Synthesis and inhibitory activity of 4-alkynyl and 4-alkenylquinazolines: Identification of new scaffolds for potent EGFR tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.08.020

The present study identified several 4-alkynyl and 4-alkenylquinazolines that serve as novel and potent EGFR tyrosine kinase inhibitors. The IC₅₀ values of these compounds are in the nanomolar range. In addition, the 4-(4-phenylbut-1-yn/en-yl)q

Full text of DOI:10.1016/j.bmcl.2007.08.020

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5863–5867
Synthesis and inhibitory activity of 4-alkynyl
and 4-alkenylquinazolines: Identification of new scaffolds
for potent EGFR tyrosine kinase inhibitors
Yasunori Kitano,* Tsuyoshi Suzuki, Eiji Kawahara and Takahisa Yamazaki
Mitsubishi Pharma Corporation, Pharmaceuticals Research Division, 1000, Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan
Received 18 June 2007; revised 7 August 2007; accepted 10 August 2007
Available online 15 August 2007
Abstract—The present study identified several 4-alkynyl and 4-alkenylquinazolines that serve as novel and potent EGFR tyrosine
kinase inhibitors. The IC50 values of these compounds are in the nanomolar range. In addition, the 4-(4-phenylbut-1-yn/en-yl)quin-
azolines provided scaffolds for potent enzyme inhibition. Chiral discrimination was observed to occur in one of the 4-alkynylqui-
nazoline derivatives with the (R)-isomer being more than 150 times as potent as the (S)-isomer.
Ó 2007 Elsevier Ltd. All rights reserved.
Epidermal growth factor receptor (EGFR) is known to
be over-expressed in a large percentage of clinical can-
cers of various types,1–3 and to be closely related to a
poor prognosis in patients.4,5 Accordingly, the EGFR
has become an important target for drug design.6 Re-
cent success in the clinical evaluation of EGFR tyrosine
kinase (TK) inhibitors such as gefitinib7 and erlotinib8
(Fig. 1) strongly suggests small molecular EGFR TK
inhibitors to be promising new anti-cancer drugs.
served for inter-comparisons of the effects of substitu-
tions at the 4-position. The preparation of 4-alkynyl
and 4-alkenylquinazolines is outlined in Scheme 1. The
4-alkynylquinazolines were synthesized by a Sonogashira
coupling reaction12 of 4-chloroquinazoline 113 with ter-
minal alkynes 3. The corresponding alkynes were made
available in several ways. Some alkynes, other than the
commercially available 3a–c and the one already known
as 3e,14 were obtained using conventional transforma-
tion of an aldehyde into an acetylene sequence.15 Other
alkynes bearing amino-functionality, except for 3i,16
were constructed from ketones and the ethynyl
Grignard reagent. Subsequent trapping in situ of the
resulting adducts with acetic anhydride yielded acetates,
which in turn upon treatment with appropriate amines
by copper-catalyzed displacement17 furnished amino-
alkynes. The 4-alkenylquinazolines were obtained via
hydro-zirconation of alkynes 3 followed by a Pd-cata-
lyzed coupling reaction18 with 1.
These quinazoline-based inhibitors have a substituted
aniline group at the 4-position of the quinazoline nu-
cleus. The 4-substitution is assumed to be optimal as
an aniline or benzylamine, with other linkers being less
effective,9 and other variations have not been well
investigated.10 During the course of our exploration of
the non-anilininoquinazoline scaffold, 4-alkynyl- and
4-alkenylquinazolines were found to be novel and
potent EGFR TK inhibitors. Herein, we report the
synthesis, inhibitory activity, and characteristic features
in SAR of this series of compounds.
Table 1 summarizes the structures and inhibitory activ-
ities of these compounds against the human EGFR TK.
In initial studies, the 6,7-dimethoxyquinazoline nucleus
present in early quinazoline-based inhibitors11 was con-
Keywords: EGFR; Tyrosine kinase inhibitor; 4-Alkynylquinazoline;
4-Alkenylquinazoline.
*
Corresponding author at present address: Mitsubishi Tanabe
Pharma Corporation, Research Division, Medicinal Chemistry
Laboratory, 16-89, Kashima 3-chome, Yodogawa-ku, Osaka 532-
8505, Japan. Tel.: +81 6 6300 2562; fax: +81 6 6300 2564; e-mail:
Kitano.Yasunori@mc.mt-pharma.co.jp
Figure 1.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.08.020

