Design and synthesis of small molecule agonists of EphA2 receptor

Article abstract of DOI:10.1016/j.ejmech.2017.10.026

Ligand-independent activation of EphA2 receptor kinase promotes cancer metastasis and invasion. Activating EphA2 receptor tyrosine kinase with small molecule agonist is a novel strategy to treat EphA2 overexpressing cancer. In this study, we performed a lead optimization of a small molecule Doxazosin that was identified as an EphA2 receptor agonist. 33 new analogs were developed and evaluated; a structure?activity relationship was summarized based on the EphA2 activation of these derivatives. Two new derivative compounds 24 and 27 showed much improved activity compared to Doxazosin. Compound 24 possesses a bulky amide moiety, and compound 27 has a dimeric structure that is very different to the parental compound. Compound 27 with a twelve-carbon linker of the dimer activated the kinase and induced receptor internalization and cell death with the best potency. Another dimer with a six-carbon linker has significantly reduced potency compared to the dimer with a longer linker, suggesting that the length of the linker is critical for the activity of the dimeric agonist. To explore the receptor binding characteristics of the new molecules, we applied a docking study to examine how the small molecule binds to the EphA2 receptor. The results reveal that compounds 24 and 27 form more hydrogen bonds to EphA2 than Doxazosin, suggesting that they may have higher binding affinity to the receptor.

Full text of DOI:10.1016/j.ejmech.2017.10.026

Accepted Manuscript
Design and synthesis of small molecule agonists of EphA2 receptor
Aaron Petty, Nethrie Idippily, Viharika Bobba, Werner J. Geldenhuys, Bo Zhong, Bin
Su, Bingcheng Wang
PII:
S0223-5234(17)30819-X
10.1016/j.ejmech.2017.10.026
EJMECH 9818
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 April 2017
Revised Date: 9 October 2017
Accepted Date: 9 October 2017
Please cite this article as: A. Petty, N. Idippily, V. Bobba, W.J. Geldenhuys, B. Zhong, B. Su, B. Wang,
Design and synthesis of small molecule agonists of EphA2 receptor, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.10.026.
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Graphical abstract

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Revision: EJMECH-D-17-00940R1
Design and Synthesis of Small Molecule Agonists of EphA2 Receptor
Aaron Pettya, Nethrie Idippilyb, Viharika Bobbab, Werner J. Geldenhuysc, Bo Zhongb,
Bin Sub*, Bingcheng Wangad*
a Rammelkamp Center for Research and Department of Medicine, MetroHealth Campus, Case Western
Reserve University School of Medicine, Cleveland, Ohio, USA,
bDepartment of Chemistry, Center for Gene Regulation in Health and Disease, College of Sciences &
Health Professions, Cleveland State University, 2121 Euclid Ave., Cleveland, Ohio, 44115, USA
cDepartment of Pharmaceutical Sciences, School of Pharmacy, Robert C. Byrd Health Sciences Center,
West Virginia University, USA
dCase Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland,
Ohio, USA
RECEIVED DATE:
TITLE RUNNING HEAD: Novel EphA2 agonist
*To whom correspondence should be addressed:
Bin Su, Ph.D.
Department of Chemistry,
Bingcheng Wang, Ph.D.
Department of Medicine
Center for Gene Regulation in Health and Disease Rammelkamp Center for Research
College of Sciences and Health Professions,
Cleveland State University,
2121 Euclid Ave., Cleveland, OH 44115, USA
Phone: 216-687-9219
Case Comprehensive Cancer Center
Case Western Reserve University School of
Medicine
10900 Euclid Avenue, Cleveland, Ohio 44106,
USA
Fax: 216-687-9298
Email: B.su@csuohio.edu
Phone: 216-778-4256
Fax: 216-778-4321
E-mail: bxw14@case.edu
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Abstract
Ligand-independent activation of EphA2 receptor kinase promotes cancer metastasis and invasion.
Activating EphA2 receptor tyrosine kinase with small molecule agonist is a novel strategy to treat EphA2
overexpressing cancer. In this study, we performed a lead optimization of a small molecule Doxazosin
that was identified as an EphA2 receptor agonist. 33 new analogs were developed and evaluated; a
structure−activity relationship was summarized based on the EphA2 activation of these derivatives. Two
new derivative compounds 24 and 27 showed much improved activity compared to Doxazosin.
Compound 24 possesses a bulky amide moiety, and compound 27 has a dimeric structure that is very
different to the parental compound. Compound 27 with a twelve-carbon linker of the dimer activated the
kinase and induced receptor internalization and cell death with the best potency. Another dimer with a
six-carbon linker has significantly reduced potency compared to the dimer with a longer linker,
suggesting that the length of the linker is critical for the activity of the dimeric agonist. To explore the
receptor binding characteristics of the new molecules, we applied a docking study to examine how the
small molecule binds to the EphA2 receptor. The results reveal that compounds 24 and 27 form more
hydrogen bonds to EphA2 than Doxazosin, suggesting that they may have higher binding affinity to the
receptor.
Keyword: EphA2, Doxazosin, small molecular ligand, dimer, internalization.
