Design, synthesis and biological evaluation of novel quinazoline-2,4-diones conjugated with different amino acids as potential chitin synthase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.05.001

A series of (2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl) acetamido) acids) (6 a-m), (7) has been designed to inhibit the action of fungus chitin synthase enzyme (CHS). The synthesis of the designed compounds was carried out in four steps starting from the reaction between 1-methylquinazoline-2,4(1H,3H)-dione and ethyl chloroacetate to yield the ethyl acetate derivative. This ester was hydrolyzed to the corresponding carboxylic acid derivative that was then utilized to couple several amino acids getting the final designed compounds. The synthesized compounds were tested for their inhibition against CHS. Compound 7 showed the highest potency among others with minimum inhibitory concentration (IC₅₀) of 0.166 mmol/L, while polyoxin B (the positive control) had IC₅₀ of 0.17 mmol/L. The synthesized compounds were also evaluated for their in vitro antifungal activity using Aspergillus fumigates, Aspergillus flavus, Crytococcus neoformans and Candida albicans. Unfortunately, the 14 synthesized compounds showed lower in vitro activity compared to the used active controls. However, compound 6m and fluconazole have synergistic effect on Aspergillus flavus; Compounds 7 and fluconazole have synergistic effects on Aspergillus fumigates.

Full text of DOI:10.1016/j.ejmech.2018.05.001

Accepted Manuscript
Design, synthesis and biological evaluation of novel quinazoline-2,4-diones
conjugated with different amino acids as potential chitin synthase inhibitors
Nada A. Noureldin, Hend Kothayer, El-Sayed M. Lashine, Mohamed M. Baraka,
Yanrong Huang, Bing Li, Qinggang Ji
PII:
S0223-5234(18)30405-7
10.1016/j.ejmech.2018.05.001
EJMECH 10411
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 October 2017
Revised Date: 15 April 2018
Accepted Date: 2 May 2018
Please cite this article as: N.A. Noureldin, H. Kothayer, E.-S.M. Lashine, M.M. Baraka, Y. Huang, B.
Li, Q. Ji, Design, synthesis and biological evaluation of novel quinazoline-2,4-diones conjugated with
different amino acids as potential chitin synthase inhibitors, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.05.001.
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ACCEPTED MANUSCRIPT
Graphical abstract
Design, Synthesis and Biological Evaluation of Novel
Quinazoline-2,4-diones Conjugated with Different Amino
Acids as Potential Chitin Synthase Inhibitors
a
a*1
a
Nada A. Noureldin *, Hend Kothayer , EL-Sayed M. Lashine ,
a
b
b
b
Mohamed M. Baraka , Yanrong Huang , Bing Li and Qinggang Ji .
a
Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, Egypt.
b
School of chemistry and chemical engineering, Southwest University, P.R.China
COOH
HOOC
HN
OH
COOH
HN
HN
H
N
COOH
S
HN
COOH
H
N
COOH
O
N
H
N
COOH
or
AA'
AA'
N
NH
NH
COOH
COOH
O
O
NH
NH
N
COOH
N
H
COOH
NH
NH
COOH
COOH
N
6
h-m
6
a-g
O
N
H
N
COOH
N
O
N
O
O
N
O
O
7
O
1
Corresponding author. Tel. 00201224578259; E-mail: hendo1311@hotmail.com or
hkelhamalawy@pharmacy.zu.edu.eg
equal contribution
*

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of Novel Quinazoline-
2
,4-diones Conjugated with Different Amino Acids as Potential
Chitin Synthase Inhibitors
a
a*1
a
Nada A. Noureldin *, Hend Kothayer , EL-Sayed M. Lashine , Mohamed M.
a
b
b
b
Baraka , Yanrong Huang , Bing Li and Qinggang Ji .
a
Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, Egypt.
b
School of chemistry and chemical engineering, Southwest University, P.R.China
Abstract: A series of (2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-
(4H)-yl) acetamido) acids) (6 a-m), (7) has been designed to inhibit the action
3
of fungus chitin synthase enzyme (CHS). The synthesis of the designed
compounds was carried out in four steps starting from the reaction between 1-
methylquinazoline-2,4(1H,3H)-dione and ethyl chloroacetate to yield the ethyl
acetate derivative. This ester was hydrolysed to the corresponding carboxylic
acid derivative that was then utilized to couple several amino acids getting the
final designed compounds. The synthesised compounds were tested for their
inhibition against CHS. Compound 7 showed the highest potency among others
with minimum inhibitory concentration (IC ) of 0.166 mmol/L, while polyoxin
5
0
B (the positive control) had IC of 0.17 mmol/L. The synthesised compounds
5
0
were also evaluated for their in vitro antifungal activity using Aspergillus
fumigates, Aspergillus flavus, Crytococcus neoformans and Candida albicans.
Unfortunately, the 14 synthesized compounds showed lower in vitro activity
compared to the used active controls. However, compound 6m and fluconazole
have synergistic effect on Aspergillus flavus; Compounds 7 and fluconazole
have synergistic effects on Aspergillus fumigates.
1
Corresponding author. Tel. 00201224578259; E-mail: hendo1311@hotmail.com or
hkelhamalawy@pharmacy.zu.edu.eg
*equal contribution

ACCEPTED MANUSCRIPT
Keywords: Quinazolinones; Amino acids; Chitin synthase inhibitor;
Antifungal.
1
. Introduction:
Fungi are eukaryotic organisms with a rigid cell wall containing beta-glucan,
chitin and mannoproteins that maintain this rigidity and also take charge of
altering host immunity leading to tissue injury and its subsequent events that
can even reach to death [1, 2]. Despite the recent medical care advances,
invasive fungal infections are still associated with a high mortality rate
commonly among immunocompromised patients. The currently available
antifungals are considered clinically insignificant concerning their poor
bioavailability, resistance development, non-selectivity and serious side effects
as nephrotoxicity [3-5]. Thus, continuous development of novel antifungal
active agents is needed indeed.
Chitin, the unique fungus cell wall component, considered as one of the
recent hopeful antifungal targets [6]. Chitin is a linear polysaccharide composed
of N-acetyl- -glucosamine (GlcNAc) units joined by β-1,4-glycoside linkage.
D
When its synthesis is interrupted fungal cell becomes osmotically unstable as it
maintains cell wall integrity. Its absence in mammalian cells makes it an
excellent target for selective novel antifungal development. Chitin biosynthesis
is mediated by chitin synthase (CHS) enzymes that utilize uridine diphosphoryl-
N-acetyl- -glucosamine (UDP-GlcNAc) (Fig.1, I) as a substrate for chitin
D
biosynthesis [7, 8].
Till the moment, the exact structure of the active site of the chitin synthase
enzyme still unknown as a result of its transmembrane position that hinder the
structural studies [9]. The well-known Polyoxins and Nikkomycins are naturally
isolated series of compounds which were previously proven for their chitin
synthase inhibitory activity, they also possess a core structure similar to that of
CHS substrate UDP-GlcNAc [10]. Despite the remarkable in vitro activity of

