem. Anal. Calcd: C,
61.52; H, 6.88; N, 17.94. Found C, 61.83; H, 6.87; N, 17.32.
4-chloro-6-(3-chloropropoxy)-7-
methoxyquinazoline (7)
6-(3-Chloropropoxy)-7-methoxyquinazolin-4(3H)-one
(6,
102 g,
0.38 mol) was added to thionyl chloride (500 mL) with magnetic
stirring. DMF (20 mL) was then slowly added dropwise, and the
mixture was heated to reflux for 4 h. Most of the excess of thionyl
chloride was then removed under reduced pressure, and the yellow
residue was dissolved in chloroform (500 mL), washed with a satu-
rated solution of sodium carbonate (2 · 100 mL) and water
(2 · 100 mL), and dried using Na2SO4. The chloroform was then
removed under reduced pressure to give off-white powder, which
was recrystallized from ethyl acetate to give the product 7 (93.5 g,
1
86% yield). Mp: 150–152 ꢀC; H-NMR d: 2.43 (tt, 2H, -CH2CH2CH2-
), 3.85 (t, 2H,-CH2Cl), 4.09 (s, 3H, -OCH3), 4.39 (t, 2H, -OCH2), 7.43
(s, 1H, HAr), 7.47 (s, 1H, HAr), 8.91 (s, 1H, HAr).
Ethyl 3-(7-methoxy-6-(3-(pyrrolidin-1-
yl)propoxy)quinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (GI-3)
As in the procedure described for GI-1, compound GI-3 was pre-
pared as yellow powder (GI-3, 0.48 g, 64% yield). Mp: 192–
194 ꢀC; H-NMR d: 1.36 (t, 3H), 1.79 (b, 4H), 2.16 (p, 2H), 2.57 (b,
Ethyl 3-(6-(3-chloropropoxy)-7-
methoxyquinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (8)
A mixture of ethyl 3-amino-1H-pyrrole-2-carboxylate hydrochloride
(2, 12.1g, 64 mmol) and 4-chloro-6-(3-chloropropoxy)-7-methoxyqui-
nazoline (7, 14.9 g, 52 mmol) in isopropanol (300 mL) was heated
to reflux for 4 h and then allowed to stand in the refrigerator over-
night; the precipitate was collected by filtration, washed with
chilled isopropanol (2 · 150 mL), and recrystallized from ethanol to
1
4H), 2.72 (t, 2H), 4.00 (s, 3H), 4.28 (t, 2H), 4.406 (q, 2H), 6.86 (t,
1H), 7.17 (s, 1H), 7.22 (s, 1H), 7.41 (t, 1H), 8.68 (s, 1H), 9.10 (b, 1H),
9.95 (b, 1H); 13C-NMR d: 14.69, 23.53, 28.56, 52.97, 54.21, 56.07,
60.05, 67.93, 101.20, 103.68, 107.88, 108.39, 109.34, 122.29,
147.13, 149.02, 153.79, 155.13, 155.24, 161.70; IR ( ⁄ cm): 3410,
3302, 3043, 2959, 2863, 1665, 1622, 1598, 1557, 1508, 1433, 1309,
1208, 1143, 1072, 1044, 896, 835, 774; m ⁄ z: 440.2 ([M+H]+, 100%);
Elem. Anal. Calcd: C, 62.85; H, 6.65; N, 15.93. Found C, 62.70; H,
6.62; N, 15.07.
1
give light yellow powder (19.1 g, 91% yield). Mp: 239–240 ꢀC; H-
NMR d: 1.43 (t, 3H), 2.41 (tt, 2H), 3.85 (t, 2H), 4.02 (s, 3H), 3.37 (t,
2H), 4.16–4.46 (m, 2H), 7.24 (s, 1H), 7.29 (s, 1H), 7.46 (d, 1H), 8.58
(b, 1H), 8.72 (d, 1H), 10.18 (b, 1H); m ⁄ z: 405.1 ([M+H]+, 100%).
Ethyl 3-(7-methoxy-6-(3-(piperidin-1-
yl)propoxy)quinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (GI-4)
Ethyl 3-(7-methoxy-6-(3-
morpholinopropoxy)quinazolin-4-ylamino)-1H-
pyrrole-2-carboxylate (GI-1)
As in the procedure described for GI-1, compound GI-4 was pre-
pared as white powder (GI-4, 0.58 g, 74% yield). Mp: 188–190 ꢀC;
1H-NMR d: 1.41 (t, 5H), 1.61 (m, 5H), 2.16 (b, 2H), 2.46 (b, 4H), 2.60
(b, 2H), 4.00 (s, 3H), 4.28 (t, 2H), 4.43 (q, 2H), 7.24 (s, 1H), 7.29 (s,
1H), 7.46 (d, 1H), 8.61 (b, 1H), 8.70 (d, 1H), 9.45–10.20 (b, 1H); 13C-
NMR d: 14.76, 24.45, 25.95, 26.45, 54.61, 55.85, 56.15, 60.19,
68.05, 101.12, 103.73, 107.92, 109.34, 109.81, 122.27, 147.18,
149.00, 153.87, 155.10, 155.27, 161.75; IR ( ⁄ cm): 3324, 2925, 1655,
1623, 1594, 1557, 1513, 1471, 1433, 1395, 1310, 1242, 1211, 1143,
1070, 1046, 980, 926, 777, 628; m ⁄ z: 452.3 ([M-H]+, 100%); Elem.
Anal. Calcd: C, 63.56; H, 6.89; N, 15.44. Found C, 63.63; H, 6.85; N,
14.69.
Ethyl 3-(6-(3-chloropropoxy)-7-methoxyquinazolin-4-ylamino)-1H-pyr-
role-2-carboxylate (8, 0.81 g, 2 mmol) and potassium iodide (0.08 g)
were added to the solution of morpholine (5 mL) in DMF (20 mL).
