Novel irreversible epidermal growth factor receptor inhibitors by chemical modulation of the cysteine-trap portion

Article abstract of DOI:10.1021/jm901558p

Irreversible EGFR inhibitors can circumvent acquired resistance to first-generation reversible, ATPcompetitive inhibitors in the treatment of non-small-cell lung cancer. They contain both a driver group, which assures target recognition, and a warhead, generally an acrylamide or propargylamide fragment that binds covalently to Cys797 within the kinase domain of EGFR. We performed a systematic exploration of the role for the warhead group, introducing different cysteine-trapping fragments at position 6 of a traditional 4-anilinoquinazoline scaffold. We found that different reactive groups, including epoxyamides (compounds 3-6) and phenoxyacetamides (compounds 7-9), were able to irreversibly inhibit EGFR. In particular, at significant lower concentrations than gefitinib (1), (2R,3R)N-(4-(3-bromoanilino)quinazolin-6-yl)- 3 -(piperidin-1 -ylmethyl)oxirane-2-carboxamide (6) inhibited EGFR autophosphorylation and downstream signaling pathways, suppressed proliferation, and induced apoptosis in gefitinib-resistant NSCLC H1975 cells, harboring the T790M mutation in EGFR.

Full text of DOI:10.1021/jm901558p

2038 J. Med. Chem. 2010, 53, 2038–2050
DOI: 10.1021/jm901558p
Novel Irreversible Epidermal Growth Factor Receptor Inhibitors by Chemical Modulation of the
Cysteine-Trap Portion
Caterina Carmi,† Andrea Cavazzoni,‡ Stefano Vezzosi,† Fabrizio Bordi,† Federica Vacondio,† Claudia Silva,†
Silvia Rivara,† Alessio Lodola,*,† Roberta R. Alfieri,‡ Silvia La Monica,‡ Maricla Galetti,‡ 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,
Dipartimento di Medicina Sperimentale, Universita degli Studi di Parma, Via Volturno 39, I-43125 Parma, Italy, and
‡
ꢀ
§Oncologia Medica, Azienda Ospedaliero—Universitaria di Parma, V.le Gramsci 14, I-43126 Parma, Italy
Received October 21, 2009
Irreversible EGFR inhibitors can circumvent acquired resistance to first-generation reversible, ATP-
competitive inhibitors in the treatment of non-small-cell lung cancer. They contain both a driver group,
which assures target recognition, and a warhead, generally an acrylamide or propargylamide fragment
that binds covalently to Cys797 within the kinase domain of EGFR. We performed a systematic
exploration of the role for the warhead group, introducing different cysteine-trapping fragments at
position 6 of a traditional 4-anilinoquinazoline scaffold. We found that different reactive groups,
including epoxyamides (compounds 3-6) and phenoxyacetamides (compounds 7-9), were able to
irreversibly inhibit EGFR. In particular, at significant lower concentrations than gefitinib (1), (2R,3R)-
N-(4-(3-bromoanilino)quinazolin-6-yl)-3-(piperidin-1-ylmethyl)oxirane-2-carboxamide (6) inhibited
EGFR autophosphorylation and downstream signaling pathways, suppressed proliferation, and
induced apoptosis in gefitinib-resistant NSCLC H1975 cells, harboring the T790M mutation in EGFR.
Introduction
The epidermal growth factor receptor (EGFRa) is a
validated target in cancer treatment strategies. EGFR is a
member of the erbB receptor family (erbB1/EGFR, erbB2/
HER2, erbB3/HER3, and erbB4/HER4) of tyrosine kinase
(TK) that is overexpressed in several human solid tumors and
is often associated with more aggressive disease and poorer
clinical outcome.1,2 Inhibition of the erbB-family receptor
tyrosine kinases represented a major advance in the treatment
of solid tumors, as demonstrated by the promising clinical
activity of the EGFR tyrosine kinase inhibitors 13 (gefitinib,
Figure 1) and N-4-(3-ethynylanilino)-6,7-bis(2-methoxyeth-
oxy)quinazoline4 (erlotinib, Figure 1). These 4-anilinoquina-
zoline tyrosine kinase inhibitors compete with ATP in a
reversible manner, binding to the kinase domain of the target.
Although these drugs have been extremely effective in specific
patient populations with tumor-containing mutated onco-
genic forms of tyrosine kinases,5 the accumulating clinical
experienceonnon-small-celllung cancers(NSCLCs) indicates
that most patients develop resistance.6 In approximately half
of NSCLC cases that showed an initial response to reversible
*To whom correspondence should be addressed. Phone: þ39 0521
905062. Fax: þ39 0521 905006. E-mail: alessio.lodola@unipr.it.
a Abbreviations: EGFR, epidermal growth factor receptor; erbB2,
human epidermal growth factor receptor 2; TK, tyrosine kinase;
NSCLC, non-small-cell lung cancer; IGF-1R, insulin-like growth factor
1 receptor; PI3K, phosphoinositide-3 kinase; mTOR, mammalian target
of rapamycin; MAPK, mitogen activated protein kinase; GSH, glu-
tathione; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; HBTU, O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
hexafluorophosphate; DCC, N,N0-dicyclohexylcarbodiimide; rmsd,
root-mean-square deviation.
Figure 1. Chemical structures of reversible and irreversible EGFR
inhibitors.
EGFR tyrosine kinase inhibitors and subsequently pro-
gressed, resistance was associated with the emergence of a
secondary mutation within the EGFR kinase domain: sub-
stitution of threonine 790 with methionine (T790M).6-8
Threonine 790 is the gatekeeper residue in EGFR; its key
r
pubs.acs.org/jmc
Published on Web 02/12/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2039
Scheme 1. Synthesis of Compounds 3-14a
location at the entrance of a hydrophobic pocket in the back
of the ATP binding cleft makes it an important determinant of
inhibitor specificity in protein kinases. Substitution of this
residue in EGFR with the bulkier methionine causes resis-
tance to tyrosine kinase reversible inhibitors, including gefiti-
nib and erlotinib, and this had been attributed to an increased
enzyme affinity for ATP.9 Additional mechanisms of resis-
tanceto reversible EGFR inhibitors have been described, such
as the activation of alternative tyrosine kinase receptors (IGF-
1R), amplification of the MET gene, and constitutive activa-
tion of signaling pathways downstream of EGFR.10,11
A second generation of 4-anilinoquinazolines and 4-anili-
no-3-cyanoquinolines12-14 carryinga Michaelacceptor group
at the 6-position, such as an acrylamide or propargylamide
fragment, that irreversibly alkylates a cysteine residue
(Cys797) close to the ATP binding site of EGFR15 has been
showntoovercome resistance.16-18 Several of theseinhibitors,
such as N-[4-(3-chloro-4-fluoroanilino)-7-[3-(4-morpholinyl)-
propoxy]quinazolin-6-yl]-2-propenamide19 (CI-1033, canertinib,
Figure 1) and (E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)-
anilino]-3-cyano-7-ethoxyquinolin-6-yl}-4-(dimethylamino)-
2-butenamide20 (HKI-272, neratinib, Figure 1), are currently
undergoing clinical testing in patients that initially responded
to erlotinib and gefitinib and subsequently relapsed.21 Irre-
versible kinase inhibitors possess several advantages over
conventional reversible ATP-competitive inhibitors. Consid-
ering that the targeted cysteine residue is conserved within the
erbB-receptor family (Cys797 inEGFR, Cys805 in erbB2, and
Cys803 in erbB4) and that only a limited group of kinases has
a cysteine at thecorresponding position, irreversibleinhibitors
are highly selective for erbB-family TK. Furthermore, cova-
lent bond formation can circumvent competition with ATP,
which under physiological conditions is in the millimolar
range. On the other hand, the intrinsic reactivity of cysteine-
reactive groups can incur augmented metabolic degradation,
toxicity, or lack of target specificity. Despite the potential
advantages of covalent binding kinase inhibitors, a draw-
back is that they might indiscriminately react with non-target-
related proteins, giving rise to increased and unexpected
toxicity.
EGFR tyrosine kinase irreversible inhibitors reported in
the literature are mostly characterized by a 4-anilinoquina-
zoline or 4-anilino-3-cyanoquinoline core structure (driver
group) carrying a reactive center (warhead) able to cova-
lently interact with the target protein by Michael addition or
alkylation with the conserved, solvent exposed cysteine
residue present in the enzyme. However, only a limited
number of reactive warheads has been considered so far,
such as acrylamide, propargylamide, vinylsulphonamide,
chloroacetamide, and lipoic acid derivatives.22-27 A more
systematic structure-activity relationship study on different
cysteine-reactive groups is required to provide new chemical
starting points for further optimization of the efficacy/
toxicity ratio.
To date, many warheads have been integrated as reactive
fragments into large collections of molecules that exhibit a
wide spectrum of biological activities.28,29 All of them estab-
lish covalent, reversible, or irreversible interactions with
nucleophilic residues exposed on the protein surface or into
inner protein cavities. There are a variety of drugs, either
approved or in clinical development, that covalently bind
therapeutic targets, including ones that target cysteine pro-
tease, such as cathepsin and caspase,30 MMP13,31 thyroid
hormone receptor,32 and FAAH.33
a (i) H2SO4, formic acid, reflux; (ii) SOCl2, dioxane, reflux; (iii) 3-
bromoaniline, i-PrOH, 60 °C; (iv) Fe, AcOH, EtOH/H2O, reflux. (v)
Method A: dichloromethylene dimethyliminium chloride, NaHCO3, 17
(for 4) or 18 (for 5), CH2Cl2, 0 °C. Method B: HBTU, DMF, 19, room
temp (for 6). Method C: PCl5, 20 (for 7) or 21 (for 8) or 22 (for 9) or 24
(for 11), CH2Cl2, reflux. Method D: DCC, 23 (for 10) or 25 (for 12) or 26
(for 13), DMF, 0 °C to room temp. Method E: t-ButOK, 27 (for 14),
DMF, microwave, 100 °C.
Considering the acrylamide moiety of 6-acrylamide-4-(3-
bromoanilino)quinazoline34 (2, PD168393, Figure 1) as a
reference warhead fragment, we explored the role of different
reactive groups that covalently interact with cysteine residues
when inserted on the 6-amino-4-(3-bromoanilino)quinazoline
driver portion35 (3, Figure 1). Combining knowledge from
existing drugs and late-stage clinical candidates that modify
their target proteins through a covalent interaction, as well as
from known EGFR tyrosine kinase inhibitors, we designed
new irreversible EGFR inhibitors 4-14 carrying different
cysteine-reactive groups at position 6 (Scheme 1 and Table 1).
In particular, we linked the 6-amino-4-(3-bromoanili-
no)quinazoline driving portion of 5 with a number of war-
heads characterized by different reaction mechanisms toward
nucleophiles: (i) nucleophilic addition (epoxides 4, 5, and 6);
(ii) nucleophilic substitution with in situ release of a leaving

2040 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Carmi et al.
