The design and synthesis of a new class of RTK/HDAC dual-targeted inhibitors

Article abstract of DOI:10.3390/molecules18066491

Over the years, the development of targeted medicines has made significant achievements. As a typical example, receptor tyrosine kinases (RTK) inhibitors have become important chemotherapy drugs for a variety of cancers. However, the effectiveness of these agents is always hindered by poor response rates and acquired drug resistance. In order to overcome these limitations, several dual-targeted inhibitors with quinazoline core were designed and synthesized. Though these compounds can simultaneously inhibit histone deacetylases (HDAC) as well as RTK, the structure-activity relationship (SAR) is still not clear enough. To further explore this type of dual-targeted inhibitors, a new class of quinazoline derivatives were designed and synthesized. Their activity evaluations include in vitro inhibitory activity of HDAC, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2). The SAR study indicated that the introduction of polar group such as hydroxamate on the 4-position of the quinazoline core is more likely to provide a potent HDACi/HER2i hybrid rather than HDACi/EGFRi molecule.

Full text of DOI:10.3390/molecules18066491

Molecules 2013, 18, 6491-6503; doi:10.3390/molecules18066491
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
The Design and Synthesis of a New Class of RTK/HDAC
Dual-Targeted Inhibitors
Xuan Zhang 1, Mingbo Su 2, Yi Chen 3, Jia Li 3 and Wei Lu 1,*
1
Institute of Drug Discovery and Development, Shanghai Engineering Research Center of Molecular
Therapeutics and New Drug Development, East China Normal University, 3663 North Zhongshan
Road, Shanghai 200062, China; E-Mail: 52113300004@student.ecnu.edu.cn
2
School of Life Sciences, East China Normal University, 3663 North Zhongshan Road,
Shanghai 200062, China; E-Mail: mbsu@mail.shcnc.ac.cn
3
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, SIBS,
Chinese Academy of Sciences, Shanghai 201203, China; E-Mails: ychen@mail.shcnc.ac.cn (Y.C.);
jli@mail.shcnc.ac.cn (J.L.)
* Author to whom correspondence should be addressed; E-Mail: wlu@chem.ecnu.edu.cn;
Tel./Fax: +86-21-6260-2475.
Received: 13 May 2013; in revised form: 21 May 2013 / Accepted: 24 May 2013 /
Published: 3 June 2013
Abstract: Over the years, the development of targeted medicines has made significant
achievements. As a typical example, receptor tyrosine kinases (RTK) inhibitors have
become important chemotherapy drugs for a variety of cancers. However, the effectiveness
of these agents is always hindered by poor response rates and acquired drug resistance. In
order to overcome these limitations, several dual-targeted inhibitors with quinazoline core
were designed and synthesized. Though these compounds can simultaneously inhibit
histone deacetylases (HDAC) as well as RTK, the structure-activity relationship (SAR) is
still not clear enough. To further explore this type of dual-targeted inhibitors, a new class
of quinazoline derivatives were designed and synthesized. Their activity evaluations
include in vitro inhibitory activity of HDAC, epidermal growth factor receptor (EGFR) and
human epidermal growth factor receptor 2 (HER2). The SAR study indicated that the
introduction of polar group such as hydroxamate on the 4-position of the quinazoline core
is more likely to provide a potent HDACi/HER2i hybrid rather than HDACi/EGFRi molecule.

Molecules 2013, 18
Keywords: receptor tyrosine kinases; histone deacetylase; antitumor; synergistic effect;
6492
dual inhibitor
1. Introduction
For decades, cytotoxic drugs such as paclitaxel and camptothecin have been the mainstay of cancer
chemotherapy. However, almost all traditional cytotoxic agents suffer from severe toxicities and other
undesirable side effects. In recent years, the development of targeted medicines has made significant
achievements. Unfortunately, though these agents can block key regulators of signaling pathways in
cancer, multiple compensatory pathways always attenuate pharmacological effect of single target
drugs. In addition, poor response rates and acquired drug resistance also represent a significant barrier
to widespread use of targeted medicines. More recently, a number of combinatorial therapies have
expanded treatment options [1–5], which can directly block several key signaling pathways and create
synergistic effect [6–9]. But in the meantime, these agents can produce adverse effects related to
pharmacokinetics, toxicity and patient compliance. Therefore, in order to overcome these barriers, a
new strategy for designing a single molecule with antitumor activities to inhibit multiple targets was
developed [10–14].