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1. Introduction
Erythropoietin-producing hepatocellular receptor 2 (EphA2) is one of the receptor tyrosine kinases
that regulate cancer cell migration. It has been well documented that EphA2 is overexpressed in many
types of human cancers, and the overexpression is correlated with malignant progression and poor
prognosis[1-3] . EphA2 in breast epithelial cells has been reported to induce morphological
transformation, while in prostate cancer and glioma cell lines, elevated EphA2 expression causes increased
chemotactic cell migration and invasion[4,5] . In contrast to its pro-oncogenic roles, other studies
indicate that EphA2 activation by its ligand, ephrin-A1, suppresses tumor progression, including induction
of apoptosis, inhibition of cell proliferation, and suppression of cell migration[5-8] . In vivo studies
demonstrate that EphA2 activation by systemically administered ephrin-A1 decreases tumorigenicity and
invasiveness of carcinoma xenografts[5,8,9] . On the other side, transgenic mice with EphA2 deletion
show increased susceptibility to carcinogen-induced skin tumorigenesis[10] . These two opposing
observations raise the question about the true role of EphA2 in tumor development and metastasis. In fact,
EphA2 plays dual roles in tumorigenesis. Under ligand stimulation, EphA2 inhibits cell migration in
keeping with the well-established repulsive roles of Eph receptors in regulating cell motility. However, in
the absence of ligand, EphA2 promotes cell migration and invision, which is correlated with its expression
level[1-3,11,12] . Unfortunately, EphA2 overexpression is often accompanied by loss of expression or
mislocalization of ephrin-A1 in breast cancer, glioma and skin tumors. The reduced ligand-dependent
activation of EphA2 coupled with increased EphA2 expression and frequent Akt activation provide a
permissive environment to promote ligand-independent pro-invasive Akt-EphA2 crosstalk. This
interaction may be in part responsible for EphA2 overexpression during tumor progression and the positive
correlation of EphA2 expression and unfavorable prognosis[1,2,13-16] .
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This hypothesis is also supported by an in vivo study demonstrating that ephrin administration
inhibited the progression of tumors with high expression of EphA2[5,8] . However, ephrins cannot cross
the blood brain barrier, which makes this endogenous ligand less useful for the treatment of gliomas with
over expression of EphA2. This fact led us to hypothesize that small molecule agonists for EphA2 can be
exploited as novel cancer therapeutics. In a previous study, we identified a small molecule EphA2 agonist
Doxazosin using a combination of structure-based virtual screening and cell-based assays[4] .
In the current study, we lead optimized Doxazosin to activate EphA2 with higher potency and reduce
the original targeting effect of Doxazosin on adrenoceptor. Several new analogs of Doxazosin showed
significantly increased potency to activate EphA2 and induce subsequent receptor internalization. A
detailed structure-activity relationship (SAR) was summarized based on the diversely modified structures
and the activities. One particular compound 27 with a dimeric structure showed superior activity within the
new compound pool. The novel symmetric structure provides us a new scaffold for further optimization.
2. Results and discussion
2.1. Lead optimization and summarization of the SAR
Our previous efforts to develop small molecule EphA2 agonist led to the identification of Doxazosin
(2-[4-(2,3-Dihydro-1,4-benzodioxine-2-carbonyl)piperazin-1-yl]-6,7-dimethoxyquinazolin-4-amine), an
α1-selective alpha blocker, used to treat high blood pressure and urinary retention associated with benign
prostatic hyperplasia (BPH) [17,18] . Doxazosin significantly activated EphA2 at 50µM in vitro, and
inhibited prostate tumor metastasis in the mouse model[4] . In the present study, a total of 33 new
derivatives based on Doxazosin were synthesized using combinatorial chemistry strategy. First, we kept
A and B moieties, and changed C moiety systematically (Figure 1). Subsequently, we synthesized several
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symmetric molecules with the best C moiety on both sides of the piperazine ring to determine if the
whole A and B parts are necessary for the activity, which is adapted from a recent report about a dimeric
EphA2 agonist showing promising activity[19] . Based on the symmetric molecular hypothesis, we also
designed larger symmetric structures as illustrated by the dimers (Figure 1). We then modified A moiety
to ethoxy, B moiety to substituted amines to generate diverse structures and enrich the SAR for the future
structure optimization.
Figure 1. Proposed modification of Doxazosin
In the newly designed derivatives, we explored other different structures at C domain including
aromatic rings and alky groups. For the aromatic rings, we also synthesized extended linkers. The
synthesis of C moiety substituted derivatives is illustrated in Scheme 1.
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Scheme 1. Synthesis of C moiety substituted Doxazosin analogs
Scheme 2. Synthesis of symmetric structure based on piperazine ring as the core structure
As illustrated in Figure 1, we also synthesized dimers with piperazine ring as the core scaffold. A and
B whole moiety was replaced by a C moiety structure. The symmetric hypothesis led us to explore the
possibility of a bigger dimer based on the whole left domain of the lead compound Doxazosin. Therefore,
we synthesized compounds 27 and 28 using different linkers as illustrated in Scheme 3.
Scheme 3. Synthesis of dimer 27 and 28
To investigate if the primary amino group on the pyrimidine ring is needed for the activity, we
modified the B moiety into different structures. First, we acetylated Doxazosin and compound 24 to
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generate compounds 29 and 30, respectively (Scheme 4).
Scheme 4. Synthesis of B moiety acetylated analogs
Furthermore, we modified this amino group to alkylated analogs as illustrated in Scheme 5.
MeO
MeO
N
Cl
N
NH
N
NH
NH
MeO
MeO
N
Cl
or
HN
N
n-BuOH
n-BuOH
Cl
MeO
MeO
N
N
Cl
N
O
N
NH
MeO
MeO
N
N
MeO
MeO
N
N
N
N
N
31 (56%)
N
O
Cl
O
O
O
N
NH
MeO
MeO
N
N
K2CO3/DMF
MeO
MeO
N
N
N
32 (60%)
N
N
N
O
Scheme 5. Synthesis of B moiety modified analogs
At last, we modified A moiety to ethoxy group to examine if methoxy is required for the activity.
The synthesis is illustrated in Scheme 6.