ACCEPTED MANUSCRIPT
polyoxins against CHS, their limited penetrability across the cell membrane and
degradation via intracellular peptidase limit their in vivo activity (Fig.1, II).
Their development is now terminated and are limited to be used against
phytopathogenic fungi [5]. These drawbacks are not considered in nikkomycins
but some of these compounds with hydroxypyridyl residue are unstable in
neutral and alkaline pH [7]. Although being tested currently in phase II clinical
trials, narrow spectrum of activity of Nikkomycin Z will limit its clinical
significance [11, 12] (Fig.1, III).
Fig. 1
Several successful studies were conducted by synthesizing non-
nucleoside organic compounds that were then tested for their CHS inhibitory
activity, showing promising inhibitory results. Part of these studies involved
replacement of the nucleoside part in Nikkomycin Z and polyoxin B by other
rings such as quinazoline-2,4-dione (Fig. 2, IV) and coumarin (Fig. 2, V), as a
trial to improve drugs hydrophobicity. These rings were designed to be attached
with a linear moiety that roughly resemble the side chain of Nikkomycin Z and
polyoxin B [13, 14]. Other attempts involved the synthesis of small non-
nucleoside organic compounds, many of which showed good inhibitory activity;
however; the exact mechanism of action of these compounds still not clear [15-
2
0] (Fig. 3).
Fig. 2
Fig. 3
In the current work, we attempted to design a series of CHS inhibitors
that keep the balance between the pharmacokinetics and pharmacodynamics.
Whereas, quinazolin-2,4-dione, which already known with its good antifungal
action, was used to improve the overall lipophilicity of the compounds in order
to improve the cellular penetration to insure their accumulation inside the

ACCEPTED MANUSCRIPT
fungus cell to the concentration that allow the significant clinical activity [13].
The 3 position in quinazolin-2,4-dione was designed to be attached to a side
chain that ends with structurally different amino acids. The side chain has
enormous polar groups required for the enzyme’s active site interactions. And
structurally variant amino acids were used to improve the bioavailability of the
target compounds.
Proline, histidine, tryptophan (containing heterocycles),
D
-alanine,
leucine, 2-aminobutyric acid, valine (with alkyl side chain), glycine, β-alanine,
GABA (unbranched amino acids), methionine (sulphur containing amino acid),
phenylalanine, tyrosine (with aromatic rings) and serine (with terminal hydroxyl
group) were used as terminal amino acids to study the structure activity
relationship of each change (Fig. 4).
Fig. 4
2
. Results and discussion:
.1. Chemistry:
2
Compounds (6a-m) and (7) were synthesized according to Scheme 1. N-
methyl anthranilic acid (1) was fused with urea to yield 1-methylquinazoline-
2
,4(1H,3H)-dione (2). Then this intermediate (2) was allowed to react with ethyl
chloroacetate to form Ethyl 2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-
(4H)-yl) acetate (3) that was then hydrolyzed to the corresponding free
3
carboxylic acid analogue (4). This acid was then converted to the acyl
benzotriazole (5) through reaction with 1H benzotriazole and SOCl at room
2
temperature in dry DCM Finally, this acyl benzotriazole semifinal intermediate
.
(
5) was allowed to react with various amino acids in acetonitrile/water mixture
using triethylamine at room temperature that yielded the novel acetamido acids
6a-m). Unexpectedly, when tyrosine (one of the amino acids) was used in such
reaction, it yielded the dimer compound (7). This could be explained by the
(

ACCEPTED MANUSCRIPT
presence of two reactive nucleophilic centers in tyrosine (the phenolic OH and
the NH groups) which both reacted with the highly reactive acyl benzotriazole
2
(5). Unfortunately, our trials to obtain the monomer product from tyrosine were
failed.
Scheme. 1
2
.2.Biological assays:
.2.1. Inhibition against yeast CHS:
2
Compounds 6c, 6l, 6m and 7 exhibited the most promising enzyme
inhibition percent as shown in (Fig. 5, table. 1) at a concentration of 300 µg/ml.
Therefore, the IC values of each of these four compounds were determined
5
0
where their IC values ranges from 0.166 mmol/L as that of polyoxin B the
50
reference drug to 0.30 mmol/L. Compound 7 with IC of 0.166 mmol/L is the
5
0
strongest inhibitor among the other compounds. While the IC values for 6c, 6l
50
and 6m were 0.30, 0.30 and 0.18 mmol/L respectively (Fig. 6, table. 1).
Fig. 5
Fig. 6
Table. 1
The unexpected dimeric structure of 7 improves the activity in an
apparent way over the other monomeric compounds. This result is completely
consistent with the two-active site mechanism hypothesis of chitin synthase
enzyme, which assumes that CHS has two active sites in close proximity to each
other. Thus, dimeric compounds generally possess higher activity than other
monomeric ones [7, 21-23].
It is also notable that compounds 6m and 6l that carry a terminal nitrogen
containing heterocycles like Nikkomycin Z showed the highest inhibitory

ACCEPTED MANUSCRIPT
activity (IC = 0.18 and 0.30 mmol/L respectively) among the synthesized
50
monomeric structures.
The superior activity of our unexpected dimeric compound (7) not only
supports the findings of Finney and Yeager [21, 22] and come against the study
of Kral etal. [24] in their debate about the advantage of dimeric chitin synthase
inhibitors, but also encourages the designing of future non-nucleoside dimeric
chitin synthase inhibitors.
2
.2.2. antifungal activity:
Four fungal strains were used to determine the in vitro antifungal activity
of the synthesized compounds Aspergillus fumigatus, Aspergillus flavus,
Cryptococcus neoformans and Candida albicans. The results are shown in
table.2.
The 14 synthesized compounds showed lower in vitro activity compared to
the two active controls as shown in table. 2. This could be attributed to their
failure to penetrate the fungal cell wall and accumulate in the concentration
required for the best activity. We may relate this result to the hydrophilicity of
the synthesised compounds that hinder their penetration across the fungal cell
wall. Future studies are suggested in which the carboxylic acid group could be
esterified to improve the lipophilicity of the target compounds and accordingly
enhance their penetrability, making a balance between the overall compound
lipophilicity and the polar groups required for the active site interactions.
Moreover, dimers could be designed and synthesised to make benefit of the
two-active site mechanism hypothesis suggested for chitin synthase activity.
Table. 2
2
.2.3. In vitro susceptibility of fungi to 6m, 7 and fluconazole alone and
in combination.

ACCEPTED MANUSCRIPT
In order to investigate whether the difficulty in penetration is the only
reason of the reduced in vitro activity or not, further study was carried out. In
this study, the effect of a combination between fluconazole and the most active
compounds 6m and 7 using the checkboard synergy analysis was evaluated
using the same four fungal strains used before. Fluconazole was especially used
as it acts through inhibition of ergosterol biosynthesis in plasma membrane,
leading to disruption in the whole cellular integrity. Thus, it was thought that
this may enhance the access of our compounds to their target (the chitin
synthase enzyme).
First, the MIC values of 6m, 7, and fluconazole were furtherly determined
as shown in table. 3. Then the MIC values of both 6m and 7 in combination
with the fluconazole together with the FICIs (Fractional Inhibitory
Concentration Index) of each combination against the four fungal strains were
calculated. Drug interactions were classified as synergistic if the FIC index was
≤
0.5, indifferent if >0.5–4, and antagonistic if >4. From the activity data,
compound 6m and fluconazole have synergistic effect on Aspergillus flavus
ATCC 16870); Compounds 7 and fluconazole have synergistic effects on
（
Aspergillus fumigates （GIMCC 3.19). The compounds 6m and 7 have no
inhibition or synergistic effect on other fungi. From these results, it is revealed
that the failure in penetration is not the only pharmacokinetic factor involved in
the reduced in vitro activity of the synthesized compounds, however, it is not a
negligible factor that if modified the activity will be varied in a positive way.
Table. 3
3
. Conclusion:
In summary, chitin synthase enzyme (CHS) is considered as a promising
target for selective novel antifungal agents’ development. A series of (2-(1-
methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl) acetamido) acids) (6 a-m),