Receptor tyrosine kinases (RTK) and histone deacetylases (HDAC) play crucial roles in numerous
biological processes. Many selective inhibitors of human epidermal growth factor receptor (HER)
family RTK, including erlotinib, gefitinib, and lapatinib, share the same quinazoline core and are
important therapeutics against multiple solid tumor cancers (Figure 1) [15,16]. By inducing histone
hyperacetylation, HDAC inhibitors (HDACi) can cause growth arrest, differentiation and apoptosis in
tumor [17–20]. Typically, a HDAC inhibitor consists of a capping group, a zinc-binding group (ZBG)
and an appropriate linker. There are various ZBGs for HDACi, including hydroxamic acids,
carboxylates, aminobenzamides and so on. Up to now, two HDAC inhibitors, vorinostat (SAHA) and
romidepsin (FK228), have been approved by FDA for the treatment of cutaneous T-cell lymphoma
(CTCL) [21–23]. However, HDACi monotherapies often have clinical limitations [24]. Recently,
several groups investigated a novel type of multi-targeted agents, RTK/HDAC dual inhibitors.
Subsequent pharmaceutical study revealed their potential ability to overcome tumour recurrence and
drug resistance [8,11,13,25]. In these pioneering studies, the zinc-binding groups such as hydroxamate
were all introduced into the hydrophilic segment (6, 7 positions of the quinazoline core). To further
explore the structure-activity relationships of these dual action inhibitors, and to find potent antitumor
agents, our group initiated a program of RTK/HDAC dual inhibitors.
Figure 1. Representative compounds of RTK inhibitors.

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In contrast to the reported RTK/HDAC hybrids, this series of novel dual action inhibitors contain
the zinc-binding group on the phenyl ring (Figure 2). To probe the effect of location of ZBG, para-
and meta-substituted compounds were synthesized. Subsequently, we have evaluated their in vitro
inhibitory activity against HDAC, EGFR and HER2.
Figure 2. Design of dual inhibitors of RTK and HDAC.
2. Results and Discussion
2.1. Chemisty
The general route for the synthesis of HDAC/RTK dual-acting inhibitors is depicted in Scheme 1.
Starting materials 1a,b were synthesized according to the published method [26]. Subsequently, 1a,b
were subjected to aromatic nucleophilic substitution with arylamines to give esters 2a–b and 3a-b,
respectively (48%–93% yield). Hydrolysis of these esters proceeded smoothly to afford the
corresponding acids 4a–b and 5a–b. Treatment of compounds 2a–b and 3a–b with H2N-OTHP in the
presence of LHMDS gave the compounds 6a–b and 7a–b, which were hydrolyzed under acidic
conditions to afford 8a–b and 9a–b. Similarly, treatment of 2a–b and 3a–b with H2N-OBn followed
by hydrogenation afforded 12a–b and 13a–b.

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Scheme 1. Synthesis of dual-acting HDAC-RTK inhibitors.
Reagents and conditions: (a) (E)-ethyl 3-(4-aminophenyl)acrylate/(E)-ethyl 3-(3-aminophenyl)acrylate,
i-PrOH, reflux; (b) LiOH, MeOH, H2O, reflux; (c) H2NOTHP, LHMDS, THF, −78 °C 1h, then 0 °C 5 h;
(d) 1N HCl (aq), MeOH; (e) H2NOBn, LHMDS, THF, −78 °C 1 h, then 0 °C 12 h; (f) 10% Pd/C, H2, MeOH.
2.2. Results and Discussion
2.2.1. In Vitro HDAC Inhibition
The inhibition of recombinant human HDAC1, HDAC3 and HDAC6 enzymes was tested first,
using SAHA as the positive control (Table 1). Generally, most compounds exhibited moderate to good
inhibitory activity against HDAC1, HDAC3 and HDAC6 (compounds 8a–b, 9a–b, 12a–b and 13a–b),
except for compounds 4a–b and 5a–b, which conformed to the reported information that hydroxamic
acid generally showed more potent HDAC inhibitory activity than carboxylic acids [27,28]. In
addition, the location of ZBGs also exerted an influence on the HDAC inhibition. Interestingly, the
saturated hydroxamates, both meta-substituted (13a,b) and para-substituted (12a,b), showed similar
anti-HDAC activity, despite their different ZBG location. The difference between meta- and para-
substitution might be reduced by the flexibility of the saturated chains. By contrast, the HDAC
inhibitory activities were quite different for unsaturated hydroxamates (compounds 8a,b and 9a,b).