The solution was stirred at 70 ꢀC for 30 min. The excess morpholine
was then removed under reduced pressure and the residue dissolved
in chloroform (60 mL), washed with water (2 · 20 mL), and then
dried using Na2SO4. The solvent was removed under vacuum. The
crude product was purified by column chromatography on silica gel,
eluting with ethyl acetate ⁄ triethylamine (20:1) to afford white pow-
1
der (GI-1, 0.78 g, 86% yield). Mp: 181–183 ꢀC; H-NMR d: 1.39 (t,
3H), 2.04–2.18 (m, 2H), 2.51 (t, 4H), 2.63 (t, 2H), 3.74 (t, 4H), 4.00 (s,
3H), 4.29 (t, 2H), 4.40 (q, 2H), 6. 90 (t, 1H), 7.20 (s, 1H), 7.24 (s, 1H),
7.45 (t, 1H), 8.71 (s, 1H), 9.10 (b, 1H), 9.95 (b, 1H); 13C-NMR d:
14.68, 26.19, 53.76, 55.44, 56.10, 60.10, 66.98, 67.76, 101.38,
103.73, 107.92, 108.41, 109.35, 122.36, 147.17, 149.01, 153.86,
155.18, 155.30, 161.83; IR ( ⁄ cm): 3417, 3311, 3147, 2968, 2847,
2193, 1657, 1621, 1559, 1509, 1464, 1393, 1318, 1238, 1208, 1122,
1072, 1040, 934, 855, 780; m ⁄ z: 456.2 ([M+H]+, 100%). Elem. Anal.
Calcd: C, 60.65; H, 6.42; N, 15.37. Found C, 59.46; H, 6.60; N, 13.69.
Ethyl 3-(6-(3-(diethylamino)propoxy)-7-
methoxyquinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (GI-5)
As in the procedure described for GI-1, compound GI-5 was pre-
pared as white powder (GI-5, 0.62 g, 76% yield). Mp: 195–196 ꢀC;
1H-NMR d: 1.05 (t, 6H), 1.40 (t, 3H), 2.09 (p, 2H), 2.57 (q, 4H), 2.71
(t, 2H), 4.00 (s, 3H), 4.28 (t, 2H), 4.43 (q, 2H), 6.90 (t, 1H), 7.21 (s,
934
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Novel Gefitinib Analogs with Improved Antitumor Activity
1H), 7.24 (s, 1H), 7.46 (t, 1H), 8.72 (s, 1H), 8.86 (b, 1H), 9.82 (b, 1H);
m ⁄ z: 483.3 ([M+H]+, 100%). Elem. Anal. Calcd: C, 62.22; H, 7.10; N,
17.41;. Found C, 62.12; H, 6.92; N, 62.12.
13C-NMR d: 11.83, 14.69, 26.62, 47.05, 49.18, 56.10, 60.10, 67.85,
100.93, 103.62, 107.79, 108.34, 109.32, 122.29, 147.07, 149.03,
153.79, 155.04, 155.23, 161.73; IR ( ⁄ cm): 2959, 2810, 1736, 1665,
1624, 1602, 1559, 1515, 1473, 1413, 1398, 1383, 1317, 1244, 1211,
1147, 1066, 928, 843, 779, 614; m ⁄ z: 442.2([M+H]+, 100%). Elem.
Anal. Calcd: C, 62.57; H, 7.08; N, 15.86. Found C, 62.44; H, 7.22; N,
14.85.
Ethyl 3-(7-methoxy-6-(3-(4-phenylpiperazin-1-
yl)propoxy)quinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (GI-9)
As in the procedure described for GI-1, compound GI-9 was pre-
pared as white powder (GI-9, 0.61 g, 59% yield). Mp: 242–243 ꢀC;
1H-NMR d: 1.37 (t, 3H), 2.16 (p, 2H), 2.68–2.72 (m, 6H), 3.23 (t, 4H),
4.00 (s, 3H), 4.30–4.43 (m, 4H), 6.82–6.93 (m, 4H), 7.25–7.28 (m,
4H), 7.46 (t, 1H), 8.72 (s, 1H), 8.85 (b, 1H); 13C-NMR d: 14.68,
26.49, 49.16, 53.30, 55.06, 56.15, 60.16, 67.82, 101.31, 103.77,
107.98, 108.43, 109.38, 116.00, 119.63, 122.31, 129.10, 147.22,
149.02, 151.38, 153.89, 155.17, 155.31, 161.79; IR ( ⁄ cm): 3423,
3334, 2947, 2879, 2822, 2733, 1663, 1624, 1601, 1600, 1472, 1436,
1413, 1397, 1317, 1242, 1211, 1148, 1066, 1044, 930, 845, 780,
690; m ⁄ z: 531.3 ([M+H]+, 100%). Elem. Anal. Calcd: C, 65.64; H,
6.46; N, 15.84. Found C, 64.86; H, 6.34; N, 15.53.
