Dimeric small molecule agonists of EphA2 receptor inhibit glioblastoma cell growth

Article abstract of DOI:10.1016/j.bmc.2020.115656

EphA2 receptor kinase could become a novel target for anti-glioblastoma treatment. Doxazosin previously identified acts like the endogenous ligand of EphA2 and induces cell apoptosis. Through lead structure modification a derivative of Doxazosin possessing unique dimeric structure showed an improvement in the activity. In the current study, we expanded the dimeric scaffold by lead optimization to explore the chemical space of the conjoining moieties and a slight variation to the core structure. 27 new derivatives were synthesized and examined with EphA2 overexpressed and wild type glioblastoma cell lines for cell proliferation and EphA2 activation. Three new compounds 3d, 3e, and 7bg showed potent and selective activities against the growth of EphA2 overexpressed glioblastoma cells. Dimer 3d modification replaces the long alkyl chain with a short polyethylene glycol chain. Dimer 7bg has a relatively longer polyethylene glycol chain in comparison to compound 3d and the length is more similar to the lead compound. Whereas dimer 3e has a rigid aromatic linker exploring the chemical space. The diversity of the linkers in the active suggest additional hydrogen binding sites has a positive correlation to the activity. All three dimers showed selective activity in EphA2 overexpressed cells, indicating the activity is correlated to the EphA2 targeting effect.

Full text of DOI:10.1016/j.bmc.2020.115656

Journal Pre-proofs
Dimeric small molecule agonists of EphA2 receptor inhibit glioblastoma cell
growth
Cody M. Orahoske, Yaxin Li, Aaron Petty, Fatma M. Salem, Jovana Hanna,
Wenjing Zhang, Bin Su, Bingcheng Wang
PII:
DOI:
Reference:
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https://doi.org/10.1016/j.bmc.2020.115656
BMC 115656
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Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
29 January 2020
2 July 2020
6 July 2020
Please cite this article as: Orahoske, C.M., Li, Y., Petty, A., Salem, F.M., Hanna, J., Zhang, W., Su, B., Wang, B.,
Dimeric small molecule agonists of EphA2 receptor inhibit glioblastoma cell growth, Bioorganic & Medicinal
Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115656
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Dimeric small molecule agonists of EphA2 receptor inhibit glioblastoma cell growth
a
a
b
a
a
a
Cody M. Orahoske , Yaxin Li , Aaron Petty , Fatma M. Salem , Jovana Hanna , Wenjing Zhang ,
a
b
Bin Su *, Bingcheng Wang *
aDepartment 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
bRammelkamp Center for Research and Department of Medicine, MetroHealth Campus, Case
Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, Ohio,
USA
RECEIVED DATE:
TITLE RUNNING HEAD: Dimeric 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,
Case Comprehensive Cancer Center
Case Western Reserve University School of
Medicine
2
121 Euclid Ave., Cleveland, OH 44115, USA
Phone: 216-687-9219
Fax: 216-687-9298
Email: B.su@csuohio.edu
10900 Euclid Avenue, Cleveland, Ohio 44106,
USA
Phone: 216-778-4256
Fax: 216-778-4321
E-mail: bxw14@case.edu
1

Abstract
EphA2 receptor kinase could become a novel target for anti-glioblastoma treatment. Doxazosin
previously identified acts like the endogenous ligand of EphA2 and induces cell apoptosis. Through lead
structure modification a derivative of Doxazosin possessing unique dimeric structure showed an
improvement in the activity. In the current study, we expanded the dimeric scaffold by lead optimization
to explore the chemical space of the conjoining moieties and a slight variation to the core structure. 27
new derivatives were synthesized and examined with EphA2 overexpressed and wild type glioblastoma
cell lines for cell proliferation and EphA2 activation. Three new compounds 3d, 3e, and 7bg showed
potent and selective activities against the growth of EphA2 overexpressed glioblastoma cells. Dimer 3d
modification replaces the long alkyl chain with a short polyethylene glycol chain. Dimer 7bg has a
relatively longer polyethylene glycol chain in comparison to compound 3d and the length is more similar
to the lead compound. Whereas dimer 3e has a rigid aromatic linker exploring the chemical space. The
diversity of the linkers in the active suggest additional hydrogen binding sites has a positive correlation
to the activity. All three dimers showed selective activity in EphA2 overexpressed cells, indicating the
activity is correlated to the EphA2 targeting effect.
Keyword: EphA2, glioblastoma, Doxazosin, agonist, dimer
2

1
. Introduction
Glioblastoma multiforme (GBM) is the most common and malignant primary neurological brain
tumor in adults.1–3 Treatment options for GBM are still very limited, and the standard available
4
chemotherapy agent for GBM is Temozolomide (TMZ), a DNA-alkylating reagent. This current
treatment often fails due to resistance.5 Exploring other pathways to inhibit malignancy growth is
critical in extending patient life expectancy. Erythropoietin-producing hepatocellular receptor 2 (EphA2)
has shown to play an important role in cancer migration.2,3,6 It has been well documented that EphA2 is
overexpressed in GBM, and the overexpression is correlated with malignant progression and poor
prognosis.7,8 Studies indicate that EphA2 activation by its ligand ephrin-A1 has the ability to suppress
tumor progression. In addition, the activation causes induction of apoptosis, inhibition of cell proliferation,
and suppression of cell migration.9–12 In vivo studies demonstrated restoring basal levels of ephrin-A1 by
systemic administration decreases tumorigenicity and invasiveness of carcinoma xenografts. 13,14
Unfortunately, EphA2 overexpression is often accompanied by loss of expression or mis-localization of
ephrin-A1 in tumor tissue.3,6,15 The reduced ligand coupled with increased EphA2 expression results in
frequent Akt activation, which promotes ligand-independent pro-invasive Akt-EphA2 crosstalk.3,16,17 This
interaction may be in part responsible for EphA2 overexpression during tumor progression and the positive
correlation of EphA2 expression and unfavorable prognosis.1,17 From a therapeutic point of view,
restoring the ligand for EphA2 could recover the tumor suppressing activity of EphA2, and provide a
new approach for the treatment of EphA2 overexpressed tumor. Unfortunately, the endogenous ligand of
EphA2, ephrin-A1, could not effectively pass blood brain barrier (BBB), which makes this endogenous
ligand less useful for the treatment of glioblastoma with overexpression of EphA2. This fact led us to
hypothesize that a small molecule agonists for EphA2 can be exploited as novel cancer therapeutic.18,19
3