The para-substituted 8a,b exhibited moderate inhibition, while the meta-substituted counterparts
(9a,b) showed much more potent activity, suggesting that appropriate rigid linker benefited the
coordination between the ZBGs and the zinc ion of the enzyme. Furthermore, the two different
hydrophilic moieties on the quinazoline core exhibited no obvious difference for the HDAC inhibitory
activity (8a vs 8b, 9a vs 9b, 12a vs 12b and 13a vs 13b). Among all these conjugates, compound 9a

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showed most potent anti-HDAC activity (HDAC-1 IC50 = 0.16 μm; HDAC-3 IC50 = 0.18 μm; HDAC-6
IC50 = 0.56 μm).
Table 1. In vitro HDAC Inhibition.
HDAC-1
HDAC-3
HDAC-6
Compound IC50 ± SD IC50 ± SD IC50 ± SD
[μM]
[μM]
[μM]
a
4a
4b
-
-
-
-
-
-
-
-
-
-
-
-
5a
5b
8a
5.57 ± 1.87 2.38 ± 0.38 1.66 ± 0.19
2.77 ± 1.02
8b
-
-
9a
0.16 ± 0.02 0.18 ± 0.05 0.56 ± 0.06
0.29 ± 0.05 0.15 ± 0.02 0.56 ± 0.05
2.83 ± 0.65 3.29 ± 0.34 5.71 ± 0.99
5.82 ± 1.15 3.90 ± 0.78 7.21 ± 0.76
1.03 ± 0.27 2.11 ± 0.34 7.63 ± 0.99
2.43 ± 0.50 1.57 ± 0.17 4.82 ± 0.37
0.25 ± 0.04 0.17 ± 0.02 0.23 ± 0.06
9b
12a
12b
13a
13b
SAHA
Lapatinib
-
-
-
a HDAC inhibition < 50% at 20 μg/mL.
2.2.2. In Vitro RTK Inhibition
Subsequently, the in vitro inhibitory activities of EGFR and HER2 were assessed by enzyme-linked
immunosorbent assay (ELISA) [29], employing lapatinib as the positive control. As shown in Table 2,
all of these derivatives showed reduced anti-RTK activity, compared with lapatinib, suggesting that the
polar groups such as hydroxamate on the phenyl group exerted negative effects on RTK inhibition [13]. On
the contrary, the hydroxamate group on the 6, 7 positions of the quinazoline core could retain their
RTK inhibition activity as reported [11,13,25]. In the light of the above results, lipophilic benzamide
seemed to be more suitable than hydroxamate to serve as the ZBG on the phenyl ring. Cinnamoyl
hydroxamates exhibited more potent inhibition against HER2 (compounds 8a,b and 9a,b). Among all
these derivatives, compound 8b showed most potent anti-EGFR and anti-HER2 activities.
Table 2. In vitro RTK Inhibition.
EGFR
HER2
Compound
%Inhibition a %Inhibition b
4a
4b
5a
5b
8a
8b
9a
6.1
20.1
20.9
19.0
63.6
70.1
9.7
18.2
0
0
2.6
74.0
84.6
47.2

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Table 2. Cont.
EGFR
HER2
Compound
%Inhibition a %Inhibition b
9b
12a
8.5
0
64.2
0
12b
44.6
42.6
20.4
0
0.1
0
13a
13b
0
SAHA
0
Lapatinib
92.7
92.0
a Inhibition ratio of EGFR, inhibitor was at 10 μg/mL. b Inhibition ratio of HER2, inhibitor was at 10 μg/mL.
3. Experimental
3.1. General
Melting points were taken on a Fisher-Johns melting point apparatus, are uncorrected and reported
1
in degrees centigrade. H-NMR spectra and 13C-NMR were recorded in CDCl3, CD3OD, D2O and
DMSO-d6 on a Bruker DRX-500 (500 MHz) or a Bruker Ascend 400 (400 MHz) using TMS as
internal standard. Chemical shifts were reported as δ (ppm) and spin-spin coupling constants as J (Hz)
values. The mass spectra (MS) were recorded on a Finnigan MAT-95 mass spectrometer. The purity of
all tested compounds was established by HPLC to be > 95%. HPLC analyses were performed on an
Agilent 1200 series instrument using an Agilent Eclipse XDB-C18 (250 mm × 4.6 mm) column.