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Scheme 6. Synthesis of A moiety modified analog
The 33 new analogs show diverse substitution on the three identified structural moieties, and the
biological results of the compounds can provide us a detail SAR to elucidate the structure requirement
for the activation of EphA2.
To evaluate the compounds, we utilized a MDA-MB-231 breast cancer cell line which overexpresses
EphA2 receptor (MDA-MB-231-A2). The ratio of phosphorylated EphA2 protein over total EphA2
protein was measured after treatment with the various compounds as the readout for receptor activation.
All the compounds were examined with doses of 2, 10, and 50 µM, and we used 50µM Doxazosin and
0.5% DMSO as the positive and negative control, respectively (Figure 2). EphA2 activation by 50µM
Doxazosin was used as the cutoff to identify more potent analogs. Compounds 1-24 all showed different
substitution at the C moiety as indicated in Scheme 1. Compounds 1 and 2 with bulky C domain seemed
to show higher activity than compound 3. However, 50 µM compounds 1 and 2 did not show a much
higher activity than the lead compound Doxazosin at 50 µM. Compounds 4-8 all have one carbon
extension at C moiety, and the activity of these compounds are comparable to Doxazosin. Compounds 9,
10, 18, 19 and 20 that have heteroaromatic ring at C moiety showed decreased activity, which rules out
other possible substitution with heteroaromatic ring in the future. Compounds 11, 12, 22 and 24 have
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slightly or very bulky and electron donating substitute group at the aromatic ring of C moiety, and
compounds 22 and 24 show similar or significantly increased activity compared to Doxazosin, whereas
compound 13 with electron withdrawing substitution group decreases the activity. However, compound
21 shows decreased activity even with an electron donating group at 4’ aromatic ring of C moiety.
Compounds 14-17 all have alky substitute groups at C moiety and show decreased activity, suggesting
that small non-aromatic substitution at C moiety diminishes the activity and should be avoided in the
future modification. Compound 23 has a long alky chain at C moiety and shows slightly decreased
activity. Based on the structure of 23, we designed dimer structures as demonstrated by compounds 27
and 28. Compound 27 shows significantly increased activity compared to Doxazosin, whereas compound
28 with short linkage shows decreased activity, suggesting that the length of the linkage of the dimer is
critical for the activity. Inspired by the dimer design, we also generated dimer structure based on the
piperazine ring as the core as illustrated by compounds 25 and 26. However, both compounds show
significantly decreased activity.
Besides C moiety, we also modified the structures of the A and B moieties. Compounds 29 and 30
were based on compounds Doxazosin and 24, respectively. The amino group was acetylated to generate
the new analogs. The results indicate that the amino group seems critical for the activity, and the
acetylation significantly diminishes the activity. In compounds 31 and 32, we replaced the amino group
with piperidine and pyrrolidine groups, respectively, and all of these changes harm the EphA2 activation.
For the A moiety, we changed the dimethoxy of compound 24 to generated compound 33. Unfortunately,
this modification diminishes the activity as well. The structure activity relationship demonstrates that A
and B moieties of Doxazosin are critical for the activity and C moiety is an open area for further
optimization. In addition, the dimer structure of compound 27 provides a new platform to design new
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core structures for a group of new analogs to activate EphA2.
Figure 2. EphA2 activation of the new derivatives. MDA-MB-231-EphA2 cells were treated with the compounds with three
doses (in 0.5% DMSO) for 30 minutes and cell lysates were subject to immunoprecipitation and immunoblot for
phosphorylated EphA/B kinases (pEphA/B) and total EphA2. Treatment with 1 µg /ml ephrin-A1-Fc (EA1-Fc) or Doxazosin
(DZ) 50 µM were served as positive controls. Treatment with 0.5% DMSO was served as vehicle control.
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2.2. EphA2 internalization
EphA2 overexpression has been identified in various different malignant cells[2,6,7,11-14,20,21] .
Decreased levels of EphA2-ligand binding in cancer cells lead to the aggressive growth of the tumor,
which can be overcome in vitro by using either monoclonal antibodies, or small molecule
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ligand[4,5,8,19,21] . In our previous studies, we have demonstrated that EphA2 ligands negatively
regulate tumor cell growth and invasiveness[4] . Ligand binding could even reverse the oncogenic
effects of EphA2 overexpression[15,16] . How did EphA2 ligands reverse the oncogenic effects of
EphA2 overexpression? Previous studies have demonstrated that ligand binding causes EphA2 to be
internalized and degraded[4,22] . Since we have developed small molecule EphA2 ligands based on
Doxazosin as the lead compound and they can effectively activate the kinase，it is necessary to determine
if the ligand could cause EphA2 internalization.
To directly demonstrate this phenomenon at the cellular level, we used immunofluorescence to
monitor localization of EphA2 following the small molecule treatment. This was performed using the
MDA-MB-231cell line. This cell line spreads well in culture, thereby facilitating immunofluorescence
detection of cell surface and intracellular EphA2 (Figure 3). Ephrin-A1-Fc was used as a positive control,
which induced nearly complete internalization of EphA2 within 60 min. Consistent with its agonistic
activities, Doxazosin also stimulated significant EphA2 internalization at 50µM. Compound 27, the most
active one in the SAR study, was chosen to perform the internalization study. The results showed that
EphA2 internalization by compound 27 even happened in very low concentration as 0.4µM, which
indicates that compound 27 has much better potency to induce EphA2 internalization than Doxazosin.
Together, these data confirm that the new analog indeed triggers EphA2 receptor internalization in keeping
with its agonistic activities.
Figure 3. Doxazosin analog compound 27 treatment causes EphA2 receptor internalization.