In previous studies, we identified a small molecule EphA2 agonist Doxazosin using a combination of
1
9
structure-based virtual screening and cell-based assays. Further lead optimization of Doxazosin to
activate EphA2 resulted in one particular compound with a unique dimeric structure and superior activity
1
8
20
compared to Doxazosin (Figure 1). Furthermore, this dimer also has the capability to cross BBB. The
novel symmetric structure provides us a new scaffold for further optimization.
NH2
OMe
N
O
N
N
OMe
N
N
MeO
MeO
N
N
O
N
NH2
Figure 1. EphA2 agonist with a dimeric structure
In this current study, we performed optimization of the dimeric lead compound. 27 new derivatives
were synthesized and examined with glioblastoma cell lines. Three new dimers 3d, 3e, and 7bg showed
similar activities compared to the lead compound (scheme 1-3). Dimer 3d exchanges the long alkyl chain
with a short polyethylene glycol linker, this modification reduces the molecular weight and introduces
hydrogen bonding acceptors into the structure allowing different interactions to happen inside the ligand
binding domain. Dimer 7bg has a relatively longer polyethylene glycol chain compared to compound 3d,
and has an additional benzamide moiety exploring the effect of the use of the polyethylene glycol chain.
Dimer 3e has a rigid aromatic linker, suggesting both the flexibility and bulkiness of the compound are
not critical to retain activity. All three dimers showed better activity in EphA2 overexpressed cells verse
wild type glioma cells, indicating the targeting effect is through EphA2 pathways. However, only 7bg
showed significant EphA2 activation, suggesting that the introduction of a benzamide moiety increases
4

the ability to bind to the EphA2 binding domain in comparison to compound 3d which also contains a
polyethylene glycol linker. Compounds 3d and 3e failed to cause EphA2 activation as effective as the
lead compound indicating that the modification may lead to other targeting effect to the pathway instead
of ephA2 activation.
2
2
. Results and discussion
.1. Development of an EphA2 overexpressed glioblastoma cell line
Targeting EphA2 to induce cell apoptosis provides a benefit due to lower systematic toxicity in
comparison to current treatment by TMZ, a chemotherapy agent for GBM. The ideal drug candidates
should selectively decrease the growth of EphA2 overexpressed cells. To examine the selectivity of the
compounds, we constructed a U251 GBM cell line with overexpression of EphA2.
The western blotting results indicate that the basal EphA2 expression in U251 cells is negligible and
similar to the empty vector transfected cells, whereas EphA2 transfected cells have a 5-fold increase
(Figure 2). Previously the lead compound resulted in inhibition of growth based on these three cell lines
wild type with an IC50 of 5.2 µM, the empty vector with an IC50 of 4.8 µM, and the EphA2
overexpressed with an IC of 2.1 µM. The results are consistent to our previous study that the dimeric
5
0
1
8
lead compound is a selective EphA2 ligand, and inhibits cell growth via activation of EphA2.
Figure 2. Construction of U251/EphA2 cell line based on U251 wild type cells. The intensities of the bands were
quantified using ImageJ (NIH) to generate the figure. The figures are representative of 3 experiments. **p <0.001 EphA2 vs.
wild type by unpaired t-test.
5

2
.2. Lead optimization and biological evaluation
Our previous efforts resulted in a new EphA2 agonist with a dimeric structure (Figure 1).18 It
significantly activated EphA2 at 2 µM in vitro, and induced EphA2 kinase internalization at 2 µM as
well. In this study, a total of 27 new derivatives were synthesized using combinatorial chemistry strategy,
mainly focused on the exploration of the dimeric structures.
First, we focused on the modification of the linker of this lead compound (Scheme1). The alkyl
linker cannot serve as either hydrogen bond acceptor or donor. To explore the ligand binding domain,
theses linkers were exchanged with various structures obsessing hydrogen bonding capability. Therefore,
we introduced polyethylene glycol linkers to overcome the possibility that introduction of a hydrogen
bond acceptor or donor could increase the binding activity (compound 3c and 3d). To explore if length
could be a contributing factor, we also generated analogs with short alkyl linkers (compounds 3a, 3b).
We also wanted to explore if flexibility and bulkiness mattered in the ligand binding domain, therefore
rigid aromatic linkers were used too (compounds 3e, 3f). The first set of new linkers for the dimmers are
listed below (Table 1).
O
O
O
O
NH2
N
N
N
MeO
MeO
Cl
Cl
MeO
MeO
N
N
N
N
OMe
OMe
2
a-f
N
N
N
N
NH
K CO , DMF,
2
3
NH2
NH2
8-16h
3a-f
1
Scheme 1. Synthesis of dimers based on 6,7-dimethoxy-2-(piperazin-1-yl) quinazolin-4-amine as the monomer
Molecular
Entry
Addition of linkers: 2a-f
% yield of 3a-f
weight 3a-f
O
Cl
1
Cl
80%
660.72
O
2
a:
6

O
O
O
O
2
3
80%
57%
674.75
676.72
Cl
Cl
Cl
Cl
2
b:
c:
O
2
O
Cl
O
4
5
O
Cl
43%
31%
692.72
858.94
O
2
d:
O
O
Cl
O
O
Cl
2
e:
O
O
Cl
6
31%
899.00
O
Cl
O
2
f:
Table 1. compound list of the original core with the new linkers.
We also modified the original core of the structure by adding benzamide moieties and varying the
aromatic nitro group (5a-5f). These nitro groups were reduced to amino group (7a-7f)(Scheme 2). To
explore if this modification retained activity these compounds were tested with the U251 cell lines. Of
these new analogs we varied the combination of nitro group with different substitutions to examine their
ability to target EphA2 (Table 2).
O
O
R
R
NH
N
Cl
MeO
MeO
N
N
MeO
MeO
N
N
4
a-f
NO2
O N
2
N
N
K CO , DMF
2
3
NH2
4h-12h
NH2
5a-f
O
R
N
NH Cl, Zn
MeO
MeO
N
N
4
MeOH, H O, THF, 2h-4h
2
NH2
N
NH2
6a-f
Scheme 2. Addition of acyl chlorides to produce a small set of monomeric compounds
7

Molecular Molecular
Addition of acyl
chlorides: 4a-f
% yield
of 5a-f
% yield
of 6a-f
Entry
weight
a-f
weight
6a-f
5
O
Cl
1
2
3
4
5
6
87%
87%
85%
72%
79%
83%
78%
89%
78%
77%
72%
77%
438.44
438.44
438.44
472.88
274.88
452.46
408.45
408.45
408.45
472.15
442.90
422.48
NO2
4
a:
b:
c:
O
O
O
O
NO2
Cl
Cl
Cl
Cl
4
4
4
NO2
NO2
Cl
d:
Cl
NO2
NO2
4
e:
O
Cl
4
f:
Table 2. compound bearing the new aromatic core as monomers.
From the new analogs we then attempted to link the two together with the same set of linkers used
beforehand (Scheme 3). Unfortunately, with the additional benzamide moiety, the formed dimers have
poor solubility in various solvents making the purification not practicable. Overall, 9 new dimers were
generated with this process (Table3).
8

O
O
O
R
Cl
Cl
N
MeO
MeO
N
N
2
a-d,g
NH2
N
K CO ,DMF
2
3
8
-16h
NH2
6a-b,e-f
O
O
R
R
O
O
N
N
MeO
MeO
N
N
N
N
OMe
OMe
N
N
H
H
N
N
NH2
NH2
7
Scheme 3. The monomeric compounds from scheme 2 introduced to the linkers from scheme 1.
%
yield of
Molecular
weight
Entry
1
Acyl chloride from 6
Addition of linker 2a-d,g
7
a-d, g
O
O
O
Cl
7ab: 70%
912.99
914.96
898.96
912.99
914.96
930.96
Cl
Cl
Cl
Cl
2
b:
NH2
6
6
a:
a:
O
O
O
Cl
2
3
4
5
6
O
7ac: 70%
7ba: 73%
7bb: 30%
7bc: 23%
7bd: 70%
2
c:
NH2
NH2
O
O
O
O
O
Cl
Cl
Cl
Cl
Cl
Cl
O
2
a:
6
6
6
6
b:
b:
b:
b:
O
O
O
O
NH2
NH2
NH2
Cl
Cl
Cl
Cl
2
b:
O
2
c:
O
Cl
O
O
Cl
O
2
d:
2
g:
O
NH2
O
Cl
7
7bg: 70%
1019.07
Cl
O
O
O
O
Cl
6
b:
O
9