Table 1. EGFR Tyrosine Kinase and Autophosphorylation Inhibition in A431 Cells
a Concentration that inhibits by 50% EGFR tyrosine kinase activity. IC50 values were measured by the phosphorylation of a peptide substrate using
time-resolved fluorometry (see Experimental Section). Mean values of three independent experiments ( SEM are reported. b Inhibition of EGFR
autophosphorylation was measured in A431 intact cells by Western blot analysis. Percent inhibition at 1 μM and IC50 values were 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
( SEM are reported.
Scheme 2. Synthesis of Compounds 15 and 16a
group (phenoxymethylamides 7, 8, and 9); (iii) carbamoylation
(carbamate 10); (iv) Pinner reaction with formation of a
thioimidate adduct (nitrile 11); (v) disulfide bond formation
(isothiazolinone 12, benzisothiazolinone 13, and thiadiazole
14). The covalent interactions could be reversible or irreversi-
ble, depending on the reaction products and conditions. More-
over, warhead reactivity was also tentatively modulated by the
introduction of electron-withdrawing groups (7 vs 8 and 9). In
order to test nonspecific toxicity of the considered warheads,
we synthesized compounds 15 and 16 (Scheme 2 and Table 1)
where two of the cysteine-trap portions within the series were
a (i) Method A: dichloromethylene dimethyliminium chloride, NaH-
linked to a naphthalene nucleus not able to recognize the
molecular target because it is lacking the structural elements
CO3, CH2Cl2, 0 °C. (ii) Method C: PCl5, CH2Cl2, reflux.
required for interaction with the ATP-binding site of EGFR.
The new compounds were tested as EGFR tyrosine kinase
inhibitors in enzyme-based and cell-based assays. Effects
on downstream signaling pathways and antiproliferative
and proapoptotic activities were also investigated in the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2041
Scheme 3. Synthesis of Warheads 17-19a
gefitinib-resistant H1975 NSCLC cell line harboring the
T790M mutation. Structure-activity relationships within
the series of synthesized compounds were evaluated in order
to identify new irreversible EGFR inhibitors active on a
mutated gefitinib-resistant cell line.
Chemistry
The amides (4-16) of Table 1 were synthesized by coupling
their precursor amines (3 or 31) with the appropriate car-
boxylic acid (17, 18, 20-26), carboxylate (19), or carboxylic
ester (27), as described in Schemes 1 and 2. 6-Amino-4-
(3-bromoanilino)quinazoline 3 was prepared in three steps
from 4-nitroanthranilonitrile 28 as previously described
(Scheme 1).35,36 Briefly, 28 was refluxed in formic acid and
sulfuric acid to give 6-nitro-4-oxoquinazoline 29, which was
chlorinated with thionyl chloride in dioxane and subsequently
treated with 3-bromoaniline to yield 6-nitro-4-(3-bromoanili-
no)quinazoline 30. Reduction of the 6-nitro group with iron
and acetic acid in aqueous ethanol gave the 6-amino-4-(3-
bromoanilino)quinazoline 3.
The epoxy derivatives 4 and 5 were synthesized by coupling
3 with carboxylic acids 17 and 18, respectively, using dichlor-
omethylene dimethyliminium chloride as the coupling reagent
(method A, Scheme 1), while 6 was obtained from 19 with
O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafl-
uorophosphate (HBTU) (method B, Scheme 1). Compounds
7-9 and 11 were prepared as described in method C
(Scheme 1) from the amine 3 and the acyl chlorides obtained
from 20 (for 7), 21 (for 8), 22 (for 9), or 24 (for 11). Derivatives
10, 12, and 13 were synthesized employing N,N0-dicyclohexyl-
carbodiimide (DCC) as the coupling reagent and 23, 25,
and 26, respectively, as carboxylic acids (method D). Finally,
amide 14 was obtained by adding potassium tert-butoxide to a
premixed mixture of ester 27 and amine 3 and exposing the
reaction mixture to microwave irradiation (method E,
Scheme 1). Amides 15 and 16 were synthesized by reacting
2-naphthylamine 31 with carboxylic acids 17 (method A)
and 20 (method C), respectively (Scheme 2).
a (i) KOH, abs EtOH, 0 °C; (ii) 5% KHSO4, room temp; (iii) NaBH4,
EtOH, 0 °C; (iv) MsCl, Et3N, CH2Cl2, 0 °C to room temp; (v) anhydrous
piperidine, KI, DMF, 0 °C to room temp.
Scheme 4. Synthesis of Warheads 25-27a
Carboxylic acids 20-24 are commercially available, while
17-19, 25, 26, and the ester 27 were prepared as shown in
Schemes 3 and 4. The (2R,3R)-epoxy diester 3237 was partially
hydrolyzed to the desired (2R,3R)-monoethyl ester 1738 or
selectively monoreduced to the hydroxyl ester 33.39 Mesyla-
tion ofthe alcohol33and substitutionwith piperidine gave the
epoxy ester 34, which was hydrolyzed to afford the desired
carboxylate 19. The (2S,3S)-monoethyl ester 18 was synthe-
sized by partial hydrolysis of the proper (2S,3S)-epoxy diethyl
ester 35.40
a (i) SO2Cl2, ClCH2CH2Cl, room temp; (ii) 1 M TFA, reflux; (iii)
BrCH2COOEt, Et3N, THF, room temp; (iv) HCl reflux; (v) CH2Cl2,
reflux; (vi) NCCOOEt, p-xylene, reflux.
The isothiazolinone derivative 25 was synthesized as shown
in Scheme 4. 3,30-Dithiodipropionamide 3641 was cyclized
with sulfuryl chloride to isothiazolinone 37,42 which was
hydrolyzed to the desired carboxylic acid 25 by refluxing in
1 M trifluoroacetic acid. The benzisothiazolinone carboxylic
acid 26 and the ethyl 3-isopropyl-1,2,4-thiadiazole-5-carboxy-
late 27 were prepared as described in Scheme 4, according to
literature methods.43,44
EGFR45 revealed that this inhibitor covalently binds the
Cys797 sulfur atom, adopting an orientation similar to that
found for several kinases in complexes with reversible quina-
zoline inhibitors.46 To find out whether the introduction of a
warhead differing from the acrylamide chemotype at the
quinazoline ring was consistent with the recognition of the
EGFR active site, docking simulations of compounds 4-14
were performed using the publicly available coordinate of the
EGFR-2 covalent adduct.45 Docking runs revealed that all
the inhibitors could be accommodated within EGFR active
site with a binding mode resembling that of the acrylamide
derivative 2 (Figure 2 and Figure S1-S11, Supporting In-
formation), indicating that the presence of a bulky warhead
directly attached, or spaced by an appropriate linker, did
not affect the crystallographic-like binding mode of the
Molecular Modeling
When irreversible enzyme inhibition is required, both war-
head reactivity and compound specificity, conferred by a
driver portion responsible for noncovalent recognition at
the binding site, are important in the search for an effective
inhibitor. Cocrystallization of 2 within the catalytic site of

2042 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Carmi et al.
epidermoid cancer cell line by Western blotting. Percent
inhibitions at 1 μM are also reported in Table 1. Results were
compared with those observed for the reference compounds 2
and 3, recognized irreversibleand reversible EGFR inhibitors,
respectively. A431 cells, which overexpress wild type EGFR,
were treated with each inhibitor for 1 h and then washed free
of drug. The degree of EGFR autophosphorylation was
measured either immediately after or 8 h after removal of
the inhibitor fromthe medium.15 Forcompoundsthatshowed
the highest inhibitory potencies toward EGFR at 1 μM, dose
dependency was investigated and IC50 determined (Figure 3
and Table 1). As previously reported,34 compounds showing
80% or greater EGFR inhibition 8 h after their removal from
the medium were designated as irreversible inhibitors while
compounds showing 20-80% inhibition were designated as
partially irreversible ones. Table 1 indicates that reference
compound 2 and several of the newly synthesized derivatives,
4-6 and 9, irreversibly inhibit EGFR activity, with inhibition
values higher than 90% after an 8 h washout. On the other
hand, compounds 7-8 and 10-14 were only partially irre-
versible under the tested conditions, with percent inhibition of
EGFR autophosphorylation between 24% and 80%. As
expected, the reference reversible compound 3, which com-
pletely inhibited EGFR activityafter 1 h of treatment at 1 μM,
did not show any inhibitory activity 8 h after its removal from
the medium.
Figure 2. Docking of compound 6 within the EGFR kinase domain.
Compound 6 (green carbons) undertakes several polar interactions
(highlighted with orange lines) at the EGFR kinase active site (black
carbons and white cartoons): (i) a direct hydrogen bond between the
quinazolineN1andthebackbone NHofMet793; (ii) awater molecule
mediated hydrogen bond between quinazoline N3 and the side chain
of Thr790; (iii) a salt bridge between Asp800 and the piperidine
˚
nitrogen. The SH group of Cys797 is at 4.13 A from the closest carbon
atom of the epoxy warhead, ready for the nucleophilic attack.
quinazoline moiety.23,47-49 Indeed, the two crucial hydrogen
bonds between the 4-(3-bromoanilino)quinazoline driving
portion and the EGFR active site, one with Met793 and the
other, through a conserved water molecule, with Thr790, were
formed by all the compounds within the series.
Compounds 4-6, which contain an epoxy group at posi-
tion 6, irreversibly inhibited EGFR at 1 μM. In fact, their
inhibitory effect persisted up to 8 h after removal from the
medium, probably because this class of compounds can
undergo nucleophilic attack on the epoxide by Cys797 to
generate an enzyme-bond addition product. This mechanism
of irreversible action through the alkylation of the Cys797 has
been reported in the literature for other warheads.23,34 Com-
pounds 4-6 showed dose-dependent inhibition of EGFR
autophosphorylation in A431 cells with IC50 values compar-
able to that of 2. In particular, the (2R,3R)-ethyl 3-(4-(3-
bromoanilino)quinazolin-6-ylcarbamoyl)oxirane-2-carboxy-
late (4) had an IC50 of 0.011 μM immediately after 1 h of
treatment and and IC50 of 0.145 μM 8 h after the removal of
the compound from the reaction medium (Figure 3C and
Table 1). Its (2S,3S)-enantiomer 5 was slightly less potent in
inhibiting EGFR after 1 h of treatment (IC50 = 0.034 μM)
and showed slightly higher potency 8 h after removal of the
compound (IC50 = 0.068 μM, Figure 3D and Table 1). While
these limited differences may not accurately establish struc-
ture-activity relationships, we surmised that the IC50 at 1 h,
together with the IC50 measured on the purified enzyme,
reflects both reversible binding to the EGFR catalytic site,
by weak interactions at the enzyme surface, and irreversible
binding, due to the formation of a covalent bond with an
exposed cysteine. On the other hand, the IC50 measured 8 h
after ligand removal wouldstem from the extent ofirreversible
covalent bonding. As observed for both acrylamide and
propargylamide derivatives,22,50 the introduction of a basic
group in the warhead-containing side chain improved inhibi-
tor potency. The amino group can act as (i) an intramolecular
catalyst for nucleophilic additions to the cysteine-reactive
center, (ii) a water-solubilizing group, since it points out of
the ATP-binding pocket toward the solvent environment, and
(iii) an additional site for hydrogen bond recognition with
acidic residues within the EGFR binding site. Although
docking of compound 6 within the EGFR active site suggests
a specific role for its piperidine fragment in ligand recognition,
Docking studies also gave information on the positions of
the warheads of the new inhibitors with respect to Cys797.