Ethyl 3-(7-methoxy-6-(3-(4-methylpiperidin-1-
yl)propoxy)quinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (GI-6)
As in the procedure described for GI-1, compound GI-6 was pre-
pared as white powder (GI-6, 0.52 g, 68% yield). Mp: 186–189 ꢀC;
1H-NMR d: 0.91 (d, 3H), 1.25–1.41 (m, 6H), 1.62 (d, 2H), 1.98 (b,
2H), 2.08 (t, 2H), 2.60 (b, 2H), 2.94 (d, 2H), 4.00 (s, 3H), 4.27 (t, 2H),
4.02 (q, 2H), 6.90 (t, 1H), 7.19 (s, 1H), 7.23 (s, 1H), 7.45 (s, 1H), 8.71
(s, 1H), 8.86 (b, 1H), 9.88 (b, 1H); 13C-NMR d: 14.72, 21.83, 26.64,
30.83, 34.38, 54.05, 55.47, 56.11, 60.13, 68.06, 101.16, 103.71,
107.89, 108.53, 109.34, 122.29, 147.15, 149.02, 153.83, 155.12,
155.26, 161.71; IR ( ⁄ cm): 2946, 2911, 1659, 1624, 1603, 1559, 1509,
1471, 1437, 1413, 1385, 1316, 1240, 1212, 1143, 1066, 1040, 923,
844, 777, 649; m ⁄ z: 467.1 ([M+H]+, 100%); Elem. Anal. Calcd: C,
64.22; H, 7.11; N, 14.98. Found C, 63.96; H, 7.11; N, 14.44.
Ethyl 3-(7-(3-chloropropoxy)-6-
methoxyquinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (9)
A mixture of ethyl 3-amino-1H-pyrrole-2-carboxylate hydrochloride
(2, 14.0 g, 74 mmol) and 4-chloro-7-(3-chloropropoxy)-6-methoxyqui-
nazoline (16.2 g, 56 mmol) in isopropanol (300 mL) was heated to
reflux for 4 h and then allowed to stand in the refrigerator over-
night; the precipitate was collected by filtration, washed with
chilled isopropanol (2 · 150 mL), and recrystallized from ethanol to
give off-white powder (18.9 g, 84% yield). Mp: 244–246 ꢀC; 1H-
NMR d: 1.42 (t, 3H), 2.38 (q, 2H), 3.80 (t, 2H), 4.07 (s, 3H), 4.33–
4.47 (m, 4H), 6.90 (s, 1H), 7.20 (s, 1H), 7.45 (d, 1H), 8.54 (b, 1H),
8.72 (d, 1H); m ⁄ z: 405.1 ([M+H]+, 100%).
Ethyl 3-(7-methoxy-6-(3-(2-methylpiperidin-1-
yl)propoxy)quinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (GI-7)
As in the procedure described for GI-1, compound GI-7 was
prepared as off-white powder (GI-7, 0.47 g, 57% yield). Mp:
131–133 ꢀC; 1H-NMR (CDCl3) d: 1.11 (d, 3H), 1.30–1.38 (m, 2H),
1.60–1.68 (m, 4H), 2.11 (p, 2H), 2.23 (t, 2H), 2.50 (b, 1H), 2.56–2.62
(m, 2H), 3.00 (p, 1H), 4.01 (s, 3H), 4.31 (t, 2H), 6.58 (t, 1H), 7.25 (s,
1H), 7.47 (d, 1H), 8.10 (s, 1H), 9.53 (s, 1H), 10.79 (b, 1H); 13C-NMR
(CDCl3) d: 18.86, 23.93, 25.47, 25.91, 26.12, 34.58, 50.37, 52.20,
54.69, 56.04, 56.25, 67.80, 101.39, 103.31, 107.69, 108.86, 109.51,
122.52, 147.74, 149.78, 153.87, 154.11, 155.28, 161.69; IR ( ⁄ cm):
3193, 2932, 1696, 1623, 1609, 1552, 1470, 1441, 1424, 1394, 1299,
1215, 1136, 1115, 1038, 866, 784, 741; m ⁄ z: 467.1 ([M+H]+, 100%).
Elem. Anal. Calcd: C, 64.22; H, 7.11; N, 14.98. Found C, 65.15; H,
6.43; N, 15.18.
Ethyl 3-(7-methoxy-6-(3-
morpholinopropoxy)quinazolin-4-ylamino)-1H-
pyrrole-2-carboxylate (GII-1)
Ethyl 3-(7-(3-chloropropoxy)-6-methoxyquinazolin-4-ylamino)-1H-pyr-
role-2-carboxyl-ate (9, 0.79 g, 2 mmol) and potassium iodide (0.08 g)
were added to the solution of morpholine (5 mL) in DMF (20 mL).
The solution was stirred at 70 ꢀC for 30 min. The excess morpholine
was then removed under reduced pressure and the residue dissolved
in chloroform (60 mL), washed with water (2 · 20 mL), and then
dried using Na2SO4. The solvent was removed under vacuum. The
crude product was purified by column chromatography on silica gel,
eluting with ethyl acetate ⁄ triethylamine (20:1) to afford white pow-
Ethyl 3-(7-methoxy-6-(3-(4-ethylpiperazin-1-
yl)propoxy)quinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (GI-8)
1
As in the procedure described for GI-1, compound GI-8 was pre-
pared as white powder (GI-8, 0.61 g, 65% yield). Mp: 175–177 ꢀC;
1H-NMR (CDCl3) d: 1.09 (t, 3H), 1.41 (t, 2H), 2.14 (p, 2H), 2.38–2.71
(m, 10H), 4.00 (s, 3H), 4.28 (t, 2H), 4.26 (q, 2H), 6.91 (t, 1H), 7.20 (s,
1H), 7.24 (s, 1H), 7.46 (t, 1H), 8.71 (s, 1H), 8.88 (b, 1H); 13C-NMR
(CDCl3) d: 11.94, 14.73, 26.52, 52.30, 52.87, 53.28, 55.09, 56.27,
60.13, 67.79, 101.28, 103.76, 107.96, 108.41, 109.35, 122.31,
147.21, 149.03, 153.85, 155.17, 155.28, 161.74; IR ( ⁄ cm): 3441,
2964, 2875, 1812, 1701, 1643, 1624, 1596, 1558, 1512, 1437, 1415,
1382, 1317, 1278, 1213, 1162, 1145, 1068, 1045, 960, 778, 639;
der (GII-1, 0.68 g, 75% yield). Mp: 202–203 ꢀC; H-NMR d: 1.37 (t,
3H), 2.03–2.14 (m, 2H), 2.47 (t, 4H), 2.56 (t, 2H), 3.71 (t, 4H), 4.05 (s,
3H), 4.23 (t, 2H), 4.40 (q, 2H), 6. 88 (t, 1H), 7.15 (s, 1H), 7.23 (s, 1Hr),
7.44 (t, 1H), 8.70 (s, 1H), 9.11 (b, 1H), 9.90 (b, 1H); 13C-NMR d:
14.65, 26.02, 53.73, 55.28, 56.25, 60.10, 66.98, 67.43, 99.95, 103.66,
108.36, 108.66, 109.21, 122.29, 147.16, 149.83, 153.82, 154.13,
155.23, 161.80; IR ( ⁄ cm): 3459, 3089, 2991, 1934, 1659, 1622, 1595,
1513, 1455, 1385, 1312, 1201, 1147, 1118, 1069, 859, 777, 621;
