Design, synthesis and evaluation of novel quinazoline-2,4-dione derivatives as chitin synthase inhibitors and antifungal agents

Article abstract of DOI:10.1016/j.bmc.2014.04.042

A series of novel 1-methyl-3-substituted quinazoline-2,4-dione derivatives were designed, synthesized, and characterized by ¹H NMR, ¹³C NMR and MS spectral data. Their inhibition against chitin synthase (CHS) and antifungal activities were evaluated in vitro. Results showed compounds 5b, 5c, 5e, 5f, 5j, 5k, 5l, and 5o had strong inhibitory potency against CHS. Compound 5c, which has the highest potency among these compounds, had a half-inhibition concentration (IC₅₀) of 0.08 mmol/L, while polyoxin B as positive drug had IC₅₀ of 0.18 mmol/L. These IC ₅₀ values of compounds 5i, 5m, 5n, and 5s were greater than 0.75 mmol/L, which revealed that those compounds had weak inhibition activity against CHS. Moreover, most of these compounds exhibited moderate to excellent antifungal activities. In detail, to Candida albicans, the activities of compound 5g and 5k were 8-fold stronger than that of fluconazole and 4-fold stronger than that of polyoxin B; to Aspergillus flavus, the activities of 5g, 5l and 5o were16-fold stronger than that of fluconazole and 8-fold stronger than that of polyoxin B; to Cryptococcus neoformans, the minimum-inhibition- concentration (MIC) values of compounds 5c, 5d, 5e and 5l were comparable to those of fluconazole and polyoxin B. The antifungal activities of these compounds were positively correlated to their IC₅₀ values against CHS. Furthermore, these compounds had negligible actions to bacteria. Therefore, these compounds were promising selective antifungal agents.

Full text of DOI:10.1016/j.bmc.2014.04.042

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of novel quinazoline-2,4-dione
derivatives as chitin synthase inhibitors and antifungal agents
a
b
b
a
a
a
Qinggang Ji a, , Dan Yang , Xin Wang , Chunyan Chen , Qiao Deng , Zhiqiang Ge , Lvjiang Yuan ,
⇑
Xiaolan Yang b, Fei Liao b,
⇑
a School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
b Unit for Analytical Probe and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing
Medical University, Chongqing 400016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 1-methyl-3-substituted quinazoline-2,4-dione derivatives were designed, synthesized,
and characterized by 1H NMR, 13C NMR and MS spectral data. Their inhibition against chitin synthase
(CHS) and antifungal activities were evaluated in vitro. Results showed compounds 5b, 5c, 5e, 5f, 5j,
5k, 5l, and 5o had strong inhibitory potency against CHS. Compound 5c, which has the highest potency
among these compounds, had a half-inhibition concentration (IC50) of 0.08 mmol/L, while polyoxin B as
positive drug had IC50 of 0.18 mmol/L. These IC50 values of compounds 5i, 5m, 5n, and 5s were greater
than 0.75 mmol/L, which revealed that those compounds had weak inhibition activity against CHS. More-
over, most of these compounds exhibited moderate to excellent antifungal activities. In detail, to Candida
albicans, the activities of compound 5g and 5k were 8-fold stronger than that of fluconazole and 4-fold
stronger than that of polyoxin B; to Aspergillus flavus, the activities of 5g, 5l and 5o were16-fold stronger
than that of fluconazole and 8-fold stronger than that of polyoxin B; to Cryptococcus neoformans, the min-
imum-inhibition-concentration (MIC) values of compounds 5c, 5d, 5e and 5l were comparable to those of
fluconazole and polyoxin B. The antifungal activities of these compounds were positively correlated to
their IC50 values against CHS. Furthermore, these compounds had negligible actions to bacteria. There-
fore, these compounds were promising selective antifungal agents.
Received 10 December 2013
Revised 19 April 2014
Accepted 21 April 2014
Available online xxxx
Keywords:
Quinazoline-2,4-dione
Chitin synthase inhibitor
Chitin synthase
Antifungal activity
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Drug affecting specific target of pathogenic fungi may lead
selective antifungal activity.12 Chitin synthase (CHS), which play
Invasive fungal infections have emerged as major causes of high
morbidity and mortality rates in patients who received antineo-
plastic chemotherapy, organ transplants or suffered AIDS.1–3
Candida, cryptococcus and aspergillus species are main fungi causing
such invasive infections.4,5 Representative antifungal drugs used in
current therapy include azoles, polyenes, echinocandins and nucle-
oside derivatives, but none of them satisfies the medical need
completely. For instance, azoles exhibit varying spectra of activity
and are vulnerable to resistance; polyenes, typified by amphoteri-
cin B, can cause serious toxicity and show poor activity against
aspergillosis; flucytosine and echinocandins are both limited in
spectra of activities and fungal strains resistant to their actions
have emerged.6–10 Moreover, these antifungal agents can kill
probiotics which are essential for human immune systems.11
Hence, new selective antifungal agents are much desired.
an important role in the process of biosynthesis of chitin that is
essential for normal growth of fungi and is a special structural
polysaccharide in fungal cell wall but absent in mammal cell, is
one of these targets. The process of biosynthesis of chitin needs
CHS to utilize UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) as a
substrate to form chitin.13,14 So CHS is a promising target for
designing selective antifungal agents.15–17 Classical CHS inhibitors
are polyoxins and nikkomycins18 (Fig. 1) whose structures resem-
ble that of UDP-GlcNAc. They act as competitive inhibitors of CHS
(from Coprinus cinereus) with inhibition constants (Ki) in 0.1–1 lM
range.19 Although many derivatives of polyoxins and nikkomycins
were prepared, none of them has better clinical significance.20–23
The dipeptide side chains of nikkomycins and polyoxins were
degraded by peptidase in the organism and consequently their
degradation made them have no or only a low level of inhibitory
activity. On the other hand, the ill-fitted lipid-water partition of
the nikkomycins and ployoxins would be a factor of the low activ-
ity towards many organism due to their extreme hydrophilicity.24
⇑
Corresponding authors. Tel./fax: +86 23 68254000.
E-mail addresses: jiqing@swu.edu.cn (Q. Ji), liaofeish@yeah.net (F. Liao).
http://dx.doi.org/10.1016/j.bmc.2014.04.042
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ji, Q.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.042

2
Q. Ji et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
OH
O
N
O
N
NH
NH
O
HO
O
OH
O
COOH
O
CH3
O
COOH
O
H2N
O
O
N
H
N
HN
HO
NH2
OH
NH2
OH OH
OH OH
polyoxin B
nikkomycin z
O
N
NH
OH
O
O
O
O
P
HO
HO
O
P
O
O
O
OH
NHAc
OH
OH OH
UDP-GlcNAc
Figure 1. The structures of polyoxin B and nikkomycin Z.
Therefore, new CHS inhibitors with better antifungal activities are
much desired.
relationship and increasing flexibilities, different amines were
introduced into the target compounds to investigate their bioactiv-
ity. Herein, a series of such derivatives were prepared and their
inhibition on CHS activities and growth of representative patholog-
ical fungi and some typical bacteria were tested in vitro. Results
revealed that most of these derivatives had stronger inhibition
activities against CHS than that of polyoxin B and better antifungal
activities than those of polyoxin B and fluconazole. Interestingly,
these compounds had little inhibition activities against bacterial.
Nitrogen-containing heterocycle can be found in a variety of
therapeutic agents. Typically, derivatives of heterocyclic quinazo-
line-2,4-dione have antifungal and antibacterial actions.25–28 The
amide moiety is an important fragment of antifungal molecules.
It was reported that the quinoline amides, azaindole amides and
pyridine amides have potent antifungal activity against Candida
albicans and Aspergillus fumigatus;29 the amide analogs of diox-
ane-triazole showed that they have higher activities in vitro than
those of fluconazole or ittaconazole30; our group’s previous study
showed that the quinolin-2(1H)-one compounds have antifungal
activity by introducing an amide moiety into the molecule.31 So,
we expect that combining the quinolin-2(1H)-one moiety and
amide moiety into a molecule would be an interesting lead
compound.
In this study, the quinazoline-2,4-dione moiety and amide moi-
eties were combined by an 3-amino-1-halopropan-2-ol moiety to
produce novel derivatives of quinazoline-2,4-dione whose struc-
tures roughly resemble that of polyoxin B (Fig. 2). The designed
molecule would not be degraded by peptidase for there is no
peptide bond in its side chain and its hydrophobicity is increasing.
These are beneficial to the molecule’s stability and membrane
permeability. For better understanding of structure–activity
2. Chemistry
Quinazoline-2,4-dione has wide range of biological activities, so
many methods have been developed for synthesizing this
heterocycle. Quinazoline-2,4-dione can be obtained by combining
anthranilic acid with urea, anthranilamide with phosgene, and
anthranilic acid with potassium cyanate or chlorosulfonyl isocya-
nate.32–34 Accordingly, compound 1 was obtained by condensation
of urea with 2-methylaminobenzoic acid under solvent-free condi-
tion at 150 °C.