ACCEPTED MANUSCRIPT
(7) has been designed, synthesised and evaluated for their inhibition activity
against the action of fungus CHS. Four compounds 6c, 6l, 6m and 7 showed
good inhibitory activity. Compound 7 with a dimeric structure was the most
active compound with IC =0.166 mmol/L, while the IC of Polyoxin B (the
50
50
reference compound) was 0.17 mmol/L. This result gives rise to the two-active
site mechanism hypothesis of chitin synthase enzyme which assume that the
enzyme has 2 active sites located in close proximity to each other making
dimeric compounds to possess better activity in comparison with monomeric
ones. The 14 synthesised compounds were also evaluated for their in vitro
antifungal activity using four fungal strains Aspergillus fumigates, Aspergillus
flavus, Crytococcus neoformans and Candida albicans. Unfortunately, the 14
synthesized compounds showed lower in vitro activity compared to the two
active controls (Fluconazole and Polyoxin B). However, compound 6m and
fluconazole have synergistic effect on Aspergillus flavus; Compounds 7 and
fluconazole have synergistic effects on Aspergillus fumigates. Further studies
are suggested in which the lipophilicity of the synthesised compounds could be
increased through esterification. Also, designing dimeric compounds regarded
as a good strategy to obtain more potent CHS inhibitors.
4
. Experimental:
4
.1. Chemistry:
Melting points (°C) were measured on a Gallenkamp melting point
apparatus (London, UK), and are uncorrected. NMR spectra were performed in
the Microanalytical Center, Faculty of Pharmacy, Cairo University, Egypt or
Microanalytical Center, Faculty of Science, Zagazig University, Egypt.
Elemental analyses and Mass spectra were performed in the Regional Center for
Mycology and Biotechnology, Al-Azhar University, Egypt. NMR spectra were
recorded on Bruker high performance Digital FT-NMR spectrometer avance III
4
00 MHz using dimethyl sulfoxide (DMSO)-d and chloroform (CDCl )-d as
6
3

ACCEPTED MANUSCRIPT
solvent and tetramethylsilane (TMS) as an internal standard (chemical shift in δ,
ppm). Mass spectra were determined using a GC/MS Shimadzu Qp-2010 plus
(Shimadzu Corporation, Tokyo, Japan). Elemental analyses were determined
using the Vario EL-III (Elementar) CHNS analyzer (Hanau, Germany). All
reactions were monitored by thin layer chromatography (TLC) using silica gel
6
0 GF245 (E-Merck, Germany) and were visualized by UV-lamp at wavelength
(
λ) 254 nm.
1
-methylquinazoline-2,4(1H,3H)-dione (2), was synthesized according to the
previously reported procedures[13]
General
method
for
synthesis
of
Ethyl2-(1-methyl-2,4-dioxo-1,2-
dihydroquinazolin-3(4H)-yl)acetate (3):
A mixture of 1-methylquinazoline-2,4(1H,3H)-dione (1, 0.0225 mole),
ethyl chloroacetate (0.0225 mole) and K CO (0.045mole) in DMF was refluxed
2
3
for 12 h. After cooling the mixture poured into excess amount of water with
stirring. The formed precipitate was filtered washed several times with water to
provide ethyl 2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl) acetate
(3) in 87.14% yield and sufficient purity to be used in the next step without
further purification.
Ethyl2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)acetate
(3):
1
(
87.14% yield). M.p. 124-127°C. H NMR (DMSO, d ) δ 1.22 (3H, t, J= 7.1
6
Hz, CH CH ), 3.56 (3H, s, NCH ), 4.14 (2H, q, J= 7.1 Hz, CH CH ), 4.71 (2H,
2
3
3
2
3
s, NCH ), 7.36 (1H, t, J= 7.5 Hz, H-6), 7.52 (1H, d, J= 8.4 Hz, H-8), 7.84 (1H,
2
t, J= 7.07 Hz, H-7), 8.08 (1H, d, J= 7.8 Hz, H-5).
General method for synthesis of 2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-
3
(4H)-yl) acetic acid (4):
A solution of NaOH (0.115 mole) in least amount of water (2ml) was
added to a solution of ethyl 2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-

ACCEPTED MANUSCRIPT
3
(4H)-yl) acetate (3, 0.0115 mole) in methanol (50 ml). The mixture was
refluxed for 2 h, then cooled and acidified with 3N HCl to pH~ 5. The formed
precipitate was filtered and washed with water and dried to obtain 2-(1-methyl-
2
,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl) acetic acid (4) in 62.5% yield in
sufficient purity to be used in the next step.
2
-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl) acetic acid (4): (62.50%
1
yield). M. p. 240-243 °C. H NMR (DMSO, d ) δ 3.55 (3H, s, NCH ), 4.63 (2H,
6
3
s, NCH ), 7.35 (1H, t, J= 7.6 Hz, H-6), 7.52 (1H, d, J= 8.4 Hz, H-8), 7.83 (1H,
2
t, J= 8.4 Hz, H-7), 8.08 (1H, d, J= 7.8 Hz, H-5), 13.06 (1H, s, COOH).
General method for synthesis of 3-(2-(1H-benzo[d][1,2,3]triazol-1-yl)-2-
oxoethyl)-1-methylquinazoline-2,4(1H,3H)-dione (5):
To a solution of 1H-benzotriazole (0.032 mole) in anhydrous
dichloromethane, thionylchloride (0.013 mole) was added and the reaction
mixture was stirred at room temperature for 30 min, then the 2-(1-methyl-2,4-
dioxo-1,2-dihydroquinazolin-3(4H)-yl) acetic acid (4, 0.0108 mole) was added
and the reaction mixture stirred at room temperature for further 4 h. The
precipitate formed was filtered off and the organic layer was extracted twice
with 5% Na CO solution, washed with water and dried over anhydrous sodium
2
3
sulphate then the solvent was removed in vacuum to obtain the desired 3-(2-
(
1H-benzo [d] [1,2,3] triazol-1-yl) -2-oxoethyl) -1-methylquinazoline-2,4
1H,3H)-dione (5) in 82.2 % yield (184-6ᵒC m. p.) and sufficient purity for use
(
in the next step.
3
2
3
-(2-(1H-benzo[d][1,2,3]triazol-1-yl)-2-oxoethyl)-1-methylquinazoline-
1
,4(1H,3H)-dione (5): (82.20% yield). M.p. 184-186°C. H NMR (CDCl ) δ
3
.69 (3H, s, NCH ), 5.32 (2H, s, NCH ), 7.31-7.37 (2H, m, ArH), 7.56 (1H, t,
3
2
J= 7.7 Hz, ArH), 7.69 (1H, t, J= 7.7 Hz, ArH), 7.78 (1H, t, J= 7.8 Hz, ArH),
8
,
.20 (1H, d, J= 8.2 Hz, ArH), 8.26 (1H, d, J= 8.2 Hz, ArH), 8.29 (1H, dd, J=7.9
1.6 Hz, ArH).