Immunofluorescence staining of MDA-MB-231 cells for EphA2 receptor (green) after treatment for 60 minutes with various
concentration of compound 27 in 0.5% DMSO. Treatment with 1 ug/mL ephrin-A1-Fc, 50uM Doxazosin and 0.5% DMSO
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served as positive and negative controls, respectively. DAPI nuclear staining is shown in blue. Scale bars, 25 um. Cells were
seeded on 24-well plates and stimulated after 24-48 hours. The experiment was repeated three times independently, and the
representative one is listed.
2.3. Compound 27 inhibits cancer cell colony formation
EphA2 is involved in tumorigenesis, growth and metastasis. Compound 27 can activate EphA2 and
induce the internalization of the receptor. Next, we examined if the compound could inhibit tumor growth
with a colony formation assay. The same MDA-MB-231 breast cancer cells were treated with compound
27 at various concentrations for two weeks (Figure 4). The results indicated that the compound inhibited
colony formation even at nanomolar concentrations. We also determined the activity of compounds 23 and
24. Compound 23 is the monomer of compound 27 and compound 24 showed potent EphA2 activation in
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the SAR study. The results revealed that compound 23 showed moderate activity to inhibit the colony
formation, and compound 24 showed slightly weaker activity compared to compound 27.
Figure 4. Compound 27 inhibits cancer cell colony formation in MDA-MB-231 cells.
MDA-MB-231 cells (1000 cells/well) were seeded in 12-well plates and cultured for 24 h, followed by treatment with various
concentrations of the Doxazosin analogs (0, 0.08, 0.4, 2 and 10 µM) for 14 days. EA1-Fc (1µg/mL) and Doxazosin (10µM and
20µM) were used as positive controls. After washing with PBS, colonies were fixed and stained with crystal violet. The
experiment was repeated three times independently, and the representative one is listed.
2.4. Docking of compounds 24 and 27 to EphA2
Doxazosin has been demonstrated as a small molecule ligand of EphA2, and the docking domain has
been identified with NMR approach and other biological assays[4,23-25] . Compounds 24 and 27
showed improved EphA2 activation compared to Doxazosin. We would like to see if the new ligand
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shows any stronger interaction to the binding domain of Doxazosin in EphA2. To further elucidate the
binding characteristics of the compounds with EphA2, we performed docking studies with Doxazosin, 24,
and 27 with the crystal structure (PDB:3FL7) of the EphA2 extracellular domain[26] . As illustrated in
Figure 5 upper panel, the aromatic amino group of Doxazosin forms hydrogen bonds with GLY51,
SER68; the oxygen of the Dioxane ring forms hydrogen bond with ARG103. In addition to the polar
interaction, the dihydrobenzo-dioxine moiety sits inside a hydrophobic pocket. Compared to Doxazosin,
compound 24 forms more hydrogen bonds (Figure 5, middle panel). The methoxy group forms hydrogen
bond with GLY51, the aromatic amino group forms hydrogen bonds with GLY51, SER68. The ketone of
the amide bond forms hydrogen bond with ARG103. The binding characteristics of compound 24 are
quite similar to Doxazosin, and the aromatic group of the amide bond sits inside the same hydrophobic
pocket. However, the aromatic group of compound 24 is bulkier than Doxazosin, which pushes the
structure a little bit out, and makes the methoxy group closer to GLY51 to form the extra hydrogen bond.
The results suggest that compound 24 may have higher binding affinity to EphA2 than Doxazosin, which
is consistent with previous EphA2 activation study. However, the most potent compound 27 has very
different structure scaffold compared to Doxazosin and compound 24. The binding manner of this ligand
looks very different to Doxazosin and compound 24 (Figure 5, low panel). Due to the bulkiness of the
structure, it does not fit into the hydrophobic pocket anymore. The molecule adapts itself into two
grooves of the protein. Besides the regular hydrophobic interaction, it forms 4 hydrogen bonds as well.
One of the nitrogens in the quinazoline ring forms two hydrogen bonds with ARG159, and one of the
ketone forms two hydrogen bonds with ARG103. To fit into the two grooves, the length of the linker
seems quite critical, and short linker could not provide enough flexibility for the structure to bind to the
groove. Compared to compound 27, compound 28 has a shorter multiple carbon linker and does not
show any activity to activate EphA2, and could not fit into the two grooves.
Figure 5. Compounds 27, 24 and Doxazosin docking with EphA2. EphA2 (PDB:3FL7) out membrane part was used to
perform the docking study. The original identified Doxazosin binding site was used to fit the compounds. Compound 24 and
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Doxazosin share the similar binding mode, whereas compound 27 shows a different binding characteristic.
2.5 Adrenoceptor binding activity
Doxazosin is an α-adrenoceptor antagonist, and is well used in clinic for the treatment of
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hypertension or benign prostate hyperplasia[17,18] . The EphA2 activation of Doxazosin is
independent of the α-adrenoceptor activity. During the lead optimization process, we would like to
gradually eliminate the α-adrenoceptor binding activity of Doxazosin, and improve the EphA2 activation
potency in the new derivatives. To examine if our new analogs still show α-adrenoceptor binding activity,
we performed the competition experiments with these ligands. We used Doxazosin as the positive control
for the binding assay, and Prazosin as the competitor to the receptor. As shown in Figure 6, Doxazosin
shows an IC50 of 0.74 ± 0.30 nM, whereas compounds 24 and 27 show IC50s of 4.28 ± 3.10 nM and 2.50
± 1.62 nM, respectively. The results suggest that these modifications actually decreased the
α-adrenoceptor binding affinity. Although we won’t be able to summarize a structure activity relationship
about the α-adrenoceptor binding activity at this stage, the lead optimization process at least showed the
feasibility to eliminate the α-adrenoceptor activity of the new derivatives in the future.