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Y. Kitano et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5863–5867
Table 1. Inhibitory activity of 4-alkynyl and 4-alkenylquinazolines
Compound
R1
EGFR TK
IC50 (nM)
No.
R
2a
Me
Me
Me
Me
Me
Me
5600
14
15
13
80
9
2b
2c
2d
2e
2f
2g
Me
33
2h
2i
2j
Me
Me
Me
3
7300
1200
Scheme 1. Reagents: (a) for 2, 8: acetylene 3, Et3N, cat. Pd(PPh3)4 or
PdCl2(PPh3)2, CuI, DMF; for 2g and 8n: followed by hydrolysis
(NaOH aq, MeOH); for 6b, 7: Cp2Zr(H)Cl, acetylene, THF then 1, cat.
Pd(PPh3)4; (b) i—CBr4, PPh3, CH2Cl2; ii—2 equiv of n-BuLi, THF;
(c) for preparation of 4, see Ref. 20; (d) ethynyl magnesium chloride,
THF then Ac2O (for 3f: then H+); (e) R5R6NH, cat. CuCl, THF; (f) see
Ref. 16; (g) H2, Pd/C, EtOH.
2k
2l
5
Me
Me
Me
200
1100
130
For comparison, gefitinib and erlotinib have an IC50 of
1.4 and 1.0 nM, respectively, in this assay system.19
The simple 4-phenylethynylquinazoline 2a showed a
modest inhibitory activity of 5.6 lM. In contrast, the
two carbon homologue 2b had a marked 400-fold incre-
ment in activity (14 nM). Its oxa-analogue 2c also
inhibited with comparable potency, implying the 4-phe-
nylbut-1-ynyl substructure to be the preferred topology.
Compounds 2d and 2e, possessing a sterically demand-
ing quaternary carbon or tertiary amine, retained their
potencies, which were substantially comparable to that
of 2b. This result indicates a degree of tolerance for ste-
ric bulk at the propargyl position.
6b
2m
7a
Me
Me
Et
4
26
15
Incorporation of functionalities by taking advantage of
the tolerance at the propargyl position was carried out
to modulate physico-chemical properties. When employ-
ing a hydroxyl (2f), di-alkylamino (2h), or amino-acid
(2g) functionality at the propargyl position, excellent
inhibitory activity was observed, especially for 2h, which
had an IC50 value in the nanomolar range. To obtain
7b
7q
Et
Et
5
140

