Pyrazolone-quinazolone hybrids: A novel class of human 4-hydroxyphenylpyruvate dioxygenase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.08.011

4-Hydroxyphenylpyruvate dioxygenase (HPPD), converting 4-hydroxyphenylpyruvate acid to homogentisate, is an important target for treating type I tyrosinemia and alkaptonuria due to its significant role in tyrosine catabolism. However, only one commercial drug, NTBC, also known as nitisinone, has been available for clinical use so far. Herein, we have elucidated the structure-based design of a series of pyrazolone-quinazolone hybrids that are novel potent human HPPD inhibitors through the successful integration of various techniques including computational simulations, organic synthesis, and biochemical characterization. Most of the new compounds displayed potent inhibitory activity against the recombinant human HPPD in nanomolar range. Compounds 3h and 3u were identified as the most potent candidates with K_ivalues of around 10 nM against human HPPD, about three-fold more potent than NTBC. Molecular modeling indicated that the interaction between the pyrazolone ring and ferrous ion, and the hydrophobic interaction of quinazolone with its surrounding residues, such as Phe347 and Phe364, contributed greatly to the high potency of these inhibitors. Therefore, compounds 3h and 3u could be potentially useful for the treatment of type I tyrosinemia and other diseases with defects in tyrosine degradation.

Full text of DOI:10.1016/j.bmc.2014.08.011

Bioorganic & Medicinal Chemistry 22 (2014) 5194–5211
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Pyrazolone–quinazolone hybrids: A novel class of human
4-hydroxyphenylpyruvate dioxygenase inhibitors
a,b,
Yu-Ling Xu a, , Hong-Yan Lin a, , Run-Jie Cao a, Ze-Zhong Ming a, Wen-Chao Yang a, , Guang-Fu Yang
⇑
⇑
a Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, PR China
b Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 30071, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
4-Hydroxyphenylpyruvate dioxygenase (HPPD), converting 4-hydroxyphenylpyruvate acid to homogen-
tisate, is an important target for treating type I tyrosinemia and alkaptonuria due to its significant role in
tyrosine catabolism. However, only one commercial drug, NTBC, also known as nitisinone, has been
available for clinical use so far. Herein, we have elucidated the structure-based design of a series of pyr-
azolone–quinazolone hybrids that are novel potent human HPPD inhibitors through the successful inte-
gration of various techniques including computational simulations, organic synthesis, and biochemical
characterization. Most of the new compounds displayed potent inhibitory activity against the recombi-
nant human HPPD in nanomolar range. Compounds 3h and 3u were identified as the most potent candi-
dates with Ki values of around 10 nM against human HPPD, about three-fold more potent than NTBC.
Molecular modeling indicated that the interaction between the pyrazolone ring and ferrous ion, and
the hydrophobic interaction of quinazolone with its surrounding residues, such as Phe347 and Phe364,
contributed greatly to the high potency of these inhibitors. Therefore, compounds 3h and 3u could be
potentially useful for the treatment of type I tyrosinemia and other diseases with defects in tyrosine
degradation.
Received 18 July 2014
Revised 7 August 2014
Accepted 8 August 2014
Available online 18 August 2014
Keywords:
Human HPPD inhibitors
Type I tyrosinemia
Pyrazolone
Quinazolones
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
tyrosinemia, type II tyrosinemia, type III tyrosinemia, hawkinsinu-
ria, and alkaptonuria.1,6–8 The most severe defect of tyrosine
The Fe(II)-dependent non-heme oxygenase, 4-hydroxyphenyl-
pyruvate dioxygenase (HPPD; E.C. 1.13.11.27) with a subunit mass
of 40–50 kDa has both agricultural and therapeutic significance
due to its catalytic role in the transformation of 4-hydroxyphenyl-
pyruvate acid (HPPA) to homogentisate (HGA).1–4 This conversion
in the presence of oxygen and ferrous ion is the second step of
the catabolic pathway, with tyrosine as the starting material
(Scheme 1). In plants, the HGA produced in this catabolic pathway
is an important aromatic precursor for the biosynthesis of the
plastoquinone and tocopherol, which are essential cofactors for
the normal growth of plants.5 Thus, in the past thirty years, a series
of particular inhibitors targeting HPPD have been discovered as
potent herbicides, due to their ability to block HPPA–HGA conver-
sion, thereafter causing the intense bleaching and even death of
weeds.
catabolism is type I tyrosinemia, attributed to the deficiency of
fumarylacetoacetase. The metabolites from the previous two steps,
fumarylacetoacetate and maleylacetoacetate, could be easily
transformed to succinylacetone and induce the symptoms that
cause type I tyrosinemia patients to suffer from liver dysfunction
and renal proximal tubular failure and to incur a high rate of mor-
tality. Type II tyrosinemia usually results in mild mental retarda-
tion at birth and corneal opacities due to the deficiency of
tyrosine aminotransferase. Type III tyrosinemia caused by the defi-
ciency of active HPPD is very similar to type II tyrosinemia in the
clinical phenotype. Hawkinsinuria is believed to be the result of a
gene mutation in HPPD resulting in the conversion of HPPA to an
alternative product that is very reactive and forms covalent
adducts with cellular thiols.9 Deficiency of HGA dioxygenase incurs
the accumulation of large quantities of quinone that readily form
the so called ochronotic pigment, the structure of which remains
unknown. Among the above diseases associated with tyrosine
catabolism, it is believed that specific inhibition of HPPD could
alleviate the symptoms of patients suffering from type I tyrosine-
mia and alkaptonuria.10–13
In the human, abnormal metabolism in the tyrosine catabolism
pathway gives rise to various diseases, including type
I
⇑
Corresponding authors. Tel.: +86 27 67867800; fax: +86 27 67867141.
E-mail addresses: tomyang@mail.ccnu.edu.cn (W.-C. Yang), gfyang@mail.ccnu.
edu.cn (G.-F. Yang).
These two authors made equal contributions.
http://dx.doi.org/10.1016/j.bmc.2014.08.011
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

Y.-L. Xu et al. / Bioorg. Med. Chem. 22 (2014) 5194–5211
5195
NH2
COOH
O
OH COOH
Plastoquinone & Tocopherol
t
COOH
n
a
l
P
HPPD
O2
human
Ochronotic pigment
(Alkaptonuria)
OH
OH
L-Tyrosine
OH
HPPA
h
u
O
HO
O
m
a
n
HGA
O
O
O
O
O
OH
OH
Succinylacetone
(Type I tyrosinemia)
Succinylacetoacetate
Scheme 1. The simple diagram for the catabolic pathways from tyrosine in plants, and alkaptonuria and type I tyrosinemia in human patients.
Over the last two decades, three main classes of potent HPPD
inhibitors have been discovered as herbicides, including triketone,
diketonitriles (DKN) and pyrazole.14,15 Among them, the triketone
herbicides are the most widely used and represent the greatest
variety of commercialized HPPD inhibitors. The major pharmaco-
phore of the triketone HPPD inhibitors is typically based around
the 2-benzoyl or 2-heteroaroyl cyclohexane-1,3-dione and its sim-
ilar derivatives. Considerably less research has been focused on the
discovery of drugs targeting human HPPD. So far, only NTBC, also
known as nitisinone (with a market name of orfadin), has been
approved as the first effective drug to clinically treat hereditary
type I tyrosinemia by the US Food and Drug Administration.7,16–18
In fact, being a simple and classical representative triketone HPPD
inhibitor, NTBC has also shown excellent herbicidal activity and
has even been used as herbicide. Inspired by this case, we intended
to design new inhibitors of human HPPD by taking advantage of the
existing pharmacophore of herbicides targeting HPPD.
As a heterocyclic aromatic compound, quinazolone has been
widely used in pesticide and pharmaceutical synthesis, and has
displayed extensive biological activities, such as antihypertensive,
antidiabetic, anti-inflammatory, anticonvulsive and antibacterial
properties.19–21 For example, by employing quinazolone as the
major active subunit, raltitrexed (ZD1964) has been developed
for effectively treating patients with advanced and previously
untreated colorectal cancer.22 Thus, we envisioned that by apply-
ing scaffold hopping, hybrid (1 in Fig. 1) of quinazolone with an
identified pharmacophore of herbicides targeting HPPD (like pyra-
zole) would lead to novel HPPD inhibitors with improved potency
against human HPPD. In addition, structure-based drug design usu-
ally provides valuable hints for the rational discovery of enzyme
inhibitors with high activity and good selectivity.23–25 Encouraged
by these ideas and by the identification of the structural differ-
ences and conformational changes of the key residues surrounding
the enzyme active site, we judiciously designed and subsequently
optimized the pyrazole–quinazolone hybrid on the basis of exper-
imental and computational studies. As anticipated, dozens of novel
HPPD inhibitors (compounds 2 and 3 in Scheme 2) were identified
with good potency in the nanomolar range in this study, whose
activities were significantly improved compared to NTBC. In addi-
tion, with the most active compound (3h) as the representative,
the structural basis of inhibitory mechanism for the title com-
pounds was determined through the use of collaborative methods,
such as molecular modeling, spectral characterization, inhibitory
kinetics and thermodynamics.
2,4-dimethyl aniline was chosen as the starting material. The acyl-
ation of 2,4-dimethyl aniline with acetic anhydride provided com-
pound 4, which was oxidized with KMnO4 to generate the
intermediate 5. The selective esterification of 5 with methanol in
the presence of concentrated H2SO4 afforded the intermediate 6,
with the concomitant deprotection of the amine group. To avoid
side reactions in the next cyclization step, the amine group of 6
was re-protected. Then the cyclization of compound 7 with various
substituted anilines was accomplished under the optimal condi-
tions to give intermediate 8 with good yields (65–85%).26 This
raw material 8 was then treated with aqueous sodium hydroxide
directly to afford carboxylic acids 9 without further purification.
To convert 9 to 10, we first tried a two-step one-pot strategy, acyl-
ating 9 to its corresponding acyl chloride form and then reacting
with pyrazol-5-ol. However, none of reactions with various regular
acylation reagents, such as SOCl2, Cl(O@)CC(@O)Cl, POCl3, PCl5,
worked for the production of 10. We next followed a reported
method, using the coupling reagent EDCI or DCC, produced 10 in
moderate yields.27 Finally, the desired products were obtained by
the rearrangement in the presence of acetone cyanohydrin and tri-
ethylamine in anhydrous CH3CN. The structures of all the target
compounds were characterized by 1H NMR, 13C NMR, elemental
analysis, and MS spectra. Their purities were also checked with
HPLC analysis and are no less than 96%.
2.2. Hit to lead optimization
As mentioned in the introduction, we envisioned that hybrid (1)
of pyrazole–quinazolone would afford novel HPPD inhibitors with
improved potency (Fig. 1). Prior to the chemical synthesis, we per-
formed molecular modeling to investigate the binding mode of
hybrid (1) with the crystal structure of human HPPD (PDB entry:
3ISQ). Since no crystal structure of human HPPD coupled with
inhibitor is available, the existing binding mode of Arabidopsis tha-
liana HPPD (PDB entry: 1SQD and 1TG5) coupled with inhibitors as
reference. As shown in the middle portion of Figure 1, the modeling
revealed that compound 1 did bind into the active center of HPPD
because of the apparent chelation with the ferrous ion, while the
quinazolone moiety occupied only a minor part of the hydrophobic
cavity, which implied that introduction of a larger group could
result in a better interaction. Bearing this consideration in mind,
we then designed alternative lead compounds (2) which employed
substituted phenyl groups on the amide nitrogen. The most com-
mon electron withdrawing and donating groups, such as halogen,
methyl, trifluoromethyl, methoxy, and trifluoromethoxyl, were
considered as the substituent groups. After chemical synthesis,
the inhibitory activity of the new compounds was evaluated
(Table 1).
