Novel quinazoline-urea analogues as modulators for Aβ-induced mitochondrial dysfunction: Design, synthesis, and molecular docking study

Article abstract of DOI:10.1016/j.ejmech.2014.07.027

A novel series of twenty-six quinazoline-urea derivatives was designed and synthesized. Their blocking activities against β-amyloid peptide (Aβ) induced mitochondrial permeability transition pore (mPTP) opening were evaluated by JC-1 assay which measured

Full text of DOI:10.1016/j.ejmech.2014.07.027

Accepted Manuscript
Novel quinazoline-urea analogues as modulators for Aβ-induced mitochondrial
dysfunction: Design, synthesis, and molecular docking study
Ahmed Elkamhawy, Jiyoun Lee, Beoung-Geon Park, Insun Park, Ae Nim Pae, Eun
Joo Roh
PII:
S0223-5234(14)00633-3
10.1016/j.ejmech.2014.07.027
EJMECH 7147
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 April 2014
Revised Date: 4 July 2014
Accepted Date: 8 July 2014
Please cite this article as: A. Elkamhawy, J. Lee, B.-G. Park, I. Park, A.N. Pae, E.J. Roh, Novel
quinazoline-urea analogues as modulators for Aβ-induced mitochondrial dysfunction: Design,
synthesis, and molecular docking study, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.07.027.
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ACCEPTED MANUSCRIPT
Graphical abstract:

ACCEPTED MANUSCRIPT
Novel quinazoline-urea analogues as modulators for Aβ-induced
mitochondrial dysfunction: Design, synthesis, and molecular
docking study
a,b
c
d,e
e,f
Ahmed Elkamhawy , Jiyoun Lee , Beoung-Geon Park , Insun Park ,
e
a,b*
Ae Nim Pae , Eun Joo Roh
a
Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), Seoul 136-791, South
Korea
b
Department of Biological Chemistry, Korea University of Science and Technology (UST), Daejeon 305-350, South
Korea
c
Department of Global Medical Science, Sungshin Women's University, Seoul 142-732, South Korea
d
School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea
e
Center of Neuromedicine, Korea Institute of Science and Technology (KIST), Seoul 136-791, South Korea
f
Department of Biotechnology, Yonsei University, Seoul 120-749, South Korea
*
Corresponding author: Eun Joo Roh
Email address: r8636@kist.re.kr
Postal address: Korea Institute of Science and Technology, Chemical Kinomics Research Center, Future
Convergence Research Division, Hwarang-ro 14-gil 5, Seongbuk-gu, Seoul 136-791, South Korea
1

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Abstract:
A novel series of twenty six quinazoline-urea derivatives was designed and synthesized.
Their blocking activities against β-amyloid peptide (Aβ) induced mitochondrial permeability
transition pore (mPTP) opening were evaluated by JC-1 assay which measured the change of
mitochondrial membrane potential. Seven compounds showed better inhibitory activities than the
standard Cyclosporin A (CsA). The most active analogues were tested by MTT assay to evaluate
their toxicity on the cellular survival; they revealed excellent cellular viability. To explain the
difference in inhibitory activity, molecular docking study using (GOLD) program was performed
for selected sets of the most active and inactive compounds on cyclophilin D (CypD) receptor as
a major component of mPTP. Moreover, ADME profiling, in silico toxicity, drug-likeness, and
drug-score studies were discussed. From these results, we report compound 31 as the most active
nonpeptidyl mPTP blocker possessing quinazoline-urea scaffold; 2 folds of CsA activity, which
would constitute a new direction for the design of novel mPTP modulators.
Keywords: Mitochondrial permeability transition pore (mPTP), Alzheimer’s disease (AD),
Quinazoline-urea, cyclophilin D (CypD), β-amyloid peptide (Aβ), Molecular docking.
2

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1
. Introduction
Dementia is caused by different diseases like stroke or arteriosclerosis in the brain,
traumatic brain injury, Parkinson disease and other neurodegenerative disorders. Alzheimer’s
disease (AD) comes to the fore as the most common cause so that it may contribute to 60–70%
of cases [1-4]. Abundant evidence suggests that an important key factor associated with
Alzheimer’s disease etiology is the excessive production of β-amyloid peptide (Aβ) and its
extracellular deposition [5]. Aβ can be found in the brains of Alzheimer’s disease patients as
plaques as well as small soluble oligomers [6]. Recent studies indicate that mitochondria are
intracellular targets for these soluble Aβ oligomers, especially, mitochondrial permeability
transition pore (mPTP) which has been reported to play a key role in mitochondrial dysfunction
induced by Aβ toxicity [7, 8]. The mPTP is a multiprotein complex found in the mitochondria
under certain pathological conditions including oxidative stress, ischemia, strock and traumatic
membrane injury [9]. It consists of three major components: a voltage-dependent anion channel
(VDAC) in the outer membrane, the adenine nucleotide translocator (ANT) in the inner
membrane, and cyclophilin D (CypD) in the mitochondrial matrix. In the normal state, the
membrane potential is regulated by reversible opening and closing of the mPTP, which maintain
intracellular calcium homeostasis. However, an accumulation of soluble Aβ oligomers in the
(AD) neuronal mitochondria leads to excessive calcium entry into cytosol. The elevated calcium
levels result in uncontrolled mPTP opening, inhibition of mitochondrial ATP production and
severe mitochondrial swelling followed by outer membrane rupture. Hence, neuronal injury
occurs due to proapoptotic proteins production from damaged mitochondria. These findings
prove the role of mitochondrial dysfunction in Alzheimer’s disease and lead to develop a new
3

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direction of drug targeting against neuronal mPTP which may be a promising therapeutic
approach for treatment of Alzheimer’s disease [7, 10].
Although some peptidic compounds including Cyclosporin A (CsA) and its analogue (N-
methyl-4-isoleucin cyclosporin (NIM811)) have been identified as mPTP inhibitors [7, 11, 12],
they are not useful as therapeutic agents because of their poor bioavailability characteristics in
addition to the hurdles of peptide delivery to the brain. Up to now, no safe mPTP inhibitors
specific to the brain have been reported for AD treatment in humans [10]. Thus, the main
objective of the present study was to design and synthesize novel nonpeptidyl small molecules
that could inhibit the activity of the mPTP. Herein, a novel series of twenty six quinazoline-urea
analogues was designed, synthesized and biologically screened against Aβ-induced mPTP
opening activity.
Since CypD is one of the three major components of mPTP protein complex, our rational
design based on its high structural similarity with cyclophilin A (CypA) because these are two
members of family highly homologous peptidyl prolyl cis-trans isomerases (PPIases) [13]. In
literature, compounds I and II (Figure. 1A) were discovered as CypA inhibitory lead compounds.
The common structural features of these compounds are the urea linker and 3-benzyloxyaryl
moiety which are responsible for the activity [14]. Recently from our institute, two papers on
compounds III & IV (Figure 1B), supported the role of the benzyloxyaryl moiety for the activity
against mPTP opening [15, 16]. Accordingly, to identify novel mPTP blockers with improved in
vitro efficacy, structural modifications were carried out on compound III as shown in Figure 1C.
Quinazoline core replaced the pyridine to investigate the quinazoline-urea as a new scaffold
4