Figure 6. Compounds 27, 24 and Doxazosin binding with α-adrenoceptor. The compounds were tested in triplication. IC50s
of the three compounds were listed as mean ± SD
120
100
80
60
DZ
Comp 24
40
Comp 27
20
0
-2
-1
0
1
2
Log[nM]
3. Conclusion
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We developed a series of EphA2 agonists based on Doxazosin as a lead compound. Several new
derivatives showed improved activity to activate EphA2 and induce receptor internalization as well. An
SAR was summarized based on the structures of the compounds and their corresponding EphA2
activation. The A and B moieties of Doxazosin are strictly limited to the current structure, and any
change in our current optimization leads to decreased or even loss of activity. C moiety seems an open
area for further structural optimization in the future. In addition, we developed compound 27 that has the
dimer structure, which is different to the main structure scaffold of most analogs. It provides a new
platform that can be used to design new dimer core structures as a group of new analogs to activate
EphA2.
The two most potent new compounds, 24 and 27, inhibit the colony formation of the cancer cells
expressing high level of EphA2. Our molecular docking study revealed that the new compounds may
possess stronger interaction to the protein than Doxazosin based on the hydrogen bonds formed between
the ligands and EphA2. Finally, the new derivatives showed relatively weaker binding affinity to
α-adrenoceptor, suggesting that our lead optimization decreased the original targeting effect of
Doxazosin.
4. Experimental section
4.1. Chemistry
Chemicals were commercially available and used as received without further purification unless
otherwise noted. Moisture sensitive reactions were carried out under a dry argon atmosphere in
flame-dried glassware. Solvents were distilled before use under argon. Thin-layer chromatography was
performed on precoated silica gel F254 plates (Whatman). Silica gel column chromatography was
performed using silica gel 60Å(Merck, 230-400 Mesh), and hexane/ethyl acetate was used as the elution
solvent. Mass spectra were obtained on the electrospray mass spectrometer at Cleveland State University
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MS facility Center. The molecular weight of the compounds was examined with LC-MS. All the NMR
1
spectra were recorded on a Bruker 400 MHz in either DMSO-d6 or CDCl3. Chemical shifts (δ) for H
NMR spectra are reported in parts per million to residual solvent protons.
Compounds 1-24, 27 and 28 were prepared as the following procedure.
The dimethoxyquinazolin and piperazine were dissolved in n-BuOH with 1 to 5 mole ratio, and the
mixture was heated to 100°C for 24 hours, then it was cooled down to room temperature. The
precipitated while solid was collected via filtration and washed with n-BuOH. After it was dried, the
solid was mixed with various different substituted acetyl chloride and K2CO3 in DMF. It was stirred until
the reaction was completed. The reaction was quenched with Na2CO3 aqueous solution and stirred
overnight, and the precipitated product was collected via filtration and washed with water and dried to
give the corresponding compounds.
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(naphthalen-2-yl)methanone(1).
1
White solid, melting point 2226-229°C, 72% yield for the last step; H-NMR (400MHz, DMSO-d6) δ
8.016 (4H, m), 7.580 (3H, m), 7.440 (1H, s), 7.167 (2H, br), 6.745 (1H, s), 3.795 (12H, m), 3.490 (2H,
13
br). C-NMR (100MHz, DMSO-d6) δ 169.642, 161.632, 158.757, 154.699, 149.117, 145.535, 133.709,
132.754, 130.970, 129.745, 128.525, 127.826, 125.353, 105.645, 104.111, 103.460, 67.487, 56.294,
66.889, 25.592. DUIS-MS calculated for C25H25N5O4 [M + H]+: 444.1, found : 443.5
[1,1'-biphenyl]-3-yl(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)methanone(2).
1
White solid, melting point 179-182°C, 73% yield for the last step; H-NMR (400MHz, DMSO-d6) δ
13
7.735 (4H, m), 7.498 (6H, m), 7.152 (2H, br), 6.740 (1H, s), 3.794 (12H, m), 3.464 (2H, m). C-NMR
(100MHz, DMSO-d6) δ 169.475, 161.620, 158.802, 154.687, 149.184, 145.519, 140.765, 139.825,
137.207, 129.509,128.279, 127.297, 126.522, 125.633, 105.669, 104.085, 103.455, 56.287, 66.879.
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DUIS-MS calculated for C27H27N5O3 [M + H]+: 470.1, found : 469.2
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(benzo[d][1,3]dioxol-5-yl)methanone (3)
1
White solid, melting point 267-269°C, 71% yield for the last step; H-NMR (400MHz, DMSO-d6) δ
7.431 (1H, s), 7.145 (2H, br), 6.981 (3H, m), 6.736 (1H, s), 6.094 (2H, s), 3.791 (10H, m), 3.541 (4H, br).
