Design, synthesis and biological evaluation of N-phenylquinazolin-4-amine hybrids as dual inhibitors of VEGFR-2 and HDAC

Article abstract of DOI:10.1016/j.ejmech.2015.12.033

A single agent that simultaneously inhibits multiple targets may offer greater therapeutic benefits in cancer than single-acting agents through interference with multiple pathways and potential synergistic action. In this work, a series of hybrids bearing N-phenylquinazolin-4-amine and hydroxamic acid moieties were designed and identified as dual VEGFR-2/HDAC inhibitors. Compound 6fd exhibited the most potent inhibitory activity against HDAC with IC₅₀ of 2.2 nM and strong inhibitory effect against VEGFR-2 with IC₅₀ of 74 nM. It also showed the most potent inhibitory activity against a human breast cancer cell line MCF-7 with IC₅₀ of 0.85 μM. Docking simulation supported the initial pharmacophoric hypothesis and suggested a common mode of interaction at the active binding sites of VEGFR-2 and HDLP ((Histone Deacetylase-Like Protein), which demonstrates that compound 6fd is a potential agent for cancer therapy deserving further researching.

Full text of DOI:10.1016/j.ejmech.2015.12.033

Accepted Manuscript
Design, synthesis and biological evaluation of N-phenylquinazolin-4-amine hybrids as
dual inhibitors of VEGFR-2 and HDAC
Fan-Wei Peng, Ji Xuan, Ting-Ting Wu, Jia-Yu Xue, Zi-Wei Ren, Da-Ke Liu, Xiu-Qi
Wang, Xin-Hang Chen, Jia-Wei Zhang, Yun-Gen Xu, Lei Shi
PII:
S0223-5234(15)30419-0
10.1016/j.ejmech.2015.12.033
EJMECH 8273
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 June 2015
Revised Date: 14 December 2015
Accepted Date: 16 December 2015
Please cite this article as: F.-W. Peng, J. Xuan, T.-T. Wu, J.-Y. Xue, Z.-W. Ren, D.-K. Liu, X.-Q.
Wang, X.-H. Chen, J.-W. Zhang, Y.-G. Xu, L. Shi, Design, synthesis and biological evaluation of N-
phenylquinazolin-4-amine hybrids as dual inhibitors of VEGFR-2 and HDAC, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2015.12.033.
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ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of N-phenylquinazolin-4-amine
hybrids as dual inhibitors of VEGFR-2 and HDAC
Fan-Wei Peng1†, Ji Xuan3†, Ting-Ting Wu1, Jia-Yu Xue2, Zi-Wei Ren1, Da-Ke Liu1,
Xiu-Qi Wang1, Xin-Hang Chen1, Jia-Wei Zhang1, Yun-Gen Xu1*, Lei Shi1*
A series of hybrids bearing N-phenylquinazolin-4-amine and hydroxamic acid
moieties were designed, synthesized, and discovered as dual VEGFR-2/HDAC
inhibitors.
*Corresponding author. E-mail address: xyg@cpu.edu.cn (Y.-G. Xu), shilei@cpu.edu.cn (L. Shi).
†Both authors contributed equally to the work.

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of N-phenylquinazolin-4-amine
hybrids as dual inhibitors of VEGFR-2 and HDAC
Fan-Wei Peng1†, Ji Xuan3†, Ting-Ting Wu1, Jia-Yu Xue2, Zi-Wei Ren1, Da-Ke Liu1,
Xiu-Qi Wang1, Xin-Hang Chen1, Jia-Wei Zhang1, Yun-Gen Xu1*, Lei Shi1*
1. Jiangsu Key Laboratory of Drug Design and Optimization, Department of
Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, P. R.
China
2. Jiangsu Provincial Key Laboratory for Plant Ex Situ Conservation, Institute of
Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, P.
R. China
3. 81 Hospital of PLA, Nanjing 210002, P. R. China
Abstract:
A single agent that simultaneously inhibits multiple targets may offer greater
therapeutic benefits in cancer than single-acting agents through interference with
multiple pathways and potential synergistic action. In this work, a series of hybrids
bearing N-phenylquinazolin-4-amine and hydroxamic acid moieties were designed
and identified as dual VEGFR-2/HDAC inhibitors. Compound 6fd exhibited the most
potent inhibitory activity against HDAC with IC50 of 2.2 nM and strong inhibitory
effect against VEGFR-2 with IC50 of 74 nM. It also showed the most potent inhibitory
activity against a human breast cancer cell line MCF-7 with IC50 of 0.85 μM. Docking
simulation supported the initial pharmacophoric hypothesis and suggested a common
mode of interaction at the active binding sites of VEGFR-2 and HDLP ((Histone
Deacetylase-Like Protein), which demonstrates that compound 6fd is a potential agent
for cancer therapy deserving further researching.
Keywords: N-phenylquinazolin-4-amine hybrid, VEGFR-2, HDAC, dual inhibitor
*Corresponding author. E-mail address: xyg@cpu.edu.cn (Y.-G. Xu), shilei@cpu.edu.cn (L. Shi).
†Both authors contributed equally to the work.
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1. Introduction
Angiogenesis, the process of new blood vessels formation from existing
vasculature, is a normal physiological event for organ development. It occurs during
tissue growth from embryonic development through to maturity. It is also activated
during wound repairment and certain stages of menstrual cycle [1,2]. However,
abnormal regulation of angiogenesis has been involved in the development of various
diseases such as rheumatoid arthritis, psoriasis, diabetic retinopathy, tumor growth,
and tumor metastasis [2-7]. Among the angiogenic factors identified to date, vascular
endothelial growth factor (VEGF) and its receptor tyrosine kinase VEGFR-2 or kinase
insert domain receptor (KDR) are the most important regulator of tumor angiogenesis
[8,9]. It is well demonstrated that upon binding its ligand, VEGFR-2
undergoes receptor dimerization and autophosphorylation and initiates downstream
signaling, ultimately leading to tumor angiogenesis, vascular permeability
enhancement, proliferation, and migration [10,11]. Consequently, inhibition of the
VEGF/VEGFR-2 signaling pathway has become a valuable approach in the treatment
of cancers. Indeed, a number of angiogenesis inhibitors of VEGFR-2 have been
approved by FDA or effectively demonstrated in preclinical and clinical settings.
Bevacizumab, a monoclonal antibody to VEGF, has been approved by the FDA for
the treatment of non-small-cell lung cancer [12] and metastatic colorectal cancer [13].
Ramucirumab, a mAb antibody (human IgG1) directed against VEGFR-2, has been
approved by FDA in 2014 for advanced gastric or gastro-esophageal
junction adenocarcinoma and metastatic non-small-cell lung carcinoma [14]. In
addition, small-molecule tyrosine kinase inhibitors of VEGFR-2, such as Sunitinib
[15], Sorafenib [16], and Vandetanib [17] have been approved for treatment of various
types of cancers including renal cell carcinoma, gastrointestinal stromal tumor (GIST),
hepatocellular carcinoma, thyroid cancer, and soft tissue sarcom.
However, a significant number of patients do not respond to VEGFR-2 targeted
therapy. Furthermore, the effectiveness of these angiogenesis inhibitors is also limited
by the drug resistance that frequently emerges following treatment [18-20]. To
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overcome the low response rate and acquired drug resistance to tyrosine kinase
inhibitors (TKIs), a number of strategies have been tested, including combination
therapies and development of multi-targeted inhibitors [21,22].
Histone deacetylases (HDACs) are a class of enzymes that catalyze the removal
of acetyl groups from the ε-amino groups of lysine residues present within the
N-terminal extension of the core histones, leading to chromatin condensation and
transcriptional repression [23,24]. In addition to regulating the acetylation state of
histones, HDACs can bind to, deacetylate and regulate the activity of a number of
non-histone proteins, including transcription factors such as p53, E2F1, and NF-κB
and proteins with diverse biological functions such as α-tubulin, Ku70 and Hsp90 [24].
Eighteen HDACs have been identified in humans, and they are subdivided into four
structurally and functionally different phylogenetic classes. Among them, Class I
HDACs (1, 2, 3, and 8) and Class II HDACs (4, 5, 6, 7, 9, and 10) are zinc dependent
proteases [25]. It has been widely recognized that zinc-containing HDACs are
promising targets for therapeutic interventions intended to reverse aberrant epigenetic
states associated with cancer [26-28]. Consequently, considerable effort has been
devoted to develop HDAC inhibitors recently [29-34].
HDAC inhibitors have been demonstrated to synergize with other antitumor
agents, including RTK inhibitors, to suppress proliferation and induce apoptosis in
tumor cells, even to overcome TKI resistance [35-39]. Recently, multi-acting
inhibitors against HDAC and RTK have been reported [40-44]. But the report of
VEGFR-2/HDAC dual inhibitors is rare [45]. In this study, a series of
N-phenylquinazolin-4-amine hybrids were rationally designed and synthesized as dual
VEGFR-2/HDAC inhibitors by combination of pharmacophores of two reference
drugs, Vandetanib and Vorinostat, which were used to achieve VEGFR-2 and HDAC
inhibition, respectively (Figure 1).