Epichlorohydrin is a versatile building block for synthesis of a
broad variety of compounds of biological interest due to the
presence of three different electrophilic carbon atoms and the
nucleophilicity of the oxygen atom.35 Epichlorohydrin reacted with
compound 1 to produce compound 2 in the presence of sodium
hydroxide in DMF. Compound 2 was then reacted with methyl-
amine to give compound 3. Intermediates 4a–4s were prepared
by combining 2-chloroacetyl chloride with various primary or sec-
ondary amines. Condensation of compound 3 with one of interme-
diates 4a–4s in 1:1.2 M ratio and in the presence of potassium
carbonate by refluxing for 5 h in CH3CN provided designed amide
derivatives (5a–5s) in good yields (Scheme 1). All these com-
pounds were purified and their structures are supported by spec-
tral data including 1H NMR, 13C NMR, MS and HRMS reported in
experimental section.
OH
O
N
NH
O
O
OH
O
O
COOH
H2N
O
O
N
H
NH
HO
NH2
OH OH
polyoxin B
N
R
O
Me
N
quinazoline-2,4-dione
O
3. Results and discussion
O
Me
OH
R1
N
N
3.1. Inhibition against yeast CHS36
N
R2
.HCl
O
CHS of Saccharomyces cerevisiae (CGMCC 2.145) was preliminar-
ily purified and inhibitory activity against CHS was assayed by
means of one-step ELISA analysis of chitin formed from UDP-Glc-
NAc with horse-radish peroxidase (HRP)-labeled wheat germ
Target compounds 5a-5s
R1,R2=hydrogen,alkyl,aryl, benzyl
Figure 2. Structure-based design of the target compounds.
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Q. Ji et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
Table 1
The MIC (mg/L) values of compounds 5a–5s against fungi in vitro
Compounds
MIC (mg/L)
No.
R1
R2
Yield %
Candida albicans
Cryptococcus neoformans
Aspergillus flavus
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-OCH3C6H5
C6H5CH2
4-CH3C6H4
2-ClC6H4
4-ClC6H4
4-CF3C6H4
3-NO2C6H4
2-NO2C6H4
4-ClC6H4CH2
C(CH3)3
4-CH3CNC6H4
2-COOMeC6H4
C6H5
4-NO2C6H4
C6H5CH2
C6H5
C3H7
C2H5
60
—
—
60
56
34
37
37
60
37
24
73
94
53
45
57
32
60
30
15
79
32
—
16
32
32
64
16
32
128
64
2.0
2.0
2.0
32
8.0
32
64
128
4.0
2.0
64
16
32
32
32
128
8.0
4.0
64
64
64
8.0
4.0
>512
64
4.0
256
32
4.0
>512
32
>512
4.0
32
32
>512
64
64
>512
64
4.0
16
32
64
C6H5CH2
CH3
C3H7
C2H5
Piperidyl
—
2.0
8.0
16
5q
5r
5s
16
8.0
512
4.0
2.0
Polyoxin B
Fluconazole
32
64
—
—
agglutinin (WGA) as the probe. Polyoxin B was the positive control
in this assay. The half-inhibition concentration of each compound
(IC50) was determined.
inhibition concentration (MIC) of each compound was estimated.
Fluconazole and polyoxin B acted as the positive controls.
Interestingly, comparing with the two controls, most designed
compounds displayed comparable or better inhibitory activities
against tested pathogenic fungi (Table 1). To Candida albicans, com-
pounds 5g and 5k whose MIC values were both 4.0 mg/L exhibited
much higher inhibitory activities than those of fluconazole and
polyoxin B whose values were 32 and 16 mg/L, respectively. Com-
pounds 5a and 5e both with MIC values of 16 mg/L were compar-
ative with that of polyoxin B. That the MIC values of 5b, 5c, 5f, 5i,
5l, 5m, and 5s all were 32 mg/L were equal to that of fluconazole.
To Aspergillus flavus, compounds 5g, 5l, and 5o which all displayed
MIC values of just 4.0 mg/L were much stronger potency than the
two positive controls whose MIC values were over 32 mg/L (32,
64 mg/L, respectively). 5f, 5k, and 5r whose MIC values all were
8.0 mg/L and 5a with MIC value of 16 mg/L had better inhibitory
activities than that of the two controls. The MIC values of 5b, 5c,
5d and 5q which were comparable with that of polyoxin B whose
MIC value was 32 mg/L, were better than that of fluconazole. To
Cryptococcus neoformans, MIC values of compounds 5c, 5d, 5e, 5l,
and 5o all were equally 2.0 mg/L, while MIC values of fluconazole
and polyoxin B were 2.0 mg/L and 4.0 mg/L, respectively. These
results supported that these designed compounds had more favor-
able activities against Candida albicans and Aspergillus flavus. There-
fore, these novel compounds may be promising leads of antifungal
agents.
Compounds 5b, 5c, 5e, 5f, 5j, 5k 5l, and 5o exhibited good inhi-
bition activities against CHS (Fig. 3) and their IC50 values were
lower than that of Polyoxin B whose IC50 was 0.18 mmol. 5c with
IC50 value of 0.08 mmol/L is the strongest inhibitor among these
compounds. The inhibitory activities of 5a, 5l and 5o were compa-
rable to that of polyoxin B. The IC50 values of 5e, 5f, and 5k were
0.16, 0.11, and 0.12 mmol/L, respectively. Compounds 5d, 5p, 5q
and 5r, however, exerted lower inhibition potency. The IC50 values
of compounds 5i, 5m, 5n, and 5s were greater than 0.75 mmol/L,
which showed these compounds had very weak inhibition activity.
In these active compounds, the use of aryl R2 as substituent pro-
duced higher inhibitory activities against CHS than the use of alkyl
R2. In aryl R2, electron-donating substituent is more favorable than
electron-withdrawing substituent on aromatic ring to enhance
inhibitory activities.
3.2. In vitro antimicrobial activity
3.2.1. In vitro antifungal activity
Most invasive infections in clinical practice are caused by
species belonging to Candida, Aspergillus, and Cryptococcus.3–5 The
inhibitions of the growth of Cryptococcus neoformans, Candida
albicans, and Aspergillus flavus by designed compounds were
determined at 37 °C. For quantitative comparison, the minimum
Meanwhile, compounds with arylamino fragments as substitu-
ents had stronger actions than those with alkylamine fragments.
Figure 3. Inhibitory effects of the test compounds⁄ against chitin synthase. ⁄The IC50 values of compounds 5i, 5m, 5n and 5s were greater than 0.75 mmol/L.
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4
Q. Ji et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Arylamine substituents bearing polar groups on the aromatic ring
showed higher activities than those without substituent groups
on aromatic ring; the position of substituent on the ring had slight
impacts on antifungal activities for compounds. Moreover, the
spectra of antifungal actions of those active compounds were
clearly wider than those of fluconazole and polyoxin B.
5d, 5e, 5l, and 5o were comparable with those of two controls.
These results suggested that these designed compounds have more
favorable activities against Candida albicans and Aspergillus flavus.
The antifungal activities of these compounds were positively corre-
lated with their inhibitory activities against CHS. Moreover, those
compounds had negligible antibacterial actions. So, those com-
pounds were promising leads that have selective antifungal agents
against a wide spectrum of pathogenic fungi.
3.2.2. Antibacterial activity
Six strains of bacteria, including Staphylococcus aureus, Bacillus
subtilis, Methicillin-resistant Staphylococcus aureus N 315 (MRSA),
Bacillus proteus, Escherichia coli JM 109, P. Aeruginosa CR56, were
tested with streptomycin sulfate as the positive control. None of
the designed compounds had MIC values lower than 512 mg/L on
those tested bacteria, while MIC values of the Streptomycin were
64 mg/L. Thus, the designed compounds specially targeted for
fungi and it indicated that their mechanism of action would be
as designed.
5. Experimental
Melting points were measured on X-6 precise micro melting
point apparatus and are uncorrected. 1H NMR and 13C NMR spectra
were recorded on Bruker Av 300 MHz or Bruker AM-400 spectrom-
eter using DMSO-d6 or CDCl3 as solvent, chemical shifts are given
in ppm with TMS as an internal reference. Mass Spectral data were
measured with a Shimadzu LCMS-2020 mass spectrometer. High-
resolution mass spectra were determined by Finnigan/AMT95.
Reactions were monitored by thin layer chromatography (TLC) on
glass plates coated with Silica Gel GF-254. All reagents and sol-
vents were used after purchasing without further purification
unless otherwise indicated.