Reference compound 2, docked in its noncovalent form,
placed the β-carbon atom of the acrylamide portion at
˚
4.15 A from the sulfhydryl group of Cys797. Equally, com-
pounds 4-14 placed the reactive center of the warhead at
similar distances from this nucleophile (Table S2, Supporting
Information). Although the formation of a covalent adduct
can be significantly affected by dynamical effects (e.g., trajec-
tory of the nucleophile on the electrophilic center), these
docking simulations qualitatively suggested that the warheads
of 4-14 may potentially undergo to a nucleophilic attack by
Cys797.
Results and Discussion
The newly synthesized 4-(3-bromoanilino)quinazoline deri-
vatives were tested as EGFR inhibitors in enzyme-based and
cell-based assays. The effects of the compounds on the kinase
activity of human EGFR were quantified by measuring the
phosphorylation of a peptide substrate, using time-resolved
fluorometry (see Experimental Section). Results are reported
in Table 1. In accordance with our docking hypothesis and with
the SAR profile of the 4-anilinoquinazoline ring,23,47-49 the
ATP-binding site of EGFR was tolerant to the introduction of
differently sized and shaped substituents at position 6. In the
tested conditions, the reference compound 2 showed an IC50 of
1.7 nM. Substitution of the acrylamide warhead of 2 with an
epoxy (4-6), a carbonylic (carbamate 10 and nitrile 11), or a
(benzo)isothiazolinonic (12 and 13) one generally produced
equally potent or more potent inhibitors. On the contrary, the
phenoxyacetamide derivatives (7-9) proved less effective than
2 but yet maintained IC50 values in the low nanomolar range.
Only the introduction of a thiadiazole warhead (14) notably
reduced EGFR tyrosine kinase inhibition potency.
The ability of the compounds to irreversibly inhibit EGFR
autophosphorylation was investigated in the A431 human

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2043
Figure 3. Irreversible inhibitory action of compounds 4-9 on EGFR autophosphorylation. A431 cells were incubated with the investigated
compound for 1 h and stimulated with EGF either immediately after or 8 h after removal of the compound from the medium. Western blot
analysis was done using monoclonal antibodies directed to p-Tyr1068. 2 and 3 were used as irreversible and reversible reference compounds,
respectively. Representative blots of three independent experiments are shown. Total EGFR is shown as loading control.
the presence of the piperidino group does not improve the
affinity toward purified EGFR enzyme with respect to the
epoxy succinate 4.
fragment in view of its intermediate reactivity toward nucleo-
philic substitutions, which can be further modulated by ring
substitution. Compounds 7-9 showed percent inhibitions at
1 μM between 92%and 97%, 1 h after treatment, and between
71% and 91%, 8 h after removal of the compound from the
medium. Complete dose-inhibition curves showed that the
introduction of a fluorine on the phenoxy aromatic ring did
not significantly affect the inhibitory effect of 8 (IC50 of 0.076
and 0.292 μM at 1 and 8 h after treatment, respectively) with
respect to the unsubsituted 7 (IC50 of 0.031 and 0.291 μM).
Moreover, the pentafluoro substitution (compound 9), while
it gave a comparable IC50 at 1 h after treatment, significantly
increased EGFR inhibition after 8 h (IC50 = 0.078 μM, lower
than the reference compound 2, Figure 3H and Table 1). Since
the introduction of the electron-withdrawing fluorine is re-
sponsible for increased reactivity toward nucleophilic substi-
tution, the remarkable potency of the pentafluoro substituted
derivative 9 under these conditions can be attributed to its
increased reactivity versus the nucleophilic Cys797, giving the
substitution product. These results indicate that this class
shows promise for chemical optimization by modification of
the stereoelectronic features of the phenolic leaving group.
On the other hand, the introduction of a piperidino group
increased the inhibitory activity of compound 6 with IC50 of
0.007 and 0.044 μM at 1 and 8 h after treatment, respectively
(Figure 3E and Table 1). The binding pose, reported in
Figure 2, shows that while the 4-anilinoquinazoline portion
forms the conventional pattern of hydrogen bonds with the
EGFR hinge region,13,45,46,51 the protonated nitrogen of the
piperidine ring can form an additional hydrogen bond with
the acidic group of Asp800.52,53 This interaction, rather than
improving the recognition of 6 at the active site, may facilitate
the nucleophilic attack at the epoxy ring by Cys797.22
The 6-phenoxyacetamide derivatives 7-9 are potentially
able to undergo a nucleophilic attack by Cys797 on their R-
methylene, with formation of a covalent adduct with the
enzyme and in situ release of a phenol leaving group. Similar
chemical approaches to EGFR inhibition have been tried with
the R-chloro- and R-methoxyacetamides,24 showing that only
the more reactive R-chloroacetamide derivatives give irrever-
sible EGFR inhibition. We introduced the phenoxyacetamide

2044 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Carmi et al.
Beyond its interest as a warhead, phenoxyacetamide deriva-
tives could also be exploited to design and optimize leaving
groups that may be pharmacologically active in a multitarget
approach.54,55
Table 2. Viability Inhibition of H1975 Gefitinib-Resistant Cell Linea
compd
H1975 cell line, IC50 (μM)
1
2
3
4
6
8
9
8.26 ( 1.11
0.61 ( 0.05
19.5 ( 2.46
6.76 ( 0.60
1.72 ( 0.47
7.82 ( 1.19
11.0 ( 1.08
Compound 10, characterized by a carbamate group linked
to the quinazoline driver portion through a glycine spacer,
gave EGFR inhibition of 95% and 80% at 1 μM at 1 and 8 h
after removal from the medium, respectively. Carbamates can
form covalent adducts by carbamoylation of serine or cysteine
residues, which can be slowly hydrolyzed to re-establish the
enzyme in the active form.29 Although EGFR average inhibi-
tion at 8 h was less than 90%, this group appeared to be
worthy of consideration as a warhead or at least as a starting
point for further chemical optimization.
a Concentration that inhibits by 50% the proliferation of NSCLC
H1975. The cell proliferation was determined by the MTT assay after
72 h of incubation with compounds (0.1-20 μM). Mean values of three
independent experiments ( SEM are reported.
The cyanoacetamide derivative 11 inhibited EGFR autop-
hosphorylation 1 h after treatment at 1 μM (95% inhibition),
while only a residual effect persisted 8 h after washout (24%
inhibition). Nitriles are known to form, through a Pinner-like
reaction, covalent reversible thioimidate ester adducts with
biological nucleophiles such as cysteine.29 Compounds 10 and
11 both containwarheads able toestablish covalent, reversible
interactions with nucleophilic cysteine residues. On the other
hand, even though irreversible inhibition was achieved by
carbamate derivative 10, only a residual effect was observed
8 h after treatment with compound 11, suggesting that the
thiocarbamate adduct is more stable than the thioimidate
ester one. Therefore, although irreversible inhibition might be
attained by chemical optimization with this class of deriva-
tives, the nitrile warhead looks less promising than the pre-
viously cited ones, with respect to the present target.
Finally, derivatives 12-14 can covalently react with Cys797
via disulfide bond formation, following ring-opening by
breaking of the sulfenamide bond.30,56 Compounds 12 and
13, bearing an isothiazolinone and a benzisothiazolinone
group, respectively, inhibited EGFR in a reversible manner,
since inhibition was significantly reduced 8 h after removal
from the medium (12, 80% and 25% inhibition at 1 and 8 h
after treatment, respectively; 13, 69% and 31%, respectively).
Only the thiadiazole derivative 14 was able to produce a
significant, yet partial, irreversible inhibition of EGFR, as
demonstrated by 67% inhibition of autophosphorylation 8 h
after treatment. This warhead has been employed to design
active-site directed inhibitors of different enzymes with a
cysteine in their active site.30
Figure 4. Effectiveness of irreversible EGFR inhibitor 6 in H1975
NSCLC cell line. (A) Antiproliferative effects of compound 6 (2) in
comparison with 1 (b) on H1975 cell line, harboring the resistance-
associated mutation T790M. Cell proliferation was determined by
MTT assay, as described in the Experimental Section. Results are
reported as the mean ( SD of three independent experiments: (/)
P < 0.01 for each dose versus 1; n = 3. (B) Comparison of compo-
unds 6, 1, and 2 in their ability to suppress EGFR autophosphor-
ylation (p-EGFR), erbB2 autophosphorylation (p-erbB2), and
phosphorylation of downstream effectors AKT (p-Akt) and MAPK
(p-p44/42) in H1975 cells. Total EGFR, erbB2, AKT, and MAPK
are shown as loading controls.
The design of a covalent inhibitor presents the risk that
enzyme inhibition is mainly the result of high warhead
reactivity, which would result in nonspecific reactions with
many possible off-targets. To test the role of the 4-anilinoqui-
nazoline driving portion, which should be to confer target
selectivity to poorly reactive warheads, we synthesized com-
pounds 15 and 16 (Scheme 2 and Table 1), where two of the
most active cysteine-trap portions of the series were linked toa
simplenaphthalenenucleus. In principle, the naphthalene ring
is not able to recognize the molecular target because it lacks
the structural elements required for interaction with the ATP-
binding site of EGFR.57 As expected, compound 15, carrying
an epoxysuccinic group analogue to compound 4, and com-
pound 16, containing the same side chain as compound 7, did
not inhibit EGFR, demonstrating that target specificity of the
series is due to driver-portion recognition within the EGFR
active site. Interestingly, compounds 15 and 16 exhibited less
than 10% inhibition of A431 cell proliferation at concentra-
tions up to 10 μM, indicating the absence of nonspecific
cellular toxicity of the considered warheads (data not shown).
Moreover, the formation of conjugates with glutathione was
evaluated in aqueous buffered solution by LC-UV and
LC-ESI-MS (see Experimental Section) toassessthe intrinsic
reactivity of the warheads toward a thiol derivative. Neither
the epoxy derivatives 4 and 15 nor the phenoxyacetamides 7
and 16 gave any measurable adduct with GSH after 1 h of
incubation (data not shown). On the other hand the acryla-
mide 2 showed conversion of 36.2 ( 1.9% of the starting

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2045
active fragments. These results suggest that this new irrever-
sible inhibitor can have both a cytostatic and a cytotoxic effect
on the H1975 cancer cell line. On the other hand, compound 6
proved to have only a weak cytotoxic profile on a cell line
where proliferation is not driven by EGFR activity, excluding
off-target general cytotoxic effects. In fact, compound 6 at
10 μM inhibited less than 50% of SW620 cell proliferation,
similar to what was observed for 1 and for the epoxysuccinic
derivative 4, while the acrylamide derivative 2 gave an IC50 of
8.23 ( 1.08 μM (Table S3, Supporting Information).
