Tyrosine Kinase Inhibitors. 20. Optimization of Substituted Quinazoline and Pyrido[3,4-d]pyrimidine Derivatives as Orally Active, Irreversible Inhibitors of the Epidermal Growth Factor Receptor Family

Article abstract of DOI:10.1021/acs.jmedchem.6b00883

Structure-activity relationships for inhibition of erbB1, erbB2, and erbB4 were determined for a series of quinazoline- and pyrido[3,4-d]pyrimidine-based analogues of the irreversible pan-erbB inhibitor, canertinib. Cyclic amine bearing crotonamides were determined to provide rapid inhibition of cellular erbB1 autophosphorylation and good metabolic stability in liver microsome and hepatocyte assays. The influence of 4-anilino substitution on pan-erbB inhibitory potency was investigated. Several anilines were identified as providing potent, reversible pan-erbB inhibition. Optimum 4- and 6-substituents with known 7-substituents provided preferred irreversible inhibitors for pharmacodynamic testing in vivo. Quinazoline 54 and pyrido[3,4-d]pyrimidine 71 were identified as clearly superior to canertinib. Both compounds possess a piperidinyl crotonamide Michael acceptor and a 3-chloro-4-fluoroaniline, indicating these as optimized 6- and 4-substituents, respectively. Pharmacokinetic comparison of compounds 54 and 71 across three species selected compound 54 as the preferred candidate. Compound 54 (PF-00299804) has been assigned the nomenclature of dacomitinib and is currently under clinical evaluation.

Full text of DOI:10.1021/acs.jmedchem.6b00883

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Article
Tyrosine Kinase Inhibitors. 20. Optimization of substituted
quinazoline and pyrido[3,4-d]pyrimidine derivatives as orally active,
irreversible inhibitors of the epidermal growth factor receptor family.
Jeff Bruce Smaill, Andrea J Gonzales, Julie A Spicer, Helen Lee, Jessica E. Reed, Karen Sexton, Irene W
Althaus, Tong Zhu, Shannon L Black, Adrian Blaser, William A. Denny, Paul A Ellis, Stephen Fakhoury,
Patricia J Harvey, Ken Hook, Florence O.J. McCarthy, Brian D. Palmer, Freddy Rivault, Kevin Schlosser,
Teresa Ellis, Andrew M. Thompson, Erin Trachet, R. Thomas Winters, Haile Tecle, and Alexander Bridges
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00883 • Publication Date (Web): 04 Aug 2016
Downloaded from http://pubs.acs.org on August 4, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Bridges, Alexander; Pfizer Global Research and Development Ann Arbor
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Tyrosine Kinase Inhibitors. 20. Optimization of substituted quinazoline and
pyrido[3,4ꢀd]pyrimidine derivatives as orally active, irreversible inhibitors of
the epidermal growth factor receptor family.
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a,b*
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Jeff B. Smaill,
Andrea J. Gonzales, Julie A. Spicer, Helen Lee, Jessica E. Reed,
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Karen Sexton, Irene W. Althaus, Tong Zhu, Shannon L. Black, Adrian Blaser,
a,b
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William A. Denny, Paul A. Ellis, Stephen Fakhoury, Patricia J. Harvey, Ken Hook,
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Florence O. J. McCarthy, Brian D. Palmer, Freddy Rivault, Kevin Schlosser, Teresa
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Ellis, Andrew M. Thompson, Erin Trachet, R. Thomas Winters, Haile Tecle and
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Alexander Bridges.
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Auckland Cancer Society Research Centre, School of Medical Sciences, The University of
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Auckland, Private Bag 92019, Auckland 1142, New Zealand. Maurice Wilkins Centre for
Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland 1142,
c
New Zealand. Pfizer Global Research and Development, Michigan Laboratories, 2800
Plymouth Rd, Ann Arbor, Michigan, 48105-1047, USA.
Abstract
Structureꢀactivity relationships for inhibition of erbB1, erbB2 and erbB4 were determined
for a series of quinazolineꢀ and pyrido[3,4ꢀd]pyrimidineꢀbased analogues of the
irreversible panꢀerbB inhibitor, canertinib. Cyclic amine bearing crotonamides were
determined to provide rapid inhibition of cellular erbB1 autophosphorylation and good
metabolic stability in liver microsome and hepatocyte assays. The influence of 4ꢀanilino
substitution on panꢀerbB inhibitory potency was investigated. Several anilines were
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identified as providing potent, reversible panꢀerbB inhibition. Optimum 4ꢀ and 6ꢀ
substituents with known 7ꢀsubstituents provided preferred irreversible inhibitors for
pharmacodynamic testing in vivo. Quinazoline 54 and pyrido[3,4ꢀd]pyrimidine 71 were
identified as clearly superior to canertinib. Both compounds possess a piperidinyl
crotonamide Michael acceptor and a 3ꢀchloroꢀ4ꢀfluoroaniline, indicating these as
optimized 6ꢀ and 4ꢀsubstituents respectively. Pharmacokinetic comparison of compounds
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4 and 71 across three species selected compound 54 as the preferred candidate.
Compound 54 (PFꢀ00299804) has been assigned the nomenclature of dacomitinib and is
currently under clinical evaluation.
Introduction
There is considerable interest in small molecule inhibitors of the human epidermal growth
factor receptor family of tyrosine kinases for the treatment of cancer. The family consists
of four members, erbB1 (EGFR, HERꢀ1), erbB2 (HERꢀ2, neu), erbB3 (HERꢀ3) and erbB4
(HERꢀ4). Hyperactivation of this family due to overexpression of the receptors, mutations
leading to constitutive activation or overexpression of the growth factors themselves
results in inappropriate signal transduction cascades driving cellular functions such as
proliferation, differentiation, migration and angiogenesis, in a process known to correlate
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ꢀ4
with poor prognosis in patients.
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, 6
The erbB1 selective, reversible ATPꢀcompetitive inhibitors gefitinib (1) and erlotinib
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(2) are approved as front line therapy for nonꢀsmall cell lung cancer (NSCLC) that has
been determined through genetic analysis to harbor somatic mutations in erbB1. In this
setting they demonstrate impressive response rates (60ꢀ80%) and significant
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improvements in progression free survival over chemotherapy. While some patients may
experience a durable response, resistance to these agents eventually emerges, with
approximately half of patients having acquired a second T790M mutation in the kinase
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, 10
active site.
Early attempts to improve the efficacy of these inhibitors was centered
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around improving their potency for inhibition of erbB2 which is known to be
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overexpressed in 25ꢀ30% of breast cancers. This was achieved by introducing large
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lipophilic substituents to the 4ꢀposition of the aniline ring, as typified by lapatinib (3),
®
which is approved for use in combination with Xeloda as the last line treatment for erbB2
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positive breast cancer.
The recognition that erbB1, 2, and 4 possess a unique unpaired cysteine residue (Cys 773,
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7
84 and 778 respectively)
at the mouth of the ATPꢀbinding domain, in a position
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normally occupied by a glutamine or serine in other kinases, has led ourselves
and
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others
to develop small molecule irreversible inhibitors of the erbB family of tyrosine
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kinases. The design concept is typified by canertinib (CIꢀ1033; 4), the first irreversible
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panꢀerbB inhibitor to enter clinical trials.
This compound bears an electrophilic 6ꢀ
acrylamide substituent ideally positioned to alkylate the key cysteine residues of erbB1, 2,
and 4, providing for low nanomolar potency against these kinases. Potent inhibition of
erbB3 signalling is also achieved through blockade of its transphosphorylation partners.
While a number of alternate Michael acceptors have been explored in the development of
irreversible erbB inhibitors, the most widely employed is the dimethylamino crotonamide
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originally developed by Tsou and colleagues. This is exemplified in the quinolineꢀ3ꢀ
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carbonitrile pelitinib (EKBꢀ569; 5), which was investigated in human clinical trials.
Here the dimethylamine group plays a critical role as an intramolecular base between
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reaction of a sulfhydryl group and the Michael acceptor through a 5ꢀmembered transition
state, increasing the reactivity of the Michael acceptor towards sulfur nucleophiles when
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compared to the unsubstituted crotonamide.
