Combined inhibition of the EGFR/AKT pathways by a novel conjugate of quinazoline with isothiocyanate

Article abstract of DOI:10.1016/j.ejmech.2016.04.002

Epidermal growth factor receptor inhibitors (EGFR-TKIs) represent a class of compounds widely used in anticancer therapy. An increasing number of studies reports on combination therapies in which the block of the EGFR-TK activity is associated with inhibition of its downstream pathways, as PI3K-Akt. Sulforaphane targets the PI3K-Akt pathway whose dysregulation is implicated in many functions of cancer cells. According to these considerations, a series of multitarget molecules have been designed by combining key structural features derived from an EGFR-TKI, PD168393, and the isothiocyanate sulforaphane. Among the obtained molecules 1-6, compound 6 emerges as a promising lead compound able to exert antiproliferative and proapoptotic effects in A431 epithelial cancer cell line by covalently binding to EGFR-TK, and reducing the phosphorylation of Akt without affecting the total Akt levels.

Full text of DOI:10.1016/j.ejmech.2016.04.002

Accepted Manuscript
Combined inhibition of the EGFR/AKT pathways by a novel conjugate of quinazoline
with isothiocyanate
Andrea Tarozzi, Chiara Marchetti, Benedetta Nicolini, Massimo D'Amico, Nicole
Ticchi, Letizia Pruccoli, Vincenzo Tumiatti, Elena Simoni, Alessio Lodola, Marco Mor,
Andrea Milelli, Anna Minarini
PII:
S0223-5234(16)30274-4
10.1016/j.ejmech.2016.04.002
EJMECH 8516
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 December 2015
Revised Date: 10 March 2016
Accepted Date: 1 April 2016
Please cite this article as: A. Tarozzi, C. Marchetti, B. Nicolini, M. D'Amico, N. Ticchi, L. Pruccoli, V.
Tumiatti, E. Simoni, A. Lodola, M. Mor, A. Milelli, A. Minarini, Combined inhibition of the EGFR/AKT
pathways by a novel conjugate of quinazoline with isothiocyanate, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.04.002.
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ACCEPTED MANUSCRIPT
Combined Inhibition of the EGFR/AKT Pathways by a Novel
Conjugate of Quinazoline with Isothiocyanate
Andrea Tarozzi,a Chiara Marchetti,b Benedetta Nicolini,a Massimo D’Amico,a Nicole Ticchi,c
Letizia Pruccoli,a Vincenzo Tumiatti,a Elena Simoni,b Alessio Lodola,d Marco Mor,d Andrea
Milelli,a*Anna Minarinib*
a Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, 47921 Rimini,
Italy
b Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna,
40126 Bologna, Italy
c Interdepartmental Center for Industrial Research, Advanced Mechanical and Materials, Alma
Mater Studiorum-University of Bologna, 47923 Rimini
d Pharmacy Department, University of Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy

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Graphical Abstract
ABSTRACT: Epidermal growth factor receptor inhibitors (EGFR-TKIs) represent a class of
compounds widely used in anticancer therapy. An increasing number of studies report on
combination therapies in which the block of the EGFR-TK activity is associated with inhibition of
its downstream pathways, as PI3K-Akt. Sulforaphane targets the PI3K-Akt pathway whose
dysregulation is implicated in many functions of cancer cells. According to these considerations, a
series of multitarget molecules have been designed by combining key structural features derived
from an EGFR-TKI, PD168393, and the isothiocyanate sulforaphane. Among the obtained
molecules 1-6, compound 6 emerges as a promising lead compound able to exert antiproliferative
and proapoptotic effects in A431 epithelial cancer cell line by covalently binding to EGFR-TK, and
reducing the phosphorylation of Akt without affecting the total Akt levels.
Corresponding Author: Dr. Andrea Milelli, Department for Life Quality Studies, Alma Mater
Studiorum-University of Bologna, C.so D’Augusto 237, 47921 Rimini, Italy, phone +390541 43
4609, andrea.milelli3@unibo.it; Prof. Anna Minarini, Department of Pharmacy and Biotecnhology,
Alma Mater Studiorum-University of Bologna, via Belmeloro 6, 40126 Bologna, Italy, phone
+39051 20 9 9709, anna.minarini@unibo.it.
KEYWORDS: EGFR-TK inhibitors, Multitarget agents, Sulforaphane, Isothiocyanate, Akt
phosphorylation.

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ABBREVIATIONS
EGFR-TK, epidermal growth factor receptor; EGFR-TKI, epidermal growth factor receptor
inhibitor; MET, mesenchymal-epithelial transition factor; PI3K, phosphatidylinositol-3-
kinase; Akt, protein kinase B; SFN, sulforaphane; ITC, isothiocyanate; DTT, dithiothreitol.

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1. Introduction
Over-expression and -activation of epidermal growth factor receptor (EGFR-TK) as well as the
dysregulation of its downstream pathways trigger a cascade of responses that drive the progression
of various human cancers.1 Hence, over the past years, EGFR-TK has been considered one of the
main anticancer target. Unfortunately, several mechanisms of resistance make most of the patients
insensitive to EGFR-TK inhibitors (EGFR-TKs), such as Gefitinib and Erlotinib (Figure 1), usually
a year after starting the treatment.2 Among others, MET amplification, which has been observed in
almost 20% of patients treated with EGFR-TKIs, leads to a persistent activation of EGFR-TK
downstream signaling pathways bypassing EGFR-TK inhibition.3 The T790M is the most frequent
mutation occurring in almost half of patients treated with Gefitinib.4 To overcome these resistance
mechanisms, new generations of EGFR-TKIs have been developed, including irreversible and
mutant-selective ligands.5 Most of the irreversible EGFR-TKIs reported in the literature, as
Dacomitinib and Afatinib (Figure 1), are structurally related to Gefitinib with the exception that
they carry a Michael acceptor group in position 6 of the quinazoline scaffold able to covalently bind
Cys797 of EGFR-TK, a residue located next to the ATP-binding site.6
Figure 1.
An increasing number of studies involves a combination therapies approach by joining a block of
the EGFR-TK activity with the inhibition of downstream pathway elements. Recently, it has been
reported that the combination of a mutant-selective EGFR-TKI, WZ4002, and a MET inhibitor,
E7050, was effective in overcoming resistance to Erlotinib induced by both T790M mutation and
MET amplification.7 Other findings confirm that inhibitors that target the major downstream nodes
of the EGFR-PI3K complex, including Akt, can overcome the EGFR-TKIs resistance in various
tumor types.8, 9
In 2006, Melchiorre and coworkers reported a multitarget ligand based on PD168393 able to
irreversibly block EGFR-TK and to induce apoptosis in A431 human epithelial cancer cells: this
compound was designed by replacing the acrylamide group in position 6 of PD168393 with a 1,2-
dithiolane ring from Lipoic acid.10 This is, indeed, able to covalently bind Cys797 and to exert
proapototic effects.11 Based on this work, we focused our attention on other chemical groups able to
react with Cys and to exert precious anticancer actions.
Isothiocyanate-based compounds, as Sulforaphane (SFN), have attracted significant consideration
as chemopreventive agents acting through a multitude of mechanisms.12, 13 SFN exerts its actions