Conclusion
We report here an exploration of possible warheads for
EGFR inhibition, extended to the most important classes of
clinically exploitable ones. We investigated warheads that
covalently react with cysteine via nucleophilic addition
(epoxides 4-6), nucleophilic substitution (phenoxyaceta-
mides 7-9), carbamoylation (carbamate 10), Pinner reaction
(nitrile 11), and disulfide bond formation (isothiazolinone 12,
benzisothiazolinone 13, thiadiazole 14).
Figure 5. Effect of compound 6 on H1975 cell death. (A) H1975
cells were treated with the indicated concentration of compound 1
and compound 6 for 72 h and then analyzed with propidium iodide/
Hoechst 33342 staining to assess cell death: (/) P < 0.01 for each
dose versus control; n = 3. (B) At the same time the cleavage of
procaspase-3 was assessed on lysate proteins by Western blotting.
The migration position of each full-length procaspase and those of
its processing products are indicated.
Compounds from different chemical classes proved effi-
cient in irreversibly inhibiting EGFR autophosphorylation.58
Epoxy derivatives 4-6 showed potencies comparable to that
ofthe acrylamidereferencecompound2. Asexpectedfrom the
structure of EGFR, the introduction of a terminal basic group
(compound 6) significantly improved inhibition of both
EGFR autophosphorylation and cell proliferation, suggest-
ing that the identified compounds could be starting points for
further structure-based optimization. Compound 6 inhibited
EGFR downstream signaling pathways and showed cyto-
static and cytotoxic activities on gefitinib-resistant NSCLC
cells (H1975). Another interesting warhead is represented by
the phenoxyacetamides (7-9), which in principle can release a
leaving group on nucleophilic substitution and may be con-
sidered for a multidrug approach in cancer treatment. Pro-
mising results were also observed with the carbamate 10 and
the thiadiazole 14.
compound to its GSH conjugate ([M þ H]þ = 676.11) under
the same conditions (mean ( SD; n = 3).
We next tested the antiproliferative activity of compounds
exhibiting highest potency as EGFR irreversible inhibitors on
the gefitinib-resistant H1975 NSCLC cell line, by MTT assay.
Resultswerecomparedwiththoseofthe reference compounds
1-3 in the same test. As reported in Table 2, compound 4
exhibited a dose-dependent inhibition of H1975 cell prolifera-
tion with an IC50 similar to that of 1 (IC50s of 6.76 and 8.26
μM, respectively). Interestingly, introduction of the basic
piperidino group on the reactive side chain made compound
6 4 times more active than 1 on H1975 cells (Figure 4A and
Table 2), with an antiproliferative IC50 of 1.72 μM. The 6-
phenoxyacetamide derivatives 8 and 9 showed IC50 on H1975
cells proliferation in the same range of the reference com-
pound 1 (IC50 of 7.82 and 11.02 μM, respectively).
We then examined the ability of compound 6 to inhibit
EGFR signaling in the NSCLC cell line H1975 harboring the
T790M mutation. 1 and 2 were used as reference compounds.
Results of Western blot analysis are illustrated in Figure 4B.
Compound 6 produced a dose-dependent inhibition of EGFR
and erbB2 autophosphorylation with complete inhibition at
1 μM. By contrast, 1 showed marginal activity on EGFR and
erbB2 in H1975 cells up to 10 μM. Compound 6 was also
considerably more effective than 1 in inhibiting the two major
signaling pathways activated by EGFR, the PI3K/AKT/
mTOR and the RAS/RAF/MAPK cascades having a central
role in controlling cell survival, cell growth, and proliferation
(Figure 4B).
In conclusion, the results described here extend the chemi-
cal diversity of new irreversible EGFR inhibitors, and similar
chemical approaches could be exploited to design compounds
targeting noncatalytic cysteines by covalent bond formation.
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 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 reac-
tions 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 spectrometer. Chemical shifts (δ scale) are reported in
parts per million (ppm) relative to the central peak of the solvent.
1H NMR spectra are reported in the following order: multiplicity,
approximate coupling constant (J value) in hertz (Hz), and
number of protons; signals were characterized as s (singlet), d
(doublet), dd (doublet of doublets), t (triplet), dt (doublet of
triplets), q (quartet), m (multiplet), br s (broad signal). Mass
spectra were recorded using an API 150 EX instrument (Applied
Biosystems/MDS SCIEX, Foster City, CA). Liquid chromatog-
raphy/mass spectrometry analysis was performed on a Agilent
1100 LC gradient system and on an Applied Biosystem 150-EX
Finally, we observed that compound 6 killed more cells
(25-30% at 5 μM) when compared with control or cells
treated with the reference compound 1 (Figure 5A) and
was associated with caspase-3 activation (Figure 5B). After
2 days of treatment at 5 μM, compound 6 significantly cleaved
this proapoptotic proteinfrom its inactive precursor form into

2046 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Carmi et al.
single quadrupole mass spectrometer equipped with a TurboIon-
Spray ion source working in positive ion mode. Compounds 1,59
2,34 and 335 were synthesized according to literature methods.
The final compounds were analyzed on ThermoQuest (Italia)
FlashEA 1112 elemental analyzer for C, H, and N. Analyses were
within (0.4% of the theoretical values (Table S1, Supporting
Information). All tested compounds were >95% pure by ele-
mental analysis.
Method A. The crude product was purified by silica gel
chromatography (CH2Cl2/MeOH, 99:1 to 97:3) to give 5
(66%) as a white solid: mp (MeOH) >230 °C; MS (APCI)
1
m/z 457.9, 459.3; H NMR (300 MHz, DMSO-d6) δ 1.26 (t,
J = 7.2 Hz, 3H), 3.81 (d, J = 1.7 Hz, 1H), 3.92 (d, J = 1.7 Hz,
1H), 4.23 (q, J = 7.1 Hz, 2H), 7.29 (d, J = 8.1 Hz, 1H), 7.35 (t,
J = 7.8 Hz, 1H), 7.80-7.87 (m, 2H), 7.89 (dd, J = 9.0, 2.2 Hz,
1H), 8.16 (s, 1H), 8.60 (s, 1H), 8.72 (s, 1H), 9.94 (br s, 1H),
10.80 (br s, 1H). Anal. (C20H17BrN4O4) C, H, N.
(2R,3S)-Ethyl 3-(Piperidin-1-ylmethyl)oxirane-2-carboxylate
(34). A 0 °C cooled solution of (2R,3S)-ethyl 3-(hydroxyme-
thyl)oxirane-2-carboxylate 3339 (405 mg, 2.77 mmol) and Et3N
(582 μL, 4.16 mmol) in anhydrous CH2Cl2 (12 mL) was stirred
for 30 min before the dropwise addition of methanesulfonyl
chloride (MsCl) (407 μL, 4.16 mmol). The mixture was then
stirred for 10 min at 0 °C and for 2 h at room temperature.
Then the solvent was evaporated. The resulting yellow paste
was dissolved in anhydrous DMF (12 mL), and KI (23 mg,
0.14 mmol) was added. The yellow solution was cooled to 0 °C
and stirred for 5 min before the dropwise addition of anhydrous
piperidine (830 μL, 8.31 mmol). The resulting suspension was
slowly warmed to room temperature and stirred for 7 days,
diluted with saturated aqueous NaHCO3 (85 mL), and extracted
with Et2O. The combined organic layers were dried and evapo-
rated to give a brown oil that was purified by silica gel chroma-
tography (CH2Cl2/MeOH, 100:0 to 96:4), furnishing 34 as
a yellow oil (30%): 1H NMR (300 MHz, CDCl3) δ 1.25 (t,
J = 7.1 Hz, 3H), 1.39 (m, 2H), 1.55 (m, 4H), 2.31 (dd, J = 13.6,
6.4 Hz, 1H), 2.36-2.55 (m, 4H), 2.71 (dd, J = 13.6, 3.4 Hz, 1H),
3.19 (d, J = 2.0 Hz, 1H), 3.29 (m, 1H), 4.18 (m, 2H).
(2R,3S)-N-(4-(3-Bromoanilino)quinazolin-6-yl)-3-(piperidin-1-
ylmethyl)oxirane-2-carboxamide (6). Method B. To a suspen-
sion of potassium (2R,3S)-3-(piperidin-1-ylmethyl)oxirane-2-
carboxylate 19 (58 mg, 0.26 mmol) in anhydrous DMF (2 mL),
O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluor-
ophosphate (HBTU) (205 mg, 0.54 mmol) was added at room
temperature. The mixture was stirred for 40 min before the
dropwise addition of 6-amino-4-(3-bromoanilino)quinazoline 3
(80 mg, 0.26 mmol) in DMF (2 mL). The reaction mixture was
stirred for 16 h, the solvent was removed, and the residue was
dissolved in CH2Cl2 and washed with saturated aqueous
Na2CO3. The organic phase was evaporated and the crude
product purified by silica gel chromatography (CH2Cl2/MeOH,
99:1 to 97:3) to give 6 as a pale-yellow solid (30%): mp >230 °C;
1
MS (APCI) m/z 482.2, 484.2; H NMR (300 MHz, CD3OD) δ
1.41-1.43 (m, 2H), 1.53-1.64 (m, 4H), 2.36 (dd, J = 13.6, 6.7 Hz,
1H), 2.48-2.56 (m, 4H), 2.80 (dd, J = 13.7, 3.4 Hz, 1H), 3.30-
3.34 (m, 1H), 3.39 (d, J = 1.89 Hz, 1H), 7.21-7.22 (m, 2H),
7.64-7.69 (m, 2H), 7.77 (dd, J = 9.0, 2.2 Hz, 1H), 8.03 (s, 1H),
8.45 (s, 1H), 8.56 (d, J = 2.1 Hz, 1H). Anal. (C23H24BrN5O2) C,
H, N.
Potassium (2R,3S)-3-(Piperidin-1-ylmethyl)oxirane-2-carbo-
xylate (19). To an ice cold stirring solution of (2R,3S)-ethyl
3-(piperidin-1-ylmethyl)oxirane-2-carboxylate 34 (170 mg,
0.80 mmol) in absolute EtOH (1 mL), a solution of KOH
(45 mg, 0.8 mmol) in absolute EtOH (1.5 mL) was added
dropwise. The resulting mixture was stirred for 2 h at 0 °C.
The solvent was removed under reduced pressure, and the
residue was washed with petroleum ether. The product 19 was
obtained as a pale-brown solid (83%): mp >230 °C; MS (APCI)
m/z 184.2; 1H NMR (300 MHz, CDCl3) δ 1.49 (m, 2H), 1.63 (m,
4H), 2.32 (dd, J = 13.4, 6.7 Hz, 1H), 2.54 (m, 4H), 2.74 (dd,
J = 13.4, 3.7 Hz, 1H), 3.02 (d, J = 2.16 Hz, 1H), 3.12 (m, 1H).