Optimisation of pelitinib focused on
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improving erbB2 potency, resulting in the irreversible erbB1/2 inhibitor neratinib (HKIꢀ
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272; 6), currently in Phase III clinical trial for erbB2ꢀpositive breast cancer.
Application of the dimethylamino crotonamide Michael acceptor to a quinazoline scaffold
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provided the potent irreversible erbB1/2 inhibitor afatinib (BIBWꢀ2992; 7), which is
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5
approved for mutant erbB1ꢀpositive NSCLC.
Irreversible inhibitors of the erbB family have been proposed for the treatment of T790Mꢀ
mediated relapse of first generation erbB1 inhibitors based on preclinical efficacy against
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T790Mꢀpositive cell lines,
and human tumor xenograft models, although this efficacy
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may be at supraphysiological concentrations.
Third generation inhibitors that are
selective for mutant forms of erbB1 (including T790M), while sparing wildꢀtype erbB1,
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have now been reported. Examples include WZ4002,
rociletinib (COꢀ1686) and
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osimertinib (AZD9291). The latter derivative demonstrates impressive efficacy in
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clinical trials against T790Mꢀpositive NSCLC, and is now approved by the FDA in this
setting.
Returning to canertinib, Phase I trials of this agent administered orally for 7 or 14 days of
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a 21ꢀday cycle established 650 mg/day or 450 mg/day as the respective maximum
tolerated doses. Diarrhea, acneiform rash, emesis and anorexia were described as the doseꢀ
limiting toxicities and are typical of the class. Other toxicities such as transient
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thrombocytopaenia and hypersensitivity reactions were also reported.
It has been
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speculated that these toxicities may be due to offꢀmechanism kinase activity. Indeed,
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canertinib is reported to be a potent inhibitor of BMX and BTK, TECꢀkinases that possess
a target cysteine in the same relative position of the ATPꢀbinding domain as erbB1, 2 and
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. Kinase screening against 317 protein kinases reveled offꢀtarget micromolar binding
affinity to approximately 12.6% of kinases tested and submicromolar affinity to 5.7%,
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with particularly strong affinity to BLK, GAK, LCK, MKNK1 and RIPK2. We herein
also identify canertinib as a potent inhibitor of JAK3 (IC50 = 128 nM), another kinase to
share an appropriately positioned cysteine in its ATPꢀbinding domain. Phase II studies of
canertinib in previously treated advanced ovarian cancer were disappointing with a lack of
4
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responses precluding further clinical development. The modest pharmacokinetic profile
of canertinib in humans, in particular a short plasma elimination halfꢀlife (4 h) has been
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proposed as limiting efficacy, while the observed lack of canertinib excretion in urine
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indicates elimination is primarily metabolic. Consistent with this, the major metabolite in
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cellular studies is the glutathione adduct of the Michael acceptor.
In the present study we describe our work to develop an improved analogue of canertinib.
We sought to identify a window of opportunity whereby the metabolic stability of the
Michael acceptor could be improved without disrupting the desired irreversible inhibition
of erbB1, 2, and 4. We argued this would minimize firstꢀpass metabolism and provide
improved pharmacokinetic parameters. Analogues were screened in vitro against
recombinant JAK3 kinase seeking to eliminate offꢀtarget potency against this enzyme,
thought to contribute to the myleotoxicity observed for canertinib. Doseꢀpotency and
duration of suppression of erbB1 autophosphorylation was assessed in nude mice bearing
tumor xenografts. Herein we discuss the strategy we adopted to develop an optimized
irreversible quinazoline or pyrido[3,4-d]pyrimidine panꢀerbB inhibitor with improved
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pharmacokinetics, tumor pharmacodynamics, tumor efficacy in animal models and
potential toxicity profile in humans, relative to canertinib itself.
Chemistry
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The synthesis of 6ꢀamide substituted quinazolines, pyrido[3,4ꢀd]pyrimidines and
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quinolineꢀ3ꢀcarbonitriles is well precedented,
and the present work proceeded along
similar lines. The substituted acrylamides (8ꢀ11) of Table 1 were prepared by reaction of
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the 6ꢀaminoquinazoline (85) with the respective acid chlorides (Scheme 1).
The 4ꢀanilinoquinazolines (12ꢀ43) of Table 2 were prepared by aniline displacement of the
4
ꢀchloro substituent of quinazoline 89. This array substrate was prepared from 7ꢀfluoroꢀ6ꢀ
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nitroquinazolone (86) by methoxide displacement of the activated fluorine to give 87,
followed by iron dust reduction of the nitro group and acylation with propionic anhydride
to give propanamide 88. Chlorination in refluxing POCl then gave 89 (Scheme 2). The
3
majority of the anilines of Table 2 were commercially available. The remainder were
previously reported (A7ꢀ10, A12) or prepared (A6, A11, A13, A14, A31, A32) by
reduction of their nitro precursors, which were obtained by standard techniques (see
Supporting Information).
The quinazolines (44ꢀ52) of Table 3 were prepared by ethoxide displacement on 86
3
followed by POCl mediated chlorination and displacement with 1ꢀbenzylꢀ1Hꢀindazolꢀ5ꢀ
amine to give 91. Nitro group reduction via hydrogenation over Raney nickel then gave
amine 92 which was acylated with the freshly prepared acid chloride of Eꢀ4ꢀbromobutꢀ2ꢀ
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enoic acid to give bromide 93. Low temperature displacement of the bromide with a range
of secondary amines then gave compounds 44ꢀ52 of Table 3 (Scheme 3).
The quinazolines (53ꢀ69) of Table 4 were prepared in an analogous manner to those of
Table 3 described above. The pyrido[3,4ꢀd]pyrimidines (70ꢀ82) of Table 4 were prepared
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similarly from 6ꢀfluoropyrido[3,4ꢀd]pyrimidinꢀ4(3H)ꢀone (95) by chlorination with
SOCl and displacement with the respective anilines to give compounds 96. Fluorine
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displacement with 4ꢀmethoxybenzylamine then gave the PMBꢀprotected amines (97),
which were deprotected using TFA to give the 6ꢀamino derivatives (98). Acylation of
these with the freshly prepared acid chloride of Eꢀ4ꢀbromobutꢀ2ꢀenoic acid gave the
bromides (99). Low temperature displacement of the bromides with a range of secondary
amines then gave compounds 70ꢀ82 of Table 4 (Scheme 4).
Results and Discussion
Kinase and Reactivity Assays. The relative reactivity towards reduced glutathione for
several Michael acceptors (AꢀE, Figure 1), on a common quinazoline scaffold, has been
22
reported. The dimethylamino crotonamide (A) was found to be more reactive than the
acrylamide (B) which was in turn more reactive than the alkylacrylamides (C and D) and
the morpholino crotonamide (E). The greater chemical reactivity of the dimethylamino
crotonamide (A) towards reduced gluthathione relative to the acrylamide (B) intrigued us
because pelitinib bears this Michael acceptor yet demonstrates a significantly longer
52
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human plasma halfꢀlife than canertinib (21.7 hours cf 4 hours ). This led us to prioritise
microsome and hepatocyte stability testing to predict compounds with improved first pass
metabolism, oral bioavailability and plasma halfꢀlife, rather than chemical glutathione
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reactivity assays. With respect to alkylacrylamides (C and D) we reasoned that these
Michael acceptors were understudied and further work was merited using the exact
canertinib scaffold. With respect to the decreased reactivity of the morpholino
crotonamide relative to the dimethylamino crotonamide (E cf A) it was unclear whether
this was due to changes in steric bulk, amine pKa or both. To investigate we therefore
targeted quinazolines and pyrido[3,4-d]pyrimidines substituted at the 6ꢀposition with
crotonamides bearing cyclic amines of varying steric bulk and pKa.
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Table 1 describes the structures and kinase inhibitory properties against erbB1, 2, and 4 of
a subset of the compounds synthesized to explore alkylacrylamide (C and D, Figure 1)
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21ꢀ
Michael acceptors on the canertinib scaffold. In previous publications we
and others
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have noted that care needs to be taken when interpreting IC50 values for putative
irreversible inhibitors. These values are essentially a composite of both the covalent
interaction with the enzyme over the time frame of any particular assay and the reversible
binding provided by the scaffold. As reference we prepared the reversible propanamide 11
for comparative purposes. Table 1 also describes the results of a timeꢀcourse washout
assay where A431 cells were treated with potential irreversible inhibitors for 1, 5, 10, 30,
6
0 and 120 minutes before being washed free of drug, stimulated with EGF, lysed and
western blotted for inhibition of erbB1 autophosphorylation. This determines the time
required for the inhibitor to completely ablate autophosphorylation of erbB1 in A431 cells,
providing an indication of the kinetics of alkylation of Cys 773 for each Michael acceptor.