4. Conclusion
Among these novel 1-methyl-3-substituted quinazoline-2,
4-dione derivatives, compounds 5a, 5c, 5e, 5f, 5k and 5l exhibited
moderate to good inhibitory activities against chitin synthase. 5c
was the strongest inhibitor among these compounds. Compounds
5b, 5e, 5f, 5k and 5j had lower IC50 value than that of control.
The inhibitory activities of 5a, 5l, and 5o against CHS were compa-
rable with that of control. The antifungal assays showed most of
these compounds inhibited the growth of Candida albicans with
potency comparable to those of fluconazole and polyoxin B. Com-
pounds 5g and 5k were the strongest inhibitors among those com-
pounds. Their inhibitory activities in vitro were 8-fold stronger
than that of fluconazole and 4-fold stronger than that of polyoxin
B. Compounds 5a and 5e were comparative with that of polyoxin
B. The antifungal activities of 5b, 5c, 5f, 5i, 5l, 5m, and 5s were
equal to that of fluconazole. To Aspergillus flavus, compounds 5g,
5l, and 5o were 8-fold stronger potency than that of polyoxin B
and at least 16-fold stronger than that of fluconazole. Compounds
5f, 5k, 5r and 5a had better inhibitory activities than the two con-
trols. The MIC values of 5b, 5c, 5d and 5q which were comparable
with that of polyoxin B were better than that of fluconazole. To
Cryptococcus neoformans, antifungal activities of compounds 5c,
5.1. Synthetic procedure for key intermediates
5.1.1. 1-Methylquinazoline-2,4(3H)-dione (1)
Urea (460 mmol) was heated to melt, then 2-methylaminoben-
zoic acid (46 mmol) was added. The mixture was stirred for 5 h at
150 °C and then cooled to below 100 °C. Water (70 mL) was added
to quench the reaction. The precipitated was collected and recrys-
tallized in a mixed solution of acetone (10 mL) and water (100 mL)
to afford compound 1 as a white powder (5.25 g, 64.3%); mp 278–
279 °C; 1H NMR (DMSO-d6) d (ppm): 3.43 (s, 3H), 7.26 (t, J = 12 Hz,
1H), 7.41 (d, J = 12 Hz, 1H), 7.75 (t, J = 15 Hz, 1H), 7.98 (d, J = 6 Hz,
1H), 11.54 (s, 1H). ESI-MS (m/z): 177 (M+H)+.
5.1.2. 1-Methyl-3-epoxypropylquinazoline-2,4-dione (2)
Epichlorohydrin (46.0 mmol) in DMF (30 mL) was added drop-
wise to a mixture of compound 1 (29.6 mmol), sodium hydroxide
(44.0 mmol), anhydrous sodium sulfate (10 g) and DMF (60 mL).
The mixture was stirred at room temperature for 1 h and heated
O
O
Cl
O
CH3NH2
b
N
NH
O
a
N
O
N
O
Me
Me
2
1
R1
N
O
N
R1
R2
Cl
CH3
R2
N
O
N
N
N
O
OH Me
O
N
N
H
4a-4s
O
HCl
OH
Me
O
c
5a-s
Me
3
H
N
R1
N
R2
R1
Cl
Cl
R2
Cl
O
d
O
Intermediate 4a-4s
Scheme 1. Synthesis of compounds 5a–s. Reagents and conditions: (a) Epichlorohydrin (46.0 mmol), 1-methylquinazoline-2,4(3H)-dione (29.6 mmol), NaOH (44.0 mmol),
DMF (60 mL), rt–60 °C, 4 h; (b) compound 2 (26.8 mmol), 25–30% methylamine (120 mL), rt, 8 h; (c) compound 3 (1.0 mmol), 4 (1.2 mmol), potassium carbonate (2.0 mmol)
CH3CN (20 mL), rt–reflux, 5–6 h; HCl; (g), CH3CN; (d) propionyl chloride (6.0 mmol), 4-methoxybenzenamine (5.0 mmol), sodium bicarbonate (12.5 mmol), CH2Cl2 (25 mL),
0 °C to rt, 1 h.
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Q. Ji et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
to 60 °C for 4 h. The mixture was cooled to room temperature and
filtrated. The filtrate was poured into water (200 mL) and stirred
for a few minutes, and then extracted with EtOAc (4 Â 50 mL).
The combined organic layer was dried by anhydrous MgSO4 and
removed under vacuum to gain the crude product that was recrys-
tallized in EtOAc to afford compound 2 as a white solid (5.11 g,
74%); mp 118–119 °C; 1H NMR (DMSO-d6) d (ppm): 2.74–2.83(m,
2H, epoxy-CH2), 3.33, (m, 1H, cepoxy-CH), 3.63(s, 3H), 4.17–
4.26(m, 1H), 4.39–4.46(m, 1H), 7.21–7.31(m, 2H), 7.68 (t,
J = 15 Hz, H), 8.23 (d, J = 9 Hz, 1H). ESI-MS (m/z): 233 (M+H)+.
5.2.7. 2-Chloro-N-(3-nitrophenyl)acetamide (4g)
Pale yellow powder, yield 75%, mp 104–105 °C, 1H NMR (CDCl3-
d1) d (ppm): 4.21 (s, 2H), 7.55 (t, J = 9 Hz, 1H), 7.98–8.06 (m, 2H),
8.45 (s, 1H), 8.48 (s, 1H). ESI-MS (m/z): 215 (M+H)+.
5.2.8. 2-Chloro-N-(2-nitrophenyl)acetamide (4h)
Brick-red needle crystal, yield 78%, mp 77–79 °C, 1H NMR
(CDCl3-d1) d (ppm): 4.26 (s, 2H), 6.08 (s, 1H), 7.26 (t, J = 7.5 Hz,
1H), 7.69 (t, J = 9 Hz, 1H), 8.26 (d, J = 6 Hz, 1H), 8.77 (d, J = 6 Hz,
1H). ESI-MS (m/z): 215 (M+H)+.
5.1.3. 1-Methyl-3(1-mehylamino-2-hydroxylpropyl)
quinazoline-2,4-dione (3)
5.2.9. 2-Chloro-N-(4-chlorobenzyl)acetamide (4i)
White powder, yield 66%, mp 112–113 °C, 1H NMR (CDCl3-d1) d
(ppm): 3.74 (s, 2H), 4.21 (s, 2H), 7.32 (d, 6 Hz, 2H), 7.58 (d, J = 6 Hz,
2H), 8.28 (s, 1H). ESI-MS (m/z): 218 (M+H)+.
Compound 2 (26.8 mmol) was added to a solution of 25–30%
methylamine in methanol (120 mL).The mixture was stirred at
room temperature for 8 h. The solvent was removed under vacuum
to obtain the crude product that was washed with water
(3 Â 30 mL). Compound 3 was obtained as a white solid (6.3 g,
87%); mp 161–162 °C; 1H NMR DMSO-d6) d (ppm): 2.25(s, 3H),
3.50 (s, 3H), 3.80–3.92 (m, 2H), 4.05–4.12 (m, 1H), 4.78 (s, 1H),
7.28 (t, J = 7.5 Hz, 1H), 7.43 (d, J = 9 Hz, 1H), 7.76 (t, J = 7.5 Hz,
1H), 8.03 (d, J = 9 Hz, 1H). ESI-MS (m/z): 249 (M+H)+.
5.2.10. N-(tert-Butyl)-2-chloroacetamide (4j)
White powder, yield 50%, mp 85–86 °C, 1H NMR (CDCl3-d1) d
(ppm): 1.68 (s, 9H), 3.95 (s, 2H), 6.38 (s, 1H). ESI-MS (m/z): 150
(M+H)+.
5.2.11. 2-Chloro-N-(4-(cyanomethyl)phenyl)acetamide (4k)
Gray powder, yield 82%, mp 129–131 °C, 1H NMR (CDCl3-d1) d
(ppm): 4.12 (s, 2H), 4.48 (s, 2H), 6.88 (s, 1H), 7.01–7.0 7(m, 2H),
7.25–7.27 (m, 2H). ESI-MS (m/z): 209 (M+H)+.
5.2. General synthetic procedure for the intermediates 4a–4s
Propionyl chloride (6.0 mmol) in dry CH2Cl2 (10 mL) was added
dropwise to a mixture of 4-methoxybenzenamine (5.0 mmol),
sodium bicarbonate (12.5 mmol) and dry CH2Cl2 (15 mL). The mix-
ture was stirred in ice water bath for 1 h and poured into water and
then extracted with CH2Cl2 (3 Â 30 mL). The combined organic
layer was washed with brine and dried over anhydrous MgSO4.
The solvent was evaporated to dryness to afford the compound
4a. Others amide derivatives 4b–4s were synthesized similarly.
Physical constants and spectral data of compounds 4a–4s are sum-
marized below.