2. Results and discussion
2.1. Chemistry
As expected, all the compounds demonstrated potent inhibitory
activity against human HPPD with inhibitory constants (Ki) at the
submicromolar level or nanomolar level. The selectivity of new
As shown in Scheme 2, an eight-step protocol was applied to
prepare the target compounds (2 and 3). Commercially available

5196
Y.-L. Xu et al. / Bioorg. Med. Chem. 22 (2014) 5194–5211
Figure 1. Design of the pyrazole–quinazolone hybrid molecules as inhibitors of human HPPD. The artificial hit compound (1) was proposed by scaffold hopping of two
commercial compounds; the actual hit compounds (2) were designed according to the simulated binding mode of artificial compound 1.
HOOC
H
N
H
N
COOH
NH2
NH2
O
O
c
b
a
O
O
e
HOOC
O
4
5
6
O
O
O
COOH
NH
O
d
O
O
f
N
N
HO
O
R2
R2
N
N
7
8
9
R1
OH
O
O
N
O
O
O
g
N
h
N
R2
N
R2
N
N
N
R1
N
2: R1 = CH3
10
: R1 = H
3
Scheme 2. Synthetic route of the title compounds. Reagents and conditions: (a) (CH3CO)2O, rt 2 h; (b) KMnO4, H2O, 90 °C for 3 h; (c) MeOH, concd H2SO4, refluxed for 8 h ; (d)
THF, Et3N, CH3COCl, ꢀ5 °C; (e) anhyd CH3CN, substituted aniline, PCl3, 50 °C; (f) THF, H2O, NaOH, rt 4–8 h; (g) anhyd CH2Cl2, DCC, DMAP, rt 24–36 h; (h) anhyd CH3CN, Et3N,
(CH3)2C(OH)CN, rt 3–8 h.

Y.-L. Xu et al. / Bioorg. Med. Chem. 22 (2014) 5194–5211
5197
Table 1
Structures of compounds 2a–s and their inhibition constants (Ki) against recombinant human HPPD
R1
O
N
O
OH
N
N
N
Compound
NTBC
R1
Ki (nM)
Compound
R1
Ki (nM)
O
O
O
NO
2
37
86
2
4
2J
4-OMe
238 45
CF
3
2a
2b
2c
2d
2e
2f
2g
2h
2i
2-Cl
2-Me
2-OMe
2-F
3-Me
3-Cl
3-Br
3-F
2k
2l
4-Br
4-Me
4-Cl
2,4–2Me
2-F, 4-Me
2-Me, 4-F
2-Br, 4-Me
2-Me, 4-Cl
2-Cl, 4-Me
130
156 10
237
1
248 91
207
79
74
3
2
2
2m
2n
2o
2p
2q
2r
2
161 20
81 15
219 12
173 16
93
9
87
177 24
109
9
90 14
247 45
4-F
2s
9
compounds for HPPD was also investigated. The addition of other
enzymes including iron-dependent enzymes, like HGD and cata-
lase, did not cause significant interference in the inhibition of
new compounds toward human HPPD. In particular, compound
interaction of the methyl substituted phenyl group, as well as via
stacking interaction with Phe347 (Fig. 2a). Furthermore, the
quinazolone part and the surrounding aromatic residues (Phe336
and Phe364) formed the sandwich that defined the stacking
p–p
p–p
2e displayed the best activity with
a
Ki value of
interaction. However, chelation between the pyrazolone part of
2e and ferrous ion appeared to decrease as the distances were
extended (2.5 and 2.7 Å). To our best understanding, this loss of
chelation may be due to the steric hindrance caused by the methyl
group at position-3 of the pyrazole ring which pushes the pyrazo-
lone away from ferrous ion, as depicted at the top left corner of
Figure 2a.
Based on the above observation, we therefore proposed that the
removal of the methyl group at position-3 of the pyrazole ring
might avoid the steric congestion. After removing the methyl
group, we synthesized a series of new compounds (3a–y) substi-
tuted by the commonly used electron-withdrawing or electron-
donating groups on the phenyl residue, such as halogen, methyl,
trifluoromethyl, methoxy and trifluoromethoxyl. We then evalu-
ated these compounds as inhibitors of human HPPD (Table 2).
Notably, all the compounds in this new set, except compounds
3r and 3w, exhibited inhibitory constants at the nanomolar level
against human HPPD, and most of the new compounds were more
potent than NTBC. We were able to draw the following conclusions
about SAR: (1) compounds containing a meta-substituent on the
0.074 0.002
0.037 0.002
l
M, which is comparable with NTBC (Ki value of
lM). For the sake of clarity, all the compounds were
divided into four groups according to the position of the substitu-
tion on the benzene ring, that is, ortho-position, meta-position,
para-position, or at both the ortho- and para-positions. We
observed some interesting points with respect to the structure–
activity relationship (SAR) about the substitution on compounds
2a–s. For compounds containing a single halogen substitution on
the phenyl group, the inhibitory activity correlated well with the
order of: ortho-substitution > meta-substitution > para-substitu-
tion. For compounds bearing a methyl substitution on the phenyl
ring, the activity order was: meta-substitution > para-substitu-
tion > ortho-substitution. For compounds with a single halogen
substitution on the phenyl ring, the introduction of a methyl as
the second substituent group had no effect on the activity
enhancement.
To achieve further optimization, we investigated the binding
mode of compound 2e through molecular modeling. Improved
binding of 2e with HPPD occurred through the strong hydrophobic
Figure 2. The simulated binding model of compounds 2e (a) and 3h (b) in the HPPD active site shown as a translucent surface. The ferrous ion is depicted as the purple
sphere; the inhibitors are displayed in cyan and the surrounding key residues are indicated in green.

5198
Y.-L. Xu et al. / Bioorg. Med. Chem. 22 (2014) 5194–5211
Table 2
phenyl ring always showed the best inhibitory activity, and most of
the compounds containing ortho-substituents on the phenyl ring
displayed better activity than those with a para-substituent; (2)
compounds containing a single halogen substitution on the phenyl
ring demonstrated better activity than those compounds contain-
ing another single substitution like methyl or methoxy on the
phenyl ring; (3) the additional substitution of small hydrophobic
group, such as halogen and methyl, generally decreased the activ-
ity. Among these newly synthesized compounds, compound 3h
and 3u were identified as the most active HPPD inhibitors with
Structures of compounds 3a–z and their inhibition constants (Ki) against recombinant
human HPPD
R1
O
N
O
OH
N
N
N
Compound
R1
Ki (nM)
Compound
R1
Ki (nM)
22
18
25
60
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
2-F
2-Cl
2-Br
2-Me
2-OMe
3-F
19
15
22
24
58
38
20
10
14
13
34
5
2
8
1
5
2
5
1
1
2
2
3n
3o
3p
3q
3r
4-Cl
4-Br
4-Me
4-OMe
4-OCF3
2-Me, 4-Cl
2-Me, 4-F
2-Br, 4-Me
3,4–2Cl
2-F, 4-Me
2-Cl, 4-Me
2,4–2Me
4
Ki values of 0.010 0.001
lM and 0.011 0.006 lM, which are
1
5
3
3-fold more potent than NTBC.
Compared with compound 2e, compound 3h showed a similar
binding mode but was better accommodated inside the HPPD
active site according to the simulated binding model (Fig. 2b). As
anticipated, the pyrazolone ring formed much stronger interac-
tions with ferrous ion (2.1 and 1.9 Å). In addition, the presence of
chlorine significantly improved the hydrophobic interaction of
3h. The above interactions may constitute the structural basis for
the greater potency of 3h.
160 10
32
40 10
3s
3t
2
3-Cl
3u
3v
3w
3x
3y
11
25
6
5
3-Br
3-Me
3-OMe
3-OCF3
4-F
3j
3k
3l
170 40
90
59
4
4
31 11
34
3m
2
2.3. Inhibitory kinetics of compound 3h
So far, several methods, including HPLC, isotope-labeling, elec-
trochemical and coupling methodology, have been established to
measure HPPD activity and to characterize the effects of inhibitors.
For the sake of safety and high-throughput convenience, a coupling
method was developed for HPPD activity assay and inhibitors
screening. This assay coupled the formation of HGA converted from
HPPA by HPPD to the production of maleylacetoacetate from HGA
via HGA dioxygenase. The absorbance peak for maleylacetoacetate
is ꢁ330 nm and is used for real-time spectrophotometric monitor-
ing. Although the purified human HPPD is known to be quite
unstable, the crude HPPD is pretty stable at 4 °C according to our
study. At 4 °C, the activity of crude HPPD can be maintained for a
week without significant decrease. While at our working tempera-
ture (30 °C), the activity reduced but the stability is sufficient for
our in vitro assay (Fig. 3).
Stability of crude human HPPD
100
80
60
40
20
Primarily, all the newly synthesized compounds were screened
for their possible activity against HGA dioxygenase. After deter-
mining their negligible inhibitory activity towards HGA dioxygen-
ase, all compounds were then evaluated as HPPD inhibitors with
the above coupling method. During the inhibitory characterization,
0
0
5
10
15
20
Incubation time (hour)
Figure 3. Stability of crude human HPPD at 30 °C.
-16
-17
-18
-19
3.5
(a)
(b)
3.0
2.5
2.0
1.5
1.0
0.0032
0.0033
0.0034
-0.05
0.00
0.05
0.10
0.15
0.20
1/T(1/K)
3h (μM)
Figure 4. (a) Dixon plots of inhibitory kinetics of compound 3h against human HPPD, with HPPA at 40
for inhibitor 3h binding to human HPPD.
lM and 80 lM, respectively. (b) van’t Hoff plot of of ln(Ki) versus 1/T

Y.-L. Xu et al. / Bioorg. Med. Chem. 22 (2014) 5194–5211
5199
the new compounds and NTBC demonstrated time-dependent
inhibition for human HPPD, and then the slow-binding equation25
seems to be more suitable for measuring Ki values. However, appli-
cation of slow-binding equation requires the assumption of
[I]t ꢂ [E]t, which means a pretty low concentration of enzyme in
the assay system. Unfortunately, the sensitivity of the established
coupling assay is not sufficient meet this requirement at low con-
centration. So, we finally chose Dixon method for our coupling
assay to characterize those title compounds (Fig. 4a).
The QIAprep Spin Plasmid Miniprep Kit, Ni–NTA resin, QG buffer,
plasmid purification kit, and QIAquick PCR purification kit were
obtained from Qiagen (Valencia, CA). NdeI and BamHI were
purchased from Promega (Madison, MI).