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thanks to isothiocyanate (ITC) moiety whose electrophilic carbon atom is able to react with
nucleophilic group of amino acids, such as the thiol of Cys, causing a covalent modification of
proteins. Several studies have recorded the ability of ITCs to inhibit the EGFR-TK activity as well
as EGFR-TK-mediated cell survival signals such as PI3K, Akt and NF-kB leading to
antiproliferative effects and induction of apoptosis of tumor cells.14, 15
Based on these premises, we designed and synthesized fused quinazoline derivatives 1-6 (Figure 2)
in which the acrylamide moiety at position 6 of PD168393 was replaced with an ITC group
(compound 6) or with polymethylene-amide linkers of different length and carrying an ITC group
(compounds 1-5). These compounds should, at least in principle, retain the biological activity of the
parent compounds, namely a) the ability to interact with EGFR-TK and covalently bind to Cys797,
and b) to induce downregulation of the phosphorylation of Akt protein. In particular, the combined
inhibition of the EGFR-TK/Akt pathways mediated by the same compound could be crucial in
abolishing the persistent activation of the Akt pathway that protects the tumor cells from apoptosis
induced by EGFR-TKI.16, 17
Figure 2.
2. Results and Discussion
Target compounds 1-6 were synthesized following the procedure depicted in Scheme 1. The N-
protected amino acids (8-12) were obtained by reacting the appropriate commercially available
amino acid with benzyl chloroformate under basic aqueous condition. Subsequent, the carboxylic
acid group of intermediates 8-12 was activated as mixed anhydride through reaction with ethyl
chloroformate in anhydrous DFM at 0°C and coupled with the common quinazoline derivative 718,
leading to intermediates 13-17, respectively. Other coupling reagents, such as EDCI and DCC, were
employed but, unfortunately, they led to poor conversions or to un-separable complex mixture.
Acidic hydrolysis (HBr in acetic acid) of the protecting group led to the primary amines (18-22).
The primary amine group of 18-22 and 7 was easily converted into the corresponding
isothiocyanate, by reacting with the commercially available 1,1’-thiocarbonyldi-2(1H)-pyridone19
in DMF at room temperature, affording the final compounds 1-5 and 6, respectively.
Scheme 1.

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Initially, compounds 1-6 were evaluated for their ability to inhibit the proliferation of the highly
EGFR-TK-expressing A431 and HaCaT cancer cells (Table 1). Both A431 and HaCaT cells were
treated for 72 h with various concentrations (0.03-30 ꢀM) of each compound and the cell
proliferation was measured by MTT assay.
In the same experimental conditions, PD168393, SFN and 7 (synthetic amino precursor of the target
compounds, see Scheme 1) were used as reference compounds. As reported in Table 1, the most
active compound of the series was compound 6 owing an IC50 value in the low micromolar range of
concentration, comparable to that of the irreversible inhibitor PD168398, in both epithelial cancer
cell lines. Inhibition of A431 and HaCaT cell proliferation is strictly dependent on the ITC group
directly linked to position 6 of the quinazoline scaffold since compound 7, bearing an amino group
in place of ITC, is nearly 6-fold less potent than compound 6. Interestingly, compound 6 did not
affect the cell proliferation of human fibroblasts, a control of normal cells, suggesting its good
selectivity.
Table 1.
Compounds 1-5, in which the ITC group is connected to the quinazoline moiety through a
polymethylene-amide linker, showed a reduction of the antiproliferative effects. Interestingly, this
loss of activity does not correlate with the length of the spacer between ITC group and quinazoline
scaffold as shown by the similar effect of compounds 1-5. Modest antiproliferative effect was also
observed with SFN. Taken together, these results show that the appropriate combination of the ITC
moiety with the quinazoline induces an increase of the antiproliferative activity compared to the
parent compounds (6 vs 7 and SFN). In parallel, the inhibitory effects of the new and reference
compounds on EGFR-TK activity were evaluated in A431 cell lysates. The inhibitory effects were
evaluated by reduction of phosphorylation, which was brought about by the receptor kinase with
EGF stimulation. As reported in Table 1, among the new compounds, 6 shows inhibition effects at
nanomolar concentration with a maximum inhibition of 89% at 10 µM. By contrast, compounds 1-5
are weak inhibitors since they induce less than 50% inhibition of the EGFR-TK activity at the
highest concentration tested (20 µM). Remarkably, compound 6 has 7- and 10 fold higher activity
than compound 7 and SFN, respectively. Interestingly, these results suggest that the
antiproliferative activity of compound 6 may involve, a minimal extent, the suppression of EGFR-
TK. In this regard, a recent study reported the ability of various alkyl ITCs, including SFN, to
inhibit EGFR-TK, but not other non-receptor kinases, in A431 cell line suggesting their reactivity
with Cys residues and inactivation of EGFR-TK.20

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As reported, ITC-based compounds are known to be unstable and their stability is affected by pH,
temperature, nucleophilic molecules. In order to rule out possible interference of these parameters,
compounds 6 (with the-NCS group directly mounted on the aromatic rings) and 4 were evaluated
for their stability in phosphate buffer solution at pH 7.2. HPLC-based analysis were performed at
single concentration, at 37 °C and at different time points (0, 0.5, 2, 4 and 24 h). Notably, no
degradation was observed for both compounds (see Supporting Informations).
Taken together these results indicate that the A431 and HaCaT antiproliferative activities as well as
the EGFR-TK inhibition are strongly dependent on the position of the ITC group compared to the
quinazoline moiety and that compound 6 emerges as the one characterized by the better biological
profile. For these reasons, this compound was selected for further studies to elucidate the nature of
the EGFR-TK inhibition that can be either reversible or irreversible. A431 cells were treated with
different concentrations of PD168393, compounds 6 and 7 for 72 h or for 4 h followed by 68 h of
recovery in compound-free medium: a significant reduction of the activity was observed for
compound 7 at 3, 10 and 30 µM concentration proving its reversible interaction with EGFR-TK
while PD168393 and compound 6 showed maintenance of the activity, suggesting their irreversible
interaction with the receptor (see Figure 3).
Figure 3.
In order to evaluate the effect of recognition at the kinase domain on the position of the ITC
warhead, compounds 1-6 were docked within EGFR-TK kinase site, starting from the coordinates
of the EGFR-TK-PD168393 covalent adduct, using the computational protocol modified from
Carmi et al.21 As quantum mechanics/molecular mechanics calculations revealed that Cys797 tends
to be deprotonated by Asp800, the thiolate/aspartic acid species was used for docking.22 In order to
explore all the possible conformations of flexible alkyl ITC of compounds 1-5, no water molecule
was included within the binding site. Docking simulations showed that compound 6 in its top-
ranked pose (Glide score= -7.84 kcal/mol) is accommodated within EGFR-TK active site with a
binding mode resembling that of PD168393 (Glide score = -7.76 kcal/mol), indicating that the
presence of an ITC warhead does not hamper the usual binding mode of the quinazoline moiety
(Figure 4). PD169383 placed the β-carbon atom of the 6-acrylamide group at 4.15 Å from the sulfur
(Figure 4A). Compound 6 placed the reactive carbon of the ITC at a distance from the nucleophile
of only 3.99 Å (Figure 4B) suggesting that it can undergo a nucleophilic attack by Cys797 leading
to formation of a covalent product. Compounds 1-5 are also well recognized at the ATP binding site
of EGFR-TK (best Glide score ranging from –8.10 to –7.42 kcal/mol). However, these simulations