O
O
Cl
O
O
O
O
Cl
Cl
8
9
O
O
7ec: 43%
7fc: 23%
983.85
943.02
Cl
Cl
Cl
Cl
2
2
c:
c:
NH2
NH2
6
e:
6
f:
Table 3. Dimeric compounds bearing the aromatic core with the new linkers.
The 27 new analogs including 15 dimers and 12 monomers show diverse substitution on the linker,
and the biological results of the compounds can provide us a detailed SAR to elucidate the structural
requirement for the targeting of EphA2.
To evaluate the compounds, we utilized the above developed EphA2 overexpressed U251 cells. To
verify the selectivity of the compounds, we also screened the compounds with wild type U251 cells with
low EphA2 expression. The selective index is calculated based on the IC s of the two cell lines (Table
5
0
4
).
All the compounds were examined with multiple doses, and the cells were treated with the
compounds for 48 hr, and cell viability was determined after. We used the lead compound and 0.1%
DMSO as the positive and negative control, respectively.
Among the first set of dimers (scheme 1 and table 1) the activity varied. Compounds 3a and 3b with
reduced length of the alkyl chain resulted in decreased activity, which is consistent with our previous
results and should be eliminated as an optimization direction in the future. Compounds 3d-3f all showed
similar or improved activity compared to the lead, which possible is due to the introduction of hydrogen
binding site or by reducing the flexibility. 3e and 3f both have a rigid and aromatic linker, and both
inhibited the growth of EphA2 overexpressed cells with IC s in the low micro mole range, suggesting
5
0
that keeping the two monomers far away from each other benefit the activity. Compounds 3c and 3d
have polyethylene glycol linkers which may contribute to the binding of the ligand to EphA2 via
1
0

hydrogen bonding. Compound 3c lost the potency but retained the selectivity for EphA2 compared to the
wild type cell lines. However, 3d has a different polyethylene glycol linker, and the potency dramatically
increased and the selectivity remained. Of the first set of dimmers compound 3d showed two-fold more
selectivity to the EphA2 overexpressed cells than the wild type and improved potency compared to the
lead.
In the next scheme we varied the core structure by adding nitro containing benzamide moieties and
then reducing the nitro group to an amino group (Scheme 2 and Table 2). For monomers 5a--6f, most of
them have the IC s above 10µM, regardless if it is the nitro group or the amino group on the benzamide
5
0
moiety. Only compounds 6d, 6e and 6f showed relatively better activity, with an IC of 3.6, 3.0 and 2.7
5
0
µM, respectively. The position of the amino group is critical to the activity and the para position has the
greatest activity of the monomers. Compound 6f had the best selectivity index among all the compounds
with a value of 17.78, suggesting the reduction of the nitro group to the amine adds the possibility of
improved ligand binding to EphA2 receptor. Overall, the trend in the reduction from a nitro group to an
amino group benefitted the activity, suggesting that hydrogen bond donors are important for the activity
on the benzamide moiety.
After synthesis of the dimers based on the amino containing benzamide, the final set of dimmers
were produced (Scheme 3 and Table 3). For the 9 dimers among this group, the activities of the majority
were not appealing with the exception of compound 7bg. It showed an IC of 1.9 µM for EphA2
5
0
overexpressed cells and 7.9 µM for wild type of cells, which results in a selective index of 4.2. Although
the potency was reduced by the addition of the benzamide moiety, it is clear that introduction of
hydrogen binding in the linker has a positive effect on the potency of the compounds. In most cases when
1
1

the polyethylene glycol linkers are used the dimeric compounds had improved activity compared to the
similar monomers. But in comparison to the original core structure, the dimers formed by monomers
with benzamide moiety did not show better activity compared to dimers 3a-3f.
Considering both of the potency and selectivity, we narrow down to compounds 3d, 3e, 7bg for
further mechanism investigation.
Table 4. Inhibition of the cell proliferation of U251/U251 EphA2 cells.
Comp
U251 EphA2
overexpressed*(µM)
2.1 ± 1.05
U251 Wild
type*(µM)
5.2 ± 2.56
Selectivity index
wild/overexpressed**
2.5 ± 2.44
Lead
3
a
6.75 ± 2.40
7.47 ± 3.98
51.86 ± 26.07
1.60 ± 0.40
2.45 ± 1.11
3.18 ± 1.44
12.70 ± 8.72
27.41 ± 15.05
7.41 ± 2.98
12.06 ± 5.98
26.43 ± 11.80
4.41 ± 2.36
11.14 ± 7.26
16.50 ± 9.16
14.60 ± 8.67
5.15 ± 3.01
3.00 ±1.26
12.62 ± 6.62
8.07 ± 4.37
1.9 ± 1.18
1.1 ± 0.82
3.1 ± 2.20
1.9 ± 0.70
3.8 ± 2.84
2.4 ± 1.66
1.0 ± .93
3
b
3
c
160 ± 80.62
3.05 ± 0.82
3
d
3
e
9.22 ± 5.56
3
f
7.48 ± 4.03
5
a
12.23 ± 8.35
24.80 ± 16.01
8.34 ± 4.42
5
b
0.9 ± .77
5
c
1.1 ± .75
5
d
14.73 ± 9.20
53.01 ± 29.78
10.77 ± 7.23
8.98 ± 4.61
1.2 ± .97
5
e
2.0 ± 1.44
2.4 ± 2.10
0.8 ± 0.67
0.5 ± 0.40
2.4 ± 2.10
3.5 ± 3.19
5.5 ± 4.21
17.8 ± 8.62
1.6 ± 1.52
5.7 ± 4.86
1.2 ± 0.97
1.0 ± 0.67
0.8 ± 0.68
5
f
6
a
6
b
8.84 ± 4.36
6
c
35.29 ± 21.98
17.91 ± 12.66
16.49 ± 10.56
47.94 ± 21.13
45.72 ± 34.86
174.01 ± 129.21
47.86 ± 26.93
128.10 ± 53.59
8.11 ± 4.42
6
d
6
e
6
f
2.70 ± 0.55
27.99 ± 14.87
30.44 ± 12.63
41.41 ± 25.89
122.30 ± 59.31
9.92 ±6.20
7
ab
ac
ba
bb
bc
7
7
7
7
1
2

7
7
bd
bg
4.99 ± 2.32
1.90 ± 0.55
3.41 ± 1.50
5.66 ± 2.94
10.12 ± 6.11
7.91 ± 2.28
2.59 ± 1.24
11.69 ± 7.36
2.0 ± 1.55
4.2 ± 1.70
0.8 ± 0.49
2.1 ± 1.69
7
ec
fc
7
The area shaded in gray represent the monomer compounds.
*
*
IC50 values are reported in µM. included Mean±SD.(n=4)
* Selectivity index is calculated by taking the wild type average/Overexpressed average included is the error of propagation
2
.3. EphA2 activation
EphA2 overexpression has been identified in various different malignant cells.2,3,24,6,7,12,13,17,21–23
Decreased levels of EphA2-ligand combined with the overexpression of EphA2 in cancer cells lead to
the aggressive growth of the tumor, which can be overcome in vitro by using endogenous EphA2 ligand
or a small molecule ligand.12,18,19,22,25 Our lead compound could induce EphA2 phosphorylation just as
the endogenous ligand ephrin-A1, and it is necessary to determine if the new analogs could cause EphA2
activation as well.18
Dimers 3e and 7bg showed better activity to induce EphA2 phosphorylation at 2µM, with only 7bg
showing statistical significance compared to control (Figure 3). To our surprise, dimer 3d did not show
clear EphA2 activation. Even 3d showed great activity and selectivity to EphA2 overexpressed cells. It is
possible that 3d targets EphA2 with some unknow different mechanism, which needs further investigation.
1
3