2-(3-Oxoisothiazolin-2-yl)acetic acid (25). A suspension of
ethyl 2-(3-oxoisothiazolin-2-yl)acetate 3742 (200 mg, 1.07 mmol)
in 1 M trifluoroacetic acid (TFA) (25 mL, 25 mmol) was refluxed
for 12 h and then evaporated. The residue was dried at 50-55 °C
in vacuo to yield 25 (98%) as a white solid: mp 171-172 °C; 1H
NMR (300 MHz, DMSO-d6) δ 4.41 (s, 2H), 6.19 (d, J = 6.2 Hz,
1H), 8.51 (d, J = 6.2 Hz, 1H).
N-(4-(3-Bromoanilino)quinazolin-6-yl)-2-phenoxyacetamide
(7). Method C. Phenoxyacetic acid 20 (361 mg, 2.37 mmol) was
added to a stirred suspension of PCl5 (490 mg, 2.37 mmol) in
CH2Cl2 (15 mL) at room temperature. The reactants were
refluxed for 30 min and then cooled to room temperature, and
6-amino-4-(3-bromoanilino)quinazoline 3 (500 mg, 1.59 mmol)
was added in 10 min. After refluxing for 2 h, the mixture was
cooled in an ice/water bath, an amount of 2.5 mL of water was
added, the mixture was stirred for 30 min, and then Na2CO3 was
added. The solid was filtered and purified by silica gel chroma-
tography (CH2Cl2/MeOH, 99:1 to 97:3) to afford 7 as a white
solid (77%): mp (EtOH/water) 212 °C; MS (APCI) m/z 449.0,
1
451.0; H NMR (300 MHz, DMSO-d6) δ 4.79 (s, 2H), 6.99 (t,
J = 7.4 Hz, 1H), 7.05 (d, J = 7.8 Hz, 2H), 7.27-7.36 (m, 4H),
7.80 (d, J = 8.9 Hz, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.95 (dd, J =
9.0, 2.1 Hz, 1H), 8.16 (s, 1H), 8.58 (s, 1H), 8.74 (d, J = 1.7 Hz,
1H), 9.93 (s, 1H), 10.44 (s, 1H). Anal. (C22H17BrN4O2) C, H, N.
N-(4-(3-Bromoanilino)quinazolin-6-yl)-2-(4-fluorophenoxy)-
acetamide (8). 6-Amino-4-(3-bromoanilino)quinazoline 3 was
reacted with 2-(4-fluorophenoxy)acetic acid 21 according to the
procedure described in Method C. The crude product was
purified by silica gel chromatography (CH2Cl2/MeOH, 99:1 to
97:3) to give 8 (67%) as a white solid: mp (EtOH/water) 224 °C;
MS (APCI) m/z 467.3, 469.1; 1H NMR (300 MHz, DMSO-d6) δ
4.78 (s, 2H), 7.07 (dd, J = 9.2, 4.3 Hz, 2H), 7.15-7.21 (m, 2H),
7.27-7.37 (m, 2H), 7.85 (m, 2H), 7.95 (dd, J = 8.2, 1.4 Hz, 1H),
8.16 (br s, 1H), 8.59 (s, 1H), 8.74 (br s, 1H), 9.93 (br s, 1H), 10.44
(br s, 1H). Anal. (C22H16BrFN4O2) C, H, N.
N-(4-(3-Bromoanilino)quinazolin-6-yl)-2-(perfluorophenoxy)-
acetamide (9). 6-Amino-4-(3-bromoanilino)quinazoline 3 was
reacted with 2-(perfluorophenoxy)acetic acid 22 according to
the procedure described in Method C. The crude product was
purified by silica gel chromatography (CH2Cl2/MeOH, 99:1 to
97:3) to give 9 (50%) as a white solid: mp 220-221 °C; MS
(APCI) m/z 539.1, 541.1; 1H NMR (300 MHz, DMSO-d6) δ 4.99
(s, 2H), 7.24-7.34 (m, 2H), 7.75-7.87 (m, 3H), 8.11 (s, 1H), 8.55
(s, 1H), 8.66 (s, 1H), 9.91 (br s, 1H), 10.50 (br s, 1H). Anal.
(C22H12BrF5N4O2) C, H, N.
(2R,3R)-Ethyl
3-(4-(3-Bromoanilino)quinazolin-6-ylcarba-
moyl)oxirane-2-carboxylate (4). Method A. Dichloromethylene
dimethyliminium chloride (356 mg, 2.19 mmol) was added to a
stirred solution of (2R,3R)-2,3-epoxysuccinic acid monoethyl
ester 1738 (350 mg, 2.19 mmol) in anhydrous CH2Cl2 (10 mL).
After the reactants were stirred for 1 h with ice-cooling, 6-
amino-4-(3-bromoanilino)quinazoline 3 (458 mg, 1.46 mmol)
and NaHCO3 (613 mg, 7.3 mmol) were added, and the suspen-
sion was stirred for 1 h at 0 °C. The mixture was washed with
water, dried, and concentrated to obtain the crude compound.
Crystallization from MeOH gave the pure product 4 as white
crystals (60%): mp (MeOH) >230 °C (dec); MS (APCI) m/z
458.3, 459.2; 1H NMR (300 MHz, DMSO-d6) δ 1.26 (t, J = 7.1
Hz, 3H), 3.82 (d, J = 1.7 Hz, 1H), 3.93 (d, J = 1.7 Hz, 1H), 4.23
(q, J = 7.1 Hz, 2H), 7.29 (d, J = 8.1 Hz, 1H), 7.35 (t, J = 7.9 Hz,
1H), 7.82-7.92 (m, 3H), 8.16 (br s, 1H), 8.60 (s, 1H), 8.72 (d, J =
1.3 Hz, 1H), 9.93 (br s, 1H), 10.80 (br s, 1H). Anal. (C20H17-
BrN4O4) C, H, N.
(2S,3S)-Ethyl 3-(4-(3-Bromoanilino)quinazolin-6-ylcarbamo-
yl)oxirane-2-carboxylate (5). 6-Amino-4-(3-bromoanilino)quin-
azoline 3 was reacted with (2S,3S)-2,3-epoxysuccinic acid
monoethyl ester 1840 according to the procedure described in
N-(4-(3-Bromoanilino)quinazolin-6-yl)-2-cyanoacetamide (11).
6-Amino-4-(3-bromoanilino)quinazoline 3 was reacted with

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2047
2-cyanoacetic acid 24 according to the procedure described in
Method C. The crude product was purified by silica gel chro-
matography (EtOAc) to give 11 (55%) as white solid: mp
compound (70%) as a white solid: mp (EtOH/water) 118-
119 °C; MS (APCI) m/z 283.3; 1H NMR (300 MHz, CDCl3) δ
1.32 (t, J = 7.1 Hz, 3H), 3.66 (d, J = 1.9 Hz, 1H), 3.85 (d, J =
2.0 Hz, 1H), 4.29 (m, 2H), 7.39-7.49 (m, 3H), 7.77-7.83 (m,
4H), 8.18 (d, J = 2.0 Hz, 1H). Anal. (C16H15NO4) C, H, N.
N-(Naphthalen-2-yl)-2-phenoxyacetamide (16). Compound
16 was synthesized by coupling the carboxylic acid 20 and 2-
naphthylamine 31 using the procedure described in Method C.
Purification by silica gel chromatography (n-hexane/EtOAc,
70:30) gave the pure compound (80%) as a pale-yellow solid:
mp (EtOH/water) 142-144 °C; MS (APCI) m/z 184.3; 1H NMR
(300 MHz, CDCl3) δ 4.65 (s, 2H), 7.00-7.09 (m, 3H), 7.33-7.55
(m, 5H), 7.77-7.82 (m, 3H), 8,25 (d, J = 1.7 Hz, 1H), 8.43 (br s,
1H). Anal. (C18H15NO2) C, H, N.
Reactivity with Reduced Glutathione. The reactivity of com-
pounds 4, 15, 7, and 16 with reduced glutathione (GSH) was
evaluated in an aqueous buffered solution (phosphate buffered
saline, PBS, pH 7.4), at 37 °C and compared to that of the
acrylamide 2. Briefly, 10 μL of the compound standard solution
in DMSO (1 mM) was diluted with 890 μL of PBS, pH 7.4. Then
an amount of 100 μL of a freshly prepared GSH solution in PBS
(20 mM) was added. Conversion of the compounds and forma-
tion of conjugates at 37 °C and at different time points were
measured by LC-UV and LC-ESI-MS. The LC column used
was a Phenomenex Synergi Fusion (2.0 mm ꢀ 100 mm, 4 μm),
and the mobile phase was a gradient of 50-10% aqueous
trifluoroacetic acid (0.05%) in methanol in 10 min at the flow
rate of 250 μL/min.
Kinase Assay. Evaluation of the effects of compounds on the
kinase activity of human EGFR was performed by measuring
the phosphorylation of the substrate Ulight-CAGAGAIETD-
KEYYTVKD (JAK1) using a human recombinant enzyme
expressed in insect cells60 and the LANCE detection method,61
employing the Cerep EGFR kinase assay.62 Briefly, the test
compound, reference compound, or water (control) was mixed
with the enzyme (0.0452 ng) in a buffer containing 40 mM
Hepes/Tris (pH 7.4), 0.8 mM EGTA/Tris, 8 mM MgCl2,
1.6 mM DTT, 0.008% Tween-20, and 100 nM poly-D-lysine.
Thereafter, the reaction was initiated by the addition of 100 nM
substrate and 10 μM ATP, and the mixture was incubated for 15
min at room temperature. For control basal measurements, the
enzyme was omitted from the reaction medium. Following
incubation, the reaction was stopped by the addition of
13 mM EDTA. After 5 min, the antiphopho-PT66 antibody
labeled with europium chelate was added. After 60 min, the
fluorescence transfer was measured at excitation wavelength of
337 nm and emission wavelength of 620 nm using a microplate
reader (Envision, Perkin-Elmer). The concentration of com-
pound that inhibited receptor phosphorylation by 50% (IC50)
was calculated from inhibition curves.
Cell Culture. The human A431 epidermoid cancer cell line was
cultured in D-MEM 4.5 g/L glucose. NSCLC cell line H1975
and SW620 cell line were cultured in RPMI. All media were
supplemented with 2 mmol/L-glutamine, 10% FCS. A431 was
from ATCC. H1975 was kindly provided by Dr. E. Giovannetti
(Department of Medical Oncology, VU University Medical
Center, Amsterdam, The Netherlands) and was maintained
under standard cell culture conditions at 37 °C in a water-
saturated atmosphere of 5% CO2 in air.
1
(EtOH/water) 263 °C; MS (APCI) m/z 382.0, 384.1; H NMR
(300 MHz, DMSO-d6) δ 4.00 (s, 2H), 7.30-7.35 (m, 2H),
7.81-7.86 (m, 3H), 8.15 (t, J = 1.7 Hz, 1H), 8.59 (s, 1H), 8.70 (s,
1H), 9.95 (s, 1H), 10.64 (s, 1H). Anal. (C17H12BrN5O 1.5H2O)
C, H, N.