5.2.12. Methyl 2-(2-chloroacetamido)benzoate (4l)
White powder, yield 80%, mp 98–99 °C, 1H NMR (CDCl3-d1) d
(ppm): 3.96 (s, 3H), 4.22 (s, 2H), 7.16 (t, J = 9 Hz, 1H), 7.26 (s,
1H), 7.57 (t, J = 7.5 Hz, 1H), 8.06 (d, J = 3 Hz, 1H), 8.70 (d, J = 9 Hz,
1H). ESI-MS (m/z): 228 (M+H)+.
5.2.13. 2-Chloro-N-phenylacetamide (4m)
White powder, yield 81%, mp 141–142 °C, 1H NMR (CDCl3-d1) d
(ppm): 4.19 (s, 2H), 7.17 (t, J = 7.5 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H),
7.55 (d, J = 12 Hz, 1H), 8.26 (s, 1H). ESI-MS (m/z): 170 (M+H)+.
5.2.1. 2-Chloro-N-(4-methoxyphenyl) acetamide (4a)
White powder, yield 87%, mp 101–102 °C, 1H NMR (CDCl3-d1) d
(ppm): 3.72 (s, 3H), 4.21 (s, 2H), 6.86 (d, J = 9 Hz, 2H), 7.46 (d,
J = 9 Hz, 2H), 8.23 (s, 1H). ESI-MS (m/z): 200 (M+H)+.
5.2.14. 2-Chloro-N-(4-nitrophenyl)acetamide (4n)
Pale yellow powder, yield 91%, mp 191–192 °C, 1H NMR (CDCl3-
d1) d (ppm): 4.31 (s, 2H), 7.78 (d, J = 6 Hz, 2H), 7.26 (d, J = 9 Hz, 1H),
8.50 (s, 1H). ESI-MS (m/z): 215 (M+H)+.
5.2.2. N-Benzyl-2-chloroacetamide (4b)
Pink powder, yield 76%, mp 95–96 °C, 1H NMR (CDCl3-d1) d
(ppm): 4.10 (s, 2H), 4.49 (d, J = 6 Hz, 2H), 6.89 (s, 1H), 7.29–7.38
(m, 5H). ESI-MS (m/z): 184(M+H)+.
5.2.15. N,N-Dibenzyl-2-chloroacetamide (4o)
Pale yellow viscous liquid, yield 57%, 1H NMR (CDCl3-d1) d
(ppm): 4.13 (s, 2H), 4.51 (s, 4H), 7.23–7.38 (m, 10H). ESI-MS (m/
z): 274 (M+H)+.
5.2.3. 2-Chloro-N-(p-tolyl)acetamide (4c)
White powder, yield 69%, mp 169–170 °C, 1H NMR (CDCl3-d1) d
(ppm): 2.36 (s, 3H), 4.18 (s, 2H), 7.15 (d, J = 6 Hz, 2H), 7.42 (d,
J = 6 Hz, 2H), 8.14 (s, 1H). ESI-MS (m/z): 184 (M+H)+.
5.2.16. 2-Chloro-N-methyl-N-phenylacetamide (4p)
Pale yellow powder, yield 81%, mp 70–71 °C, 1H NMR (CDCl3-d1)
d (ppm): 3.38 (s, 3H), 3.86 (s, 2H), 7.25 (d, J = 6 Hz, 2H), 7.39–7.48
(m, 1H). ESI-MS (m/z):184 (M+H)+.
5.2.4. 2-Chloro-N-(2-chlorophenyl)acetamide (4d)
Pink powder, yield 76%, mp 74–75 °C, 1H NMR (CDCl3-d1) d
(ppm): 4.21 (s, 2H), 7.11 (t, J = 7.5 Hz, 1H), 7.30 (t, J = 12 Hz, 1H),
7.40 (d, J = 9 Hz, 1H), 8.37 (d, J = 6 Hz, 1H), 8.94 (s, 1H). ESI-MS
(m/z): 204 (M+H)+.
5.2.17. 2-Chloro-N,N-dipropylacetamide(4q)
Pink liquid, yield 68%, 1H NMR (CDCl3-d1) d (ppm): 1.11 (t,
J = 4.5 Hz), 1.71–1.75 (m, 4H), 3.46 (t, J = 3 Hz, 4H), 4.20 (s, 2H).
ESI-MS (m/z): 178 (M+H)+.
5.2.5. 2-Chloro-N-(4-chlorophenyl)acetamide (4e)
5.2.18. 2-Chloro-N,N-diethylacetamide (4r)
Pale yellow liquid, yield 70%, 1H NMR (CDCl3-d1) d (ppm): 1.17
(t, J = 6 Hz, 6H), 3.47 (t, J = 6 Hz, 4H), 4.19 (s, 2H). ESI-MS (m/z):150
(M+H)+.
White needle crystal, yield 82%, mp 180–181 °C, 1H NMR
(CDCl3-d1) d (ppm): 4.10 (s, 2H), 7.32 (d, J = 9 Hz, 2H), 7.51 (d,
J = 12 Hz, 2H), 8.23 (s, 1H). ESI-MS (m/z): 204 (M+H)+.
5.2.6. 2-Chloro-N-(4-(trifluoromethyl)phenyl)acetamide (4f)
White needle crystal, yield 96%, mp 161–162 °C, 1H NMR
(CDCl3-d1) d (ppm): 4.22 (s, 2H), 7.62 (d, J = 12 Hz, 2H), 7.69 (d,
J = 9 Hz,2H), 8.36 (s, 1H). ESI-MS (m/z): 238 (M+H)+.
5.2.19. 2-Chloro-1-(piperidin-1-yl)ethanone (4s)
Colorless liquid, yield 70%, 1H NMR (CDCl3-d1) d (ppm): 1.61–
1.67 (m, 6H), 3.52 (t, J = 6 Hz, 4H), 4.16 (s, 2H). ESI-MS (m/z): 162
(M+H)+.
Please cite this article in press as: Ji, Q.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.042

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Q. Ji et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5.3. General synthetic procedure for the target compounds 5a–
5s
116.52(1C), 114.99(1C), 63.61(1C), 59.26(1C), 57.12(1C),
45.17(1C), 42.11(1C), 31.18(1C). ESI-MS (m/z): 431 (M+H)+; HRMS:
Calcd for (C21H23ClN4O4+H)+: 431.1486; Found: 431.1480.
One intermediate of 4a–4s (1.2 mmol) was added to a mixture
of compound 3 (1.0 mmol), potassium carbonate (2.0 mmol) and
dry CH3CN (20 mL). The mixture was reflux for 5–6 h. After cooling,
the resulting mixture was filtrated. The filtrate was evaporated to
dryness, and the residue was chromatographed with silica gel col-
umn chromatography (eluting with ethyl acetate/petroleum
ether = 2/1), then the product was salified with HCl gas in CH3CN
to afford the target compound. The yield, melting point and spec-
tral data of compounds 5a–5s are given below.
5.3.5. N-(4-Chlorophenyl)-2-((2-hydroxy-3-(1-methyl-2,4-
dioxo-1,2-dihydroquinazolin-3(4H)-
yl)propyl)(methyl)amino)acetamide hydrochloride (5e)
Yield 37%; mp 126–127 °C. 1H NMR (CDCl3-d1) d (ppm): 2.42 (s,
3H), 2.54–2.59 (m, 2H), 3.32 (s, 2H), 3.69 (s, 3H), 3.89–4.00 (m, 2H),
4.12–4.19 (m, 1H), 4.65 (s, 1H), 7.06–7.29 (m, 3H), 7.47–7.71 (m,
4H), 7.99–8.03 (m, 1H). 13C NMR (DMSO-d6): d = 168.79(1C),
161.58(1C), 151.09(1C), 140.75(1C), 135.76(1C), 133.05,(1C),
132.29(1C), 128.77(2C), 128.22(1C), 123.26(1C), 19.69(2C),
115.58(1C), 115.69(1C), 65.78(1C), 62.37(1C), 59.98(1C),
45.70(1C), 42.69(1C), 31.34(1C). ESI-MS (m/z): 431 (M+H)+; HRMS:
Calcd for (C21H23ClN4O4+H)+: 431.1486; Found: 431.1478.