ACCEPTED MANUSCRIPT
General method for synthesis of (2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-
3
(4H)-yl) acetamido) acids) (6 a-m), (7):
A solution of N-acylbenzotriazole derivative (5, 0.005 mole) and the
appropriate amino acid (0.005 mole) in acetonitrile/water mixture (3:1)
containing triethylamine (0.0075 mole) was stirred at room temperature for 24
h, then acetonitrile was evaporated under reduced pressure and the residual
water solution was then acidified with 1N HCl to pH~5 and the resultant
precipitate was then filtered, dried and crystalized from methanol to give the
titled compounds (6 a-m and 7) in 32.18-78.47 % yield.
2
-(2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)acetamido)acetic acid
1
(
6a): (32.18% yield). M.p. 250-253°C. H NMR (DMSO, d ) δ 3.53 (3H, s,
6
NCH ), 3.79 (2H, d, J= 6 Hz, NHCH ), 4.61 (2H, s, NCH ), 7.34 (1H, t, J= 7.6
3
2
2
Hz, H-6), 7.51 (1H, d, J= 8.4 Hz, H-8), 7.82 (1H, t, J= 7.2 Hz, H-7), 8.05 (1H,
dd, J= 7.6 Hz, H-5), 8.49 (1H, t, J= 5.6 Hz, CONH), 12.60 (1H, bs, COOH).
1
3
C NMR (DMSO, d + CDCl , d) δ 30.85 (NCH ), 41.13 (NHCH ), 43.76
6
3
3
2
(
NCH ), 114.04 (ArCH), 115.29 (ArCH), 122.93 (ArCH), 128.56 (ArCH),
2
1
35.42 (ArC), 140.69 (ArC), 150.68 (ArC), 161.41 (ArC), 167.51 (ArC), 171.34
+
(
ArC). MS, m/z: 291 (M ). Analysis calcd. for C H N O : C, 53.61; H, 4.50;
1
3
13
3
5
N, 14.43. Found: C, 53.52; H, 4.62; N, 14.73.
3
-(2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)acetamido)propanoic
1
acid (6b): (76.92% yield). M.p. 231-233 °C. H NMR (DMSO, d ) δ 2.39 (2H, t,
6
J= 6.8 Hz, CH CO), 3.27 (2H, t, J= 6.8 Hz, NCH CH CO), 3.53 (3H, s, NCH ),
2
2
2
3
4
.52 (2H, 2H, s, NCH ), 7.34 (1H, t, J= 7.2 Hz, H-6), 7.51 (1H, d, J= 8.4 Hz,
2
H-8), 7.82 (1H, t, J= 7.2 Hz, H-7), 8.05 (1H, dd, J= 8 Hz, H-5), 8.22 (1H, t, J=
1
3
5
3
.6 Hz, CONH), 12.24 (1H, s, COOH), C NMR (DMSO, d + CDCl , d) δ
6
3
0.86 (NCH ), 34.14 (CH CO), 35.27 (NCH CH CO), 43.89 (NCH ), 114.17
3
2
2
2
2

ACCEPTED MANUSCRIPT
(ArCH), 115.33 (ArCH), 122.92 (ArCH), 128.48 (ArCH), 135.43 (ArC), 140.73
(ArC), 150.68 (ArC), 161.40 (ArC), 167.19 (ArC), 173.37 (ArC). MS, m/z: 306
+
(
M ). Analysis calcd. for C H N O : C, 55.08; H, 4.95; N, 13.76. Found: C,
14
15
3
5
5
5.47; H, 5.03; N, 14.03.
4
-(2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)acetamido)butanoic
1
acid (6c): (75.79% yield). M.p. 273-275°C. H NMR (DMSO, d ) δ 1.60-1.66
6
(
2H, m, NHCH CH CH ), 2.23 (2H, t, J= 7.6 Hz, NHCH CH CH ), 3.05-3.10
2
2
2
2
2
2
(
2H, m, NHCH CH CH ), 3.54 (3H, s, NCH ), 4.52 (2H, s, NCH ), 7.34 (1H, t,
2
2
2
3
2
J= 7.6 Hz, H-6), 7.49 (1H, d, J= 8.4 Hz, H-8), 7.82 (1H, t, J= 7.2 Hz, H-7),
.05 (1H, dd, J= 8 Hz, H-5), 8.12 (1H, t, J= 6.5 Hz, CONH), 12.05 (1H, bs,
8
1
3
COOH), C NMR (DMSO, d + CDCl , d) δ 24.81 (NHCH CH CH ), 30.85
6
3
2
2
2
(
NCH ), 31.44 (NHCH CH CH ), 38.63 (NHCH CH CH ), 43.99 (NCH ),
3
2
2
2
2
2
2
2
1
1
14.02 (ArCH), 115.41 (ArCH), 122.89 (ArCH), 128.55 (ArCH), 135.37 (ArC),
40.73 (ArC), 150.64 (ArC), 161.49 (ArC), 167.18 (ArC), 174.87 (ArC). MS,
m/z: 319 (M+). Analysis calcd. for C H N O : C, 56.42; H, 5.37; N, 13.16.
15
17
3
5
Found: C, 56.90; H, 5.50; N, 13.42.
2
-(2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)acetamido)propanoic
1
acid (6d): (54.95% yield). M.p. 245-247°C. H NMR (DMSO, d ) δ 1.29 (3H,
6
d, J= 7.6, CHCH ), 3.53 (3H, s, NCH ), 4.19-4.27 (1H, m, CHCH ), 4.53-4.65
3
3
3
(
2H, m, NCH ), 7.34 (1H, t, J= 7.2 Hz, H-6), 7.50 (1H, d, J= 8.8 Hz, H-8), 7.82
2
(1H, t, J= 7.2 Hz, H-7), 8.05 (1H, dd, J= 8 Hz, H-5), 8.50 (1H, d, J= 7.2 Hz,
1
3
CONH), 12.63 (1H, bs, COOH). C NMR (DMSO, d + CDCl , d) δ 18.15
6
3
(
CHCH ), 30.83 (NCH ), 43.77 (NCH ), 48.08 (CHCH ), 113.88 (ArCH),
3
3
2
3
1
1
15.25 (ArCH), 122.92 (ArCH), 128.65 (ArCH), 135.37 (ArC), 140.62 (ArC),
50.71 (ArC), 161.45 (ArC), 166.75 (ArC), 174.47 (ArC). MS, m/z: 306 (M+).
Analysis calcd. for C H N O : C, 55.08; H, 4.95; N, 13.76. Found: C, 54.96;
1
4
15
3
5
H, 5.11; N, 14.09.