3
N-[2-[(4-(3-Bromoanilino)quinazolin-6-yl)amino]-2-oxoethyl]
Phenylcarbamate (10). Method D. 2-(Phenoxycarbonylamino)-
acetic acid 23 (375 mg, 1.92 mmol) and N,N0-dicyclohexylcar-
bodiimide (DCC) (416 mg, 1.04 mmol) were added to a solution
of 3 (400 mg, 1.28 mmol) in anhydrous DMF (6 mL) at 0 °C. The
mixture was stirred 16 h at room temperature, the solid was
removed by filtration, and the filtrate was evaporated in vacuo
to obtain the crude product, which was purified by silica gel
chromatography (EtOAc). The pure product 10 (55%) ap-
peared as a white solid: mp (EtOH/water) >250 °C (dec); MS
1
(APCI) m/z 492.1; H NMR (300 MHz, DMSO-d6) δ 4.20 (s,
2H), 6.73-6.78 (m, 3H), 7.15 (t, J = 8.6 Hz, 2H), 7.32 (dt, J =
8.1, 1.6 Hz, 1H), 7.37 (t, J = 7.9 Hz, 1H), 7.82-7.93 (m, 3H),
8.21 (s, 1H), 8.50 (s, 1H), 8.57 (d, J = 1.7 Hz, 1H), 8.70 (s, 1H),
9.32 (s, 1H), 9.98 (br s, 1H). Anal. (C25H20BrN3O3 0.5H20) C,
H, N.
3
N-(4-(3-Bromoanilino)quinazolin-6-yl)-2-(3-oxoisothiazolin-2-
yl)acetamide (12). 6-Amino-4-(3-bromoanilino)quinazoline 3
was reacted with 2-(3-oxoisothiazolin-2-yl)acetic acid 25 ac-
cording to the procedure described in Method D. The crude
product was purified by silica gel chromatography (EtOAc/
MeOH, 99:1 to 90:10) to give 12 (40%) as a white solid: mp
(EtOH/water) >230 °C; MS (APCI) m/z 456.1, 458.3; 1H NMR
(300 MHz, DMSO-d6) δ 4.65 (s, 2H), 6.24 (d, J = 6.2 Hz, 1H),
7.28 (d, J = 8.0 Hz, 1H), 7.33 (t, J = 7.9 Hz, 1H), 7.79-7.85 (m,
3H), 8.10 (s, 1H), 8.55 (d, J = 6.2 Hz, 1H), 8.55 (s, 1H), 8.71 (s,
1H), 9.94 (br s, 1H), 10.69 (br s, 1H). Anal. (C19H14BrN5O2S
1.5H2O) C, H, N.
3
N-(4-(3-Bromoanilino)quinazolin-6-yl)-2-(3-oxobenzo[d]isoth-
iazolin-2-yl)acetamide (13). 6-Amino-4-(3-bromoanilino)quinazo-
line 3 was reacted with 2-(3-oxobenzo[d]isothiazolin-2-yl)acetic
acid 26 according to the procedure described in Method D. The
crude product was purified by silica gel chromatography (EtOAc/
n-hexane, 99:1) to afford 13 (50%) as a white solid: mp (MeOH)
238 °C; MS (APCI) m/z 506.1, 508.1; 1H NMR (300 MHz,
DMSO-d6) δ 4.75 (s, 2H), 7.25-7.31 (m, 2H), 7.43 (t, J = 7.5 Hz,
1H), 7.69 (t, J = 7.8 Hz, 1H), 7.76-7.83 (m, 3H), 7.88 (d, J =
7.9 Hz, 1H), 7.98 (d, J = 8.5 Hz, 1H), 8.09 (s, 1H), 8.54 (s, 1H),
8.72 (s, 1H), 9.89 (br s, 1H), 10.66 (br s, 1H). Anal. (C23H16BrN5-
O2S 0.5H2O) C, H, N.
3
N-(4-(3-Bromoanilino)quinazolin-6-yl)-3-isopropyl-1,2,4-thia-
diazole-5-carboxamide (14). Method E. Potassium tert-butox-
ide (84 mg, 0.75 mmol) was added to a premixed mixture of ethyl
3-isopropyl-1,2,4-thiadiazole-5-carboxylate 2744 (150 mg,
0.75 mmol) and 6-amino-4-(3-bromoanilino)quinazoline 3
(235 mg, 0.75 mmol) in anhydrous DMF (1 mL). The reaction
mixture was microwaved (150 W) to 100 °C for 8 min. The
solution was diluted with water and extracted with EtOAc. The
organic phase was evaporated to obtain the crude compound,
which was purified by silica gel chromatography (CH2Cl2/
MeOH, 99:1 to 97:3) to give 14 (50%) as pale-yellow crystals:
1
mp (CH2Cl2) 224 °C; MS (APCI) m/z 469.0, 471.0; H NMR
Antibodies and Reagents. Media were from Euroclone, and
FBS was purchased from Gibco-BRL (Grand Island, NY).
Monoclonal anti-EGFR, polyclonal anti-phospho-EGFR
(Tyr1068), monoclonal anti-erbB2, monoclonal anti-phospho-
erbB2 (Tyr 1221/1222), polyclonal Akt, polyclonal anti-phos-
pho-Akt (Ser473), monoclonal anti-p44/42 MAPK, monoclonal
anti-phospho-p44/42 MAPK (Thr202/Tyr204), and poly-
clonal anticaspase-3 antibodies were from Cell Signaling Tech-
nology (Beverly, MA). Horseradish peroxidase-conjugated
(HRP) secondary antibodies were from Pierce. The enhanced
chemiluminescence system (ECL) wasfrom Millipore (Millipore,
(300 MHz, DMSO-d6) δ 1.47 (d, J = 6.9 Hz, 6H), 3.43 (m, 1H),
7.34 (dt, J = 8.2, 1.5 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 7.90 (d,
J = 8.9 Hz, 1H), 7.93 (d, J = 7.9 Hz, 1H), 8.20-8.23 (m, 2H),
8.68 (s, 1H), 8.93 (d, J = 1.8 Hz, 1H), 10.01 (s, 1H), 11.33 (br s,
1H). Anal. (C20H17BrN6OS 0.5H2O) C, H, N.
3
(2R,3R)-Ethyl 3-(Naphthalen-2-ylcarbamoyl)oxirane-2-carb-
oxylate (15). Compound 15 was synthesized by coupling the
carboxylic acid 17 and 2-naphthylamine 31 following the pro-
cedure described in Method A. Purification by silica gel chro-
matography (n-hexane/EtOAc, 90:10 to 70:30) gave the pure

2048 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Carmi et al.
MA). Reagents for electrophoresis and blotting analysis were
obtained from, respectively, BIO-RAD Laboratories and Milli-
pore.
Supporting Information Available: Combustion analytical
data; docking of compounds 2, 4, 5, and 7-14 within the EGFR
active site; SW620 cells viability inhibition. This material is
available free of charge via the Internet at http://pubs.acs.org.
Western Blot Analysis. Procedures for protein extraction,
solubilization, and protein analysis by 1D PAGE are described
elsewhere.63 50-100 μg proteins from lysates were resolved
by 5-15% SDS-PAGE and transferred to PVDF membranes
(Millipore). The membranes were then incubated with
primary antibody, washed, and then incubated with HRP-
antimouse or HRP-antirabbit antibodies. Immunoreactive
bands were visualized using an enhanced chemiluminescence
system.
Autophosphorylation Assay. Inhibition of EGFR autopho-
sphorylation was determined as previously described using
specific antiphosphotyrosine and antitotal EGFR antibodies
by Western blot analysis.15
Cell Growth Inhibition. Cell viability was assessed after 3 days
of treatment by tetrazolium dye [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT), Sigma, Dorset, U.K.]
assay as previously described.63 Representative results of at least
three independent experiments were used for evaluationof 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. The compounds
were renewed every 24 h.
Cell Death. Cell death was assessed by morphology on stained
(Hoechst 33342, propidium iodide) or unstained cells using
light, phase contrast, and fluorescence microscopy.55 Activation
of caspase-3 was evaluated by Western blotting procedure as
previously described.55
References
(1) Yarden, Y.; Sliwknowski, M. X. Untangling the ErbB signalling
network. Nat. Rev. Mol. Cell. Biol. 2001, 2, 127–137.
(2) Teman, S.; Kawaguchi, H.; El-Naggar, A. K.; Jelinek, J.; Tang, H.;
Liu, D. D.; Lang, W.; Issa, J. P.; Lee, J. J.; Mao, L. Epidermal
growth factor receptor copy number alterationscorrelate with poor
clinical outcome in patients with head and neck squamous cancer.
J. Clin. Oncol. 2007, 25, 2164–2170.
(3) Barker, A. J.; Gibson, K. H.; Grundy, W.; Godfrey, A. A.; Barlow,
J. J.; Healy, M. P.; Woodburn, J. R.; Ashton, S. E.; Curry, B. J.;
Sarlett, L.; Henthorn, L.; Richards, L. Studies leading to the
identification of ZD1839 (Iressa): an orally active, selective epider-
mal growth factor receptor tyrosine kinase inhibitor targeted to the
treatment of cancer. Bioorg. Med. Chem. Lett. 2001, 11, 1911–1914.
(4) Moyer, J. D.; Barbacci, E. G.; Iwata, K. K.; Arnold, L.; Boman, B.;
Cunningham, A.; Di Orio, C.; Doty, J.; Morin, M. J.; Moyer, M.
P.; Neveu, M.; Pollack, V. A.; Pustilnick, L. R.; Reynolds, M. M.;
Sloan, D.; Theleman, A.; Miller, P. Induction of apoptosis and cell
cycle arrest by CP-358,774, an inhibitor of epidermal growth factor
receptor tyrosine kinase. Cancer Res. 1997, 57, 4838–4848.
(5) 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.
(6) Kobayashi, S.; Boggon, T. J.; Dayaram, T.; Janne, P. A.; Kocher,
O.; Meyerson, M.; Johnson, B. E.; Eck, M. J.; Tenen, D. G.;
Halmos, B. EGFR mutation and resistance of non-small-cell lung
cancer to gefitinib. N. Engl. J. Med. 2005, 352, 786–792.
(7) Pao, W.; Miller, V. A.; Politi, K. A.; Riely, G. J.; Somwar, R.;
Zakowski, M. F.; Heelan, R. T.; Kris, M. G.; Varmus, H. E. KRAS
mutations and primary resistance of lung adenocarcinomas to
gefitinib or erlotinib. PLoS Med. 2005, 2, e17.
(8) Engelman, J. A.; Janne, P. A. Mechanisms of acquired resistance to
epidermal growth factor receptor tyrosine kinase inhibitors in non-
small cell lung cancer. Clin. Cancer Res. 2008, 14, 2895–2899.
(9) Yun, C.-H.; Mengwasser, K. E.; Toms, A. V.; Woo, M. S.;
Greulich, H.; Wong, K.-K.; Meyerson, M.; Eck, M. J. The
T790M mutation in EGFR kinase causes drug resistance by
increasing the affinity for ATP. Proc. Natl. Acad. Sci. U.S.A.