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against mPTP opening activity. Variations of substitution patterns were performed on the
benzyloxy moiety to evaluate the structure activity relationship. Moreover, the size of the aryl
segment attached to the urea linker has been assessed through different substitutions of aryl
groups.
<Insert Figure 1 here>
2
. Results and discussion
2
.1.Chemistry
The target quinazoline-urea derivatives 6–31 were prepared according to the sequence of
reactions depicted in Scheme 1. Heating 2,4-dinitrophenol 1 with appropriate benzylbromide in
the presence of anhydrous K CO and catalytic amount of potassium iodide in acetonitrile
2
3
afforded the corresponding dinitrobenzene derivatives 2a–2f which by catalytic hydrogenation at
room temperature using 10% Pt/C in methanol gave the diamine analogues 3a–3f. The
dicarbamate derivatives 4a–4f were obtained by stirring compounds 3a–3f with ethyl
chloroformate in the presence of triethylamine (TEA) in tetrahydrofuran (THF) at room
temperature. Cyclization to quinazolineamines 5a–5f was carried out by treatment of
intermediates 4a–4f with hexamethylenetetramine (HMTA) in the presence of trifluoroacetic
acid (TFA) at room temperature, and subsequent heating with 10% aqueous-ethanolic KOH (1:1)
3 6
and K Fe(CN) [17]. Reaction of the amino groups in compounds 5a–5f with appropriate
isocyanate derivatives in THF at 85 °C provided the target quinazoline-urea derivatives 6–31.
<Insert Scheme 1 here>
5

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2
2
.2. Biological evaluation
.2.1. JC-1 assay (in vitro screening)
The ability of the new quinazoline-urea derivatives to inhibit the mitochondrial membrane
potential loss is summarized in Table 1. Their activity to block the Aβ-induced mPTP opening
was evaluated at a single dose concentration of 5 µM using a cell-based JC-1 assay by measuring
the change of mitochondrial membrane potential [18, 19]. The color shift from green to red as
the membrane potential increases indicates the recovery of mitochondrial function responding to
applied Aβ toxicity. In fact, compounds III & IV exhibited 57% and 41% of increased green to
red ratio, respectively, which means that they were able to reduce 43% and 59% of Aβ-induced
mitochondrial damage [15, 16]. CsA was used as a positive standard; it exerted 46% of increased
green to red ratio.
<
Insert Table 1 here>
It was noted that when R
2
was 2-fluorophenyl as in 25 and 31, or 3-fluorophenyl as in 9, 11,
1
6, and 30, they exhibited better inhibitory activities than those with 1-naphtyl (7), 4-
trifluoromethylphenyl (12, 17) or 4-methylphenyl (10, 18). It can be deduced that the relatively
bulky substituents linked to urea moiety are not well tolerated at the target site, whereas it is
more favorable for the ligand to have R as phenyl group substituted with small size halogen.
2
However, compounds 13 and 14 possessing bulky substituents showed good inhibitory activities.
1
Of particular significance was the observation that compounds having R as para-
chlorobenzyloxy groups 26–31 showed improved activities than their corresponding analogues
6

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with para-fluorobenzyloxy groups 17–22. These results could suggest that the size of the
halogen groups on the benzyloxy moiety is critical for activity.
The pharmacological results indicated that seven compounds including 9, 11, 13, 14, 25, 30
and 31 exhibited mPTP opening inhibitory activity superior to that of CsA. They revealed 38, 27,
2
6.7, 39.4, 31, 29, and 24% of increased green to red ratio. Compound 31 possessing para-
chlorobenzyloxy moiety and 3-fluorophenyl segment attached to the urea linker was the most
active compound in this series.
2
.2.2. MTT assay (Cell viability screening)
Since it is essential for the active compounds to modulate the activity of mPTP opening
without exhibiting toxic effects on the cellular viability, the most active compounds were
selected to measure their toxicity on the cellular activity by MTT assay at a single dose of 5 µM.
As shown in Table 2, all the tested compounds revealed promising values ranging from 83 to
1
31%. In this event, compound 31 exerted excellent cell survival (102%), while cellular viability
of 90% was achieved by the standard CsA.
<
Insert Table 2 here>
2
.2.3. Molecular docking
To explain the difference in inhibitory activity of the target compounds, Genetic Optimization
for Ligand Docking (GOLD) [20] was used as flexible docking program to predict the different
7

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molecular binding modes of CypD with compound III as well as selected sets of the most active
and inactive compounds based upon X-ray cocrystal structure of CypD receptor with its inhibitor
CsA (PDB: 2Z6W) [21]. In CypD−CsA complex; Residues of five amino acids (Trp121, Arg55,
Gln63, Asn102, and Gly72) are involved in H-bond interactions. As shown in Figure 2;
prediction showed that Compound III may bind to the active site through two H-bond
interactions with Gln63 and Asn102 amino acid residues, in addition to a relatively weak π-π
stacking interaction formed with Phe113 phenyl moiety. The current default fitness scoring
function in Gold Suite-5.2 (CHEMPLP score) was used in the present study since recent
validation tests have shown it to be generally more effective than the other scoring functions for
both pose prediction and virtual screening [20]. In this event, Compound III exerted CHEMPLP
score of 61.8714.
<
Insert Figure 2>
We then clarified the different binding modes of CypD with selected sets of the most active
and inactive analogues, exploiting docking calculations and CHEMPLP fitness scoring functions
to get an explanation for the in vitro results of JC-1 assay against mPTP opening activity. The
docking results showed that two possible binding modes may be energetically favored for CypD
with the active set including compounds 9, 11, 25, 30, and 31. The active ligands achieved
effectively two to four H-bond interactions with CypD which led to remarkably increase of
CHEMPLP scores ranging from 62.0435 to 68.9412 as listed in Table 3.
<
Insert Table 3 here>
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As representative hypothetical model for the first binding mode, docking of compound 25 is
presented in Figure 3. It was noted that the quinazoline-urea scaffold paved the way for the
ligand to interfere more efficiently with the active site so that three H-bonds were formed with
Asn102, Gly109 and Gln111 residues, in addition to stronger π-π stacking interaction with
Phe113 phenyl moiety. This well-established interaction revealed high CHEMPLP score
(68.94120). The second binding mode presented by compound 31, was characterized by a ligand
rotation in the active site where the benzyloxy moiety changed its position with the phenyl urea
segment, however, this orientation kept three H-bond interactions with Arg55 and Gln63
residues through the urea linker as shown in Figure 4 with high CHEMPLP score (65.5608).
<
Insert Figure 3 & 4 here>
On the other hand, most binding modes obtained for CypD with the inactive set including
compounds 17, 18, 20, 22, and 29 clearly suggested an energetically unfavorable binding
orientation characterized by perpendicular flipping of the ligand out of the binding site.
Compounds 18, 22, and 29 couldn’t establish any H-bond interaction with the target protein
while both of compounds 17 and 20 achieved H-bonds with only one amino acid residue as listed
in Table 3. This inefficient binding orientation resulted in relatively low CHEMPLP fitness
scores ranging from 54.8588 to 58.4236. That could potentially be attributed to steric hindrance
factors of the substituted groups on the phenyl urea ring. Docking models of compounds 17, 18
and 29 possessing 4-trifluoromethylphenyl, 4-methylphenyl, and 3,4-dichlorophenyl moieties are
presented in Figure 5.
9

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<Insert Figure 5 here>
Results of these docking study show a great variability of binding modes which may provide
a possible explanation for the in vitro results previously discussed for JC-1 assay; compounds
bearing R as small halogen 9, 11, 25, 30, and 31 fitted well in the active site of CypD with high
2
CHEMPLP scores vice versa compounds 17 and 18 with bulky substituents. In addition,
Compounds 29−31 possessing para-chlorobenzyloxy groups showed better CHEMPLP fitness
scores than those with para-fluorobenzyloxy groups (17, 18, 20, and 22). Therefore, it is worth
emphasizing that the relation between mPTP blocking activity and CHEMPLP scoring function
was highly correlated as illustrated in Table 4.
<
Insert Table 4 here>
2
.2.4. Molar refractivity, Total polar surface area, and ADME profiling
Since the size of the different substituents played a major role in the activity and the
docking fitness scores, we investigated the influence of the molar refractometry (MR, steric
factor) on the activity for the compounds with the most active analogues as listed in Table 5.
Generally, it was noted that compounds 11, 25, 30, and 31 with low MR values exerted higher
activities against mPTP opening comparing to that of compound 14 with the highest MR value.
Moreover, In case of compounds 13 and 14 with the same 2-fluorobenzyloxy moiety, the MR
value of 13 possessing 4-methylphenyl segment was lower than 14 with 1-naphtylphenyl moiety
1
0