ACCEPTED MANUSCRIPT
2
-(2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)acetamido)butanoic
1
acid (6e): (71.49% yield). M.p. 258-259°C. H NMR (DMSO, d ) δ 0.90 (3H, t,
6
J= 7.4, CH CH ), 1.56-1.67 (1H, m, CH CH ), 1.71-1.78 (1H, m, CH CH ),
2
3
2
3
2
3
3
7
.53 (1H, s, NCH ), 4.14-4.20 (1H, m, NHCHCO), 4.57-4.67 (2H, m, NCH ),
3
2
.33 (1H, t, J= 7.2 Hz, H-6), 7.50 (1H, d, J= 8.8 Hz, H-8), 7.82 (1H, t, J= 7.2
Hz, H-7), 8.05 (1H, dd, J= 8 Hz, H-5), 8.44 (1H, d, J= 8 Hz, CONH), 12.62
1
3
(
1H, bs, COOH). C NMR (DMSO, d + CDCl , d) δ 9.97 (CH CH ), 25.43
6
3
2
3
(
CH CH ), 30.82 (NCH ), 43.78 (NCH ), 53.51 (NHCHCO), 113.86 (ArCH),
2
3
3
2
1
1
15.28 (ArCH), 122.90 (ArCH), 128.66 (ArCH), 135.35 (ArC), 140.64 (ArC),
+
50.72 (ArC), 161.45 (ArC), 166.97 (ArC), 173.83 (ArC). MS, m/z: 321 (M ).
Analysis calcd. for C H N O : C, 56.42; H, 5.37; N, 13.16. Found: C, 56.79;
1
5
17
3
5
H, 5.23 N, 13.58.
3
-methyl-2-(2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)acetamido)
1
butanoic acid (6f): (78.47% yield). M.p. 241-243°C. H NMR (DMSO, d ) δ
6
0
.90 (6H, dd, J= 6.8 Hz, CH(CH ) ), 2.02-2.09 (1H, m, CH(CH ) ), 3.53 (3H, s,
3
2
3 2
NCH ), 4.16-4.20 (1H, m, NHCHCO), 4.61-4.70 (2H, m, NCH ), 7.33 (1H, t,
3
2
J= 7.6 Hz, H-6), 7.50 (1H, d, J= 8.4 Hz, H-8), 7.82 (1H, t, J= 7.2 Hz, H-7),
.05 (1H, dd, J= 7.6 Hz, H-5), 8.40 (1H, d, J= 8.4 Hz, CONH), 12.68 (1H, bs,
8
1
3
COOH), C NMR (DMSO, d + CDCl , d) δ 17.97 (CH(CH ) ), 19.22
6
3
3 2
(
CH(CH ) ), 30.81 (NCH ), 31.07 (CH(CH ) ), 43.78 (NCH ), 57.40
3
2
3
3 2
2
(NHCHCO), 113.85 (ArCH), 115.27 (ArCH), 122.88 (ArCH), 128.66 (ArCH),
1
35.33 (ArC), 140.64 (ArC), 150.72 (ArC), 161.45 (ArC), 167.16 (ArC), 173.41
+
(
ArC). MS, m/z: 334 (M ). Analysis calcd. for C H N O : C, 57.65; H, 5.75;
1
6
19
3
5
N, 12.61. Found: C, 57.22; H, 5.68; N, 12.97.
4
-methyl-2-(2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)acetamido)
1
pentanoic acid (6g): (39.22% yield). M.p. 213-215°C. H NMR (DMSO, d ) δ
6
0
.86 (3H, d, J= 6.5, CH(CH ) ), 0.92 (3H, d, J= 6.6 Hz, CH(CH ) ), 1.52 (2H, t,
3
2
3 2
J= 7.6 Hz, CHCH CH), 1.63-1.69 (1H, m, CH(CH ) ), 3.53 (3H, s, NCH ),
2
3 2
3

ACCEPTED MANUSCRIPT
4
.22-4.28 (1H, m, NHCHCO), 4.55-4.64 (2H, m, NCH ), 7.33 (1H, t, J= 7.6
2
Hz, H-6), 7.50 (1H, d, J= 8.4 Hz, H-8), 7.82 (1H, t, J= 8.4 Hz, H-7), 8.05 (1H,
1
3
dd, J= 8 Hz, H-5), 8.43 (1H, d, J= 8 Hz, CONH), 12.54 (1H, bs, COOH). C
NMR (DMSO, d + CDCl , d) δ 21.93 (CH(CH ) ), 23.04 (CH(CH ) ), 24.68
6
3
3 2
3 2
(
CH(CH ) ), 30.83 (NCH ), 41.22 (CHCH CH), 43.70 (NCH ), 50.83
3
2
3
2
2
(NHCHCO), 113.97 (ArCH), 115.30 (ArCH), 122.88 (ArCH), 128.58 (ArCH),
1
35.36 (ArC), 140.68 (ArC), 150.68 (ArC), 161.41 (ArC), 167.03 (ArC), 174.49
+
(
ArC). MS, m/z: 348 (M ). Analysis calcd. for C H N O : C, 58.70; H, 6.09;
1
7
21
3
5
N, 12.10. Found: C, 59.01; H, 6.21; N, 12.37.
3
-hydroxy-2-(2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)acetamido)
1
propanoic acid (6h): (38.23% yield). M.p. 227-230°C. H NMR (DMSO, d ) δ
6
3
4
.53 (3H, s, NCH ), 3.60-3.64 (1H, m, CH OH), 3.69-3.73 (1H, m, CH OH),
3
2
2
.28-4.32 (1H, m, NHCHCO), 4.61-4.71 (2H, m, NCH ), 7.34 (1H, t, J= 7.6
2
Hz, H-8), 7.48 (1H, d, J= 8.4 Hz, H-6), 7.82 (1H, t, J= 7.2 Hz, H-7), 8.05 (1H,
1
3
dd, J= 7.6 Hz, H-5), 8.42 (1H, d, J= 7.6 Hz, CONH), 12.59 (1H, s, COOH), C
NMR (DMSO, d + CDCl , d) δ 30.85 (NCH ), 43.84 (NCH ), 54.98
6
3
3
2
(
NHCHCO), 62.39 (CH OH), 114.06 (ArCH), 115.25 (ArCH), 122.97 (ArCH),
2
1
1
28.58 (ArCH), 135.44 (ArC), 140.69 (ArC), 150.71 (ArC), 161.41 (ArC),
+
67.13 (ArC), 174.14 (ArC). MS, m/z: 321 (M ). Analysis calcd. for
C H N O : C, 52.34; H, 4.71; N, 13.08. Found: C, 52.57; H, 4.84; N, 13.29.
1
4
15
3
6
2
-(2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)acetamido)-4-
1
(
(
(
(
methylthio)butanoic acid (6i): (41.82% yield). M.p. 229-231°C. H NMR
DMSO, d ) δ 1.80-1.91 (1H, m, CHCH ), 1.95-1.98 (1H, m, CHCH ), 2.05
6
2
2
3H, s, SCH ), 2.51 (2H, t, J= 6 Hz, CH CH S), 3.54 (3H, S, NCH ), 4.33-4.38
3
2
2
3
1H, m, NHCHCO), 4.60 (2H, s, NCH ), 7.34 (1H, t, J= 7.6 Hz, H-6), 7.51 (1H,
2
d, J= 8.4 Hz, H-8), 7.82 (1H, t, J= 8.4 Hz, H-7), 8.05 (1H, d, J= 7.6 Hz, H-5),
1
3
8
.47 (1H, d, J= 8 Hz, CONH), 12.72 (1H, bs, COOH), C NMR (DMSO, d +
6
CDCl , d) δ 15.30 (SCH ), 30.09 (CH CH S), 30.82 (NCH ), 31.84 (CHCH ),
3
3
2
2
3
2