2. Results and discussion
2.1. Chemistry
The synthetic route to obtain the desired target compounds is outlined in Scheme
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1 according to the literature with some modifications [40]. Compounds 3a-3f were
prepared
through
the
coupling
of
substituted
anilines
with
4-chloro-7-methoxyquinazolin-6-yl acetate (2), which was prepared through
chlorination of 7-methoxy-4-oxo-3,4-dihydroquinazolin-6-yl acetate (1). Hydrolysis
of the acetyl group on compounds 3a-3f using lithium hydroxide gave corresponding
phenol intermediates 4a-4f. Alkylation of the phenolic hydroxyl group on compounds
4a-4f with various chain lengths of ethyl bromoalkanoate gave ethyl ester
intermediates 5aa-5fd. Conversion of ethyl esters 5aa-5fd to hydroxamic acids using
freshly prepared hydroxylamine furnished target N-phenylquinazolin-4-amine hybrids
6aa-6fd.
2.2. Biological evaluation
The in vitro enzymatic inhibitory activities of the target compounds against
VEGFR-2 and HDAC were evaluated. Additionally, the in vitro antiproliferative
effects of the targeted compounds against MCF-7, a human breast adenocarcinoma
cell line, were also tested. The results were summarized in Table 1.
2.2.1. VEGFR-2 inhibition
As shown in Table 1, the target compounds 6aa-6fd exhibited mild to moderate
VEGFR-2 inhibitory activities compared to the reference compound Vandetanib.
Among them, compounds 6fa-6fd with para-bromo substituent on the phenyl ring
exhibited potent inhibitory activity against VEGFR-2 kinase with IC50 values ranging
from 74 nM to 153 nM, which was comparable to the positive drug Vandetanib (IC50
= 54 nM). Among them, compound 6fb exhibited the most potent VEGFR-2
inhibitory activity with IC50 of 59 nM. It seems that the change of the length of
carbon chain (n = 2 to 5) does not influence VEGFR-2 inhibition significantly. On the
contrary, the type and position of halogen substituent on the phenyl ring play
important roles in the VEGFR-2 inhibition. Compared with compounds 6fa-6fd, the
inhibitory activity of compounds 6ea-6ed with ortho-bromo substituent decreased
dramatically with IC50 over 10000 nM against VEGFR-2. It is likely that ortho-Br
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restricts the free rotation of the benzene ring. This is supported by the fact that the
rank order of the VEGFR-2 inhibitory activity is ortho-Br < ortho-Cl <= ortho-F <
ortho-H. Compounds 6ba-6bd with para-fluoro substituent showed stronger
inhibitory activity against VEGFR-2 (IC50 ranged from 270 nM to 524 nM) than
compounds 6aa-6ad with ortho-fluoro substituent (IC50 ranged from 754 nM to 918
nM). Similarly, compounds 6cd-6dd with para-chloro substituent showed weaker
inhibition against VEGFR-2 (IC50 ranged from 364 nM to 857 nM) than compounds
6da-6dd with ortho-fluoro substituent (IC50 ranged from 182 nM to 313 nM). All the
above results indicated that introduction of bromo substituent at para position on the
phenyl ring is favorable for the VEGFR-2 inhibition. It is worth noting that
7-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyheptanamide
(CUDC-101) reported by Cai et.al. exhibited potent inhibitory activities against
HDAC, EGFR, and HER2 but weak inhibitory activity against VEGFR-2 [40]. The
main difference between our synthesized compounds and CUDC-101 is the
substituent and its position on the phenyl ring. The substituent on the phenyl ring of
CUDC-101 is meta-ethynyl group while of our synthesized compounds is para- or
ortho- substituted halogen.
2.2.2. HDAC inhibition
Data in Table 1 illustrated that the target compounds 6aa-6fd exhibited moderate
to potent HDAC inhibitory activities, which suggested that the
N-phenylquinazolin-4-amine moiety of the target compounds is a suitable cap group
for HDAC inhibitor. Among the compounds tested, compound 6fd exhibited the most
potent HDAC inhibitory activity with IC50 of 2.2 nM, which was 7-fold higher than
the reference compound Vorinostat (IC50 = 15 nM). Obviously, increasing the length
of the hydroxamic acid side chain increases HDAC inhibitory activity by comparing
the HDAC inhibitory activity of compounds 6fa-6fd with the same substituent
(para-bromo) on the phenyl ring. The shortest chain hybrid 6fa (n = 2) showed the
weakest HDAC inhibitory activity with IC50 of 545 nM. Moderate HDAC inhibition
was observed when the carbon chain length between the quinazoline C6 oxygen and
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the hydroxamic acid carbonyl group reached 4 carbons (n = 3, compound 6fb, IC50 =
68 nM). The HDAC inhibitory activity further increased with the carbon chain length
of 5 carbons (n=4, compound 6fc, IC50 = 68 nM). The optimal carbon chain length is
6 carbons (n = 5, compound 6fd, IC50 = 2.2 nM). This discipline was also found in
other para or ortho halogen substituted hybrids. In addition, halogen substituent on
the phenyl ring affects the HDAC inhibitory to some extent. The inhibitory activities
against HDAC of compounds 6bd, 6dd and 6fd with different para-halogen
substituents increased in the following order: fluoro substituted compound 6bd, (IC50
= 8.5 nM) < chloro substituted compound 6bd (IC50 = 3.2 nM) < bromo substituted
compound 6bd (IC50 = 2.2 nM). This rule was also found in other para or ortho
halogen substituted compounds with the same carbon chain length. Besides, the
position of halogen substituent has certain impact on HDAC inhibition. In general,
compounds with halogen substituent at para position on the phenyl ring showed more
potent activity against HDAC than compounds with the same carbon chain length and
the same halogen substituent at ortho position.
In order to further investigate the HDAC isoforms selectivity, compound 6fd
with the most potent HDAC inhibitory activity was chosen to perform enzyme
inhibitory assays against a series of HDAC isoenzymes, including HDAC1, HDAC2,
HDAC6 and HDAC8. Data in Table 2 indicated that compound 6fd showed potent
inhibitory activities against HDAC1, HDAC2, HDAC6 and HDAC8, with IC50 of 1.8
nM, 3.3 nM, 16.4 nM and 4.6 nM, respectively.
2.2.3. Cell growth inhibition
To further explore the antitumor effect of these VEGFR-2/HDAC inhibitors, all
the synthesized target compounds 6aaꢀ6fd were evaluated for their anticancer
activities against a human breast cancer cell line MCF-7 by MTT assay. The results
were also summarized in Table 1. Among the tested compounds, compound 6fd with
the most potent HDAC inhibitory activity and strong VEGFR-2 inhibitory activity
also exhibited the most potent anticancer activities with the IC50 value of 0.85 μM
against MCF-7, which showed greater potency than the reference compounds
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Vandetanib (IC50 = 18.5 μM) and Vorinostat (IC50 = 4.2 μM).
It seemed that the SARs analysis result of antiproliferation activities of the tested
compounds were more consistent with their HDAC inhibitory activities. This was
probably due to their potent inhibitory activities against HDAC but moderate
inhibitory activities against VEGFR-2.
2.3. Molecular docking studies
The possible binding modes of the synthesized target compounds on VEGFR-2
and HDAC were explored with the representative compound 6fd using the Discovery
Studio 3.1/CDOCKER protocol.
Figure 2 demonstrates the compound 6fd docking into the ATP-binding cavity of
VEGFR-2 kinase (PDB code: 2QU5) [46]. In the binding model, the carbonyl oxygen
atom of compound 6fd forms two hydrogen bonds with the amino hydrogen atoms of
Asn923 (O...H-N: 2.2 Å). Besides, the terminal hydroxyl hydrogen atom of compound
6fd forms another hydrogen bond with the carbonyl oxygen atom of Leu840 (O...H-O:
1.9 Å). Both the pyrimidine ring and the phenyl ring of quinazoline moiety form
π-cation interactions (distance: 4.6 Å for pyrimidine ring and 5.7 Å for phenyl ring)
with the terminal amino cation of Lys868.
The binding model of compound 6fd into the binding site of HDLP (Histone
Deacetylase-Like Protein, PDB code: 1C3S) [47] is depicted in Figure 3. In this
binding model, the carbonyl oxygen atom of compound 6fd forms a hydrogen bond
with the phenolic hydroxyl hydrogen atom of Tyr297 (O...H-O: 2.1 Å). In addition,
the hydrogen atom of amide group forms a hydrogen bond with the imidazole
nitrogen atom of His131 (N...H-N: 2.1 Å). Apart from these, the terminal hydroxyl
hydrogen atom forms a hydrogen bond with the carbonyl oxygen atom of Gly129
(O...H-O: 2.2 Å). It is obvious that the N-phenylquinazolin moiety at the other end of
the aliphatic chain is well tolerated.