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showed that the reactive centers of 1-5 remained far away from Cys797 sulfur atom (Csp2 – S
distance > 5.7 Å). Linker flexibility allowed, in some cases, the ITC group to be accommodated
closer to the thiolate one, but the corresponding conformations had worse docking scores (Figure
5). This can disfavor the formation of thiocarbamate adducts, thus reducing inhibitor potency,
compared to compound 6.
Figure 4.
Figure 5.
To confirm the irreversibility of the compound-receptor interaction, we evaluated the effect of
dithiothreitol (DTT) on the inhibition of A431 cell proliferation induced by compound 6.23 Cells
were incubated with compound 6 (0.03-30 µM) for 4 h, after which they were allowed to recover
for 68 h in compound-free medium in the absence or presence of DTT. As reported in Figure 6,
DTT induced a reduction of the antiproliferative activity of compound 6, in a dose-dependent
manner, with a significant difference with respect to basal values. These experiments demonstrated
the establishment of a covalent interaction between the ITC group of compound 6 and the Cys797
of the receptor leading to an irreversible block of the EGFR-TK activity.
However, the significant abrogation of the antiproliferative effects of compound 6 recorded after
co-treatment with high DTT concentration can be explained only in part by the inhibition of the
EGFR-TK activity. Indeed, compared to reference compound PD168393, compound 6 showed
similar antiproliferative effects but less inhibitor effects on EGFR-TK activity.
Figure 6.
In this context, the ITC group of compound 6 could inhibit the survival kinase activity involved in
the EGFR-TK downstream signaling pathway by its ability to covalently bind Cys. Recent studies
show that various ITCs reduce the EGFR-TK activity and downstream signaling, including the
suppression in the phosphorylation cascade of PI3K, PDK1 and Akt.14, 15 In particular, targeting the
PI3K-Akt pathway has become attractive in cancer therapy considering its dysregulation in many
cancer cell functions including growth, survival, differentiation and invasion24.
As reported in Figure 7, the treatment of A431 cells with 2 µM concentration of compound 6 and
PD168393 significantly inhibited the increase of phospho-Akt (Ser473) elicited by EGF. By
contrast to PD168393, compound 6 completely abolishes the Akt phosphorylation suggesting its

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ability to directly inhibit this survival kinase. In similar experimental conditions, 6 did not modify
the total Akt levels confirming an inhibitory activity restricted to Akt phosphorylation (data not
shown).
Figure 7.
In addition, the 24 h treatment of A431 cells with the same concentration of compound 6 led to
apoptosis, in terms of DNA fragmentation (Figure 8). The increase of apoptosis (40%) was lower
than that of camptothecin (CAM, 65%), used as a positive control, but similar to that of the
selective inhibitor PD168393 (48%). These findings obtained with compound 6 are particularly
significant, since the phosphorylation of Akt at Ser473 is also associated with tumor resistance to
chemotherapy and radiotherapy.25, 26 In this regard, several studies suggest that a combinatory
therapy of PI3K-Akt pathway inhibitors with EGFRIs provides a promising way to overcome
EGFR-TK resistance.8, 9, 17
Figure 8.
3. Conclusions
In summary, we have developed a novel quinazoline-ITC conjugate 6 able to inhibit the activation
of EGFR-TK and Akt as well as to exert antiproliferative and proapoptotic effects on A431
epithelial cancer cell line. Interestingly, the ITC moiety is responsible for compound 6’s ability to
irreversibly inhibit the receptor and to inhibit survival of the Akt pathway, promoting the cell death.
The combined inhibition of the EGFR-TK/Akt pathways may be a promising approach to overcome
EGFR-TKIs resistance. Our results provide a foundation for further studies to improve the
pharmacological properties of compound 6 through its structural modification and to confirm the
additional inhibitory activity on Akt pathway in vitro and in vivo EGFR-TKI resistance tumor
models.
4. Materials and Methods
4.1 Chemistry
Uncorrected melting points were taken in glass capillary tubes on a Buchi SMP-20 apparatus. ESI-
MS spectra were recorded on Perkin Elmer 297 and Waters ZQ 4000. 1H NMR and 13C NMR were
recorded on Varian VRX 200 and 400 MHz instruments. Chemical shifts are reported in parts per

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million (ppm) relative to peak of tetramethylsilane (TMS) and spin multiplicities are given as s
(singlet), brs (broad singlet), d (doublet), t (triplet), q (quartet) or m (multiplet). The elemental
analysis was performed with Perkin Elmer elemental analyzer 2400 CHN. From all new compounds
satisfactory elemental analyses were obtained, confirming >95% purity. Chromatographic
separations were performed on silica gel columns by flash (Kieselgel 40, 0.040 e 0.063 mm, Merck)
column chromatography. Reactions were followed by thin layer chromatography (TLC) on Merck
(0.25 mm) glass-packed precoated silica gel plates (60 F254) and then visualized in an iodine
chamber or with a UV lamp.
4.1.1 General procedure for the synthesis of 1-6: To a solution of the appropriate compound 18-22
(1 eq) or 7 (1 eq) in anhydrous DMF under nitrogen atmosphere was added a solution of 1,1’-
thiocarbonyldi-2(1H)-pyridone (1 eq). The reaction mixture was stirred overnight at room
temperature. Following solvent removal, the residue was purified by flash chromatography eluting
with a mixture of CH2Cl2/MeOH 9.5/0.5 to give 1-5 or with a mixture of petroleum
ether/CH2Cl2/MeOH 6/3.5/0.5 to give 6.
4.1.1.1 N-(4-((3-bromophenyl)amino)quinazolin-6-yl)-3-isothiocyanatopropanamide (1): Yellow
solid: 29% yield, mp 185°C, 1H NMR (400 MHz, DMSO-d6) δ 2.86 (t, 2H, J = 6.2 Hz), 4.01 (t, 2H,
J = 6.8 Hz), 7.29-7.35 (m, 2H), 7.78-7.88 (m, 3H), 8.17 (s, 1H), 8.59 (s, 1H), 8.74 (s, 1H), 9.94
(brs, 1H, exch D2O), 10.48 (brs, 1H, exch D2O); 13C NMR (100 MHz, DMSO) δ 31.46, 58.17,
120.68, 121.12, 121.21, 123.61, 124.65, 130.33, 130.43, 135.48, 137.29, 154.52, 155.63, 157.51,
167.59, 168.15; ESI-MS (m/z): 429 [M+H]+; Elemental analysis for C18H14BrN5OS Calculated: C,
50.48; H, 3.29; N, 16.35; Found: C, 50.36; H, 3.37; N, 15.99.
4.1.1.2 N-(4-((3-bromophenyl)amino)quinazolin-6-yl)-4-isothiocyanatobutanamide (2): Yellow
1
solid: 41% yield, mp 160°C, H NMR (400 MHz, DMSO-d6) δ 1.97-2.04 (m, 2H), 2.50- 2.54 (m,
2H), 3.79 (t, 2H, J = 6.6 Hz), 7.27-7.36 (m, 2H), 7.75-7.78 (s, 1H), 7.82-7.87 (m, 2H), 8.16 (t, 1H, J
= 1.8 Hz), 8.57 (s, 1H), 8.71 (s, 1H), 9.88 (brs, 1H, exch D2O), 10.35 (brs, 1H, exch D2O); 13C
NMR (100 MHz, DMSO) δ 25.09, 32.78, 44.41, 109.54, 111.75, 115.42, 120.83, 121.09, 124.26,
125.80, 127.18, 130.27, 136.82, 141.16, 146.56, 152.95, 157.23, 170.16; ESI-MS (m/z): 443
[M+H]+; Elemental analysis for C19H16BrN5OS Calculated: C, 51.59; H, 3.65; N, 15.83; Found: C,
51.49; H, 3.77; N, 15.67.