Relative to canertinib (4) compounds 8ꢀ10 showed a modest loss of potency (3 to 11ꢀfold)
for inhibition of erbB1 in the isolated enzyme assay and a greater loss of potency in the
cellular erbB1 assay (13 to 38ꢀfold). Compounds 8-10 further showed a considerable loss
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of potency against erbB2 (> 31ꢀfold) and erbB4 (>37ꢀfold) in cellular and biochemical
assays, respectively. Compounds 8 and 9 also gave incomplete inhibition of erbB1
autophosphorylation after 2 h drug exposure in the washout assay, although compound 10
was a little more encouraging in this regard. In general, the data for 8ꢀ10 was comparable
to that for 11, with the erbB1 selective nature of the compounds indicating inefficient
alkylation of the target cysteines of erbB1, 2 and 4. It became apparent from this study
that in order to reduce the reactivity of the Michael acceptor while retaining potent panꢀ
erbB activity it may be first be preferable to increase the reversible binding affinity of the
kinase inhibitor scaffold to erbB2 and 4. A study to investigate this was undertaken prior
to an examination of the type E Michael acceptors (Figure 1).
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The SAR for erbB2 potency of 4ꢀaniline substituents of quinazoline and quinolineꢀ3ꢀ
carbonitrile inhibitors has been reasonably well explored during the development of both
5
3ꢀ56
24
lapatinib
and neratinib. In this latter study however, we felt it was difficult to
separate the relative contribution made to the inhibitory potency of the test compounds
from the aniline substituent versus that from the Michael acceptor. Further, to our
knowledge, no erbB4 data has been reported for an SAR study around 4ꢀaniline
substituents. For these reasons we undertook such a study in a reversible 7ꢀmethoxyꢀ6ꢀ
propanamide series. The aniline pharmacophore outlined in Figure 2 was explored to
determine its influence on panꢀerbB potency. This approach had the added advantage that
each aniline studied required only one synthetic step to provide final compounds for
testing (aniline displacement of the 4ꢀchloroquinazoline 89). Approximately 95 anilines
were explored in this series. Table 2 shows the erbB1, 2 and 4 isolated enzyme activities
for the 32 that provided the most potent panꢀerbB activity (compounds 12ꢀ43). We
reasoned that any compound with erbB1, 2 and 4 activity of < 500 nM would be
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sufficiently interesting to synthesise comparative irreversible analogues (see Tables 3 and
4
). Of the 32 compounds, 6 fitted this profile (A1, A4, A5, A10, A17, A24 of Table 2). A
further 8 of the 32 had erbB1, 2 and 4 activity of < 1 ꢀM (A6, A11, A12, A13, A25, A26,
A30, A32 of Table 2).
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11
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44
45
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53
54
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56
57
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59
60
The erbB1 selective nature of the 1Hꢀindolꢀ5ꢀamine (A2) and the 1Hꢀindazolꢀ5ꢀamine
(A3) are readily apparent, while the 3ꢀchloroꢀ4ꢀfluoroaniline (A1) is 203ꢀfold erbB1
selective (over erbB2) but possesses acceptable erbB2 and 4 potency (< 500 nM). As
53
previously described introduction of a benzyl substituent to the Nꢀ1 position of the
indole (A4) and indazoleamines (A5) provided a significant improvement in the erbB2
and 4 potency giving a potent panꢀerbB scaffold. Nꢀ1ꢀPhenylsulfonylindole (A10) was
also potent in this regard, while only the 2″ꢀpyridomethyl substituted indole (A6) was
tolerated by erbB1 and 4 (and to a lesser extent erbB2) with the 3″ꢀ and 4″ꢀpyridine
isomers (A7, A8) showing a considerable loss of erbB1 potency. The erbB1 preference for
a 2″ꢀpyridyl isomer is noted for the other pyridines in this study where a direct comparison
could be made (A21, A22 and A26, A27), consistent with the preferred 2″ꢀ
24
pyridomethyloxy substituent of neratinib (6). The 1ꢀ(2ꢀfurylmethyl)ꢀ1Hꢀindolꢀ5ꢀamine
(A11) and indoline (A12, A13) analogues were detrimental to overall potency when
compared to the analogous benzylindole (A4) and the need for an aromatic substituent at
the indole Nꢀ1 position was confirmed by the lack of potency for the Nꢀ1ꢀisoꢀbutyl
derivative (A14). The benzylamine (A15) and αꢀmethylbenzylamine (A16) substituents
were demonstrated to be erbB1 selective. The 4ꢀphenoxyaniline (A17) showed panꢀerbB
potency as did the 4ꢀ(benzyloxy)aniline derivatives (A24, A25), typical of that present in
5
6
lapatinib. The 4ꢀphenylsulfonamide (A30) was also considered to be of interest. Several
representative anilines with panꢀerbB activity (erbB1, 2 and 4 mostly < 500 nM) from
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Table 2 (A1, A5, A10, A17, A21, A25, A26, A30) were selected for studies incorporating
a range of Michael acceptors and 7ꢀsubstituents (see Table 4).
Variations in the terminal dialkylamino group of the crotonamide Michael acceptor (e.g. A
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22
and E; Figure 1) have been widely reported in the quinazoline and quinolineꢀ3ꢀ
2
3, 24
carbonitrile
scaffolds. However, to our knowledge only morpholine has been directly
22
studied and determined to be less reactive towards reduced gluthathione. For this
example, it is not clear if it is the reduced amine pKa or increased steric demand of the
cyclic amine that is responsible for this attenuated reactivity. To investigate the influence
of amine structure on the reactivity of the crotonamide Michael acceptor both
metabolically and towards covalent reaction with the target Cysꢀ773 of erbB1, we utilized
the most potent panꢀerbB Nꢀ1ꢀbenzylindazole (A5) scaffold of Table 2. Table 3 shows the
erbB1, 2 and 4 isolated enzyme data, inhibition of erbB1 cellular autophosphorylation,
timeꢀcourse washout data and rat and human liver microsome and hepatocyte stability for
9
amine variations of the solubilized crotonamide Michael acceptor. Isolated enzyme
inhibition of JAK3 kinase is included as an important offꢀtarget counterscreen. The most
striking observation was that the dimethylamine (44), diisopropylamine (45), pyrrolidine
(
46), piperidine (47), morpholine (49), 4ꢀmethylpiperazine (50) and 3,5ꢀ
dimethylpiperazine (51) crotonamides were all potent low nanomolar inhibitors of erbB1,
and 4 that irreversibly inhibited 100% of erbB1 cellular autophosphorylation after only
2
one minute of compound exposure.The 2,6ꢀdimethyl substituted piperidine (48) and
piperazine (52), possessing additional steric bulk around the amine nitrogen, showed an
approximate 10ꢀfold reduction in erbB1 and 2 isolated enzyme activity, consistent with
24
reported observations of poorer performance. These compounds were not progressed to
further studies. Of the compounds advanced to rat and human liver microsome and
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hepatocyte stability assays only the diisopropylamine (45) was considered unstable. All of
the compounds of Table 3 showed a favourable selectivity profile for the erbB family
relative to JAK3 (approximately 100 to 1000ꢀfold). The cyclic amine crotonamides tested
displayed excellent metabolic stability and this in conjunction with rapid inhibition of
cellular erbB1and a favourable profile against JAK3 kinase indicated this class of Michael
acceptor warranted further investigation in combination with preferred anilines of Table 2.