ACCEPTED MANUSCRIPT
4
3.83 (NCH ), 51.52 (NHCHCO), 113.89 (ArCH), 115.30 (ArCH), 122.90
2
(ArCH), 128.63 (ArCH), 135.36 (ArC), 140.65 (ArC), 150.69 (ArC), 161.44
+
(
ArC), 167.20 (ArC), 173.44 (ArC). MS, m/z: 365 (M ). Analysis calcd. for
C H N O S: C, 52.59; H, 5.24; N, 11.50. Found: C, 52.41; H, 5.36; N, 11.54.
1
6
19
3
5
2
-(2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)acetamido)-3-
1
phenylpropanoic acid (6j): (65.45% yield). M.p. 238-241°C. H NMR (DMSO,
d ) δ 2.89-2.94 (1H, m, CHCH ), 3.01-3.06 (1H, m, CHCH ), 3.53 (3H, s,
6
2
2
NCH ), 4.39-4.44 (1H, m, CH-CH ), 4.54-4.62 (2H, m, NCH ), 7.25 (3H, d, J=
3
2
2
7
.2 Hz, ArH), 7.28-7.35 (3H, m, ArH), 7.50 (1H, d, J= 8 Hz, H-8), 7.81 (1H, t,
J= 7.2 Hz, H-7), 8.04 (1H, dd, J= 7.6 Hz, H-5), 8.54 (1H, d, J= 8 Hz, CONH),
1
3
1
2.77 (1H, s, COOH). C NMR (DMSO, d + CDCl , d) δ 30.83 (NCH ), 37.46
6
3
3
(
CHCH ), 43.84 (NCH ), 53.70 (NHCHCH ), 113.91 (ArCH), 115.27 (ArCH),
2
2
2
1
22.92 (ArCH), 126.63 (ArCH), 128.23 (ArCH), 128.68 (ArCH), 129.62
(ArCH), 135.38 (ArC), 136.85 (ArC), 140.64 (ArC), 150.68 (ArC), 161.41
+
(
ArC), 166.90 (ArC), 172.88 (ArC). MS, m/z: 381 (M ). Analysis calcd. for
C H N O : C, 62.99; H, 5.02; N, 11.02. Found: C, 62.56; H, 4.70; N, 11.21.
2
0
19
3
5
3
-(1H-indol-3-yl)-2-(2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)
1
acetamido)propanoic acid (6k): (69.84% yield). M.p. 237-239°C. H NMR
(
(
(
DMSO, d ) δ 3.03-3.08 (1H, m, CHCH ), 3.15-3.20 (1H, m, CHCH ), 3.54
6
2
2
3H, s, NCH ), 4.47-4.52 (1H, m, NHCHCO), 4.57-4.66 (2H, m, NCH ), 6.99
3
2
1H, t, J= 7.6 Hz, ArH), 7.07 (1H, t, J= 8 Hz, ArH), 7.17 (1H, d, J= 2.4 Hz,
CHNH), 7.34 (2H, t, J= 7.6 Hz, ArH), 7.49-7.55 (2H, m, ArH), 7.82 (1H, t, J=
8
1
.4 Hz, H-7), 8.06 (1H, dd, J= 7.6 Hz, H-5), 8.51 (1H, d, J= 7.6 Hz, CONH),
1
3
0.90 (1H, s, CHNH), 12.70 (1H, bs, COOH). C NMR (DMSO, d + CDCl ,
6
3
d) δ 27.48 (CHCH ), 30.83 (NCH ), 43.91 (NCH ), 53.33 (NHCHCO), 109.51
2
3
2
(ArCH), 111.38 (ArCH), 113.92 (ArCH), 115.26 (ArCH), 118.69 (ArCH),
1
1
18.84 (ArCH), 121.21 (ArCH), 122.91 (ArCH), 123.95 (ArCH), 127.81 (ArC),
28.65 (ArC), 135.36 (ArC), 136.31 (ArC), 140.64 (ArC), 150.71 (ArC), 161.44

ACCEPTED MANUSCRIPT
ArC), 166.87 (ArC), 173.40 (ArC). MS, m/z: 420 (M ). Analysis calcd. for
C H N O : C, 62.85; H, 4.79; N, 13.33. Found: C, 63.13; H, 4.68; N, 13.52.
+
(
2
2
20
4
5
3
-(1H-imidazol-5-yl)-2-(2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-
1
yl)acetamido)propanoic acid (6l): (43.63% yield). M.p. 185-188°C. H NMR
(
DMSO, d ) δ 3.06-3.12 (1H, m, CHCH ), 3.21-3.26 (1H, m, CHCH ), 3.52
6
2
2
(
3H, s, NCH ), 4.63-4.71 (3H, m, NHCHCO+ NCH ), 7.19-7.25 (3H, m, H-6 +
3
2
H-8 +CCHN), 7.66 (1H, t, J= 6.8 Hz, H-5), 8.05 (1H, dd, J= 7.6 Hz, H-5), 8.52
1H, d, J= 8 Hz, CONH), 8.66 (1H, d, J= 6.8 Hz, NHCHN), 11.19 (1H, s,
(
1
3
COOH), 14.42 (1H, bs, NHCHN), C NMR (DMSO, d6) δ 26.77 (NCH ),
3
3
0.92 (CHCH ), 43.86 (NCH ), 51.62 (CHCH ), 114.08 (ArCH), 115.23
2
2
2
(ArCH), 117.16 (ArCH), 122.99 (ArCH), 128.55 (ArCH), 129.45 (ArCH),
1
32.91 (ArC), 135.47 (ArC), 140.64 (ArC), 150.63 (ArC), 161.39 (ArC), 167.48
+
(
ArC), 171.91 (ArC). MS, m/z: 371 (M ). Analysis calcd. for C H N O : C,
1
7
17
5
5
5
4.98; H, 4.61; N, 18.86. Found: C, 55.12; H, 4.73; N, 18.49.
1
-(2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)acetyl)pyrrolidine-2-
1
carboxylic acid (6m): (34.69% yield). M.p. 217-219°C. H NMR (DMSO, d ) δ
6
1
2
.86-1.93 (1H, m, NCHCH CH CH ), 1.98-2.03 (2H, m, NCHCH CH CH ),
2
2
2
2
2
2
.15-2.18 (1H, m, NCHCH CH CH ), 3.54 (1H, s, NCH ), 3.64-3.74 (2H, m,
2
2
2
3
NCHCH CH CH ), 4.24 (1H, dd, J= 8.8 Hz, NCHCH CH CH ), 4.66-4.90 (2H,
2
2
2
2
2
2
m, NCH CO), 7.34 (1H, t, J= 7.5 Hz, H-6), 7.49 (1H, d, J= 8.4 Hz, H-8), 7.83
2
(1H, t, J= 7.2 Hz, H-7), 8.05 (1H, dd, J= 7.8 Hz, H-5), 12.51 (1H, bs, COOH),
1
3
C NMR (CDCl , d) δ 24.84 (NCHCH CH CH ), 27.76 (NCHCH CH CH ),
3
2
2
2
2
2
2
3
0.96 (NCH3), 42.95 (NCH2), 46.85 (NCHCH CH CH ), 60.00
2
2
2
(
NCHCH CH CH ), 113.75 (ArCH), 115.20 (ArCH), 123.19 (ArCH), 129.19
2
2
2
(ArCH), 135.49 (ArC), 140.63 (ArC), 150.79 (ArC), 161.58 (ArC), 167.81
+
(
ArC), 173.09 (ArC). MS, m/z: 331 (M ). Analysis calcd. for C H N O : C,
1
6
17
3
5
5
8.00; H, 5.17; N, 12.68. Found: C, 58.34; H, 5.54; N, 12.63.