3.2. Chemistry
3.2.1. General Procedure for the Synthesis of Compounds 2a, 2b, 3a, 3b
Substituted 4-chloroquinazoline (1.0 eq) and corresponding arylamine (2.0 eq) were refluxed in
isopropanol for 5 h. After cooling to room temperature, the mixture was treated with Et2O. The
resulting solid was filtered and washed with EtOAc.
Ethyl (E)-3-(4-((7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)amino)phenyl)acrylate (2a).
Starting from 1a and (E)-ethyl 3-(4-aminophenyl)acrylate, 48.3% of 2a was obtained as a white solid
1
according to the aforementioned procedure, mp: 198–200 °C. H-NMR (500 MHz, CDCl3) δ 1.34 (t,
J = 7.1 Hz, 3H), 2.10–2.15 (m, 2H), 2.40–2.50 (m, 4H), 2.58 (t, J = 7.1 Hz, 2H), 3.70–3.75 (m, 4H),
4.00 (s, 3H), 4.21 (t, J = 6.6 Hz, 2H), 4.27 (q, J = 7.1 Hz, 2H), 6.39 (d, J = 16.0 Hz, 1H), 7.14 (s, 1H),
7.47(s, 1H), 7.57 (d, J = 8.5 Hz, 2H), 7.68 (d, J = 16.0, 1H), 7.79 (d, J = 8.5 Hz, 2H), 8.69(s, 1H) ;
HRMS (ESI): m/z calcd for C27H33N4O5 (M+H+): 493.2446, found: 493.2452.
Ethyl (E)-3-(4-((6,7-bis(2-methoxyethoxy)quinazolin-4-yl)amino)phenyl)acrylate (2b). Starting from
1b and (E)-ethyl 3-(4-aminophenyl)acrylate, 81.2% of 2b was obtained as a white solid according to
1
the aforementioned procedure, mp: 233–236 °C. H-NMR (500 MHz, D2O-DMSO-d6) δ 1.20 (t,
J = 7.2 Hz, 3H), 3.31 (s, 3H), 3.33 (s, 3H), 3.70–3.78 (m, 4H), 4.12 (q, J = 7.2 Hz, 2H), 4.18–4.25

Molecules 2013, 18
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(m, 4H), 6.35 (d, J = 16.0 Hz, 1H), 7.06 (s, 1H), 7.43–7.60 (m, 5H), 7.75 (s, 1H), 8.53 (s, 1H); HRMS
(ESI): m/z calcd for C25H30N3O6 (M+H+): 468.2129, found: 468.2134.
Ethyl (E)-3-(3-((7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)amino)phenyl)acrylate (3a).
Starting from 1a and (E)-ethyl 3-(3-aminophenyl)acrylate, 85.3% of 3a was obtained as a white solid
1
according to the aforementioned procedure, mp: 104–105 °C. H-NMR (400 MHz, DMSO) δ1.27 (t,
J = 7.1 Hz, 3H), 1.92–2.07 (m, 2H), 2.35–2.43 (m, 4H), 2.46–2.49 (m, 2H), 3.55–3.62 (m, 4H), 3.94
(s, 3H), 4.14–4.27 (m, 4H), 6.60 (d, J = 16.0 Hz, 1H), 7.20 (s, 1H), 7.41–7.54 (m, 2H), 7.67 (d,
J = 16.0 Hz, 1H), 7.81–7.90 (m, 2H), 8.06 (s, 1H), 8.48 (s, 1H), 9.55 (s, 1H). HRMS (ESI): m/z calcd
for C27H33N4O5 (M+H+): 493.2450, found: 493.2452.
Ethyl (E)-3-(3-((6,7-bis(2-methoxyethoxy)quinazolin-4-yl)amino)phenyl)acrylate (3b). Starting from
1b and (E)-ethyl 3-(3-aminophenyl)acrylate, 93.6% of 3b was obtained as a white solid according to
1
the aforementioned procedure, mp: 178–181 °C. H-NMR (500 MHz, D2O-DMSO-d6) δ 1.51 (t,
J = 7.0 Hz, 3H), 3.62 (s, 3H), 3.63 (s, 3H), 4.00–4.08 (m, 4H), 4.42–4.50 (m, 6H), 6.70 (d, J = 15.8 Hz,
1H), 7.31 (s, 1H), 7.67–7.70 (m, 2H), 7.77 (d, J = 15.8 Hz, 1H), 7.85 (d, J = 6.5 Hz, 1H), 7.99 (s, 2H),
8.76(s, 1H); HRMS (ESI): m/z calcd for C25H30N3O6 (M + H+): 468.2129, found: 468.2141.