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4.1.1.3 N-(4-((3-bromophenyl)amino)quinazolin-6-yl)-5-isothiocyanatopentanamide (3): Yellow
1
solid: 43% yield, mp 139°C, H NMR (400 MHz, DMSO-d6) δ 1.68-1.87 (m, 4H), 2.41-2.43 (m,
2H), 3.69 (t, 2H, J = 7 Hz), 7.23-7.32 (m, 2H), 7.72 (d, 1H, J = 8.8 Hz), 7.79 (t, 2H, J = 8.0 Hz),
8.12 (t, 1H, J = 1.8 Hz), 8.52 (s, 1H), 8.68 (s, 1H), 9.85 (brs, 1H, exch D2O), 10.22 (brs 1H, exch
13
D2O); C NMR (100 MHz, DMSO) δ 22.08, 28.85, 35.23, 44.58, 120.92, 121.12, 124.34, 125.86,
125.87, 127.20, 130.31, 136.95, 141.16, 146.47, 151.00, 152.92, 171.02; ESI-MS (m/z): 479
[M+Na]+; Elemental analysis for C20H18BrN5OS Calculated: C, 52.64; H, 3.98; N, 15.35; Found: C,
52.50; H, 4.08; N, 15.14.
4.1.1.4 N-(4-((3-bromophenyl)amino)quinazolin-6-yl)-6-isothiocyanatohexanamide (4): Yellow
1
solid: 55% yield, mp 134 °C, H NMR (400 MHz, DMSO-d6) δ 1.41-1.47 (m, 2H), 1.66-1.69 (m,
4H), 2.41 (t, 2H, J = 7.2 Hz), 3.69 (t, 2H, J = 7.0 Hz), 7.29-7.34 (m, 2H), 7.71 (d, 1H, J = 8.8 H),
7.76 (m, 2H), 8.16 (s, 1H), 8.56 (s, 1H), 8.71 (s, 1H), 9.88 (brs, 1H, exch D2O), 10.24 (brs, 1H,
exch D2O); 13C NMR (100 MHz, DMSO) δ 25.29, 26.25, 28.30, 29.54, 45.14, 111.97, 115.85,
121.30, 121.52, 124.73, 126.27, 127.59,128.73, 130.70, 137.46, 141.58, 146.82, 153.28, 157.69,
171.87; ESI-MS (m/z): m/z 471 [M+H]+; Elemental analysis for C21H20BrN5OS Calculated: C,
53.62; H, 4.29; N, 14.89; Found: C, 53.88; H, 4.21; N, 14.70.
4.1.1.5 N-(4-((3-bromophenyl)amino)quinazolin-6-yl)-7-isothiocyanatoheptanamide (5): Yellow
1
solid: 57% yield, mp 129 °C, H NMR (400 MHz, DMSO-d6) δ 1.36 (m, 4H), 1.62-1.65 (m, 4H),
2.38 (t, 2H, J = 7.4 Hz), 3.64 (t, 2H, J = 6.4 Hz), 7.24-7.31 (m, 2H), 7.73-7.82 (m, 3H), 8.14 (s,
13
1H), 8.54 (s, 1H), 8.69 (s, 1H), 9.86 (brs, 1H, exch D2O), 10.20 (brs, 1H, exch D2O); C NMR
(100 MHz, DMSO) δ 24.90, 25.86, 27.91, 29.15, 35.81, 44.75, 111.59, 115.46, 120.92, 121.14,
124.35, 125.89, 127.20, 127.30, 130.32, 137.07, 141.19, 146.43, 152.90, 157.30, 171.48; ESI-MS
(m/z): 508 [M+Na]+; Elemental analysis for C22H22BrN5OS Calculated: C, 54.55; H, 4.58; N, 14.46;
Found: C, 54.26; H, 4.84; N, 14.20.
4.1.1.6 N-(3-bromophenyl)-6-isothiocyanatoquinazolin-4-amine (6): White solid: 21% yield, mp
212°C, 1H NMR (400 MHz, DMSO-d6) δ 7.27-7.36 (m, 2H), 7.78 (d, 1H, J= 8.8Hz), 7.83-7.87 (m,
2H), 8.16 (s, 1H), 8.57 (s, 1H), 8.67 (s, 1H), 9.88 (brs, 1H, exch D2O), 10.28 (brs, 1H, exch D2O);
13C NMR (100 MHz, DMSO) 112.20, 115.87, 121.27, 121.55, 124.68, 126.24, 127.63, 128.84,
130.74, 137.41, 141.64, 146.97, 153.38, 157.68, 168.94; ESI-MS (m/z): 380 [M+Na]+; Elemental
analysis for C15H9BrN4S Calculated: C, 50.43; H, 2.54; N, 15.68; Found: C, 50.32; H, 2.35; N,
15.50.

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4.1.2 General procedure for the synthesis of 18-22: To a cooled solution (0°C) of 13-17 (1 eq) in
AcOH, 33% HBr in AcOH (36 eq) was added dropwise. The reaction mixture was allowed to stir
for 3 h at room temperature. Et2O was added and the resulting solid was washed many times with
Et2O. The solid was dissolved in H2O and the solution was made basic with NaHCO3. After solvent
removal, the residue was purified by flash chromatography eluting with a mixture of
CH2Cl2/MeOH/33% aq.NH4OH 9/1/0.1 to give 18-22.
4.1.2.1 3-amino-N-(4-((3-bromophenyl)amino)quinazolin-6-yl)propanamide (18): Yellow solid:
1
56% yield, H-NMR (400 MHz, CD3OD) δ 2.68 (t, 2H, J = 6.4 Hz), 3.08-3.09 (m, 2H), 7.21-7.22
13
(m, 2H), 7.62-7.68 (m, 3H), 8.04 (d, 1H, J = 0.8 Hz), 8.43 (s, 1H), 8.52 (s, 1H); C-NMR (100
MHz, CD3OD) δ 37.45, 38.11, 112.36, 116.72, 122.07, 123.04, 126.25, 128.06, 131.15, 138.11,
141.74, 147.22, 154.38, 159.16, 172.39; ESI-MS (m/z): 387 [M+H]+.
4.1.2.2 4-amino-N-(4-((3-bromophenyl)amino)quinazolin-6-yl)butanamide (19): Yellow solid: 74%
1
yield. H-NMR (400 MHz, CD3OD) δ 1.97-2.10 (m, 2H), 2.65 (t, 2H, J= 6.8Hz), 3.08 (t, 2H, J =
7.6 Hz), 7.31-7.34 (m, 2H), 7.78-7.85 (m, 3H), 8.10 (s, 1H), 8.57 (s, 1H), 8.69 (s, 1H); ESI-MS
(m/z): 401 [M+H]+.
4.1.2.3 5-amino-N-(4-((3-bromophenyl)amino)quinazolin-6-yl)pentanamide (20): Yellow solid:
76% yield. 1H-NMR (400 MHz, CD3OD) δ 1.57-1.61 (m, 2H), 1.74-1.78 (m, 2H), 2.46 (t, 2H, J =
7.4 Hz), 2.73 (t, 2H, J = 7.4 Hz), 7.25- 7.26 (m, 2H), 7.71-7.73 (m, 3H), 8.07 (s, 1H), 8.48 (s, 1H),
13
8.61 (s, 1H); C-NMR (100 MHz, CD3OD) δ 23.85, 32.06, 37.46, 41.76, 112.36, 116.75, 122.05,
123.05, 126.22, 128.05, 128.13, 131.15, 138.27, 141.76, 147.21, 154.37, 159.19, 174.36; ESI-MS
(m/z): 415 [M+H]+.
4.1.2.4 6-amino-N-(4-((3-bromophenyl)amino)quinazolin-6-yl)hexanamide (21): Yellow solid: 81%
1
yield, H-NMR (400 MHz, CD3OD) δ 1.46-1.52 (m, 2H), 1.67-1.77 (m, 4H), 2.49 (t, 2H, J =
7.4Hz), 2.93 (t, 2H, J = 7.8 Hz), 7.22-7.28 (m, 2H), 7.66 (t, 1H), 7.85 (d, 1H), 8.03 (t, 1H, J = 1.6
13
Hz), 8.50 (s, 1H), 8.57 (d, 1H, J = 2.0 Hz); C-NMR (100 MHz, CD3OD) δ 26.00, 27.00, 28.25,
37.50, 40.63, 112.54, 116.16, 122.52, 122.96, 126.62, 128.71, 128.90, 131.19, 138.87, 143.99,
153.19, 159.47, 174.43; ESI-MS (m/z): 429 [M+H]+.
4.1.2.5 7-amino-N-(4-((3-bromophenyl)amino)quinazolin-6-yl)heptanamide (22): Yellow solid:
69% yield, 1H-NMR (400 MHz, CD3OD) δ 1.23-1.44 (m, 4H), 1.64-1.74 (m, 4H), 2.46 (t, 2H, J =

