Irreversible inhibition of epidermal growth factor receptor activity by 3-aminopropanamides

Article abstract of DOI:10.1021/jm201507x

Irreversible epidermal growth factor receptor (EGFR) inhibitors contain a reactive warhead which covalently interacts with a conserved cysteine residue in the kinase domain. The acrylamide fragment, a commonly employed warhead, effectively alkylates Cys797 of EGFR, but its reactivity can cause rapid metabolic deactivation or nonspecific reactions with off-targets. We describe here a new series of irreversible inhibitors containing a 3-aminopropanamide linked in position 6 to 4-anilinoquinazoline or 4-anilinoquinoline-3- carbonitrile driving portions. Some of these compounds proved to be as efficient as their acrylamide analogues in inhibiting EGFR-TK (TK = tyrosine kinase) autophosphorylation in A549 lung cancer cells. Moreover, several 3-aminopropanamides suppressed proliferation of gefitinib-resistant H1975 cells, harboring the T790M mutation in EGFR, at significantly lower concentrations than did gefitinib. A prototypical compound, N-(4-(3-bromoanilino)quinazolin-6- yl)-3-(dimethylamino)propanamide (5), did not show covalent binding to cell-free EGFR-TK in a fluorescence assay, while it underwent selective activation in the intracellular environment, releasing an acrylamide derivative which can react with thiol groups.

Full text of DOI:10.1021/jm201507x

Article
pubs.acs.org/jmc
Irreversible Inhibition of Epidermal Growth Factor Receptor Activity
by 3-Aminopropanamides
Caterina Carmi,† Elena Galvani,‡ Federica Vacondio,† Silvia Rivara,† Alessio Lodola,† Simonetta Russo,†
Stefania Aiello,§ Fabrizio Bordi,† Gabriele Costantino,† Andrea Cavazzoni,‡ Roberta R. Alfieri,‡
Andrea Ardizzoni,∥ Pier Giorgio Petronini,‡ and Marco Mor*,†
†Dipartimento Farmaceutico, Universita
̀
degli Studi di Parma, V.le G.P. Usberti 27/A, I-43124 Parma, Italy
degli Studi di Parma, Via Volturno 39, I-43125 Parma, Italy
‡Dipartimento di Medicina Sperimentale, Universita
̀
§Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, Universita
I-90123 Palermo, Italy
∥Oncologia Medica, Azienda Ospedaliero-Universitaria di Parma, V.le Gramsci 14, I-43125 Parma, Italy
̀
degli Studi di Palermo, Via Archirafi 32,
S
* Supporting Information
ABSTRACT: Irreversible epidermal growth factor receptor (EGFR) inhibitors contain a reactive warhead which covalently
interacts with a conserved cysteine residue in the kinase domain. The acrylamide fragment, a commonly employed warhead,
effectively alkylates Cys797 of EGFR, but its reactivity can cause rapid metabolic deactivation or nonspecific reactions with off-
targets. We describe here a new series of irreversible inhibitors containing a 3-aminopropanamide linked in position 6 to 4-
anilinoquinazoline or 4-anilinoquinoline-3-carbonitrile driving portions. Some of these compounds proved to be as efficient
as their acrylamide analogues in inhibiting EGFR-TK (TK = tyrosine kinase) autophosphorylation in A549 lung cancer
cells. Moreover, several 3-aminopropanamides suppressed proliferation of gefitinib-resistant H1975 cells, harboring the T790M
mutation in EGFR, at significantly lower concentrations than did gefitinib. A prototypical compound, N-(4-(3-
bromoanilino)quinazolin-6-yl)-3-(dimethylamino)propanamide (5), did not show covalent binding to cell-free EGFR-TK in a
fluorescence assay, while it underwent selective activation in the intracellular environment, releasing an acrylamide derivative
which can react with thiol groups.
have been extensively investigated and employed as antitumor
agents.
INTRODUCTION
■
The epidermal growth factor receptor (EGFR, erbB1) is a
member of the erbB family of receptor tyrosine kinases (RTKs),
also including erbB2/HER2, erbB3/HER3, and erbB4/HER4.1,2
Upon ligand binding, EGFR forms homo- or heterodimers with
other members of the erbB family and undergoes autophosphor-
ylation of specific tyrosine residues in its intracellular domain.
This event activates a series of downstream signals and regulates
biological processes such as cell proliferation, apoptosis, angio-
genesis, and differentiation.3,4 Deregulation of EGFR signaling
has been observed in many human cancers, including lung, head
and neck, colorectal, ovarian, breast, and bladder cancers,5,6 and
it has been associated with more aggressive disease and poorer
clinical outcome.7 In this light, inhibitors targeting the EGFR
The EGFR inhibitors 18 (gefitinib, Figure 1) and 4-
(3-ethynylanilino)-6,7-bis(2-methoxyethoxy)quinazoline9 (er-
lotinib, Figure 1) and the EGFR/erbB2 inhibitor 210 (lapatinib,
Figure 1) belong to the chemical class of 4-anilinoquinazolines,
designed to bind the ATP binding pocket of the kinase domain
of the target. Although 1 is effective in the treatment of non-
small-cell lung cancer (NSCLC) in patients having activating
mutations within the EGFR tyrosine kinase domain,11 ac-
cumulating clinical experience indicates that most patients develop
resistance after repeated treatments.12 In half of NSCLC cases,
Received: November 8, 2011
Published: January 26, 2012
© 2012 American Chemical Society
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Figure 1. Chemical structures of reversible and irreversible EGFR inhibitors.
resistance was associated with the emergence of a single
amino acid substitution in the catalytic domain of EGFR: con-
version of the gatekeeper threonine 790 with methionine
(T790M).12−15
(such as glutathione) than the reference acrylamide 316
(PD168393, Table 1) proved efficient in irreversibly inhibiting
EGFR autophosphorylation.
In our ongoing search for functional groups that are able to
covalently interact with cysteine residues of EGFR,35−37 we
focused on a new chemical entity containing a 3-aminopro-
panamide moiety. Aryl β-aminoethyl ketones (Mannich bases)
have been described as irreversible inhibitors of enzymes having
a crucial cysteine residue in the active site or at regulatory
positions.38−40 These compounds possess a particular profile of
reactivity, being nonreactive directly, but able to covalently
modify their biological target after bioconversion via β-elimination
to the corresponding α,β-unsaturated carbonyl compound.
Maresso and co-workers38 reported aryl β-aminoethyl ketones
as irreversible inhibitors of the bacterial enzyme sortase, a target
for the development of antibacterial therapeutics. They demo-
nstrated through mass spectrometry and X-ray analysis that
enzyme inhibition proceeds via a reactive olefin intermediate,
generated in situ by amine elimination, which forms a covalent
adduct with a specific cysteine residue within the protein active
site. Aryl β-aminoethyl ketones have also been reported as
inhibitors of intracellular kinases, such as Janus kinase
3 (Jak3),41 interleukin-2 inducible tyrosine kinase (ITK),42
and Bruton’s tyrosine kinase (BTK),42 as well as weak EGFR
tyrosine kinase inhibitors in enzymatic assay.43 Recently, β-
aminolactones have also been described as novel anticancer
agents, endowed with high oral bioavailability in rats. β-
Aminolactone derivatives can undergo retro-Michael addition
reaction to generate the parent α-methylenelactones that inter-
act with nucleophilic sites on biological macromolecules.44 In
principle, masking the acrylamide warhead of irreversible in-
hibitors should reduce the risk of covalent interactions with off-
targets, due to limited systemic exposure. Of course, this would
result in a specific advantage only if the antiproliferative potency
and efficacy of masked agents are equivalent or similar to those
of corresponding compounds having a free acrylamide fragment.
In the present study, we describe the synthesis and bio-
logical evaluation of new EGFR-TK inhibitors containing a
3-aminopropanamide side chain linked to a 4-anilinoquinazo-
line (5−9, A-series, and 11−13, B-series, in Table 1) or 4-
anilinoquinoline-3-carbonitrile (15−17 and 19−21, C- and
Covalent alkylation of a cysteine residue (Cys797), posi-
tioned at the entrance of the ATP binding site,16,17 was the
additional mechanism of second-generation irreversible EGFR
inhibitors, which had been shown to overcome resistance to
gefitinib.18−20 Several inhibitors of this class, such as N-(4-
(3-chloro-4-fluoroanilino)-7-(3-morpholinopropoxy)-
quinazolin-6-yl)acrylamide21 (CI-1033, canertinib, Figure 1),
N-(4-(3-chloro-4-fluoroanilino)-7-(tetrahydrofuran-3-yloxy)-
quinolin-6-yl)-4-(dimethylamino)but-2-enamide22 (BIBW-
2992, Figure 1), and N-(4-(3-chloro-4-(pyridin-2-ylmethoxy)-
anilino)-3-cyano-7-ethoxyquinolin-6-yl)-4-(dimethylamino)but-2-
enamide23 (HKI-272, neratinib, Figure 1), have progressed to
clinical investigation in patients that initially responded to 1 and
subsequently relapsed.24 EGFR irreversible covalent inhibitors are
characterized by a heterocyclic core structure (driving portion)
carrying at a proper position an electrophilic functionality
(warhead) that covalently interacts with the specific cysteine
residue in the target protein.25−29 The ability of the warhead to
covalently interact with nucleophiles is responsible for the
advantages of EGFR irreversible inhibitors over conventional
ATP-competitive ones: (i) overcoming secondary resistance,19
(ii) selectivity for erbB-family TK,30 (iii) long-lasting effect.16
On the other hand, the intrinsic reactivity of the warhead is also
associated with augmented metabolic degradation and toxicity,
due to reactions with nontarget proteins.31,32 Systematic
analysis of cysteine residues present in the nucleotide binding
site of kinases had recently shown that combination of a driving
portion and an electrophilic warhead could also be applied in a
more general way to target different cysteines in different
kinases.33 To this aim, the availability of a panel of cysteine-
reactive groups with different reactivities could be beneficial for
the development of new druglike inhibitors.