5.3.1. 2-((2-Hydroxy-3-(1-methyl-2,4-dioxo-1,2-
dihydroquinazolin-3(4H)-yl)propyl)(methyl)amino)-N-(4-
methoxyphenyl)acetamide hydrochloride (5a)
Yield 60%; mp 159–160 °C. 1H NMR (DMSO-d6) d (ppm): 2.33 (s,
3H), 2.43–2.49 (m, 2H), 3.20 (s, 2H), 3.61 (s, 3H), 3.72 (s, 2H), 3.91–
3.94 (m, 1H), 4.10–4.16 (m, 2H), 5.14 (s, 1H), 6.87 (d, J = 9 Hz, 2H),
7.29 (t, J = 6 Hz, 1H), 7.41 (d, J = 6 Hz, 1H), 7.55 (d, J = 6 Hz, 2H),
7.76 (t, J = 6 Hz, 1H), 8.04 (d, J = 6 Hz, 1H, quinazolone-8H). 13C
NMR (DMSO-d6): d = 168.91(1C), 161.86(1C), 155.73(1C),
151.06(1C), 140.84(1C), 135.60(1C), 132.26(1C), 128.27(1C),
125.07(1C), 121.26(2C), 115.56(1C), 114.89(1C), 114.20(2C),
65.82(1C), 62.41(1C), 61.98(1C), 66.60(1C), 46.90(1C), 45.60(1C),
31.06(1C). ESI-MS (m/z): 427(M+H)+; HRMS: Calcd for (C22H26N4-
O5+H)+: 427.1981; Found: 427.1990.
5.3.6. 2-((2-Hydroxy-3-(1-methyl-2,4-dioxo-1,2-
dihydroquinazolin-3(4H)-yl)propyl)(methyl)amino)-N-(4-
(trifluoromethyl)phenyl)acetamide hydrochloride (5f)
Yield 60%; mp 161–162 °C. 1H NMR (DMSO-d6) d (ppm): 2.41 (s,
3H), 2.63–2.67 (m, 2H), 3.42 (s, 2H), 3.72 (s, 3H), 3.91–3.9 (m, 1H),
4.02–4.05 (m, 2H), 4.63 (s, 1H), 7.23–7.26 (m, 1H), 7.60–7.64 (m,
5H), 7.79–7.81 (m, 1H), 7.96–7.98 (m, 1H). 13C NMR (DMSO-d6):
d = 164.26(1C), 161.29(1C), 151.00(1C), 142.11(1C), 140.26(1C),
135.77(2C), 128.28(1C), 126.67(2C), 123.56(1C), 123.16(1C),
119.69(2C), 116.51(1C), 114.96(1C), 63.67(1C), 59.32(1C),
57.64(1C), 45.25(1C), 42.69(1C), 31.16(1C). ESI-MS (m/z): 465
(M+H)+; HRMS: Calcd for (C22H23F3N4O4+H)+: 465.1750; Found:
465.1761.
5.3.2. N-Benzyl-2-((2-hydroxy-3-(1-methyl-2,4-dioxo-1,2-
dihydroquinazolin-3(4H)-yl)propyl)(methyl) amino)acetamide
hydrochloride (5b)
Yield 56%; mp 121–122 °C. 1H NMR (DMSO-d6) d (ppm): 2.31 (s,
3H), 2.38–2.43 (m, 2H), 3.26 (s, 2H), 3.45 (s, 3H), 3.84–3.89 (m, 1H),
4.02–4.05 (m, 2H), 4.26 (s, 2H), 5.82 (s, 1H), 7.23–7.29 (m, 7H), 7.40
(d, J = 9 Hz, 1H), 7.75 (t, J = 9 Hz, 1H), 7.98 (d, J = 3 Hz, 1H). 13C NMR
(DMSO-d6): d = 164.66(1C), 161.32(1C), 150.91(1C), 140.90(1C),
138.87(1C), 135.80(1C), 128.79(2C), 128.29(1C) 127.79(2C),
127.16(1C), 123.12(1C), 116.29(1C), 114.99(1C), 63.64(1C),
59.20(1C), 56.71(1C), 45.16(1C), 42.60(1C), 40.53(1C), 31.19(1C).
ESI-MS (m/z): 411 (M+H)+; HRMS: Calcd for (C22H26N4O4+H)+:
411.2032; Found: 411.2040.
5.3.7. 2-((2-Hydroxy-3-(1-methyl-2,4-dioxo-1,2-
dihydroquinazolin-3(4H)-yl)propyl)(methyl)amino)-N-(3-
nitrophenyl)acetamide hydrochloride (5g)
Yield 37%; mp 216–217 °C. 1H NMR (DMSO-d6) d (ppm): 2.46 (s,
3H), 2.66–2.69 (m, 2H), 3.22–3.31 (m, 1H), 3.40 (s, 2H), 3.72 (s, 3H),
3.88–3.99 (m, 1H), 4.22–4.31 (m, 1H), 7.20 (t, J = 4.5 Hz, 1H), 7.55
(d, J = 6 Hz, 1H), 7.59–7.62 (m, 1H), 7.68 (t, J = 4.5 Hz, 1H), 7.86–
7.94 (m, 3H), 8.60 (s, 1H). 13C NMR (DMSO-d6): d = 164.33(1C),
161.86(1C), 150.95(1C), 148.25(1C), 140.81(1C), 139.62(1C),
135.72(1C), 130.69(1C), 128.24(1C), 126.76(1C), 123.12(1C),
118.91(1C), 115.22(1C), 114.89(1C), 113.87(1C), 63.62(1C),
59.51(1C), 57.56(1C), 45.12(1C), 42.11(1C), 31.14(1C). ESI-MS (m/
z): 442(M+H)+; HRMS: Calcd for (C21H23N5O6+H)+: 442.1727;
Found: 442.1740.
5.3.3. 2-((2-Hydroxy-3-(1-methyl-2,4-dioxo-1,2-
dihydroquinazolin-3(4H)-yl)propyl)(methyl)amino)-N-(p-
tolyl)acetamide hydrochloride (5c)
Yield 35%; mp 164–165 °C. 1H NMR (DMSO-d6) d (ppm): 2.18 (s,
3H), 3.12 (s, 3H), 3.40–3.59 (m, 7H), 4.08–4.13 (m, 1H), 4.24–4.44
(m, 2H), 4.63 (s, 1H), 6.90 (d, J = 3 Hz, 2H), 7.11 (d, J = 3 Hz, 2H),
7.39–7.45 (m, 2H), 7.52–7.58 (m, 1H), 8.02–8.05 (m, 1H). 13C
NMR (DMSO-d6): d = 167.91(1C), 161.58(1C), 151.06(1C),
140.58(1C), 136.34(1C), 135.76(1C), 132.42(1C), 128.92(2C),
127.65(1C), 123.26(1C), 120.88(2C), 115.56(1C), 114.97(1C),
63.22(1C), 59.54(1C), 56.93(1C), 45.20(1C), 42.76(1C), 31.16(1C),
20.79(1C). ESI-MS (m/z): 411 (M+H)+; HRMS: Calcd for (C22H26N4
O4+H)+: 411.2032; Found: 411.2036.
5.3.8. 2-((2-Hydroxy-3-(1-methyl-2,4-dioxo-1,2-
dihydroquinazolin-3(4H)-yl)propyl)(methyl)amino)-N-(2-
nitrophenyl)acetamide hydrochloride (5h)
Yield 24%; mp 119–120 °C. 1H NMR (DMSO-d6) d (ppm): 2.69 (s,
3H), 2.70–2.72 (m, 2H), 3.42 (s, 2H), 3.65 (s, 3H), 3.67–3.72 (m, 1H),
4.14–4.17 (m, 1H), 4.32–4.51 (m, 1H), 4.70 (s, 1H), 7.13–7.23 (m,
3H), 7.52–7.61 (m, 2H), 7.90 (d, J = 6 Hz, 1H), 8.04–8.09 (m, 2H).
13C NMR (DMSO-d6): d = 168.46(1C), 161.95(1C), 151.00(1C),
142.36(1C), 140.35(1C), 132.25(1C), 130.53(2C), 128.27(1C),
124.96(1C), 124.92(1C), 124.87(1C), 123.21(1C), 115.06(1C),
114.99(1C), 63.96(1C), 59.72(1C), 57.73(1C), 45.78(1C), 42.67(1C),
31.19(1C). ESI-MS (m/z): 442 (M+H)+; HRMS: Calcd for (C21H23N5-
O6+H)+: 442.1727; Found: 442.1720.
5.3.4. N-(2-Chlorophenyl)-2-((2-hydroxy-3-(1-methyl-2,4-
dioxo-1,2-dihydroquinazolin-3(4H)-
yl)propyl)(methyl)amino)acetamide hydrochloride (5d)
Yield 37%; mp 132–133 °C. 1H NMR (DMSO-d6) d (ppm): 2.35 (s,
3H), 2.39–2.43 (m, 2H), 3.21 (s, 2H), 3.71 (s, 3H), 3.46–3.51 (m, 1H),
4.01–4.04 (m, 2H), 4.54 (s, 1H), 7.2–7.27 (m, 3H), 7.39 (d, J = 6 Hz,
1H), 7.43–7.47 (m, 2H), 7.73 (t, J = 9 Hz, 1H), 7.98 (d, J = 4.5 Hz, 1H).