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44
45
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49
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52
53
54
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57
58
59
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To prepare an optimized 4ꢀanilinoquinazoline or 4ꢀanilinopyrido[3,4ꢀd]pyrimidine,
preferred 4ꢀ, 6ꢀ, and 7ꢀsubstituents were selected. At the 4ꢀposition, the most potent panꢀ
erbB anilines of Table 2 (A1, A5, A10, A17, A21, A25, A26, A30). At the key 6ꢀposition,
optimized cyclic amineꢀbearing crotonamide Michael acceptors of Table 3, with a number
of dimethylamino crotonamides prepared for comparison, while at the 7ꢀposition (where
present), alkoxy substituents selected from those of gefitinib (OMe), neratinib (OEt) and
erlotinib (OCH CH OMe) were included. Table 4 shows the erbB1, 2 and 4 isolated
2
2
enzyme activity, erbB1 cellular autophosphorylation inhibitory activity and JAK3 isolated
enzyme inhibitory activity as an offꢀtarget counter screen, for these compounds (44, 46,
4
7, 50, 55ꢀ82). A number of trends were apparent. The anilines that generally provided the
best panꢀerbB potency on either the quinazoline or pyrido[3,4ꢀd]pyrimidine scaffolds,
when prepared with a crotonamide Michael acceptor at the 6ꢀposition, were the 3ꢀchloroꢀ
4
ꢀfluoroaniline (A1), the 1ꢀbenzylꢀ1Hꢀindazolꢀ5ꢀamine (A5) and the lapatinib aniline
(A25). Within this set of anilines ranging the dialkylamine substituted crotonamide had
very little influence on the potencies against the isolated erbB1, 2, and 4 enzymes, or the
cellular erbB1 potency. The anilines that generally provided less potent panꢀerbB activity,
when prepared with a crotonamide Michael acceptor at the 6ꢀposition, were the 1ꢀ
(phenylsulfonyl)ꢀ1Hꢀindolꢀ5ꢀamine (A10), the 4ꢀphenoxyaniline (A17), the 4ꢀ(2ꢀ
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pyridinylmethyl)aniline (A21), the 4ꢀ(2ꢀpyridinylmethoxy)aniline (A26) and the Nꢀ(4ꢀ
aminophenyl)benzenesulfonamide (A30). Within this set of anilines ranging the
dialkylamine substituted crotonamide had more influence on the erbB1, 2, and 4 isolated
enzyme potency, and the cellular erbB1 potency, with the most basic amines
(dimethylamine, piperidine and pyrrolidine) providing the superior potency when
compared to the less basic cyclic amines (morpholine and 4ꢀmethylpiperazine) (compare
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8, 59 with 60; 65 with 66; 67, 68 with 69; 78, 79 with 80). Where a comparison of 7ꢀ
substituent could be made between 7ꢀOEt and 7ꢀOCH CH OMe, the overall potencies
2
2
were comparable (compare 44, 46 and 50 with 55, 56 and 57). With the exception of
compound 71 (JAK3 IC50 = 0.793 ꢀM) all of the compounds of Table 4 were micromolar
inhibitors of isolated JAK3.
A selection of compounds from Table 4 were investigated in the erbB1 cellular
autophosphorylation washꢀout assay and the rat and human liver microsome and
hepatocyte stability assays. The results are shown in Table 5. With the exception of
compound 69, all of the compounds gave rapid and complete shutdown of cellular
erbB1autophosphorylation after 1 to 5 minutes of drug exposure, indicating rapid
alkylation of Cysꢀ773. With the exception of compound 53, all of the compounds tested
were stable in rat and human microsomes for the duration of the assay (40 min) and gave
comparable or improved rat and human hepatocyte stability relative to canertinib (4).
Pharmacodynamic testing in vivo. The majority of the compounds of Table 4
demonstrated potent panꢀerbB activity in enzyme and cellular assays, an acceptable
counterꢀscreening profile against JAK3 and good metabolic stability in microsome and
hepatocycte assays. It appeared likely that physicochemical and pharmacokinetic
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differences between analogues would be the main determinant of in vivo efficacy. We
therefore decided to advance most of the compounds of Table 4 to in vivo
pharmacodynamic testing, where it was anticipated analogues optimal in this regard would
show superior target modulation. Mice (3 per cohort) bearing NIH3T3 tumors were dosed
orally with each test compound of Table 4 at 30, 65 and 130 mg/kg twice daily (QDx2).
Tumors were harvested 6 and 24 h after the final dose, digested and Western blotted for
the ability of each compound to inhibit the autophosphorylation of erbB1 (Table 6).
Gefitinib (1) and canertinib (4) were included in this assay as reference compounds. As
expected, the irreversible inhibitor canertinib showed superior inhibition of erbB1
autophosphorylation when compared to gefitinib, which only gave complete inhibition at
the earliest time point and highest dose (6h after 130 mg/kg dose). In contrast canertinib
showed some return of phosphorylated erbB1 24 h after the lowest dose (30 mg/kg), while
all other doses and time points gave complete inhibition.
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We sought to identify compounds of Table 6 with pharmacodynamic profiles comparable
to or better than canertinib for more advanced investigation. Very few compounds
qualified, despite being potent inhibitors of both isolated enzyme and cellular erbB1 (for
example 44, 46, 47, 50, 58, 59, 60, 62, 67, 74, 75, 79, 80). We attribute this poor target
modulation in vivo to a combination of factors including poor solubility, permeability,
bioavailability, pharmacokinetics and tissue penetration. We note that when dialkylamino
crotonamide Michael acceptors are compared across scaffolds, the higher molecular
weight compounds, bearing the more structurally complex anilines, are generally less
effective at providing doseꢀpotent and prolonged inhibition of erbB1 autophosphorylation.
Quinazoline 54 and pyrido[3,4ꢀd]pyrimidines 71 and 73 stood out as having superior
activity to canertinib in the erbB1 pharmacodynamic assay, providing complete inhibition
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of phosphorylated erbB1 24 h after the lowest dose tested (30 mg/kg). These compounds
possess the same 3ꢀchloroꢀ4ꢀfluoroaniline (A1) of canertinib (gefitinib, peltinib and
afatinib) and a piperidine (54, 71) or methylpiperazine (73) solubilized crotonamide
Michael acceptor. We propose for simplicity that the common features of 54 and 71
represent the optimal 4ꢀ and 6ꢀsubstituents for irreversible inhibition of erbB1 in vivo.
These optimized substituents appear to be equally effective on a quinazoline or
pyrido[3,4ꢀd]pyrimidine scaffold.
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An improvement in the duration of in vivo activity for a cyclic amine bearing crotonamide
relative to a dimethylamino crotonamide is evident when comparing dimethylamine (74)
and pyrrolidine (75). These compounds are identical in terms of reversible scaffold,
isolated enzyme erbB1, 2 and 4 activity and erbB1 cellular activity. Both compounds were
effective inhibitors of erbB1 in vivo at the highest dose tested (130 mg/kg) while only the
cyclic amine derivative 75 was effective 24 h after the middle and lower doses (65 and 30
mg/kg respectively). It was however slow to inhibit erbB1 at the 30 mg/kg dose with 2 of
3
mice showing considerable erbB1 autophosphorylation at the 6 h time point which had
been completely inhibited after 24 h.
Pharmacokinetic comparison of compounds 54 and 71 in rat, dog and monkey. A
pharmacokinetic comparison of compounds 54 and 71 was undertaken in three preclinical
species (rat, dog and monkey) following a single oral and intravenous dose (Table 7). As
57
previously described compound 54 displayed an excellent pharmacokinetic profile
across all three species demonstrating high oral bioavailability (>50%), a long plasma
halfꢀlife (t1/2 > 12 hours) and a large volume of distribution (>17 L/kg). Indeed compound
5
4 was determined to have significantly improved pharmacokinetics relative to
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canertinib. Compound 71 however was considerably more variable across species,
showing a volume of distribution (24.8 L/kg) and oral bioavailability that was acceptable
in rat (60%), but with considerably poorer parameters in dog and particularly monkey,
where plasma halfꢀlife following intravenous administration (0.7 hours) and oral
bioavailability (4%) were disappointing. This pharmacokinetic variability was deemed to
be of concern, sufficiently so to recommend compound 54 to advance to human trial over
compound 71.