4.1.1. General procedure for intermediate 8
To a solution of the 2-acetamido-5-(methoxycarbonyl)benzoic
acid (6 mmol) in CH3CN was added a solution of the substituted
aniline (8 mmol) in CH3CN (10 mL) at room temperature to give
a white suspension. PCl3 (12 mmol) was added via syringe, and
the resulting mixture was warmed to 50 °C and stirred for an addi-
tional 6–10 h. The mixture was cooled to room temperature and
diluted with EtOAc. The mixture was quenched by the addition
of 1 N aqueous HCl. The organic layer was separated, washed with
15% aqueous NaHCO3 solution, and evaporated under vacuum to
give a solid, which crystallized on standing. The crude products
were recrystallized from ethyl acetate/petroleum ether to give
the desired products of 8a–x.
2.4. van’t Hoff analysis of the inhibition constant Ki
By measuring Ki values at a series of temperatures, the van’t
Hoff Eq. 1 may be used to determine the individual contributions
of
shows a plot of ln(Ki) versus 1/T based on the van’t Hoff equation
with good correlation (R2 = 0.99). Thus,
H and S were calculated
DH and DS for the enzyme-inhibitor dissociation. Figure 4b
D
D
to be about ꢀ86.90 kJ/mol and ꢀ438.39 J/(mol K), respectively, val-
ues that correspond to the HPPD–3h complex dissociation process.
The benchtop mixing kinetics showed that binding of compound
3h to HPPD had a favorable enthalpy value and an unfavorable
entropy value, illustrating that the binding process is enthalpy-
driven. Usually, an enthalpy-driven process correlates with abun-
dant electrostatic interactions and van der Waals interactions. The
4.1.2. 2-Methyl-4-oxo-3-p-tolyl-3,4-dihydroquinazoline-6-
carboxylate (8a)
White solid, yield: 71%. Mp 175–177 °C. 1H NMR (600 MHz,
CDCl3) d 8.93 (s, 1H), 8.40 (d, J = 8.4 Hz, 1H), 7.84 (s, 1H), 7.39 (d,
J = 7.2 Hz, 2H), 7.15 (d, J = 7.2 Hz, 2H), 3.97 (s, 3H), 2.46 (s, 3H),
2.36 (s, 3H). MS (EI) m/z: 308.24(M)+.
free energy at physiological temperature is
D
G = ꢀ48.99 kJ/mol.
ꢀ
ꢁ
D
Hbinding
R
1
T
DSbinding
4.1.3. 2-Methyl-4-oxo-3-(3-(trifluoromethoxy)phenyl)-3,4-
dihydroquinazoline-6-carboxylate (8b)
lnðKiÞ ¼ ꢀ
þ
ð1Þ
R
White solid, yield: 85%. Mp 138–140 °C. 1H NMR (600 MHz,
CDCl3) d 8.93 (s, 1H), 8.41 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 8.4 Hz,
1H), 7.64 (t, J = 8.4 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.25 (s, 1H),
7.20 (s, 1H), 3.97 (s, 3H), 2.29 (s, 3H). MS (EI) m/z: 378.26(M)+.
3. Conclusions
In summary, a series of hybrids of pyrazolone–quinazolone as
human HPPD inhibitors were rationally designed through scaffold
hopping, and subsequently optimized by combining computational
simulations and experimental studies, leading to the discovery of
several novel human HPPD inhibitors with excellent inhibitory
potency. In particular, compounds 3h, characterized by a pyrazo-
lone moiety linked to a substituted quinazolone, was identified
as the most potent human HPPD inhibitor with Ki values of ꢁ10
nM, over three-fold more potent than NTBC. The binding mode of
the 3h in HPPD was further investigated by a series of experiments,
such as spectral studies, inhibitory kinetics, thermodynamics, and
computational modeling. Based the aforementioned results,
hybrids of pyrazolone–quinazolone could be novel lead com-
pounds for human HPPD inhibitors that are worthy of further study
in drug discovery.
4.1.4. 3-(4-Fluoro-2-methylphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate (8c)
White solid, yield: 63%. Mp 178–180 °C. 1H NMR (600 MHz,
CDCl3) d 8.95 (s, 1H), 8.41 (d, J = 9.0 Hz, 1H), 7.73 (d, J = 8.4 Hz,
1H), 7.15 (m, 2H), 7.10 (m, 1H), 3.97 (s, 3H), 2.22 (s, 3H), 2.13 (s,
3H). MS (EI) m/z: 326.28(M)+.
4.1.5. 3-(2-Chloro-4-methylphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate(8d)
White solid, yield: 73%. Mp 191–193 °C. 1H NMR (600 MHz,
CDCl3) d 8.95 (s, 1H), 8.40 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 8.4 Hz,
1H), 7.45 (s, 1H), 7.28 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 7.8 Hz, 1H),
3.96 (s, 3H), 2.45 (s, 3H), 2.26 (s, 3H). MS (EI) m/z: 342.20(M)+.
4.1.6. 3-(2-Fluoro-4-methylphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate(8e)
4. Experimental section
4.1. Synthetic chemistry
White solid, yield: 85%. Mp 138–140 °C. 1H NMR (600 MHz,
CDCl3) d 8.94 (d, J = 1.2 Hz, 1H), 8.40 (dd, J = 8.4, 1.8 Hz, 1H), 7.75
(d, J = 8.4 Hz, 1H), 7.13–7.20 (m, 3H), 3.97 (s, 3H), 2.47 (s, 3H),
2.33 (s, 3H). MS (EI) m/z: 326.27(M)+.
Unless otherwise noted, all chemical and biological reagents
were commercially available and treated with standard methods
before use. Solvents were dried in a routine way and redistilled
before use. 1H NMR and 1C NMR spectra were recorded on a VAR-
IAN Mercury-Plus 600 or 400 spectrometer in CDCl3 or DMSO-d6
with TMS as the internal reference. Mass spectral data were
obtained on a ThermoFisher Mass platform DSQII by electrospray
ionization (ESI-MS). Elemental analyses were performed on a Vario
EL III elementary analysis instrument. Melting points were taken
on a Buchi B-545 melting point apparatus and were uncorrected.
All chemical yields are unoptimized and generally represent the
result of a single experiment. Cloned pfx DNA polymerase and DpnI
endonuclease were obtained from Invitrogen. All oligonucleotides
were synthesized by Integrated DNA Technologies (Coralville, IA).
4.1.7. 3-(2-Bromo-4-methylphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate(8f)
White solid, yield: 75%. Mp 195–197 °C. 1H NMR (600 MHz,
CDCl3) d 8.95 (d, J = 1.8 Hz, 1H), 8.40 (dd, J = 8.4, 1.8 Hz, 1H), 7.74
(d, J = 8.4 Hz, 1H), 7.62 (s, 1H), 7.33 (d, J = 7.8 Hz, 1H), 7.23 (d,
J = 7.8 Hz, 1H), 3.96 (s, 3H), 2.45 (s, 3H), 2.26 (s, 3H). MS (EI) m/z:
386.17(M)+.
4.1.8. 3-(3,4-Dichlorophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate (8g)
White solid, yield: 77%. Mp 204–205 °C. 1H NMR (600 MHz,
CDCl3) d 8.92 (d, J = 1.8 Hz, 1H), 8.41 (dd, J = 8.4, 1.8 Hz, 1H), 7.73

5200
Y.-L. Xu et al. / Bioorg. Med. Chem. 22 (2014) 5194–5211
(d, J = 8.4 Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.43 (d, J = 1.8 Hz, 1H),
7.16 (dd, J = 8.4, 2.4 Hz, 1H), 3.97 (s, 3H), 2.31 (s, 3H). MS (EI) m/
z: 362.20(M)+.
J = 8.4 Hz, 1H), 7.57 (s, 2H), 7.44 (t, J = 8.4 Hz, 2H), 3.91 (s, 3H),
2.18 (s, 3H). MS (EI) m/z: 312.12(M)+.
4.1.18. 3-(4-Chlorophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate (8q)
4.1.9. 2-Methyl-4-oxo-3-(4-(trifluoromethoxy)phenyl)-3,4-
dihydroquinazoline-6-carboxylate (8h)
White solid, yield: 84%. Mp 132–134 °C. 1H NMR (600 MHz,
White solid, yield: 69%. Mp 135–136 °C. 1H NMR (600 MHz,
CDCl3) d 8.93 (d, J = 1.8 Hz, 1H), 8.41 (dd, J = 8.4, 1.8 Hz, 1H), 7.76
(d, J = 8.4 Hz, 1H), 7.44 (d, J = 9.0 Hz, 2H), 7.34 (d, J = 9.0 Hz, 2H),
3.97 (s, 3H), 2.31 (s, 3H). MS (EI) m/z: 378.27(M)+.
DMSO-d6) d 8.65 (s, 1H), 8.33 (d, J = 8.4 Hz, 1H), 7.77 (d,
J = 8.4 Hz, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 9.0 Hz, 2H),
3.91 (s, 3H), 2.17 (s, 3H). MS (EI) m/z: 328.08(M)+.
4.1.19. 3-(2-Methoxyphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate (8r)
4.1.10. 3-(4-Chloro-2-methylphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate (8i)
White solid, yield: 65%. Mp 180–181 °C. 1H NMR (600 MHz,
CDCl3) d 8.94 (d, J = 1.8 Hz, 1H), 8.38 (dd, J = 9.0, 1.8 Hz, 1H), 7.73
(d, J = 8.4 Hz, 1H), 7.50 (t, J = 7.2 Hz, 1H), 7.25 (dd, J = 7.8, 1.2 Hz,
1H), 7.15–7.10 (m, 2H), 3.96 (s, 3H), 3.81 (s, 3H), 2.26 (s, 3H). MS
(EI) m/z: 324.37(M)+.
White solid, yield: 78%. Mp 163–165 °C. 1H NMR (600 MHz,
CDCl3) d 8.94 (s, 1H), 8.41 (d, J = 9.0 Hz, 1H), 7.74 (d, J = 8.4 Hz,
1H), 7.43 (s, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.12 (d, J = 8.4 Hz, 1H),
3.97 (s, 3H), 2.23 (s, 3H), 2.12 (s, 3H). MS (EI) m/z: 342.32(M)+.
4.1.11. Methyl 2-methyl-4-oxo-3-phenyl-3,4-
dihydroquinazoline-6-carboxylate (8j)
4.1.20. 3-(4-Methoxyphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate (8s)
White solid, yield: 77%. Mp 178–179 °C. 1H NMR (600 MHz,
CDCl3) d 8.94 (s, 1H), 8.41 (d, J = 8.4 Hz, 1H), 7.82 (s, 1H), 7.61–
7.56 (m, 3H), 7.28 (d, J = 7.2 Hz, 2H), 3.97 (s, 3H), 2.34 (s, 3H). MS
(EI) m/z: 294.26(M)+.
White solid, yield: 75%. Mp 165–167 °C. 1H NMR (400 MHz,
CDCl3) d 8.93 (s, 1H), 8.38 (dd, J = 8.4, 1.6 Hz, 1H), 7.71 (d,
J = 8.4 Hz, 1H), 7.18 (d, J = 8.8 Hz, 2H), 7.07 (d, J = 8.8 Hz, 2H),
3.96 (s, 3H), 3.88 (s, 3H), 2.29 (s, 3H). MS (EI) m/z: 324.20(M)+.