13C NMR (DMSO-d6): d = 164.12(1C), 161.90(1C), 150.99(1C),
140.87(1C), 135.82(1C), 134.15(1C), 130.12(1C), 128.29(1C),
127.99(1C), 127.93(1C), 127.81(1C), 127.19(1C), 123.19(1C),
5.3.9. N-(4-Chlorobenzyl)-2-((2-hydroxy-3-(1-methyl-2,4-dioxo-
1,2-dihydroquinazolin-3(4H)-yl)propyl)
(methyl)amino)acetamide hydrochloride (5i)
Yield 73%; mp 100–101 °C. 1H NMR (DMSO-d6) d (ppm): 2.56 (s,
3H), 2.67–2.71 (m, 2H), 3.43 (s, 2H), 3.72 (s, 3H), 3.97–4.10 (m, 2H),
4.05–4.10 (m, 1H), 4.26 (s, 2H), 7.08 (t, J = 6 Hz, 1H), 7.22–7.28 (m,
Please cite this article in press as: Ji, Q.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.042

Q. Ji et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
4H), 7.40 (d, J = 6 Hz, 1H), 7.72 (t, J = 6 Hz, 1H), 7.97 (d, J = 3 Hz, 1H).
5.3.14. 2-((2-Hydroxy-3-(1-methyl-2,4-dioxo-1,2-
dihydroquinazolin-3(4H)-yl)propyl)(methyl)amino)-N-(4-
nitrophenyl)acetamide hydrochloride (5n)
13C NMR (DMSO-d6): d = 164.66(1C), 161.32(1C), 150.91(1C),
140.90(1C), 138.87(1C), 135.80(1C), 128.79(2C), 128.29(1C)
127.79(2C), 127.16(1C), 123.12(1C), 116.29(1C), 114.99(1C),
63.64(1C), 59.20(1C), 56.71(1H), 45.16(1C), 42.60(1C), 40.53(1C),
31.19(1C). ESI-MS (m/z): 445 (M+H)+; HRMS: Calcd for (C22H25-
ClN4O4+H)+: 445.1643; Found: 445.1641.
Yield 32%; mp 121–122 °C. 1H NMR (DMSO-d6) d (ppm): 2.60 (s,
3H), 2.68–2.71 (m, 2H), 3.40–3.45 (m, 2H), 3.52 (s, 3H), 3.89–4.06
(m, 2H), 4.32–4.37 (m, 1H), 5.90 (s, 1H), 7.28 (t, J = 6 Hz, 1H),
7.43 (d, J = 6 Hz, 1H), 7.76 (t, J = 6 Hz, 1H), 7.82–7.89 (m, 2H),
8.01 (d, J = 6 Hz, 1H), 8.20 (d, J = 6 Hz, 2H). 13C NMR (DMSO-d6):
d = 164.62(1C), 161.90(1C), 151.00(1C), 144.66(1C), 143.16(1C),
140.87(1C), 135.77(1C), 128.29(1C), 126.40(2C), 123.17(1C),
119.63(2C), 115.33(1C), 114.97(1C), 63.66(1C), 59.36(1C),
57.67(1C), 46.14(1C), 42.65(1C), 31.17(1C). ESI-MS (m/z): 442
(M+H)+; HRMS: Calcd for (C21H23N5O6+H)+: 442.1727; Found:
442.1722.
5.3.10. N-(tert-Butyl)-2-((2-hydroxy-3-(1-methyl-2,4-dioxo-1,2-
dihydroquinazolin-3(4H)-yl)propyl)(methyl) amino)acetamide
hydrochloride (5j)
Yield 94%; mp 258–259 °C. 1H NMR (DMSO-d6) d (ppm): 1.17 (s,
9H), 2.46 (s, 3H), 2.57–2.61 (m, 2H), 3.37 (s, 2H), 3.68 (s, 3H), 3.70–
3.74 (m, 2H), 3.99–4.02 (m, 1H), 7.26 (t, J = 6 Hz, 1H), 7.39 (d,
J = 3 Hz, 1H), 7.78 (t, J = 4.5 Hz, 1H), 7.98 (d, J = 6 Hz, 1H). 13C NMR
(DMSO-d6): d = 167.44(1C), 161.92(1C), 151.01(1C), 140.27(1C),
135.80(1C), 128.28(1C), 123.20(1C), 115.51(1C) 114.98(1C),
64.99(1C), 60.81(1C), 60.25(1C), 50.72(1C), 45.61(1C), 42.95(1C),
31.15(1C), 28.74(3C). ESI-MS (m/z): 377(M+H)+; HRMS: Calcd for
(C19H28N4O4+H)+: 377.2189; Found: 377.2180. Element analysis:
Calcd for (C19H29ClN4O4): C, 55.27; H, 7.08; Found: C 55.14; H,7.13.
5.3.15. N,N-Dibenzyl-2-((2-hydroxy-3-(1-methyl-2,4-dioxo-1,2-
dihydroquinazolin-3(4H)-yl)propyl) (methyl) amino)acetamide
hydrochloride (5o)
Yield 60%; mp 119–120 °C. 1H NMR (DMSO-d6) d (ppm): 2.39 (s,
3H), 2.40–2.44 (m, 2H), 3.46 (s, 2H), 3.65 (s, 3H), 3.89–4.01 (m, 2H),
4.31–4.66 (m, 5H), 7.11–7.42 (m, 12H), 7.73 (t, J = 3 Hz, 1H), 7.99
(d, J = 6 Hz, 1H). 13C NMR (DMSO-d6): d = 165.61(1C), 161.92(1C),
150.99(1C), 140.86(1C), 136.81(1C), 135.99(1C), 135.25(1C),
129.31(2C), 128.96(2C), 128.22(1C), 128.17(2C), 128.12(1C),
127.80(1C), 127.63(2C), 123.22(1C), 115.29(1C), 115.02(1C),
64.29(1C), 59.62(1C), 56.74(1C), 49.76(2C), 45.14(1C), 42.62(1C),
31.16(1C). ESI-MS (m/z): 501 (M+H)+; HRMS: Calcd for (C29H32N4-
O4+H)+: 501.2502; Found: 501.2511.
5.3.11. N-(4-(Cyanomethyl)phenyl)-2-((2-hydroxy-3-(1-methyl-
2,4-dioxo-1,2-dihydroquinazolin-3(4H)-
yl)propyl)(methyl)amino)acetamide hydrochloride (5k)
Yield 53%; mp 144–145 °C. 1H NMR (DMSO-d6) d (ppm): 2.46 (s,
3H), 2.65–2.68 (m, 2H), 3.23 (s, 2H), 3.65 (s, 3H), 3.68–3.71 (m, 2H),
4.01–4.05 (m, 1H), 4.26 (s, 2H), 720–7.25 (m, 3H), 7.38 (d, J = 6 Hz,
1H), 7.54 (d, J = 9 Hz, 2H), 7.72 (t, J = 6 Hz, 1H), 7.96 (d, J = 3 Hz,
1H). 13C NMR (DMSO-d6): d = 167.74(1C), 161.95(1C), 151.01(1C),
140.86(1C), 137.94(1C), 135.84(1C), 129.29(1C), 129.01(1C),
128.29(1C), 127.06(1C), 123.21(1C), 120.23(1C), 119.80),
115.66(1C), 115.32(1C), 115.202(1C), 63.91(1C), 59.48(1C),
67.72(1C), 45.17(1C), 42.49(1C), 31.18(1C), 22.32(1C). ESI-MS (m/
z): 436 (M+H)+; HRMS: Calcd for (C23H25N5O4+H)+: 436.1985;
Found: 436.1981.
5.3.16. 2-((2-Hydroxy-3-(1-methyl-2,4-dioxo-1,2-
dihydroquinazolin-3(4H)-yl)propyl)(methyl)amino)-N-methyl-
N-phenylacetamide hydrochloride (5p)
Yield 30%; mp 110–111 °C. 1H NMR (DMSO-d6) d (ppm): 2.29 (s,
3H), 2.50–2.54 (m, 2H), 3.35 (s, 2H), 3.53 (s, 6H), 3.84–4.03 (m, 3H),
4.28 (s, 1H), 7.32 (t, J = 6 Hz, 1H), 7.40–7.61 (m, 6H), 7.78 (t,
J = 6 Hz, 1H), 8.06 (d, J = 6 Hz, 1H). 13C NMR (DMSO-d6):
d = 164.16(1C), 161.90(1C), 151.00(1C), 141.49(1C), 140.91(1C),
135.86(1C), 130.61(2C), 129.07(1C) 128.31(2C), 127.76(1C),
123.23(1C), 115.56(1C), 115.04(1C), 63.94(1C), 59.52(1C),
56.69(1C), 45.11(1C), 42.98(1C), 37.61(1C), 31.20(1C). ESI-MS (m/
z): 411 (M+H)+; EI-HRMS: Calcd for (C22H26N4O4+H)+: 411.2032;
Found: 411.2026.