The nice binding model of compound 6fd with VEGFR-2 and HDLP indicates
that compound 6fd could be a potent dual VEGFR-2/HDAC inhibitor.
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3. Conclusions
A series of hybrids bearing N-phenylquinazolin-4-amine and hydroxamic acid
side chain moieties have been designed, synthesized, and evaluated in vitro as dual
inhibitors against VEGFR-2 and HDAC that displayed great antiproliferation potency
against a solid tumor cell line (MCF-7). Compound 6fd exhibited the most potent
inhibitory activity against HDAC with IC50 of 2.2 nM and it showed potent inhibition
against VEGFR-2 kinase as well with IC50 of 74 nM. In vitro cell growth inhibition
assays indicated that compound 6fd also exhibited the greatest inhibitory activities
against a human breast cancer cell line MCF-7 with IC50 of 0.85 μM. Molecular
docking of the compound 6fd into the active binding sites of VEGFR-2 and HDLP
was performed and the result suggested that compound 6fd could bind well with these
two sites. The above results demonstrated that compound 6fd could be a potential
anticancer agent and deserved further research.
4. Experimental Section
4.1. Chemistry
All solvents and reagents were commercially available and used without further
purification. Melting points (uncorrected) were determined on a RY-1 MP apparatus.
ESI-MS spectra were recorded on an Agilent/HP 1100 Series LC/MSD Trap SL Mass
1
spectrometer, and H NMR spectra were recorded on a Bruker AV-300 spectrometer
at 25 °C with TMS and solvent signals allotted as internal standards. Chemical shifts
were reported in ppm (δ). Elemental analyses were performed on a CHN-O-Rapid
instrument.
4.1.1. Preparation of 4-chloro-7-methoxyquinazolin-6-yl acetate (2)
A suspension of 7-methoxy-4-oxo-3,4-dihydroquinazolin-6-yl acetate (1) (2.34 g)
in SOCl2 (30 mL) and DMF (0.5 mL) was refluxed for 3h. After that, the mixture was
concentrated under reduced pressure to give 4-chloro-7-methoxyquinazolin-6-yl
1
acetate (2). Yellow powder, yield: 77%. H-NMR (300 MHz; d6-DMSO): 2.08 (s,
8

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3H); 3.73 (s, 3H); 7.55 (s, 1H); 7.50 (s, 1H); 9.44(s, 1H). MS (ESI+) m/z 253.6
(M+H)+.
4.1.2.
General
procedure
for
the
preparation
of
the
7-methoxy-4-(phenylamino)quinazolin-6-yl acetate derivatives 3a-3f
A mixture of 4-chloro-7-methoxyquinazolin-6-yl acetate (2, 10 mmol) and
appropriate substituted anilines (15 mmol) in isopropanol (80 mL) was stirred at
reflux for 4 h. The mixture was cooled to room temperature and the resultant
precipitate was collected by filtration and washed with isopropanol. The solid was
dried to give compounds 3a-3f.
4-(2-Fluorophenylamino)-7-methoxyquinazolin-6-yl acetate (3a)
1
White powder, yield: 64%. H-NMR (300 MHz; CDCl3): 2.11 (s, 3H); 3.69 (s,
3H); 7.27 (s, 1H); 7.42-7.68 (m, 4H); 8.11 (s, 1H); 8.61 (s, 1H). MS (ESI+) m/z 328.1
(M+H)+.
4-(4-Fluorophenylamino)-7-methoxyquinazolin-6-yl acetate (3b)
1
White powder, yield: 64%. H-NMR (300 MHz; CDCl3): 2.14 (s, 3H); 3.70 (s,
3H); 7.26 (s, 1H); 7.43 (d, J = 8.1 Hz, 2H); 7.69 (s, 1H); 8.11 (d, J = 8.1 Hz, 2H);
8.61 (s, 1H), 11.18 (s, 1H). MS (ESI+) m/z 328.1 (M+H)+.
4-(2-Chlorophenylamino)-7-methoxyquinazolin-6-yl acetate (3c)
1
White powder, yield: 65%. H-NMR (300 MHz; CDCl3): 2.13 (s, 3H); 3.70 (s,
3H); 7.25 (s, 1H); 7.43-7.66 (m, 4H); 7.75 (s, 1H); 8.12 (s, 1H); 8.61 (s, 1H), 11.22 (s,
1H). MS (ESI+) m/z 344.1 (M+H)+.
4-(4-Chlorophenylamino)-7-methoxyquinazolin-6-yl acetate (3d)
1
White powder, yield: 65%. H-NMR (300 MHz; CDCl3): 2.12 (s, 3H); 3.68 (s,
3H); 7.21 (s, 1H); 7.37 (d, J = 8.4 Hz, 2H); 7.75 (s, 1H); 8.10 (d, J = 8.4 Hz, 2H);
8.59 (s, 1H). MS (ESI+) m/z 344.1 (M+H)+.
4-(2-Bromophenylamino)-7-methoxyquinazolin-6-yl acetate (3e)
1
Yellow powder, yield: 62%. H-NMR (300 MHz; CDCl3): 2.11 (s, 3H); 3.72 (s,
3H); 7.32 (s, 1H); 7.43-7.56 (m, 3H); 7.75 (s, 1H); 8.14 (d, J = 8.4 Hz, 1H); 8.63 (s,
1H); 11.05 (s, 1H). MS (ESI+) m/z 388.0 (M+H)+.
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4-(4-Bromophenylamino)-7-methoxyquinazolin-6-yl acetate (3f)
1
Yellow powder, yield: 63%. H-NMR (300 MHz; CDCl3): 2.16 (s, 3H); 3.71 (s,
3H); 7.16 (s, 1H); 7.41 (d, J = 8.7 Hz, 2H); 7.84 (s, 1H); 8.12 (d, J = 8.7 Hz, 2H);
8.60 (s, 1H); 11.27 (s, 1H). MS (ESI+) m/z 388.0 (M+H)+.
4.1.3.
General
procedure
for
the
preparation
of
the
7-methoxy-4-(phenylamino)quinazolin-6-ol derivatives 4a-4f
A mixture of 7-methoxy-4-(phenylamino)quinazolin-6-yl acetate derivatives
(3a-3f, 6 mmol) and LiOH·H2O (19.8 mmol) in methanol (190 mL) and H2O (190 mL)
o
was stirred at 30 C for 1 h. The mixture was neutralized by the addition of 1.0 M
hydrochloric acid. The resultant precipitate was collected by filtration and dried to
give compounds 4a-4f.
4-(2-Fluorophenylamino)-7-methoxyquinazolin-6-ol (4a)
1
Yellow powder, yield: 53%. H-NMR (300 MHz; CDCl3): 3.70 (s, 3H); 7.18 (s,
1H); 7.40-7.71 (m, 4H); 8.19 (s, 1H,); 8.62 (s, 1H); 9.43 (s, 1H); 9.52(s, 1H). MS
(ESI+) m/z 286.0 (M+H)+.
4-(4-Fluorophenylamino)-7-methoxyquinazolin-6-ol (4b)
1
Yellow powder, yield: 60%. H-NMR (300 MHz; CDCl3): 3.71 (s, 3H); 7.19 (s,
1H); 7.41 (d, J =8.4 Hz, 2H); 7.82 (d, J = 8.1 Hz, 2H); 8.21 (s, 1H,); 8.58 (s, 1H);
9.46 (s, 1H) . MS (ESI+) m/z 286.0 (M+H)+.
4-(2-Chlorophenylamino)-7-methoxyquinazolin-6-ol (4c)
1
Yellow powder, yield: 55%. H-NMR (300 MHz; CDCl3): 3.73 (s, 3H); 7.26 (s,
1H); 7.44-7.56 (m, 2H); 7.76-7.86 (m, 2H); 8.13 (s, 1H,); 8.66 (s, 1H); 9.39 (s, 1H);
9.59 (s, 1H) . MS (ESI+) m/z 302.0 (M+H)+.
4-(4-Chlorophenylamino)-7-methoxyquinazolin-6-ol (4d)
1
Yellow powder, yield: 53%. H-NMR (300 MHz; CDCl3): 3.71 (s, 3H); 7.26 (s,
1H); 7.43 (d, J = 8.4 Hz, 2H); 7.84 (d, J = 8.7 Hz, 2H); 8.12 (s, 1H,); 8.61 (s, 1H);
9.45 (s, 1H). MS (ESI+) m/z 302.0 (M+H)+.
4-(2-Bromophenylamino)-7-methoxyquinazolin-6-ol (4e)
1
Yellow powder, yield: 52%. H-NMR (300 MHz; CDCl3): 3.69 (s, 3H); 7.12 (s,
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1H); 7.37 (d, J =8.4 Hz, 2H); 7.78 (d, J = 8.1 Hz, 1H); 7.82 (d, J = 8.4 Hz, 1H); 8.11
(s, 1H,); 8.58 (s, 1H); 9.52 (s, 1H) . MS (ESI+) m/z 346.0 (M+H)+.