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vice versa the potency. From these results, we can conclude that MR and bulkiness are inversely
proportional to the activity.
<
Insert Table 5 here>
In this present report, the total polar surface area (TPSA) was also calculated since it is a
key property that has been linked to drug bioavailability. Thus, passively absorbed molecules
with a TPSA > 140 are thought to have low oral bioavailability [22]. All the evaluated
2
compounds have TPSA = 76.14
oral absorption.
Å
(Table 5) suggesting that they have a possible good passive
Approximately 40% of drug candidates fail in clinical trials due to their poor ADME
absorption, distribution, metabolism, and elimination), we assessed the compliance of the most
(
active compounds to the Lipinski rule of five [23]. Violation of more than one of these rules may
result in bioavailability problems. Predictions of ADME properties using Osiris program [24] are
given in Table 5. The results showed that all the studied compounds comply with these rules,
consequently, good ADME properties are theoretically guaranteed.
2
.2.5. In silico toxicity, drug-likeness, and drug score
Osiris program was used for prediction of the overall toxicity of the most active derivatives
as the prediction process relies on a predetermined set of structural fragments that give rise to
toxicity alerts in case they are encountered in the structure. Interestingly, all compounds
presented low in silico possible toxicity risks in most cases as shown in Table 6, only except
1
1

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compound 14 with the bulky naphthyl moiety, showed a high risk of possible tumorigenic
toxicity. These theoretical data suggest that the safety of analogues bearing small size halogen or
methyl on the phenyl ring attached to the urea linker is better than the corresponding derivatives
bearing bulky naphthyl group.
<
Insert Table 6 here>
Currently, there are many approaches to assess a compound drug-likeness based on
topological descriptors, fingerprints of molecular drug-likeness structure keys or other properties
such as clog P and molecular weight [25]. In this work, Osiris program was also used for
calculating the fragment-based drug-likeness of the most active compounds as illustrated in
Table 6. Compounds 30 and 31 showed improvement of drug-likeness values comparing to other
derivatives and lead compounds I, II, and III. Herein, compound 31 demonstrated the highest
drug-likeness value.
Of special interest, the drug-scores of the active compounds have also been determined in the
present study as shown in Figure 6. The results showed that compound 31 exerted the highest
value comparing to all series derivatives as well as all lead compounds. The in silico toxicity
profiles, drug-likeness, and drug-score data of compound 31 make it a promising lead for future
development of safe and efficient mPTP opening inhibitors.
<Insert Figure 6 here>
3
. Conclusion
1
2

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Based on this study, we report compound 31 as the most active nonpeptidyl mPTP blocker
possessing quinazoline-urea scaffold; 2 folds of CsA potency, which would constitute a new
direction for the design of novel mPTP modulators as recent therapeutic approach for the
treatment of Alzheimer’s disease. Extensive SAR (structure-activity relationship) study as well
as detailed pharmacological profile for the active analogues with in vivo study on Alzheimer’s
disease animal model will be our next plan.
4
. Experimental
4
.1. Chemistry
All reactions and manipulations were performed in nitrogen atmosphere using standard
Schelenk techniques. Reagents and solvents were purchased from Aldrich Co. and Tokyo
Chemical Industry Co. and used without purification. Thin-layer chromatography was performed
with Merk silica gel 60 F254 pre-coated glass sheets. Column chromatography was performed on
Merck Silica Gel 60 (230-400 mesh) and the eluting solvents are noted as mixed solvent with
1
13
given volume-to-volume ratios. NMR (400 MHz for H and 100 MHz for C) was measured on
Bruker Avance 400, and chemical shifts and coupling constants are presented in parts per million
relative to Me Si and hertz, respectively. Abbreviations are as follows: s, singlet; d, doublet; t,
4
triplet; q, quartet; m, multiplet; br, broad. Low-resolution spectra were carried out on Agilent
1
200 Series−API 3200 LC/MS/MS System. High-resolution spectra were performed on Waters
ACQUITY UPLC BEH C18 1.7µ−Q-TOF SYNAPT G2-Si High Definition Mass Spectrometry.
4
.1.1. General procedure for preparation of compounds 2a–2f:
1
3

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2 3
To a solution of 2,4-dinitrophenol (0.92 g, 5 mmol) in acetonitrile (15 mL), anhydrous K CO
(2.07 g, 15 mmol) was added. The mixture was stirred for 30 minutes at room temperature.
Catalytic amount of potassium iodide (0.17 g, 1 mmol) and the appropriate benzylbromide (5
mmol) were added. The reaction was stirred at 75 °C for 8 h. Water (125 mL) was added to the
reaction mixture and the organics were extracted with ethyl acetate (3 × 125 mL). The organic
layer extracts were washed with brine and dried over anhydrous Na SO . The solvent was
2
4
evaporated under reduced pressure and the residue was purified by flash column chromatography
silica gel, hexane/ethyl acetate 3:1 v/v).
(
4
.1.1.1.1-(benzyloxy)-2,4-dinitrobenzene (2a)
1
Yield 58%; H NMR (400 MHz, Acetone-d
6
) δ [ppm]: 8.75 (d, J = 2.8 Hz, 1H), 8.52 (dd, J = 9.3,
2
.8 Hz, 1H), 7.71 (d, J = 9.3 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.45–7.38 (m, 3H), 5.53 (s, 2H).
4
.1.1.2.1-(3-fluorobenzyloxy)-2,4-dinitrobenzene (2b)
1
Yield 70%; H NMR (400 MHz, CDCl
3
) δ [ppm]: 8.80 (d, J = 2.7 Hz, 1H), 8.43 (dd, J = 9.2, 2.7
Hz, 1H), 7.42 (dd, J = 13.6, 7.8 Hz, 1H), 7.25 (d, J = 9.2 Hz, 2H), 7.20 (d, J = 9.2 Hz, 1H), 7.10
td, J = 8.2, 1.9 Hz, 1H), 5.38 (s, 2H).
(
4
.1.1.3.1-(2-fluorobenzyloxy)-2,4-dinitrobenzene (2c)
1
Yield 75%; H NMR (400 MHz, DMSO-d
6
) δ [ppm]: 8.79 (d, J = 2.6 Hz, 1H), 8.56 (dd, J = 9.3,
2
7
1
.6 Hz, 1H), 7.78 (d, J = 9.3 Hz, 1H), 7.61 (t, J = 7.3 Hz, 1H), 7.49 (dd, J = 13.4, 6.36 Hz, 1H),
13
.30 (dd, J = 11.7, 5.1 Hz, 2H), 5.54 (s, 2H). C NMR (100 MHz, DMSO-d ) ꢀ 161.99, 159.54,
6
55.76, 140.43, 139.25, 131.51, 130.88, 129.81, 125.16, 122.52, 121.72, 116.52, 66.70.
1
4