ACCEPTED MANUSCRIPT
2
-(2-(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)acetamido)-3-(4-(2-
(1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)acetoxy)phenyl)propanoic
1
acid (7): (34.49% yield). M.p. 250-253°C. H NMR (DMSO, d ) δ 2.90-2.96
6
(
(
(
1H, m, NHCHCH ), 3.02-3.06 (1H, m, NHCHCH ), 3.55 (3H, s, NCH ), 3.59
2
2
3
3H, s, NCH ), 4.30-4.43 (1H, m, NHCHCH ), 4.58 (2H, s, NCH CONH), 5.01
3
2
2
2H, s, NCH COO), 7.08 (2H, d, J= 8.4 Hz, ArH), 7.28-7.40 (4H, m, ArH),
2
7
.49 (1H, d, J= 8.8 Hz, ArH), 7.55 (1H, d, J= 8.4 Hz, ArH), 7.81 (1H, t, J= 8.8
Hz, ArH), 7.86 (1H, t, J= 7.2 Hz, ArH), 8.06 (1H, dd, J= 8.6 Hz, ArH), 8.13
1H, dd, J= 7.6 Hz, ArH), 8.53 (1H, d, J= 8 Hz, CONH), 12.83 (1H, bs,
(
1
3
COOH). C NMR (DMSO, d6) δ 31.08 (NCH ), 31.26 (NCH ), 36.54
3
3
(
NHCHCH ), 43.13 (NCH COO), 43.73 (NCH CONH), 54.07 (NHCHCH ),
2
2
2
2
1
14.79 (ArCH), 115.09 (ArCH), 115.45 (ArCH), 121.58 (ArCH), 123.31
(ArCH), 123.70 (ArCH), 128.31 (ArCH), 128.38 (ArCH), 130.90 (ArCH),
1
1
35.80 (ArCH), 135.93 (ArCH), 136.37 (ArCH), 140.85 (ArC), 149.21 (ArC),
50.50 (ArC), 150.62 (ArC), 161.28 (ArC), 161.34 (ArC), 167.03 (ArC), 167.51
+
(
ArC), 172.97 (ArC). MS, m/z: 613 (M ). Analysis calcd. for C H N O : C,
3
1
27
5
9
6
0.68; H, 4.44; N, 11.41. Found: C, 60.89; H, 4.75; N, 11.84.
4
.2. Biology:
.2.1. Extraction of chitin synthase [25]:
4
A single colony of Saccharomyces cerevisiae (CGMCC2.145) was inserted
into of YPAD liquid medium, and cultured at 30°C for 48 h with oscillating at
5 rpm. The yeast cells were collected through centrifugation at 1500 g for 15
6
min at 4 °C and discard the supernatant. The pellet was lysed with sonication
treatment by being suspended in 20 mL 100mM Tris-HCl media at pH7.0 which
contained 40µL fungal protease inhibitor cocktail and 50µL solution of 200mM
Phenylmethanesulfonyl fluoride. Cell wall and fragment were removed by
centrifugation at 1500 g for 15 min at 4°C and the supernatant was carefully
aspirated and placed in a 2 volume of 10% (w / w) sucrose in 100 mM Tris–HCl

ACCEPTED MANUSCRIPT
at pH 7.5 buffer and then centrifuged at 55000 g for 2 h at 4 °C. After
centrifugation, the pellet was re-suspended in 1.6 mL/g Cytoplasmic matrix (50
mM Tris–HCl at pH 7.0 and 33% glycerol) to serve as CHS sample stock
solution which could be diluted with 50 mM Tris-HCl buffer at pH 7.5 to get a
series of enzyme diluent (1:1, 1:2, 1:4, 1:8, 1:16).
4
.2.2. Determination the activity of Chitin Synthase:
2
00 µL 50 µg/mL WGA buffer solutions in 50 mM Tris–HCl at pH 7.5 was
added to each microtiter plate of 96-well plates and oscillation cultured at 37 °C
for 2 h. The plates were flushed and washed three times with 50 mM Tris-HCl
buffer at pH 7.5, and then 300µL of 10 mg/mL borine serum albumin (50 mM
Tris-HCl at pH7.5) buffer solution was added, oscillation incubated for 2 h at 37
°
C, which could be stored at -20°C. The plates were emptied and washed at
least three times with 50 mM Tris–HCl buffer at pH 7.5. Reaction substrate
100 µL ,40 mM GlcNAc, 2mM UDP–GlcNAc, in 0.1M Tris-Maleic acid buffer
at pH 6.8), 50µL CHS diluent and 50 µL of 0.1M Tris- Maleic acid buffer of pH
.8 were added to appropriate wells. The plates were incubated at 37 °C for 1 h.
(
6
The wells were emptied and washed three times with 50 mM Tris–HCl buffer at
pH 7.5. Each well was added 200 µL of 50 mM pH 7.5 in Tris-HCl buffer with
1
µg/mL WGA-HRP and incubated at 37 °C for 15 min with shaking, and then
washed five times with 50 mM Tris–HCl buffer at pH 7.5. Finally, 150 µL
peroxidase substrate solutions (0.8 mM TMB, 2 mM H O , 50 mM Na HPO -
2
2
2
4
Citric acid buffer at pH3.7) was added for lucifuge reaction time of 30 min at 25
C. The reaction was stopped with 50 µL 2M H SO and measured with Tecan
°
2
4
Infinite 200 PRO Microplate reader at 450 nm. Draw the curve of concentration
with (OD -B ), and finally determine activity of the CHS.
0
0
[
OD : Blank absorbance without inhibitor, substrate, and CHS.
0
B : Add CHS and substrate, without inhibition of HRP absorbance.]
0

ACCEPTED MANUSCRIPT
4
.2.3. Determination of inhibitors IC assay:
50
The stock solution of test compound was prepared by dissolving 10 mg of
the test compound in 1 mL of DMSO solution. The stock solution was diluted
to 300, 150, 75, 37.5, 18.75 µg/mL with 50mM Tris–HCl buffer at pH 7.5 as
test solutions.
Plates stored at −20 °C were thawed at room temperature, emptied by
pipettes and washed three times with 50 mM Tris–HCl buffer at pH 7.5.
Reaction substrate (100 µL ,40 mM GlcNAc, 2mM UDP–GlcNAc, in 0.1M
Tris-Maleic acid buffer at pH 6.8), 50µL test solution of compound and 50 µL
diluted solution of CHS were added to appropriate wells. The positive control
was added with 50 µL 0.1 M Tris-maleic acid buffer at pH 6.8 and 50 µL of
CHS diluted solution. Blank control was added with 100 µL 0.1 M Tris- Maleic
acid buffer at H 6.8. The plates were incubated at 37 °C for 1 h. The followed
steps were the same as determination the activity of Chitin Synthase. Inhibition
rate = (B -B )/(B -OD . The curve of log c and inhibition rate is plotted by
o
n
0
0)
IC .
5
0
B : Add enzyme and substrate, without inhibition of HRP absorbance.
0
B : Absorbance of HRP at a certain concentration
n
OD : Blank absorbance without inhibitor, substrate, and enzyme.
0
4
.2.4. Antifungal activity assays [6]:
Antifungal activity was screened against four main pathogenic fungal species
(
C. albicans CMCC 76615, A. fumigatus GIMCC 3.19, C. neoformans ATCC
3
2719 and A. flavus ATCC 16870) in clinic. Fluconazole and polyoxin B used
as standard antifungal drugs. DMSO was used as a solvent control. A spore
suspension in sterile distilled water was prepared from 1-day old culture of the
fungi growing on the media containing 1% peptone, 2% Glucose and solid