2
.5
.0
.5
.0
.5
.0
*
2
*
1
1
0
0
O
d
e
b
d
3
3
g
S
a
7
e
l
M
D
Figure 3. EphA2 activation of the new derivatives. U251 EphA2 cells were treated with the compounds with 2 µM (in 0.1%
DMSO) for 1hr and cell lysates were subject to immunoprecipitation and immunoblot for phosphorylated EphA/B kinases
(pEphA/B) and total EphA2. Treatment with 2 µM of lead was served as positive control. Treatment with 0.1% DMSO was
served as vehicle control. The band intensities of the bands were quantified using ImageJ (NIH) to generate the figure. The
figures are representative of 3 experiments. *p <0.05 treatment vs. DMSO by unpaired t-test.
2
.4. Log P value
Since the compounds are designed to be used for the treatment of GBM, whether they can cross
BBB is very critical for the potential application. Hansch and Leo found that blood-brain barrier
penetration is optimal when the LogP values of the compounds are in the range of 1.5-2.7, with the mean
value of 2.1. 26,27 Therefore, we measured LogP value of compounds 3d, 3e, and 7bg to predict the
potential capability of these compounds to cross BBB, and the lead compound was measured as a
positive control. The results are listed in Table 5. The data reported here showed that dimers 3e and 7bg
showed similar LogP values compared to the lead compounds. Although the value is out of the range of
the preferred LogP values indicated by Hansch, the lead compound has the ability to cross the BBB
shown in our previous study. 20 We speculate that for these types of dimeric structures, may cross the
BBB with an unknown mechanism instead of passive lipid penetration.
1
4

Compounds
Log P ( Mean±SD)
1.28±0.03
0.75±0.01
3
3
7
d
e
bg
0.78±0.03
Lead comp
0.71±0.02
Table 5. The measurement was repeated three times with water and 1-octanol as the two solvents, and the results are
presented as Mean±SD.
3
. Conclusion
Based on the pervious structure activity relationship, the dimeric structure of Doxazosin analog
showed promising ligand binding activity to EphA2. To further study this unique structure a variety of
structural moieties examined if they could further improve the activity, we synthesized 27 new
derivatives and 15 which retain the dimeric structure. The dimers consist of several different monomers
and various linkers including alkyl, polyethylene glycol and aromatic or rigid structures. Two
compounds 7bg and 3d showed great potency and selectivity toward EphA2 overexpressed GBM cells.
They also stimulated EphA2 phosphorylation, which demonstrated their targeting effect to EphA2
receptor. The LogP values of the two identified dimers are not in the ideal range for their capability to
cross the BBB. However, the lead compound has a similar LogP value and does cross the BBB in our
20
previous study, suggesting that these types of compounds may possess a novel mechanisms to cross the
BBB other than passive lipid penetration, which will need further investigation.
4
4
. Experimental section
.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
1
5

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 ethanol/dichloromethane was used as the
elution solvent. Mass spectra were obtained on the electrospray mass spectrometer at Cleveland State
University MS facility Center. The molecular weight of the compounds was examined with LC-MS. All
1
the NMR spectra were recorded on a Bruker 400 MHz in DMSO-d . Chemical shifts () for H NMR
6
spectra are reported in parts per million to residual solvent protons.
Compounds 3a-3f 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 linkers and K CO in DMF. It was stirred until the reaction was
2
3
completed. The reaction was quenched with Na CO aqueous solution and stirred overnight, and the
2
3
precipitated product was collected via filtration and washed with water and dried to give the
corresponding compounds.
1
,4-bis(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)butane-1,4-dione (3a)
1
Light pink solid, 80% yield for the last step; H-NMR (400MHz, DMSO-d ) δ 7.44 (2H, s), 7.14 (4H, b),
6
1
3
6
.76 (2H, s), 3.84 (6H, s), 3.81(10H, s) 3.70 (4H, s), 3.55-3.52 (4H, s), 2.63 (4H, s). C-NMR (400MHz,
DMSO-d ) δ 170.67, 161.62, 159.10, 158.82, 154.73, 149.35, 149.26, 145.50, 145.39, 105.73, 104.20,
6
+
1
6
03.44, 56.33, 55.89, 45.33, 44.25, 43.93, 41.73, 28.15. DUIS-MS calculated for C H N O [M + H] :
3
2
40 10
6
60.31, found : 661.10
1
6

1
,5-bis(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)pentane-1,5-dione (3b).
1
Light pink solid, 80% yield for the last step; H-NMR (400MHz, DMSO-d ) δ 7.43 (2H, s), 7.14 (4H, b),
6
6
.75 (2H, s), 3.84 (6H, s), 3.80 (6H, s), 3.75 (4H, s), 3.70 (4H, s), 3.52 (8H, s), 2.43-2.40 (3H, t, J=7.1
1
3
Hz), 1.78 (2H, t, J= 14.0 Hz). C-NMR (400MHz, DMSO-d ) δ 171.09, 161.62, 159.10, 158.79, 154.73,
6
1
3
2
49.35, 149.26, 145.50, 145.39, 105.72, 104.19, 103.44, 56.33, 55.89, 45.34, 44.33, 44.18, 43.94, 41.54,
+
2.41, 21.09. DUIS-MS calculated for C H N O [M + H] : 674.33, found : 675.15
3
3
42 10
6
,2'-oxybis(1-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)ethanone) (3c)
1
Light pink solid, 72% yield for the last step; H-NMR (400MHz, DMSO-d ) δ 7.43 (2H, s), 7.15 (4H, b),
6
1
3
6
.76 (4H, s), 4.31 (4H, s), 3.84 (6H, s), 3.79 (6H, s), 3.75(8H,s) 3.52 (8H, s) C-NMR (400MHz,
DMSO-d ) ) δ 167.62, 161.63, 158.76, 154.73, 149.25, 145.52, 105.74, 104.18, 103.45, 69.64, 56.33,
6
+
5
6
5.89, 44.72, 44.28, 43.87, 41.66. DUIS-MS calculated for C H N O [M + H] : 676.31, found :
3
2
40 10
7
77.20
ethane-1,2-diyl bis(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazine-1-carboxylate) (3d)
1
Light pink solid, 43% yield for the last step; H-NMR (400MHz, DMSO-d ) δ 7.43 (2H, s), 7.14 (4H, b),
6
1
3
6
.75 (4H, s), 4.27 (4H, s), 3.83 (6H, s), 3.79 (6H, s), 3.78(8H, s), 3.45 (8H, s) C-NMR (400MHz,
DMSO-d ) δ 162.77, 161.63, 158.76, 155.00, 149.22, 145.52, 105.72, 104.19, 103.46, 63.67, 56.32,
6
+
5
5.87, 43.98, 43.80. DUIS-MS calculated for C H N O [M + H] : 692.30, found : 693.15
3
2
40 10
8
propane-2,2-diylbis(4,1-phenylene)bis(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazine-1-carb
oxylate) (3e)
1
light pink solid, 31% yield for the last step; H-NMR (400MHz, DMSO-d ) δ 7.45 (2H, s), 7.24-7.18
6
(8H, m), 7.07-7.06 (4H, m), 6.78(2H, s) 3.84 (9H, s), 3.80 (9H, s), 3.64 (4H, m) 3.50 (4H, m), 1.66 (6H,
1
3
s).
C-NMR (400MHz, DMSO-d ) δ 161.67, 158.68, 154.77, 153.60, 149.51, 149.09, 147.45, 145.58,
6
1
7