4-(4-Bromophenylamino)-7-methoxyquinazolin-6-ol (4f)
1
Yellow powder, yield: 62%. H-NMR (300 MHz; CDCl3): 3.71 (s, 3H); 7.26 (s,
1H); 7.42 (d, J = 8.1 Hz, 2H); 7.83 (d, J = 8.1 Hz, 2H); 8.13 (s, 1H,); 8.71 (s, 1H);
9.42 (s, 1H); 9.69 (s, 1H). MS (ESI+) m/z 302.0 (M+H)+.
4.1.4. General procedure for the preparation of the ethyl esters 5aa-4fd
A mixture of 7-methoxy-4-(phenylamino)quinazolin-6-ol derivatives 4a-4f (0.87
mmol), appropriate ethyl bromoalkanoate (3.48 mmol), and potassium carbonate
(0.3g, 2.18 mmol) in DMF (20 mL) was stirred at 40 oC for 4 h. The reaction mixture
was filtered, and the filtrate was poured into ice water (200 mL) and extracted with
ethyl acetate (3×50 mL). The combined organic extract was washed with brine, dried
over Na2SO4, and filtered and the filtrate was evaporated in vacuo. The residue was
washed with diethyl ether and dried to give compounds 5aa-5fd.
Ethyl 4-(4-(2-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)butanoate (5aa)
Yellow powder, yield: 67%. 1H-NMR (300 MHz; d6-DMSO): 1.28 (t, J = 6.9 Hz,
3H); 1.57-1.62 (m, 2H); 2.30 (t, J = 6.9 Hz, 2H); 3.88 (s, 3H); 4.15-4.20 (m, 4H); 7.19
(s, 1H ); 7.42-7.84 (m, 5H); 8.13 (s, 1H); 8.45 (s, 1H). MS (ESI+) m/z 400.1 (M+H)+.
Ethyl 5-(4-(2-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)pentanoate (5ab)
1
Pale yellow powder, yield: 66%. H-NMR (300 MHz; d6-DMSO): 1.31 (t, J =
6.3 Hz, 3H); 1.91-2.02 (m, 4H); 2.44 (t, J = 6.3 Hz, 2H); 3.93 (s, 3H); 4.13 (t, J = 6.6
Hz, 2H); 4.24 (t, J = 6.9 Hz, 2H); 7.48-7.59 (m, 4H); 7.84 (s, 1H); 8.12 (s, 1H); 8.26
(s, 1H); 8.48 (s, 1H). MS (ESI+) m/z 414.2 (M+H)+.
Ethyl 6-(4-(2-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)hexanoate (5ac)
Yellow powder, yield: 70%. 1H-NMR (300 MHz; d6-DMSO): 1.31 (t, J = 6.6 Hz,
3H); 1.32-1.35 (m, 2H), 1.64-1.72 (m, 4H); 2.44 (t, J = 6.6 Hz, 2H); 3.93 (s, 3H);
4.13-4.24 (m, 4H); 7.48-7.59 (m, 4H); 7.84 (s, 1H); 8.12 (s, 1H); 8.26 (s, 1H); 8.46 (s,
1H). MS (ESI+) m/z 428.2 (M+H)+.
Ethyl 7-(4-(2-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)heptanoate (5ad)
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Yellow powder, yield: 74%. 1H-NMR (300 MHz; d6-DMSO): 1.38 (t, J = 6.3 Hz,
3H); 1.35-1.37 (m, 2H), 1.47-1.53 (m, 4H); 1.65-1.67 (m, 2H); 2.46 (t, J = 6.9 Hz,
2H); 3.97 (s, 3H); 4.15-4.18 (m, 4H); 7.23 (s, 1H); 7.45-7.84 (m, 5H); 8.18 (s, 1H);
8.56 (s, 1H). MS (ESI+) m/z 442.1 (M+H)+.
Ethyl 4-(4-(4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)butanoate (5ba)
Yellow powder, yield: 62%. 1H-NMR (300 MHz; d6-DMSO): 1.26 (t, J = 6.6 Hz,
3H); 1.70-1.78 (m, 2H); 2.45 (t, J = 6.6 Hz, 2H); 3.94 (s, 3H); 4.14 (t, J = 6.3 Hz, 2H);
4.23 (t, J = 6.9 Hz, 2H); 7.28 (d, J = 8.1 Hz, 2H); 7.35 (s, 1H ); 7.45 (d, J = 8.7 Hz,
2H); 7.85 (s, 1H); 8.16 (s, 1H); 8.64(s, 1H). MS (ESI+) m/z 400.1 (M+H)+.
Ethyl
(5bb)
5-(4-(4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)pentanoate
1
Pale yellow powder, yield: 65%. H-NMR (300 MHz; d6-DMSO): 1.23 (t, J =
6.6 Hz, 3H); 1.97-2.02 (m, 4H); 2.45 (t, J = 6.3 Hz, 2H); 3.94 (s, 3H); 4.13-4.22 (m,
4H); 7.23 (s, 1H); 7.43 (d, J = 8.4 Hz, 2H); 7.84 (d, J = 8.4 Hz, 2H); 8.14 (s, 1H);
8.26 (s, 1H); 8.63 (s, 1H). MS (ESI+) m/z 414.1 (M+H)+.
Ethyl 6-(4-(4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)hexanoate (5bc)
Yellow powder, yield: 70%. 1H-NMR (300 MHz; d6-DMSO): 1.24 (t, J = 6.3 Hz,
3H); 1.60-1.68 (m, 4H); 1.83-1.86 (m, 2H); 2.44 (t, J = 6.6 Hz, 2H); 3.93 (s, 3H);
4.14-4.17 (m, 4H); 7.24 (s, 1H); 7.48 (d, J = 8.1 Hz, 2H); 7.92 (d, J = 8.1 Hz, 2H);
8.15 (s, 1H); 8.23 (s, 1H); 8.64 (s, 1H). MS (ESI+) m/z 428.1 (M+H)+.
Ethyl
(5bd)
7-(4-(4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)heptanoate
Yellow powder, yield: 73%. 1H-NMR (300 MHz; d6-DMSO): 1.24 (t, J = 6.6 Hz,
3H); 1.34-1.38 (m, 2H); 1.53-1.57 (m, 2H); 1.63-1.73 (m, 4H); 2.43 (t, J = 6.6 Hz,
2H); 3.95 (s, 3H); 4.14-4.23(m, 4H); 7.25 (s, 1H); 7.45 (d, J = 8.1 Hz, 2H); 7.86 (d, J
= 8.4 Hz, 2H); 7.95 (s, 1H); 8.16 (s, 1H); 8.53 (s, 1H). MS (ESI+) m/z 442.1 (M+H)+.
Ethyl 4-(4-(2-chlorophenylamino)-7-methoxyquinazolin-6-yloxy)butanoate (5ca)
Yellow powder, yield: 66%. 1H-NMR (300 MHz; d6-DMSO): 1.31 (t, J = 6.9 Hz,
3H); 1.57-1.61 (m, 2H); 2.43 (t, J = 6.3 Hz, 2H); 3.92 (s, 3H); 4.15-4.22 (m, 4H); 7.24
(s, 1H); 7.43-7.56 (m, 4H); 7.83 (d, J = 8.7 Hz, 1H); 8.14 (s, 1H); 8.64 (s, 1H). MS
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(ESI+) m/z 416.1 (M+H)+.
Ethyl
(5cb)
5-(4-(2-chlorophenylamino)-7-methoxyquinazolin-6-yloxy)pentanoate
Yellow powder, yield: 65%. 1H-NMR (300 MHz; d6-DMSO): 1.31 (t, J = 6.3 Hz,
3H); 2.12-2.18 (m, 4H); 2.46 (t, J = 6.6 Hz, 2H); 3.93 (s, 3H); 4.14-4.23 (m, 4H); 7.24
(s, 1H); 7.73-7.85 (m, 2H); 7.89-7.96 (m, 3H); 8.14 (s, 1H); 8.32 (s, 1H); 8.63 (s, 1H).
MS (ESI+) m/z 430.1 (M+H)+.
Ethyl 6-(4-(2-chlorophenylamino)-7-methoxyquinazolin-6-yloxy)hexanoate (5cc)
1
Pale yellow powder, yield: 72%. H-NMR (300 MHz; d6-DMSO): 1.31 (t, J =
6.3 Hz, 3H); 1.46-1.56 (m, 2H); 1.64-1.68 (m, 4H); 2.43 (t, J = 6.3 Hz, 2H); 3.94 (s,
3H); 4.13-4.22 (m, 4H); 7.23 (s, 1H); 7.42-7.45 (m, 2H); 7.42-7.47(m, 2H); 7.87 (s,
1H); 8.13 (s, 1H); 8.52 (s, 1H). MS (ESI+) m/z 444.1 (M+H)+.