Y. Kitano et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5863–5867
5865
Table 1 (continued)
Compound
EGFR TK
IC50 (nM)
No.
R1
R
8n
Et
10
12
1.8
8o
8p
Et
Et
further structural requirements for the most potent com-
pound 2h, related compounds 2i–l were examined. With
respect to the length of the side chain, the shorter (2i)
and the longer methylene linker (2j) resulted in a three
digits in magnitude drop in activity. These results reveal
the optimal number of atoms between the acetylene
b-carbon and the phenyl portion to be two [i.e., 4-(4-
phenylbut-1-ynyl)quinazoline scaffold], as is consistent
with the simple case of 2a–c. The reduced activity
observed with replacement of the phenyl portion in 2h
with pyridyl (2k) and cyclohexyl (2l) serves to highlight
the importance of the phenyl aromatic nature.
Figure 2. Docking of 2b (yellow), 6b (red) and erlotinib (white) with
the EGFR TK catalytic domain. The hydrophobic pocket (Leu764-
Thr766 and Lys721) is shown in green. The residues Val693-Ser696,
Val702, Tyr703, and Ile720, and a structural water molecule have been
removed for greater clarity of viewing.
erlotinib is located. Thus, this overlay model is different
from our initial expectation, but explains the potent
inhibition of 2b and 6b as well as their contact with
the protein.
The results with 2b prompted us to postulate that shifting
of the ethylene in 2b into part of the ring system could
lead to potent inhibition through rigidity (Fig. 3). Com-
pound 2m, showing the incorporation of a benzene ring,
was found to exhibit excellent potency against EGFR
TK, proving this modification to be fruitful.
To gain further insight into the structural requirements
of EGFR TK inhibition, the saturation state of acety-
lene with respect to inhibitory activity was examined.
It should be noted that compound 5 (full saturation at
the triple bond of 2h) showed significantly reduced activ-
ity, while the potency of compound 6b, which possesses
an (E)-double bond, was greater than that of the parent
compound 2b. It is of interest that the 4-(4-phenyl-but-
1-enyl)quinazoline scaffold exhibited potent activity,
although the 4-alkenyl group was anticipated to be
placed in a different orientation with either the 4-alkynyl
group or the aniline group of the 4-anilinoquinazolines.
In order to understand the potent activities and interac-
tions of these compounds with EGFR TK, a docking
model of compounds 2b and 6b based on the crystal
structure of EGFR TK complexed with erlotinib21
(PDB code 1M17) was developed (Fig. 2).22 This model
shows that the quinazoline cores of 2b and 6b slightly
deviate from that of erlotinib, but they bind in substan-
tially the same fashion as erlotinib. It can thus be
expected that each quinazoline N-1 atom of 2b and 6b
forms a hydrogen bond to the backbone NH of
Met769 in the hinge region, and each N-3 atom binds
to a structural water molecule, which in turn is associ-
ated with the OH of Thr766 by another hydrogen bond
(for clarity, the water molecule is not shown in Fig. 2),
since the putative distance of each hydrogen bond is
With the results of 6b and 2m in hand, their derivatives
(7, 8) were briefly examined. Since it has been reported
that the potency of the 6,7-diethoxyquinazoline nucleus
is greater than that of the 6,7-dimethoxy substitution,13b
the 6,7-diethoxyquinazoline system was employed
henceforth. Compound 7b exhibited excellent activity
at 5 nM, while direct connection of the phenyl ring with
the ethenyl portion (7a) showed comparable to slightly
detrimental results and the longer methylene linker
(7q) led to decreased activity. A similar tendency was
observed for the 4-alkynyl-6,7-dimethoxyquinazoline
series, but even 7a and 7q were still potent at less than
micromolar concentrations.
Replacement of the central phenyl portion in compound
2m with a 5-membered heteroaryl such as pyrrole (8n),
imidazole (8o), or pyrazole (8p) was generally effective
and all compounds showed relatively potent inhibition.
Among them, the pyrazole derivative 8p, possessing a
˚
estimated to be ca. 3 A. This model also shows there is
a
relatively large hydrophobic pocket containing
Leu764-Thr766 and Lys721, which may lead to toler-
ance of the steric bulk at the propargyl position of the
4-alkynylquinazolines. A key feature of this model is
that the phenyl portions of 2b and 6b point in similar
directions and are buried deep in the hydrophobic pock-
et, where the acetylene moiety appended to the aniline of
Figure 3.