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4
.1.1.4.1-(4-fluorobenzyloxy)-2,4-dinitrobenzene (2d)
1
Yield 68%; H NMR (400 MHz, CDCl ) δ [ppm]: 8.79 (d, J = 2.7 Hz, 1H), 8.44 (dd, J = 9.2, 2.7
3
Hz, 1H), 7.47 (dd, J = 8.3, 5.2 Hz, 2H), 7.26 (s, 1H), 7.14 (t, J = 8.6 Hz, 2H), 5.35 (s, 2H).
4
.1.1.5.1-(2-chlorobenzyloxy)-2,4-dinitrobenzene (2e)
1
Yield 75%; H NMR (400 MHz, DMSO-d
6
) δ [ppm]: 8.80 (d, J = 2.6 Hz, 1H), 8.55 (dd, J = 9.2,
2
.6 Hz, 1H), 7.75 (d, J = 9.2 Hz, 1H), 7.64 (t, J = 4.0 Hz, 1H), 7.55 (t, J = 4.0 Hz, 1H), 7.45–7.43
(m, 2H), 5.55 (s, 2H).
4
.1.1.6.1-(4-chlorobenzyloxy)-2,4-dinitrobenzene (2f)
1
Yield 62%; H NMR (400 MHz, Acetone-d
6
) δ [ppm]: 8.75 (d, J = 2.8 Hz, 1H), 8.52 (dd, J = 9.3,
2
2
1
.8 Hz, 1H), 7.69 (d, J = 9.3 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 5.53 (s,
1
3
H). C NMR (100 MHz, Acetone-d ) δ [ppm]: 156.83, 141.33, 140.21, 135.07, 134.73, 130.17,
6
30.10, 129.84, 129.66, 129.59, 122.21, 116.65, 72.03.
4
.1.2. General procedure for preparation of compounds 3a–3f:
To a solution of compound 2a–2f (4.2 mmol) in methanol (70 mL), 10% Pt/C (0.42 mmol) was
added and the reaction mixture was stirred under hydrogen atmosphere at room temperature for 6
h. The mixture was filtered through celite and the filtrate was evaporated under reduced pressure
and the residue was purified by flash column chromatography (silica gel, hexane/ethyl acetate
1
:1 v/v) to afford the desired diamines.
1
5

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4
.1.2.1.4-(benzyloxy)benzene-1,3-diamine (3a)
1
Yield 85%; H NMR (400 MHz, CDCl ) δ [ppm]: 7.46–7.34 (m, 5H), 6.71 (d, J = 8.4 Hz, 1H),
3
6
.17 (d, J = 2.6 Hz, 1H), 6.06 (dd, J = 8.4, 2.6 Hz, 1H), 5.01 (s, 2H).
4
.1.2.2.4-(3-fluorobenzyloxy)benzene-1,3-diamine (3b)
1
Yield 88%; H NMR (400 MHz, CDCl ) δ [ppm]: 7.33 (dd, J = 13.0, 7.5 Hz, 1H), 7.17 (dd, J =
3
1
6
3.0, 7.5 Hz, 2H), 7.01 (t, J = 8.4 Hz, 1H), 6.65 (d, J = 8.4 Hz, 1H), 6.14 (d, J = 2.0 Hz, 1H),
.03 (dd, J = 8.4, 2.0 Hz, 1H), 4.98 (s, 2H).
4
.1.2.3.4-(2-fluorobenzyloxy)benzene-1,3-diamine (3c)
1
Yield 90%; H NMR (400 MHz, CDCl ) δ [ppm]: 7.40 (t, J = 6.5 Hz, 1H), 7.21 (d, J = 6.5 Hz,
3
1
2
1
H), 7.07–6.98 (m, 2H), 6.63 (d, J = 8.7 Hz, 1H), 5.9 (s, 2H), 4.95 (s, 2H), 3.76 (s, 2H), 3.35 (s,
1
3
H). C NMR (100 MHz, CDCl ) δ [ppm]: 161.85, 141.84, 139.55, 138.10, 130.20, 129.86,
3
24.86, 124.30, 115.41, 115.15, 104.93, 103.60, 67.12.
4
.1.2.4.4-(4-fluorobenzyloxy)benzene-1,3-diamine (3d)
1
Yield 92%; H NMR (400 MHz, CDCl ) δ [ppm]: 7.41 (dd, J = 8.5, 5.5 Hz, 2H), 7.08 (t, J = 8.5
3
Hz, 2H), 6.68 (d, J = 8.5 Hz, 1H), 6.17 (d, J = 2.5 Hz, 1H), 6.07 (dd, J = 8.5, 2.5 Hz, 1H), 4.96 (s,
2
H).
4
.1.2.5.4-(2-chlorobenzyloxy)benzene-1,3-diamine (3e)
1
6

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1
Yield 95%; H NMR (400 MHz, DMSO-d
6
) δ [ppm]: 7.65–7.63 (m, 1H), 7.50–7.47 (m, 1H),
7
1
.39–7.36 (m, 2H), 6.57 (d, J = 8.3 Hz, 1H), 5.97 (d, J = 2.3 Hz, 1H), 5.75 (dd, J = 8.3, 2.3 Hz,
H), 4.95 (s, 2H), 4.53 (s, 2H), 4.42 (s, 2H).
4
.1.2.6.4-(4- chlorobenzyloxy)benzene-1,3-diamine (3f)
1
Yield 75%; H NMR (400 MHz, CDCl ) δ [ppm]: 7.37 (br, 2H), 7.36 (br, 2H), 7.34–7.29 (m,
3
3
H), 4.70 (s, 2H).
4
.1.3. General procedure for preparation of compounds 4a–4f:
A mixture of diamine 3a–3f (4 mmol), ethyl chloroformate (16 mmol), and TEA (16 mmol) in
anhydrous THF (40 mL) was stirred at room temperature for 2 h. The solid was filtered off and
the solution was evaporated under reduced pressure. The residue was dissolved in EtOAc (100
mL) and the organic phase was washed with water (2 x 100 mL). The organic phase was
evaporated under reduced pressure and the residue was purified by flash column chromatography
(silica gel, hexane/ethyl acetate 4:1 v/v) to yield the corresponding dicarbamates 4a–4f.
4
.1.3.1.Diethyl 4-(benzyloxy)-1,3-phenylenedicarbamate (4a)
1
Yield 86%; H NMR (400 MHz, Acetone-d
6
) δ [ppm]: 8.46 (s, 1H), 8.28 (d, J = 2.5 Hz, 1H),
7
4
1
1
.59 (s, 1H), 7.49 (d, J = 8.3 Hz, 2H), 7.42–7.34 (m, 4H), 6.99 (d, J = 8.9 Hz, 1H), 5.12 (s, 2H),
13
3
.17 (q, J = 7.1 Hz, 4H), 1.25 (t, J = 7.1 Hz, 6H). C NMR (100 MHz, CDCl ) δ [ppm]: 153.93,
53.27, 143.15, 137.09, 132.99, 128.53, 128.35, 127.90, 113.14, 112.39, 110.38, 70.86, 60.75,
4.07.
1
7