5.3.12. Methyl 2-(2-((2-hydroxy-3-(1-methyl-2,4-dioxo-1,2-
dihydroquinazolin-3(4H)-yl)propyl)(methyl)
amino)acetamido)benzoate hydrochloride (5l)
Yield 45%; mp 170–171 °C. 1H NMR (DMSO-d6) d (ppm): 2.46 (s,
3H), 2.55–2.59 (m, 2H), 2.98 (s, 2H), 3.28–3.41 (m, 2H), 3.46 (s, 3H),
3.77 (s, 3H), 3.93–4.01 (m, 1H), 5.91 (s, 1H), 7.21–7.26 (m, 2H), 7.39
(d, J = 3 Hz, 1H), 7.64–7.68 (m, 1H), 7.73–7.78 (m, 3H), 7.97–7.99
(d, J = 6 Hz, 1H). 13C NMR (DMSO-d6): d = 167.46(1C), 164.00(1C),
161.91(1C), 151.01(1C), 140.55(1C), 137.25(1C), 135.30(1C),
133.72(1C), 130.75(2C), 128.29(1C), 125.69(1C), 125.52 (1C),
123.18(1C), 115.66(1C), 114.98(1C), 63.99(1C), 59.24(1C),
57.69(1C), 52.37(1C), 43.19(1C), 42.46(1C), 31.17(1C). ESI-MS (m/
z): 455 (M+H)+; HRMS: Calcd for (C23H26N4O6+H)+: 455.1931;
Found: 455.1923.
5.3.17. 2-((2-Hydroxy-3-(1-methyl-2,4-dioxo-1,2-
dihydroquinazolin-3(4H)-yl)propyl)(methyl)amino)-N,N-
dipropylacetamide hydrochloride (5q)
Yield 15%; mp 131–132 °C. 1H NMR (DMSO-d6) d (ppm): 0.94–
1.02 (m, 6H), 1.56–1.62 (m, 4H), 2.39 (s, 3H), 2.45–2.51 (m, 2H),
3.24 (s, 2H), 3.26–3.33 (m, 4H), 3.67 (s, 3H), 4.12–4.57 (m, 3H),
4.57 (s, 1H), 7.22 (t, J = 12 Hz, 2H), 7.68 (t, J = 6 Hz, 1H), 8.16 (d,
J = 3 Hz, 1H). 13C NMR (DMSO-d6): d = 164.50(1C), 161.76(1C),
150.81(1C), 140.31(1C), 135.77(1C), 128.32(1C), 123.56(1C),
115.25(1C), 114.93(1C), 64.79(1C), 59.30(2C), 51.93(2C),
48.55(1C), 45.52(1C), 31.14(1C), 21.03(2C), 11.49(2C). ESI-MS (m/
z): 405 (M+H)+; HRMS: Calcd for (C21H32N4O4+H)+: 405.2502;
Found: 405.2508.
5.3.13. 2-((2-Hydroxy-3-(1-methyl-2,4-dioxo-1,2-
dihydroquinazolin-3(4H)-yl)propyl)(methyl)amino)-N-
phenylacetamide hydrochloride (5m)
Yield 57%; mp 141–142 °C. 1H NMR (DMSO-d6) d (ppm): 2.61 (s,
3H), 2.70–2.571 (m, 2H), 3.14–3.17 (m, 2H), 3.40 (s, 2H), 3.62 (s,
3H), 3.94–4.06 (m, 1H), 4.30–4.33 (s, 1H), 5.89 (s, 1H), 7.09 (t,
J = 6 Hz, 1H), 7.30–7.34 (m, 3H), 7.46 (d, J = 3 Hz, 1H), 7.61 (d,
J = 6 Hz, 2H), 7.79 (t, J = 4.5 Hz, 1H), 8.05 (d, J = 6 Hz, 1H). 13C
NMR (DMSO-d6): d = 167.35(1C), 161.92(1C), 151.05(1C),
140.89(1C), 138.62(1C), 135.79(1C), 129.26(2C), 128.51(1C),
124.34(1C), 123.19(1C), 119.82(2C), 115.57(1C), 114.99(1C),
64.21(1C), 59.87(1C), 58.21(1C), 45.32(1C), 42.52(1C), 31.16(1C).
ESI-MS (m/z): 431 (M+H)+; HRMS: Calcd for (C21H24N4O4+H)+:
397.1876; Found: 397.1866.
5.3.18. N,N-Diethyl-2-((2-hydroxy-3-(1-methyl-2,4-dioxo-1,2-
dihydroquinazolin-3(4H)-yl)propyl) (methyl) amino)acetamide
hydrochloride (5r)
Yield 79%; mp 130–131 °C. 1H NMR (DMSO-d6) d (ppm): 0.82–
0.97 (m, 6H), 2.35 (s, 3H), 2.38–2.42 (m, 2H), 3.23 (s, 2H), 3.28–
3.35 (m, 4H), 3.67 (s, 3H), 4.09–4.13 (m, 1H), 4.34–4.38 (m, 1H),
4.50 (m, 1H), 4.56 (s, 1H), 7.20 (t, J = 6 Hz, 1H), 7.25 (d, J = 6 Hz,
1H), 7.67 (t, J = 6 Hz, 1H), 8.16 (d, J = 6 Hz, 1H). 13C NMR (DMSO-
d6):
d = 164.57(1C),
161.79(1C),
150.88(1C),
140.29(1C),
Please cite this article in press as: Ji, Q.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.042

8
Q. Ji et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
135.82(1C), 128.28(1C), 123.32(1C), 115.28(1C), 115.20(1C),
64.81(1C), 59.24(2C), 48.65(1C), 45.20(1C), 43.15(2C), 31.09(1C),
11.65(1C), 11.42(1C). ESI-MS (m/z): 377(M+H)+; HRMS: Calcd for
(C19H28N4O4+H)+: 377.2189; Found: 377.2193.
5.5. Antimicrobial assay
5.5.1. Antifungal activity assay
Cryptococcus neoformans extracted from patient, Candida albi-
cans ATCC 76615, and Aspergillus flavus were used for evaluating
the antifungal activity of all designed compounds. The media was
modified Sabouraud-chloramphenicol agar containing 1% peptone,
2% Glucose and solid media as well as 15% agar. All target
compounds were dissolved in sterile water as stock solutions.
The antifungal activity was evaluated by disc diffusion method.
The inhibition action was tested after incubating for 48 h at 37 °C
and the minimum inhibitory concentrations (MICs, mg/L) were
recorded. Polyoxin B and fluconazole were used as the positive
controls.
5.3.19. 3-(2-Hydroxy-3-(methyl(2-oxo-2-(piperidin-1-
yl)ethyl)amino)propyl)-1-methylquinazoline-2,4(1H,3H)-dione
hydrochloride (5s)
Yield 60%; mp 105–106 °C. 1H NMR (DMSO-d6) d (ppm):
1.66–1.69 (m, 6H), 2.33 (s, 3H), 2.40–2.44 (m, 2H), 3.30 (s, 2H),
3.42–3.47 (m, 4H), 3.70 (s, 3H), 4.10–4.15 (m, 2H), 4.56 (m, 1H),
4.71 (s, 1H), 7.21 (t, J = 6 Hz, 1H), 7.25 (d, J = 6 Hz, 1H), 7.67 (t,
J = 6 Hz, 1H), 8.16 (d, J = 6 Hz, 1H). 13C NMR (DMSO-d6):
d = 167.56(1C), 161.58(1C), 151.01(1C), 140.85(1C), 135.87(1C),
128.18(1C), 123.26(1C), 115.42(1C), 115.39(1C), 63.99(1C),
59.13(1C), 58.96(1C), 48.59(2C), 47.87(1C), 45.52(1C), 31.14(1C),
24.93(2C), 22.97(1C). ESI-MS (m/z): 389 (M+H)+; HRMS: Calcd for
(C20H28N4O4+H)+: 389.2189; Found: 389.2182.
5.5.2. Antibacterial assay
Six strains of bacteria Staphylococcus aureus ATCC 25923, Bacil-
lus subtilis ATCC 6633, Methicillin-resistant Staphylococcus aureus N
315 (MRSA), Bacillus proteus, Escherichia coli JM 109, P. aeruginosa
CR56 were tested with streptomycin sulfate as the positive control.
The media used in this assay was Beef Extract-peptone media
containing 1% peptone, 0.3% beef extract, 0.5% sodium chloride in
distilled water, the solid media as well as 15% agar. All compounds
were dissolved in sterile water. The antibacterial activity was eval-
uated by disc diffusion method. Tests were incubated overnight at
37 °C prior to recording MICs.