Recently, our group has reported a systematic exploration of
the role and reactivity of warheads for EGFR-TK inhibition by
introducing different cysteine-trap portions on a 4-anilino-
quinazoline recognition scaffold.34 Compounds with different
functional groups that were less reactive toward bionucleophiles
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Table 1. EGFR Tyrosine Kinase and Autophosphorylation Inhibition in A549 Cells: Viability Inhibition of the H1975
Gefitinib-Resistant Cell Line
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Table 1. continued
a
Concentration to inhibit by 50% EGFR-wt tyrosine kinase activity. IC50 values were measured by the phosphorylation of a peptide substrate using
b
time-resolved fluorometry (see the Experimental Section). Mean values of three independent experiments SEM are reported. Inhibition of EGFR
autophosphorylation was measured in A549 intact cells by Western blot analysis. The percent inhibition at 1 μM concentration was measured
immediately after and 8 h after removal of the compound from the medium (1 h of incubation). Mean values of at least two independent experiments
c
SEM are reported. Concentration to inhibit by 50% the proliferation of NSCLC H1975. The cell proliferation was determined by the MTT assay
d
after 72 h of incubation with compounds (0.1−20 μM). Mean values of three independent experiments SEM are reported. Data from ref 34.
D-series, respectively, in Table 1) driving portion. Reference
acrylamide derivatives for each series were also prepared (3, 10,
14, and 18, Table 1).
CHEMISTRY
■
Compounds of series A (5−9) of Table 1 were synthesized by
coupling their precursor amines (4 or 26) with the proper
carboxylic acid and substituting the terminal chlorine with
various amines, as described in Scheme 1. The 6-amino-4-
(3-bromoanilino)quinazoline (4) was prepared in three steps
from 5-nitroanthranilonitrile (22) as previously described
(Scheme 1).45,46 Condensation of 4 with 3-chloropropionyl
chloride gave the 3-chloropropanamide intermediate 27a,
which underwent substitution with the proper amine to obtain
the secondary 3-aminopropanamides 5−9. Alternatively, 6-amino-
quinazoline 4 was converted to its N-methyl analogue 2647 by
carbamoylation with ethyl chloroformate in pyridine followed
by reduction of the resulting carbamate 25 with sodium
bis(2-methoxyethoxy)aluminum hydride (Red-Al). The (N-
methylamino)quinazoline 26 thus generated was first con-
densed with 3-chloropropionyl chloride to 27b and then
substituted with dimethylamine to obtain the tertiary 3-
aminopropanamide derivative 9 (Scheme 1).
The new compounds were tested as EGFR tyrosine kinase
inhibitors in enzyme-based and cell-based assays. Antiprolifer-
ative activity of the new compounds was also investigated in the
gefitinib-resistant H1975 NSCLC cell line, harboring the
T790M mutation. Moreover, for the most active agents, effects
on mutated (T790M) EGFR autophosphorylation and on
ErbB2 tyrosine kinase activity were evaluated. We hypothesized
that the observed long-lasting effect on EGFR autophosphor-
ylation was the result of an irreversible covalent interaction
between the 3-aminopropanamide side chain and Cys797 with-
in the active site of the enzyme. To evaluate this hypothesis, we
performed a series of in vitro chemical stability assays, reactivity
studies in the presence of thiol nucleophiles, and reactivity
studies toward EGFR tyrosine kinase. Pharmacological data and
reactivity study results were combined and evaluated to identify
new irreversible EGFR inhibitors with lower intrinsic reactivity
and optimized efficacy/toxicity profile compared to those of the
other cysteine-reactive species described to date.
4-(3-Chloro-4-fluoroanilino)-7-ethoxyquinazolino com-
pounds of series B (10−13) of Table 1 were synthesized as
described in Scheme 2. 6-Aminoquinazoline intermediate 3448
was obtained following a slightly modified procedure49 from
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a
Scheme 1. Synthesis of Compounds 4−9
a
Reagents and conditions: (a) H2SO4, formic acid, reflux; (b) SOCl2, dioxane, reflux; (c) 3-bromoaniline, i-PrOH, 60 °C; (d) Fe, AcOH, EtOH/
H2O, reflux; (e) ClCOOEt, anhydrous pyridine, 0 °C to room temperature; (f) Red-Al, THF, room temperature; (g) ClCH2CH2COCl, THF,
50 °C; (h) R2R3NH, KI, absolute EtOH, reflux.
a
sodium ethoxide, and the resulting 7-ethoxy derivative 31 was
first chlorinated by heating in phosphorus oxychloride and then
substituted with 3-chloro-4-fluoroaniline to obtain the inter-
mediate 33. Reduction of the nitro group of 33 with iron and
acetic acid gave the 6-aminoquinazoline 34. The 6-acrylamide
derivative 10 was then obtained from 34 with acryloyl chloride
in the presence of N,N-diisopropylethylamine (DIPEA). Finally,
3-aminopropanamides 11−13 were synthesized from 34 by
reaction with 3-chloropropionyl chloride to 35 and then
substitution of the chlorine with the proper secondary amine
(Scheme 2).
Scheme 2. Synthesis of Compounds 10−13
4-Anilinoquinoline-3-carbonitriles of series C and D (14−17
and 18−21, respectively) of Table 1 were synthesized as de-
scribed in Scheme 3. The key N-(4-chloro-3-cyano-7-ethoxy-
quinolin-6-yl)acetamide intermediate (40) was synthesized in
six steps from 2-amino-5-nitrophenol (36), as previously re-
ported.50 The quinoline intermediate 40 was reacted with the
proper aniline (to 41a and 41b) and deacetylated in aqueous
hydrochloric acid to amines 42a51 and 42b.52 Aniline derivative
45 was synthesized by reaction of 4-amino-2-chlorophenol (44)
and picolyl chloride in the presence of benzaldehyde as de-
scribed in Scheme 3.53 The acrylamides 14 and 18 were sub-
sequently synthesized from amine precursors 42a and 42b,
respectively, with acryloyl chloride in the presence of a base
(DIPEA). The 3-aminopropanamides 15−17 and 19−21 were
synthesized in two steps from amines 42 by condensation with
3-chloropropionyl chloride and substitution with a secondary
amine (Scheme 3).
RESULTS AND DISCUSSION
■
a
Reagents and conditions: (a) formamidine acetate, 2-methoxyetha-
Kinase and Cellular Inhibitory Activities. Compounds
3−21 (Table 1) were evaluated in enzyme-based and cell-based
assays for their ability to inhibit EGFR tyrosine kinase activity.
Their inhibitor potency on human EGFR in a cell-free envi-
ronment was measured on the phosphorylation of a peptide sub-
strate using time-resolved fluorometry (see the Experimental
Section). In these conditions, the reference compound 3 had an
IC50 of 1.7 nM.34 Within the series of quinazoline derivatives
with the same driving portion as 3 (series A in Table 1),
substitution of the acrylamide warhead with a substituted
3-aminopropanamide (5−8) produced a generalized increase in
nol, 130 °C; (b) H2SO4, HNO3, 100 °C; (c) NaOEt, anhydrous
EtOH, reflux; (d) POCl3, reflux; (e) 3-chloro-4-fluoroaniline, i-PrOH,
reflux; (f) Fe, AcOH, EtOH/H2O, reflux; (g) acryloyl chloride,
DIPEA, DMF, 0−50 °C; (h) ClCH2CH2COCl, 50 °C; (i) R2R3NH,
KI, absolute EtOH, reflux.