4.1.12. 2-Methyl-4-oxo-3-o-tolyl-3,4-dihydroquinazoline-6-
carboxylate (8k)
4.1.21. 3-(3-Methoxyphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate (8t)
White solid, yield: 77%. Mp 148–150 °C. 1H NMR (600 MHz,
CDCl3) d 8.96 (s, 1H), 8.42 (d, J = 8.4 Hz, 1H), 7.85 (s, 1H), 7.45 (d,
J = 8.4 Hz, 2H), 7.41 (t, J = 7.8 Hz, 1H) 7.18 (d, J = 7.8 Hz, 1H), 3.97
(s, 3H), 2.29 (s, 3H), 2.14 (s, 3H). MS (EI) m/z: 308.29(M)+.
White solid, yield: 64%. Mp 171–173 °C. 1H NMR (400 MHz,
cdcl3) d 8.93 (s, 1H), 8.39 (dd, J = 8.4, 2.0 Hz, 1H), 7.73 (d,
J = 8.4 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 6.86
(d, J = 7.8 Hz, 1H), 6.80 (s, 1H), 3.96 (s, 3H), 3.85 (s, 3H), 2.32 (s,
3H). MS (EI) m/z: 324.30(M)+.
4.1.13. 3-(2-Fluorophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate (8l)
4.1.22. 3-(3-Chlorophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate (8u)
White solid, yield: 78%. Mp 162–163 °C. 1H NMR (600 MHz,
CDCl3) d 8.94 (s, 1H), 8.41 (d, J = 9.0 Hz, 1H), 7.74 (d, J = 8.4 Hz,
1H), 7.55 (d, J = 6.6 Hz, 1H), 7.38–7.32 (m, 3H), 3.97 (s, 3H), 2.32
(s, 3H). MS (EI) m/z: 312.27(M)+.
White solid, yield: 81%. Mp 204–205 °C. 1H NMR (600 MHz,
CDCl3) d 8.93 (s, 1H), 8.41 (d, J = 6.6 Hz, 1H), 7.75 (d, J = 8.4 Hz,
1H), 7.53 (d, J = 4.8 Hz, 2H), 7.32 (s, 1H), 7.19 (s, 1H), 3.97 (s, 3H),
2.31 (s, 3H). MS (EI) m/z: 328.26(M)+.
4.1.14. 3-(2-Bromophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate (8m)
4.1.23. 3-(2-Chlorophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate (8v)
White solid, yield: 71%. Mp 172–174 °C. 1H NMR (600 MHz,
CDCl3) d 8.96 (s, 1H), 8.41 (d, J = 7.2 Hz, 1H), 7.81 (d, J = 7.8 Hz,
1H), 7.77 (d, J = 5.4 Hz, 1H), 7.55 (t, J = 7.2 Hz, 1H), 7.43 (t,
J = 7.2 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 3.97 (s, 3H), 2.27 (s, 3H).
MS (EI) m/z: 372.16(M)+.
White solid, yield: 81%. Mp 160–162 °C. 1H NMR (600 MHz,
CDCl3) d 8.95 (d, J = 1.8 Hz, 1H), 8.42 (dd, J = 8.4, 1.8 Hz, 1H), 7.78
(d, J = 9.0 Hz, 1H), 7.64–7.65 (m, 1H), 7.53–7.49 (m, 2H), 7.36 (dd,
J = 6.6, 2.4 Hz, 1H), 3.97 (s, 3H), 2.28 (s, 3H). MS (EI) m/z: 328.19(M)+.
4.1.15. 3-(2,4-Dimethylphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate (8n)
4.1.24. 3-(3-Fluorophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate (8w)
White solid, yield: 70%. Mp 162–163 °C. 1H NMR (600 MHz,
CDCl3) d 8.96 (s, 1H), 8.40 (d, J = 8.4 Hz, 1H), 7.74 (d, J = 8.4 Hz,
1H), 7.23 (s, 1H), 7.19 (d, J = 7.8 Hz, 1H), 7.04 (d, J = 7.8 Hz, 1H),
3.96 (s, 3H), 2.41 (s, 3H), 2.23 (s, 3H), 2.09 (s, 3H). MS (EI) m/z:
322.28(M)+.
White solid, yield: 79%. Mp 198–200 °C. 1H NMR (600 MHz,
CDCl3) d 8.93 (s, 1H), 8.40 (d, J = 8.4 Hz, 1H), 7.74 (d, J = 9.0 Hz,
1H), 7.57 (dd, J = 14.4, 7.8 Hz, 1H), 7.31–7.23 (m, 2H), 7.10 (d,
J = 7.8 Hz, 1H), 7.05 (d, J = 9.0 Hz, 1H), 3.97 (s, 3H), 2.31 (s, 3H).
MS (EI) m/z: 312.23(M)+.
4.1.16. 3-(4-Bromophenyl)-2-methyl-4-oxo-3,4-
4.1.25. 3-(3-Bromophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate (8o)
dihydroquinazoline-6-carboxylate (8x)
White solid, yield: 78%. Mp 226–227 °C. 1H NMR (600 MHz,
DMSO-d6) d 8.65 (s, 1H), 8.32 (dd, J = 8.4, 1.2 Hz, 1H), 7.80 (d,
J = 8.4 Hz, 2H), 7.77 (d, J = 8.4 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H),
3.91 (s, 3H), 2.17 (s, 3H). MS (EI) m/z: 372.17(M)+.
White solid, yield: 77%. Mp 215–217 °C. 1H NMR (600 MHz,
CDCl3) d 8.92 (s, 1H), 8.40 (d, J = 8.4 Hz, 1H), 7.76 (d, J = 7.8 Hz,
1H), 7.69 (d, J = 7.8 Hz, 1H), 7.47 (t, J = 7.8 Hz, 2H), 7.25 (d,
J = 7.8 Hz, 1H), 3.97 (s, 3H), 2.32 (s, 3H). MS (EI) m/z: 374.18(M)+.
4.1.17. 3-(4-Fluorophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylate (8p)
4.1.26. 2-Methyl-4-oxo-3-m-tolyl-3,4-dihydroquinazoline-6-
carboxylate (8y)
White solid, yield: 87%. Mp 158–160 °C. 1H NMR (600 MHz,
White solid, yield: 75%. Mp 152–154 °C. 1H NMR (400 MHz,
CDCl3) d 8.94 (s, 1H), 8.39 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 8.8 Hz,
DMSO-d6)
d 8.64 (s, 1H), 8.33 (d, J = 6.6 Hz, 1H), 7.77 (d,

Y.-L. Xu et al. / Bioorg. Med. Chem. 22 (2014) 5194–5211
5201
1H), 7.46 (t, J = 8.0 Hz, 1H), 7.34 (d, J = 7.2 Hz, 1H), 7.08 (d,
4.1.35. 2-Methyl-4-oxo-3-(4-(trifluoromethoxy)phenyl)-3,4-
dihydroquinazoline-6-carboxylic acid (9h)
J = 8.4 Hz, 2H), 3.96 (s, 3H), 2.44 (s, 3H), 2.29 (s, 3H). MS (EI) m/z:
308.28(M)+.
White solid, yield: 82%. Mp 314–316 °C. 1H NMR (600 MHz,
DMSO-d6) d 13.37 (s, 2H), 8.64 (s, 1H), 8.32 (d, J = 8.4 Hz, 1H),
7.75 (d, J = 8.4 Hz, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.4 Hz,
2H), 2.17 (s, 3H). MS (EI) m/z: 364.29(M)+.
4.1.27. General synthesis of intermediate 9
To a solution of the intermediate 8 (10 mmol) in THF (50 mL)/
H2O (50 mL) was added a 0.8 g (20 mmol) NaOH, and the mixture
was stirred at room temperature for 4–8 h, followed by evapora-
tion of THF under reduced pressure. To the residue solution,
30 mL H2O was added, and then 6 N hydrochloric acid was utilized
to adjust the pH value to 2–3. The resulting precipitate was col-
lected by filtration, washed with water and dried to afford the
intermediates 9a–y.
4.1.36. 3-(4-Methoxyphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid(9i)
White solid, yield: 86%. Mp 238–240 °C. 1H NMR (600 MHz,
DMSO-d6) d 13.28 (s, 1H), 8.64 (s, 1H), 8.29 (d, J = 9.0 Hz, 1H),
7.72 (d, J = 9.0 Hz, 1H), 7.39 (d, J = 8.4 Hz, 2H), 7.11 (d, J = 8.4 Hz,
2H), 3.84 (s, 3H), 2.17 (s, 3H). MS (EI) m/z: 310.24(M)+.
4.1.28. 2-Methyl-4-oxo-3-phenyl-3,4-dihydroquinazoline-6-
carboxylic acid (9a)
4.1.37. 3-(4-Bromophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9j)
White solid, yield: 90%. Mp 317–319 °C. 1H NMR (400 MHz,
CDCl3) d 13.27 (s, 1H), 8.65 (s, 1H), 8.30 (d, J = 7.2 Hz, 1H), 7.74
(d, J = 8.4 Hz, 1H), 7.58 (d, J = 7.2 Hz, 2H), 7.54 (d, J = 6.8 Hz, 1H),
7.48 (d, J = 6.8 Hz, 2H), 2.16 (s, 3H). MS (EI) m/z: 280.07(M)+.
White solid, yield: 85%. Mp 318–320 °C. 1H NMR (600 MHz,
DMSO-d6) d 13.31 (s, 1H), 8.64 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H),
7.80 (d, J = 7.8 Hz, 2H), 7.74 (d, J = 8.4 Hz, 1H), 7.48 (d, J = 7.2 Hz,
2H), 2.16 (s, 3H). MS (EI) m/z: 360.31(M)+.
4.1.29. 3-(3-Bromophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9b)
4.1.38. 2-Methyl-4-oxo-3-p-tolyl-3,4-dihydroquinazoline-6-
carboxylic acid (9k)
White solid, yield: 91%. Mp 308–310 °C. 1H NMR (600 MHz,
DMSO-d6) d 13.29 (s, 1H), 8.64 (d, J = 2.4 Hz, 1H), 8.31 (dd, J = 8.4,
1.8 Hz, 1H), 7.85 (s, 1H), 7.80–7.70 (m, 2H), 7.56 (dd, J = 3.6,
18 Hz, 2H), 2.17 (s, 3H). MS (EI) m/z: 358.16(M)+.
White solid, yield: 92%. Mp 290–291 °C. 1H NMR (400 MHz,
DMSO-d6) d 13.27 (s, 1H), 8.64 (s, 1H), 8.30 (d, J = 8.4 Hz, 1H),
7.73 (d, J = 8.4 Hz, 1H), 7.36 (dd, J = 16.8, 8.0 Hz, 4H), 2.41 (s, 3H),
2.16 (s, 3H). MS (EI) m/z: 294.23(M)+.