ACCEPTED MANUSCRIPT
media as well as 15% agar. The final spore concentration was 1-5 ×10 spore
3
-1
mL All target compounds were dissolved in DMSO to prepare the stock
solutions. The tests were made resulting in twelve wanted concentrations (0.25-
5
12 mg/mL). These dilutions were incubated at 37 °C for 24 h. The MIC values
of antifungal activity in mg/mL were summarized in Table 2.
4
.2.5. The checkboard synergy analysis[26, 27]:
Two-dimensional broth microdilution checkboard assay between 6m and
7
with fluconazole was carried out according to CLSI M27-A3 guidelines[28].
Each test was performed twice using 96-well plates. The plates were incubated
for 48 hrs. at 35°C. It is important to say that the MIC values showed in Table. 3
were determined again in this investigation to allow fair comparison.
Using the obtained data, the FIC index for each combination was
calculated. FIC index is calculated using the formula: FIC index = [(MIC in
A
combination)/ MIC alone] + [(MIC in combination)/ MIC alone)]. Drug
A
B
B
interactions were classified as synergistic if the FIC index was ≤0.5, indifferent
if >0.5–4, and antagonistic if >4.
Declaration of interest
The authors report no declarations of interest.
Funding:
This research did not receive any specific grant from funding agencies in
the public, commercial, or not-for-profit sectors
.
References:

ACCEPTED MANUSCRIPT
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Tables
Design, Synthesis and Biological Evaluation of Novel Quinazoline-
2
,4-diones Conjugated with Different Amino Acids as Potential
Chitin Synthase Inhibitors
a
a*1
a
Nada A. Noureldin *, Hend Kothayer , EL-Sayed M. Lashine , Mohamed M.
a
b
b
b
Baraka , Yanrong Huang , Bing Li and Qinggang Ji .
a
Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, Egypt.
b
School of chemistry and chemical engineering, Southwest University, P.R.China
1
Corresponding author. Tel. 00201224578259; E-mail: hendo1311@hotmail.com or
hkelhamalawy@pharmacy.zu.edu.eg
equal contribution
*

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COMP.
IP（%） IC (mmol/L) COMP.
IP
IC (mmol/L)
5
0
50
（
%）
6
6
6
6
6
6
6
6
a
b
c
d
e
f
22.6
47.5
68.2
47.8
13.2
18.5
52.8
56.2
-
-
6i
6j
6k
6l
6m
7
43.1
50.5
43.8
61.5
75.5
65.3
-
-
-
0.30
-
-
-
-
0.30
0.18
0.166
0.17
g
h
Polyoxin B 63.9
Table. 1. The inhibition percent (IP) of compounds at concentration of
3
00µg/mL and IC values of some compounds.
50
Compound
Aspergillus
fumigates
Aspergillus
flavus
Crytococcus
neoformans
Candida
albicans
6
6
6
6
6
6
6
6
6
6
6
6
6
7
a
b
c
d
e
f
>1.75
0.83
0.80
0.83
0.80
0.76
0.73
0.79
1.40
>1.34
>1.21
1.37
0.77
0.41
0.05
0.87
0.87
0.83
0.80
0.83
0.80
0.76
>1.47
>1.59
0.70
>1.34
0.60
0.68
0.77
0.41
0.10
>1.75
1.67
1.67
1.60
1.60
0.83
1.67
1.60
>1.60
>1.53
>1.47
>1.59
>1.40
>1.34
>1.21
>1.37
>1.54
0.83
>1.53
>1.47
1.59
g
h
i
>1.40
>1.34
0.60
j
k
l
>1.37
1.54
m
0.41
Fluconazole
0.10
0.20
Polyoxin B
0.12
0.12
0.06
0.12
Table. 2. The MIC value（mmol/L）of compounds 6a-m and 7 against fungi in
vitro.

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a
MIC(mmol/L)
b
FIC
Index
FIC Index
(7)
Alone
7
In combination
Fluconazole
b
fungi
6m
fluconazole
0.104
6m
7
Fluconazole
(with 7)
0.013
(
6m)
(with 6m)
Candida
albicans ATCC
0.773
0.417
0.386
0.417
0.013
0.625
0.3125
1.0625
1.5
1.125
0.625
7
6615
Aspergillus
flavus ATCC
0.773
1.545
0.773
0.417
0.834
0.417
0.104
0.209
0.104
0.193
1.545
0.386
0.209
0.209
0.026
0.007
0.013
0.104
0.026
0.052
0.052
1
6870
Aspergillus
fumigates
GIMCC 3.19
Crytococcus
neofonmans
ATCC 32719
0.3125
0.5625
Table 3. In vitro susceptibility of fungi to 6m, 7 and fluconazole alone and
in combination.
a
All experiments were performed in duplicate. b Fractional inhibitory concentration
index. Drug interactions were classified as synergistic if the FIC index was ≤0.5, indifferent
if >0.5–4, and antagonistic if >4. From the activity data.

ACCEPTED MANUSCRIPT
Figures and scheme
Design, Synthesis and Biological Evaluation of Novel Quinazoline-
2
,4-diones Conjugated with Different Amino Acids as Potential
Chitin Synthase Inhibitors
a
a*1
a
Nada A. Noureldin *, Hend Kothayer , EL-Sayed M. Lashine , Mohamed M.
a
b
b
b
Baraka , Yanrong Huang , Bing Li and Qinggang Ji .
a
Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, Egypt.
b
School of chemistry and chemical engineering, Southwest University, P.R.China
O
O
N
O
N
HO
NH
NH
OH
NH
HO
O
OH
O
COOH
CH
3
O
COOH
O
O
P
O
P
N
O
O
HO
HO
O
O
N
N
H
O
O
H
2
N
O
N
O
O
O
NHAc
H
OH OH
OH NH2
II)
OH NH
(III)
2
OH OH
OH OH
OH OH
(
(
I)
Figure. 1: (I) UDP-GlcNAc, (II) Polyoxin B, (III) Nikkomycin Z
O
N
OH
O
OH
O
H
H
N
N
HO
N
N
N
N
OCH3
OH CH3
O
O
OH
O
CH3
O
CH3
(
V)
(
IV)
Figure. 2: Non-nucleoside organic compounds designed to roughly
resemble UDP-GlcNAc.
1
Corresponding author. Tel. 00201224578259; E-mail: hendo1311@hotmail.com or
hkelhamalawy@pharmacy.zu.edu.eg
equal contribution
*

ACCEPTED MANUSCRIPT
CH3
CH3
O
R1
O
N
O
CN
Cl
N
N
O
R1= H or CH3
N
NH2
NH
N
N
O
N
O
N
O
N
O
H
H
H
RO-41-0986
RO-09-3024
RO-09-3143
Figure. 3: Some examples on non-nucleoside small organic compounds
with promising chitin synthase inhibitory activity.

ACCEPTED MANUSCRIPT
O
N
O
H
N
H
O
N
COOH
H
N
N
N
COOH
N
COOH
N
O
O
O
O
N
O
O
6c
6b
6
a
O
O
O
H
H
N
H
N
COOH
COOH
N
COOH
N
N
N
O
O
O
N
O
N
O
N
O
6f
6e
6d
O
O
N
O
H
N
H
N
H
N
COOH
COOH
OH
COOH
S
N
g
N
N
O
O
O
O
O
N
O
O
O
O
N
O
6
6h
6i
O
O
O
H
N
H
N
H
COOH
COOH
NH
N
COOH
N
N
N
H
N
O
O
N
O
N
N
O
N
6
j
6k
6l
HOOC
O
O
N
H
COOH
N
N
N
O
N
N
O
N
O
N
O
O
O
7
6m
Figure.4: The diverse structures of the synthesized compounds (6a-m and 7)