4-chloroanthranilic acid (28). Briefly, cyclization of 28 with
formamidine acetate followed by nitration gave a mixture of iso-
mers from which pure 30 was obtained after recrystallization
from acetic acid. The 7-chlorine group was substituted with
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a
Scheme 3. Synthesis of Compounds 14−21
a
Reagents and conditions: (a) Ac2O, AcOH, 60 °C; (b) EtBr, DMF, K2CO3, 60 °C; (c) H2, Pd/C, THF, room temperature; (d) toluene, 90 °C; (e)
Dowterm, 250 °C; (f) POCl3, diglyme, 100 °C; (g) 3-chloro-4-fluoroaniline or 45, pyridine hydrochloride, i-PrOH, reflux; (h) HCl, reflux; (i)
acryloyl chloride, DIPEA, DMF, 0−50 °C; (j) ClCH2CH2COCl, THF, 50 °C; (k) R2R3NH, KI, absolute EtOH, reflux; (l) PhCHO, K2CO3, picolyl
chloride hydrochloride, DMF, 50 °C.
inhibitor potency, with IC50 values in the subnanomolar range.
These values are likely to result from a complex inhibition
mechanism, including reversible competition with ATP and
covalent interaction with EGFR in different proportions for
different compounds. We therefore consider these values as
rough measures of the relative propensity to interact with the
target. Methylation of the amide nitrogen of 5 to the tertiary
both the EGFR and erbB2 binding sites of the quinazoline/
quinoline-3-carbonitrile driver portions.
The ability of the compounds to inhibit EGFR autophos-
phorylation was investigated in the A549 human lung cancer
cell line by Western blotting. Percent inhibitions at 1 μM con-
centration are reported in Table 1. Results were compared to
those observed for the reference compounds 3 and 4, recog-
nized as irreversible and reversible EGFR inhibitors, respec-
tively. A549 cells, which express wild-type EGFR, were treated
for 1 h with inhibitor and then washed with drug-free medium.
The degree of EGFR autophosphorylation was measured either
immediately after or 8 h after removal of the inhibitor.16 As
previously reported,54 80% or greater EGFR inhibition 8 h after
removal of the inhibitor from the medium was considered a
sign of irreversible inhibition. Within the A-series of 4-
anilinoquinazolines, the irreversible acrylamide 3 showed 86%
inhibition after the 8 h washout. Substitution of the reactive
acrylamide warhead with 3-(dimethylamino)propanamide in 5,
3-piperidinopropanamide in 6, or 3-morpholinopropanamide in
7 gave similar results on EGFR autophosphorylation, both im-
mediately after and 8 h after washout, with inhibition of EGFR
autophosphorylation over 95% and in the range of 89−93%
at 1 and 8 h, respectively. The 4-methylpiperazine analogue 8
strongly inhibited EGFR autophosphorylation after 1 h (>99%
inhibition), while it showed a slight decrease in inhibitory acti-
vity 8 h after removal from the medium (79% inhibition). The
tertiary amide 9 poorly inhibited EGFR (47% inhibition at 1 h)
with only 10% residual inhibition 8 h after treatment, probably
because of reduced affinity at the recognition site. As expected,
the 6-amino derivative 4 completely inhibited EGFR activity
after 1 h of treatment and did not show any inhibitory effect 8 h
after its removal from the medium.
amide 9 notably reduced EGFR-TK inhibition potency (IC50
=
17 nM). B-series quinazoline compounds 10−13, carrying 4-
(4-fluoro-3-chloroanilino) and 7-ethoxy substituents, did
not show significant changes in kinase inhibition potencies
when the electrophilic warhead in 10 was replaced with 3-
(dimethylamino)propanamide in 11, 3-piperidinopropanamide
in 12, or 3-morpholinopropanamide in 13, all compounds having
subnanomolar IC50 values. Considering the two series
of quinoline-3-carbonitriles C and D, compounds 14−17
(C-series) were only slightly less potent in inhibiting EGFR-
TK compared to the parent B-series quinazoline derivatives,
while compounds 17−21 (D-series), carrying a 3-chloro-4-
(3-fluorobenzyloxy)anilino substituent at position 4 similar to
that of the EGFR/erbB2 inhibitor 2 (Figure 1), were up to 60-
fold less potent than the A- and B-series quinazolines. In view
of the similarity between the D-series compounds (Table 1)
and the EGFR/erbB2 inhibitor 2, the effect of 19 and 20 was
also evaluated on erbB2-TK. Compound 2 is a slowly re-
versible, noncovalent inhibitor of tyrosine kinase that binds the
enzyme in an inactive-like conformation, thus reducing the rate
of inhibitor dissociation, enabling prolonged actions in bio-
logical systems.10 Results of enzyme-based assay for erbB2 in-
hibition by time-resolved fluorometry were compared to that
of the 3-(dimethylamino)propanamide 5, belonging to the
A-series of quinazoline compounds (Supporting Information,
Table S2). As expected, while 5 did not inhibit erbB2 up to
10 μM concentration, compounds 19 and 20 showed IC50
values for erbB2 tyrosine kinase inhibition in the submicro-
molar range (IC50 = 0.97 and 0.98 μM, respectively). These
data show that the 3-aminopropanamide is well tolerated at
We further introduced the 3-aminopropanamido fragment on
quinazoline or quinoline-3-carbonitrile scaffolds with 3-chloro-
4-fluoro substitution on the 4-aniline ring and a 7-ethoxy group
on the heterocyclic nucleus (B- and C-series, Table 1) since
previous work had suggested that this substitution pattern led
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to optimal activity.25,51 The acrylamide derivative 10, belong-
ing to the B-series of quinazolines, completely inhibited
EGFR autophosphorylation 1 and 8 h after treatment at 1 μM
concentration (96% and 99% inhibition, respectively). Sub-
stitution of the electrophilic warhead of 10 with a
3-aminopropanamide side chain led to compounds 11
(dimethylamino), 12 (piperidino), and 13 (morpholino),
which gave EGFR percent inhibition 1 and 8 h after removal
from the medium between 92% and 100%. In the C-series of
compounds, the acrylamide 14 proved efficient in inhibiting
EGFR at 1 μM concentration after 1 h of treatment (92%
inhibition), but produced only partial inhibition 8 h after
washout (33% inhibition). This unexpected behavior may be
due to the reduced solubility of compound 14 in the reaction
medium (data not shown). In fact, among 3-aminopropana-
mides 15−17, only the piperidino derivative 16 gave complete
and irreversible EGFR inhibition (92% and 82% inhibition 1
and 8 h after removal from the medium, respectively), while
dimethylamino (15) and morpholino (17) compounds in-
hibited EGFR at 1 h, but produced only a partial, yet significant,
irreversible inhibition 8 h after treatment, as demonstrated by
67% and 48% inhibition of autophosphorylation, respectively.
Partial inhibition of EGFR autophosphorylation in A459 cells
was also shown by the other series of quinoline-3-carbonitrile
derivatives (D-series, 19−21). In this series, the acrylamide 18
was quite effective as an irreversible inhibitor of EGFR (100%
and 83% inhibition 1 and 8 h after treatment, respectively),
while 3-(dimethylamino)propanamide 19, 3-piperidinopropa-
namide 20, and 3-morpholinopropanamide 21 produced in-
complete EGFR inhibition after 1 h of treatment at 1 μM
concentration (inhibitions between 48% and 71%) with only
partial inhibition 8 h after removal from the medium (19, 59%
inhibition at 8 h; 20, 58% inhibition at 8 h; 21, 39% inhibition
at 8 h).
derivatives (IC50 of 0.7 μM), 11 times more potent than 1 in
the same test.
We further examined the ability of compounds 5 and 20 to
inhibit EGFR autophosphorylation in the NSCLC cell line
H1975 harboring the T790M mutation on EGFR. Results of
Western blot analysis are shown in Figure 4. The compounds
produced dose-dependent inhibition of EGFR autophosphor-
ylation, indicating that the 3-aminopropanamide side chain had
a positive effect on EGFR-T790M inhibition (compound 1 had
marginal activity on EGFR in H1975 cells up to 10 μM).34 In
particular, quinazoline derivative 5 showed an IC50 of 0.07 μM,
and the potent antiproliferative quinoline-3-carbonitrile de-
rivative 20 had an IC50 of 0.15 μM. No correlation was found
between enzyme activities and the ability of these compounds
to inhibit the growth of the H1975 cell line nor between wild-
type EGFR and T790M-mutated EGFR autophosphorylation
inhibition. However, this was not an unexpected result, as
factors other than EGFR-TK inhibiting potency (e.g., cell per-
meability of our compounds) may account for the results
observed.
Reactivity Studies. Compound 5, carrying a 3-
(dimethylamino)propanamidic side chain at position 6 on a
4-(3-bromoanilino)quinazoline nucleus, was chosen as the
prototype of the new series and tested for its chemical (buffered
solutions at pH 7.4 and 9.0) and biological (80% (v/v) rat
plasma) stability, as well as for its reactivity against low molecular
weight (LMW) thiol nucleophiles. Results were compared to
those obtained for the acrylamide 3 (Tables 2 and 3).