ACCEPTED MANUSCRIPT
4
.1.3.2.Diethyl 4-(3-fluorobenzyloxy)-1,3-phenylenedicarbamate (4b)
1
Yield 83%; H NMR (400 MHz, CDCl ) δ [ppm]: 7.99 (s, 1H), 7.38 (dd, J = 13.2, 7.4 Hz, 1H),
3
7
1
.18 (d, J = 8.3 Hz, 2H), 7.13 (d, J = 8.4 Hz, 1H), 7.07 (t, J = 8.6 Hz, 1H), 6.84 (d, J = 8.9 Hz,
H), 6.23 (s, 1H), 5.09 (s, 2H), 4.24 (q, J = 7.0 Hz, 4H), 1.33 (t, J = 7.0 Hz, 6H). HRMS
+
(
ES+): m/z calculated for C H FN O : 399.1332 [M+Na] . Found 399.1326.
19
21
2
5
4
.1.3.3.Diethyl 4-(2-fluorobenzyloxy)-1,3-phenylenedicarbamate (4c)
1
Yield 70%; H NMR (400 MHz, CDCl ) δ [ppm]: 8.07 (s, 1H), 7.38–7.28 (m, 3H), 7.13–7.04 (m,
3
1
3
2
H), 6.85 (d, J = 8.4 Hz, 1H), 5.07 (s, 2H), 4.17 (q, J = 6.5 Hz, 4H), 1.25 (t, J = 6.5 Hz, 6H). C
NMR (100 MHz, CDCl ) δ [ppm]: 158.75, 152.03, 151.48, 140.55, 130.57, 128.73, 128.21,
3
1
25.61, 122.28, 121.60, 113.56, 111.07, 110.47, 107.92, 63.28, 60.23, 12.53.
4
.1.3.4.Diethyl 4-(4-fluorobenzyloxy)-1,3-phenylenedicarbamate (4d)
1
Yield 80%; H NMR (400 MHz, CDCl ) δ [ppm]: 7.99 (d, J = 2.2 Hz, 1H), 7.39 (dd, J = 8.7, 5.4
3
Hz, 2H), 7.18 (s, 1H), 7.11 (t, J = 8.7 Hz, 2H), 6.87 (d, J = 8.7 Hz, 1H), 6.58 (s, 1H), 5.05 (s, 2H),
4
3
2 5
.23 (q, J = 7.0 Hz, 4H), 1.32 (t, J = 7.0 Hz, 6H). HRMS (ES+): m/z calculated for C19H21FN O :
+
99.1332 [M+Na] . Found 399.1328.
4
.1.3.5.Diethyl 4-(2-chlorobenzyloxy)-1,3-phenylenedicarbamate (4e)
1
Yield 88%; H NMR (400 MHz, DMSO-d
6
) δ [ppm]: 9.46 (s, 1H), 8.32 (s, 1H), 7.79 (s, 1H),
7
1
.65 (s, 1H), 7.52−7.50 (m, 1H), 7.40−7.38 (m, 2H), 7.14 (d, J = 7.7 Hz, 1H), 7.01 (d, J = 8.9 Hz,
H), 5.15 (s, 2H), 4.06 (q, J = 6.3 Hz, 4H), 1.22 (t, J = 6.3 Hz, 6H).
1
8

ACCEPTED MANUSCRIPT
4
.1.3.6.Diethyl 4-(4-chlorobenzyloxy)-1,3-phenylenedicarbamate (4f)
1
Yield 88%; H NMR (400 MHz, Acetone-d ) δ [ppm]: 8.46(s, 1H), 8.18 (d, J = 2.4 Hz, 1H), 7.60
6
(s, 1H), 7.54 (d, J = 8.5 Hz, 2H), 7.44 (d, J = 8.5 Hz, 2H), 7.28 (d, J = 7.2 Hz, 1H), 7.01 (d, J =
8
.9 Hz, 1H), 5.17 (s, 2H), 4.15 (q, J = 6.8 Hz, 4H), 1.26 (t, J = 6.8 Hz, 6H). HRMS
+
(
ES+): m/z calculated for C H ClN O : 415.1037 [M+Na] . Found 415.1034.
19
21
2
5
4
.1.4. General procedure for preparation of compounds 5a–5f:
A mixture of dicarbamate 4a–4f (5 mmol) and HMTA (35 mmol) in TFA (35 mL) was refluxed
for 1 h. The mixture was then cooled to room temperature and diluted with 4 M HCl (200 mL).
The undissolved residue was filtered off and the solution was evaporated under reduced pressure.
2
The residue was dissolved in aqueous ethanolic (H O/EtOH, 1/1) 10% KOH (300 mL), added of
K
3
Fe(CN) (12.5 g, 38 mmol) and refluxed for 4 h. After cooling, the mixture was diluted with
6
water (300 mL), extracted with ethyl acetate (500 mL), and the organic phase was evaporated
under reduced pressure. The residue was purified by flash column chromatography (silica gel,
methylene chloride/methanol 25:1 v/v).
4
.1.4.1.6-(benzyloxy)quinazolin-7-amine (5a)
1
Yield 30%; H NMR (400 MHz, CDCl ) δ [ppm]: 9.03 (s, 1H), 8.99 (s, 1H), 7.49–7.38 (m, 5H),
3
7
.08 (d, J = 1.9 Hz, 2H), 5.25 (s, 2H), 4.75 (s, 2H). LC/MS (ESI+): m/z calculated for
+
C H N O: 252.1 [M+H] . Found 252.1.
15
13
3
4
.1.4.2.6-(3-fluorobenzyloxy)quinazolin-7-amine (5b)
1
9

ACCEPTED MANUSCRIPT
1
Yield 20%; H NMR (400 MHz, CDCl
3
) δ [ppm]: 9.03 (s, 1H), 8.98 (s, 1H), 7.39 (dd, J = 7.6,
5
.8 Hz, 1H), 7.24 (s, 1H), 7.19 (d, J = 9.2 Hz, 1H), 7.09–7.04 (m, 3H), 5.24 (s, 2H), 4.75 (s, 2H).
4
.1.4.3.6-(2-fluorobenzyloxy)quinazolin-7-amine (5c)
1
Yield 25%; H NMR (400 MHz, CDCl
3
) δ [ppm]: 9.06 (s, 1H), 9.03 (s, 1H), 7.52 (t, J = 8.8 Hz,
1
4
1
H), 7.42 (dd, J = 13.6, 5.9 Hz, 1H), 7.25–7.18 (m, 2H), 7.15 (s, 1H), 7.12 (s, 1H), 5.34 (s, 2H),
1
3
.45 (s, 2H). C NMR (100 MHz, CDCl ) δ [ppm]: 163.01, 156.42, 154.22, 148.43, 147.51,
3
44.87, 130.62, 130.06, 124.52, 122.75, 119.62, 116.15, 106.64, 104.47, 64.73.
4
.1.4.4.6-(4-fluorobenzyloxy)quinazolin-7-amine (5d)
1
Yield 32%; H NMR (400 MHz, CDCl ) δ [ppm]: 9.06(s, 1H), 9.02 (s, 1H), 7.48 (dd, J = 8.5, 5.3
3
Hz, 2H), 7.15 (t, J = 8.5 Hz, 2H), 7.11 (d, J = 4.6 Hz, 2H), 5.23 (s, 2H), 4.74 (s, 2H).
4
.1.4.5.6-(2-chlorobenzyloxy)quinazolin-7-amine (5e)
1
Yield 27%; H NMR (400 MHz, DMSO-d ) δ [ppm]: 9.02 (s, 1H), 8.83 (s, 1H), 7.82–7.78 (m,
6
1
H), 7.57–7.53 (m, 1H), 7.48–7.42 (m, 3H), 6.95 (s, 1H), 6.27−6.26 (m, 2H), 5.36 (s, 2H).
4
.1.4.6.6-(4-chlorobenzyloxy)quinazolin-7-amine (5f)
1
Yield 37%; H NMR (400 MHz, CDCl ) δ [ppm]: 9.04 (s, 1H), 8.99 (s, 1H), 7.43 (br, 4H), 7.12
3
1
3
(
s, 1H), 7.07 (s, 1H), 5.22 (s, 2H), 4.80 (s, 2H). C NMR (100 MHz, CDCl ) δ 156.28, 154.20,
3
1
48.39, 147.52, 144.82, 134.55, 134.09, 129.12, 119.56, 106.59, 104.47, 70.10.
4
.1.5. General procedure for preparation of compounds 6–31:
2
0