2008, 105, 2070–2075.
Statistical Analysis. Statistical significances of differences
between data were estimated using the two-tailed Student’s t
test.
Computational Studies. Docking simulations were performed
with Glide 5.064 employing the crystal structure of the EGFR
kinase domain covalently bound to 2 (PDB code 2BJF).45
Molecular models of compounds 4-14 were built using Maes-
tro,65 and their geometries were optimized by energy minimiza-
tion using the OPLS2003 force field66 in combination with GB/
SA67 model for implicit solvent representation (water) to an
˚
energy gradient of 0.01 kcal/(mol A). The EGFR crystal struc-
3
ture was submitted to the protein preparation procedure of
Maestro, which includes addition of missing side chains and
hydrogens, assignment of tautomeric state of histidines max-
imizing the number of hydrogen bonds, and geometric optimi-
zation of the whole system to a root-mean-square displacement
(10) Camp, E. R.; Summy., J.; Bauer, T. V.; Liu, W.; Gallick, G. E.;
Ellis, L. M. Molecular mechanisms of resistance to therapies
targeting the epidermal growth factor receptor. Clin. Cancer Res.
2005, 11, 397–405.
(11) Engelman, J. F.; Settleman, J. Acquired resistance to tyrosine
kinase inhibitors during cancer therapy. Curr. Opin. Gene Dev.
2008, 18, 73–79.
˚
(rmsd) value of 0.3 A. Docking simulations were performed
starting from minimum energy conformations of compounds
4-14 placed in an arbitrary position within a region centered on
the covalent inhibitor 2, using enclosing and bounding boxes of
(12) Mukherji, D.; Spicer, J. Second-generation epidermal growth
factor tyrosine kinase inhibitors in non-small cell lung cancer.
Expert Opin. Invest. Drugs 2009, 18, 293–301.
(13) Bikker, J. A.; Brooijmans, N.; Wissner, A.; Mansour, T. S. Kinase
domain mutations in cancer: implications for small molecule drug
design strategies. J. Med. Chem. 2009, 52, 1493–509.
(14) Zhang, J.; Yang, P. L.; Gray, N. S. Targeting cancer with small
molecule kinase inhibitors. Nat. Rev. Cancer 2009, 9, 28–39.
(15) Fry, D. W.; Bridges, A. J.; Denny, W. A.; Doherty, A.; Greis,
K. D.; Hicks, J. L.; Hook, K. E.; Keller, P. R.; Leopold, W. R.;
Loo, J. A.; McNamara, D. J.; Nelson, J. M.; Sherwood, V.; Smaill,
J. B.; Trumpp-Kallmeyer, S.; Dobrusin, E. M. Specific, irreversible
inactivation of the epidermal growth factor receptor and erbB2,
by a new class of tyrosine kinase inhibitor. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 12022–12027.
˚
20 and 14 A on each side, respectively. van der Waals radii of the
protein atoms were not scaled, while van der Waals radii of the
ligand atoms with partial atomic charges lower than |0.15| were
scaled by 0.8. Standard precision (SP) mode was applied, and
the first 10 poses with the highest G-score values were collected
for each docked compound. The resulting binding poses were
reranked according to the rmsd of the 4-anilinoquinazoline
heavy atoms, taking the crystallized compound 2 as a reference
structure.
(16) Kwak, E. L.; Sordella, R.; Bell, D. W.; Godin-Heymann, N.;
Okimoto, R. A.; Branningan, B. W.; Harria, P. L.; Driscoli, D.
R.; Fidias, P.; Lynch, T. J.; Rabindran, S. K.; McGinnis, J. P.;
Sharma, S. V.; Isselbacher, K. J.; Settleman, J.; Haber, D. A.
Irreversible inhibitors of the EGF receptor may circumvent ac-
quired resistance to gefitinib. Proc. Natl. Acad. Sci. U.S.A. 2005,
102, 7665–7670.
(17) Carter, T. A.; Wodicka, L. M.; Shah, N. P.; Velasco, A. M.;
Fabian, M. A.; Treiber, D. K.; Milanov, Z. V.; Atteridge, C. E.;
Biggs, V. H.; Edeen, P. T.; Floyd, M.; Ford, J. M.; Grotzfeld, R.
M.; Herrgard, S.; Insko, D. E.; Mehta, S. A.; Patel, H. K.; Pao, W.;
Acknowledgment. Grant support was from the following:
Ministero della Salute (Programma Straordinario di Ricerca
Oncologica 2006), Regione Emilia Romagna; AIRC
(Associazione Italiana per la Ricerca sul Cancro); Associa-
zione Marta Nurizzo, Brugherio MI; Associazione Chiara
Tassoni, Parma; A.VO.PRO.RI.T., Parma; Associazione Da-
vide Rodella, Montichiari BS; Lega Italiana per la Lotta
contro i Tumori, Sezione di Parma; and CONAD, Bologna.

Article
Sawyers, C. L.; Varmus, H.; Zarrinkar, P. P.; Lockhart, D. J.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2049
epidermal growth factor receptor. J. Med. Chem. 1999, 42, 1803–
1815.
Inhibition of drug-resistant mutants of ABL, KIT, and EGF
receptor kinases. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 11011–
11016.
(35) Rewcastle, G. W.; Denny, W. A.; Bridges, A. J.; Zhou, H.; Cody,
D. R.; McMichael, A.; Fry, D. W. Tyrosine kinase inhibitors:
synthesis andstructure-activity relationships for 4-[(phenylmethyl)-
amino]- and 4-[(phenylamino)quinazolines as potent adenosine 50-
triphosphate binding site inhibitors of the tyrosine kinase domain
of the epidermal growth factor receptor. J. Med. Chem. 1995, 38,
3482–3487.
(18) Godin-Heymann, N.; Ulkus, L.; Brannigan, B. W.; McDermott,
U.; Lamb, J.; Maheswaran, S.; Settleman, J.; Haber, D. A. The
T790M “gatekeeper” mutation in EGFR mediates resistance to
low concentration of an irreversible EGFR inhibitor. Mol. Cancer
Ther. 2008, 7, 874–879.
(19) Slichenmyer, W. J.; Elliott, W. L.; Fry, D. W. CI-1033, a pan-erbB
tyrosine kinase inhibitor. Semin. Oncol. 2001, 28, 80–85.
(20) Rabindran, S. K.; Discafani, C. M.; Rosfjord, E. C.; Baxter, M.;
Floyd, M. B.; Golas, J.; Hallett, W. A.; Johnson, B. D.; Nilakan-
tan, D.; OverBeek, E.; Reich, M. F.; Shen, R.; Shi, X.; Tsou, H. R.;
Wang, Y. F.; Wissner, A. Antitumor activity of HKI-272, an orally
active, irreversible inhibitor of the Her-2 tyrosine kinase. Cancer
Res. 2004, 64, 3958–3965.
(36) Roth, G. A.; Tai, J. J. A new synthesis of aryl substituted
quinazolin-4(1H)-ones. J. Heterocycl. Chem. 1996, 33, 2051–2053.
(37) Saito, S.; Komada, K.; Morowake, T. Diethyl (2S,3R)-2-(N-tert-
butoxycarbonyl)amino-3-hydroxysuccinate. Org. Synth. 1995, 73,
184–200.
€
(38) Korn, A.; Rudolph-Bohner, S.; Moroder, L. A convenient synth-
esis of optically pure (2R,3R)-2,3-epoxysuccinyl-dipeptides. Tetra-
hedron 1994, 50, 8381–8392.
(21) Gill, A. L.; Verdonk, M.; Boyle, R. G.; Taylor, R. A comparison of
physicochemical property profiles of marketed oral drugs and
orally bioavailable anti-cancer protein kinase inhibitors in clinical
development. Curr. Top. Med. Chem. 2007, 7, 1408–1422.
(22) Tsou, H.-R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber,
B. C.; Ye, F.; Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.;
Davis, R.; Koehn, F. E.; Greenberger, L. M.; Wang, Y.-F.;
Wissner, A. 6-Substituted-4-(3-bromophenylamino)quinazolines
as putative irreversible inhibitors of the epidermal growth factor
receptor (EGFR) and human epidermal growth factor receptor
(HER-2) tyrosine kinases with enhanced antitumor activity.
J. Med. Chem. 2001, 44, 2719–2734.
(23) Smaill, J. B.; Showalter, H. D. H.; Zhou, H.; Bridges, A. J.;
McNamara, D. J.; Fry, D. W.; Nelson, J. M.; Sherwood, V.;
Vincent, P. W.; Roberts, B. J.; Elliott, W. L.; Denny, W. A.
Tyrosine kinase inhibitors. 18. 6-Substituted 4-anilinoquinazolines
and 4-anilinopyrido[3,4-d]pyrimidines as soluble, irreversible in-
hibitors of the epidermal growth factor receptor. J. Med. Chem.
2001, 44, 429–440.
(24) Mishani, E.; Abourbeh, G.; Jacobson, O.; Dissoki, S.; Daniel,
R. B.; Rozen, G.; Shaul, M.; Levitzki, A. High-affinity epidermal
growth factor receptor (EGFR) irreversible inhibitors with dimin-
ished chemical reactivities as positron emission tomography
(PET)-imaging agent candidates of EGFR overexpressing tumors.
J. Med. Chem. 2005, 48, 5337–5348.
(25) Antonello, A.; Tarozzi, A.; Morroni, F.; Cavalli, A.; Rosini, M.;
Hrelia, P.; Bolognesi, M. L.; Melchiorre, C. Multitarget-directed
drug design strategy: a novel molecule designed to block epidermal
growth factor receptor (EGFR) and to exert proapoptotic effects.
J. Med. Chem. 2006, 49, 6642–6645.
(39) Hayashi, Y.; Shoji, M.; Mukaiyama, T.; Gotoh, H.; Yamaguchi,
S.; Nakata, M.; Kakeya, H.; Osada, H. First asymmetric total
synthesis of synerazol, an antifungal antibiotic, and determination
of its absolute stereochemistry. J. Org. Chem. 2005, 70, 5643–5654.
(40) Tamai, M.; Yokoo, C.; Murata, M.; Oguma, K.; Sota, K.; Sato, E.;
Kanaoka, Y. Efficient synthetic method for ethyl (þ)-(2S,3S)-
3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butylcarbamoyl]-2-
oxiranecarboxylate (EST), a new inhibitor of cysteine proteinase.
Chem. Pharm. Bull. 1987, 35, 1098–1104.
(41) Clerici, F.; Contini, A.; Gelmi, M. L.; Pocar, D. Isothiazoles. Part
14: 3-Aminosubstituted isothiazole dioxides and their mono- and
dihalogeno derivatives. Tetrahedron 2003, 59, 9399–9408.
(42) Lewis, S. N.; Miller, G. A.; Hausman, M.; Szamborski, E. C. 4-
Isothiazolin-3-ones. A general synthesis from 3,30-dithiodipropio-
namides. J. Heterocycl. Chem. 1971, 8, 571–580.