5.4. Inhibition on chitin synthase36
Yeast cells (Saccharomyces cerevisiae CGMCC2.145) were col-
lected through centrifugation at 1500 g for 15 min at 4 °C, then
washed with water, and precipitated. The precipitates were sus-
pended in 20 mL 50 mM Tris–HCl solution at pH 7.0 which con-
tains 20
ll fungal protease inhibitor cocktail (Sigma–Aldrich) and
50 l solution of 200 mM Phenylmethanesulfonyl fluoride (PMSF,
l
Acknowledgements
Genview) in DMSO, and were lysed by sonication treatment at
4 °C for 80 min. Insoluble materials were removed. The superna-
tant was mixed with twofold volume solution of 10% sucrose in
50 mM Tris–HCl (w/v) at pH 7.5, and centrifuged at 55,000 g for
2 h at 4 °C. The pellet was re-suspended in 50 mM Tris–HCl at pH
7.0 to serve as CHS sample, and 33% glycerol was added for storage
at À80 °C.
This work was supported by National Natural Science Founda-
tion of China (No. 81071427), the Education Ministry of China
(No. 20125503110007) and Natural Science Foundation Project of
CQ (CSTC2012JJA0057).
References and notes
Stock solutions of candidate compounds were made with
DMSO for no less than 10 g/L and those compounds that did
not dissolved in DMSO were dissolved in the water. Each reaction
1. Hossain, M. A.; Ghannoum, M. A. Expert Opin. Investig. Drugs 2000, 9, 1797.
2. Bastert, J.; Schaller, M.; Korting, H. C.; Evans, E. G. Int. J. Antimicrob. Agents 2001,
17, 81.
3. Pfaller, M. A.; Diekema, D. J. Clin. Microbiol. Rev. 2007, 20, 133.
4. Chamilos, G.; Kontoyiannis, D. P. Drug Resist. Updat. 2005, 8, 344.
5. Antinori, S. ISRN AIDS. http://dx.doi.org/10.1155/2013/471363.
6. Masiá, C. M.; Gutiérrez, R. F. Lancet. Infect. Dis. 2002, 2, 550.
7. Perlin, D. S. Drug Resist. Updat. 2007, 10, 121.
system in a well of microplate contained 50
UDP-GlcNAc (Sigma–Aldrich) plus 320 mM GlcNAc (Aladdin
reagent Co., Ltd), 50 l solution of a candidate compound diluted
with 50 mM Tris–HCl buffer at pH 7.0 for final DMSO below 3%,
and 100 l of CHS sample to make up to a total of 200 l. Micro-
ll solution of 16 mM
l
l
l
8. Verweij, P. E.; Snelders, E.; Kema, G. H.; Mellado, E.; Melchers, W. J. Lancet
Infect. Dis. 2009, 9, 789.
plates were kept at 37 °C in an incubator for 45 min after being
mildly shaken for 2 min on Qilinbeier QB-9001 shaker. After reac-
tion for a indicated time, unbound components were removed
and wells were washed with the washing buffer (50 mM Tris–
9. Walker, L. A.; Gow, N. A.; Munro, C. A. Fungal. Genet. Biol. 2010, 47, 117.
10. Kathiravan, M. K.; Salake, A. B.; Chothe, A. S.; Dudhe, P. B.; Watode, R. P.;
Mukta, M. S.; Gadhwe, S. Bioorg. Med. Chem. 2012, 20, 5678.
11. Santamaría, R.; Rizzetto, L.; Bromley, M.; Zelante, T.; Lee, W.; Cavalieri, D.;
Romani, L.; Miller, B.; Gut, I.; Santos, M.; Pierre, P.; Bowyer, P.; Kapushesky, M.
S. Immunobiology 2011, 216, 1212.
HCl plus 2% Tween 20 at pH 7.0). To each well, 150
ll solution
of 0.2 g/mL WGA-HRP (Sangon, Shanghai, China) diluted in
l
12. Georgopapadakou, N. H. Curr. Opin. Microbiol. 1998, 1, 547.
13. Munro, C. A.; Gow, N. A. Med. Mycol. 2001, 39, 41.
10 mM sodium phosphate buffer was added. After being gently
shaken for 2.0 min, microplates were further kept at 37 °C for
15 min, and then washed five times with the washing buffer.
Finally, 150 ll peroxidase substrate reagent (TMB 0.2 mM, H2O2
0.3% v/v, 100 mM Na2HPO4-Citric acid, pH 3.7, Sangon, Shanghai,
China) was added for exact reaction time of 30 min at 37 °C. The
reaction was stopped with 50 ll 2 M H2SO4 and measured with
Biotek ELX 800 Microplate reader at 450 nm. Standard chitin
(Sigma–Aldrich) of 0.50 g/L was used to construct a response of
absorbance at 450 nm with logarithmic quantities of chitin in
wells. There was a linear response of absorbance at 450 nm to
logarithmic quantities of chitin from 3.1 to 50 mg/L in a total of
14. Feng, Y. A.; Cui, Z. F. Microbiol. Commun. 2008, 35, 267.
15. Lóránd, T.; Kocsis, B. Mini Rev. Med. Chem. 2007, 7, 900.
16. Ruiz-Herrera, J.; San-Blas, G. Curr. Drug Targets Infect. Disord. 2003, 3, 77.
17. Lenardon, M. D.; Munro, C. A.; Gow, N. A. Curr. Opin. Microbiol. 2010, 13, 416.
18. Zhang, D.; Miller, M. J. Curr. Pharm. Des. 1999, 5, 73.
19. Georgopapadakou, N. H.; Tkacz, J. S. Trends Microbiol. 1995, 3, 98.
20. Emmer, G.; Ryder, N. S.; Grassberger, M. A. J. Med. Chem. 1985, 28, 278.
21. Khare, R. K.; Becker, J. M.; Naider, F. R. J. Med. Chem. 1988, 31, 650.
22. Cabib, E. Antimicrob. Agents Chemother. 1991, 35, 170.
23. Liao, G.; Li, J.; Li, L.; Yang, H.; Tan, H. Microb. Cell Fact. 2010, 9, 6.
24. Decker, H.; Zähner, H.; Heitsch, H.; König, W. A.; Fiedler, H. P. J. Gen. Microbiol.
1991, 1805, 137.
25. Kumar, N.; Shalini, K.; Drabu, S. Biointerface Res. Appl. Chem. 2011, 1, 203.
26. Zhu, L.; Jin, J.; Liu, C.; Zhang, C.; Sun, Y.; Guo, Y.; Fu, D.; Chen, X.; Xu, B. Bioorg.
Med. Chem. 2011, 19, 2797.
27. Ryu, C. K.; Kim, Y. H.; Im, H. A.; Kim, J. Y.; Yoon, J. H.; Kim, A. Bioorg. Med. Chem.
Lett. 2012, 22, 500.
200 ll solution in microplate wells. Parameters of such a linear
response showed good consistency during independent repetitive
assays. Chitin in each well was calculated accordingly to estimate
half-inhibition concentration (IC50) of a test compound.
28. Oppegard, L. M.; Streck, K. R.; Rosen, J. D.; Schwanz, H. A.; Drlica, K.; Kerns, R. J.;
Hiasa, H. Antimicrob. Agents Chemother. 2010, 54, 3011.
Please cite this article in press as: Ji, Q.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.042

Q. Ji et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
29. Nakamoto, K.; Tsukada, I.; Tanaka, K.; Matsukura, M.; Haneda, T.; Inoue, S.;
Murai, N.; Abe, S.; Ueda, N.; Miyazaki, M.; Watanabe, N.; Asada, M.;
Yoshimatsu, K.; Hata, K. Bioorg. Med. Chem. Lett. 2010, 20, 4624.
30. Uchida, T.; Kagoshima, Y.; Konosu, T. Bioorg. Med. Chem. Lett. 2009, 19, 2013.
31. Liu, H. B.; Tang, H.; Yang, D.; Deng, Q.; Yuan, L. J.; Ji, Q. G. Bioorg. Med. Chem.
Lett. 2012, 22, 5845.
32. Li, F.; Feng, Y.; Meng, Q.; Li, W.; Li, Z.; Wang, Q.; Tao, F. ARKIVOC 2007, 1, 40.
33. Li, J.; Chen, X.; Shi, D.; Ma, S.; Li, Q.; Zhang, Q.; Tang, J. Org. Lett. 2009, 11, 1193.
34. Shi, D. Q.; Dou, G. L.; Li, Z. Y.; Ni, S. N.; Li, X. Y.; Wang, X. S.; Wu, H.; Ji, S. J.
Tetrahedron 2007, 63, 9764.
35. Singh, G. S.; Mollet, K.; D’hooghe, M.; DeKimpe, N. Chem. Rev. 2013, 113, 1441.
36. Lucero, H. A.; Kuranda, M. J.; Bulik, D. A. Anal. Biochem. 2002, 305, 97.
Please cite this article in press as: Ji, Q.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.042