3.2.2. General Procedure for the Synthesis of Compounds 4a, 4b, 5a, 5b
Ester (1.0 eq) and LiOH (4.0 eq) were refluxed in a mixture MeOH and water (5:2) for 2h. After
cooling to 0 °C, the resulting solid was filtered off and washed with water followed by EtOH.
(E)-3-(4-((7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)amino)phenyl)acrylic
acid
(4a).
Starting from 2a, 89.0% of 4a was obtained as a yellow solid according to the aforementioned
procedure, mp: 242–245 °C. 1H-NMR (500 MHz, DMSO-d6) δ 2.30–2.35 (m, 2H), 3.01–3.50 (m, 6H),
3.75–3.85 (m, 2H), 3.90–3.95 (m, 2H), 3.99 (s, 3H), 4.35–4.40 (m, 2H), 6.55 (d, J = 16.0 Hz, 1H), 7.39
(s, 1H), 7.61 (d, J = 16.0 Hz, 1H), 7. 78 (d, J = 8.5 Hz, 2H), 7.87 (d, J = 8.5 Hz, 2H), 8.59 (s, 1H),
8.85(s, 1H); HRMS (ESI): m/z calcd for C25H29N4O5 (M+H+): 465.2132, found: 465.2156.
(E)-3-(4-((6,7-bis(2-methoxyethoxy)quinazolin-4-yl)amino)phenyl)acrylic acid (4b). Starting from 2b,
65.0% of 4b was obtained as a yellow solid according to the aforementioned procedure, mp:
1
298–300 °C (decomp.). H-NMR (500 MHz, DMSO-d6) δ 3.34 (s, 3H), 3.35 (s, 3H), 3.70–3.78
(m, 4H), 4.35–4.40 (m, 4H), 6.42 (d, J = 16.0 Hz, 1H), 7.20 (s, 1H), 7.34 (d, J = 16.0 Hz, 1H), 7.56
(d, J = 8.5 Hz, 2H), 7.87 (d, J = 8.5 Hz, 2H), 7.94 (s, 1H), 8.46 (s, 1H), 9.69 (s, 1H); HRMS (ESI): m/z
calcd for C23H26N3O6 (M + H+): 440.1816, found: 440.1867.
(E)-3-(3-((7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)amino)phenyl)acrylic
acid
(5a).
Starting from 3a, 80.1% of 5a was obtained as a white solid according to the aforementioned
procedure, mp: 195–197 °C. 1H-NMR (500 MHz, DMSO-d6) δ 2.30–2.37 (m, 2H), 3.07–3.17 (m, 2H),
3.30–3.35 (m, 2H), 3.45–3.53 (m, 2H), 3.78–3.85 (m, 2H), 3.92–3.98 (m, 2H), 4.00 (s, 3H), 4.35–4.42
(m, 2H), 6.55 (d, J = 16.0 Hz, 1H), 7.41 (s, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.53–7.62 (m, 2H), 7.80 (d,
J = 8.0 Hz, 1H), 8.07 (s, 1H), 8.61 (s, 1H), 8.84(s, 1H), 11.05–11.15 (br, 1H), 11.76 (s, 1H); HRMS
(ESI): m/z calcd for C25H29N4O5 (M+H+): 465.2132, found: 465.2134.

Molecules 2013, 18
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(E)-3-(3-((6,7-bis(2-methoxyethoxy)quinazolin-4-yl)amino)phenyl)acrylic acid (5b). Starting from 3b,
70.0% of 5b was obtained as a white solid according to the aforementioned procedure, mp:
1
219–222 °C. H NMR (500 MHz, DMSO-d6) δ 3.35 (s, 3H), 3.37 (s, 3H), 3.70–3.85 (m, 4H),
4.25–4.32 (m, 4H), 6.51 (d, J = 15.8 Hz, 1H), 7.23 (s, 1H), 7.40–7.45 (m, 2H), 7.60 (d, J = 15.8 Hz,
1H), 7.88 (s, 2H), 8.03 (s, 1H), 8.46 (s, 1H), 9.53 (s, 1H), 11.5–12.5 (br, 1H); HRMS (ESI): m/z calcd
for C23H26N3O6 (M + H+): 440.1816, found: 440.1840.
3.2.3. General Procedure for the Synthesis of Compounds 6a, 6b, 7a, 7b
Ester (1.0 eq) and O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (3.0 eq) were dissolved in THF.