1
27.75, 121.74. 105.66, 104.23, 103.48, 56.34, 55.91, 43.78, 42.38, 31.02. DUIS-MS calculated for
+
C H N O [M + H] : 858.38, found : 859.15
4
5
50 10
8
cyclohexane-1,1-diylbis(4,1-phenylene)bis(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazine-1-c
arboxylate) (3f)
1
light pink solid, 31% yield for the last step; H-NMR (400MHz, DMSO-d ) δ 7.45 (2H, s), 7.31 (2H,
6
s),7.28 (2H, s), 7.18(4H, b) 7.06 (2H, s) , 7.04 (2H, s), 6.77 (2H, s) 3.84 (6H, s), 3.80 (13H, s), 3.63 (5H,
1
3
m) 3.50 (6H, m), 2.25 (4H, s), 1.47 (6H, s). C-NMR (400MHz, DMSO-d ) δ 161.66, 158.64, 154.78,
6
1
4
53.56, 149.26, 149.04, 145.59, 145.42, 128.04, 121.91, 105.64, 104.24, 103.47, 56.35, 55.92, 45.52,
+
3.78, 36.80, 26.17, 22.97. DUIS-MS calculated for C H N O [M + H] : 898.41, found : 899.25
4
8
54 10
8
Compounds 5a-5f were prepared as the following procedure.
The dimethoxyquinazolin and piperazine product was mixed with various different substituted acetyl
chloride and K CO in THF / H O one to one mixture. It was stirred until the reaction was completed.
2
3
2
The reaction was quenched with Na CO aqueous solution and stirred overnight, and the precipitated
2
3
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)(4-nitrophenyl)methanone (5a)
1
Light yellow solid, 87% yield for the step; H-NMR (400MHz, DMSO-d ) 8.30, 8.28, (1H, d, J= 7.9 Hz),
6
8
7
3
1
1
.25-8.23 (1H, d, J= 7.9), 8.13-8.11 (1H, d, J= 8.1Hz), 7.74-7.72 (1H, d, J = 8.0 Hz), 7.47-7.45 (1H, d, J=
.0 Hz) 7.19 (2H, b), 6.76-6.75( 1H, d, J= 5.9 Hz), 3.94 (2H, s), 3.83 (4H, s), 3.80 (3H, s), 3.73 (2H, s)
1
3
.35 (1H, s), 3.12 ( 1H, s). C-NMR (400MHz, DMSO-d ) δ 167.73, 167.39, 161.74, 161.65, 158.66,
6
58.35, 154.78, 154.73, 149.38, 149.05, 148.95, 148.29, 145.78, 145.58, 142.80, 130.77, 128.86, 124.25,
23.64, 105.70, 105.64, 104.19, 103.61, 103.48, 56.33, 55.90, 47.43, 44.22, 43.77, 43.08, 42.16, 41.36.
1
8

+
DUIS-MS calculated for C H N O [M + H] : 438.17, found : 439.15
2
1
22
6
5
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(3-nitrophenyl)methanone (5b)
1
Light yellow solid, 80% yield for the step; H-NMR (400MHz, DMSO-d ) ) δ 8.33-8.31 (1H, d, J= 8.2
6
Hz), 8.27 (1H, s), 7.92-7.90 (1H, d, J= 7.6 Hz), 7.79-7.75(1H, t, J= 15.9 Hz) 7.15(2H, b), 6.75 (1H, s),
1
3
3
1
5
.84 (4H, s), 3.80 (4H, s), 3.74 (3H, m), 3.40 (1H, m). C-NMR (400MHz, DMSO-d ) δ 167.39, 161.65,
6
58.78, 154.75, 149.22, 148.21, 145.58, 138.01, 133.92, 130.71, 124.72, 122.46, 105.73, 104.20, 103.50,
+
6.33, 55.89, 44.19, 42.23. DUIS-MS calculated for C H N O [M + H] : 438.17, found : 439.10
2
1
22
6
5
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(2-nitrophenyl)methanone (5c)
1
Light yellow solid, 85% yield for the step; H-NMR (400MHz, DMSO-d ) δ 8.23-8.212, (1H, d, J= 8.0
6
Hz), 7.87 (1H, t, J= 6.9 Hz), 7.72 (1H, t, J= 7.3 Hz), 7.60-7.58 (1H, d, J= 7Hz) 7.44 (1H, s), 7.17 (2H, b)
6
1
1
4
.74 (1H, s), 3.83 (5H, s), 3.79 (4H, s), 3.72 (3H, m), 3.26 (2H, m)-NMR (400MHz, DMSO-d ) δ
6
66.02, 161.65, 158.77, 154.72, 149.16, 145.91, 145.59, 133.36, 132.89, 130.77, 128.62, 125.23, 105.71,
+
04.15, 103.51,56.31, 55.89, 46.95, 43.95, 43.65, 41.88. DUIS-MS calculated for C H N O [M + H] :
2
1
22
6
5
38.17, found : 439.10
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(4-chloro-3-nitrophenyl)methanone (5d)
1
Light yellow solid, 72% yield for the step; H-NMR (400MHz, DMSO-d ) δ 8.18, (1H, s), 7.88-7.87 (1H,
6
s), 7.80 (1H, s), 7.44 (1H, s) 7.17 (2H, b), 6.74 (1H, s), 3.83 (6H, s), 3.79 (5H, s), 3.73-3.60 (4H, m), 3.41
1
3
(
2H, m.) C-NMR (400MHz, DMSO-d ) δ 166.45, 161.63, 158.71, 154.72, 149.12, 148.03, 145.56,
6
1
36.69, 132.76, 132.39, 126.40, 124.90, 105.66, 104.12, 103.46, 56.30, 55.89, 47.55, 44.18, 43.71, 42.32.
+
DUIS-MS calculated for C H ClN O [M + H] : 472.13, found : 472.95
2
1
21
6
5
1
9

(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(2-chloro-4-nitrophenyl)methanone (5e)
1
Light yellow solid, 79% yield for the step; H-NMR (400MHz, DMSO-d ) δ 8.42 ( 1H, s), 8.30-8.28 (1H,
6
d, J= 7.5 Hz), 7.76-7.74 (1H, d, J= 7.8) 7.43 (1H, s), 7.17 (2H, b), 6.76-6.74 (1H, d, J= 7.0), 3.83 (4H, s),
1
3
3
1
1
.79 (4H, s), 3.73 (3H, s), 3.22 (1H, s) . C-NMR (400MHz, DMSO-d ) δ 164.61, 161.64, 161.58,
6
59.09, 158.68, 154.72, 154.67, 149.33, 149.13, 148.60, 145.60, 145.37, 145.31, 130.86, 129.71, 125.15,
23.39, 105.70, 104.20, 104.12, 103.50, 103.39, 56.31, 55.89, 46.62, 44.21, 44.17, 43.76, 41.81.
+
DUIS-MS calculated for C H ClN O [M + H] : 472.13, found : 473.00
2
1
21
6
5
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(4-methyl-3-nitrophenyl)methanone (5f)
1
Light yellow solid, 83% yield for the step; H-NMR (400MHz, DMSO-d ) δ 8.05, (1H, s), 7.70 (1H, s),
6
7
.60 (1H, s), 7.44 (1H, s) 7.16 (2H, b), 6.74 (1H, s), 3.83 (4H, s), 3.80 (4H, s), 3.74 (4H, m), 3.42 (2H,
1
3
m), 2.56(3H, s). C-NMR (400MHz, DMSO-d ) δ 167.37, 161.63, 158.76, 154.72, 149.28, 149.19,
6
1
4
45.55, 135.45, 134.52, 133.47, 132.12, 123.67, 105.70, 104.10, 103.48, 56.30, 55.88, 47.65, 44.19,
+
3.82, 42.23, 19.82. DUIS-MS calculated for C H N O [M + H] : 452.18, found : 453.15
2
2
24
6
5
Compounds 6a-6f was prepared as the following procedure.
Last step products with the nitro group were reduced by using NH Cl 5 molar equivalence and Zn 15
4
molar equivalence to the nitro compounds. This was monitored by TLC in DCM/MeOH until reaction
was complete it was then stopped by using an Acid base extraction. Acid was added until all zinc was
dissolved then filtered to remove any undissolved zinc or other contaminates. Follow by readjusting the
pH to a basic 12 by adding sodium carbonate aqueous solution. This mixture was then extracted by DCM
and ethyl acetate then was dried with MgSO and washed with diethyl ether and hexane to give the
4
2
0