4.1.30. 3-(2-Fluorophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9c)
4.1.39. 3-(4-Fluorophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9l)
White solid, yield: 80%. Mp 298–300 °C. 1H NMR (600 MHz,
DMSO-d6) d 13.35 (s, 1H), 8.66 (d, J = 1.8 Hz, 1H), 8.34 (dd, J = 8.4,
1.8 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H),
7.67–7.57 (m, 1H), 7.53 (t, J = 9.0 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H),
2.22 (s, 3H). MS (EI) m/z: 298.23(M)+.
White solid, yield: 80%. Mp 253–255 °C. 1H NMR (400 MHz,
DMSO-d6) d 13.28 (s, 1H), 8.64 (s, 1H), 8.31 (d, J = 9.2 Hz, 1H),
7.74 (d, J = 9.2 Hz, 1H), 7.56 (dd, J = 8.8, 5.2 Hz, 2H), 7.43 (t,
J = 8.8 Hz, 2H), 2.16 (s, 3H). MS (EI) m/z: 298.12(M)+.
4.1.40. 3-(4-Chlorophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9m)
4.1.31. 3-(2-Methoxyphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9d)
White solid, yield: 90%. Mp 287–289 °C. 1H NMR (600 MHz,
DMSO-d6) d 13.30 (s, 1H), 8.64 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H),
7.74 (d, J = 7.8 Hz, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.4 Hz,
2H), 2.16 (s, 3H). MS (EI) m/z: 314.10(M)+.
White solid, yield: 75%. Mp 286–288 °C. 1H NMR (600 MHz,
DMSO-d6) d 13.26 (s, 1H), 8.64 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H),
7.74 (d, J = 8.4 Hz, 1H), 7.54 (t, J = 8.4 Hz, 1H), 7.44 (d, J = 7.8 Hz,
1H), 7.28 (d, J = 8.4 Hz, 1H), 7.15 (t, J = 7.8 Hz, 1H), 3.78 (s, 3H),
2.14 (s, 3H). MS (EI) m/z: 310.25(M)+.
4.1.41. 3-(2-Fluoro-4-methylphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9n)
4.1.32. 3-(2-Bromophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9e)
White solid, yield: 94%. Mp 245–247 °C. 1H NMR (600 MHz,
DMSO-d6) d 13.32 (s, 1H), 8.65 (d, J = 1.8 Hz, 1H), 8.34 (td, J = 8.4,
1.8 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.53 (t, J = 7.8 Hz, 1H), 7.35
(d, J = 10.8 Hz, 1H), 7.24 (d, J = 7.8 Hz, 1H), 2.43 (s, 3H), 2.22 (s,
3H). MS (EI) m/z: 312.23(M)+.
White solid, yield: 74%. Mp 290–292 °C. 1H NMR (600 MHz,
DMSO-d6) d 13.32 (s, 1H), 8.66 (s, 1H), 8.34 (dd, J = 9.0, 1.6 Hz,
1H), 7.92 (d, J = 7.8 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.76–7.70 (m,
1H), 7.64 (t, J = 7.8 Hz, 1H), 7.53 (t, J = 7.8 Hz, 1H), 2.14 (s, 3H).
MS (EI) m/z: 358.44(M)+.
4.1.42. 3-(4-Fluoro-2-methylphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9o)
4.1.33. 3-(2-Chloro-4-methylphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9f)
White solid, yield: 92%. Mp 277–279 °C. 1H NMR (600 MHz,
DMSO-d6) d 8.66 (d, J = 1.8 Hz, 1H), 8.34 (dd, J = 8.4, 1.8 Hz, 1H),
7.79 (d, J = 8.4 Hz, 1H), 7.52 (dd, J = 9.0, 6.0 Hz, 1H), 7.37 (dd,
J = 9.6, 2.4 Hz, 1H), 7.26 (m, 1H), 2.17 (s, 3H), 2.07 (s, 3H). MS (EI)
m/z: 312.24(M)+.
White solid, yield: 91%. Mp 274–275 °C. 1H NMR (600 MHz,
DMSO-d6) d 13.33 (s, 1H), 8.66 (d, J = 1.8 Hz, 1H), 8.34 (dd, J = 8.4,
1.8 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.61 (s, 1H), 7.59 (d,
J = 7.8 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H), 2.43 (s, 3H), 2.16 (s, 3H).
MS (EI) m/z: 328.10(M)+.
4.1.43. 3-(4-Chloro-2-methylphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9p)
4.1.34. 2-Methyl-4-oxo-3-(3-(trifluoromethoxy)phenyl)-3,4-
dihydroquinazoline-6-carboxylic acid (9g)
White solid, Yield: 81%. Mp 308–310 °C. 1H NMR (600 MHz,
DMSO-d6) d 13.31 (s, 1H), 8.66 (s, 1H), 8.33 (d, J = 8.4 Hz, 1H),
7.76 (d, J = 8.4 Hz, 1H), 7.60 (s, 1H), 7.50 (s, 2H), 2.14 (s, 3H), 2.05
(s, 3H). MS (EI) m/z: 328.21(M)+.
White solid, yield: 88%. Mp 240–242 °C. 1H NMR (600 MHz,
DMSO-d6) d 8.65 (s, 1H), 8.34 (d, J = 8.4 Hz, 1H), 7.79 (t, J = 8.4 Hz,
1H), 7.75 (t, J = 8.4 Hz, 1H), 7.72 (s, 1H), 7.59 (t, J = 7.2 Hz, 2H),
2.19 (s, 3H). MS (EI) m/z: 364.23(M)+.

5202
Y.-L. Xu et al. / Bioorg. Med. Chem. 22 (2014) 5194–5211
4.1.44. 3-(2-Bromo-4-methylphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9q)
4.1.53. General procedure for the synthesis of intermediate 10
Intermediate 9 (4 mmol), dissolved in dry dichloromethane
(60 mL), and DCC (4.8 mmol) were mixed together and stirred at
room temperature for 1 h. Then 1,3-dimethyl-1H-pyrazol-5-ol
(4 mmol) and DMAP (0.4 mmol) were added to the above solution,
and stirred for a further 24–36 h at room temperature. After adding
dichloromethane (30 mL), the resultant mixture was washed
sequentially with 2 N hydrochloric acid (100 mL), saturated
NaHCO3 and saturated brine and dried over anhydrous sodium sul-
fate. The residue solution was evaporated in vacuum and purified
by silica gel column chromatography using gradient elution of
ethyl acetate/hexane (V/V = 1/2) to obtain the desired intermediate
10.
White solid, yield: 81%. Mp 257–259 °C. 1H NMR (600 MHz,
DMSO-d6) d 13.24 (s, 1H), 8.65 (s, 1H), 8.33 (d, J = 8.4 Hz, 1H),
7.77 (d, J = 8.4 Hz, 1H), 7.74 (s, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.43
(d, J = 7.8 Hz, 1H), 2.42 (s, 3H), 2.15 (s, 3H). MS (EI) m/z:
372.19(M)+.
4.1.45. 3-(3,4-Dichlorophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9r)
White solid, yield: 83%. Mp 302–304 °C. 1H NMR (600 MHz,
DMSO-d6) d 8.64 (s, 1H), 8.35 (d, J = 8.4 Hz, 1H), 7.95 (s, 1H), 7.90
(d, J = 8.4 Hz, 1H), 7.82 (d, J = 9.0 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H),
2.26 (s, 3H). MS (EI) m/z: 348.21(M)+.
4.1.54. 1,3-Dimethyl-1H-pyrazol-5-yl-3-(4-fluorophenyl)-2-
methyl-4-oxo-3,4-dihydroquinazoline-6-carboxylate
White solid, yield: 41%. Mp 212–214 °C. 1H NMR (600 MHz,
4.1.46. 3-(2,4-Dimethylphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9s)
White solid, Yield: 93%. Mp 189–190 °C. 1H NMR
DMSO-d6) d 8.80 (s, 1H), 8.50 (d, J = 8.4 Hz, 1H), 7.84 (d,
(400 MHz, DMSO-d6)
d
13.28 (s, 1H), 8.66 (s, 1H), 8.31
J = 8.4 Hz, 1H), 7.58 (s, 2H), 7.44 (t, J = 8.4 Hz, 2H), 6.06 (s, 1H),
(d, J = 7.8 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.27 (s, 2H), 7.20
(d, J = 8.0 Hz, 1H), 2.37 (s, 3H), 2.12 (s, 3H), 2.00 (s, 3H). MS (EI)
m/z: 308.25(M)+.
3.66 (s, 3H), 2.20 (s, 3H), 2.15 (s, 3H). MS (EI) m/z: 392.30(M)+.
4.1.55. 1-methyl-1H-pyrazol-5-yl-3-(4-fluorophenyl)-2-methyl-
4-oxo-3,4-dihydroquinazoline-6-carboxylate
4.1.47. 3-(3-Fluorophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9t)
White solid, yield: 47%. Mp 220–222 °C. 1H NMR (400 MHz,
CDCl3) d 9.02 (s, 1H), 8.46 (dd, J = 8.4, 1.6 Hz, 1H), 7.78 (d,
J = 8.4 Hz, 1H), 7.46 (s, 1H), 7.30 (s, 2H), 7.28 (s, 2H), 6.27 (s, 1H),
3.81 (s, 3H), 2.30 (s, 3H). MS (EI) m/z: 378.30(M)+.
White solid, yield: 81%. Mp 288–290 °C. 1H NMR (600 MHz,
DMSO-d6) d 13.28 (s, 1H), 8.64 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H),
7.74 (d, J = 8.4 Hz, 1H), 7.64 (dd, J = 15.0, 7.8 Hz, 1H), 7.50 (d,
J = 9.0 Hz, 1H), 7.41 (t, J = 8.4 Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H), 2.19
(s, 3H). MS (EI) m/z: 298.22(M)+.
4.1.56. 1,3-Dimethyl-1H-pyrazol-5-yl-2-methyl-4-oxo-3-m-
tolyl-3,4-ihydroquinazoline-6-carboxylate
White solid, yield: 52%. Mp 218–220 °C. 1H NMR (600 MHz,
CDCl3) d 9.08 (d, J = 1.8 Hz, 1H), 8.48 (dd, J = 8.4, 1.2 Hz, 1H), 7.80
(d, J = 8.4 Hz, 1H), 7.48 (t, J = 7.8 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H),
7.09 (s, 1H), 7.08 (d, J = 8.4 Hz, 1H), 6.08 (s, 1H), 3.74 (s, 3H), 2.45
(s, 3H), 2.32 (s, 3H), 2.28 (s, 3H). MS (EI) m/z: 388.26(M)+.
4.1.48. 3-(3-Chlorophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9u)
White solid, yield: 84%. Mp 312–313 °C. 1H NMR (600 MHz,
DMSO-d6) d 13.30 (s, 1H), 8.64 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H),
7.75 (d, J = 8.4 Hz, 1H), 7.72 (s, 1H), 7.63 (d, J = 4.8 Hz, 2H), 7.51
(d, J = 4.2 Hz, 1H), 2.18 (s, 3H). MS (EI) m/z: 314.19(M)+.