Both compounds 3 and 5 were stable at pH 7.4, with per-
centages of remaining compound over 97% after 24 h of
incubation at 37 °C in buffer (Table 2). Under alkaline pH con-
Table 2. In Vitro Stability Studies on Compounds 3 and 5
percentage of compd remaining
Activity on Gefitinib-Resistant H1975 Cells. Com-
pounds 5−21 were evaluated for their ability to inhibit the
growth of the gefitinib-resistant H1975 NSCLC cell line
harboring the T790M mutation by MTT assay. Results were
compared with those of the reference compounds 1, 3, and 4 in
the same assay. As reported in Table 1, the 3-(dimethylamino)-
propanamide 5 showed the best antiproliferative activity on
H1975 cells within the A-series of quinazoline compounds with
an IC50 of 3.7 μM, being 2 times more potent than 1 (IC50 of
8.3 μM)34 and 5-fold more active than 4 (IC50 of 19.5 μM).34
Substitution of the dimethylamino group with a piperidine
made compound 6 equally as potent as 1 (IC50 of 6.7 μM),
while 3-morpholinopropanamide 7, 3-(4-methylpiperazino)-
propanamide 8, and 3-(dimethylamino)-N-methylpropanamide
9 gave IC50 values over 13 μM. B-series 3-aminoamides 11−13
proved 3−4 times more effective than 1 in inhibiting H1975
proliferation and showed IC50 values between 1.6 and 2.2 μM,
in the same range as that of the corresponding irreversible
acrylamide 10 (IC50 of 1.6 μM). Compounds characterized by a
quinoline-3-carbonitrile driving portion (C- and D-series) were
more potent than 1 in inhibiting H1975 cell proliferation. In
particular, 3-aminopropanamides 15−17 (C-series) showed
IC50 values between 2.4 and 4.0 μM, comparable to that of the
corresponding acrylamide derivative 14. Finally, D-series
quinoline-3-carbonitriles 19−21 gave similar results, with the
compounds displaying IC50 values in the low micromolar or
submicromolar range. The 3-piperidinopropanamide derivative
20 proved to have the most potent antiproliferative effect
on H1975 cells within all the series of 3-aminopropanamide
a
(1 μM, 24 h)
conditions
pH 7.4
3
5
102.3 3.1
105.2 3.7
21.8 7.4
97.5 0.1
77.0 0.8
98.7 4.6
pH 9.0
80% (v/v) rat plasma
a
Percentage of parent compound detected by LC−UV and LC−ESI-
MS after incubation for the indicated time at 37 °C (see the
Experimental Section). Mean SD, n = 3.
Table 3. In Vitro Reactivity Studies on Compounds 3 and 5
a
percentage of compd remaining (1 μM, 1 h)
conditions
3
5
DTT
100.0 2.3
63.8 1.9
85.6 3.1
20.1 0.4
99.2 8.1
101.4 2.8
100.4 2.1
100.0 1.1
GSH
NAC
cysteamine
a
Percentage of parent compound detected by LC−UV and LC−ESI-
MS after incubation in the presence of LMW thiols (2 mM) for
the indicated time at pH 7.4, 37 °C (see the Experimental Section).
Mean SD, n = 3.
ditions (pH 9.0), while 3 was stable over the entire incubation
time, 77% of intact 3-aminopropanamide 5 was recovered, with
acrylamide 3 (19%) and amine 4 (4%) as the major degrada-
tion products (see the Supporting Information, Figure S1, for
details). In the presence of rat plasma, compound 5 showed
high stability, while only 22% of acrylamide 3 was detected after
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24 h of incubation. The reactivity of compounds 3 and 5
against thiol nucleophiles was evaluated according to literature
procedures.55,56 While for the acrylamide derivative 3 sig-
nificant formation of conjugates was detected in the presence of
cysteamine, glutathione (GSH), and N-acetylcysteine (NAC)
(Table 3 and Supporting Information, Figure S2), compound 5
gave no measurable adducts with thiol derivatives up to 24 h
of incubation.
Intracellular concentrations of 5, 3, and 4 were measured by
HPLC−ESI-MS in A549 lung cancer cells under the conditions
of the EGFR autophosphorylation assay, i.e., immediately after
or 8 h after incubation for 1 h with the compound at 1 μM
(see the Experimental Section and Supporting Information,
Table S1).57 Both 4 and 5 were detected in the intra-
cellular extracts after incubation (0.105 0.010 and 0.050
0.010 nmol/mg of protein, respectively), while their levels
dropped below the limit of detection (LOD) of the LC−MS
method when measured 8 h after incubation. Intracellular
concentrations of 3 were below the limit of detection both 1
and 8 h after incubation. This result is probably due to the re-
activity of the acrylamide group in 3, interacting covalently with
cellular components.
The reactivity of 5 was also tested in the presence of purified
EGFR-TK by a fluorescence-based assay for evaluation of ir-
reversible kinase inhibition.58,59 Fluorescent molecules are very
sensitive to solvent polarity and dipolar perturbation from their
environments.60,61 Moreover, reversible interactions,62 such as
solvation, hydrogen bonding, charge transfer, and redox, as well
as irreversible interactions,63 such as Michael addition of thiols
to electron-deficient alkenes, significantly influence the fluo-
rescence spectra of fluorophores. In particular, quinazoline
and quinoline fluorophores had been shown to significantly
enhance fluorescence emission after covalent reaction with
Cys797 of EGFR-TK.58 Thus, the 3-aminopropanamide 5 was
added to a buffered solution containing EGFR-TK (see the
Experimental Section and Figure 2A for details), samples were
excited at 390 nm, and fluorescence emission at 420 nm was
monitored over time. The results were compared to those of
the irreversible 3 and the reversible N-(4-(3-bromoanilino)-
quinazolin-6-yl)acetamide64 (46) (Figure 2B). Upon addition
of 3 to EGFR-TK, covalent bond formation with the cysteine
sulfhydryl group resulted in a time-dependent saturable in-
crease in emission intensity at 420 nm, whereas a significantly
lower fluorescence change was observed over 50 min when the
reversible 6-acetyl compound 46 or the 3-aminopropanamide 5
was added to EGFR-TK. This result suggests that 5 did not
covalently react with EGFR under these experimental con-
ditions, behaving more like the acetamide derivative than like
the acrylamide one.
Figure 2. Fluorescence-based assay for irreversible kinase inhibition.
(A) Compounds 3, 5, and 46 (1 μM) were added to purified EGFR-
TK (0.5 μM) in the presence of ATP (1 mM), and fluorescence
emission was monitored at 420 nm in real time over 50 min
(excitation 390 nm). ΔEm420 nm = Emcompd+EGFR solution − Emcompd solution
− EmEGFR solution. (B) Chemical structures of compounds 3, 5, and 46.
Finally, compounds 3 and 5 were tested for their reactivity in
A549 cell lysate. The formation of conjugates with cysteine
added in molar excess (2 mM) was evaluated in cell lysate by
LC−HRMS employing an LTQ-Orbitrap mass analyzer.56
Compound 3 quickly reacted with the thiol derivative to form
the corresponding cysteine conjugate (data not shown). When
compound 5 was added to the cell lysate containing cysteine, a
peak corresponding to the acrylamide derivative 3 was detected
after 1 h ([M + H]+ = 369.03, tR = 8.87; Figure 3A), as well as a
peak corresponding to the adduct of cysteine to the acrylamide
fragment ([M + H]+ = 490.05, tR = 6.61 min; Figure 3B). After
1 h of incubation, 25.5% of the starting acrylamide 3 had been
converted to the corresponding cysteine conjugate, compared
to a 1.1% conversion observed for 5. The concentration of
Figure 3. HPLC−HRMS extracted ion chromatograms of A549 cell
lysate after 1 h of incubation in the presence of compound 5 (10 μM)
and cysteine (2 mM): (A) formed acrylamide derivative (m/z =
369.03, [M + H]+, tR = 8.81 min), (B) acrylamide−cysteine conjugate
(m/z = 490.03, [M + H]+, tR = 6.61 min).
cysteine conjugate doubled for both 3 and 5 at the second time
point (t = 4 h), being 54.4% (3) and 2.0% (5) of the starting
concentrations, respectively. The unique identity of the
conjugate formed by compounds 3 and 5 with cysteine was
further confirmed by high-resolution mass spectrometry
(Supporting Information, Figure S4). On the other hand, the
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We therefore first tested if the long-lasting inhibition observed
for the new 3-aminopropamides of Table 1 could be explained
by their accumulation in cells. The absence of a detectable
amount of 5 in A549 cells after the 8 h period, and the presence
of 1 in the same conditions,57 suggested that 5 is not accumu-
lated in the A549 cell line. Moreover, data from fluorescence-
based assay for irreversible enzyme inhibition (Figure 2) ruled
out direct interaction between the 3-aminopropanamide 5 and
purified EGFR-TK in the chosen time period. On the other
hand, reactivity studies on 5 indicated that the compound re-
generated significant amounts of the acrylamide 3 only in the
presence of cell lysate (Figure 3) while it did not under cell-free
conditions (Tables 2 and 3). The results demonstrate that 5
can act as a prodrug of 3, releasing the acrylamide fragment in
the intracellular environment of A549 cells.