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Conclusions
We have described an approach to develop an irreversible inhibitor of erbB1, 2 and 4 with
an improved pharmacokinetic and toxicity profile relative to canertinib. We have
investigated the reactivity of alkyl substituted acrylamides and dialkylamino crotonamides
in microsome and hepatocyte assays, while ascertaining their relative rate of reaction with
Cysꢀ773 of erbB1. This has allowed identification of drug candidates with the potential for
rapid irreversible inhibition of the target erbB family, minimized glutathione conjugation
and therefore improved firstꢀpass metabolism, oral bioavailability, plasma halfꢀlife and
tumor pharmacodynamics. We have counterꢀscreened analogues against JAK3 kinase to
identify compounds with minimized potency against this enzyme, an offꢀtarget event
thought to contribute to the undesirable thrombocytopenia observed during human trials of
canertinib.
Cyclic amine solubilized crotonamides have been discovered to be optimal in terms of
their rapid alkylation of erbB1 and desirable metabolic stability. We have characterized
the panꢀerbB potency imparted by a range of 4ꢀaniline substituents to a reversible
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quinazoline scaffold and established one of the broadest SAR datasets for the inhibition of
erbB4 reported to date. We have determined that 3ꢀchloroꢀ4ꢀfluoroaniline, 5ꢀaminoꢀNꢀ1ꢀ
benzylindole, 5ꢀaminoꢀNꢀ1ꢀbenzylindazole, 5ꢀaminoꢀNꢀ1ꢀphenylsulfonylindole, 4ꢀ
phenoxyaniline and 4ꢀbenzyloxyaniline all provide sufficiently potent panꢀerbB isolated
enzyme inhibition (IC < 500 nM). Compounds with cyclic amine solubilized
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crotonamides and panꢀerbB anilines on a quinazoline or pyrido[3,4ꢀd]pyrimidine scaffold
have been prepared. These compounds have been tested for their ability to inhibit erbB1, 2
and 4 isolated enzyme activity and erbB1 cellular activity. They have further been
analyzed in functional assays to determine their erbB1 alkylation kinetics and
pharmacodynamic inhibition of erbB1 in vivo. Compounds 54 and 71 were identified as
clear lead candidates for advanced studies, having demonstrated selective, potent,
irreversible inhibition of erbB1 in vitro and prolonged inhibition of erbB1
autophosphorylation in vivo. The sustained target modulation for 54 in vivo is consistent
with dramatically improved pharmacokinetics relative to canertinib in rat, dog and
57
monkey, a consequence of the metabolic stability of the piperidinyl crotonamide
Michael acceptor. We propose that this Michael acceptor at the 6ꢀposition, particularly
with a 3ꢀchloroꢀ4ꢀfluoroanilino group at the 4ꢀposition, provides an optimum
pharmacophore for irreversible inhibition of erbB1, 2 and 4 on both quinazoline and
pyrido[3,4ꢀd]pyrimidine scaffolds. Pharmacokinetic comparison of compounds 54 and 71
in rat, dog and monkey identified compound 54 over (the more variable) compound 71 as
the preferred clinical candidate.
Compound 54 (PFꢀ00299804), has now been widely studied in our laboratory and by
several other groups. The irreversible nature of erbB1 inhibition was confirmed at the
isolated enzyme level employing an ELISAꢀbased enzyme assay and at the cellular level
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by assaying inhibition of erbB1autophosphorylation following drug exposure time course
57
and extensive washꢀout in A431 cells. This was further supported by solution state
protein mass spectrometry experiments and an erbB1 T790M Xꢀray coꢀcrystal structure at
1
.8 Å resolution, clearly showing the presence of PFꢀ00299804 covalently linked to the
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target cysteine. PFꢀ00299804 has been screened against a panel of 38 protein kinases to
assess offꢀtarget interactions and was found to be particularly selective for the erbB family
with only Lck, Src and JAK3 returning isolated enzyme inhibitory IC50’s < 10 ꢀM (IC50
5
9
=
0.094, 0.110 and 3.57 ꢀM, respectively).
Growth inhibitory activity has been observed for PFꢀ00299804 in several cancer cell lines
including models of squamous cell carcinoma of the head and neck that exhibit low
60
response to cetuximab, biliary tract cancer cell lines either as a single agent or in
61
combination with gemcitabine, erbB2ꢀamplified breast cancer cell lines resistant to
6
2
63
trastuzumab and lapatinib and erbB2ꢀamplified gastric cancer cell lines.
PFꢀ00299804 has shown excellent distribution to human tumor xenografts in nude mice
5
7
and admirable pharmacokinetic properties across species. Features resulting in efficacy
superior to canertinib, demonstrating marked tumor regression in several xenograft models
including the wildꢀtype erbB1 overꢀexpressing A431 epidermoid carcinoma xenograft, the
erbB2/3 expressing H125 nonꢀsmallꢀcell lung cancer xenograft and the erbB2 overꢀ
57
expressing SKOV3 ovarian carcinoma xenograft. The striking erbB1 pharmacodynamics
reported herein is matched by greater than 99% inhibition of phosphoꢀerbB2 observed in
5
7
SKOV3 tumor xenografts following a well tolerated dose. In addition, PFꢀ00299804 has
been shown preꢀclinically to be a potent inhibitor of erbB1ꢀactivating mutations (L858R,
exon 19 deletions) as well as the erbB1 T790M resistance mutation, both in vitro and in
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vivo. Robust antiꢀtumor activity is observed in the NCIꢀH1975 and engineered HCC827ꢀ
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7, 59
del/T790M NSCLC xenograft models that are resistant to erlotinib/gefitinib.
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65
Phase I trials of PFꢀ00299804 have now been conducted in the USA, South Korea and
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Japan providing broadly similar results. Doseꢀlimiting toxicities include rash, diarrhea,
paronychia, dehydration and stomatitis. The maxium tolerated daily oral dose has been
established to be 45 mg and the mean human plasma halfꢀlife (t1/2) was 59 – 85 h, with
dosing ranging from 30 to 60 mg. The improved human halfꢀlife relative to canertinib (~ 4
h) and the elimination of thrombocytopenia as a problematic toxicity validates the drug
optimisation approach reported herein.
The efficacy of PFꢀ00299804 has to date most widely been assessed in advanced or
67, 68
metastatic NSCLC.
Phase II studies evaluating PFꢀ00299804 as salvage therapy in
advanced NSCLC patients who progressed following chemotherapy and erlotinib
65, 69
demonstrated preliminary evidence of activity,
however a doubleꢀblind, randomised
Phase III trial (BRꢀ26) in this setting determined PFꢀ00299804 did not improve overall
7
0
survival compared with placebo (NCT01000025). Phase II evaluation of PFꢀ00299804
as a secondꢀline therapy for NSCLC in comparison to erlotinib, successfully met the
primary endpoint of a statistically significant improvement in progressionꢀfree survival
71
over erlotinib. Unfortunately, the doubleꢀblind, randomised phase III trial (ARCHER
009) comparing the efficacy of PFꢀ00299804 to erlotinib as secondꢀ or thirdꢀline NSCLC
therapy failed to establish superiority of PFꢀ00299804 over erlotinib in this unselected
1
72
patient population (NCT01360554). A subanalysis of this trial has since demonstrated
superiority of PFꢀ00299804 over erlotinib among NSCLC patients harbouring erbB1
73
mutations, while Phase II evaluation of PFꢀ00299804 in a firstꢀline setting in this patient
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population has demonstrated significant activity with an objective response rate of 74%.
A randomised phase III study of PFꢀ00299804 versus gefitinib (ARCHER 1050) in this
setting is onꢀgoing (NCT01774721). Outside of lung cancer, PFꢀ00299804 has also
demonstrated promising efficacy in Phase II trial as a firstꢀline treatment in recurrent
10
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and/or metastatic squamousꢀcell carcinoma of the head and neck where 12.7% of patients
75
achieved a partial response and 57.1% of patients experienced stable disease.
In summary, PFꢀ00299804, now assigned the nomenclature of dacomitinib, has
73,76
demonstrated encouraging antiꢀtumor activity in human trials.
A large multiꢀcenter
randomised Phase III trial in the appropriately selected NSCLC EGFR mutant positive
patient population is currently in progress. Only when this is complete will we further
understand the role of this second generation irreversible panꢀerbB inhibitor for the
treatment of NSCLC.