ACCEPTED MANUSCRIPT
7.4 Hz), 2.90 (t, 2H, J = 6.8 Hz), 7.26-7.27 (m, 2H), 7.70 (m, 2H), 7.78 (d, 1H, J = 8.8 Hz), 8.07 (t,
13
1H, J = 3.4 Hz), 8.49 (s, 1H), 8.59 (d, 1H, J = 2.0 Hz); C-NMR (100 MHz, CD3OD) δ 26.50,
27.52, 29.97, 31.88, 37.78, 41.85, 121.79, 122.95, 125.94, 127.81, 128.01, 128.64, 121.00, 138.15,
141.66, 147.03, 154.20, 158.89, 174.54; ESI-MS (m/z): 443 [M+H]+.
4.1.3 General procedure for the synthesis of 13-17: To a solution of the appropriate compound 8-12
(1 eq) in anhydrous DMF was added Et3N (1.4 eq). After 10 min, to the stirring and cooled solution
(5°C), ethyl chloroformate (1.1 eq) was added dropwise. The white suspension formed was allowed
to reach the room temperature and the stirring was continued for 30 min, after that a solution of 7 (1
eq) in anhydrous DMF was added. The resulting mixture was stirred for 72 h at room temperature.
After solvent removal, the residue was purified by flash chromatography eluting with a mixture of
CHCl3/toluene/MeOH 8.5/1/0.5 to obtain 13-17.
4.1.3.1 Benzyl (3-((4-((3-bromophenyl)amino)quinazolin-6-yl)amino)-3-oxopropyl)carbamate (13):
1
Yellow solid: 25% yield, H NMR (400 MHz, DMSO-d6) δ 2.69-2.73 (m, 2H), 3.51-3.56 (m, 2H),
5.09 (s, 2H), 7.29-7.39 (m, 7H), 7.72-7.82 (m, 3H), 8.07 (s, 1H), 8.25 (brs, 1H, exch D2O), 8.49 (s,
1H), 8.66 (s, 1H), 9.88 (brs, 1H, exch D2O), 10.2 (brs, 1H, exch D2O); ESI-MS (m/z): 521 [M+H]+.
4.1.3.2 Benzyl (4-((4-((3-bromophenyl)amino)quinazolin-6-yl)amino)-4-oxobutyl)carbamate (14):
1
Yellow solid: 28% yield, H NMR (400 MHz, DMSO-d6) δ 1.72-1.79 (m, 2H), 2.38 (t, 2H, J= 7.6
Hz), 3.04-3.09 (m, 2H), 4.98 (s, 2H), 7.24-7.35 (m, 7H), 7.73 (d, 1H, J = 9.2 Hz), 7.78-7.83 (m,
2H), 8.12 (s, 1H), 8.28 (brs, 1H, exch D2O), 8.53 (s, 1H), 8.69 (s, 1H), 9.89 (brs, 1H, exch D2O),
10.2 (brs, 1H, exch D2O); ESI-MS (m/z): 535 [M+H]+.
4.1.3.3 Benzyl (5-((4-((3-bromophenyl)amino)quinazolin-6-yl)amino)-5-oxopentyl)carbamate (15):
1
Yellow solid: 37% yield, H NMR (400 MHz, DMSO-d6) δ 1.39-1.53 (m, 4H), 2.26-2.31 (m, 2H),
2.98-3.06 (m, 2H), 5.01 (s, 2H), 7.24-7.36 (m, 7H), 7.77 (d, 1H, J = 8.8 Hz), 7.84-7.87 (m, 2H),
8.17 (s, 1H), 8.31 (brs, 1H, exch D2O), 8.57 (s, 1H), 8.72 (s, 1H), 9.90 (brs, 1H, exch D2O), 10.2
(brs, 1H, exch D2O); ESI-MS (m/z): 549 [M+H]+.
4.1.3.4 Benzyl (6-((4-((3-bromophenyl)amino)quinazolin-6-yl)amino)-6-oxohexyl)carbamate (16):
1
Yellow solid: 48% yield, H-NMR (400 MHz, DMSO-d6) δ 1.30-1.38 (m, 2H), 1.48-1.50 (m, 2H),
1.64-1.70 (m, 2H), 2.40 (t, 2H, J = 7.4 Hz), 3.00-3.05 (m, 2H), 5.01 (s, 2H), 7.25-7.38 (m, 7H), 7.78

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(d, 1H, J = 8.8 Hz), 7.84-7.88 (m, 2H), 8.18 (s, 1H), 8.32 (brs, 1H, exch D2O), 8.58 (s, 1H), 8.74 (s,
1H), 9.90 (brs, 1H, exch D2O), 10.2 (br s, 1H, exch D2O); ESI-MS (m/z): 563 [M+H]+.
4.1.3.5 Benzyl (7-((4-((3-bromophenyl)amino)quinazolin-6-yl)amino)-7-oxoheptyl)carbamate (17):
1
Yellow solid: 56% yield, H-NMR (200 MHz, DMSO-d6) δ 1.24-1.64 (m, 8H), 2.38 (t, 2H, J = 7.4
Hz), 2.92-3.34 (m, 2H), 5.00 (s, 2H), 7.19-7.23 (m, 7H), 7.76 (d, 1H, J = 8.8 Hz), 7.84-7.87 (m,
2H), 8.16 (s, 1H), 8.30 (brs, 1H, exch D2O), 8.57 (s, 1H), 8.72 (s, 1H), 9.89 (brs, 1H, exch D2O),
10.2 (brs, 1H, exch D2O).;ESI-MS (m/z): 577 [M+H]+.
4.1.4 General procedure for the synthesis of 8-12: To a cooled (0°C) solution of the appropriate
amino acid (1 eq) in NaOH 2N, a solution of benzyl chloroformate (1.1 eq) was added dropwise.
The mixture was kept under vigorous stirring for 30 min. The solution was then washed with Et2O,
acidified to pH = 2 with HCl 3N and extracted with EtOAc. The organic phase was dried over
Na2SO4 and evaporated under vacuo to give 8-12.
1
4.1.4.1 3-(((benzyloxy)carbonyl)amino)propanoic acid (8): White solid: 52% yield, H-NMR (400
MHz, CDCl3) δ 2.51-2.59 (m, 2H), 3.44 (t, 2H, J = 5.7 Hz), 5.07 (s, 2H), 5.28 (brs, 1H, exch D2O),
7.23-7.32 (m, 5H), 11.97 (brs, 1H, exch D2O); ESI-MS (m/z): 224 [M+H]+.
1
4.1.4.2 4-(((benzyloxy)carbonyl)amino)butanoic acid (9): White solid: 52% yield, H NMR (400
MHz, CDCl3) δ 1.82-1.89 (m, 2H), 2.40-2.43 (m, 2H), 3.27 (t, 2H, J = 6.4 Hz), 4.91 (brs, 1H, exch
D2O), 5.13 (s, 2H), 7.27-7.37 (m, 5H), 11.92 (brs, 1H, exch D2O); ESI-MS (m/z): 238 [M+H]+.
4.1.4.2 5-(((benzyloxy)carbonyl)amino)pentanoic acid (10): White solid: 70% yield, 1H NMR (400
MHz, DMSO) δ 1.38-1.52 (m, 4H), 2.20-2.27 (m, 2H), 2.98 (t, 2H, J = 6.38 Hz), 5.00 (s, 2H), 7.23
(brs, 1H, exch D2O), 7.28-7.38 (m, 5H), 11.98 (brs, 1H, exch D2O); ESI-MS (m/z): 252 [M+H]+.
1
4.1.4.3 6-(((benzyloxy)carbonyl)amino)hexanoic acid (11): White solid: 81% yield, H NMR (400
MHz, DMSO) δ 1.35-1.42 (m, 2H), 1.50-1.58 (m, 2H), 1.63-1.70 (m, 2H), 2.36-2.39 (m, 2H), 3.21
(m, 2H, J = 7.4 Hz), 4.87 (brs, 1H, exch D2O), 5.13 (s, 2H), 7.33-7.39 (m, 5H), 9.36 (brs, 1H, exch
D2O); ESI-MS (m/z): 266 [M+H]+.
4.1.4.4 7-(((benzyloxy)carbonyl)amino)heptanoic acid (12): White solid: 88% yield, 1H NMR (400
MHz, DMSO) δ 1.29-1.36 (m, 4H), 1.52-1.60 (m, 4H), 2.30-2.37 (m, 2H), 3.18 (t, 2H, J = 6 Hz),