m ⁄ z: 456.2 ([M+H]+, 100%). Elem. Anal. Calcd: C, 60.65; H, 6.42; N,
15.37. Found C, 60.22; H, 6.40; N, 14.69.
Chem Biol Drug Des 2011; 78: 932–940
935

Wu et al.
Ethyl 3-(6-methoxy-7-(3-(4-methylpiperazin-1-
yl)propoxy)quinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (GII-2)
7.17 (s, 1H), 7.25 (s, 1H), 7.46 (t, 1H), 8.70 (d, 1H), 8.81 (b, 1H),
9.86 (b, 1H); 13C-NMR d: 11.87, 14.69, 26.72, 47.03, 49.29, 56.32,
60.15, 67.77, 99.98, 103.78, 108.74, 109.20, 109.79, 122.58, 147.30,
149.94, 153.82, 154.35, 155.30, 161.87; IR ( ⁄ cm): 3345, 319, 2968,
1697, 1659, 1623, 1597, 1556, 1495, 1453, 1409, 1382, 1313, 1287,
1242, 1199, 1138, 1070, 1044, 930, 863, 775, 632; m ⁄ z: 442.1
([M+H]+, 100%). Elem. Anal. Calcd: C, 62.57; H, 7.08; N, 15.86.
Found C, 62.20; H, 6.93; N, 15.46.
As in the procedure described for GII-1, compound GII-2 was pre-
pared as white powder (GII-2, 0.56 g, 67% yield). Mp: 183–
1
185 ꢀC; H-NMR d: 1.40 (t, 3H), 2.11 (p, 2H), 2.30 (s, 3H), 2.48–2.61
(m, 1H), 4.06 (s, 3H), 4.23 (t, 2H), 4.40 (q, 2H), 6.89 (s, 1H), 7.15 (s,
1H), 7.21 (d, 1H), 7.44 (t, 1H), 8.70 (s, 1H), 9.21 (b, 1H), 9.94 (b, 1H);
13C-NMR d: 14.68, 26.36, 45.98, 53.19, 54.81, 55.18, 56.27, 60.10,
67.44, 99.98, 103.68, 108.37, 108.70, 109.22, 122.29, 147.21,
149.87, 153.82, 154.19, 155.25, 161.80; IR ( ⁄ cm): 3422, 3337, 3113,
2939, 2797, 1656, 1620, 1592, 1513, 1454, 1386, 1314, 1204, 1151,
1074, 1042, 1013, 895, 826, 777; m ⁄ z: 469.3 ([M+H]+, 100%). Elem.
Anal. Calcd: C, 61.52; H, 6.88; N, 17.94. Found C, 60.91; H, 6.84; N,
17.10.
Ethyl 3-(6-methoxy-7-(3-(4-methylpiperidin-1-
yl)propoxy)quinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (GII-6)
As in the procedure described for GII-1, compound GII-6 was pre-
pared as white powder (GII-6, 0.51 g, 69% yield). Mp: 176–
1
178 ꢀC; H-NMR d: 0.92 (d, 3H), 1.26 (m, 2H), 1.40 (m, 3H), 1.63 (d,
2H), 1.98 (b, 2H), 2.13 (t, 2H), 2.58 (b, 2H), 2.95 (d, 2H), 4.04 (s, 3H),
4.23 (t, 2H), 4.21 (q, 2H), 6.88 (s, 1H), 7.12 (s, 1H), 7.43 (t, 1H), 8.69
(s, 1H), 9.14 (s, 1H), 9.87 (b, 1H); 13C-NMR d: 14.70, 21.80, 26.50,
30.84, 34.36, 54.03, 55.25, 56.29, 60.10, 67.58, 99.92, 103.66,
108.25, 108.32, 109.18, 122.30, 147.98, 150.87, 153.80, 154.15,
155.20, 161.79; IR ( ⁄ cm): 3165, 3157, 2945, 2930, 2841, 2071, 1698,
1655, 1623, 1597, 1586, 1558, 1511, 1493, 1452, 1411, 1382, 1313,
1276, 1201, 1143, 1070, 1044, 770; m ⁄ z: 467.1 ([M+H]+, 100%).
Elem. Anal. Calcd: C, 64.22; H, 7.11; N, 14.98. Found C, 64.06; H,
6.92; N, 15.50.