13C-NMR (100MHz, DMSO-d6) δ 169.133, 161.610, 158.769, 154.682, 149.187, 148.700, 147.605,
145.499, 130.049, 121.990, 108.458, 105.666, 104.094, 103.440, 101.901, 56.503, 56.283, 55.878,
44.074, 19.023. DUIS-MS calculated for C22H23N5O5 [M + H]+: 438.1, found : 437.5
1-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)-2-(4-methoxyphenyl)ethanone(4)
White solid, melting point 240-243°C, 65% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.422 (1H, s), 7.168 (4H, m), 6.881 (2H, d, J= 8.8 Hz), 6.734 (1H, s), 3.831 (3H, s), 3.788 (3H, s), 3.729
(3H, s), 3.671 (4H, m), 3.601 (2H, br), 3.532 (4H, br). 13C-NMR (100MHz, DMSO-d6) δ 169.795,
161.584, 158.522, 154.669, 149.169, 145.485, 130.419, 128.163, 114.233, 105.668, 104.061, 103.412,
56.276, 55.662, 45.928, 44.254, 43.886, 41.754. DUIS-MS calculated for C23H27N5O4 [M + H]+: 438.1,
found : 437.5
1-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)-2-(3-methoxyphenyl)ethanone(5)
White solid, melting point 198-201°C, 60% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.423 (1H, s), 7.231 (1H, m), 7.143 (1H, br), 6.817 (3H, m), 6.737 (1H, s), 3.830 (3H, s), 3.788 (3H, s),
3.738 (5H, m), 3.598 (9H, m). 13C-NMR (100MHz, DMSO-d6) δ 169.382, 161.590, 159.707, 158.745,
154.676, 149.159, 145.495, 137.832, 129.834, 121.631, 115.114, 112.281, 105.662, 104.060, 103.416,
67.488, 56.275, 55. 643, 45.977, 44.237, 43.878, 41.783. DUIS-MS calculated for C23H27N5O4 [M + H]+:
438.1, found : 437.5
1-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)-2-phenylethanone(6)
22

ACCEPTED MANUSCRIPT
White solid, melting point 221-223°C, 96% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.420 (1H, s), 7.272 (5H, m), 7.140 (2H, br), 6.733 (1H, s), 3.829 (3H, s), 3.786 (3H, s), 3.775 (2H, s),
3.607 (8H, m). 13C-NMR (100MHz, DMSO-d6) δ 169.500, 161.590, 158.759, 154.674, 149.173, 145.492,
136.385, 129.469, 128.807, 126.828, 105.672, 104.064, 103.419, 56.279, 55.877, 45.955, 44.239, 43.883,
41.778. DUIS-MS calculated for C22H25N5O3 [M + H]+: 408.1, found : 407.2
1-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)-2-(naphthalen-2-yl)ethanone(7)
White solid, melting point 236-238°C, 70% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.862 (3H, m), 7.774 (1H, s), 7.436 (4H, m), 7.133 (2H, br), 6.725 (1H, s), 3.957 (2H, s), 3.823 (3H, s),
3.782 (3H, s), 3.610 (8H, m). 13C-NMR (100MHz, CDCl3-d6) δ 169.688, 160.521, 158.510, 155.158,
149.759, 145.970, 133.577, 132.459, 128.540, 127.446, 126.517, 125.780, 105.814, 102.901, 101.134,
56.160, 46.256, 43.913, 41.707. DUIS-MS calculated for C26H27N5O3 [M + H]+: 458.1, found : 457.5
1-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)-2-phenylpropan-1-one(8)
White solid, melting point 207-209°C, 82% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.400 (1H, s), 7.306 (5H, m), 7.112 (2H, br), 6.704 (1H, s), 4.155 (1H, q, J= 6.8 Hz), 3.816 (3H, s),
3.775 (3H, s), 3.680-3.399 (6H, m), 3.007 (1H, m), 1.316 (3H, d, J= 6.8 Hz). 13C-NMR (100MHz,
CDCl3-d6) δ 172.254, 160.514, 158.541, 155.142, 149.783, 145.917, 142.085, 128.993, 127.214, 126.849,
105.782, 102.866, 101.177, 56.091, 45.570, 43.777, 42.118, 20.719. DUIS-MS calculated for
C22H23N5O5 [M + H]+: 422.1, found : 421.5
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(pyridin-2-yl)methanone (9)
White solid, melting point 250-252°C, 42% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
8.625 (1H, d, J= 4.4 Hz), 7.952 (1H, t, J= 8 Hz), 7.615 (1H, d, J= 8 Hz), 7.507 (1H, m), 7.432 (1H, s),
13
7.156 (2H, br), 6.738 (1H, s), 3.831-3.726 (12H, m), 3.469 (2H, m). C-NMR (100MHz, DMSO-d6) δ
23

ACCEPTED MANUSCRIPT
167.378, 161.623, 158.737, 154.599, 149.175, 148.901, 145.529, 137.864, 125.097, 123.712, 105.685,
104.109, 103.456, 56.299, 55.886, 47.042, 44.409, 43.866, 42.193. DUIS-MS calculated for C20H22N6O3
[M + H]+: 395.1, found : 394.4
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(pyridin-4-yl)methanone(10)
White solid, melting point 235-237°C, 32% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
8.697 (2H, d, J= 4.8 Hz), 7.444 (3H, m), 7.163 (2H, br), 6.735 (1H, s), 3.830-3.710 (14H, m) (Note: it
seems that two protons are missing but they may overlap with the peak from water). 13C-NMR (100MHz,
DMSO-d6) δ 167.422, 161.632, 158.709, 154.711, 150.553, 149.161, 145.559, 143.952, 121.794,
105.669, 104.104, 103.463, 56.301, 55.896, 47.319, 44.215, 43.737, 41.990. DUIS-MS calculated for
C20H22N6O3 [M + H]+: 395.1, found : 394.4
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(4-ethylphenyl)methanone(11)
White solid, melting point 240-242°C, 84% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.433 (1H, s), 7.370 (2H, d, J= 8 Hz), 7.302 (2H, d, J= 8 Hz), 7.154 (2H, br), 6.735 (1H, s), 3.831-3.445
(14H, m), 2.660 (2H, q, J= 7.6 Hz), 1.214 (3H, t, J= 7.6 Hz). 13C-NMR (100MHz, DMSO-d6) δ 169.768,