Experimental Section
Combustion analyses were performed by the Microchemical Laboratory, University of
Otago, Dunedin, NZ, or by the Analytical Department, Pfizer Global Research and
Development, Michigan Laboratories. Purity of the compounds was confirmed to be
≥
95% by combustion analysis with found values for carbon, hydrogen and nitrogen being
within 0.4% of the calculated values for the proposed formula. The compounds were
recrystallized from ethyl acetate unless otherwise stated. Melting points were determined
using either an Electrothermal Model 9200 or a Gallenkamp digital melting point
1
apparatus, and are as read. H NMR spectra were measured either on a Bruker
Avanceꢀ400, or a Varian INOVA 400 MHz spectrometer, and are referenced to Me Si.
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High resolution mass spectra were recorded on a Finnigan MAT 900Q or a Varian VGꢀ
7
0SE spectrometer at nominal 5000 resolution. Liquid chromatographyꢀmass spectrometry
(LCMS) was performed either on an Agilent 1100 LC system interfaced with an Agilent
MSD mass detector or on a Micromass Platform LC mass spectrometer. In both cases,
mass detection was performed with an APCI source, using simultaneous positive and
negative ion acquisition. Unless otherwise indicated, compounds were purified by flash
column chromatography on Silica gel 60 support (Scharlau, 230ꢀ400 mesh ASTM), using
the indicated eluants.
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General Procedure 1. Synthesis of Quinazolines of Scheme 1
18
The appropriate acid chloride (1.25 eq) was added to a solution of 6ꢀaminoquinazoline
(85) (1.0 eq) and triethylamine (1.25 eq) in THF. The reaction was stirred at room
temperature until tlc indicated the reaction is complete. The reaction was then diluted
with water and extracted into EtOAc. The organic extract was dried over MgSO , filtered,
4
concentrated and purified by column chromatography eluting with a gradient of
2 2
MeOH/CH Cl .
N-{4-(3-Chloro-4-fluoroanilino)-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl}-2-
methylacrylamide (8). Reaction of amine 85 with methacryloyl chloride according to
General Procedure 1, gave the title compound 8 as a cream solid (6%), m.p. 137ꢀ138ºC.
1
3 2
H NMR [(CD ) SO] δ 9.78 (s, 1 H), 9.19 (s, 1 H), 8.71 (s, 1 H), 8.51 (s, 1 H), 8.12 (dd, J
= 2.7, 6.8 Hz, 1 H), 7.78 (m, 1 H), 7.39 (t, J = 9.3 Hz, 1 H), 7.25 (s, 1 H), 5.91 (s, 1 H),
5
.56 (s, 1 H), 4.20 (t, J = 6.1 Hz, 2 H), 3.54 (t, J = 4.6 Hz, 4 H), 2.44 (m, 4 H), 2.33 (br s,
1
2
H), 1.99 (s, 3 H), 1.93 (m, 2 H). Anal. (C H N O FCl.1 / H O) C, H, N.
25
27
5
3
5
2
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9
(
2E)-N-{4-(3-Chloro-4-fluoroanilino)-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl}-
2
-butenamide (9). Reaction of amine 85 with crotonyl chloride according to General
1
Procedure 1, gave the title compound 9 as a cream solid (1%), m.p. 117ꢀ118ºC. H NMR
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
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42
43
44
45
46
47
48
49
50
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52
53
54
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59
60
[(CD ) SO] δ 9.76 (s, 1 H), 9.33 (s, 1 H), 8.83 (s, 1 H), 8.49 (s, 1 H), 8.10 (dd, J = 2.6, 6.8
3
2
Hz, 1 H), 7.39 (t, J = 9.3 Hz, 1 H), 7.24 (s, 1 H), 6.70 (m, 1H), 6.39 (m, 1H), 4.20 (t, J =
6
.3 Hz, 1 H), 3.54 (t, J = 4.6 Hz, 4 H), 2.44 (m, 4 H), 2.33 (br s, 2 H), 1.99 (s, 2 H), 1.85
1
(
m, 3 H) ( H NMR contains 1 equivalent of crotonic acid). Anal.
1
(
C H N O FCl.C H O .1 / H O) C, H, N.
25 27
5
3
4
6
2
5
2
N-{4-(3-Chloro-4-fluoroanilino)-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl}-1-
cyclopentene-1-carboxamide (10). Reaction of amine 85 with 1ꢀcyclopentenecarboxylic
acid chloride according to General Procedure 1, gave the title compound 10 as a cream
1
solid (42%), m.p. 165ꢀ166ºC. H NMR [(CD ) SO] δ 9.76 (s, 1 H), 8.97 (s, 1 H), 8.74 (s, 1
3
2
H), 8.48 (s, 1 H), 8.07 (m, 1 H), 7.75 (m, 1 H), 7.38 (t, J = 9.0 Hz, 1 H), 7.20 (s, 1 H), 6.72
(s, 1 H), 4.19 (m, 2 H), 3.52 (m, 4 H), 2.57 (m, 2 H), 2.42 (m, 6 H), 1.93 (m, 4 H). Anal.
1
(C
27
H
29
N
5
O
3
FCl.1 /10
2
H O) C, H, N.
N-{4-(3-Chloro-4-fluoroanilino)-7-[3-(4-morpholinyl)propoxy]-6-
quinazolinyl}propanamide (11). Reaction of amine 85 with propionyl chloride according
1
to General Procedure 1, gave the title compound 11 as a cream solid, m.p. 201ꢀ202ºC. H
3 2
NMR [(CD ) SO] δ 9.73 (s, 1 H), 9.24 (s, 1 H), 8.70 (s, 1 H), 8.47 (s, 1 H), 8.10 (m, 1 H),
7.75 (m, 1 H), 7.36 (t, J = 9.0 Hz, 1 H), 7.20 (s, 1 H), 4.19 (t, J = 6.3 Hz, 2 H), 3.54 (m, 4
H), 2.44 (m, 8 H), 1.90 (m, 2 H), 1.09 (t, J = 7.6 Hz, 3 H). Anal. (C H N O FCl) C, H,
24
27
5
3
N.
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General Procedure 2. Synthesis of Quinazolines of Scheme 2
7
-Methoxy-6-nitro-4(3H)-quinazolinone (87). Sodium (2.3 g, 0.1 mol) was dissolved in
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
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7
8
9
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7
8
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9
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9
0
methanol (150 mL). This solution was then added to 7ꢀfluoroꢀ6ꢀnitroꢀ4(3H)ꢀ
77
quinazolinone 86 (4.01 g, 19.1 mmol) in a sealed pressure vessel and heated to 100ºC
over night. The solution was neutralised with AcOH and diluted with water. The solid was
filtered off to give the title compound 87 as light brown solid (3.63 g, 86%), m.p. (EtOH)
o
1
287 C (dec). H NMR [(CD SO] δ 12.4 (br s, 1 H), 8.53 (s, 1 H), 8.23 (s, 1 H), 7.43 (s, 1
3
)
2
H), 4.05 (s, 3 H). Anal. (C H N O ) C, H, N.
9
7 3 4
N-(7-Methoxy-4-oxo-3,4-dihydro-6-quinazolinyl)propanamide (88). Compound 87
5.1 g, 23.2 mmol) was suspended in EtOH (200 mL) and water (100 mL) and heated to
100ºC. After addition of glacial acetic acid (3 mL), Fe dust (activated by 1N HCl wash,
.8 g, 121 mmol) was added and the mixture was heated at 100ºC for 1 h. The hot solution
(
6
was basified with 25% ammonia solution and filtered through a pad of Celite, then cooled
to r.t. The pH of the solution was adjusted to pH 6 (HCl) and basified again to pH 8
3
(NaHCO ). Ethanol was removed under reduced pressure, then water was added and the
78
solid was collected by filtration to give the amine as a brown solid (3.98 g, 90%). The
amine was mixed with propionic anhydride (200 mL) and heated at 120ºC. Once the
solution turned dark brown it was cooled down and water (250 mL) was added. The
mixture was then heated to quench the excess anhydride. The cooled solution was
neutralised with 25% ammonia solution followed by saturated NaHCO
3
solution. Filtration
of the solid gave the title compound 88 as light brown solid (4.63 g, 90%), m.p. (EtOH)
o
1
2
3 2
98ꢀ301 C. H NMR [(CD ) SO] δ 12.0 (br s, 1 H), 9.23 (s, 1 H), 8.76 (s, 1 H), 7.99 (s, 1
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H), 7.17 (s, 1 H), 4.16 (s, 3 H), 2.45 (q, J = 7.5 Hz, 2 H), 1.09 (t, J = 7.5 Hz, 3 H). Anal.