Ethyl 3-(6-methoxy-7-(3-(pyrrolidin-1-
yl)propoxy)quinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (GII-3)
As in the procedure described for GII-1, compound GII-3 was pre-
pared as light yellow powder (GII-3, 0.47 g, 59% yield). Mp: 189–
1
191 ꢀC; H-NMR d: 1.39 (t, 3H), 1.82 (b, 4H), 2.16 (p, 2H), 2.59 (b,
4H), 2.74 (t, 2H), 4.06 (s, 3H), 4.21 (t, 2H), 4.44 (q, 2H), 6.85 (t, 1H),
7.11 (s, 1H), 7.14,(s, 1H), 7.37 (t, 1H), 8.67 (s, 1H), 9.44 (b, 1H), 9.78
(b, 1H); 13C-NMR d: 14.71, 23.54, 28.35, 52.76, 54.15, 56.27, 60.08,
67.39, 99.89, 103.66, 108.33, 108.67, 109.19, 122.28, 147.20,
149.83, 153.80, 154.11, 155.17, 161.80; IR ( ⁄ cm): 3423, 3323, 3067,
2952, 2879, 1658, 1622, 1595, 1557, 1511, 1453, 1387, 1314, 1206,
1143, 1072, 1045, 895, 831, 779, 639; m ⁄ z: 440.3 ([M+H]+, 100%).
Elem. Anal. Calcd: C, 62.85; H, 6.65; N, 15.93. Found C, 62.14; H,
6.64; N, 15.20.
Ethyl 3-(6-methoxy-7-(3-(2-methylpiperidin-1-
yl)propoxy)quinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (GII-7)
As in the procedure described for GII-1, compound GII-7 was pre-
pared as off-white powder (GII-7, 0.48 g, 58% yield). Mp: 227–
230 ꢀC; 1H-NMR d: 1.10 (d, 3H), 1.57–1.67 (m, 4H), 2.10 (m, 2H),
2.30 (t, 1H), 2.40 (b, 1H), 2.56 (t, 1H), 2.92–3.00 (m, 2H), 3.00 (p,
1H), 4.01 (s, 3H), 4.25 (t, 2H), 4.45 (q, 2H), 6.58 (t, 1H), 7.25 (s, 1H),
7.47 (d, 1H), 8.10 (s, 1H), 9.53 (s, 1H), 10.79 (b, 1H); 13C-NMR d:
18.61, 23.77, 25.44, 25.93, 26.12, 34.67, 50.28, 52.01, 54.58, 55.89,
56.39, 67.81, 101.20, 103.13, 107.66, 108.78, 109.44, 122.81,
147.64, 149.81, 153.56, 154.06, 155.09, 161.72; IR ( ⁄ cm): 3125,
2902, 1699, 1622, 1608, 1574, 1489, 1445, 1382, 1300, 1213, 1132,
1116, 1042, 869, 786, 745; m ⁄ z: 467.1 ([M+H]+, 100%). Elem. Anal.
Calcd: C, 64.22; H, 7.11; N, 14.98. Found C, 65.06; H, 6.73; N,
15.16.
Ethyl 3-(6-methoxy-7-(3-(piperidin-1-
yl)propoxy)quinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (GII-4)
As in the procedure described for GII-1, compound GII-4 was pre-
pared as white powder (GII-4, 0.61 g, 72% yield). Mp: 199–
202 ꢀC; 1H-NMR d: 1.38–1.48 (m, 5H), 1.60 (t, 4H), 2.10 (p, 2H),
2.44 (b, 4H), 2.55 (d, 2H), 4.06 (s, 3H), 4.22 (t, 2H), 4.41 (q, 2H),
6.62 (d, 1H), 6.88 (s, 1H), 7.19 (s, 1H), 7.42 (t, 1H), 8.69 (s, 1H), 9.19
(b, 1H), 9.80 (b, 1H); 13C-NMR d: 14.69, 24.47, 26.00, 26.39, 54.63,
55.62, 56.29, 60.11, 67.63, 99.96, 103.70, 108.34, 108.72, 109.20,
122.27, 147.24, 149.89, 153.81, 154.20, 155.23, 161.77; IR ( ⁄ cm):
3061, 2932, 1657, 1622, 1595, 1558, 1512, 1454, 1411, 1382, 1312,
1245, 1211, 1143, 1070, 1045, 922, 898, 827, 777, 628; m ⁄ z: 454.3
([M+H]+, 100%). Elem. Anal. Calcd: C, 63.56; H, 6.89; N, 15.44.
Found C, 63.57; H, 6.86; N, 14.85.
Ethyl 3-(6-methoxy-7-(3-(4-ethylpiperazin-1-
yl)propoxy)quinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (GII-8)
As in the procedure described for GII-1, compound GII-8 was pre-
pared as white powder (GII-8, 0.63 g, 67% yield). Mp: 236–
1
Ethyl 3-(7-(3-(diethylamino)propoxy)-6-
methoxyquinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (GII-5)
238 ꢀC; H-NMR d: 1.08 (t, 3H), 1.40 (t, 2H), 2.17 (p, 2H), 2.41 (q,
3H), 2.55–2.66 (m, 10), 4.04 (s, 3H), 4.23 (q, 2H), 4.42 (q, 2H), 6.64
(t, 1H), 7.11 (s, 1H), 7.23 (s, 1H), 7.50 (t, 1H), 8.69 (s, 1H), 9.24 (b,
1H); 13C-NMR d: 11.91, 14.70, 26.33, 52.27, 52.81, 53.24, 54.87,
56.37, 60.11, 67.43, 99.90, 103.66, 108.18, 108.65, 109.67, 122.34,
147.65, 149.84, 153.02, 154.15, 155.20, 161.98; IR ( ⁄ cm): 3452,
3167, 2929, 1700, 1620, 1602, 1552, 1494, 1394, 1277, 1210, 1107,
As in the procedure described for GII-1, compound GII-5 was pre-
pared as white powder (GII-5, 0.63 g, 76% yield). Mp: 175–
1
177 ꢀC; H-NMR d: 1.04 (t, 6H), 1.41 (t, 3H), 2.10 (p, 2H), 2.55 (q,
4H), 2.67 (t, 2H), 4.06 (s, 3H), 4.23 (t, 2H), 4.42 (q, 2H), 6.91 (t, 1H),
936
Chem Biol Drug Des 2011; 78: 932–940

Novel Gefitinib Analogs with Improved Antitumor Activity
948, 897, 780, 738; m ⁄ z: 483.3 ([M+H]+, 75%). Elem. Anal. Calcd: C,
62.22; H, 7.10; N, 17.41. Found C, 61.90; H, 6.77; N, 17.94.
group at position 6, and different basic side chains at position 7 of
the quinazoline nucleus. For all new compounds, the pyrrole ring
had an ethyl formate substitutional group. The reason we chose a
carboxylic group but not halides as a substituent was that carboxylic
group was also an electron-withdrawing group but rarely reported in
previous gefitinib analogs, with the hope to obtain more data for
SAR. Structures of the new compounds are illustrated in Figure 2.