4.1.57. 1-Methyl-1H-pyrazol-5-yl-2-methyl-4-oxo-3-m-tolyl-
3,4-dihydroquinazoline-6-carboxylate
4.1.49. 3-(3-Methoxyphenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9v)
White solid, yield: 41%. Mp 196–198 °C. 1H NMR (600 MHz,
CDCl3) d 9.09 (s, 1H), 8.49 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 7.8 Hz,
1H), 7.48 (t, J = 7.8 Hz, 2H), 7.36 (d, J = 7.8 Hz, 1H), 7.08 (d,
J = 12 Hz, 2H), 6.28 (s, 1H), 3.82 (s, 3H), 2.45 (s, 3H), 2.35 (s, 3H).
MS (EI) m/z: 373.20(M-1)+.
White solid, yield: 83%. Mp 270–272 °C. 1H NMR (600 MHz,
DMSO-d6) d 13.30 (s, 1H), 8.64 (s, 1H), 8.30 (d, J = 9.0 Hz, 1H),
7.73 (d, J = 8.4 Hz, 1H), 7.49 (t, J = 7.8 Hz, 1H), 7.13 (s, 1H), 7.10
(d, J = 9.0 Hz, 1H), 7.04 (d, J = 7.8 Hz, 1H), 3.80 (s, 3H), 2.19 (s,
3H). MS (EI) m/z: 310.22(M)+.
4.1.58. 1,3-Dimethyl-1H-pyrazol-5-yl-3-(4-bromophenyl)-2-
methyl-4-oxo-3,4-dihydroquin azoline-6-carboxylate
4.1.50. 3-(2-Chlorophenyl)-2-methyl-4-oxo-3,4-
dihydroquinazoline-6-carboxylic acid (9w)
White solid, yield: 41%. Mp 237–239 °C. 1H NMR (600 MHz,
CDCl3) d 9.07 (s, 1H), 8.49 (d, J = 9.0 Hz, 1H), 7.82 (d, J = 8.4 Hz,
1H), 7.74 (d, J = 7.2 Hz, 2H), 7.17 (d, J = 7.2 Hz, 2H), 6.10 (s, 1H),
3.77 (s, 3H), 2.32 (s, 3H), 2.29 (s, 3H). MS (EI) m/z: 453.94(M)+.
White solid, yield: 88%. Mp 269–270 °C. 1H NMR (600 MHz,
DMSO-d6) d 13.32 (s, 1H), 8.67 (d, J = 1.8 Hz, 1H), 8.34 (dd, J = 8.4,
1.8 Hz, 1H), 7.78 (dd, J = 8.4, 3.6 Hz, 2H), 7.75–7.68 (m, 1H), 7.62
(dd, J = 5.4, 2.4 Hz, 2H), 2.16 (s, 3H). MS (EI) m/z: 314.18(M)+.
4.1.59. 1-Methyl-1H-pyrazol-5-yl-3-(4-bromophenyl)-2-methyl-
4-oxo-3,4-dihydroquinazoline-6-carboxylate
4.1.51. 2-Methyl-4-oxo-3-o-tolyl-3,4-dihydroquinazoline-6-
carboxylic acid (9x)
White solid, yield: 40%. Mp 250–252 °C. 1H NMR (600 MHz,
DMSO-d6)
d 8.82 (s, 1H), 8.52 (d, J = 8.4 Hz, 1H), 7.86 (d,
White solid, yield: 81%. Mp 255–257 °C. 1H NMR (600 MHz,
DMSO-d6) d 13.32 (s, 1H), 8.66 (s, 1H), 8.33 (d, J = 8.4 Hz, 1H),
7.77 (d, J = 9.0 Hz, 1H), 7.54–7.44 (m, 2H), 7.42 (d, J = 6.6 Hz, 2H),
2.13 (s, 3H), 2.05 (s, 3H). MS (EI) m/z: 294.24(M)+.
J = 8.4 Hz, 1H), 7.81 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H),
7.45 (s, 1H), 6.26 (d, J = 1.8 Hz, 1H), 3.75 (s, 3H), 2.20 (s, 3H). MS
(EI) m/z: 438.01(M)+.
4.1.60. 1-Methyl-1H-pyrazol-5-yl-2-methyl-4-oxo-3-phenyl-3,4-
dihydroquinazoline-6-carboxylate
4.1.52. 2-Methyl-4-oxo-3-m-tolyl-3,4-dihydroquinazoline-6-
carboxylic acid (9y)
White solid, yield: 45%. Mp 213–215 °C. 1H NMR (600 MHz,
CDCl3) d 9.09 (s, 1H), 8.50 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 9.0 Hz,
1H), 7.61 (t, J = 7.2 Hz, 2H), 7.56 (d, J = 6.6 Hz, 1H), 7.47 (s, 1H),
7.29 (d, J = 7.2 Hz, 2H), 6.28 (s, 1H), 3.82 (s, 3H), 2.32 (s, 3H). MS
(EI) m/z: 360.13(M)+.
White solid, yield: 88%. Mp 274–276 °C. 1H NMR (400 MHz,
DMSO-d6) d 13.26 (s, 1H), 8.64 (s, 1H), 8.29 (dd, J = 8.4, 1.6 Hz,
4H), 7.73 (d, J = 8.8 Hz, 1H), 7.36 (dd, J = 17.2, 8.4 Hz, 4H), 2.41 (s,
3H), 2.16 (s, 3H). MS (EI) m/z: 294.24(M)+.

Y.-L. Xu et al. / Bioorg. Med. Chem. 22 (2014) 5194–5211
5203
4.1.61. 1,3-Dimethyl-1H-pyrazol-5-yl-3-(4-chlorophenyl)-2-
methyl-4-oxo-3,4-dihydroquina zoline-6-carboxylate
4.1.69. 1-Methyl-1H-pyrazol-5-yl-3-(2-fluoro-4-methylphenyl)-
2-methyl-4-oxo-3,4-dihydroquinazoline-6-carboxylate
White solid, yield: 65%. Mp 193–195 °C. 1H NMR (600 MHz,
CDCl3) d 9.09 (d, J = 1.8 Hz, 1H), 8.49 (dd, J = 8.4, 2.4 Hz, 1H), 7.81
(d, J = 9.0 Hz, 1H), 7.47 (d, J = 1.8 Hz, 1H), 7.12–7.22 (m, 3H), 6.27
(d, J = 1.8 Hz, 1H), 3.82 (s, 3H), 2.47 (s, 3H), 2.34 (s, 3H). MS (EI)
m/z: 392.55(M)+.
White solid, yield: 47%. Mp 214–216 °C. 1H NMR (400 MHz,
CDCl3) d 9.02 (s, 1H), 8.46 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.4 Hz,
1H), 7.57 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H), 6.06 (s, 1H),
3.73 (s, 3H), 2.30 (s, 3H), 2.26 (s, 3H). MS (EI) m/z: 408.14(M)+.
4.1.62. 1-Methyl-1H-pyrazol-5-yl-3-(4-chlorophenyl)-2-methyl-
4-oxo-3,4-dihydroquina zoline-6-carboxylate
4.1.70. 1-Methyl-1H-pyrazol-5-yl-3-(4-fluoro-2-methylphenyl)-
2-methyl-4-oxo-3,4-dihydroquinazoline-6-carboxylate
White solid, yield: 51%. Mp 212–214 °C. 1H NMR (600 MHz,
CDCl3) d 9.10 (s, 1H), 8.51 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.4 Hz,
1H), 7.47 (d, J = 1.8 Hz, 1H), 7.17 (t, J = 9.0 Hz, 2H), 7.11 (t,
J = 8.4 Hz, 1H), 6.27 (d, J = 1.8 Hz, 1H), 3.82 (s, 3H), 2.25 (s, 3H),
2.14 (s, 3H). MS (EI) m/z: 392.66(M)+.
White solid, yield: 53%. Mp 226–228 °C. 1H NMR (400 MHz,
CDCl3) d 9.01 (d, J = 1.6 Hz, 1H), 8.46 (dd, J = 8.4, 2.0 Hz, 1H), 7.78
(d, J = 8.8 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 1.6 Hz, 1H),
7.25 (d, J = 8.4 Hz, 2H), 6.26 (d, J = 2.0 Hz, 1H), 3.81 (s, 3H), 2.30
(s, 3H). MS (EI) m/z: 394.21(M)+.
4.1.63. 1-Methyl-1H-pyrazol-5-yl-3-(4-methoxyphenyl)-2-
methyl-4-oxo-3,4-dihydroquinazoline-6-carboxylate
White solid, yield: 46%. Mp 207–209 °C. 1H NMR (400 MHz,
cdcl3) d 9.09 (s, 1H), 8.48 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 8.4 Hz,
1H), 7.47 (s, 1H), 7.19 (d, J = 8.8 Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H),
6.27 (s, 1H), 3.89 (s, 3H), 3.82 (s, 4H), 2.33 (s, 3H). MS (EI) m/z:
390.56(M)+.
4.1.71. 1,3-Dimethyl-1H-pyrazol-5-yl-3-(2-fluoro-4-
methylphenyl)-2-methyl-4-oxo-3,4-dihydroquinazoline-6-
carboxylate
White solid, yield: 61%. Mp 170–172 °C. 1H NMR (600 MHz,
CDCl3) d 9.08 (s, 1H), 8.48 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 9.0 Hz,
1H), 7.17 (m, 3H), 6.06 (s, 1H), 3.74 (s, 3H), 2.47 (s, 3H), 2.34 (s,
3H), 2.27 (s, 3H). MS (EI) m/z: 406.26(M)+.
4.1.64. 1,3-Dimethyl-1H-pyrazol-5-yl-3-(4-methoxyphenyl)-2-
methyl-4-oxo-3,4-dihydro quinazoline-6-carboxylate
White solid, yield: 41%. Mp 203–205 °C. 1H NMR (400 MHz,
CDCl3) d 9.01 (d, J = 2.0 Hz, 1H), 8.43 (dd, J = 8.8, 2.0 Hz, 1H), 7.75
(d, J = 8.8 Hz, 1H), 7.17 (d, J = 8.8 Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H),
6.04 (s, 1H), 3.86 (s, 3H), 3.71 (s, 3H), 2.29 (s, 3H), 2.24 (s, 3H).
MS (EI) m/z: 404.36(M)+.
4.1.72. 1,3-Dimethyl-1H-pyrazol-5-yl-3-(4-fluoro-2-
methylphenyl)-2-methyl-4-oxo-3,4-dihydroquinazoline-6-
carboxylate
White solid, yield: 47%. Mp 206–208 °C. 1H NMR (600 MHz,
CDCl3) d 9.09 (s, 1H), 8.50 (d, J = 9.0 Hz, 1H), 7.82 (d, J = 8.4 Hz,
1H), 7.16 (d, J = 6.0 Hz, 2H), 7.12 (t, J = 7.8 Hz, 1H), 6.07 (s, 1H),
3.75 (s, 3H), 2.28 (s, 3H), 2.26 (s, 3H), 2.14 (s, 3H). MS (EI) m/z:
406.32(M)+.
4.1.65. 1-Methyl-1H-pyrazol-5-yl-2-methyl-4-oxo-3-p-tolyl-3,4-
dihydroquinazoline-6-carboxylate
White solid, yield: 42%. Mp 219–221 °C. 1H NMR (400 MHz,
CDCl3) d 9.06 (s, 1H), 8.46 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.4 Hz,
1H), 7.47 (t, J = 7.6 Hz, 2H), 7.35 (d, J = 7.6 Hz, 1H), 7.09 (d,
J = 8.8 Hz, 2H), 6.27 (s, 1H), 3.81 (s, 3H), 2.45 (s, 3H), 2.31 (s, 3H).