12 13 3 3
(C H N O ) C, H, N.
N-(4-Chloro-7-methoxy-6-quinazolinyl)propanamide (89). Compound 88 (6.14 g, 25
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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mmol) was suspended in POCl (80 mL) and heated at reflux for 90 min. The POCl was
3
3
distilled off under reduced pressure and the resulting yellowꢀcream solid dissolved in
EtOAc (700 mL), then treated with a mixture of ice and saturated NaHCO solution. The
aqueous layer was further extracted with EtOAc (100 mL), then the combined organic
fractions were washed with brine (2x250 mL) and dried (Na SO ). The solvent was
3
2
4
removed under reduced pressure to afford the title compound 89 as a pale yellow solid
o
1
(
5.64 g, 86%), m.p. 165ꢀ168 C. H NMR [(CD
s, 1 H), 7.52 (s, 1 H), 4.10 (s, 3 H), 2.55 (q, J = 7.5 Hz, 2 H), 1.11 (t, J = 7.5 Hz, 3 H).
) C, H, N.
3 2
) SO] δ 9.59 (s, 1 H), 9.08 (s, 1 H), 8.90
(
3 2
Anal. (C12H12ClN O
N-[4-(3-Chloro-4-fluoroanilino)-7-methoxy-6-quinazolinyl]propanamide (12).
Compound 89 (50 mg, 0.19 mmol) and 3ꢀchloroꢀ4ꢀfluoroaniline (64 mg, 0.44 mmol) in
isopropanol (10 mL) with 1 drop of conc. HCl was heated to 100ºC. After 45 min TEA
was added until the solution was pH 7ꢀ8, then the solvent was removed under reduced
pressure. The resulting solid was purified by chromatography on silica gel (2ꢀ10%
MeOH/EtOAc as eluant) to afford the title compound 12 as a white solid (64 mg, 90%),
o
1
m.p. 253ꢀ256 C. H NMR [(CD ) SO] δ 9.78 (s, 1 H), 9.43 (s, 1 H), 8.84 (s, 1 H), 8.52 (s,
3
2
1 H), 8.12 (dd, J = 6.9, 2.5 Hz, 1 H), 7.79, (ddd, J = 9.0, 6.9, 2.5 Hz, 1 H), 7.42 (t, J = 9.1
Hz, 1 H), 7.39 (s, 1 H), 4.01 (s, 3 H), 2.48 (q, J = 7.5 Hz, 2 H), 1.13 (t, J = 7.5 Hz, 3 H).
Anal. (C H ClFN O ) C, H, N.
18
16
4
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N-[4-(1H-Indol-5-ylamino)-7-methoxy-6-quinazolinyl]propanamide (13). Reaction of
chloride 89 with 1Hꢀindolꢀ5ꢀamine according to General Procedure 2, gave the title
1
compound 13 as a cream solid (62%), m.p. 256ꢀ258ºC. H NMR [(CD
3
)
2
SO] δ 10.89 (s, 1
H), 9.42 (s, 1 H), 9.22 (s, 1 H), 8.67 (s, 1 H), 8.24 (s, 1 H), 7.71 (d, J = 1.6 Hz, 1 H), 7.26ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7.19 (m, 3 H), 7.08 (s, 1 H), 6.29 (m, 1 H), 3.86 (s, 3 H), 2.36 (q, J = 7.5 Hz, 2 H), 1.00 (t,
1
J = 7.5 Hz, 3 H). Anal. (C20
H
19
N
5
O
2
. /
8
MeOH) C, H, N.
N-[4-(1H-Indazol-5-ylamino)-7-methoxy-6-quinazolinyl]propanamide (14). Reaction
of chloride 89 with 5ꢀaminoindazole according to General Procedure 2, gave the title
1
compound 14 as a cream solid (39%), m.p. 280ꢀ282ºC. H NMR [(CD ) SO] δ 12.98 (s, 1
3
2
H), 9.70 (s, 1 H), 9.37 (s, 1 H), 8.81 (s, 1 H), 8.40 (s, 1 H), 8.09 (d, J = 1.8 Hz, 1 H), 8.04
(d, J = 1.4 Hz, 1 H), 7.60 (m, 1 H), 7.50 (d, J = 9.0 Hz, 1 H), 7.21 (s, 1 H), 3.98 (s, 3 H),
4
2
.44 (q, J = 7.4 Hz, 2 H), 1.11 (t, J = 7.6 Hz, 3 H). Anal. (C19
H
18
N
6
O
2
.1 /
5
H
2
O) C, H, N.
N-{4-[(1-Benzyl-1H-indol-5-yl)amino]-7-methoxy-6-quinazol0inyl}propanamide (15).
Reaction of chloride 89 with 1ꢀbenzylꢀ1Hꢀindolꢀ5ꢀamine according to General Procedure
1
2
, gave the title compound 15 as a cream solid (62%), m.p. 208ꢀ212ºC. H NMR
[
(CD
3 2
) SO] δ 9.57 (s, 1 H), 9.34 (s, 1 H), 8.80 (s, 1 H), 8.36 (s, 1 H), 7.87 (d, J = 1.8 Hz, 1
H), 7.50 (d, J = 3.1 Hz, 1 H), 7.21ꢀ7.42 (m, 8 H), 6.48 (d, J = 3.1 Hz, 1 H), 5.42 (s, 2 H),
3
.98 (s, 3 H), 2.45 (q, J = 7.5 Hz, 2 H), 1.12 (t, J = 7.5 Hz, 3 H). Anal. (C H N O ) C, H,
27 25 5 2
N.
N-{4-[(1-Benzyl-1H-indazol-5-yl)amino]-7-methoxy-6-quinazolinyl}propanamide
4
79
(
16). Reaction of N ꢀ(1ꢀbenzylꢀ1Hꢀindazolꢀ5ꢀyl)ꢀ7ꢀmethoxyꢀ4,6ꢀquinazolinediamine
with propionyl chloride according to General Procedure 1, gave the title compound 16 as a
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1
cream solid (26%), m.p. 211ꢀ212ºC. H NMR [(CD
3
)
2
SO] δ 9.69 (s, 1 H), 9.38 (s, 1 H),
8
3
.77 (s, 1 H), 8.36 (s, 1 H), 8.05 (s, 2 H), 7.60 (m, 2 H), 7.29ꢀ7.18 (m, 6 H), 5.61 (s, 2 H),
.94 (s, 3 H), 1.19 (d, J = 13.4 Hz, 2 H), 1.07 (t, J = 7.6 Hz, 3 H). Anal.
1
(
C H N O FCl.1 / H O) C, H, N.
2
5
29
5
2
5
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N-(7-Methoxy-4-{[1-(2-pyridinylmethyl)-1H-indol-5-yl]amino}-6-
quinazolinyl)propanamide (17). Reaction of chloride 89 with 1ꢀ(2ꢀpyridinylmethyl)ꢀ1Hꢀ
indolꢀ5ꢀamine (Supporting Information) according to General Procedure 2, gave the title
1
compound 17 as a cream solid (91%), m.p. 219ꢀ221ºC. H NMR [(CD ) SO] δ 9.57 (s, 1
3 2
H), 9.36 (s, 1 H), 8.79 (s, 1 H), 8.55 (m, 1 H), 8.36 (s, 1 H), 7.87 (d, J = 1.8 Hz, 1 H), 7.72
(dt, J = 7.7, 1.8 Hz, 1 H), 7.50 (d, J = 3.1 Hz, 1 H), 7.26ꢀ7.39 (m, 3 H), 7.19 (s, 1 H), 6.99
(d, J = 7.7 Hz, 1 H), 6.50 (d, J = 3.1 Hz, 1 H), 5.51 (s, 2 H), 3.98 (s, 3 H), 2.44 (q, J = 7.4
1
Hz, 2 H), 1.12 (t, J = 7.4 Hz, 3 H). Anal. (C26
H
24
N
6
O
2
. /
2
H
2
O) C, H, N.