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5.09 (brs, 1H, exch D2O), 6.03 (s, 2H), 7.38-7.47 (m, 5H), 11.0 (brs, 1H, exch D2O); ESI-MS (m/z):
280 [M+H]+.
4.2 Biology
4.2.1 Cell lines. The effects of the compounds were evaluated in A431 and HaCaT cells, a
malignant and benign human epithelial cancer cell line, respectively. The normal fibroblasts derived
from gingival (HGFs) were used as control.
4.2.2 Cell cultures. A431 and HaCaT cells were routinely grown at 37°C in a humidified incubator
with 5% CO2 in DMEM medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine,
50 U/ml penicillin and 50 ꢀg/ml streptomycin.
HGFs were cultured from biopsies of healthy gingiva of permanent molars. Informed consent was
obtained from the donors. The biopsies were stored at 4 °C for 24 h in DMEM/F12 medium
supplemented with 50 U/ml penicillin and 50 ꢀg/ml streptomycin. The tissue samples were
fragmented in small pieces, placed into tissue culture Petri dishes and grown in DMEM/F12
medium supplemented with antibiotics and 10% FCS at 37 ◦C and 5% CO2. When outgrowth of
cells was observed, the medium was replaced twice weekly until cells reached confluence.
To determine the EGFR tyrosine kinase activity, the A431 cells were seeded in 100-mm culture
dishes at 4x106/dish. To determine the cell proliferation the A431, HaCaT and HGF cells were
seeded in 96-well plates at 1x104 cells/well. To determine the apoptosis the A431 cells were seeded
in 96-well plates at 5x103 cells/well. All experiments were performed after 24 h of incubation at
37°C in 5% CO2.
4.2.3 Determination of cell proliferation. The cell proliferation was evaluated with the MTT [3-
(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay, as described by Antonello
et al.25 Briefly, A431, HaCaT and HGF cells were treated with compounds (0.03-30 µM) for 72 h at
37°C in 5% CO2. After treatment, the cells were washed with phosphate buffered saline (PBS) and
then incubated with MTT (5 mg/ml) in PBS for 4 h. After removal of MTT and further washing, the
formazan crystals were dissolved with isopropanol. The amount of formazan was measured (405
nm) with a multilabel plate reader (VICTORTM X, Perkin Elmer Inc., MA, USA). To determine the
reversibility of antiproliferative activity, the A431 cells were treated with compounds for 4 h, after
which they were allowed to recover for 68 h in compound-free medium. In one set, dithiothreitol
(DTT), a sulfhydryl reducing agent, was added to the compound-free medium.

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The cell viability was expressed as percentage of control cells and calculated by the formula Ft/Fnt x
100, where Ft = absorbance of treated cells and Fnt = absorbance of untreated cells. At least three
independent dose-response curves were plotted and the concentration of compound resulting in
50% inhibition of cell proliferation (IC50) calculated.
4.2.4 Determination of EGFR tyrosine kinase activity. The epidermal growth factor receptor
(EGFR-TK) activity was evaluated in A431 cells treated with epidermal grow factor (EGF, 100
nM) for 60 min in the absence or presence of various concentration (0.01-20 mM) of compounds
according to the manufacturer’s instructions for the Kinase Assay Kit (Millipore, CA, USA).
Tyrosine Kinase Assay Kit is a direct ELISA system for the colorimetric detection of protein
tyrosine phosphotransferase activity using a biotinylated peptide substrate and a monoclonal anti-
phosphotyrosine-HRP conjugate detection antibody. The results are expressed as concentration of
compound resulting in 50% inhibition of tyrosine kinase activity (IC50) in A431 cell stimulated with
EGF.
4.2.5 Determination of phospho- and total-AKT level. The phosphorylated Akt levels were
evaluated in A431 cells treated with EGF (100 nM) for 2 h in the absence or presence of 2 mM
compound according to manufacturer’s instructions for the ab126433 ELISA Kit (abcam®, MA,
USA). The ab126433 Kit is an enzyme-linked immunosorbent assay for the measurement of human
phosphor-Akt (Ser473) and total Akt. The values are expressed as absolute optical density units.
4.2.6 Determination of apoptosis. Apoptosis in terms of DNA fragmentation was determined using
Cell Death Detection ELISAplus (Roche Diagnostics GmbH, Mannheim, Germany) according to the
manufacturer’s instructions. Briefly, A431 cells were treated with 2 mM compound for 24 h at
37°C in 5% CO2. After treatment, the cells were washed, 200 ml lysis buffer was added to each
well for 30 min, followed by centrifugation at 2200 rpm for 10 min. Supernatant (20 ml) was then
incubated in a streptavidin-coated 96-well plate with a mixture of two monoclonal antibodies anti-
histone (biotin-labeled) and anti-DNA (peroxidase-conjugated). After washing the wells 3 times
with incubation buffer, 100 ml of peroxidase substrate solution (ABTS®) was added to each well for
20 min. The amount of colored product was measured (405 nm) with a multilabel plate reader
(VICTORTM X, Perkin Elmer Inc., MA, USA). The values are expressed as fold increase of
apoptosis and calculated by the formula: absorbance of treated sample/ absorbance of untreated
sample.