In principle, activation of 3-aminopropanamides to acryl-
amides in the intracellular environment could be affected by
the nature of the heterocyclic nucleus (i.e., quinazoline or
quinoline-3-carbonitrile), since a specific enzymatic trans-
formation is likely to occur. However, the similar behavior of
quinazolines (A- and B-series) and quinoline-3-carbonitriles
(C- and D-series) on EGFR autophosphorylation at 8 h, as well
as previous data on the in vivo activity of Mannich bases (see
the Introduction), suggests that activation of the β-amino-
carbonyl fragment to a Michael acceptor is a rather general
process. In this context, masking the electrophilic warhead
may provide some improvements in the pharmacokinetic or
pharmacodynamic profile of antiproliferative agents. Although
not conclusive evidence of specific advantages, the observation
that some 3-aminopropanamide derivatives in the quinazoline
and quinoline-3-carbonitrile series showed inhibition potencies
on H1975 cell lines close to those of the corresponding acryl-
amides encourages further evaluation of the biological proper-
ties of these compounds.
Figure 4. Effect of compounds 5 and 20 on EGFR autophosphor-
ylation in the H1975 cell line harboring the T790M mutation. (A)
Percent inhibition of EGFR autophosphorylation for compounds
5 ( ) and 20 ( ) on H1975 cells. Results are reported as the mean
SD of three independent experiments. (B) Representative Western
blot analysis for EGFR inhibition by 5 and 20. Analysis was done using
monoclonal antibodies directed to p-Tyr1068 (see the Experimental
Section). Total EGFR is shown as the loading control.
■
●
effect of acrylamide released by the 3-aminopropionamide
derivative can be enhanced by higher persistence of the prodrug
5, which has detectable intracellular levels in A549 cells 1 h
after incubation (see above).
Evidence for Irreversible Binding to EGFR. Mannich
bases are versatile synthetic intermediates used in various trans-
formations to prepare Michael acceptors via elimination of the
amino moiety.65 As reported in the literature, aryl β-aminoethyl
ketones can irreversibly inhibit enzymes by covalent interaction
with cysteine residues38−40 after bioconversion to the corres-
ponding α,β-unsaturated carbonyl compound. The new 3-
aminopropanamides, characterized by a quinazoline (5−7 and
11−13) or quinoline-3-carbonitrile (15−17 and 19−21)
driving portion, showed inhibition of EGFR autophosphor-
ylation in A549 cells after 1 h of incubation at 1 μM con-
centration, and the effect generally persisted up to 8 h after re-
moval of the compounds from the reaction medium (Table 1).
In principle, the long-lasting effect observed on EGFR
autophosphorylation could be ascribed to different phenomena:
(i) accumulation of the inhibitor in cells, as previously demo-
nstrated for some reversible quinazolines;66 (ii) conversion of
the competitive inhibitor into an irreversible one at the active
site of the enzyme (mechanism-based inhibition), as described
for other β-aminocarbonyl compounds;38 (iii) generation of
the corresponding reactive acrylamide, as described for aryl
β-aminoethyl ketones that have potential application as pro-
drugs of unsaturated ketones.67
CONCLUSION
■
We report here a new series of EGFR inhibitors containing a
3-aminopropanamide linked to a 4-anilinoquinazoline (5−7
and 11−13) or 4-anilinoquinoline-3-carbonitrile (15−17 and
19−21) nucleus. The newly synthesized 3-aminopropanamides
proved efficient in inhibiting EGFR-TK activity, showing a
long-lasting effect on the enzyme autophosphorylation in A549
lung cancer cells. Notably, several 3-aminopropanamides sup-
pressed proliferation of gefitinib-resistant NSCLC cells
(H1975) at significantly lower concentration than 1. Further-
more, compounds 5 and 20 blocked mutated (T790M) EGFR-
TK activity on the resistant cellular model.
Finally, a combined approach, based on (i) in vitro chemical
stability assays, (ii) reactivity studies in the presence of thiol
nucleophiles, and (iii) reactivity studies toward EGFR tyrosine
kinase and in the presence of cell lysate, showed that 3-
(dimethylamino)propanamide 5 acts as a prodrug, releasing the
acrylamide derivative 3 in the intracellular environment,
although it is stable in other conditions.
In conclusion, these findings expand the chemical diversity of
irreversible inhibitors of EGFR, and similar strategies might be
applied to the design of compounds able to form a covalent bond
with a peripheral cysteine residue within a biological target.
As previously described,54,66 some reversible quinazoline
EGFR inhibitors are sequestered in cells generating false-
positive results in the autophosphorylation assay based on the
8 h washout protocol. As an example, the fully reversible com-
pound 1, which is strongly sequestered in cells, produced
46.4% 6.7% inhibition of EGFR autophosphorylation (A459
cells) at 1 μM concentration 8 h after removal from the medium.
EXPERIMENTAL SECTION
Reagents were obtained from commercial suppliers and used without
further purification. Solvents were purified and stored according to
standard procedures. Anhydrous reactions were conducted under a
■
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positive pressure of dry N2. Reactions were monitored by TLC, on
Kieselgel 60 F 254 (DC-Alufolien, Merck). Final compounds and
intermediates were purified by flash chromatography (SiO2 60, 40−
63 μm). Microwave reactions were conducted using a CEM Discover
synthesis unit (CEM Corp., Matthews, NC). Melting points were not
corrected and were determined with a Gallenkamp melting point
apparatus. The 1H NMR spectra were recorded on a Bruker 300 MHz
Avance or on a Bruker 400 MHz Avance spectrometer; chemical shifts
(δ scale) are reported in parts per million relative to the central peak of
Silica gel chromatography purification (CH2Cl2/MeOH, 98:2 to
90:10) afforded 9 as a white solid (68%): mp (CH2Cl2/n-hexane)
169−172 °C; MS (APCI) m/z 430.4, 431.4, 432.4; 1H NMR
(CD3OD, 300 MHz) δ 2.14 (s, 6H), 2.39 (br s, 2H), 2.67 (br s, 2H),
3.40 (s, 3H), 7.34 (m, 2H), 7.79 (m, 1H), 7.85 (dd, J = 8.6, 2.3 Hz,
1H), 7.93 (d, J = 8.0 Hz, 1H), 8.17 (s, 1H), 8.42 (s, 1H), 8.66 (s, 1H).
Anal. (C20H22BrN5O) C, H, N.
N-(4-(3-Chloro-4-fluoroanilino)-7-ethoxyquinazolin-6-yl)-
acrylamide (10). Acryloyl chloride (25 μL, 0.3 mmol) in anhydrous
THF (0.5 mL) was added dropwise to a solution of 6-aminoquinazo-
line 34 (100 mg, 0.3 mmol) and N,N-diisopropylethylamine (DIPEA)
(52 μL, 0.3 mmol) in anhydrous DMF (2.5 mL) at 0 °C. The reaction
mixture was stirred for 1 h, then the solvent was removed under re-
duced pressure, and the residue was purified by silica gel chrom-
atography (CH2Cl2/MeOH, 95:5) to afford 10 as a white solid (25%):
1
the solvent. H NMR spectra are reported in the following order:
multiplicity, approximate coupling constant (J value) in hertz, and
number of protons; signals are characterized as s (singlet), d (doublet),
dd (doublet of doublets), t (triplet), dt (doublet of triplets),
q (quartet), m (multiplet), and br s (broad signal). Mass spectra
were recorded using an API 150 EX instrument (AB/SCIEX, Toronto,
Canada). Compounds 1,68 3,54 and 469 were synthesized according to
literature methods. The final compounds were analyzed on a Thermo-
Quest (Italia) FlashEA 1112 elemental analyzer for C, H, and N.
Analyses were within 0.4% of the theoretical values (Supporting
Information, Table S3). All tested compounds were >95% pure by
elemental analysis.