Ethyl 3-(6-methoxy-7-(3-(4-phenylpiperazin-1-
yl)propoxy)quinazolin-4-ylamino)-1H-pyrrole-2-
carboxylate (GII-9)
As in the procedure described for GII-1, compound GII-9 was pre-
pared as white powder (GII-9, 0.61 g, 59% yield). Mp: 221–
223 ꢀC; 1H-NMR d: 1.41 (t, 3H), 2.17 (t, 2H), 2.63–2.69 (m, 6H),
3.23 (t, 4H), 4.07 (s, 3H), 4.27 (t, 2H), 4.42 (q, 2H), 6.85 (t, 1H), 6.90
(d, 2H), 6.95 (s, 1H), 7.17 (s, 1H), 7.23–7.29 (m, 4H), 7.46 (t, 1H),
8.71 (s, 1H), 8.85 (b, 1H); 13C-NMR d: 14.69, 26.33, 49.16, 53.28,
54.96, 56.31, 60.16, 67.45, 99.97, 103.77, 105.45, 109.25, 116.09,
119.64, 122.27, 129.08, 147.19, 149.88, 151.37, 153.05, 153.83,
154.18, 155.27, 161.80; IR ( ⁄ cm): 3423, 3326, 3249, 3136, 2937,
2820, 1693, 1659, 1624, 1599, 1583, 1558, 1499, 1453, 1394, 1383,
1312, 1238, 1211, 1201, 1141, 1071, 1043, 1010, 991, 926, 863,
780, 650; m ⁄ z: 531.3 ([M+H]+, 100%). Elem. Anal. Calcd: C, 65.64;
H, 6.46; N, 15.84. Found C, 64.84; H, 6.13; N, 15.35.
Detailed synthetic routes of 18 4-pyrrylamino quinazoline derivatives
in series A (GI-1-9) and series B (GII-1-9) are depicted in
Schemes 1 and 2, respectively. The intermediate 4-chloro-6-(3-chlo-
ropropoxy)-7-methoxyquinazoline (7) was obtained using the same
method we reported previously (7). Briefly, methyl 3-hydroxy-4-meth-
oxybenzoate as starting material was alkylated with 1-bromo-3-chlo-
ropropane to give 3 in 95% yield. Nitration of 3 with nitric acid in
acetic acid afforded 4, which was then reduced by powdered iron
in acetic acid to give 5 in satisfactory yield (77%). In contrast, cata-
lytic hydrogenation using Raney ⁄ Ni or 5% Pd ⁄ C gave incomplete
conversions, even after a long reaction time. Cyclization of 5 with
formamidine acetate and then chlorination with thionyl chloride
gave 7. Then, aminolysis of 7 was performed using excess ethyl 3-
amino-1H-pyrrole-2-carboxylate hydrochloride (2) to afford 8 in 91%
yield. The final step was nucleophilic displacement of the chlorine
atom with different aliphatic amines to yield the corresponding tar-
get compound series A GI-1-9. The material ethyl 3-amino-1H-pyr-
role-2-carboxylate hydrochloride (2) was synthesized ahead by the
route described in Scheme 3: Ethyl formate was condensed with
acetonitrile under catalysis of sodium hydride to give 2-formylaceto-
nitrile, which was very active and unstable. Without separation, 2-
formylacetonitrile was then condensed with diethyl 2-aminomalo-
nate to afford 1. Cyclization of 1 under catalysis of sodium ethylate
and then salifying obtained 2 in 82% yield.
Cell cytotoxicity assay
Human pancreatic cancer cell line Miapaca2 and prostate cancer
cell line DU145 were purchased from American Type Culture Collec-
tion and cultured in high-glucose DMEM (HyClone, Logan, UT, USA)
supplemented with 10% fetal bovine serum (FBS; HyClone) in a 5%
CO2 humidified incubator at 37 ꢀC. The medium was also supple-
mented with 100 units ⁄ mL penicillin and 100 lg ⁄ mL streptomycin.
Cells were seeded in 96-well culture plates (5000 cells ⁄ well) and
treated with serially diluted testing compounds in triplicate. After
96-h incubation, cell growth medium was removed, and proliferation
reagent WST-8 (Sigma) was added to each well and incubated at
37 ꢀC for 1–3 h. Absorbance was measured using a plate reader at
450 nm with correction at 650 nm. The results were expressed as
the percentage of absorbance of treated wells versus that of vehi-
cle control. IC50, the drug concentration causing 50% growth inhibi-
tion, was calculated via sigmoid curve fitting using GRAPHPAD PRISM
5.0 (GraphPad, San Diego, CA, USA) as we described previously (7).
Starting from methyl 4-hydroxy-3-methoxybenzoate (Scheme 2), ser-
ies B compounds GII-1-9 could be obtained via the same synthetic
methods as described earlier. We are the first to replace the ben-
zene ring of gefitinib with pyrrole ring to design new compounds.
All target compounds are completely new [except compound GI-1
reported in our previous paper (17)] and obtained in satisfactory
yields (Figure 2).