The mixture was cooled to −78 °C and 1 M LHMDS in THF (6.0 eq) was dropped into the flask. After
stirring at this temperature for 1 h, it was warmed to 0 °C and reacted for another 5 h. Subsequently it
was extracted with EtOAc and concentrated in vacuo. The crude product was crystallized in a mixture
of EtOAc and petroleum ether and used directly in the next step.
3.2.4. General Procedure for the Synthesis of Compounds 8a, 8b, 9a, 9b
Compound 6a/6b/7a/7b in MeOH was treated with 1 M HCl aq (10 eq). The mixture was stirred at
room temperature overnight and the solvent was removed under reduced pressure. Crystallization of
the crude product with EtOH gave the pure title compounds.
(E)-N-hydroxy-3-(4-((7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)amino)phenyl)acrylamide
(8a). Starting from 6a, 39.2% of 8a was obtained as a yellow solid according to the aforementioned
procedure, mp: 222–224 °C. 1H-NMR (500 MHz, DMSO-d6) δ 2.28–2.35 (m, 2H), 3.09–3.50(m, 6H),
3.70–3.80 (m, 2H), 3.90–3.95 (m, 2H), 3.99 (s, 3H), 4.35–4.40 (m, 2H), 6.49 (d, J = 15.8 Hz, 1H), 7.39
(s, 1H), 7.46 (d, J = 15.8 Hz, 1H), 7.62–7.87 (m, 4H), 8.56 (s, 1H), 8.83 (s, 1H), 10.80–10.95 (m, 2H),
11.55–11.62 (br, 1H); HRMS (ESI): m/z calcd for C25H30N5O5 (M + H+): 480.2241, found: 480.2254.
(E)-3-(4-((6,7-bis(2-methoxyethoxy)quinazolin-4-yl)amino)phenyl)-N-hydroxyacrylamide
(8b).
Starting from 6b, 65.8% of 8b was obtained as a yellow solid according to the aforementioned
1
procedure, mp: 237–240 °C. H-NMR (500 MHz, DMSO-d6) δ 3.35 (s, 6H), 3.45–3.55 (m, 4H),
4.30–4.40 (m, 4H), 6.50 (d, J = 15.0 Hz, 1H), 7.38 (s, 1H), 7.48 (d, J = 15.0 Hz, 1H), 7.60–7.85 (m,
4H), 8.37 (s, 1H), 8.83 (s, 1H), 10.80–10.85 (br, 1H), 11.41 (s, 1H); HRMS (ESI): m/z calcd for
C23H27N4O6 (M+H+): 455.1925, found: 455.1943.
(E)-N-hydroxy-3-(3-((7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)amino)phenyl)acrylamide
(9a). Starting from 7a, 86.2% of 9a was obtained as a white solid according to the aforementioned
procedure, mp: 230–233 °C. 1H-NMR (500 MHz, DMSO-d6) δ 2.30–2.35 (m, 2H), 3.08–3.15 (m, 2H),
3.30–3.50 (m, 4H), 3.78–3.83 (m, 2H), 3.92–3.96 (m, 2H), 3.98 (s, 3H), 4.34–4.40 (m, 2H), 6.54 (d,
J = 15.5 Hz, 1H), 7.37–7.50 (m, 4H), 7.70–7.75 (m, 1H), 7.92 (s, 1H), 8.57 (s, 1H), 8.83 (s, 1H),
10.80–11.10 (m, 2H), 11.73 (br, 1H); HRMS (ESI): m/z calcd for C25H30N5O5 (M + H+): 480.2241,
found: 480.2244.

Molecules 2013, 18
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(E)-3-(3-((6,7-bis(2-methoxyethoxy)quinazolin-4-yl)amino)phenyl)-N-hydroxyacrylamide
(9b).
Starting from 7b, 90.8% of 9b was obtained as a white solid according to the aforementioned
1
procedure, mp: 214–217 °C. H-NMR (500 MHz, DMSO-d6) δ 3.35 (s, 6H), 3.68–3.78 (m, 4H),
4.30–4.38 (m, 4H), 6.52 (d, J = 16.0 Hz, 1H), 7.35–7.70 (m, 5H), 7.86 (s, 1H), 8.25–8.35 (m, 1H), 8.81
(s, 1H), 10.80–10.90 (br, 1H), 11.25–11.45 (m, 1H); HRMS (ESI): m/z calcd for C23H27N4O6 (M+H+):
455.1925, found: 455.1933.