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4.3 Chemical Stability Studies
Test solutions (2 ꢀM) were prepared by dissolving 2 and 6 in DMSO (1 mg/mL) and by diluting
with pH 7.2 phosphate buffer. Solutions were maintained at 37°C and aliquots (20 ꢀL) were
analyzed at different incubation times by HPLC. Chromatographic separations were performed
using a 1260 Infinity Binary Pump System (Agilent, Palo Alto, CA) equipped with a diode array
detector and a Phenomenex phenyl hexyl (15 mm × 4.6 mm, particle size 5 ꢀm, 100Å). The
selected wavelength was 230 nm. The mobile phase used consisted of acetonitrile and water (15/85
v/v) on isocratic conditions for 10 minutes. The flow rate was 0.6 mL/min with an injection volume
of 20 ꢀL. All reagents and solvents were analytical grade and deionized water was purified through
a Milli Q system (Millipore). Time interval points considered were 0.5, 1, 2, 4 and 24 h.
4.4 Computational Studies
Docking simulations were performed with Glide 6.250 employing the crystal structure of the EGFR
kinase domain covalently bound to PD168393 (PDB code 2J5E).47 Molecular models of PD168393
and 1-6 were built using Maestro 9.7,51 and their geometries were optimized by energy
minimization using the OPLS2005 force field52 in implicit solvent (water) to an energy gradient of
0.01 kcal/(mol Å). The crystal structure of the kinase domain of EGFR-TK was prepared using
procedure implemented in Maestro.53 This included addition of missing side chains, hydrogens, as
well the assignment of tautomeric state of histidines.54 We modeled the tautomer state which
maximized the number of hydrogen bonds. The side chains of Cys797 and Asp800 were modeled in
there thiolate and aspartic acid species, respectively. All the water molecules present in the X-ray
structure were deleted. As a final step, a geometric optimization of the whole system was performed
to resolve steric clashes to a root-mean-square displacement value of 0.3 Å. Docking simulations
were performed starting from minimum energy conformations of PD168393 and compounds 1-6,
using a Glide grid centered on the X-ray structure of PD168393 and featured by enclosing and
bounding boxes of 20 and 14 Å 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.
Acknowledgment

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A.M. and C.M. thank Davide Giorgini for excellent technical support. This work was supported by
University of Bologna (RFO) and Regione Emilia Romagna, Italy (Funding POR FESR 2007-
2013).
Figure and Scheme captions
F
HN
O
HN
Cl
O
O
N
O
O
O
O
N
N
N
N
Gefitinib
Erlotinib
F
F
HN
N
Cl
HN
N
Cl
H
N
H
N
N
N
N
O
O
O
N
O
Dacomitinib
Afatinib
O
Figure 1. Chemical structure of reversible and irreversible EGFR-TK inhibitors.
Figure 2. Drug Design Leading to compounds 1-6.

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Figure 3. Reversibility of antiproliferative effects of 7 (a), 6 (b) and PD168393 (c) in A431 cells.
Cells were incubated with various concentrations of compounds for 4 h (open circles), after which
they were allowed to recover for 68 h in compound-free medium, or continuously for 72 h (closed
circles). Cell proliferation was determined by MTT assay (as described in the experimental section).
Results are reported as mean ± SD of three independent experiments (*p < 0.05, **p < 0.01, vs.
samples treated for 72 h, ANOVA/Bonferroni's test).
Figure 4. Best docking poses for PD168393 (grey carbon atoms, Gscore = -7.76 kcal/mol) and for
compound 6 (white carbon atoms, Gscore = -7.84 kcal/mole) within the ATP binding site of EGFR-
TK. In evidence the hinge region of EGFR-TK (white carbon atoms and cyan ribbons) and the
inhibitor warheads, which lay close to the thiolate group of Cys797.
Figure 5. Docking poses for compound 1 (white carbon atoms) within the ATP binding site of
EGFR. On panel A, the best Glide solution is reported (Gscore = -8.10 kcal/mol), while on

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panel B a docking solution where the ITC group points toward Cys797 is reported (Gscore = -
7.03 kcal/mol),
Figure 6. Effect of DTT on the inhibition of A431 cell proliferation by 6. Cells were incubated with
6 (0.03-30 µM) for 4 h, after which they were allowed to recover for 68 h in compound-free
medium in the absence or presence of DTT. Cell proliferation was determined by the MTT assay
(as described in the supporting information). (**p < 0.01, vs. untreated samples by DTT, ANOVA
with Dunnett's test).
Figure 7. Phospho-Ser473 Akt levels induced by EGF in the presence of 6, PD168393 and SFN in
A431 cells. Cells were treated with compounds (2 ꢀM) for 2 h, after which phospho-Akt levels
were determined by ELISA (as described in the supporting information). (*p<0.05, **p<0.01, vs.
samples treated with EGF, ANOVA with Bonferroni’s test).
Figure 8. Apoptotic effects of SNF, 6, PD168393 and CAM in A431 cells. Cells were treated with
compounds (2 ꢀM) for 24 h, after which apoptosis in terms of DNA fragmentation was determined
by the Cell Death Detection ELISAplus (as described in the supporting information). Results are
reported as mean ± SD of two independent experiments.
13

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Scheme 1. i) Benzyl chloroformate, NaOH 2N, rt, 30 min., 52-88% yields; ii) ethyl chloroformate,
7, DMF, 72h, 0 °C, then rt, 72 h, 25-56 yields; iii) 33% HBr in CH3COOH, rt, 3h, 56-81% yields;
iv) 1,1'-thiocarbonyldi-2(1H)-pyridinone), DMF, N2, rt, overnight, 25-57% yields.
Table 1. Effects of compounds 1-6 and reference compounds PD168393, SFN and 7 on epithelial
cancer (A431 and HaCaT) and normal (HGFs) cell proliferation and EGFR-TK activitya
A431
HaCaT
HGFs
EGFR-TK
IC50 (µM)c
0.036 ± 0.005 (99%)f
9.00 ± 0.36 (69%)f
n.d. e
Compounds
IC50 (µM)b
1.96 ± 0.63
15.76 ± 1.89
17.06 ± 1.87
17.06 ± 1.87
16.28 ± 1.79
14.38 ± 1.50
16.08 ± 2.09
2.04 ± 0.22
11.14 ± 1.44
IC50 (µM)b
IC50 (µM)b
PD168393
SFN
-
-
-
-
-
18.88 ± 1.54
1
2
3
4
5
6
7
16.34 ± 1.02 n.d. d
n.d. e
18.22 ± 1.24
-
n.d. e
16.48 ± 1.65 n.d. d
n.d. e
17.28 ± 1.75
-
n.d. e
2.62 ± 0.31 n.d. d
14.32 ± 1.24 n.d. d
0.88 ± 0.035 (89%)f
6.10 ± 0.183 (66%)f
aThe values are mean ± SD of at least two independent experiments. Concentration of compound
b
c
resulting in 50% inhibition of cell proliferation. Concentration of compound resulting in 50%
inhibition of EGFR-TK activity in A431 cell lysates. dn.d. = IC50 not determined because less than
e
50% inhibition was observed at the highest concentration tested (30 µM). n.d. = IC50 not
determined because less than 50% inhibition was observed at the highest concentration tested (20
µM). fValues in parentheses are the percent inhibition observed at the highest concentration tested.