Ethyl
(5cd)
7-(4-(2-chlorophenylamino)-7-methoxyquinazolin-6-yloxy)heptanoate
1
Pale yellow powder, yield: 45%. H-NMR (300 MHz; d6-DMSO): 1.32 (t, J =
6.3 Hz, 3H); 1.44-1.62 (m, 6H); 1.71-1.75 (m, 2H); 2.47 (t, J = 6.3 Hz, 2H); 3.94 (s,
3H); 4.14-4.24 (m, 4H); 7.23 (s, 1H); 7.45-7.63 (m, 4H); 7.86 (s, 1H); 8.22 (s, 1H);
8.73 (s, 1H). MS (ESI+) m/z 458.1 (M+H)+.
Ethyl 4-(4-(4-chlorophenylamino)-7-methoxyquinazolin-6-yloxy)butanoate (5da)
Yellow powder, yield: 52%. 1H-NMR (300 MHz; d6-DMSO): 1.28 (t, J = 6.6 Hz,
3H); 1.54-1.58 (m, 2H); 2.41 (t, J = 6.6 Hz, 2H); 3.93 (s, 3H); 4.13 (t, J = 6.3 Hz, 2H);
4.23 (d, J = 6.0 Hz, 2H); 7.24 (s, 1H); 7.44 (d, J = 8.1 Hz, 2H); 7.85 (d, J = 8.4 Hz,
2H); 8.14 (s, 1H); 8.23 (s, 1H); 8.56 (s, 1H). MS (ESI+) m/z 416.1 (M+H)+.
Ethyl
(5db)
5-(4-(4-chlorophenylamino)-7-methoxyquinazolin-6-yloxy)pentanoate
1
Pale yellow powder, yield: 72%. H-NMR (300 MHz; d6-DMSO): 1.26 (t, J =
6.3 Hz, 3H); 1.86-1.97 (m, 4H); 2.34 (t, J = 6.9 Hz, 2H); 3.92 (s, 3H); 4.13 (t, J = 6.3
Hz, 2H); 4.23 (d, J = 6.6 Hz, 2H); 7.23 (s, 1H); 7.46 (d, J = 8.4 Hz, 2H); 7.82 (d, J =
8.1 Hz, 2H); 8.12 (s, 1H); 8.16 (s, 1H); 8.679 (s, 1H). MS (ESI+) m/z 430.2 (M+H)+.
Ethyl 6-(4-(4-chlorophenylamino)-7-methoxyquinazolin-6-yloxy)hexanoate (5dc)
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1
Pale yellow powder, yield: 70%. H-NMR (300 MHz; d6-DMSO): 1.19 (t, J =
6.3 Hz, 3H); 1.64-1.71(m, 6H); 2.41 (t, J = 6.6 Hz, 2H); 3.94 (s, 3H); 4.15-4.23 (m,
4H); 7.24 (s, 1H); 7.48 (d, J = 8.4 Hz, 2H); 7.83 (s, 1H); 8.02 (s, 1H); 8.14 (d, J = 8.4
Hz, 2H); 8.64 (s, 1H). MS (ESI+) m/z 444.1 (M+H)+.
Ethyl
(5dd)
7-(4-(4-chlorophenylamino)-7-methoxyquinazolin-6-yloxy)heptanoate
Yellow powder, yield: 53%. 1H-NMR (300 MHz; d6-DMSO): 1.38 (t, J = 6.3 Hz,
3H); 1.45-1.49 (m, 2H); 1.57-1.61 (m, 2H); 1.64-1.81 (m, 4H); 2.44 (t, J = 6.6 Hz,
2H); 3.92 (s, 3H); 4.12-4.17 (m, 4H); 7.24 (s, 1H); 7.46 (d, J = 8.7 Hz, 2H); 7.85 (d, J
= 8.7 Hz, 2H); 8.15 (s, 1H); 8.58 (s, 1H); 8.62(s, 1H). MS (ESI+) m/z 458.1 (M+H)+.
Ethyl 4-(4-(2-bromophenylamino)-7-methoxyquinazolin-6-yloxy)butanoate (5ea)
Yellow powder, yield: 61%. 1H-NMR (300 MHz; d6-DMSO): 1.31 (t, J = 6.9 Hz,
3H); 1.76-1.80 (m, 2H); 2.45 (t, J = 6.9 Hz, 2H); 3.94 (s, 3H); 4.12 (t, J = 6.6 Hz, 2H);
4.23 (d, J = 6.0 Hz, 2H); 7.24 (s, 1H); 7.45-7.52(m, 3H); 7.85 (t, J = 8.4 Hz, 1H); 7.96
(s, 1H); 8.15 (d, J = 8.4 Hz, 1H); 8.53 (s, 1H). MS (ESI+) m/z 460.1 (M+H)+.
Ethyl
(5eb)
5-(4-(2-bromophenylamino)-7-methoxyquinazolin-6-yloxy)pentanoate
Yellow powder, yield: 63%. 1H-NMR (300 MHz; d6-DMSO): 1.31 (t, J = 6.6 Hz,
3H); 1.87-1.92 (m, 4H); 2.45 (t, J = 6.9 Hz, 2H); 3.92 (s, 3H); 4.12-4.23 (m, 4H); 7.25
(s, 1H); 7.43-7.68 (m, 3H); 7.74 (s, 1H); 8.22 (s, 1H); 8.35 (s, 1H); 8.56 (s, 1H). MS
(ESI+) m/z 474.1 (M+H)+.
Ethyl 6-(4-(2-bromophenylamino)-7-methoxyquinazolin-6-yloxy)hexanoate (5ec)
1
Pale yellow powder, yield: 45%. H-NMR (300 MHz; d6-DMSO): 1.31 (t, J =
6.9 Hz, 3H); 1.57-1.66 (m, 4H); 1.71-1.73 (m, 2H); 2.33 (t, J = 6.6 Hz, 2H); 3.92 (s,
3H); 4.13-4.22 (m, 4H); 7.23 (s, 1H); 7.48-7.82 (m, 4H); 7.99 (s, 1H); 8.16 (s, 1H);
8.63 (s, 1H). MS (ESI+) m/z 488.1 (M+H)+.
Ethyl
(5ed)
7-(4-(2-bromophenylamino)-7-methoxyquinazolin-6-yloxy)heptanoate
1
Pale yellow powder, yield: 70%. H-NMR (300 MHz; d6-DMSO): 1.37 (t, J =
6.6 Hz, 3H); 1.45-1.47 (m, 2H); 1.56-1.74 (m, 6H); 2.46 (t, J = 6.3 Hz, 2H); 3.98 (s,
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ACCEPTED MANUSCRIPT
3H); 4.16-4.22 (m, 4H); 7.261 (s, 1H); 7.46 (d, J = 8.4 Hz, 2H); 7.58-7.63 (m, 2H);
7.95 (s, 1H); 8.19 (s, 1H); 8.641 (s, 1H). MS (ESI+) m/z 488.1 (M+H)+.
Ethyl 4-(4-(4-bromophenylamino)-7-methoxyquinazolin-6-yloxy)butanoate (5fa)
1
Pale yellow powder, yield: 63%. H-NMR (300 MHz; d6-DMSO): 1.30 (t, J =
6.9 Hz, 3H); 1.56-1.60 (m, 2H); 2.43 (d, J = 6.6 Hz, 2H); 3.91 (s, 3H); 4.12 (t, J = 6.6
Hz, 2H); 4.18-4.22 (m, 2H); 7.23 (s, 1H); 7.43 (d, J = 8.7 Hz, 2H); 7.84 (d, J = 8.7 Hz,
2H); 8.15 (s, 1H); 8.52 (s, 1H). MS (ESI+) m/z 460.2 (M+H)+.
Ethyl
(5fb)
5-(4-(4-bromophenylamino)-7-methoxyquinazolin-6-yloxy)pentanoate
Yellow powder, yield: 66%. 1H-NMR (300 MHz; d6-DMSO): 1.33 (t, J = 6.6 Hz,
3H); 1.71-1.76 (m, 4H); 2.42 (t, J = 6.3 Hz, 2H); 3.94 (s, 3H); 4.13-4.18 (m, 4H); 7.24
(s, 1H); 7.45 (d, J = 8.4 Hz, 2H); 7.87 (d, J = 8.4 Hz, 2H); 8.13 (s, 1H); 8.66 (s, 1H).
MS (ESI+) m/z 474.1 (M+H)+.
Ethyl 6-(4-(4-bromophenylamino)-7-methoxyquinazolin-6-yloxy)hexanoate (5fc)
1
Pale yellow powder, yield: 68%. H-NMR (300 MHz; d6-DMSO): 1.30 (t, J =
6.6 Hz, 3H); 1.56-1.63 (m, 4H); 1.82-1.84 (m, 2H); 2.44 (t, J = 6.9 Hz, 2H); 3.92 (s,
3H); 4.15-4.23 (m, 4H); 7.24 (s, 1H); 7.45 (d, J = 8.7 Hz, 2H); 7.84 (d, J = 8.7 Hz,
2H); 7.91(s, 1H); 8.16 (s, 1H); 8.55 (s, 1H). MS (ESI+) m/z 488.1 (M+H)+.