ACCEPTED MANUSCRIPT
A mixture of quinazolinamines 5a–5f (1.0 mmol) and the appropriate isocyanate derivative (1.2
mmol) in anhydrous THF (10 mL) was stirred at 85 °C overnight. The mixture was evaporated
under reduced pressure and the residue was purified by flash column chromatography (silica gel,
methylene chloride/ethyl acetate 1:4 v/v).
4
.1.5.1.1-(6-(benzyloxy)quinazolin-7-yl)-3-p-tolylurea (6)
1
Yield 64%; H NMR (400 MHz, Acetone-d ) δ [ppm]: 9.17 (s, 1H), 9.06 (s, 1H), 8.97 (s, 1H),
6
8
.89 (s, 1H), 8.53 (s, 1H), 7.67–7.56 (m, 3H), 7.46–7.39 (m, 5H), 7.11 (d, J = 8.4 Hz, 2H), 5.43
+
(
s, 2H), 2.29 (s, 3H). HRMS (ES+): m/z calculated for C H N O : 407.1484 [M+Na] . Found
2
3
20
4
2
4
07.1493.
4
.1.5.2.1-(6-(benzyloxy)quinazolin-7-yl)-3-(naphthalen-1-yl)urea (7)
1
Yield 72%; H NMR (400 MHz, DMSO-d ) δ [ppm]: 9.78 (s, 1H), 9.30 (s, 1H), 9.26 (s, 1H),
6
9
1
1
1
.06 (s, 1H), 8.80 (s, 1H), 8.21 (d, J = 8.0 Hz, 1H), 7.98 (t, J = 7.6 Hz, 2H), 7.72 (d, J = 8.0 Hz,
1
3
6
H), 7.67 (s, 1H), 7.63–7.45 (m, 8H), 5.54 (s, 2H). C NMR (100 MHz, DMSO-d ) δ [ppm]:
62.80, 157.62, 154.30, 153.30, 147.80, 147.11, 137.70, 136.44, 134.24, 134.12, 129.15, 128.87,
28.69, 128.27, 127.14, 126.51, 126.31, 124.43, 122.41, 121.00, 119.62, 113.41, 106.14, 70.94.
+
LC/MS (ESI+): m/z calculated for C H N O : 421.16 [M+H] . Found 421.13.
2
6
20
4
2
4
.1.5.3.1-(6-(benzyloxy)quinazolin-7-yl)-3-(3,4-dichlorophenyl)urea (8)
1
Yield 58%; H NMR (400 MHz, DMSO-d ) δ [ppm]: 10.09 (s, 1H), 9.26 (s, 1H), 9.08 (s, 1H),
6
8
8
.86 (s, 1H), 8.73 (s, 1H), 7.96–7.93 (m, 1H), 7.67 (s, 1H), 7.60 (d, J = 7.1 Hz, 2H), 7.56 (d, J =
13
.8 Hz, 1H), 7.45 (t, J = 7.1 Hz, 2H), 7.40–7.34 (m, 2H), 5.48 (s, 2H). C NMR (100 MHz,
2
1

ACCEPTED MANUSCRIPT
DMSO-d
6
) δ [ppm]: 162.80, 157.72, 154.39, 152.38, 147.66, 147.00, 139.81, 137.03, 136.29,
1
31.68, 131.26, 129.13, 128.75, 128.46, 124.28, 121.17, 120.01, 119.02, 113.41, 106.20, 71,00.
+
LC/MS (ESI+): m/z calculated for C H Cl N O : 439.07 [M+H] . Found 439.06.
2
2
16
2
4
2
4
.1.5.4.1-(6-(benzyloxy)quinazolin-7-yl)-3-(3-fluorophenyl)urea (9)
1
Yield 40%; H NMR (400 MHz, DMSO-d ) δ [ppm]: 10.01 (s, 1H), 9.26 (s, 1H), 9.07 (s, 1H),
6
8
2
.87 (s, 1H), 8.75 (s, 1H), 7.66 (s, 1H), 7.61–7.55 (m, 3H), 7.46 (t, J = 7.8 Hz, 2H), 7.40–7.35 (m,
H), 7.16 (d, J = 8.1 Hz, 1H), 6.86 (t, J = 8.1 Hz, 1H), 5.49 (s, 2H). HRMS (ES+): m/z calculated
+
for C H FN O : 389.1414 [M+H] . Found 389.1410.
2
2
17
4
2
4
.1.5.5.1-(6-(3-fluorobenzyloxy)quinazolin-7-yl)-3-p-tolylurea (10)
1
Yield 75%; H NMR (400 MHz, DMSO-d ) δ [ppm]: 9.67 (s, 1H), 9.25 (s, 1H), 9.07 (s, 1H),
6
8
7
.77 (d, J = 4.8 Hz, 2H), 7.65 (s, 1H), 7.51–7.40 (m, 5H), 7.22 (t, J = 7.9 Hz, 1H), 7.14 (d, J =
.9 Hz, 2H), 5.49 (s, 2H), 2.27 (s, 3H). HRMS (ES+): m/z calculated for C H FN O : 425.1390
2
3
19
4
2
+
[
M+Na] . Found 425.1396.
4
.1.5.6.1-(6-(3-fluorobenzyloxy)quinazolin-7-yl)-3-(3-fluorophenyl)urea (11)
1
Yield 66%; H NMR (400 MHz, DMSO-d
6
) δ [ppm]: 9.98 (s, 1H), 9.26 (s, 1H), 9.08 (s, 1H),
8
.86 (s, 1H), 8.75 (s, 1H), 7.65 (s, 1H), 7.56 (d, J = 11.6 Hz, 1H), 7.53–7.42 (m, 3H), 7.37 (dd, J
=
15.4, 7.8 Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H), 7.16 (d, J = 7.8 Hz, 1H), 6.85 (t, J = 7.8 Hz, 1H),
+
5
4
.49 (s, 2H). HRMS (ES+): m/z calculated for C H F N O : 429.1139 [M+Na] . Found
2
2
16
2
4
2
29.1143.
2
2

ACCEPTED MANUSCRIPT
4
.1.5.7.1-(6-(2-fluorobenzyloxy)quinazolin-7-yl)-3-(4-(trifluoromethyl)phenyl)urea (12)
1
Yield 69%; H NMR (400 MHz, DMSO-d ) δ [ppm]: 10.17 (s, 1H), 9.31 (s, 1H), 9.09 (s, 1H),
6
8
2
.84 (s, 1H), 8.77 (s, 1H), 7.76–7.69 (m, 6H), 7.50–7.48 (m, 1H), 7.36–7.30 (m, 2H), 5.50 (s,
+
H). HRMS (ES+): m/z calculated for C H F N O : 479.1107 [M+Na] . Found 479.1109.
2
3
16
4
4
2
4
.1.5.8.1-(6-(2-fluorobenzyloxy)quinazolin-7-yl)-3-p-tolylurea (13)
1
Yield 53%; H NMR (400 MHz, MeOD) δ [ppm]: 9.19 (s, 1H), 8.99 (s, 1H), 8.87 (s, 1H), 7.64 (t,
J = 7.4 Hz, 1H), 7.57 (s, 1H), 7.47 (dd, J = 12.5, 5.0 Hz, 1H), 7.32 (d, J = 8.2 Hz, 2H), 7.29–7.21
(m, 2H), 7.09 (d, J = 8.2 Hz, 2H), 5.45 (s, 2H), 2.29 (s, 3H). HRMS (ES+): m/z calculated for
+
C H FN O : 425.1390 [M+Na] . Found 425.1390.
23
19
4
2
4
.1.5.9.1-(6-(2-fluorobenzyloxy)quinazolin-7-yl)-3-(naphthalen-1-yl)urea (14)
1
Yield 33%; H NMR (400 MHz, DMSO-d ) δ [ppm]: 9.76 (s, 1H), 9.30 (s, 1H), 9.18 (s,1H), 9.09
6
(s, 1H), 8.82 (s, 1H), 8.17 (d, J = 8.2 Hz, 1H), 7.97–7.95 (m, 2H), 7.74–7.69 (m, 3H), 7.63–7.48
4 2
(m, 4H), 7.36–7.29 (m, 2H), 5.55 (s, 2H). LC/MS (ESI+): m/z calculated for C26H19FN O :
+
4
39.16 [M+H] . Found 439.13.
4
.1.5.10.
1-(3-chlorophenyl)-3-(6-(2-fluorobenzyloxy)quinazolin-7-yl)urea (15)
1
Yield 53%; H NMR (400 MHz, DMSO-d
6
) δ [ppm]: 9.98 (s, 1H), 9.30 (s, 1H), 9.09 (s, 1H),
.75 (br, 2H), 7.76 (d, J = 9.5 Hz, 2H), 7.70 (t, J = 7.6 Hz, 1H), 7.49 (dd, J = 13.2, 5.8 Hz, 1H),
.36–7.26 (m, 4H), 7.07 (d, J = 7.6 Hz, 1H), 5.50 (s, 2H). HRMS (ES+): m/z calculated for
8
7
+
+
C H ClFN O : 423.1024 [M+H] . Found 423.1063; 445.0844 [M+Na] . Found 445.0886.
22
16
4
2
2
3