3.2.5. General Procedure for the Synthesis of Compounds 10a, 10b, 11a, 11b
Ester (1.0 eq) and O-benzylhydroxylamine hydrochloride (3.0 eq) were dissolved in THF. The
mixture was cooled to −78 °C and 1M LHMDS in THF (10.0 eq) was dropped in. After stirring at this
temperature for 1 h, it was warmed to room temperature and reacted for another 12 h. Then it was
extracted with DCM and concentrated in vacuo. The crude product was crystallized in a mixture of
EtOAc and petroleum ether and used directly in the next step.
3.2.6. General Procedure for the Synthesis of Compounds 12a, 12b, 13a, 13b
A mixture of compound 10a/10b/11a/11b and 10% Pd/C in MeOH was hydrogenated at room
temperature for 48 h. The catalyst was then filtered off and the filtrate was concentrated in vacuo.
Crystallization of the crude product from EtOH gave the pure title compounds.
N-hydroxy-3-(4-((7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)amino)phenyl)propanamide (12a).
Starting from 10a, 36.6% of 12a was obtained as a white solid according to the aforementioned
procedure, mp: 155–157 °C. 1H-NMR (400 MHz, DMSO-d6) δ 1.94–2.06 (m, 2H), 2.29 (t, J = 7.4 Hz,
2H), 2.36–2.48 (m, 6H), 2.82 (t, J = 7.4 Hz, 2H), 3.53–3.63 (m, 4H), 3.93 (s, 3H), 4.18 (m, 2H),
7.10–7.26 (m, 3H), 7.65 (d, J = 8.1 Hz, 2H), 7.83 (s, 1H), 8.41 (s, 1H), 8.68 (s, 1H), 9.40 (s, 1H), 10.37
(s, 1H). HRMS (ESI): m/z calcd for C23H32N5O5 (M + H+): 482.2398, found: 482.2405.
3-(4-((6,7-bis(2-methoxyethoxy)quinazolin-4-yl)amino)phenyl)-N-hydroxypropanamide (12b). Starting
from 10b, 15.9% of 12b was obtained as a white solid according to the aforementioned procedure, mp:
188–191 °C. 1H-NMR (400 MHz, DMSO-d6) δ 2.28 (t, J = 7.6 Hz, 2H), 2.82 (t, J = 7.6 Hz, 2H), 3.35
(s, 3H), 3.37 (s, 3H), 3.69–3.84 (m, 4H), 4.23–4.35 (m, 4H), 7.14–7.26 (m, 3H), 7.67 (d, J = 7.9 Hz,
2H), 7.85–7.99 (m, 1H), 8.41 (s, 1H), 8.68 (s, 1H), 9.41 (br, 1H), 10.37 (s, 1H). HRMS (ESI): m/z
calcd for C23H29N4O6 (M+H+): 457.2082, found: 457.2087.
N-hydroxy-3-(3-((7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)amino)phenyl)propanamide (13a).
Starting from 11a, 52.1% of 13a was obtained as a pale yellow solid according to the aforementioned
procedure, mp: 118–120 °C. 1H-NMR (400 MHz, DMSO-d6) δ 1.92–2.08 (m, 2H), 2.26–2.34 (m, 2H),
2.36–2.48 (m, 6H), 2.84 (t, J = 7.6 Hz, 2H), 3.53–3.65 (m, 4H), 3.93 (s, 3H), 4.19 (t, J = 6.3 Hz, 2H),
6.96 (d, J = 7.2 Hz, 1H), 7.18 (s, 1H), 7.29 (t, J = 7.8 Hz, 1H), 7.56 (s, 1H), 7.69 (d, J = 8.5 Hz, 1H),
7.84 (s, 1H), 8.39–8.48 (m, 1H), 8.68 (s, 1H), 9.40 (s, 1H), 10.37 (s, 1H). HRMS (ESI): m/z calcd for
C23H32N5O5 (M+H+): 482.2398, found: 482.2410.