(43) Bordi, F.; Catellani, P. L.; Morini, G.; Plazzi, P. V.; Silva, C.;
Barocelli, E.; Chiavarini, M. 4-(3-Oxo-1,2-benzisothiazolin-2-yl)-
alkanoic, -phenylalkanoic, and -phenoxyalkanoic acids: synthesis
and anti-inflammatory, analgesic and antipyretic properties. Farmaco
1989, 44, 795–807.
(44) Buck, W. Herbicidal[1,2,4]thiadiazoles. U.S. Patent 5,583,092,
1996.
(45) Blair, J. A.; Rauh, D.; Kung, C.; Yun, C.-H.; Fan, Q.-W.; Rode,
H.; Zhang, C.; Eck, M. J.; Weiss, W. A.; Shokat, K. M. Structure
guided development of affinity probes for tyrosine kinases using
chemical genetics. Nat. Chem. Biol. 2007, 3, 229–238.
(46) Stamos, J.; Sliwkowski, M. X.; Eigenbrot, C. Structure of the
epidermal growth factor receptor kinase domain alone and in
complex with a 4-anilinoquinazoline inhibitor. J. Biol. Chem.
2002, 277, 46265–46272.
(26) Wissner, A.; Tsou, H.-R.; Jonson, B. D.; Hamann, P. R.; Zhang, N.
Substituted Quinazoline Derivatives and Their Use as Tyrosine
Kinase Inhibitors. PCT Int. Appl. WO99/09016, 1999.
(27) Ban, S. H.; Usui, T.; Nabeyama, W.; Morita, H.; Fukuzawa, K.;
Nakamura, H. Discovery of boron-conjugated 4-anilinoquinazo-
line as a prolonged inhibitor of EGFR tyrosine kinase. Org.
Biomol. Chem. 2009, 7, 4415–4427.
(28) Potashman, M. H.; Duggan, M. E. Covalent modifiers: an ortho-
gonal approach to drug design. J. Med. Chem. 2009, 52, 1231–1246.
(29) Powers, J. C.; Asgian, J. L.; Dogan Ekici, O.; Ellis James, K.
Irreversible inhibitors of serine, cysteine, and threonine proteases.
Chem. Rev. 2002, 102, 4639–4750.
(47) Domarkas, J.; Dudouit, F.; Williams, C.; Qiyu, Q.; Banerjee, R.;
Brahimi, F.; Jean-Claude, B. J. The Combi-targeting concept:
synthesis of stable nitrosoureas designed to inhibit the epidermal
growth factor receptor (EGFR). J. Med. Chem. 2006, 49, 3544–
3552.
(48) Tsou, H.-R.; Overbeek-Klumpers, E. G.; Hallett, W. A.; Reich, M.
F.; Floyd, M. B.; Johnson, B. D.; Michalak, R. S.; Nilakantan, R.;
Discafani, C.; Golas, J.; Rabindran, S. K.; Shen, R.; Shi, X.; Wang,
Y.-F.; Upeslacis, J.; Wissner, A. Optimization of 6,7-disubstituted-
4-(arylamino)quinoline-3-carbonitriles as orally active, irreversible
inhibitors of human epidermal growth factor receptor-2 kinase
activity. J. Med. Chem. 2005, 48, 1107–1131.
(30) Leung-Toung, R.; Zhao, Y.; Li, W.; Tam, T. F.; Karimian, K.;
Spino, M. Thiol proteases: inhibitors and potential therapeutic
targets. Curr. Med. Chem. 2006, 13, 547–581.
(31) Overall, C. M.; Kleifeld, O. Towards third generation matrix
metalloproteinase inhibitors for cancer theraphy. Br. J. Cancer
2006, 94, 941–946.
(32) Arnold, L. A.; Kosinski, A.; Estebanez-Perpina, E.; Fletterick,
R. J.; Guy, R. K. Inhibitors of the interaction of a thyroid hormone
receptor and coactivators: preliminary structure-reactivity rela-
tionships. J. Med. Chem. 2007, 50, 5269–5280.
(33) Mor, M.; Lodola, A.; Rivara, S.; Vacondio, F.; Duranti, A.;
Tontini, A.; Sanchini, S.; Piersanti, G.; Clapper, J. R.; King,
A. R.; Tarzia, G.; Piomelli, D. Synthesis and structure-reactivity
relationship of fatty acid amide hydrolase inhibitors: modulation at
the N-portion of biphenyl-3-yl alkylcarbamates. J. Med. Chem.
2008, 51, 3484–3498.
(34) Smaill, J. B.; Palmer, B. D.; Rewcastle, G. W.; Denny, V. A.;
McNamara, D. J.; Dobrusin, E. M.; Bridges, A. J.; Zhou, H.;
Showalter, H. D. H.; Winters, R. T.; Leopold, V. R.; Fry, D. V.;
Nelson, J. M.; Slintak, V.; Elliot, V. L.; Roberts, B. J.; Vincent,
P. W.; Patmore, S. J. Tyrosine kinase inhibitors. 15. 4-(Phenyla-
mino)quinazoline and 4-(phenylamino)pyrido[d]pyrimidine acry-
lamides as irreversible inhibitors of the ATP binding site of the
(49) Fry, D. W. Mechanism of action of erb tyrosine kinase inhibitors.
Exp. Cell Res. 2003, 284, 131–139.
(50) Wissner, A.; Overbeek, E.; Reich, M. F.; Floyd, M. B.; Johnson, B.
D.; Mamuya, N.; Rosfjord, E. C.; Discafani, C.; Davis, R.; Shi, X.;
Rabindran, S. K.; Gruber, B. C.; Ye, F.; Hallet, W. A.; Nilakantan,
R.; Shen, R.; Wang, Y.-F.; Greenberger, L. M.; Tsou, H.-R.
Synthesis and structure-activity relationships of 6,7-disubstituted
4-anilinoquinazoline-3-carbonitriles. The design of an orally
active, irreversible inhibitor of the tyrosine kinase activity of
the epidermal growth factor receptor (EGFR) and the human
epidermal growth factor receptor-2 (HER-2). J. Med. Chem.
2003, 46, 49–63.
(51) Johnson, L. N. Protein kinase inhibitors: contributions from
structure to clinical compounds. Q. Rev. Biophys. 2009, 19, 1–40.
(52) Rachid, Z.; Brahimi, F.; Qiu, Q.; Williams, C.; Hartley, J. M.;
Hartley, J. A.; Jean-Claude, B. J. Novel nitrogen mustard-armed
combi-molecules for the selective targeting of epidermal growth
factor receptor overexperessing solid tumors: discovery of an
unusual structure-activity relationship. J. Med. Chem. 2007, 50,
2605–2608.
(53) Smaill, J. B.; Rewcastle, J. W.; Loo, J. A.; Greis, K. D.; Chan, O.
H.; Reyner, E. L.; Lipka, E.; Showalter, H. D. H.; Vincent, P. W.;
Elliott, W. L.; Denny, W. A. Tyrosine kinase inhibitors. 17.

2050 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Carmi et al.
ꢀ
Irreversible inhibitors of the epidermal growth factor receptor:
4-(phenylamino)quinazoline- and 4-(phenylamino)pyrido[3,2-d]-
pyrimidine-6-acrylamides bearing additional solubilizing func-
tions. J. Med. Chem. 2000, 43, 1380–1397.
EGFR con Attivita Antiproliferativa. Italian Patent Application
MI2008A002336, 2008.
(59) Gilday, J. P.; David, M. Process for the Preparation of 4-(30-
Chloro-40-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quin-
azoline. PCT Int. Appl.WO2004/024703, 2004.
(54) Matheson, S. L.; McNamee, J.; Jean-Claude, B. J. Design of
a chimeric 3-methyl-1,2,3-triazene with mixed receptor tyrosine
kinase and DNA damaging properties: a novel tumour targeting
strategy. J. Pharmacol. Exp. Ther. 2001, 296, 832–840.
(55) Cavazzoni, A.; Alfieri, R.; Carmi, C.; Zuliani, V.; Galetti, M.;
Fumarola, C.; Frazzi, R.; Bonelli, M.; Bordi, F.; Lodola, A.; Mor,
M.; Petronini, P. G. Dual mechanism of action of the 5-benzyli-
dene-hydantoin UPR1204 on lung cancer cell lines. Mol. Cancer
Ther. 2008, 7, 361–370.
(60) Weber, W.; Bertics, P. J.; Gill, G. N. Immunoaffinity purification
of the epidermal growth factor receptor. Stoichiometry of binding
and kinetics of self-phosphorylation. J. Biol. Chem. 1984, 259,
14631–14636.
(61) Olive, D. M. Quantitative methods for the analysis of protein
phosphorylation in drug development. Expert Rev. Proteomics
2004, 1, 327–341.
(62) www.cerep.fr.
(56) Alvarez-Sanchez, R.; Basketter, D.; Pease, C.; Lepoittevin, J.-P.
Studies of chemical selectivity of hapten, reactivity, and skin
sensitization potency. 3. Synthesis and studies on the reactivity
toward model nucleophiles of the 13C-labeled skin sensitizers, 5-
chloro-2-methylisothiazol-3-one (MCI) and 2-methylisothiazol-3-
one (MI). Chem. Res. Toxicol. 2003, 16, 627–636.
(57) Palmer, B. D.; Trumpp-Kallmeyer, S.; Fry, D. W.; Nelson, J. M.;
Showalter, H. D. H.; Denny, W. A. Tyrosine kinase inhibitors. 11.
Soluble analogues of pyrrolo- and pyrazoloquinazolines as epidermal
growth factor receptor inhibitors: synthesis, biological evaluation, and
modeling of the mode of binding. J. Med. Chem. 1997, 40, 1519–1529.
(58) Mor, M.; Bordi, F.; Carmi, C.; Vezzosi, S.; Lodola, A.; Petronini,
P. G.; Alfieri, R.; Cavazzoni, A. Composti Inibitori Irreversibili di
(63) Cavazzoni, A.; Petronini, P. G.; Galetti, M.; Roz, L.; Andriani, F.;
Carbognani, P.; Rusca, M.; Fumarola, C.; Alfieri, R.; Sozzi, G.
Dose-dependent effect of FHIT-inducible expression in Calu-1
lung cancer cell line. Oncogene 2004, 23, 8439–8446.
€
(64) Glide, version 5.0; Schrodinger, LLC: New York, 2005.
€
(65) Maestro, version 8.5; Schrodinger, LLC: New York, 2008.
(66) Jorgensen, W. L.; Maxwell, D. S.; Tirado-Rives, J. Development
and testing of the OPLS all-atom force field on conformational
energetics and properties of organic liquids. J. Am. Chem. Soc.
1996, 118, 11225–11236.
(67) Ghosh, A.; Sendrovic Rapp, C.; Friesner, R. A. Generalized Born
model based on a surface integral formulation. J. Phys. Chem. B
1998, 102, 10983–10990.