MS (EI) m/z: 374.67(M)+.
4.1.73. 1-Methyl-1H-pyrazol-5-yl-3-(3-chlorophenyl)-2-methyl-
4-oxo-3,4-dihydro quinazoline-6-carboxylate
White solid, yield: 52%. Mp 212–214 °C. 1H NMR (600 MHz,
CDCl3) d 9.08 (s, 1H), 8.50 (d, J = 9.0 Hz, 1H), 7.83 (d, J = 8.4 Hz,
1H), 7.56 (s, 2H), 7.48 (s, 1H), 7.33 (s, 1H), 7.21 (s, 1H), 6.28 (s,
1H), 3.83 (s, 3H), 2.34 (s, 3H). MS (EI) m/z: 394.44(M)+.
4.1.66. 1,3-Dimethyl-1H-pyrazol-5-yl-2-methyl-4-oxo-3-p-tolyl-
3,4-dihydroquinazoline-6-carboxylate
4.1.74. 1,3-Dimethyl-1H-pyrazol-5-yl-3-(3-chlorophenyl)-2-
methyl-4-oxo-3,4-dihydro quinazoline-6-carboxylate
White solid, yield: 49%. Mp 245–247 °C. 1H NMR
(400 MHz, CDCl3) d 9.05 (s, 1H), 8.48 (d, J = 8.4 Hz, 1H), 7.80 (d,
J = 8.4 Hz, 1H), 7.55 (d, J = 4.4 Hz, 2H), 7.33 (s, 1H), 7.21 (s, 1H),
6.08 (s, 1H), 3.75 (s, 3H), 2.33 (s, 3H), 2.27 (s, 3H). MS (EI) m/z:
408.39(M)+.
White solid, yield: 48%. Mp 204–205 °C. 1H NMR (400 MHz,
CDCl3) d 9.05 (s, 1H), 8.46 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.4 Hz,
1H), 7.38 (d, J = 8.0 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H), 6.06 (s, 1H),
3.74 (s, 3H), 2.46 (s, 3H), 2.31 (s, 3H), 2.27 (s, 3H). MS (EI) m/z:
388.26(M)+.
4.1.67. 1,3-Dimethyl-1H-pyrazol-5-yl-3-(2,4-dimethylphenyl)-2-
methyl-4-oxo-3,4-dihydro quinazoline-6-carboxylate
4.1.75. 1-Methyl-1H-pyrazol-5-yl-3-(3-methoxyphenyl)-2-
methyl-4-oxo-3,4-dihydro quinazoline-6-carboxylate
White solid, yield: 48%. Mp 212–214 °C. 1H NMR (600 MHz,
CDCl3) d 9.09 (s, 1H), 8.49 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 8.4 Hz,
1H), 7.50 (t, J = 7.8, Hz, 1H), 7.47 (s, 1H), 7.08 (d, J = 7.8 Hz, 1H),
6.87 (d, J = 7.2 Hz, 1H), 6.81 (s, 1H), 6.28 (s, 1H), 3.86 (s, 3H), 3.82
(s, 3H), 2.35 (s, 3H). MS (EI) m/z: 293.28(M-97)+.
White solid, yield: 44%. Mp 201–203 °C. 1H NMR (400 MHz,
CDCl3) d 9.09 (s, 1H), 8.49 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.8 Hz,
1H), 7.25 (d, J = 9.6 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.05 (d,
J = 7.6 Hz, 1H), 6.07 (s, 1H), 3.75 (s, 3H), 2.42 (s, 3H), 2.28 (s, 3H),
2.26 (s, 3H), 2.10 (s, 3H). MS (EI) m/z: 291.30(M-111)+.
4.1.68. 1-Methyl-1H-pyrazol-5-yl-3-(2,4-dimethylphenyl)-2-
methyl-4-oxo-3,4-dihydro quinazoline-6-carboxylate
White solid, yield: 41%. Mp 189–191 °C. 1H NMR (400 MHz,
CDCl3) d 9.11 (d, J = 2.0 Hz, 1H), 8.51 (dd, J = 8.4, 2.0 Hz, 1H), 7.89
(d, J = 8.4 Hz, 1H), 7.48 (d, J = 2.0 Hz, 1H), 7.25 (s, 1H), 7.21 (d,
J = 8.0 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 6.28 (d, J = 2.0 Hz, 1H),
3.83 (s, 3H), 2.43 (s, 3H), 2.30 (s, 3H), 2.10 (s, 3H). MS (EI) m/z:
388.26(M)+.
4.1.76. 1-Methyl-1H-pyrazol-5-yl-2-methyl-4-oxo-3-(4-
(trifluoromethoxy)phenyl)-3,4-dihydroquinazoline-6-
carboxylate
White solid, yield: 33%. Mp 202–203 °C. 1H NMR (600 MHz,
CDCl3) d 9.08 (s, 1H), 8.51 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.4 Hz,
1H), 7.46 (t, J = 9.6 Hz, 3H), 7.35 (d, J = 7.8 Hz, 2H), 6.28 (s, 1H),
3.83 (s, 3H), 2.33 (s, 3H). MS (EI) m/z: 347.27(M-97)+.

5204
Y.-L. Xu et al. / Bioorg. Med. Chem. 22 (2014) 5194–5211
4.1.77. 1-Methyl-1H-pyrazol-5-yl-2-methyl-4-oxo-3-(3-
(trifluoromethoxy)phenyl)-3,4-dihydroquinazoline-6-
carboxylate
4.1.85. 1-Methyl-1H-pyrazol-5-yl-3-(2-bromo-4-methylphenyl)-
2-methyl-4-oxo-3,4-dihydro quinazoline-6-carboxylate
White solid, yield: 40%. Mp 205–207 °C. 1H NMR (600 MHz,
CDCl3) d 9.11 (s, 1H), 8.51 (d, J = 9.0 Hz, 1H), 7.84 (d, J = 8.4 Hz,
1H), 7.64 (s, 1H), 7.47 (s, 1H), 7.35 (d, J = 7.2 Hz, 1H), 7.24 (d,
J = 6.6 Hz, 1H), 6.27 (s, 1H), 3.83 (s, 3H), 2.47 (s, 3H), 2.30 (s, 3H).
MS (EI) m/z: 452.43(M)+.
White solid, yield: 35%. Mp 169–171 °C. 1H NMR (600 MHz,
CDCl3) d 9.08 (s, 1H), 8.51 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 9.0 Hz,
1H), 7.66 (t, J = 7.8 Hz, 1H), 7.48 (s, 1H), 7.44 (d, J = 8.4 Hz, 1H)
7.27 (s, 1H), 7.20 (s, 1H), 6.28 (s, 1H), 3.83 (s, 3H), 2.32 (s, 3H).
MS (EI) m/z: 444.26(M)+.
4.1.86. 1,3-Dimethyl-1H-pyrazol-5-yl-3-(2-bromo-4-
methylphenyl)-2-methyl-4-oxo-3,4-dihydroquinazoline-6-
carboxylate
4.1.78. 1-Methyl-1H-pyrazol-5-yl-3-(3-fluorophenyl)-2-methyl-
4-oxo-3,4-dihydroquinazoline-6-carboxylate
White solid, yield: 31%. Mp 217–218 °C. 1H NMR (600 MHz,
CDCl3) d 9.08 (s, 1H), 8.50 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.4 Hz,
1H), 7.59 (dd, J = 14.4,7.8 Hz, 1H), 7.47 (s, 1H), 7.29 (t, J = 8.64 Hz,
1H), 7.11 (d, J = 8.4 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 6.28 (s, 1H),
3.83 (s, 3H), 2.34 (s, 3H). MS (EI) m/z: 378.30(M)+.
White solid, yield: 61%. Mp 212–214 °C. 1H NMR (600 MHz,
CDCl3) d 9.10 (s, 1H), 8.49 (d, J = 9.0 Hz, 1H), 7.81 (d, J = 8.4 Hz,
1H), 7.64 (s, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 7.8 Hz, 1H),
6.06 (s, 1H), 3.74 (s, 3H), 2.46 (s, 3H), 2.29 (s, 3H), 2.27 (s, 3H).
MS (EI) m/z: 466.27(M)+.
4.1.79. 1,3-Dimethyl-1H-pyrazol-5-yl-3-(3-fluorophenyl)-2-
methyl-4-oxo-3,4-dihydroquina zoline-6-carboxylate
4.1.87. 1-Methyl-1H-pyrazol-5-yl-3-(3,4-dichlorophenyl)-2-
methyl-4-oxo-3,4-dihydro quinazoline-6-carboxylate
White solid, yield: 52%. Mp 247–249 °C. 1H NMR (600 MHz,
CDCl3) d 9.06 (s, 1H), 8.50 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 8.4 Hz,
1H), 7.69 (d, J = 8.4 Hz, 1H), 7.47 (s, 1H), 7.45 (s, 1H), 7.18 (d,
J = 8.4 Hz, 1H), 6.28 (s, 1H), 3.83 (s, 3H), 2.34 (s, 3H). MS (EI) m/z:
428.66(M)+.
White solid, yield: 31%. Mp 237–239 °C. 1H NMR (600 MHz,
CDCl3) d 9.07 (d, J = 1.8 Hz, 1H), 8.50 (dd, J = 8.4, 1.8 Hz, 1H), 7.81
(d, J = 8.4 Hz, 1H), 7.59 (m, 1H), 7.29 (dd, J = 8.4, 1.8 Hz, 1H), 7.11
(d, J = 7.8 Hz, 1H), 7.08–7.02 (m, 1H), 6.07 (s, 1H), 3.74 (s, 3H),
2.33 (s, 3H), 2.28 (s, 3H). MS (EI) m/z: 392.04(M)+.
4.1.80. 1-Methyl-1H-pyrazol-5-yl-3-(3-bromophenyl)-2-methyl-
4-oxo-3,4-dihydroquinazoline-6-carboxylate
4.1.88. 1-Methyl-1H-pyrazol-5-yl-3-(2-chlorophenyl)-2-methyl-
4-oxo-3,4-dihydroquina zoline-6-carboxylate
White solid, yield: 40%. Mp 220–221 °C. 1H NMR (600 MHz,
CDCl3) d 9.07 (s, 1H), 8.50 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 8.4 Hz,
1H), 7.70 (d, J = 8.4 Hz, 1H), 7.48 (t, J = 7.8 Hz, 3H), 7.25 (s, 1H),
6.28 (s, 1H), 3.82 (s, 3H), 2.33 (s, 3H). MS (EI) m/z: 341.20(M-97)+.
White solid, yield: 41%. Mp 179–181 °C. 1H NMR (600 MHz,
CDCl3) d 9.11 (d, J = 1.8 Hz, 1H), 8.51 (dd, J = 8.4, 1.8 Hz, 1H), 7.84
(d, J = 8.4 Hz, 1H), 7.66 (dd, J = 7.8
, 1.8 Hz, 1H), 7.59–7.49
(m, 2H), 7.47 (d, J = 1.8 Hz, 1H), 7.37 (dd, J = 6.6, 1.8 Hz, 1H), 6.28
(d, J = 1.8 Hz, 1H), 3.82 (s, 3H), 2.30 (s, 3H). MS (EI) m/z:
394.64(M)+.