Ethyl
(5fd)
7-(4-(4-bromophenylamino)-7-methoxyquinazolin-6-yloxy)heptanoate
1
Pale yellow powder, yield: 72%. H-NMR (300 MHz; d6-DMSO): 1.28 (t, J =
6.6 Hz, 3H,); 1.34-1.37 (m, 2H); 1.53-1.67 (m, 6H); 2.54 (t, J = 6.7 Hz, 2H); 3.95 (s,
3H); 4.12 (t, J = 6.3 Hz, 2H); 4.28 (d, J = 6.6 Hz, 2H); 7.33 (s, 1H); 7.60 (d, J = 8.4
Hz, 2H); 7.78 (d, J = 8.7 Hz, 2H); 7.90 (s, 1H); 8.15 (s, 1H); 8.62 (s, 1H). MS (ESI+)
m/z 502.1 (M+H)+.
4.1.5. General procedure for the preparation of the title compounds 6aa-6fd
A solution of potassium hydroxide (5.6 g, 100 mmol) in methanol (14 mL) was
added to a stirred solution of hydroxylamine hydrochloride (4.67 g, 67.2 mmol) in
o
o
methanol (24 mL) at 0 C. The reaction mixture was stirred at 0 C for 1 h. The
15

ACCEPTED MANUSCRIPT
precipitate was removed by filtration and the filtrate was collected to provide fresh
hydroxylamine solution which was stored in a refrigerator before use. The appropriate
esters 5aa-5fd (10 mmol) was added to the above freshly prepared hydroxylamine
solution (30 mL) at 0 oC. The reaction mixture was stirred at room temperature for 24
h. The reaction mixture was neutralized with acetic acid. The formed precipitate was
collected by filtration, washed with water, and dried in vacuo to give the title
compounds.
4-(4-(2-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxybutanamid
e (6aa)
o
1
Pale yellow powder, yield: 36%, mp: 195-197 C. H-NMR (300 MHz;
d6-DMSO): 2.06 (t , J = 6.9 Hz, 2H); 2.21 (t, J = 6.9 Hz, 2H); 3.94 (s, 3H); 4.15 (t, J =
5.1 Hz); 7.20 (s, 1H); 7.43 (d, J = 8.4 Hz, 2H); 7.88 (t, J = 8.4 Hz, 2H); 8.48 (s, 1H);
8.72 (s, 1H); 9.52 (s, 1H); 10.47 (s, 1H). MS (ESI+) m/z 387.2 (M+H)+. Anal. Calcd
for C19H19FN4O4: C, 59.06; H, 4.96; N, 14.50; Found: C, 59.34; H, 4.92; N, 14.58.
5-(4-(2-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxypentanami
de (6ab)
o
1
Pale yellow powder, yield: 32%, mp: 235-237 C. H-NMR (300 MHz;
d6-DMSO): 1.70-1.83 (m, 4H); 2.07 (t, J = 6.4 Hz, 2H); 4.02 (s, 3H); 4.21 (t, J = 6.3
Hz, 2H); 7.32-7.59 (m, 5H); 8.25 (s, 1H); 8.79 (s, 1H); 10.429 (s, 1H). MS (ESI+) m/z
401.2 (M+H)+. Anal. Calcd for C20H21FN4O4: C, 59.99; H, 5.29; N, 13.99; Found: C,
59.78; H, 5.33; N, 14.02.
6-(4-(2-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyhexanamid
e (6ac)
o
1
Pale yellow powder, yield: 40%, mp: 220-223 C. H-NMR (300 MHz;
d6-DMSO): 1.25-1.30 (m, 2H); 1.44-1.46 (m, 2H); 1.58-1.62 (m, 2H); 2.00 (t, J = 6.9
Hz, 2H); 4.01 (s, 3H); 4.20 (t, J = 6.2 Hz, 2H); 7.32-7.59 (m, 5H); 8.25 (s, 1H); 8.79
(s, 1H); 10.40 (s, 1H). MS (ESI+) m/z 415.2 (M+H)+. Anal. Calcd for C21H23FN4O4: C,
60.86; H, 5.59; N, 13.52; Found: C, 61.13; H, 5.64; N, 13.60.
7-(4-(2-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyheptanami
de (6ad)
16

ACCEPTED MANUSCRIPT
o
1
Pale yellow powder, yield: 35%, mp: 165-167 C. H-NMR (300 MHz;
d6-DMSO): 1.26-1.56 (m, 6H); 1.83 (s, 2H); 1.97 (t, J = 6.6 Hz, 2H); 4.01 (s, 3H);
4.18 (t, J = 6.4 Hz, 2H); 7.35-7.58 (m, 5H); 8.22 (s, 1H); 8.78 (s, 1H); 10.36 (s, 1H).
MS (ESI+) m/z 429.2 (M+H)+. Anal. Calcd for C22H25FN4O4: C, 61.67; H, 5.88; N,
13.08; Found: C, 61.55; H, 5.85; N, 13.14.
4-(4-(4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxybutanamid
e (6ba)
o
1
Yellow powder, yield: 52%, mp: 216-218 C. H-NMR (300 MHz; d6-DMSO):
2.06 (t, J = 6.3 Hz, 2H); 2.22 (t, J = 6.0 Hz, 2H); 3.94 (s, 3H); 4.07-4.15 (m, 2H);
7.19-7.25 (m, 3H); 7.76-7.86 (m, 2H); 8.41 (s, 1H); 8.72 (s, 1H); 9.50 (s, 1H); 10.47
(s, 1H). MS (ESI+) m/z 387.2 (M+H)+. Anal. Calcd for C19H19FN4O4: C, 59.06; H,
4.96; N, 14.50; Found: C, 58.73; H, 4.93; N, 14.46.
5-(4-(4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxypentanami
de (6bb)
o
1
Pale yellow powder, yield: 54%, mp: 236-238 C. H-NMR (300 MHz;
d6-DMSO): 1.70-1.82 (m, 4H); 2.05 (t, J = 6.3 Hz, 2H); 3.94 (s, 3H); 4.15 (t, J = 6.0
Hz, 2H); 7.19-7.26 (m, 3H); 7.75-7.85 (m, 2H); 8.45 (s, 1H); 8.70 (s, 1H); 9.71 (s,
1H).10.39 (s, 1H). MS (ESI+) m/z 401.2 (M+H)+. Anal. Calcd for C20H21FN4O4: C,
59.99; H, 5.29; N, 13.99; Found: C, 60.25; H, 5.32; N, 14.04.
6-(4-(4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyhexanamid
e (6bc)
o
1
Pale yellow powder, yield: 52%, mp: 198-200 C. H-NMR (300 MHz;
d6-DMSO): 1.45 (s, 2H); 1.60 (t, J = 6.9 Hz, 2H); 1.82 (d, J = 6.6 Hz, 2H); 2.00 (t, J =
6.9 Hz, 2H); 3.97 (s, 3H); 4.16 (t, J = 6.3 Hz, 2H); 7.24 (s, 1H); 7.49 (d, J = 8.7 Hz,
2H); 7.81 (d, J = 8.7 Hz, 2H); 7.98 (s, 1H); 8.63 (s, 1H); 10.39 (s, 1H). MS (ESI+) m/z
415.2 (M+H)+. Anal. Calcd for C21H23FN4O4: C, 60.86; H, 5.59; N, 13.52; Found: C,
61.16; H, 5.56; N, 13.45.
7-(4-(4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyheptanami
de (6bd)
o
1
Yellow powder, yield: 43%, mp: 251-253 C. H-NMR (300 MHz; d6-DMSO):
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1.34-1.56 (m, 6H); 1.81 (d, J = 6.9 Hz, 2H); 1.97 (t, J = 6.9 Hz, 2H); 4.00 (s, 3H);
4.20 (t, J = 6.3 Hz, 2H); 7.30-7.36 (m, 3H); 7.68-7.73 (m, 2H); 8.23 (s, 1H); 8.79 (s,
1H); 10.36 (s, 1H). MS (ESI+) m/z 429.3 (M+H)+. Anal. Calcd for C22H25FN4O4: C,
61.67; H, 5.88; N, 13.08; Found: C, 61.89; H, 5.91; N, 13.14.
4-(4-(2-chlorophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxybutanamid
e (6ca)
o
1
Pale yellow powder, yield: 57%, mp: 210-212 C. H-NMR (300 MHz;
d6-DMSO): 2.08 (t, J = 6.6 Hz, 2H); 2.22 (d, J = 6.6 Hz, 2H); 4.00 (s, 3H); 4.22 (t, J =
6.2 Hz, 2H); 7.32 (s, 1H); 7.64-7.79 (m, 4H); 8.19 (s, 1H); 8.78 (s, 1H); 10.48 (s, 1H).
MS (ESI+) m/z 403.1 (M+H)+. Anal. Calcd for C19H19ClN4O4: C, 56.65; H, 4.75; N,
13.91; Found: C, 56.79; H, 4.71; N, 13.86.