corresponding compounds.
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(4-aminophenyl)methanone (6a)
1
Light yellow solid, 78% yield for the step; H-NMR (400MHz, DMSO-d ) δ 7.43 (1H, s), 7.19-7.17 (3H,
6
m), 6.74 (1H, s), 6.59 (1H, s) 6.56 (1H, d), 5.53 (2H, s), 3.83 (3H, s), 3.79 (3H, s), 3.76 (4H, s) 3.56 (4H,
1
3
s). C-NMR (400MHz, DMSO-d ) 170.55, 161.61, 158.82, 154.70, 150.99, 149.24, 145.48, 129.80,
6
1
22.44, 113.19, 105.70, 104.16, 103.44, 56.31, 55.88, 44.22. DUIS-MS calculated for C H N O [M +
21 24 6 3
+
H] : 408.19, found : 409.15
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(3-aminophenyl)methanone (6b)
1
Light yellow solid, 89% yield for the last step; H-NMR (400MHz, DMSO-d ) δ 7.43 (1H, s), 7.15 (2H,
6
b), 7.10-7.06 (1H, t, J= 7.7 Hz), 6.74 (1H, s), 6.64-6.2 (1H, d, J= 8.0 Hz), 6.59 (1H, s), 6.53-6.51 (1H, d,
J= 7.4 Hz), 3.83 (3H, s), 3.79 (3H, s), 3.75 (4H, m), 3.62-3.59 (2H, t, J= 16.5 Hz), 3.42 (2H, b).
1
3
C-NMR (400MHz, DMSO-d ) δ 170.30, 161.61, 158.80, 154.72, 149.20, 145.54, 137.13, 129.32,
6
1
15.25, 114.43, 112.63 105.71, 104.18, 103.47, 56.33, 55.89, 44.17. DUIS-MS calculated for
+
C H ClN O [M + H] : 408.19, found : 409.05
2
1
21
6
5
(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)(2-aminophenyl)methanone (6c)
1
Light yellow solid, 78% yield for the step; H-NMR (400MHz, DMSO-d ) δ 7.43 (1H, s), 7.15-7.11 (3H,
6
m), 7.04 (1H, s), 6.74 (2H, s) 6.58 (1H, b), 5.19 (2H, s), 3.83 (4H, s), 3.79 (7H, s), 3.53 (4H,
1
3
m). C-NMR (400MHz, DMSO-d ) δ 169.28, 161.61, 158.81, 154.70, 149.22, 146.32, 145.50, 130.50,
6
1
28.29, 119.80, 116.01, 105.70, 104.14, 103.45, 56.31, 55.88, 44.20, 30.89. DUIS-MS calculated for
2
1

+
C H N O [M + H] : 408.19, found : 409.15
2
1
24
6
3
(3-amino-4-chlorophenyl)(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)methanone (6d)
1
Light yellow solid, 77% yield for the step; H-NMR (400MHz, DMSO-d ) δ 7.44 (1H, s), 7.25 (1H, s),
6
7
.18, (2H, s), 6.84 (1H, s), 6.74 (1H, s), 6.57 (1H, s) 5.57 (2H, b), 3.83 (3H, s), 3.79 (5H, m), 3.64 (5H,
1
3
m). C-NMR (400MHz, DMSO-d ) δ 169.27, 161.62, 158.78, 154.69, 149.17, 145.52, 145.22, 135.92,
6
1
29.46, 118.40, 115.58, 114.29, 105.68, 104.12, 103.48, 56.30, 55.89, 47.59, 44.17, 42.17. DUIS-MS
+
calculated for C H N O [M + H] : 442.15, found : 443.10
1
7
23
5
3
(4-amino-2-chlorophenyl)(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)methanone (6e)
1
Light yellow solid, 72% yield for the step; H-NMR (400MHz, DMSO-d ) δ 7.43 (1H, s), 7.14, (2H, b),
6
7
.01-6.99, (1H, d, J= 7.5 Hz), 6.73 (1H, s) 6.63 (1H, s), 6.55- 6.53 ( 1H, d, J= 7.5 Hz), 5.63 (2H, s) 3.83
1
3
(
4H, s) 3.79 (5H, s), 3.65-3.60 (8H, m), 3.25 (2H, s), 3.17(2H, s) C-NMR (400MHz, DMSO-d ) δ
6
1
1
+
67.25, 161.61, 158.77, 154.70, 151.04, 149.17, 145.54, 130.48, 129.40, 122.63, 113.56, 112.90, 105.70,
04.13, 103.47, 56.32, 55.90, 49.07, 46.91, 44.34, 43.94, 41.83. DUIS-MS calculated for C H N O [M
1
9
21
5
4
+
H] : 442.15, found : 443.05
(3-amino-4-methylphenyl)(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)methanone (6f)
1
Light yellow solid, 77% yield for the step; H-NMR (400MHz, DMSO-d ) δ 7.43 (1H, s), 7.14 (2H, b),
6
6
.99-6.97 (1H, d, J= 7.7 Hz), 6.74 (1H, s), 6.67 (1H, s), 6.50-6.49 (1H, s, J= 3 Hz), 5.02 (2H, b) 3.83
(
3H, s), 3.80 (4H, m), 3.723-3.40 (8H, m), 2.08(3H, s). 13C-NMR (400MHz, DMSO-d ) δ 170.41,
6
1
5
61.65, 154.82, 147.06, 145.76, 134.53, 130.16, 123.04, 1114.97, 112.95, 104.51, 103.31, 56.45, 56.10,
+
5.98, 44.37, 17.80. DUIS-MS calculated for C H N O [M + H] : 422.21, found : 423.10
2
2
26
6
3
2
2