N-(4-(3-Bromoanilino)quinazolin-6-yl)-3-(dimethylamino)-
propanamide (5). A 33% (v/v) solution of dimethylamine in ab-
solute EtOH (0.8 mL, 4.46 mmol) was added over 15 min to a stirred
suspension of N-(4-(3-Bromoanilino)quinazolin-6-yl)-3-chloropropa-
namide (27a) (145 mg, 0.36 mmol) and KI (42 mg, 0.25 mmol) in
absolute EtOH (10 mL), and the resulting mixture was refluxed for
8 h. After being cooled to 0 °C, the mixture was basified with KOH
pellets (0.74 g) and stirred for 1 h at 0 °C. The solvent was evaporated
under reduced pressure, and the solid residue was dissolved with
EtOAc and washed with brine. The organic phase was dried, the
solvent evaporated, and the residue purified by silica gel chromatog-
raphy (CH2Cl2/MeOH, 99:1 to 70:30) to give 5 as a pale yellow solid
(86%): mp (EtOAc/n-hexane) 170−172 °C; MS (atmospheric pres-
sure chemical ionization, APCI) m/z 414.4, 416.4; 1H NMR (CD3OD,
300 MHz) δ 2.35 (s, 6H), 2.65 (t, J = 6.5 Hz, 2H), 2.78 (t, J = 6.5 Hz,
2H), 7.29−7.31 (m, 2H), 7.74 (m, 3H), 8.12 (br s, 1H), 8.53 (s, 1H),
8.66 (br s, 1H). Anal. (C19H20BrN5O) C, H, N.
1
mp (EtOH/H2O) 228.5−230 °C; MS (APCI) m/z 387.3, 389.3; H
NMR (acetone-d6, 400 MHz) δ 1.52 (t, J = 6.9 Hz, 3H), 4.35 (q, J =
6.9 Hz, 2H), 5.83 (dd, J = 10.1, 1.7 Hz, 1H), 6.45 (dd, J = 16.8, 1.7 Hz,
1H), 6.70 (dd, J = 16.8, 10.1 Hz, 1H), 7.29 (s, 1H), 7.32 (t, J = 9.0 Hz,
1H), 7.86 (ddd, J = 9.0, 4.1, 2.8 Hz, 1H), 8.25 (dd, J = 6.8, 2.6 Hz,
1H), 8.57 (s, 1H), 9.04 (s, 1H). Anal. (C19H16ClFN4O2) C, H, N.
N-(4-(3-Chloro-4-fluoroanilino)-7-ethoxyquinazolin-6-yl)-3-
(dimethylamino)propanamide (11). 3-Chloropropanamide 35 was
reacted with dimethylamine according to the procedure described for
compound 5. The product was purified by silica gel chromatography
(AcOEt/MeOH, 99:1 to 85:15) to obtain 11 as a white solid (85%):
1
mp (EtOH/H2O) 182−183 °C; MS (APCI) m/z 432.5, 434.3; H
NMR (CD3OD, 300 MHz) δ 1.54 (t, J = 7.0 Hz, 3H), 2.37 (s, 6H),
2.69 (m, 4H), 4.26 (q, J = 7.0 Hz, 2H), 7.11 (s, 1H), 7.21 (t, J =
9.0 Hz, 1H), 7.62 (ddd, J = 9.0, 4.1, 2.7 Hz, 1H), 7.95 (dd, J = 6.7,
2.6 Hz, 1H), 8.41 (s, 1H), 8.83 (s, 1H). Anal. (C21H23ClFN5O2·2/3H2O)
C, H, N.
N-(4-(3-Chloro-4-fluoroanilino)-7-ethoxyquinazolin-6-yl)-3-
piperidin-1-ylpropanamide (12). 3-Chloropropanamide 35 was
reacted with anhydrous piperidine according to the procedure
described for compound 5. The product was purified by silica gel
chromatography (AcOEt/MeOH, 99:1 to 93:7) to give 12 as a white
solid (60%): mp (EtOH/H2O) 106.5−108 °C; MS (APCI) m/z
1
472.2, 474.2; H NMR (DMSO-d6, 400 MHz) δ 1.54 (t, J = 6.9 Hz,
N-(4-(3-Bromoanilino)quinazolin-6-yl)-3-piperidin-1-ylpro-
panamide (6). 27a was reacted with anhydrous piperidine according
to the procedure described for compound 5. The product was purified
by silica gel chromatography (CH2Cl2/MeOH, 95:5) to give 6 as a
white solid (78%): mp (Et2O) 184−186 °C; MS (APCI) m/z 454.1,
5H), 1.68 (4H, m), 2.56 (s, 4H), 2.75 (m, 4H), 4.38 (q, J = 6.9 Hz,
2H), 7.22 (s, 1H), 7.25 (t, J = 8.9 Hz, 1H), 7.65 (ddd, J = 9.0, 3.7,
2.8 Hz, 1H), 7.99 (dd, J = 6.6, 2.5 Hz, 1H), 8.47 (s, 1H), 8.83 (s, 1H).
Anal. (C24H27ClFN5O2·H2O) C, H, N.
N-(4-((3-Chloro-4-fluorophenyl)amino)-7-ethoxyquinazolin-
6-yl)-3-morpholinopropanamide (13). 3-Chloropropanamide 35
was reacted with morpholine according to the procedure described for
compound 5. The product was purified by silica gel chromatography
(AcOEt/MeOH, 99:1 to 96:4) to obtain 13 as a light yellow solid
(96%): mp (EtOH/H2O) 108−110 °C; MS (APCI) 474.0, 476.3 °C;
1H NMR (DMSO-d6, 400 MHz) δ 1.54 (t, J = 7.0 Hz, 3H), 2.60 (m,
1
456.2; H NMR (DMSO-d6, 300 MHz) δ 1.61 (m, 2H), 1.74−1.82
(m, 10H), 2.68−2.73 (m, 2H), 7.18−7.32 (m, 3H), 7.67 (d, J = 6.8
Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.97 (s, 1H), 8.08 (s, 1H), 8.71
(s, 1H), 8.89 (s, 1H), 12.04 (s, 1H). Anal. (C22H24BrN5O·3/4H2O) C,
H, N.
N-(4-(3-Bromoanilino)quinazolin-6-yl)-3-morpholinopropa-
namide (7). 27a was reacted with morpholine according to the
procedure described for compound 5. The product was purified by
silica gel chromatography (CH2Cl2/MeOH, 95:5) to give 7 as a yellow
solid (70%): mp (Et2O/n-hexane) 196−198 °C; MS (APCI) m/z
456.2, 458.4; 1H NMR (CDCl3, 300 MHz) δ 2.59−2.77 (m, 8H), 3.89
(m, 4H), 7.17−7.25 (m, 3H), 7.62 (d, J = 7.4 Hz, 1H), 7.76 (d, J = 8.9
Hz, 1H), 7.90 (s, 1H), 8.16 (br s, 1H), 8.67 (s, 1H), 8.93 (d, J = 1.9
Hz, 1H), 11.40 (s, 1H). Anal. (C21H22BrN5O2·1/3H2O) C, H, N.
N-(4-(3-Bromoanilino)quinazolin-6-yl)-3-(4-methylpipera-
zin-1-yl)propanamide (8). 27a was reacted with N-methylpiperazine
according to the procedure described for compound 5. The product
was purified by silica gel chromatography (CH2Cl2/MeOH, 95:5) to
give 8 as a white solid (77%): mp (Et2O) 196 °C; MS (APCI) m/z
469.3, 471.3; 1H NMR (CDCl3, 300 MHz) δ 2.33 (s, 3H), 2.42−2.49
(m, 12H), 6.94−7.07 (m, 3H), 7.44−7.51 (m, 2H), 7.58 (s, 1H),
8.50 (s, 1H), 8.68 (m, 1H), 8.88 (s, 1H), 11.55 (s, 1H). Anal.
(C22H25BrN6O·1/2H2O) C, H, N.
4H), 2.74 (t, J = 6.0, 2H), 2.81 (t, J = 5.8 Hz, 2H), 3.77 (t, J = 4.6 Hz,
4H), 4.37−4.41 (m, 2H), 7.18−7.26 (m, 2H), 7.63−7.65 (m, 1H),
7.97−7.99 (m, 1H), 8.46 (d, J = 2.9 Hz, 1H), 8.83 (d, J = 4.7 Hz, 1H).
Anal. (C23H25ClFN5O3·2/3H2O) C, H, N.
N-(4-(3-Chloro-4-fluoroanilino)-3-cyano-7-ethoxyquinolin-
6-yl)acrylamide (14). The product was obtained by coupling the
amino intermediate 42a with acryloyl chloride as described for com-
pound 10. Silica gel chromatography purification (EtOAc/n-hexane,
60:40) afforded 14 (40%) as a white solid: mp (EtOH/H2O) >
1
230 °C; MS (APCI) m/z 411.0; H NMR (DMSO-d6, 300 MHz) δ
1.48 (t, J = 6.9 Hz, 3H), 4.33 (q, J = 6.9 Hz, 2H), 5.81 (d, J = 10.7 Hz,
1H), 6.29 (d, J = 16.5 Hz, 1H), 6.77 (dd, J = 16.5, 9.7 Hz, 1H), 7.25
(m, 1H), 7.41 (m, 3H), 8.53 (s, 1H), 8.98 (s, 1H), 9.60 (s, 1H), 9.75
(br s, 1H). Anal. (C21H16ClFN4O2·1/2H2O) C, H, N.
