washout experiments, mass spectrometry, and mutagenesis assays [77]. Among the
synthetized derivatives, PD168393 (31), substituted with an acrylamide moiety at
quinazoline C6, presented an IC 50 of 0.70 nM and a faster EGFR kinetic inactivation
in comparison with the C7-substituted regioisomer 32, which had shown a similar
inhibitory potency (IC 50 ¼ 0.45 nM).
Additionally, even though covalent inhibitors usually raise some questions
concerning selectivity, safety, and the possibility of adduct formation with
off-targets, PD168393 (31) was demonstrated to be selective for EGFR, showing
no inhibitory activity against several other PKs, e.g., platelet-derived growth factor
receptor (PDGFR), fibroblast growth factor receptor (FGFR), protein kinase C
(PKC), and insulin receptor [78].
Subsequently, a novel series of quinazoline (33) and pyrido[d]pyrimidine
(34–35) EGFR covalent inhibitors was described (Fig. 10), and despite the similar
enzymatic inhibitory potencies, the quinazoline derivative 33 showed selectivity to
inhibit the proliferation of A431 cell line, which overexpresses EGFR, while pyrido
[d]pyrimidine derivatives 34–35 were equipotent or even selective to the MDA-MD435 cell line, overexpressing HER-2. Compound 33 presented promising preclinical
results in tumor xenograft animal models, but solubility issues had hampered its
clinical development considering the relevance of this molecular property for pharmacokinetics and in vivo activity [78]. To overcome these issues, the same research
group in Parke Davis Laboratory designed novel EGFR covalent inhibitors bearing
basic moieties at quinazoline C7, which would be easily protonated in vivo, keeping
the acrylamide moiety at C6 [79].
According to enzymatic and cellular assays, the propoxymorpholine substituent
(36–37) was identified as the most promising among the evaluated structural modifications (Fig. 10). The pyrido[d]pyrimidine derivative 37 showed lower hydrolysis
stability and a higher reactivity to glutathione in comparison with quinazoline 36.
The more stable and less reactive derivative 36 also demonstrated a higher potency
both in cellular and mouse xenograft models [79]. Compound PD183805 (36), later
named canertinib, was then selected for clinical development as a pan-ErbB family
covalent inhibitor, but it was discontinued during phase II clinical trials due to
unacceptable skin toxicity [80].
Simultaneously, researchers from Wyeth Pharmaceuticals were working on a
series of compounds harboring basic sites at the C6 substituent close to the covalent
reactive group (Fig. 11) aiming to optimize their aqueous solubility [81]. Moreover,
the basic tertiary amines at quinazoline C6 would act as in situ catalysts for cysteine
side chain thiol group deprotonation improving the reactivity of this atom to covalent
bond formation; and a protonated basic group would increase, by inductive effect,
the electrophilicity of the β-carbonyl nucleophilic carbon [82].
Several tertiary amines linked to butynamides (38), crotonamide (39) and
butenamide (40) were evaluated in enzymatic and cellular assays [81]. According
to the oncogene addiction phenomenon, which describes the dependency of certain
tumor cells on a specific oncogenic protein, if the corresponding target or signaling
pathway is inhibited, an antiproliferative effect would be observed [83]. The cellular
assays employed A431 EGFR overexpressing cells; SKBR3 HER-2 overexpressing
Case Study on Receptor Tyrosine Kinases EGFR, VEGFR, and PDGFR
169
synthetized derivatives, PD168393 (31), substituted with an acrylamide moiety at
quinazoline C6, presented an IC 50 of 0.70 nM and a faster EGFR kinetic inactivation
in comparison with the C7-substituted regioisomer 32, which had shown a similar
inhibitory potency (IC 50 ¼ 0.45 nM).
Additionally, even though covalent inhibitors usually raise some questions
concerning selectivity, safety, and the possibility of adduct formation with
off-targets, PD168393 (31) was demonstrated to be selective for EGFR, showing
no inhibitory activity against several other PKs, e.g., platelet-derived growth factor
receptor (PDGFR), fibroblast growth factor receptor (FGFR), protein kinase C
(PKC), and insulin receptor [78].
Subsequently, a novel series of quinazoline (33) and pyrido[d]pyrimidine
(34–35) EGFR covalent inhibitors was described (Fig. 10), and despite the similar
enzymatic inhibitory potencies, the quinazoline derivative 33 showed selectivity to
inhibit the proliferation of A431 cell line, which overexpresses EGFR, while pyrido
[d]pyrimidine derivatives 34–35 were equipotent or even selective to the MDA-MD435 cell line, overexpressing HER-2. Compound 33 presented promising preclinical
results in tumor xenograft animal models, but solubility issues had hampered its
clinical development considering the relevance of this molecular property for pharmacokinetics and in vivo activity [78]. To overcome these issues, the same research
group in Parke Davis Laboratory designed novel EGFR covalent inhibitors bearing
basic moieties at quinazoline C7, which would be easily protonated in vivo, keeping
the acrylamide moiety at C6 [79].
According to enzymatic and cellular assays, the propoxymorpholine substituent
(36–37) was identified as the most promising among the evaluated structural modifications (Fig. 10). The pyrido[d]pyrimidine derivative 37 showed lower hydrolysis
stability and a higher reactivity to glutathione in comparison with quinazoline 36.
The more stable and less reactive derivative 36 also demonstrated a higher potency
both in cellular and mouse xenograft models [79]. Compound PD183805 (36), later
named canertinib, was then selected for clinical development as a pan-ErbB family
covalent inhibitor, but it was discontinued during phase II clinical trials due to
unacceptable skin toxicity [80].
Simultaneously, researchers from Wyeth Pharmaceuticals were working on a
series of compounds harboring basic sites at the C6 substituent close to the covalent
reactive group (Fig. 11) aiming to optimize their aqueous solubility [81]. Moreover,
the basic tertiary amines at quinazoline C6 would act as in situ catalysts for cysteine
side chain thiol group deprotonation improving the reactivity of this atom to covalent
bond formation; and a protonated basic group would increase, by inductive effect,
the electrophilicity of the β-carbonyl nucleophilic carbon [82].
Several tertiary amines linked to butynamides (38), crotonamide (39) and
butenamide (40) were evaluated in enzymatic and cellular assays [81]. According
to the oncogene addiction phenomenon, which describes the dependency of certain
tumor cells on a specific oncogenic protein, if the corresponding target or signaling
pathway is inhibited, an antiproliferative effect would be observed [83]. The cellular
assays employed A431 EGFR overexpressing cells; SKBR3 HER-2 overexpressing
Case Study on Receptor Tyrosine Kinases EGFR, VEGFR, and PDGFR
169