In vitro kinase assays
Cytotoxicity
In vitro kinase inhibitory ability was determined using the HTScan
EGFR Kinase Assay Kit (Cell Signaling Technology, Danvers, MA,
USA), following the manufacturer's instructions.
The cytotoxicity of 18 compounds, as well as the parental com-
pound gefitinib, in human pancreatic cancer cell line Miapaca2 and
human prostate cancer cell line DU145, was investigated by the
MTT (3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide)-
based cytotoxicity assay. As shown in Table 1, the 50% inhibitory
concentration (IC50) of all new compounds against the two cancer
cell lines was much lower than that of gefitinib, indicating that the
in vitro antitumor activity of these compounds was significantly
improved. Interestingly, all compounds show greater inhibitory effect
on Miapaca2 cells compared with DU145 cells, while gefitinib
shows minor effect between the two cell lines (both IC50 values
around 40 lM).
Results and Discussion
Chemistry
Based on the structure–activity relationships (SAR) and quantitative
structure–activity relationships of the 4-anilinoquinazolines reported
previously (8–16), and using gefitinib as a leading compound, we
designed two series of novel analogs: series A, replacement of the
benzene ring with pyrrole ring, different basic side chains at position
6, and methoxy group at position 7 of the quinazoline nucleus; ser-
ies B, replacement of the benzene ring with pyrrole ring, methoxy
With further inspection, we found that the cytotoxicity of compound
GI-1 was the weakest among the 18 compounds but still more
Chem Biol Drug Des 2011; 78: 932–940
937

Wu et al.
H
N
H
N
C2H5OOC
HN
C2H5OOC
HN
R
N
N
MeO
N
R
N
series B
series A
Cpd.
Yield
(%)
86
Cpd.
R
N
N
Yield
(%)
75
R
O
O
GI-1
GI-2
O
O
N
O
GII-1
GII-2
71
67
O
N
CH3
N
N
CH3
64
74
76
68
57
59
72
76
69
58
O
N
GI-3
GI-4
GI-5
GI-6
GII-3
GII-4
GII-5
GII-6
O
N
N
O
N
N
O
O
O
N
O
N
CH3
O
N
CH3
H3C
N
H3C
N
GI-7
GI-8
GII-7
GII-8
O
O
65
59
67
59
O
O
N
N
N CH2CH3
O
O
N
N
N CH2CH3
GI-9
GII-9
N
N
Figure 2: Structure and yield of new compounds.
Table 1: Cytotoxicity against cancer cell lines and EGFR inhibitory activity of gefitinib and new compounds
Cytotoxicity (IC50, lM)a
Cytotoxicity (IC50, lM)a
Compound
Miapaca2
DU145
EGFR inhibition (%)b,c
Compound
Miapaca2
DU145
EGFR inhibition (%)b,c
Gefitinib
GI-1
GI-2
GI-3
GI-4
GI-5
GI-6
GI-7
GI-8
GI-9
38.81
15.36
9.897
7.392
5.855
7.914
7.241
7.670
9.206
8.690
42.17
39.28
16.09
27.13
21.41
17.02
12.89
18.34
16.87
19.24
99.18
18.32
31.46
26.58
33.91
40.50
51.23
32.67
37.81
29.83
GII-1
GII-2
GII-3
GII-4
GII-5
GII-6
GII-7
GII-8
GII-9
14.35
7.273
10.51
8.721
7.752
6.498
13.17
5.844
5.460
27.52
16.83
14.63
10.59
16.58
7.947
20.09
13.45
12.94
23.27
38.72
36.57
53.20
43.28
66.54
27.64
58.53
56.31
EGFR, epidermal growth factor receptor.
aValues are averages of three independent experiments, SD < 10%.
bValues are averages of two independent experiments, SD < 10%.
cCompounds tested at concentration of 10 lM.
potent than gefitinib. The structure modification of gefitinib to design
GI-1 is that pyrrole ring was substituted for the benzene ring. This
result reveals that replacement of benzene ring with pyrrole ring
increases in vitro antitumor activity. The ethyl formate substitutional
group may also have synergic effect. The structure difference among
GI-1 and other compounds in series A was the basic side chain at
938
Chem Biol Drug Des 2011; 78: 932–940

Novel Gefitinib Analogs with Improved Antitumor Activity
Cl
O
O
COOCH3
Cl
O
O
COOCH3
HO
O
COOCH3
c
a
b
NO2
3
4
Cl
O
Cl
O
O
Cl
O
Cl
O
COOCH3
NH2
N
NH
e
d
N
O
N
O
7
6
5
H
N
H
N
C2H5OOC
C2H5OOC
g
HN
HN
f
R
Cl
O
O
N
N
MeO
N
N
8
GI-1-9
Scheme 1: Reagents and conditions: (a) ClCH2CH2CH2Br, K2CO3, 70 ꢀC; (b) HNO3, AcOH, Ac2O, 0–5 ꢀC; (c) Fe, AcOH, MeOH, N2, 50 ꢀC;
(d) formamidine acetate, EtOH, reflux; (e) SOCl2, DMF, reflux; (f) 2, i-PrOH, reflux; (g) aliphatic amine, KI, DMF, 70 ꢀC.
O
O
COOCH3
O
O
COOCH
b3
O
COOCH3
c
a
Cl
NO2
Cl
Cl
HO
O
N
O
O
O
O
O
O
COOCH3
NH2
e
N
NH
d
Cl
N
Cl
Cl
H
H
N
N
C2H5OOC
C2H5OOC
HN
N
HN
N
g
f
O
O
N
N
Cl
R
GII-1-9
9
Scheme 2: Reagents and conditions: (a) ClCH2CH2CH2Br, K2CO3, 70 ꢀC; (b) HNO3, AcOH, Ac2O, 0–5 ꢀC; (c) Fe, AcOH, MeOH, N2, 50 ꢀC;
(d) formamidine acetate, EtOH, reflux; (e) SOCl2, DMF, reflux; (f) 2, i-PrOH, reflux; (g) aliphatic amine, KI, DMF, 70 ꢀC.