N-(4-(3-Chloro-4-fluoroanilino)-3-cyano-7-ethoxyquinolin-
6-yl)-3-(dimethylamino)propanamide (15). 3-Chloropropana-
mide 43a was reacted with dimethylamine according to the pro-
cedure described for compound 5. Silica gel chromatography (CH2Cl2/
MeOH, 99:1 to 97:3) gave 15 as a light yellow solid (69%):
N-(4-(3-Bromophenylamino)quinazolin-6-yl)-3-(dimethyla-
mino)-N-methylpropanamide (9). N-(4-((3-Bromophenyl)amino)-
quinazolin-6-yl)-3-chloro-N-methylpropanamide (27b) was reacted with
dimethylamine according to the procedure described for compound 5.
1
mp (EtOH) 168−171 °C; H NMR (DMSO-d6, 300 MHz) δ 1.48
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(t, J = 7.0 Hz, 3H), 2.29 (s, 6H), 2.57 (m, 4H), 4.32 (q, J = 7.0 Hz,
2H), 7.19 (m, 1H), 7.40 (m, 3H), 8.54 (s, 1H), 9.06 (s, 1H), 9.68
(br s, 1H), 10.98 (s, 1H). Anal. (C23H23ClFN5O2·H2O) C, H, N.
N-(4-(3-Chloro-4-fluoroanilino)-3-cyano-7-ethoxyquinolin-
6-yl)-3-piperidin-1-ylpropanamide (16). 3-Chloropropanamide
43a was reacted with piperidine according to the procedure described
for compound 5. Silica gel chromatography (CH2Cl2/MeOH, 96:4)
gave 16 as a white solid (45%): mp (EtOH) 180−184 °C; MS (APCI)
In Vitro Stability and Reactivity Assays. Chemical (pH 7.4, pH
9.0) and rat plasma stability was investigated at 37 °C according to
procedures reported previously.70 (See the Supporting Information for
details.) The reactivity of 3 and 5 toward cysteamine, NAC, dithio-
threitol (DTT), and reduced GSH was evaluated by adding a freshly
prepared thiol solution (2 mM) to PBS (pH 7.4) containing the test
compound (1 μM). Formation of conjugates with LMW thiols after
1 h of incubation at 37 °C was measured by HPLC−UV and HPLC−
ESI-MS. The reactivity of compounds 3 and 5 in A549 cell lysate was
tested in the presence of cysteine, to trap thiol-reactive compounds.
A549 cells were seeded at 1 000 000 cells/dish (66 cm2); after 2 days,
the cells were lysed mechanically by the freeze/thaw method. Stock
solutions (1 mM) of 3 and 5 were prepared in DMSO immediately
before use and added to cell lysate at a final concentration of 10 μM
(equal percentage of DMSO in controls), where 2 mM cysteine had
been previously added. Incubations were carried out at 37 °C. At
stated time points (t = 0, 1, and 4 h) aliquots were sampled, a double
volume of acetonitrile was added, and the resulting solutions were
centrifuged (4 °C, 10000g, 10 min) and directly analyzed by LC−
HRMS employing an LTQ-Orbitrap mass analyzer, working in
positive ion mode (PIM). (See the Supporting Information for
details.)
Determination of Intracellular Concentrations. A549 cells
were plated at a 1 000 000 cells/dish (25 cm2) density. After 24 h, test
compounds (3−5) were added to the medium (final concentration
1 μM, final DMSO concentration 0.1%). the cells were incubated for
1 h at 37 °C and tested either immediately after or 8 h after removal of
the compound from the extracellular medium by washing the cells
three times with 1 mL aliquots of fresh medium. Compounds were
then extracted from the cells with absolute EtOH (1.1 mL at 4 °C),
and the cell extracts were centrifuged (4 °C, 10000g, 5 min) and
collected. A fixed volume of ethanolic extract was evaporated to
dryness, dissolved in HPLC eluent, and injected into the LC−MS sys-
tem. Calibration curves were built in the concentration range from
5 μM to 50 nM for compounds 3−5 (limit of quantification (LOQ) of
50 nM, LOD = 20 nM). Cell proteins were quantified after solu-
bilization in NaOH (0.5 N) (2 mL/25 cm2 dish) by the Bradford
method.71
1
m/z 496.4, 497.3, 499.4; H NMR (DMSO-d6, 400 MHz) δ 1.46 (t,
J = 7.0 Hz, 3H); 1.49 (m, 2H), 1.56 (m, 4H), 2.44 (m, 4H), 2.60 (s,
4H), 4.39 (t, J = 7.0 Hz, 2H), 7.22 (m, 1H), 7.41 (m, 3H), 8.54
(s, 1H), 8.97 (s, 1H), 9.68 (br s, 1H), 10.24 (s, 1H). Anal.
(C26H27ClFN5O2·1/2C2H6O) C, H, N.
N-(4-(3-Chloro-4-fluoroanilino)-3-cyano-7-ethoxyquinolin-
6-yl)-3-morpholinopropanamide (17). 3-Chloropropanamide 43a
was reacted with morpholine according to the procedure described for
compound 5. Silica gel chromatography (CH2Cl2/MeOH, 98:2)
afforded 17 as a white solid (52%): mp (EtOH/H2O) 195−198 °C;
MS (APCI) m/z 498.3, 499.2, 500.3; 1H NMR (DMSO-d6, 400 MHz)
δ 1.45 (t, J = 7.0 Hz, 3H), 2.46 (m, 4H), 2.63 (m, 4H), 3.62 (t, J = 4.2
Hz, 4H), 4.36 (q, J = 7.1 Hz, 2H), 7.20 (br s, 1H), 7.39 (m, 3H), 8.53
(br s, 1H), 8.94 (s, 1H), 9.69 (br s, 1H), 9.89 (s,1H). Anal.
(C25H25ClFN5O3·1/2C2H6O) C, H, N.
N-(4-(3-Chloro-4-(pyridin-2-ylmethoxy)anilino)-3-cyano-7-
ethoxyquinolin-6-yl)acrylamide (18). The product was obtained
by coupling the amino intermediate 42b with acrylic acid as described
for compound 10. Silica gel chromatography purification (CH2Cl2/
MeOH, 98:2) gave 18 as a white solid (35%): mp (EtOH/H2O) > 230 °C;
1
MS (APCI) m/z 500.1; H NMR (DMSO-d6, 300 MHz) δ 1.53 (t,
J = 6.9 Hz, 3H), 4.39 (q, J = 6.9 Hz, 2H), 5.32 (s, 2H); 5.79 (dd, J =
10.1, 1.9 Hz, 1H), 6.39 (dd, J = 16.9, 1.9 Hz, 1H), 6.68 (dd, J = 16.9,
10.1 Hz, 1H), 7.30 (m, 3H), 7.41 (s, 1H), 7.48 (d, J = 2.3 Hz, 1H),
7.68 (d, J = 7.8 Hz, 1H), 7.87 (td, J = 7.8, 1.8 Hz, 1H), 8.50 (s, 1H),
8.60 (d, J = 4.5 Hz, 1H), 9.05 (s, 2H), 9.16 (s, 1H). Anal.
(C27H22ClN5O3) C, H, N.
N-(4-(3-Chloro-4-(pyridin-2-ylmethoxy)anilino)-3-cyano-7-
ethoxyquinolin-6-yl)-3-(dimethylamino)propanamide (19). 3-
Chloropropanamide 43b was reacted with dimethylamine according to
the procedure described for compound 5. Silica gel chromatography
(CH2Cl2/MeOH, 99:1 to 90:10) gave 19 as a white solid (73%): mp
(EtOH) 235−237 °C; MS (APCI) m/z 545.4, 547.3, 502.7, 500.2; 1H
NMR (DMSO-d6, 300 MHz) δ 2.29 (s, 6H), 2.54−2.61 (m, 4H), 4.31
(q, J = 7.0 Hz, 2H), 5.28 (s, 2H), 7.17 (dd, J = 7.7, 1.7 Hz, 1H), 7.25
(d, J = 8.8 Hz, 1H), 7.38−7.40 (m, 3H), 7.59 (d, J = 7.9 Hz, 1H), 7.88
(dt, J = 7.7, 1.7 Hz, 1H), 8.47 (s, 1H), 8.60 (d, J = 4.0 Hz, 1H), 9.06
(s, 1H), 9.58 (br s, 1H), 10.92 (br s, 1H). Anal.
(C29H29ClN6O3·1/2H2O) C, H, N.
Analytical Method. A linear gradient elution was set up to
evaluate in the same chromatographic run the disappearance of com-
pounds 3 and 5 and the formation of corresponding degradation
products. An Agilent 1100 HPLC gradient system (Agilent Tech-
nologies, Santa Clara, CA) with an RP-C18 Supelco Discovery
(Supelco, Bellefonte, PA), 5 μm, 150 × 4.6 mm i.d. column and an
AB/SCIEX API 150-EX single-quadrupole mass spectrometer equip-
ped with an ESI ion source acquiring in positive ion mode were
employed. (See the Supporting Information for details.)