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Y. Kitano et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5863–5867
halogenated phenyl ring, was the most potent, with
an IC50 of 1.8 nM. This observation suggests an
opportunity for further optimization by decorating
the phenyl ring and/or refining the heteroaromatic
ring. These results, combined with that obtained for
compound 2m, demonstrate that a 1-ethynyl-2-phenyl-
aryl/heteroaryl substructure constitutes a new subclass
of 4-alkynylquinazolines.
criminates the enantiopairs by more than 150-fold. With
this result, future efforts will be directed toward under-
standing the interactions of the enantiomers from a
structural point of view.
In summary, non-anilinoquinazolines were explored in
the search for EGFR TK inhibitors and 4-(4-phenyl-
but-1-yn/en-yl)quinazolines were identified as new and
potential scaffolds. In addition, 1-ethynyl-2-phenyl-
aryl/heteroaryl was found to be a novel substructure at
the 4-position of quinazolines. To our knowledge, these
chemotypes represent the first illustration of carbon-
substituted quinazolines at the 4-position with potent
inhibitory activity and serve as new starting points for
a structurally diverse class of EGFR TK inhibitors.
Finally, we turned our attention to assessing whether
inhibition was enantio-selective, since the potent com-
pounds 2f–h had an asymmetric center. The compound
8g was chosen and both enantiomers were successfully
prepared, as outlined in Scheme 2. The racemic precur-
sor rac-9 was resolved by HPLC separation on a chiral
stationary column23 followed by hydrolysis to produce
the enantiomers (+)- and (ꢀ)-8g, each with >99% ee.23
To determine the absolute configuration of 8g thus
obtained, a stereorational synthesis of 8g was carried
out. It was found that the acetylene 3g formed a
crystalline salt with (2R,3R)-(ꢀ)-di-O-benzoyltartaric
acid [L-(ꢀ)-DBTA] in acetone.24 These crystals were
determined by X-ray crystallographic analysis to be
(R)-3g Æ L-(ꢀ)-DBTA. The optical rotation and mobility
profile of 8g prepared from (R)-3g on chiral column
chromatography correlated well with those of (+)-8g,
thus proving that (R) corresponds to (+) in 8g. Table
2 summarizes the optical purities, specific rotations,
and inhibitory activities of racemic and optically active 8g.
Acknowledgment
We thank Dr. Hideo Kubodera for helpful suggestions
on the docking study.
References and notes
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It can be seen from the table that (R)-8g had a potent
activity of 4.2 nM while the activity of the (S)-isomer
was markedly reduced, revealing that EGFR TK dis-
5. Delarue, J. C.; Terrier, P.; Terrier-Lacombe, M. J.;
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Scheme 2. Preparation of optically active 8g. Reagents: (a) 1b, cat.
Pd(PPh3)4, CuI, Et3N, DMF; (b) HPLC separation; (c) NaOH aq
MeOH; (d) L-(ꢀ)-DBTA, acetone, re-crystallization.
Table 2. Inhibitory activities and physicochemical properties of 8g
20
Compound EGFR TK IC50 (nM) ½aꢁD (c in CHCl3) % eea
rac-8g
(R)-8g
(S)-8g
11.7
4.2
—
+34.4° (0.99)
ꢀ35.7° (1.02)
15. Corey, E. J.; Fuchs, P. L. Tetrahedron Lett. 1972, 3769;
For a review, see Eymery, F.; Iorga, B.; Savignac, P.
Synthesis 2000, 185.
>99
>99
662
a Determined by HPLC analysis using a chiral stationary column.23
16. Frey, H.; Kaupp, G. Synthesis 1990, 931.

Y. Kitano et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5863–5867
5867
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19. TK assay was performed using a partially purified EGFR-
TK isolated from human epidermoid carcinoma A431
cells according to the procedure of Pike et al. with minor
modification Pike, L. J.; Gallis, B.; Casnellie, J. E.;
Bornstein, P.; Krebs, E. G. Proc. Nat. Acad. Sci. USA
1982, 79, 1433. IC50 values are representative of at least
two independent experiments.
nyanm, C.U.S. Patent 5,569,655, 1996; Compound 4p:
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22. The conformational ensemble and the local energy min-
imization of the poses (ꢂ1 kcal/mol) for 2b and 6b were
performed using the Insight II software (Accelrys Software
Inc.).
23. Separation and determination of optical purity were
performed using CHIRALPAK AD (Daicel Chemical
Industries, Ltd).
20. Compound 4d: Purohit, V. G.; Subramanian, R. Chem.
´
Ind. 1978, 731; Compound 4n: Ghosez, L.; Cecile, F.;
Frederic, D.; Cuisinier, C.; Touillaux, R. Can. J. Chem.
´ ´
24. Re-crystallization from acetone three times and liberation
yielded optically active 3g with 96% ee with a 17%
2001, 79, 1827; Compound 4n: Dority, J.A. Jr.; Earley,
W.G.; Kumar, V.; Mallamo, J.P.; Miller, M.S.; Subrama-
18
theoretical yield. (R)-3g: ½aꢁD ¼ ꢀ12:7ꢃ (c 1.36, CHCl3).