Molecules 2013, 18
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3-(3-((6,7-bis(2-methoxyethoxy)quinazolin-4-yl)amino)phenyl)-N-hydroxypropanamide (13b). Starting
from 11b, 39.8% of 13b was obtained as a pale yellow solid according to the aforementioned
1
procedure, mp: 119–122 °C. H-NMR (500 MHz, CD3OD) δ 2.46 (t, J = 7.5 Hz, 2H), 3.01 (t,
J = 7.5 Hz, 2H), 3.49 (s, 3H), 3.50 (s, 3H), 3.87–3.92 (m, 4H), 4.35–4.43 (m, 4H), 7.23 (d, J = 7.5 Hz,
1H), 7.27 (s, 1H), 7.42 (t, J = 7.5 Hz, 1H), 7.52–7.60 (m, 2H), 8.03 (s, 1H), 8.67 (s, 1H); HRMS (ESI):
m/z calcd for C23H29N4O6 (M+H+): 457.2082, found: 457.2080.
3.3. Evaluation of Enzyme Activities
3.3.1. HDAC Enzymatic Assay in Vitro
Recombinant human HDAC1, HDAC3 and HDAC6 were cloned and expressed in insect High5
cells using the baculovirus expression system, and purified using Ni-NTA (QIAGEN). The inhibitory
activity of HDAC1 and HDAC3 was determined with the HDAC substrate (Ac-Lys-Tyr-Lys
(ε-acetyl)-AMC) while the anti-HDAC6 activity was assayed with another HDAC substrate (Boc-Lys
(ε-acetyl)-AMC). The reaction was carried out in black 384-well plates (OptiPlateTM-384F,
PerkinElmer) at room temperature. The typical inhibition assay was carried out in 25 μl system
containing 25 mM Hepes, 137 mM NaCl, 2.7 mM KCl and 4.9 mM MgCl2, pH 8.0, HDAC protein
(20–200 nM), HDAC substrate (5–50 μM) and 20 μg/mL individual compounds. Positive controls
contained all the above components except the inhibitor. The negative controls contained neither
enzyme nor inhibitor. After incubated for 24 h and 3 h respectively, the reaction of HDAC1, HDAC3
and HDAC6 was quenched with the addition of 25 l Trypsin (diluted to final concentration 0.3125%).
The plates were incubated for 30 min at room temperature to allow the fluorescence signal to develop.
The fluorescence generated was monitored at a 355nm (excitation) and 460nm (emission) in the
EnVision multilabel plate reader (PerkinElmer Life Sciences, Boston, MA, USA).
3.3.2. EGFR and HER2 Inhibition Assay
EGFR and HER2 kinase activity were assessed using HTScan EGFR and HER2 kinase assay kits
(Cell Signaling Technology, Danvers, MA, USA). In short, the GST-EGFR fusion protein was
incubated with synthetic biotinylated peptide substrate and 10 μg/mL inhibitors in the presence of 400
μM ATP. Phosphorylated substrate was captured with strapavidin-coated 96-well plates. The level of
phosphorylation was monitored by anti-phosphotyrosine and europium-labeled secondary antibodies
(DELFIA, Perkin-Elmer). The enhancement solution was added at the end of the assay and enzyme
activity was measured in the Wallac Victor II 1420 microplate reader at 615 nM.
4. Conclusions
In summary, a new series of quinazoline derivatives was designed based on the reported synergistic
effect between RTK and HDAC inhibitors. These hybrids featured the key moieties of gefitinib or
erlotinib as well as the ZBG of HDACi. The inhibitory activities against HDAC, EGFR and HER2
under cell-free conditions were evaluated. Almost all hybrids with hydroxamate segment exhibited
potent HDAC inhibitory activity. In addition, the appropriate rigid linker facilitated the binding of
hydroxamate segment with zinc ion at the bottom of the active site. Compared with SAHA, compound

Molecules 2013, 18
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9a showed similar anti-HDAC activities against HDAC1 (IC50 = 0.16 μm), HDAC3 (IC50 = 0.18 μm)
and HDAC6 (IC50 = 0.56 μm). However, these hydrids exhibited reduced inhibitory activities against
EGFR and HER2. The primary SAR study indicated that the introduction of polar group such as
hydroxamate on the 4-position of the quinazoline core is more likely to provide a potent HDAC/HER2
dual inhibitor rather than HDACi/EGFRi hybrid molecule. To further improve anti-RTK activity,
lipophilic ZBG such as aminobenzamide would be worthwhile investigating. The in-depth SAR
analysis provided concrete foundations for future optimization.
Acknowledgments
This work was supported by the State Key Laboratory of Drug Research (No. SIMM1203KF-10),
the grants of The National Natural Science Foundation of China (No. 81172936 and No. 21102046),
and grants of The Fundamental Research Funds for the Central Universities.
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 4a,b, 5a,b, 8a,b, 9a,b, 12a,b and 13a,b are available
from the authors.
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