N-(7-Methoxy-4-{[1-(3-pyridinylmethyl)-1H-indol-5-yl]amino}-6-
quinazolinyl)propanamide (18). Reaction of chloride 89 with 1ꢀ(3ꢀpyridinylmethyl)ꢀ1Hꢀ
80
indolꢀ5ꢀamine according to General Procedure 2, gave the title compound 18 as a cream
1
solid (88%), m.p. 213ꢀ217ºC. H NMR [(CD
3
)
2
SO] δ 9.57 (s, 1 H), 9.35 (s, 1 H), 8.79 (s, 1
H), 8.54 (d, J = 1.7 Hz, 1 H), 8.46 (dd, J = 4.8, 1.6 Hz, 1 H), 8.37 (s, 1 H), 7.88 (d, J = 1.8
Hz, 1 H), 7.58 (m, 1 H), 7.54 (d, J = 3 .1 Hz, 1 H), 7.47 (d, J = 8.8 Hz, 1 H), 7.32ꢀ7.38 (m,
2
7
H), 7.21 (s, 1 H), 6.50 (dd, J = 3.1, 0.7 Hz, 1 H), 5.47 (s, 2 H), 3.98 (s, 3 H), 2.45 (q, J =
+
.6 Hz, 2 H), 1.13 (t, J = 7.6 Hz, 3 H). HRMS (FAB ) calcd for C26
H
25
N
6
O
2
: 453.2039
+
(MH )
.
Found 453.2034.
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4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N-(7-Methoxy-4-{[1-(4-pyridinylmethyl)-1H-indol-5-yl]amino}-6-
quinazolinyl)propanamide (19). Reaction of chloride 89 with 1ꢀ(4ꢀpyridinylmethyl)ꢀ1Hꢀ
80
indolꢀ5ꢀamine according to General Procedure 2, gave the title compound 19 as a cream
1
solid (92%), m.p. 168ꢀ171ºC. H NMR [(CD ) SO] δ 9.58 (s, 1 H), 9.35 (s, 1 H), 8.79 (s, 1
3
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H), 8.49 (m, 2 H), 8.37 (s, 1 H), 7.91 (m, 1 H), 7.51 (d, J = 3.1 Hz, 1 H), 7.36 (d, J = 1.3
Hz, 2 H), 7.21 (s, 1 H), 7.10 (m, 2 H), 6.53 (d, J = 3.1 Hz, 1 H), 5.50 (s, 2 H), 3.97 (s, 3
1
H), 2.45 (q, J = 7.6 Hz, 2 H), 1.12 (t, J = 7.6 Hz, 3 H). Anal. (C26
24
H N
6
O
2
.1 /
2
H
2
O) C, H,
N.
N-(7-Methoxy-4-{[1-(2-pyridinylmethyl)-1H-indazol-5-yl]amino}-6-
4
quinazolinyl)propanamide (20). Reaction of 7ꢀmethoxyꢀN ꢀ(1ꢀ(pyridinꢀ2ꢀylmethyl)ꢀ1Hꢀ
81
indazolꢀ5ꢀyl)quinazolineꢀ4,6ꢀdiamine with propionyl chloride according to General
1
Procedure 1, gave the title compound 20 as a cream solid (58%). H NMR [(CD
3
)
2
SO] δ
11.47 (s, 1 H), 9.65 (s, 1 H), 9.11 (s, 1 H), 8.55 (d, J = 4.1 Hz, 1 H), 8.17 (s, 1 H), 7.93 (d,
J = 1.5 Hz, 1 H), 7.85 (t, J = 7.5 Hz, 1 H), 7.75 (d, J = 9.0 Hz, 1 H), 7.55 (dd, J = 1.7, 8.8
Hz, 2 H), 7.40 (t, J = 5.6 Hz, 2 H), 7.12 (d, J = 7.8 Hz, 1 H), 5.83 (s, 2 H), 4.01 (s, 3 H),
2
.44 (q, J = 1.7 Hz, 2 H), 1.07 (t, J = 7.6 Hz, 3 H). Anal. (C25
23 7 2
H N O .3HCl) C, N. H:
calcd, 4.7; found, 5.2.
N-(7-Methoxy-4-{[1-(phenylsulfonyl)-1H-indol-5-yl]amino}-6-
quinazolinyl)propanamide (21). Reaction of chloride 89 with 1ꢀ(phenylsulfonyl)ꢀ1Hꢀ
24
indolꢀ5ꢀamine according to General Procedure 2, gave the title compound 21 as a cream
1
solid (85%), m.p. 270ꢀ275ºC. H NMR [(CD
3
)
2
SO] δ 9.69 (s, 1 H), 9.37 (s, 1 H), 8.81 (s, 1
H), 8.42 (s, 1 H), 7.99 (m, 3 H), 7.91 (d, J = 8.9 Hz, 1 H), 7.79 (d, J = 3.7 Hz, 1 H), 7.70ꢀ
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
7.59 (m, 4 H), 7.23 (s, 1 H), 6.86 (dd, J = 3.7, 0.6 Hz, 1 H), 3.99 (s, 3 H), 2.45 (q, J = 7.5
1
Hz, 2 H), 1.12 (t, J = 7.5 Hz, 3 H). Anal. (C26
H
23
N
5
O
4
S. /
2
MeOH) C, H, N.
N-(4-{[1-(2-Furylmethyl)-1H-indol-5-yl]amino}-7-methoxy-6-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
quinazolinyl)propanamide (22). Reaction of chloride 89 with 1ꢀ(2ꢀfurylmethyl)ꢀ1Hꢀ
indolꢀ5ꢀamine (Supporting Information) according to General Procedure 2, gave the title
1
compound 22 as a cream solid (82%), m.p. 221ꢀ224ºC. H NMR [(CD
3
)
2
SO] δ 9.58 (s, 1
H), 9.36 (s, 1 H), 8.80 (s, 1 H), 8.37 (s, 1 H), 7.85 (d, J = 1.8 Hz, 1 H), 7.58 (m, 1 H), 7.53
(d, J = 8.8 Hz, 1 H), 7.39 (m, 2 H), 7.21 (s, 1 H), 6.40ꢀ6.47 (m, 3 H), 5.41 (s, 2 H), 3.99 (s,
1
3 H), 2.45 (q, J = 7.5 Hz, 2 H), 1.12 (t, J = 7.5 Hz, 3 H). Anal. (C H N O . / THF) C, H,
2
5
23
5
3
3
N.
N-{4-[(1-Benzyl-2,3-dihydro-1H-indol-5-yl)amino]-7-methoxy-6-
quinazolinyl}propanamide (23). Reaction of chloride 89 with 1ꢀbenzylꢀ2,3ꢀdihydroꢀ1Hꢀ
24
indolꢀ5ꢀylamine according to General Procedure 2, gave the title compound 23 as a
1
yellow solid (80%), m.p. 169ꢀ171ºC. H NMR [(CD
3
)
2
SO] δ 9.39 (s, 1 H), 9.33 (s, 1 H),
8
4
.73 (s, 1 H), 8.35 (s, 1 H), 7.22ꢀ7.41 (m, 7 H), 7.19 (s, 1 H), 6.57 (d, J = 8.4 Hz, 1 H),
.27 (s, 2 H), 3.97 (s, 3 H), 3.26 (t, J = 8.2 Hz, 2 H), 2.92 (t, J = 8.2 Hz, 2 H), 2.44 (q, J =
1
7.5 Hz, 2 H), 1.12 (t, J = 7.5 Hz, 3 H). Anal. (C H N O . / EtOAc) C, H, N.
27
27
5
2
4
N-(7-Methoxy-4-{[1-(2-pyridinylmethyl)-2,3-dihydro-1H-indol-5-yl]amino}-6-
quinazolinyl)propanamide (24). Reaction of chloride 89 with 1ꢀ(2ꢀpyridinylmethyl)ꢀ2,3ꢀ
dihydroꢀ1Hꢀindolꢀ5ꢀylamine (Supporting Information) according to General Procedure 2,
1
gave the title compound 24 as a brown solid (79%), m.p. 90ꢀ94ºC. H NMR [(CD ) SO] δ
3
2
9
.40 (s, 1 H), 9.34 (s, 1 H), 8.72 (s, 1 H), 8.54 (d, J = 4.0 Hz, 1 H), 8.35 (s, 1 H), 7.78 (dt,
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