N-(4-(3-Chloro-4-(pyridin-2-ylmethoxy)anilino)-3-cyano-7-
ethoxyquinolin-6-yl)-3-piperidin-1-ylpropanamide (20). 3-
Chloropropanamide 43b was reacted with piperidine according to
the procedure described for compound 5. Silica gel chromatography
(CH2Cl2/MeOH, 97:3 to 95:5) gave 20 as a light yellow solid (69%):
Real-Time Detection of Covalent Bond Formation in
Microtiter Plates. The assay was carried out in black 384-well
small-volume plates, and fluorescence emission was measured over
time by using a multilabel plate reader (Victor, PerkinElmer), as pre-
viously described.58 EGFR-TK was purchased from Sigma-Aldrich.
The sample wells were excited at 390 nm, and the changing emission
intensity was monitored at 420 nm. The reaction was monitored im-
mediately after combination of equal volumes (7 μL) of EGFR-TK
(0.5 μM) in buffer (50 mM Hepes, 200 mM NaCl, 0.05% Triton-
X100, pH 7.4) containing ATP (1 mM) and inhibitor (1 μM, 0.2%
1
mp (EtOH) 190−193 °C; MS (APCI) m/z 585.2, 587.5, 588.5; H
NMR (DMSO-d6, 400 MHz) δ 1.45 (t, J = 6.8 Hz, 3H), 1.50 (m, 2H),
1.56 (m, 4H), 2.44 (m, 4H), 2.60 (s, 4H), 4.36 (q, J = 7.0 Hz, 2H),
5.28 (s, 2H), 7.18 (d, J = 8.2 Hz, 1H), 7.25 (d, J = 8.4 Hz, 1H), 7.39
(m, 3H), 7.58 (d, J = 7.2 Hz, 1H), 7.88 (t, J = 7.3 Hz, 1H), 8.47
(s, 1H), 8.60 (d, J = 4.3 Hz, 1H), 8.95 (s, 1H), 9.59 (s, 1H), 10.21 (s,
1H). Anal. (C32H33ClN6O3·3/2H2O) C, H, N.
DMSO) solution in each well. Data were reported as ΔEm420
,
nm
calculated as the difference in emission intensity between the samples
(inhibitor + EGFR) and controls (inhibitor in buffer solution and
EGFR in buffer solution).
N-(4-(3-Chloro-4-(pyridin-2-ylmethoxy)anilino)-3-cyano-7-
ethoxyquinolin-6-yl)-3-morpholinopropanamide (21). 3-Chlo-
ropropanamide 43b was reacted with morpholine according to the
procedure described for compound 5. Silica gel chromatography
(CH2Cl2/MeOH, 99:1 to 97:3) gave 21 as a light yellow solid (74%):
Kinase Assay. Evaluation of the effects of compounds on the
kinase activity of human EGFR was performed by measuring the phos-
phorylation of the substrate Ulight-CAGAGAIETDKEYYTVKD (JAK1)
using a human recombinant enzyme expressed in insect cells72 and the
LANCE detection method,73 employing the Cerep EGFR kinase assay74
as previously described.34
Cell Culture. The human NSCLC cell lines A549 and H1975 were
cultured as recommended. Medium was supplemented with L-glutamine
(2 mmol/L) and fetal calf serum (FCS) (10%), and the cells were
1
mp (EtOH) 224−228 °C; MS (APCI) m/z 587.3, 589.5; H NMR
(CDCl3, 400 MHz) δ 1.49 (t, J = 6.9 Hz, 3H), 2.55 (m, 4H), 2.67 (m,
4H), 3.77 (s, 4H), 4.30 (q, J = 6.8 Hz, 2H), 5.19 (s, 2H), 6.72 (s, 2H),
7.61 (d, J = 7.8 Hz, 2H), 7.75 (t, J = 7.7 Hz, 1H), 8.16 (s, 1H), 8.32
(s, 1H), 8.58 (d, J = 4.4 Hz, 1H), 9.19 (s,1H), 10.08 (s, 1H). Anal.
(C31H31ClN6O4) C, H, N.
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Journal of Medicinal Chemistry
Article
maintained under standard cell culture conditions at 37 °C in a water-
saturated atmosphere of 5% CO2 in air. The A549 cell line was from the
American Type Culture Collection (Manassas, VA), and H1975 was
kindly provided by Dr. E. Giovannetti (Department of Medical Oncology,
VU University Medical Center, Amsterdam, The Netherlands).
Antibodies and Reagents. Medium was from Euroclone, and fetal
bovine serum (FBS) was purchased from Gibco-BRL (Grand Island,
NY). Monoclonal anti-EGFR and polyclonal anti-phospho-EGFR
(Tyr1068) antibodies were from Cell Signaling Technology (Beverly,
MA). Horseradish peroxidase (HRP)-conjugated secondary antibodies
were from Pierce. The enhanced chemiluminescence (ECL) system was
from Millipore (Millipore, MA). Reagents for electrophoresis and blotting
analysis were obtained from, respectively, BIO-RAD Laboratories and
Millipore.
Forensic Medicine at the University of Parma) for support in
the fluorescence-based kinase assay.
ABBREVIATIONS USED
■
EGFR, epidermal growth factor receptor; erbB2, human
epidermal growth factor receptor 2; TK, tyrosine kinase;
ATP, adenosine triphosphate; NSCLC, non-small-cell lung
cancer; GSH, glutathione; NAC, N-acetylcysteine; DTT,
dithiothreitol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide; Red-Al, sodium bis(2-methoxye-
thoxy)aluminum hydride; DIPEA, N,N-diisopropylethyl-
amine; PBS, phosphate-buffered saline
Western Blot Analysis. Procedures for protein extraction,
solubilization, and protein analysis by 1-D polyacrylamide gel electro-
phoresis (PAGE) are described elsewhere.75 Samples of 40−50 μg of
proteins from lysates were resolved by 7.5% sodium dodecyl sulfate
(SDS)−PAGE and transferred to poly(vinylidene fluoride) (PVDF)
membranes (Millipore). The membranes were then incubated with
primary antibody, washed, and then incubated with HRP−antirabbit
antibody. Immunoreactive bands were visualized using an enhanced
chemiluminescence system.
Autophosphorylation Assay. Inhibition of EGFR autophosphor-
ylation was determined as previously described using specific anti-
phosphotyrosine and anti total EGFR antibodies by Western blot
analysis.34
Cell Growth Inhibition. Cell viability was assessed after 3 days of
treatment by tetrazolium dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT), Sigma, Dorset, U.K.] assay as pre-
viously described.75 Representative results of at least three independent
experiments were used for evaluation of dose−response curves,
calculated from experimental points using Graph Pad Prism5 software.
The concentration that inhibits 50% (IC50) (e.g., the point at which
viability is 50%) was extrapolated from the dose−response curves.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures for compounds 25−27a,b, 29−35,
41b, 42b, 43a,b, and 45, combustion analytical data for com-
pounds 3−21, experimental procedures for in vitro stability
assays and HPLC−ESI-MS analysis on compounds 3−5,
HPLC−MS chromatogram of 5 incubated at pH 9.0 for 24
h, HPLC−MS chromatograms of 3 incubated in the presence
of LMW thiols, HR−MS chromatograms of conjugates formed
by 3 and 5 with cysteine in A549 cell lysate, quantification of
the intracellular concentration of compounds 3−5 in A549
cells, and ErbB2 tyrosine kinase inhibition for compounds 3, 5,
19, and 20. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
(11) Lynch, T. J.; Bell, D. W.; Sordella, R.; Gurubhagavatula, S.;
Okimoto, R. A.; Brannigan, B. W.; Harris, P. L.; Haserlat, S. M.; Supko,
J. G.; Haluska, F. G.; Louis, D. N.; Christiani, D. C.; Settleman, J.;
Haber, D. A. Activating mutations in the epidermal growth factor
receptor underlying responsiveness of non-small-cell lung cancer to
gefitinib. N. Engl. J. Med. 2004, 350, 2129−2139.
*Phone: +39 0521 905059. Fax: +39 0521 905006. E-mail:
marco.mor@unipr.it.
Notes
The authors declare no competing financial interest.
(12) Kobayashi, S.; Boggon, T. J.; Dayaram, T.; Janne, P. A.; Kocher,
̈
ACKNOWLEDGMENTS
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(13) Pao, W.; Miller, V. A.; Politi, K. A.; Riely, G. J.; Somwar, R.;
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or erlotinib. PLoS Med. 2005, 2, e17.
Grant support for this work was from the Ministero della Salute
(Programma straordinario di ricerca oncologica 2006), Regione
Emilia Romagna, AIRC (Associazione Italiana per la Ricerca sul
Cancro), Associazione Chiara Tassoni, Parma, and Associa-
zione Davide Rodella, Montichiari BS. We thank Prof. Prisco
Mirandola (Department of Anatomy, Pharmacology and
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