Compounds 7aa-7fc were prepared as the following procedure.
The corresponding reduced benzamides 6a-6f were mixed with different dichloride linkers. They
were dissolved in anhydrous DMF in a 2 to 1 ratio under argon protection. The dichloride linkers where
diluted in anhydrous DMF and added slowly. It was stirred overnight and monitored by TLC after 24
hours. The reaction was stopped by reducing down the solvent and purified with PTLC in DCM/EtOH.
1
5
N ,N -bis(4-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazine-1-carbonyl)phenyl)glutaramide
7ab)
Light yellow solid, 70% yield for the last step; H-NMR (400MHz, DMSO-d ) 10.15 (2H, s), 7.71-7.69
(
1
6
(4H, m), 7.44-7.40 (7H, d), 7.16 (6H, m), 6.74 (3H, d), 3.83 (10H, m), 3.79 (13H, m), 3.56 (9H, m) 2.44
1
3
(
4H, s) 1.93 (2H, s). C-NMR (400MHz, DMSO-d ) δ 171.61, 169.51, 161.62, 158.78, 154.70, 154.52,
6
1
4
2
49.20, 145.51, 140.99, 130.58, 128.94, 128.60, 120.88, 118.94, 105.69, 104.15, 103.45, 56.31, 55.88,
+
4.17, 36.06, 21.29. DUIS-MS calculated for C H N O [M + H] : 912.40, found : 913.20
4
7
52 12
8
,2'-oxybis(N-(4-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazine-1-carbonyl)phenyl)acetamid
e) (7ac)
1
Light yellow solid, 70% yield for the last step; H-NMR (400MHz, DMSO-d ) δ 10.27 (2H, s), 7.78 (2H,
6
s), 7.77 (2H, s), 7.47 (2H, s), 7.45 (2H, s) 7.43 (2H, s), 7.15 (4H, m) , 6.74 (2H, s), 4.33 (4H, s) 3.83
1
3
(
10H, s), 3.79 (11H, m) 3.61-3.56 (8H, m). C-NMR (400MHz, DMSO-d ) δ 169.40, 168.88, 161.62,
6
1
5
58.77, 154.71, 149.18, 145.52, 139.99, 131.34, 128.64, 119.61, 105.68, 104.15, 103.45, 71.33, 56.31,
+
5.88, 44.13. DUIS-MS calculated for C H N O [M + H] : 914.38, found : 915.15
4
6
50 12
9
1
4
N ,N -bis(3-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazine-1-carbonyl)phenyl)succinamide
(7ba)
2
3

1
Light yellow solid, 70% yield for the last step; H-NMR (400MHz, DMSO-d ) δ 10.32 (2H, s), 8.55 (4H,
6
s), 7.83(2H, s), 7.70 (2H, s) 7.64-7.62 (2H, d, J= 7.0 Hz), 7.42-7.38(2H, t, J= 14 Hz), 7.12-7.10 (2H, d,
1
3
J= 7.8 Hz), 3.92 (8H, m), 3.86 (8H, s), 3.84 (7H, s) 3.76 (4H, m) 3.53 (5H, m) 2.71 (4H, s). C-NMR
(
400MHz, DMSO-d ) δ 171.18, 169.60, 161.74, 155.59, 147.08, 139.91, 129.38, 121.93, 120.57, 118.05,
6
+
1
05.29, 102.39, 56.68, 56.43, 45.00, 31.68. DUIS-MS calculated for C H N O [M + H] : 898.39,
4
6
50 12
8
found : 899.20
1
5
N ,N -bis(3-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazine-1-carbonyl)phenyl)glutaramide
(7bb)
1
Light yellow solid, 70% yield for the last step; H-NMR (400MHz, DMSO-d ) 10.09 (2H, s), 7.45 (2H,
6
s), 7.64-7.62 (2H, m), 7.43 (2H, s) 7.40-7.38 (4H, m), 7.14-7.10(6H, m), 6.74 (2H, d), 3.92 (8H, m), 3.83
1
3
(
9H, m), 3.79 (18H, m) 2.41 (4H, s) 1.93 (2H, s). C-NMR (400MHz, DMSO-d ) δ 171.57, 169.44,
6
1
1
9
2
61.62, 158.79, 154.72, 149.18, 145.55, 139.77, 136.78, 129.33, 121.98, 120.50, 118.15, 105.71, 104.15,
+
03.48, 56.31, 55.89, 47.55, 44.06, 36.03, 21.28. DUIS-MS calculated for C H N O [M + H] :
4
7
52 12
8
12.40, found : 913.20
,2'-oxybis(N-(3-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazine-1-carbonyl)phenyl)acetamid
e) (7bc)
1
Light yellow solid, 73% yield for the last step; H-NMR (400MHz, DMSO-d ) δ 10.26 (2H, s), 7.82 (2H,
6
s), 7.74-7.73 (2H, d, J= 6.7 Hz), 7.44 (4H, m) 7.15 (5H, b), 6.74 (2H, d), 4.31 (3H, s) 3.82 (8H, s), 3.79
1
3
(
9H, m). C-NMR (400MHz, DMSO-d ) δ 169.30, 168.82, 162.78, 161.62, 158.80, 154.71, 149.19,
6
1
4
45.55, 138.83, 129.44, 122.71, 121.16, 118.79, 105.71, 104.17, 103.49, 71.31, 56.32, 55.88, 47.64,
+
4.18, 42.26, 36.24, 31.25. DUIS-MS calculated for C H N O [M + H] : 914.38, found : 915.10
4
6
50 12
9
ethane-1,2-diylbis((3-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazine-1-carbonyl)phenyl)carb
2
4

amate) (7bd)
light yellow solid, 30% yield for the last step; H-NMR (400MHz, DMSO-d ) δ 9.94 (2H, s), 7.57-7.54
1
6
(4H, m), 7.43 (3H, s), 7.37 (2H, s) 7.14 (5H, b), 7.07-7.05 (2H, d), 6.74 (3H, s) 4.37 (4H, s) 3.83 (11H,
1
3
s), 3.79 (11H, m) . C-NMR (400MHz, DMSO-d ) δ 169.38, 161.62, 158.78, 154.71, 153.81, 149.18,
6
1
5
9
45.54, 139.58, 136.91, 129.44, 121.52, 119.75, 117.30, 105.70, 104.15, 103.47, 63.27, 62.49, 56.31,
+
5.88, 47.56, 44.12, 42.18, 25.95. DUIS-MS calculated for C H N O [M + H] : 930.38, found :
4
6
50 12 10
31.20
(ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl)
bis((3-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazine-1-carbonyl)phenyl)carbamate) (7bg)
1
Light yellow solid, 23% yield for the last step; H-NMR (400MHz, DMSO-d ) δ 9.92 (2H, s), 7.57-7.53
6
(4H, m), 7.43 (2H, s), 7.37-7.36 (2H, s) 7.14 (4H, b), 7.06-7.04 (2H, d), 6.74 (2H, s) 4.22 (3H, s) 3.83
1
3
(
9H, s), 3.79 (10H, m), 3.67 (4H, s), 3.59 (4H, s). C-NMR (400MHz, DMSO-d ) δ 169.41, 162.77,
6
1
1
61.62, 158.78, 154.70, 153.96, 149.19, 145.53, 139.70, 136.90, 129.41, 121.38, 119.61, 117.15, 105.71,
04.14, 103.47, 70.18, 69.15, 64.12, 60.69, 56.31, 55.88, 47.64, 44.17, 42.24, 36.24, 31.25. DUIS-MS
+
calculated for C H N O [M + H] : 1018.43, found : 1019.20
5
0
58 12 12
2
,2'-oxybis(N-(4-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazine-1-carbonyl)-3-chlorophenyl)
acetamide) (7ec)
1
Light yellow solid, 43% yield for the last step; H-NMR (400MHz, DMSO-d ) δ 10.32 (2H, s), 7.97 (2H,
6
s), 7.68-7.66 (2H, d, J= 8.0 Hz), 7.44 (2H, s), 7.41-7.39 (2H, s), 7.18 (5H, m) , 6.74 (3H, s), 4.33 (4H, s)
1
3
3
1
1
.83 (13H, s), 3.75 (11H, s), 3.72 (11H, m) 3.24 (6H, m). C-NMR (400MHz, DMSO-d ) δ 169.09,
6
66.09, 161.63, 158.64, 154.74, 148.97, 145.60, 145.48, 140.35, 131.20, 129.89, 129.02, 120.15, 118.86,
05.61, 105.42, 104.27, 104.16, 103.48, 103.35, 71.13, 56.32, 55.90, 46.78, 44.31, 44.16, 43.88, 41.79.
2
5