5-(4-(2-chlorophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxypentanami
de (6cb)
o
1
Pale yellow powder, yield: 52%, mp: 168-170 C. H-NMR (300 MHz;
d6-DMSO): 1.71-1.83 (m, 4H); 2.09 (s, 2H); 3.98 (s, 3H); 4.17 (s, 2H); 7.25 (s, 1H);
7.40-7.49 (m, 2H); 7.55-7.64 (m, 2H); 7.99 (s, 1H); 8.53 (s, 1H); 10.36 (s, 1H). MS
(ESI+) m/z 417.2 (M+H)+. Anal. Calcd for C20H21ClN4O4: C, 57.62; H, 5.08; N, 13.44;
Found: C, 57.85; H, 5.12; N, 13.49.
6-(4-(2-chlorophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyhexanamid
e (6cc)
o
1
Pale yellow powder, yield: 48%, mp: 193-194 C. H-NMR (300 MHz;
d6-DMSO): 1.45 (d, J = 5.2 Hz, 2H); 1.60-2.09 (m, 6H); 3.96 (s, 3H); 4.12 (s, 2H);
7.21 (s, 1H); 7.36-7.47 (m, 2H); 7.54-7.62 (m, 2H); 8.42 (s, 1H); 8.68 (s, 1H); 9.98 (s,
1H); 10.36 (s, 1H). MS (ESI+) m/z 431.1 (M+H)+. Anal. Calcd for C21H23ClN4O4: C,
58.54; H, 5.38; N, 13.00; Found: C, 58.32; H, 5.42; N, 12.96.
7-(4-(2-chlorophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyheptanami
de (6cd)
o
1
Pale yellow powder, yield: 36%, mp: 185-187 C. H-NMR (300 MHz;
d6-DMSO): 1.34-1.56 (m, 6H); 1.82 (d, J = 6.6 Hz, 2H); 1.97 (t, J = 6.9 Hz, 2H); 4.00
(s, 3H); 4.17 (t, J = 6.3 Hz, 2H); 7.29 (s, 1H); 7.44-7.68 (m, 4H); 8.09 (s, 1H); 8.67 (s,
18

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1H); 10.35 (s, 1H). MS (ESI+) m/z 445.1 (M+H)+. Anal. Calcd for C22H25ClN4O4: C,
59.39; H, 5.66; N, 12.59; Found: C, 59.64; H, 5.61; N, 12.53.
4-(4-(4-chlorophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxybutanamid
e (6da)
o
1
Yellow powder, yield: 64%, mp: 245-247 C. H-NMR (300 MHz; d6-DMSO):
2.06 (t, J = 6.9 Hz, 2H); 2.22 (t, J = 7.2 Hz, 2H); 3.94 (s, 3H); 4.15 (t, J = 5.4 Hz, 2H);
7.20 (s, 1H); 7.43 (d, J = 8.4 Hz, 2H); 7.85-7.91 (m, 2H); 8.48 (s, 1H); 8.72 (s, 1H);
9.52 (s, 1H); 10.47 (s, 1H). MS (ESI+) m/z 403.2 (M+H)+. Anal. Calcd for
C19H19ClN4O4: C, 56.65; H, 4.75; N, 13.91; Found: C, 56.82; H, 4.80; N, 13.98.
5-(4-(4-chlorophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxypentanami
de (6db)
o
1
Pale yellow powder, yield: 63%, mp: 235-237 C. H-NMR (300 MHz;
d6-DMSO): 1.70-1.83 (m, 4H); 2.07 (t, J = 7.2 Hz, 2H); 3.99 (s, 3H); 4.20 (t, J = 6.0
Hz, 2H); 7.26 (s, 1H); 7.52 (d, J = 8.7 Hz, 2H); 7.78 (d, J = 8.7 Hz, 2H); 8.07 (s, 1H);
8.72 (s, 1H); 10.40 (s, 1H). MS (ESI+) m/z 417.2 (M+H)+. Anal. Calcd for
C20H21ClN4O4: C, 57.62; H, 5.08; N, 13.44; Found: C, 57.36; H, 5.03; N, 13.51.
6-(4-(4-chlorophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyhexanamid
e (6dc)
o
1
Pale yellow powder, yield: 45%, mp: 218-220 C. H-NMR (300 MHz;
d6-DMSO): 1.46 (d, J = 6.6 Hz, 2H); 1.60 (t, J = 6.9 Hz, 2H); 1.82 (t, J = 6.6 Hz, 2H);
2.00 (t, J = 6.9 Hz, 2H); 3.97 (s, 3H); 4.16 (t, J = 6.3 Hz, 2H); 7.24 (s, 1H), 7.49 (d, J
= 8.7 Hz, 2H); 7.81 (d, J = 8.7 Hz, 2H); 7.98 (s, 1H); 8.63 (s, 1H); 10.37 (s, 1H). MS
(ESI+) m/z 431.2 (M+H)+. Anal. Calcd for C21H23ClN4O4: C, 58.54; H, 5.38; N, 13.00;
Found: C, 58.91; H, 5.43; N, 13.06.
7-(4-(4-chlorophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyheptanami
de (6dd)
o
1
Pale yellow powder, yield: 51%, mp: 238-240 C. H-NMR (300 MHz;
d6-DMSO): 1.34-1.53 (m, 6H); 1.83 (s, 2H); 1.96 (t, J = 7.2 Hz, 2H); 4.00 (s, 3H);
4.17 (t, J = 5.4 Hz, 2H); 7.28 (s, 1H); 7.55 (d, J = 8.4 Hz, 2H); 7.74 (d, J = 8.7 Hz,
2H); 8.12 (s, 1H); 8.79 (s, 1H); 10.34 (s, 1H). MS (ESI+) m/z 445.2 (M+H)+. Anal.
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Calcd for C22H25ClN4O4: C, 59.39; H, 5.66; N, 12.59; Found: C, 59.14; H, 5.63; N,
12.64.
4-(4-(2-bromophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxybutanami
de (6ea)
o
1
Yellow powder, yield: 63%, mp: 183-186 C. H-NMR (300 MHz; d6-DMSO):
2.06 (d, J = 7.2 Hz, 2H); 2.21 (d, J = 6.6 Hz, 2H); 3.97 (s, 3H); 4.14 (s, 2H);
7.22-7.54 (m, 4H); 7.70-7.79 (m, 1H); 7.94-7.99 (m, 1H); 8.46 (s, 1H); 10.45 (s, 1H).
MS (ESI+) m/z 447.1 (M+H)+. Anal. Calcd for C19H19BrN4O4: C, 51.02; H, 4.28; N,
12.53; Found: C, 50.75; H, 4.30; N, 12.58.
5-(4-(2-bromophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxypentanami
de (6eb)
o
1
Yellow powder, yield: 46%, mp: 195-197 C. H-NMR (300 MHz; d6-DMSO):
1.73-1.82 (m, 4H); 2.09 (s, 2H); 3.94 (s, 3H); 4.13 (s, 2H); 7.19 (s, 1H); 7.28 (d, J =
7.2 Hz, 1H); 7.47-7.52 (m, 1H); 7.75 (d, J = 7.8Hz, 1H); 7.85 (s, 1H); 8.33 (s, 1H);
8.69 (s, 1H); 9.63 (s, 1H); 10.38 (s, 1H). MS (ESI+) m/z 461.1 (M+H)+. Anal. Calcd
for C20H21BrN4O4: C, 52.07; H, 4.59; N, 12.15; Found: C, 51.82; H, 4.63; N, 12.10.
6-(4-(2-bromophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyhexanami
de (6ec)
o
1
Yellow powder, yield: 50%, mp: 170-172 C. H-NMR (300 MHz; d6-DMSO):
1.46 (d, J = 6.6 Hz, 2H); 1.60 (t, J = 6.9 Hz, 2H); 1.82 (d, J =6.6 Hz, 2H); 2.00 (t, J =
6.9 Hz, 2H); 3.97 (s, 3H); 4.16 (t, J = 6.3 Hz, 2H); 7.24 (s, 1H); 7.49 (d, J = 8.7 Hz,
2H); 7.81 (d, J = 8.7 Hz, 2H); 7.98(s, 1H); 8.63 (s, 1H); 10.37 (s, 1H). MS (ESI+) m/z
475.1 (M+H)+. Anal. Calcd for C21H23BrN4O4: C, 53.06; H, 4.88; N, 11.79; Found: C,
53.32; H, 4.92; N, 11.84.
7-(4-(2-bromophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyheptanami
de (6ed)
o
1
Yellow powder, yield: 44%, mp: 188-190 C. H-NMR (300 MHz; d6-DMSO):
1.23-1.55 (m, 6H); 1.81 (d, J = 6.9 Hz, 2H); 1.95 (d, J = 7.2 Hz, 2H); 4.00 (s, 3H);
4.16 (t, J = 6.0 Hz, 2H); 7.31-7.38 (m, 2H); 7.43 (s, 2H); 7.81 (d, J = 8.1 Hz, 1H);
8.05 (d, J = 8.1 Hz, 1H); 8.67 (s, 1H); 10.35 (s, 1H). MS (ESI+) m/z 489.1 (M+H)+.