ACCEPTED MANUSCRIPT
4
.1.5.11.
1-(6-(2-fluorobenzyloxy)quinazolin-7-yl)-3-(3-fluorophenyl)urea (16)
1
Yield 63%; H NMR (400 MHz, DMSO-d ) δ [ppm]: 9.99 (s, 1H), 9.30 (s, 1H), 9.09 (s, 1H),
6
8
3
.18 (s, 1H), 8.76 (s, 1H), 7.75 (s, 1H), 7.70 (t, J = 7.5 Hz, 1H), 7.56–7.47 (m, 2H), 7.38–7.28 (m,
H), 7.14 (d, J = 8.1 Hz, 1H), 6.85 (td, J = 6.3, 1.9 Hz, 1H), 5.50 (s, 2H). LC/MS (ESI+): m/z
+
calculated for C H F N O : 407.13 [M+H] . Found 407.13.
2
2
16
2
4
2
4
.1.5.12.
1-(6-(4-fluorobenzyloxy)quinazolin-7-yl)-3-(4-(trifluoromethyl)phenyl)urea (17)
1
Yield 81%; H NMR (400 MHz, Acetone-d ) δ [ppm]: 9.22 (br, 2H), 9.08 (s, 1H), 8.96 (s, 1H),
6
8
.62(s, 1H), 7.78 (d, J = 8.6 Hz, 2H), 7.70–7.63 (m, 5H), 7.23 (t, J = 8.6 Hz, 2H), 5.47 (s, 2H).
+
HRMS (ES+): m/z calculated for C H F N O : 457.1287 [M+H] . Found 457.1285; 479.1107
2
3
16
4
4
2
+
[
M+Na] . Found 479.1103.
4
.1.5.13.
1-(6-(4-fluorobenzyloxy)quinazolin-7-yl)-3-p-tolylurea (18)
1
Yield 78%; H NMR (400 MHz, CDCl ) δ [ppm]: 9.15 (s, 1H), 9.08 (s, 1H), 8.94 (s, 1H), 7.95 (s,
3
1
H), 7.32–7.29 (m, 2H), 7.15 (d, J = 8.2 Hz, 2H), 7.10 (s, 1H), 7.05 (t, J = 8.5 Hz, 2H), 6.98 (d, J
=
4 2
8.2 Hz, 2H), 5.08 (s, 2H), 2.29 (s, 3H). HRMS (ES–): m/z calculated for C23H19FN O :
–
4
01.1414 [M–H] . Found 401.1416.
4
.1.5.14.
1-(3-chlorophenyl)-3-(6-(4-fluorobenzyloxy)quinazolin-7-yl)urea (19)
1
Yield 64%; H NMR (400 MHz, Acetone-d
6
) δ [ppm]: 9.21 (s, 1H), 9.11 (s, 1H), 9.08 (s, 1H),
8
7
.95 (s, 1H), 8.60 (s, 1H), 7.83 (t, J = 2.0 Hz, 1H), 7.68 (dd, J = 8.6, 5.5 Hz, 2H), 7.63 (s, 1H),
.39–7.30 (m, 2H), 7.23 (t, J = 8.8 Hz, 2H), 7.07 (dt, J = 7.8, 1.2 Hz, 1H), 5.47 (s, 2H). HRMS
–
(
ES–): m/z calculated for C H ClFN O : 421.0868 [M–H] . Found 421.0849.
22
16
4
2
2
4

ACCEPTED MANUSCRIPT
4
.1.5.15.
1-(3,4-dichlorophenyl)-3-(6-(4-fluorobenzyloxy)quinazolin-7-yl)urea (20)
1
Yield 83%; H NMR (400 MHz, Acetone-d ) δ [ppm]: 9.27 (s, 1H), 9.21 (s, 1H), 9.07 (s, 1H),
6
8
.92 (s, 1H), 8.63 (s, 1H), 7.97 (d, J = 2.3 Hz, 1H), 7.70–7.65 (m, 2H), 7.63 (s, 1H), 7.50–7.42
(
m, 2H), 7.23 (t, J = 8.8 Hz, 2H), 5.47 (s, 2H). HRMS (ES–): m/z calculated for C22 FN O :
H
15Cl
2
4 2
–
4
55.0478 [M–H] . Found 455.0470.
4
.1.5.16.
1-(6-(4-fluorobenzyloxy)quinazolin-7-yl)-3-(3-fluorophenyl)urea (21)
1
Yield 66%; H NMR (400 MHz, CDCl
3
) δ [ppm]: 9.12 (s, 1H), 9.04 (s, 1H), 8.88(s, 1H), 8.09 (s,
1
6
H), 8.02 (s, 1H), 7.36 (dd, J = 8.2, 5.4 Hz, 2H), 7.19 (d, J = 10.8 Hz, 1H), 7.10–7.01 (m, 4H),
.93 (d, J = 7.7 Hz, 1H), 6.71 (t, J = 7.7 Hz, 1H), 5.09 (s, 2H). LC/MS (ESI+): m/z calculated
+
for C H F N O : 407.13 [M+H] . Found 407.13.
2
2
16
2
4
2
4
.1.5.17.
1-(6-(4-fluorobenzyloxy)quinazolin-7-yl)-3-(2-fluorophenyl)urea (22)
1
Yield 68%; H NMR (400 MHz, Acetone-d
6
) δ [ppm]: 9.21 (s, 1H), 9.07 (s, 1H), 9.02 (s, 1H),
8
1
.98 (s, 1H), 8.69 (s, 1H), 8.35 (td, J = 8.2, 1.6 Hz, 1H), 7.66 (dd, J = 8.5, 5.4 Hz, 2H), 7.62 (s,
H), 7.25–7.16 (m, 4H), 7.14–7.07 (m, 1H), 5.45 (s, 2H). HRMS (ES+): m/z calculated for
+
C H F N O : 429.1139 [M+Na] . Found 429.1135.
22
16
2
4
2
4
.1.5.18.
1-(6-(2-chlorobenzyloxy)quinazolin-7-yl)-3-(4-(trifluoromethyl)phenyl)urea (23)
1
Yield 76%; H NMR (400 MHz, DMSO-d ) δ [ppm]: 10.19 (s, 1H), 9.31 (s, 1H), 9.10 (s, 1H),
6
8
.86 (s, 1H), 8.78 (s, 1H), 7.72–7.67 (m, 6H), 7.61–7.59 (m, 1H), 7.46–7.44 (m, 2H), 5.52 (s,
2
5