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References and notes
1.
Yarden, Y.; Sliwkowski, M. X. Untangling the ErbB signalling network. Nat Rev Mol Cell
Biol 2001, 2, 127-37.
2.
Wheeler, D. L.; Dunn, E. F.; Harari, P. M. Understanding resistance to EGFR inhibitors-
impact on future treatment strategies. Nat Rev Clin Oncol 2010, 7, 493-507.
3.
Engelman, J. A.; Zejnullahu, K.; Mitsudomi, T.; Song, Y.; Hyland, C.; Park, J. O.; Lindeman,
N.; Gale, C. M.; Zhao, X.; Christensen, J.; Kosaka, T.; Holmes, A. J.; Rogers, A. M.; Cappuzzo, F.;
Mok, T.; Lee, C.; Johnson, B. E.; Cantley, L. C.; Jänne, P. A. MET amplification leads to gefitinib
resistance in lung cancer by activating ERBB3 signaling. Science 2007, 316, 1039-43.
4.
Yano, S.; Wang, W.; Li, Q.; Matsumoto, K.; Sakurama, H.; Nakamura, T.; Ogino, H.;
Kakiuchi, S.; Hanibuchi, M.; Nishioka, Y.; Uehara, H.; Mitsudomi, T.; Yatabe, Y.; Sone, S.
Hepatocyte growth factor induces gefitinib resistance of lung adenocarcinoma with epidermal
growth factor receptor-activating mutations. Cancer Res 2008, 68, 9479-87.
5.
in cancer. Nat Med 2013, 19, 1389-400.
6. Carmi, C.; Lodola, A.; Rivara, S.; Vacondio, F.; Cavazzoni, A.; Alfieri, R. R.; Ardizzoni, A.;
Chong, C. R.; Jänne, P. A. The quest to overcome resistance to EGFR-targeted therapies
Petronini, P. G.; Mor, M. Epidermal growth factor receptor irreversible inhibitors: chemical
exploration of the cysteine-trap portion. Mini Rev Med Chem 2011, 11, 1019-30.
7.
Nakagawa, T.; Takeuchi, S.; Yamada, T.; Nanjo, S.; Ishikawa, D.; Sano, T.; Kita, K.;
Nakamura, T.; Matsumoto, K.; Suda, K.; Mitsudomi, T.; Sekido, Y.; Uenaka, T.; Yano, S.
Combined therapy with mutant-selective EGFR inhibitor and Met kinase inhibitor for
overcoming erlotinib resistance in EGFR-mutant lung cancer. Mol Cancer Ther 2012, 11,
2149-57.
8.
Sun, Z.; Li, Q.; Zhang, S.; Chen, J.; Huang, L.; Ren, J.; Chang, Y.; Liang, Y.; Wu, G. NVP-
BEZ235 overcomes gefitinib-acquired resistance by down-regulating PI3K/AKT/mTOR
phosphorylation. Onco Targets Ther 2015, 8, 269-77.
9.
Li, H.; Schmid-Bindert, G.; Wang, D.; Zhao, Y.; Yang, X.; Su, B.; Zhou, C. Blocking the
PI3K/AKT and MEK/ERK signaling pathways can overcome gefitinib-resistance in non-small
cell lung cancer cell lines. Adv Med Sci 2011, 56, 275-84.
10.
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, 6642-5.
11.
van de Mark, K.; Chen, J. S.; Steliou, K.; Perrine, S. P.; Faller, D. V. Alpha-lipoic acid
induces p27Kip-dependent cell cycle arrest in non-transformed cell lines and apoptosis in
tumor cell lines. J Cell Physiol 2003, 194, 325-40.
12.
genotoxic potential versus chemoprevention. Mutat Res 2012, 750, 107-31.
13. Milelli, A.; Fimognari, C.; Ticchi, N.; Neviani, P.; Minarini, A.; Tumiatti, V. Isothiocyanate
Fimognari, C.; Turrini, E.; Ferruzzi, L.; Lenzi, M.; Hrelia, P. Natural isothiocyanates:
Synthetic Analogs: Biological Activities, Structure-Activity Relationships and Synthetic
Strategies. Mini Rev Med Chem (14) 2014 963-77.
14.
overexpressing ovarian cancer cells. Mol Cancer Ther (6) 2007, 6, 334-45.
15. H.J. Chen, C.M. Lin, C.Y. Lee, N.C. Shih, S. Amagaya, Y.C. Lin, J.S. Yang, Phenethyl
D. Chaudhuri, S.Orsulic, B.T. Ashok, Antiproliferative activity of sulforaphane in Akt-
isothiocyanate suppresses EGF-stimulated SAS human oral squamous carcinoma cell invasion
by targeting EGF receptor signaling. Int J Oncol (43) 2013 629-37.
16.
H. Hirai, H. Sootome, Y. Nakatsuru, K. Miyama, S. Taguchi, K. Tsujioka, Y. Ueno, H. Hatch,
P.K. Majumder, B.S. Pan, H. Kotani, MK-2206, an allosteric Akt inhibitor, enhances antitumor

ACCEPTED MANUSCRIPT
efficacy by standard chemotherapeutic agents or molecular targeted drugs in vitro and in
vivo. Mol Cancer Ther (9) 2010 1956-67.
17.
M. Puglisi, P. Thavasu, A. Stewart, J.S. de Bono, M.E. O'Brien, S. Popat, J. Bhosle, U.
Banerji, AKT inhibition synergistically enhances growth-inhibitory effects of gefitinib and
increases apoptosis in non-small cell lung cancer cell lines. Lung Cancer (85) 2014, 85, 141-6.
18.
H.R. Tsou, N. Mamuya, B.D. Johnson, M.F. Reich, B.C. Gruber, F. Ye, R. Nilakantan, R.
Shen, C. Discafani, R. DeBlanc, R. Davis, F.E. Koehn, L.M. Greenberger, Y.F. Wang, A Wissner, 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 (44) 2001 2719-34.
19.
S. Kim, K.Y. Yi, 1,1'-Thiocarbonyldi-2,2'-pyridone - a new useful reagent for functional
group convertion under essentially neutral conditions Journal of Organic Chemistry (51) 1986
2613-2615.
20.
T. Nomura, Y. Uehara, H. Kawajiri, K. Ryoyama, T. Yamori, Y. Fuke, Alkyl
isothiocyanates suppress epidermal growth factor receptor kinase activity but augment
tyrosine kinase activity. Cancer Epidemiol (33) 2009 288-92.
21.
C. Carmi, A. Cavazzoni, S. Vezzosi, F. Bordi, F. Vacondio,C. Silva, S. Rivara, A. Lodola, R.R.
Alfieri, S. La Monica, M. Galetti, A. Ardizzoni, P.G. Petronini, M. Mor, Novel irreversible
epidermal growth factor receptor inhibitors by chemical modulation of the cysteine-trap
portion. J Med Chem (53) 2010 2038-50.
22.
L. Capoferri, A. Lodola, S. Rivara, M. Mor, Quantum mechanics/molecular mechanics
modeling of covalent addition between EGFR-cysteine 797 and N-(4-anilinoquinazolin-6-yl)
acrylamide. J Chem Inf Model (55) 2015 589-99.
23.
H.S. Youn, Y.S. Kim, Z.Y. Park, S.Y. Kim, N.Y. Choi, S.M. Joung, J.A. Seo, K.M. Lim, M.K.
Kwak, D.H. Hwang, J.Y. Lee, Sulforaphane suppresses oligomerization of TLR4 in a thiol-
dependent manner. J Immunol (184) 2010 411-9.
24.
pathway in cancer. Nat Rev Drug Discov (8) 2009 627-44.
25. E. Tokunaga, A. Kataoka, Y. Kimura, E. Oki, K. Mashino, K. Nishida, T. Koga, M. Morita, Y.
P. Liu, H. Cheng, T.M. Roberts, J.J. Zhao, Targeting the phosphoinositide 3-kinase
Kakeji, H. Baba, S. Ohno, Y. Maehara, The association between Akt activation and resistance to
hormone therapy in metastatic breast cancer. Eur J Cancer (42) 2006 629-35.
26.
T.J. Kim, J.W. Lee, S.Y. Song, J.J. Choi, C.H. Choi, B.G. Kim, J.H. Lee, D.S. Bae, Increased
expression of pAKT is associated with radiation resistance in cervical cancer. Br J Cancer (94)
2006 1678-82.

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• The combined inhibition of the EGFR-TK/Akt pathways may be a promising
approach to overcome EGFR-TKIs resistance.
• We report on new multitarget agents obtained by combining key structural
features from EGFR-TK inhibitor PD168393 and Sulforaphane.
• Novel conjugate 6 emerges as promising lead compound able to exert
proapoptotic and antiproliferative activity in A451 lung cancer cell line
• Compound 6 inhibits the activation of EGFR-TK and completely abolishes Akt
phosphorylation without affecting the total Akt levels.