20

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Anal. Calcd for C22H25BrN4O4: C, 54.00; H, 5.15; N, 11.45; Found: C, 54.26; H, 5.19;
N, 11.50.
4-(4-(4-bromophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxybutanami
de (6fa)
o
1
Yellow powder, yield: 54%, mp: 225-227 C. H-NMR (300 MHz; d6-DMSO):
2.06 (t, J = 6.3 Hz, 2H); 2.21 (t, J = 6.6 Hz, 2H); 4.00 (s, 3H); 4.22 (s, 2H); 7.32 (s,
1H); 7.64-7.75(m, 4H); 8.19 (s, 1H); 8.78 (s, 1H); 10.48(s, 1H). MS (ESI+) m/z 447.1
(M+H)+. Anal. Calcd for C19H19BrN4O4: C, 51.02; H, 4.28; N, 12.53; Found: C, 51.26;
H, 4.31; N, 12.49.
5-(4-(4-bromophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxypentanami
de (6fb)
o
1
Yellow powder, yield: 35%, mp: 212-213 C. H-NMR (300 MHz; d6-DMSO):
1.71-1.82 (m, 4H); 2.08 (s, 2H); 3.94 (s, 3H); 4.14 (d, J = 6.0 Hz, 2H); 7.20 (s, 1H);
7.56 (d, J = 8.7 Hz, 2H); 7.82 (d, J = 7.2 Hz, 2H); 8.47 (s, 1H); 8.69 (s, 1H); 9.50 (s,
1H); 10.39 (s, 1H). Anal. Calcd for C20H21BrN4O4: C, 52.07; H, 4.59; N, 12.15;
Found: C, 52.30; H, 4.57; N, 12.11.
6-(4-(4-bromophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyhexanami
de (6fc)
o
1
Yellow powder, yield: 61%, mp: 225-226 C. H-NMR (300 MHz; d6-DMSO):
1.45 (s, 2H); 1.61 (s, 2H); 1.83 (s, 2H); 1.99 (d, J = 6.9 Hz, 2H); 3.94 (s, 3H); 4.13 (s,
2H); 7.20 (s, 1H); 7.56 (d, J = 8.7 Hz, 2H); 7.81 (d, J = 6.0 Hz, 2H); 8.47 (s, 1H);
8.68 (s, 1H); 9.51 (s, 1H,) ;10.38 (s, 1H). MS (ESI+) m/z 475.1 (M+H)+. Anal. Calcd
for C21H23BrN4O4: C, 53.06; H, 4.88; N, 11.79; Found: C, 53.29; H, 4.85; N, 11.74.
7-(4-(4-bromophenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyheptanami
de (6fd)
o
1
Yellow powder, yield: 45%, mp: 235-237 C. H-NMR (300 MHz; d6-DMSO):
1.34 (d, J = 6.3 Hz, 2H); 1.46-1.54 (m, 4H); 1.83 (s, 2H) ; 1.95 (d, J = 6.9 Hz, 2H);
4.00 (s, 3H); 4.19 (s, 2H); 7.28 (s, 1H); 7.69 (s, 4H); 8.12 (s, 1H); 8.78 (s, 1H); 10.35
(s, 1H). MS (ESI+) m/z 489.1 (M+H)+. Anal. Calcd for C22H25BrN4O4: C, 54.00; H,
5.15; N, 11.45; Found: C, 54.24; H, 5.18; N, 11.41.
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4.2. VEGFR-2 Inhibition Assay
VEGFR-2 kinase inhibitory activity was measured using HTScan VEGF
Receptor 2 Kinase Assay Kit (Cell Signaling Technology, Inc.) by colorimetric
ELISA assay according to the manufacturer’s instructions, as described previously by
us [48]. Briefly, 12.5 ꢀL of the 4× reaction cocktail containing 100 ng VEGFR-2 was
incubated with 12.5 ꢀL of various concentrations of tested compounds for 5 min at
room temperature. 25 ꢀL of 2× ATP/gastrin precursor (Tyr87) biotinylated peptide
cocktail was then added to the pre-incubated reaction cocktail. After incubation at
room temperature for 30 min, 50 ꢀL of stop buffer (50 mM EDTA, pH 8) were added
to each well to stop the reaction. After that, 25 ꢀL of each reaction were transferred
into a 96-well streptavidin-coated plate containing 75 ꢀL H2O/well and were
incubated at room temperature for 60 min. Next, the wells were washed three times
with 200 ꢀL/well PBS/T (0.05% Tween 20), after which 100 ꢀL of primary antibody
[phosphorylated tyrosine monoclonal antibody (pTyr-100), 1:1000 in PBS/T with 1%
bovine serum albumin (BSA)] was added per well. Following incubation at room
temperature for 60 min, the wells were washed three times with 200 ꢀL/well of
PBS/T. Next, 100ꢀL secondary antibody (HRP labeled anti-mouse IgG, 1:500 in
PBS/T with 1% BSA) was added per well. Following incubation at room temperature
for 30 min, the wells were washed five times with 200 ꢀL/well of PBS/T.
Subsequently, 100 ꢀL of TMB substrate were added per well, and the plate was
incubated at room temperature for 15 min. After that, 100 ꢀL/well of stop solution
was added, and the wells were mixed and incubated at room temperature for 20 min.
The plate was then read at 450 nm with an ELISA microplate reader.
4.3. HDAC Inhibition Assay
HDAC inhibitory activity was measured using the Fluor de Lys HDAC
fluorometric activity assay kit (Enzo Life Sciences, Inc.) as described elsewhere [44]
with some modifications. Briefly, HeLa cell nuclear extracts or HDAC isoforms were
incubated with various concentrations of compounds for 15 min at room temperature,
and HDAC reaction was initiated by addition of Fluor de Lys substrate. The resulting
22

ACCEPTED MANUSCRIPT
mixture was incubated for 1 h at room temperature, followed by adding developer to
stop the reaction. Fluorescence was measured by a microplate reader (Synergy Mx)
with excitation at 355 nm and emission at 460 nm. The assay was repeated three times
independently.
4.4. Cell proliferation Assay
The antiproliferative activities of the prepared N-phenylquinazolin-4-amine
hybrids against MCF-7 cells were evaluated by MTT assay, as described previously
by us [48]. Briefly, MCF-7 cells were grown to log phase in RPMI 1640 medium
supplemented with 10% fetal bovine serum. After diluting to 2×104 cells mL-1 with
the complete medium, 100 µL of the obtained cell suspension was added to each well
of 96-well culture plates. The subsequent incubation was permitted at 37 °С, 5% CO2
atmosphere for 24 h before the antiproliferative assessments. Tested compounds at
pre-set concentrations were added to 6 wells. After 48 h exposure period, 40 µL of
PBS containing 2.5 mg mL-1 of MTT was added to each well. 100 µL extraction
solution (10% SDS-5% isobutyl alcohol-0.010M HCl) was added after 4 h. After an
overnight incubation at 37 °С, the optical density was measured at a wavelength of
570 nm on an ELISA microplate reader. The inhibition rate of tumor cells for each
compound with different concentrations was calculated following the formula:
inhibition rate (%) = (1 - ODcompound exposure/ODcontrol) × 100. The IC50 value was
defined as the concentration at which 50% survival of cells was occurred.
4.5. Molecular docking
Molecular docking of compound 6fd into the three dimensional X-ray structure
of VEGFR-2 (PDB code: 2QU5) and HDLP (HDAC homologue, PDB code: 1C3S)
was carried out using the Discovery Stutio (version 3.1) as implemented through the
graphical user interface DiscoveryStudio CDOCKER protocol.
The three-dimensional structure of the compound 6fd was constructed using
ChemBio 3D Ultra 11.0 software [Chemical Structure Drawing Standard; Cambridge
Soft corporation, USA (2008)], then it was energetically minimized by using
MMFF94 with 5000 iterations and minimum RMS gradient of 0.10. The crystal
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ACCEPTED MANUSCRIPT
structures of VEGFR-2 kinase and HDLP were retrieved from the RCSB Protein Data
Bank (http://www.rcsb.org/pdb/home/home.-do). All bound waters and ligands were
eliminated and the polar hydrogen was added. The whole 2QU5 or 1C3S was defined
as a receptor and the site sphere was selected based on the binding site of 2QU5 or
1C3S. Compound 6fd were placed during the molecular docking procedure. Types of
interactions of the docked protein with ligand were analyzed after the end of
molecular docking.
Acknowledgements
This work was supported by National Natural Science Foundation of China
(Grant No. 21102182), Natural Science Foundation of Jiangsu Province (Grant No.
BK2012760), Research Fund of young scholars for the Doctoral Program of Higher
Education of China (Grant No. 20110096120008), College Students Innovation
Project for the R&D of Novel Drugs (Grant No. J1030830) and Youth development
projects of Army Medical Technology (Grant No. 15QNP021).
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