+
DUIS-MS calculated for C H C N O [M + H] : 982.30, found : 983.10
4
6
48 l2 12
9
2
,2'-oxybis(N-(5-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazine-1-carbonyl)-2-methylphenyl
)acetamide) (7fc)
1
Light yellow solid, 23% yield for the last step; H-NMR (400MHz, DMSO-d ) δ 9.62 (2H, s), 7.60 (2H,
6
s), 7.43 (2H, s), 7.34-7.32 (2H, d, J= 7.0 Hz), 7.21-7.19 (2H, d, J= 7.6 Hz), 7.15 (4H, b) , 6.74 (2H, s),
1
3
4
1
1
.33 (4H, s) 3.83 (9H, s), 3.79 (11H, s), 2.29 (6H, m) 1.36 (3H, s). C-NMR (400MHz, DMSO-d ) δ
6
69.16, 168.46, 161.62, 158.77, 154.71, 149.20, 145.52, 135.90, 134.08, 133.91, 130.91, 124.73, 124.29,
05.71, 104.15, 103.46, 71.09, 56.31, 55.88, 44.16, 34.85, 30.90, 18.09. DUIS-MS calculated for
+
C H N O [M + H] : 942.41, found : 943.20
4
8
54 12
9
4
.2. LogP measurement
Compounds 3d, 3e, 7gb and lead compound were used in the determination of their log P values.
This method follows the OECD test guidelines for the slow stir method, with slight modification due to
the data collection method. The data was collected on a UV-Vis spectrophotometer Shimadzu UV-1280
and standard UV Quartz 10 mm cuvette was used. The chemical was purchased from VWR brand
EMPLURA 1-octanol 99% purity. The Flask where equipped with Teflon coated magnetic stirrers and
Coring PC420D stir plates where used. The 1-octanol and DDH O where placed into two separate
2
containers containing both phases to become mutual saturated by allowing them to stir for 48 h. Then the
two phases where separated by a separation funnel. Compounds 3d, 3e, 7gb and lead now were dissolved
in 1-octanol pre-saturated with water. These stock solutions are then centrifuged at 5,000 rpm for 5 min.
The supernatant was taken to be the working solution. Take 5 mL working solution of each into new
clear flask, and add identical volume water saturated with 1-octanol, then stir at room temperature for 48
h. After that, stop stirring; take two-phase solution out, centrifuge at 5,000 rpm for 5 min to separate this
2
6

two-phase system. Removal of the 1-octanol phase from the solution then transferred for UV
spectrophotometry to achieve absorbance at the correct wavelength. According to the procedure,
concentrations were determined by UV measurement so, equation written as:
Ax
Log P= log
A0  Ax
Where A and A are the initial and final absorbance values of organic layer.
0
x
HPLC-PDA analysis of the purity of the compounds.
Reversed-phase HPLC analysis of compounds were conducted on Beckman HPLC system with
AutoSampler (table 6). The chromatographic separation was performed on a C18 column (2.0 mm × 150
mm, 5 μm) from Phenomenex (Torrance, CA, USA). one mobile phase was employed for isocratic
elution with a flow rate of 0.2 mL/min. The injection volume was 6 µL and the PDA detector was set up
3
46nm
Table 6. Purity of the compounds
H O/MeOH (20%/80%)
2
Compound Retention time (R ) Purity
t
in minutes
3
a
1.90
1.90
1.98
1.88
1.86
1.92
1.75
1.76
1.76
1.76
99.99%
99.99%
97.59%
99.99%
99.61%
90.71%
99.97%
99.95%
99.91%
99.90%
3
b
3
c
3
d
3
e
3
f
5
a
5
b
5
c
5
d
2
7

5
e
1.75
1.76
1.74
1.75
1.75
1.75
1.78
1.74
1.79
1.82
1.86
1.86
1.84
1.85
1.83
1.84
1.85
99.75%
99.95%
99.95%
99.95%
99.94%
99.77%
99.54%
97.79%
99.29%
98.93%
98.95%
98.88%
97.08%
97.23%
98.15%
98.01%
99.44%
5
f
6
a
6
b
6
c
6
d
6
e
6
f
7
ab
ac
ba
bb
bc
bd
bg
ec
fc
7
7
7
7
7
7
7
7
4
4
.3. Biological studies
.3.1. Cell Culture and Antibodies.
U251 cells were obtained from ATCC (Rockville, MD). The cells were maintained in Dulbecco’s
Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mg/ml
L-Glutamine, 100 U/mL penicillin-streptomycin, and 0.1 mg/ml streptomycin. EphA2 overexpression
cells were grown in the presence of 1 µg/ml puromycin. Cell cultures were grown at 37 ºC, in a
humidified atmosphere of 5% CO in a Thermo CO incubator (Grand Island, NY). Antibodies used
2
2
included rabbit polyclonal antibodies against pEphA/B (Santa Cruz, Santa Cruz, CA), as well as mouse
2
8

monoclonal antibody to EphA2 (clone D7, Millipore, Billerica, MA).
4
.3.2. Mammalian cell viability analysis
The MTT assay was used to examine the effect of the compounds on the growth of U251 and U251
over expression EphA2 cells in four replications. 3000 cells per well were seeded with RPMI1640
medium in 96-well flat-bottomed plates for 24 h and were then exposed to various concentrations of test
compounds dissolved in DMSO (final concentration 0.1%) in medium for 48 h. Controls received
DMSO at a same concentration as that in drug-treated cells. Cells were incubated in 200 µL of 0.5
mg/mL of MTT reagent diluted in fresh media at 37°C for 3 h. Supernatants were removed from the
wells, and the reduced MTT dye was solubilized with 200 µL/well DMSO. Absorbance at 570 nm was
determined on a SpectraMax Plus384 spectrophotometer (Molecular Devices). Data obtained with
quadruplication were normalized and fitted to a dose–response curve using GraphPad Prism v.5
(GraphPad).
4
.3.3. EphA2 activation.
U251 EphA2-overexpressing cells were plated in 6-well dishes at a density of 100,000 cells/well and
grown for 24 hours prior to stimulation with appropriate compounds for 30 min. 0.1% DMSO was used as
vehicle control. Following treatment, cells were washed and lysed in modified RIPA Buffer (20 mM
Tris-HCl pH 7.4, 20 mM NaF, 150 mM NaCl, 10% glycerol, 0.1% SDS, 0.5% DCA, 2 mM EDTA, 1%
Triton X-100, 2 mM Na VO , and protease inhibitors, including 1 mM phenylmethylsulphonyl fluoride,
3
4
and 2 µg/ml each of aprotinin and leupeptin) for 20 min, followed by immunoprecipitation. Samples were
boiled 5 min and run on 4-12% Bis-Tris Plus gels (Thermo Fisher), followed by transfer to Immobilon-P
PVDF membranes (Millipore, Billerica, MA). Membranes were blocked 1 hr at room temperature in 3%
BSA in TBS containing 0.05% Tween-20 (TBS-T) followed by overnight incubation with pEphA/B
(
1:500) and EphA2 (Santa Cruz) (1:1000) primary antibodies. Membranes were washed in TBS-T and
2
9