161.614, 158.774, 154.687, 149.181, 145.696, 133.742, 128.206, 127.749, 105.667, 104.088, 103.453,
56.284, 55.877, 28.458, 15.801. DUIS-MS calculated for C23H27N5O3 [M + H]+: 422.1, found : 421.5
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(4-iodophenyl)methanone(12)
White solid, melting point 254-258°C, 85% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.843 (2H, d, J= 8 Hz), 7.423 (1H, s), 7.252 (2H, d, J= 8 Hz), 7.144 (2H, br), 6.735 (1H, s), 3.841-3.716
(14H, m). 13C-NMR (100MHz, DMSO-d6) δ 168.865, 161.618, 158.734, 154.704, 149.161, 145.533,
137.730, 135.857, 129.657, 105.670, 104.110, 103.454, 96.865, 56.301, 55.893. DUIS-MS calculated for
C21H22IN5O3 [M + H]+: 520.0, found : 519.3
24

ACCEPTED MANUSCRIPT
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(4-chloro-3-nitrophenyl)methanone(13)
White solid, melting point 268-272°C, 91% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
8.179 (1H, s), 7.883 (1H, d, J= 8 Hz), 7.792 (1H, d, J= 8 Hz), 7.434 (1H, s), 7.159 (2H, br), 6.740 (1H,
s), 3.833-3.401 (14H, m). 13C-NMR (100MHz, DMSO-d6) δ 166.451, 161.632, 158.722, 154.699,
149.143, 148.029, 145.547, 136.683, 132.589, 126.400, 124.905, 105.664, 104.089, 103.456, 56.293,
55.892. DUIS-MS calculated for C21H21ClN6O5 [M + H]+: 473.0, found : 472.9
1-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)ethanone(14)
White solid, melting point 244-247°C, 88% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.426 (1H, s), 7.143 (2H, br), 6.745 (1H, s), 3.836-3.684 (10H, m), 3.490 (4H, m), 2.050 (3H, s).
13C-NMR (100MHz, DMSO-d6) δ 168.845, 161.606, 158.768, 154.682, 149.209, 145.469, 105.675,
104.106, 103.410, 56.289, 55.885, 46.158, 44.261, 43.872, 41.361, 21.803. DUIS-MS calculated for
C16H21N5O3 [M + H]+: 332.1, found : 331.4
1-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)propan-1-one(15)
White solid, melting point 269-272°C, 63% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.428 (1H, s), 7.141 (2H, br), 6.746 (1H, s), 3.837-3.686 (10H, m), 3.507 (4H, m), 2.366 (2H, q, J= 7.2
Hz), 1.017 (3H, t, J= 7.2 Hz). 13C-NMR (100MHz, DMSO-d6) δ 171.950, 161.602, 158.795, 154.682,
149.227, 145.466, 105.680, 104.110, 103.413, 56.289, 55.879, 45.251, 44.304, 43.930, 41.538, 26.125,
9.897. DUIS-MS calculated for C17H23N5O3 [M + H]+: 346.1, found : 345.4
1-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)butan-1-one(16)
White solid, melting point 259-262°C, 72% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.426 (1H, s), 7.142 (2H, br), 6.743 (1H, s), 3.836 (3H, s), 3.791 (3H, s), 3.735 (2H, m), 3.683 (2H, m),
13
3.507 (4H, m), 2.337 (2H, t, J=7.2 Hz), 1.555 (2H, m), 0.914 (3H, t, J= 7.6 Hz). C-NMR (100MHz,
25

ACCEPTED MANUSCRIPT
DMSO-d6) δ 171.13, 161.60, 158.78, 154.68, 149.23, 145.47, 105.68, 104.11, 103.41,67.49, 56.29, 55.86,
45.41, 44.37, 43.96, 41.49, 34.76, 25.59, 18.69, 14.31. DUIS-MS calculated for C18H25N5O3 [M + H]+:
360.1, found : 359.4
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(cyclohexyl)methanone(17)
White solid, melting point 220-222°C, 89% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.429 (1H, s), 7.138 (2H, br), 6.740 (1H, s), 3.837 (3H, s), 3.792 (3H, s), 3.703 (4H, m), 3.520 (4H, m),
2.627 (1H, m), 1.701 (5H, m), 1.336 (4H, m), 1.174 (1H, m). 13C-NMR (100MHz, DMSO-d6) δ 174.107,
161.596, 158.774, 154.691, 149.220, 145.480, 105.684, 104.138, 103.432, 56.299, 55.879, 45.298,
44.640, 43.992, 41.567, 29.631, 26.081, 25.656, 19.020. DUIS-MS calculated for C21H29N5O3 [M + H]+:
400.2, found : 399.5
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(furan-2-yl)methanone(18)
White solid, melting point 230-233°C, 66% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.869 (1H, d, J= 0.8 Hz), 7.438 (1H, s), 7.162 (2H, br), 7.037 (1H, d, J= 3.2 Hz), 6.752 (1H, s), 6.652
(1H, dd, J= 0.8, 3.2 Hz), 3.795 (14H, m). 13C-NMR (100MHz, DMSO-d6) δ 161.633, 158.862, 154.712,
149.213, 147.474, 145.521, 145.205, 116.020, 111.775, 105.700, 104.137, 103.463, 67.487, 56.302,
55.886, 44.153, 25.591. DUIS-MS calculated for C19H21N5O4 [M + H]+: 384.1, found : 383.4
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(thiophen-2-yl)methanone(19)
White solid, melting point 222-224°C, 56% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.784 (1H, d, J= 4.8 Hz), 7.475 (1H, d, J= 3.2 Hz), 7.438 (1H, s), 7.161 (3H, m), 6.750 (1H, s),
13
3.811(10H, m), 3.761 (4H, br). C-NMR (100MHz, DMSO-d6) δ 162.930, 161.637, 158, 732, 154.715,
149.215, 145.523, 137.835, 130.013, 129.650, 127.626, 105.698, 104.143, 103.465, 56.308, 55.892,
44.090. DUIS-MS calculated for C19H21N5O3S [M + H]+: 400.1, found : 399.5
26

ACCEPTED MANUSCRIPT
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(isoxazol-5-yl)methanone (20)
White solid, melting point 249-251°C, 43% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
8.782 (1H, s), 7.442 (1H, s), 7.198 (2H, br), 6.984 (1H, s), 6.758 (1H, s), 3.839-3.611 (14H, m).