O
H
H
N
COOC2H5
COOC2H5
O
c
N
a
O
.HCl
b
CH3CN
+
CN
O
O
NH2
N
2
1
Scheme 3: Reagents and conditions: (a) NaH, C6H5CH3; (b) NH2CH(COOC2H5)2, AcONa; (c) (i) EtOH, EtONa, (ii) HCl, CH3COCH3.
position 6 of quinazoline nucleus, which was reported to affect the
physical properties of compound in vivo (16). As cytotoxicity assay
was carried out in vitro, the inhibitory effect variance among these
compounds could be attributed to the hydrophobicity and permeability
changes resulting from different basic side chains.
are no longer specific EGFR tyrosine kinase inhibitors. We are cur-
rently carrying out more experiments to access inhibitory activity on
other RTKs, hoping to figure out the exact target(s) and details on
the mechanism of action.
Taken the biological data together, we noticed that compounds with
stronger cytotoxicity also show better EGFR inhibitory potency. This
data may suggest that in vitro antitumor activity of these com-
pounds results from their kinase inhibitory activity. We also found
that shifting basic amine side chain at position 6 (series A) to posi-
tion 7 (series B) results in a minor increased cytotoxicity and more
obviously higher EGFR inhibitory potency in most cases.
Kinase inhibitory activity
Because gefitinib is a selective EGFR inhibitor, we determined the
inhibitory activity on EGFR of these gefitinib analogs. As also shown
in Table 1, the inhibitory potency of the new compounds is much
lower than that of gefitinib, which indicates that these compounds
Chem Biol Drug Des 2011; 78: 932–940
939

Wu et al.
Compounds GI-6, GII-4, GII-6, GII-8, and GII-9 that possess
relatively stronger cytotoxicity against the two cancer cell lines are
more potent RTK inhibitors and will be chosen for further evaluation
of in vitro and in vivo activities.
8. Rewcastle G.W., Denny W.A., Bridges A.J., Zhou H.R., Cody
D.R., Mcmichael A., Fry D.W. (1995) Tyrosine kinase inhibitors
.5. Synthesis and structure-activity-relationships for 4-[(Phenylm-
ethyl)Amino]-quinazolines and 4-(Phenylamino)quinazolines as
potent adenosine 5¢-triphosphate binding-site inhibitors of the
tyrosine kinase domain of the epidermal growth-factor receptor.
J Med Chem;38:3482–3487.
Conclusion
9. Bridges A.J., Zhou H., Cody D.R., Rewcastle G.W., McMichael
A., Showalter H.D.H., Fry D.W., Kraker A.J., Denny W.A. (1996)
Tyrosine kinase inhibitors .8. An unusually steep structure-activ-
ity relationship for analogues of 4-(3-bromoanilino)-6,7-dimeth-
oxyquinazoline (PD 153035), a potent inhibitor of the epidermal
growth factor receptor.. J Med Chem;39:267–276.
10. Smaill J.B., Palmer B.D., Rewcastle G.W., Denny W.A., McNamara
D.J., Dobrusin E.M., Bridges A.J., et al. (1999) Tyrosine kinase
inhibitors. 15. 4-(phenylamino)quinazoline and 4-(phenylamino)pyr-
ido[d] pyrimidine acrylamides as irreversible inhibitors of the ATP
binding site of the epidermal growth factor receptor. J Med
Chem;42:1803–1815.
11. Smaill J.B., Rewcastle G.W., Loo J.A., Greis K.D., Chan O.H.,
Reyner E.L., Lipka E., Showalter H.D., Vincent P.W., Elliott W.L.,
Denny W.A. (2000) Tyrosine kinase inhibitors. 17. Irreversible
inhibitors of the epidermal growth factor receptor: 4-(phenylami-
no)quinazoline- and 4-(phenylamino)pyrido[3,2-d]-pyrimidine-6-
acrylamides bearing additional solubilizing functions (vol 43, pg
1380, 2000). J Med Chem;43:3180–1397.
12. Wissner A., Berger D.M., Boschelli D.H., Floyd M.B. Jr, Greenber-
ger L.M., Gruber B.C., Johnson B.D., et al. (2000) 4-Anilino-6,7-
dialkoxyquinoline-3-carbonitrile inhibitors of epidermal growth
factor receptor kinase and their bioisosteric relationship to the
In our study, using gefitinib as a leading compound, two series of
novel 4-pyrrylamino quinazoline derivatives were designed and syn-
thesized. Cytotoxicity against two cancer cell lines Miapaca2 and
DU145 and EGFR inhibitory activity of these compounds were exam-
ined. The preliminary evaluation result demonstrates that all new
compounds possess improved in vitro antitumor activity compared
with the parental gefitinib, especially the compounds GI-6, GII-4,
GII-6, GII-8, and GII-9. Based on the investigation result, certain
SAR were proposed: Replacement of benzene ring with pyrrole ring
contributes to the improvement of antitumor activity; basic side
chain at position 6 or 7 of quinazoline nucleus also affects the
cytotoxicity of the compound. Our study provides useful information
for developing new and more potent antitumor agents.
Acknowledgments
This study was supported in part by China Scholarship Council and
NIH grants CA121830, CA128220 and CA134655 (L. X.) and by NIH
through the University of Michigan's Cancer Center Support Grant
(P30 CA46592).
4-anilino-6,7-dialkoxyquinazoline inhibitors.
3244–3256.
J Med Chem;43:
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