ACCEPTED MANUSCRIPT
+
2
4
3 4 2
H). HRMS (ES+): m/z calculated for C23H16ClF N O : 473.0992 [M+H] . Found 473.0987;
+
95.0812 [M+Na] . Found 495.0808.
4
.1.5.19.
1-(6-(2-chlorobenzyloxy)quinazolin-7-yl)-3-p-tolylurea (24)
1
Yield 54%; H NMR (400 MHz, DMSO-d
6
) δ [ppm]: 9.68 (s, 1H), 9.29 (s, 1H), 9.08 (s, 1H),
8
.77 (s, 1H), 8.71 (s, 1H), 7.73–7.69 (m, 2H), 7.59 (d, J = 6.4 Hz, 1H), 7.46–7.44 (m, 2H), 7.38
(d, J = 7.8 Hz, 2H), 7.13 (d, J = 7.8 Hz, 2H), 5.50 (s, 2H), 2.26 (s, 3H). HRMS
+
(
ES+): m/z calculated for C H ClN O : 441.1095 [M+Na] . Found 441.1095.
23
19
4
2
4
.1.5.20.
1-(6-(2-chlorobenzyloxy)quinazolin-7-yl)-3-(2-fluorophenyl)urea (25)
1
Yield 81%; H NMR (400 MHz, DMSO-d ) δ [ppm]: 9.69 (s, 1H), 9.30 (s, 1H), 9.27 (s, 1H),
6
9
7
.09 (s, 1H), 8.78 (s, 1H), 8.17 (td, J = 8.2, 1.4 Hz, 1H), 7.71–7.68 (m, 2H), 7.60–7.57 (m, 1H),
.48–7.42 (m, 2H), 7.29–7.24 (m, 1H), 7.17 (t, J = 7.7 Hz, 1H), 7.10–7.07 (m, 1H), 5.52 (s, 2H).
+
HRMS (ES+): m/z calculated for C H ClFN O : 423.1024 [M+H] . Found 423.1023.
2
2
16
4
2
4
.1.5.21.
1-(6-(4-chlorobenzyloxy)quinazolin-7-yl)-3-(4-(trifluoromethyl)phenyl)urea (26)
1
Yield 75%; H NMR (400 MHz, Acetone-d ) δ [ppm]: 9.29 (s, 1H), 9.20 (s, 1H), 9.08 (s, 1H),
6
8
7
1
1
.95 (s, 1H), 8.65 (s, 1H), 7.78 (d, J = 8.0 Hz, 2H), 7.65 (dd, J = 8.5, 2.0 Hz, 4H), 7.61 (s, 1H),
1
3
6
.49 (d, J = 8.0 Hz, 2H), 5.48 (s, 2H). C NMR (100 MHz, Acetone-d ) δ [ppm]: 157.18, 154.13,
51.78, 147.50, 147.37, 143.07, 136.68, 134.95, 133.69, 129.88, 128.74, 126.05, 123.75, 123.43,
21.17, 118.32, 113.82, 105.19, 70.23.
4
.1.5.22.
1-(6-(4-chlorobenzyloxy)quinazolin-7-yl)-3-p-tolylurea (27)
2
6

ACCEPTED MANUSCRIPT
1
Yield 73%; H NMR (400 MHz, Acetone-d
6
) δ [ppm]: 9.19 (s, 1H), 9.06 (s, 1H), 8.97 (s, 1H),
8
.79 (s, 1H), 8.51 (s, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.59 (s, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.43 (d,
J = 8.2 Hz, 2H), 7.12 (d, J = 8.2 Hz, 2H), 5.46 (s, 2H), 2.35 (s, 3H). HRMS
+
(
ES+): m/z calculated for C H ClN O : 419.1275 [M+H] . Found 419.1287; 441.1095
2
3
19
4
2
+
[
M+Na] . Found 441.1104.
4
.1.5.23.
Yield 79%; H NMR (400 MHz, DMSO-d
.84 (s, 1H), 8.74 (s, 1H), 7.78 (d, J = 1.8 Hz, 1H), 7.66 (s, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.51 (d,
1-(6-(4-chlorobenzyloxy)quinazolin-7-yl)-3-(3-chlorophenyl)urea (28)
1
6
) δ [ppm]: 9.96 (s, 1H), 9.26 (s, 1H), 9.08 (s, 1H),
8
J = 8.4 Hz, 2H), 7.38–7.25 (m, 2H), 7.10−7.08 (m, 1H), 5.47 (s, 2H). HRMS
+
(
ES+): m/z calculated for C H Cl N O : 461.0548 [M+Na] . Found 461.0375.
22
16
2
4
2
4
.1.5.24.
1-(6-(4-chlorobenzyloxy)quinazolin-7-yl)-3-(3,4-dichlorophenyl)urea (29)
1
Yield 84%; H NMR (400 MHz, DMSO-d ) δ [ppm]: 10.05 (s, 1H), 9.26 (s, 1H), 9.08 (s, 1H),
6
8
.85 (s, 1H), 8.74 (s, 1H), 7.94 (d, J = 2.5 Hz, 1H), 7.66 (s, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.56 (d,
J = 8.8 Hz, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.35 (dd, J = 8.8, 2.5 Hz, 1H), 5.47 (s, 2H). HRMS
–
(
ES–): m/z calculated for C H Cl N O : 471.0183 [M–H] . Found 471.0176.
22
15
3
4
2
4
.1.5.25.
1-(6-(4-chlorobenzyloxy)quinazolin-7-yl)-3-(3-fluorophenyl)urea (30)
1
Yield 77%; H NMR (400 MHz, DMSO-d ) δ [ppm]: 9.97 (s, 1H), 9.26 (s, 1H), 9.08 (s, 1H),
6
8
8
2
.85 (s, 1H), 8.75 (s, 1H), 7.66 (s, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.57–7.54 (m, 1H), 7.52 (d, J =
.4 Hz, 2H), 7.39−7.33 (m, 1H), 7.16 (d, J = 8.2 Hz, 1H), 6.86 (td, J = 8.2, 2.0 Hz, 1H), 5.48 (s,
+
H). HRMS (ES+): m/z calculated for C22
H16ClFN
4
O
2
: 445.0844 [M+Na] . Found 445.0843.
2
7

ACCEPTED MANUSCRIPT
4
.1.5.26.
1-(6-(4-chlorobenzyloxy)quinazolin-7-yl)-3-(2-fluorophenyl)urea (31)
1
Yield 86%; H NMR (400 MHz, Acetone-d ) δ [ppm]: 9.20 (s, 1H), 9.07 (s, 1H), 9.04 (s, 1H),
6
8
2
.98 (s, 1H), 8.76 (s, 1H), 8.34 (td, J = 8.3, 1.6 Hz, 1H), 7.63−7.59 (m, 3H), 7.47 (d, J = 6.7 Hz,
H), 7.21–7.14 (m, 2H), 7.09–7.06 (m, 1H), 5.46 (s, 2H). HRMS (ES+): m/z calculated for
+
+
C H ClFN O : 423.1024 [M+H] . Found 423.1031; 445.0844 [M+Na] . Found 445.0854.
22
16
4
2
4
4
.2.Evaluation of the biological activity
.2.1. Cell culture
HT-22 (mouse hippocampal cells) cells were grown in Dulbecco’s Modified Eagle’s Medium
(DMEM, GIBCO) supplemented with 10% (v/v) FBS and antibiotics (100 µg/mL
penicillin/streptomycin mix) in a humidified atmosphere at 37 °C with 5% CO
2
.
4
.2.2. JC-1 mitochondrial membrane potential assay
HT-22 cells (30,000 per well) were seeded into a clear 96-well plate (FALCON) at 200 µL
per well one day prior to assay. 750 µM of JC-1 (Stratagene) in DMSO stock solution was
dissolved into phenol red-free Opti-MEM (GIBCO) medium to make final concentration of 7.5
µM JC-1 per well. Medium was removed from the plate, and 100 µL per well of JC-1 was added.
Plates were incubated for 1 h and 15 min at 37 °C and washed twice with 100 µL per well PBS.
Subsequently, cells were treated with 25 µL solution of each compound at 5 µM in Opti-MEM
and incubated at 37 °C for 10 min followed by addition of 25 µL of Aβ (American peptide, 1–42)
solution at 5 µM. Fluorescence was measured at every 1 h for 3 h at ex/em 530 nm/580 nm (‘red’)
and ex/em 485 nm/530 nm (‘green’). The ratio of green to red fluorescence was recorded and the
2
8