13C-NMR (100MHz, DMSO-d6) δ 161.661, 157.432, 151.435, 145.615, 106.739, 104.129, 103.462,
56.310, 55.916. DUIS-MS calculated for C18H20N6O4 [M + H]+: 385.1, found : 384.4
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(4-methoxyphenyl)methanone (21)
White solid, melting point 229-232°C, 85% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.420 (3H, m), 7.148 (2H, br), 7.007 (2H, d, J= 8.4 Hz), 6.738 (1H, s), 3.813 (13H, m), 3.557 (4H, m).
13C-NMR (100MHz, DMSO-d6) δ 169.598, 161.623, 160.689, 158.790, 154.705, 149.211, 145.517,
129.628, 128.371, 114.131, 105.691, 104.134, 103.458, 56.302, 55.803, 44.140. DUIS-MS calculated for
C22H25N5O4 [M + H]+: 424.1, found : 423.5
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(4-bromophenyl)methanone (22)
White solid, melting point 128-130°C, 83% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.665 (2H, d, J= 8.4 Hz), 7.440 (1H, s), 7.407 (2H, d, J= 8.4 Hz), 7.164 (2H, br), 6.744 (1H, s), 3.832
(3H, s), 3.793 (3H, s), 3.725 (6H, m), 3.388 (2H, br). 13C-NMR (100MHz, DMSO-d6) δ 168.663,
161.624, 158.747, 154.710, 149.178, 145.538, 136.589, 131.933, 129.770, 123.409, 105.679, 104.115,
103.4584, 56.303, 55.895. DUIS-MS calculated for C21H22N5O3Br [M + H]+: 474.0, found : 472.5
1-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)hexan-1-one (23)
White solid, melting point 170-172°C, 73% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.427 (1H, s), 7.146 (2H, br), 6.743 (1H, s), 3.834 (3H, s), 3.790 (3H, s), 3.731 (2H, m), 3.679 (2H, m),
3.500 (4H, m), 2.335 (2H, t, J= 7.2 Hz), 1.516 (2H, m), 1.287 (4H, m), 0.872 (3H, t, J= 6.4 Hz).
13C-NMR (100MHz, DMSO-d6) δ 171.287, 161.603, 158.789, 154.690, 149.226, 145.478, 105.679,
27

ACCEPTED MANUSCRIPT
104.123, 103.424, 56.294, 55.878, 45.433, 44.380, 43.959, 41.508, 32.789, 31.497, 24.984, 22.452,
14.356. DUIS-MS calculated for C20H29N5O3 [M + H]+: 388.1, found : 387.5
1-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)-2-(4-(benzyloxy)phenyl)ethanone (24)
White solid, melting point 230-234°C, 89% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.381 (6H, m), 7.169 (4H, m), 6.958 (2H, d, J= 8.4 Hz), 6.740 (1H, s), 5.071 (2H, s), 3.830 (3H, s),
3.789 (3H, s), 3.693-3.535 (10H, m). 13C-NMR (100MHz, DMSO-d6) δ 169.755, 161.602, 158.781,
157.409, 154.701, 149.206, 145.510, 137.636, 130.455, 128.410, 115.151, 105.703, 104.125, 103.441,
69.643, 67.493, 56.302, 55.879, 45.937, 44.274, 43.903, 41.764, 25.596. DUIS-MS calculated for
C29H31N5O4 [M + H]+: 514.2, found : 513.6
1,12-bis(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)dodecane-1,12-dione (27)
White solid, melting point 218-225°C, 68% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.425 (2H, s), 7.145 (4H, br), 6.740 (2H, s), 3.831 (6H, s), 3.787 (6H, s), 3.700 (8H, m), 3.495 (8H, br),
2.331 (4H, t, J= 7.2 Hz), 1.504 (4H, br), 1.267 (12H, br). 13C-NMR (100MHz, DMSO-d6) δ 171.277,
161.596, 158.786, 154.682, 149.221, 145.467, 105.680, 104.119, 103.417, 56.293, 55.880, 45.427,
44.380, 43.958, 41.504, 32.834, 29. 358, 25.306. DUIS-MS calculated for C40H56N10O6 [M + H]+: 774.0,
found : 772.9
1,6-bis(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)hexane-1,6-dione (28)
White solid, melting point 279-280°C, 77% yield for the last step; 1H-NMR (400MHz, DMSO-d6) δ
7.424 (2H, s), 7.138 (4H, br), 6.743 (2H, s), 2.846-2.507 (28H, m), 2.393 (4H, br), 1.567 (4H, br).
13C-NMR (100MHz, DMSO-d6) δ 171.217, 161.602, 158.789, 154.683, 149.218, 145.471, 105.679,
104.105, 103.413, 56.293, 55.882, 45.433, 44.386, 43.962, 41.521, 32.759, 25.050. DUIS-MS calculated
for C34H44N10O6 [M + H]+: 689.1, found : 688.8
28