4.1.81. 1,3-Dimethyl-1H-pyrazol-5-yl-3-(3-bromophenyl)-2-
methyl-4-oxo-3,4-dihydro quinazoline-6-carboxylate
White solid, yield: 44%. Mp 249–251 °C. 1H NMR (600 MHz,
CDCl3) d 9.06 (s, 1H), 8.49 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 8.4 Hz,
1H), 7.70 (d, J = 7.8 Hz, 1H), 7.49 (t, J = 8.4 Hz, 2H), 7.25 (s, 1H),
6.09 (s, 1H), 3.75 (s, 3H), 2.33 (s, 3H), 2.28 (s, 3H). MS (EI) m/z:
452.48(M)+.
4.1.89. 1,3-Dimethyl-1H-pyrazol-5-yl-3-(2-chlorophenyl)-2-
methyl-4-oxo-3,4-dihydro quinazoline-6-carboxylate
White solid, yield: 39%. Mp 199–201 °C. 1H NMR (600 MHz,
cdcl3) d 9.09 (d, J = 1.8 Hz, 1H), 8.50 (dd, J = 8.4, 1.8 Hz, 1H), 7.82
(d, J = 8.4 Hz, 1H), 7.66 (dd, J = 6.6, 1.8 Hz, 1H), 7.57–7.45 (m, 2H),
7.37 (d, J = 6.6, 1.8 Hz, 1H), 6.07 (s, 1H), 3.74 (s, 3H), 2.29 (s, 3H),
2.27 (s, 3H). MS (EI) m/z: 408.08(M)+.
4.1.82. 1-Methyl-1H-pyrazol-5-yl-3-(2-chloro-4-methylphenyl)-
2-methyl-4-oxo-3,4-dihydro quinazoline-6-carboxylate
White solid, yield: 51%. Mp 211–213 °C. 1H NMR (600 MHz,
CDCl3) d 9.10 (d, J = 1.8 Hz, 1H), 8.50 (d, J = 9.0 Hz, 1H), 7.82 (d,
J = 8.4 Hz, 1H), 7.46 (s, 2H), 7.29 (d, J = 8.4 Hz, 1H), 7.23 (d,
J = 7.8 Hz, 1H), 6.26 (d, J = 1.8 Hz, 1H), 3.82 (s, 3H), 2.46 (s, 3H),
2.29 (s, 3H). MS (EI) m/z: 408.49(M)+.
4.1.90. 1,3-Dimethyl-1H-pyrazol-5-yl-3-(2-fluorophenyl)-2-
methyl-4-oxo-3,4-dihydro quinazoline-6-carboxylate
White solid, yield: 51%. Mp 172–174 °C. 1H NMR (600 MHz,
CDCl3) d 9.09 (d, J = 1.8 Hz, 1H), 8.50 (dd, J = 8.4, 1.8 Hz, 1H), 7.82
(d, J = 8.4 Hz, 1H), 7.56 (dd, J = 8.4 , 2.0 Hz, 1H), 7.47 (d, J = 1.8 Hz,
1H), 7.41–7.30 (m, 3H), 6.27 (d, J = 1.8 Hz, 1H), 3.82 (s, 3H), 2.34
(s, 3H). MS (EI) m/z: 378.57(M)+.
4.1.83. 1-Methyl-1H-pyrazol-5-yl-3-(4-chloro-2-methylphenyl)-
2-methyl-4-oxo-3,4-dihydro quinazoline-6-carboxylate
White solid, yield: 60%. Mp 226–228 °C. 1H NMR (600 MHz,
CDCl3) d 9.10 (s, 1H), 8.51 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.4 Hz,
1H), 7.47 (d, J = 15 Hz, 2H), 7.40 (d, J = 8.4 Hz, 1H), 7.14 (d,
J = 8.4 Hz, 1H), 6.28 (s, 1H), 3.83 (s, 3H), 2.27 (s, 3H), 2.14 (s, 3H).
MS (EI) m/z: 408.44(M)+.
4.1.91. 1,3-Dimethyl-1H-pyrazol-5-yl-3-(2-fluorophenyl)-2-
methyl-4-oxo-3,4-dihydro quinazoline-6-carboxylate
White solid, yield: 40%. Mp 205–207 °C. 1H NMR (600 MHz,
CDCl3) d 9.08 (s, 1H), 8.49 (d, J = 9.0 Hz, 1H), 7.81 (d, J = 8.4 Hz,
1H), 7.56 (d, J = 6.0 Hz, 1H), 7.47–7.29 (m, 3H), 6.08 (s, 1H), 3.75
(s, 3H), 2.34 (s, 3H), 2.28 (s, 3H). MS (EI) m/z: 392.13(M)+.
4.1.84. 1,3-Dimethyl-1H-pyrazol-5-yl-3-(2-chloro-4-
methylphenyl)-2-methyl-4-oxo-3,4-dihydroquinazoline-6-
carboxylate
4.1.92. 1-Methyl-1H-pyrazol-5-yl-3-(2-methoxyphenyl)-2-
methyl-4-oxo-3,4-dihydroquina zoline-6-carboxylate
White solid, yield: 52%. Mp 183–185 °C. 1H NMR (600 MHz,
CDCl3) d 9.10 (s, 1H), 8.50 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.4 Hz,
1H), 7.47 (s, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 7.8 Hz, 1H),
6.07 (s, 1H), 3.75 (s, 3H), 2.47 (s, 3H), 2.30 (s, 3H), 2.28 (s, 3H).
MS (EI) m/z: 311.21(M-111)+.
White solid, yield: 45%. Mp 195–197 °C. 1H NMR (600 MHz,
CDCl3) d 9.10 (s, 1H), 8.48 (d, J = 9.0 Hz, 1H), 7.81 (d, J = 8.4 Hz,
1H), 7.52 (t, J = 7.8 Hz, 1H), 7.47 (d, J = 1.8 Hz, 1H), 7.23 (d,
J = 7.8 Hz, 1H), 7.18–7.09 (m, 2H), 6.27 (d, J = 1.8 Hz, 1H), 3.82 (s,
2H), 3.82 (s, 3H), 2.29 (s, 3H). MS (EI) m/z: 389.44(M-1)+.

Y.-L. Xu et al. / Bioorg. Med. Chem. 22 (2014) 5194–5211
5205
4.1.93. 1-Methyl-1H-pyrazol-5-yl-2-methyl-4-oxo-3-o-tolyl-3,4-
dihydroquinazoline-6-carboxylate
4.1.100. 6-(5-Hydroxy-1,3-dimethyl-1H-pyrazole-4-carbonyl)-2-
methyl-3-o-tolylquinazolin-4(3H)-one (2b)
White solid, yield: 49%. Mp 199–200 °C. 1H NMR (600 MHz,
CDCl3) d 9.11 (d, J = 1.8 Hz, 1H), 8.50 (dd, J = 8.4, 1.8 Hz, 1H), 7.84
(d, J = 9.0 Hz, 1H), 7.44 (m, 4H), 7.18 (d, J = 7.2 Hz, 1H), 6.27 (d,
J = 1.8 Hz, 1H), 3.82 (s, 3H), 2.26 (s, 3H), 2.15 (s, 3H). MS (EI) m/z:
374.35(M)+.
White solid, mp 181–184 °C. Yield: 38.5%. 1H NMR (600 MHz,
CDCl3) d 8.57 (s, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 7.8 Hz,
1H), 7.43 (s, 2H), 7.41 (d, J = 7.2 Hz, 1H), 7.19 (d, J = 6.6 Hz, 1H),
3.65 (s, 3H), 2.24 (s, 3H), 2.14 (s, 3H), 2.08 (s, 3H). 13C NMR
(100 MHz, CDCl3)
d 190.91, 160.96, 160.23, 156.36, 149.61,
146.68, 136.24, 136.16, 135.07, 133.52, 131.58, 129.73, 127.69,
127.15, 120.07, 102.25, 32.49, 23.94, 17.32, 15.78. MS (EI) m/z:
388.37(M)+. Anal. Calcd for C22H20N4O3: C, 68.03; H, 5.19; N,
14.42. Found: C, 68.13; H, 5.40; N, 14.13.
4.1.94. 1,3-Dimethyl-1H-pyrazol-5-yl-2-methyl-4-oxo-3-o-tolyl-
3,4-dihydroquinazoline-6-carboxylate
White solid, yield: 50%. Mp 222–224 °C. 1H NMR (600 MHz,
CDCl3) d 9.10 (d, J = 1.8 Hz, 1H), 8.49 (dd, J = 8.4, 1.8 Hz, 1H), 7.81
(d, J = 9.0 Hz, 1H), 7.42 (m, 3H), 7.18 (d, J = 7.8 Hz, 1H), 6.07 (s,
1H), 3.74 (s, 3H), 2.27 (s, 3H), 2.25 (s, 3H), 2.15 (s, 3H). MS (EI)
m/z: 388.26(M)+.
4.1.101. 6-(5-Hydroxy-1,3-dimethyl-1H-pyrazole-4-carbonyl)-3-
(2-methoxyphenyl)-2-methylquinazolin-4(3H)-one (2c)
White solid, mp 219–221 °C. Yield: 59.4%. 1H NMR (600 MHz,
CDCl3) d 8.55 (s, 1H), 8.03 (d, J = 9.0 Hz, 1H), 7.85 (s, 1H), 7.51 (t,
J = 8.4 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.15 (t, J = 7.8 Hz, 1H), 7.11
(d, J = 9.0 Hz, 1H), 3.82 (s, 3H), 3.65 (s, 3H), 2.31 (s, 3H), 2.09 (s,
3H). 13C NMR (100 MHz, CDCl3) d 190.92, 161.20, 160.18, 157.28,
154.28, 149.43, 146.81, 136.01, 133.44, 131.11, 129.05, 127.75,
126.92, 125.49, 121.42, 120.15, 112.28, 102.27, 55.71, 32.51,
23.46, 15.82. MS (EI) m/z: 404.32(M)+. Anal. Calcd for
C22H20N4O4: C, 65.34; H, 4.98; N, 13.85. Found: C, 65.62; H, 4.89;
N, 13.75.
4.1.95. Methyl-1H-pyrazol-5-yl-3-(2-bromophenyl)-2-methyl-4-
oxo-3,4-dihydroquinazoline-6-carboxylate
White solid, yield: 41%. Mp 187–188 °C. 1H NMR (600 MHz,
CDCl3) d 9.11 (s, 1H), 8.52 (d, J = 8.4 Hz, 1H), 7.83 (dd, J = 9.0,
4.8 Hz, 2H), 7.57 (t, J = 7.2 Hz, 1H), 7.48 (s, 1H), 7.45 (t, J = 7.8 Hz,
1H), 7.38 (d, J = 7.8 Hz, 1H), 6.28 (s, 1H), 3.83 (s, 3H), 2.29 (s, 3H).
MS (EI) m/z: 439.32(M)+.
4.1.96. 1-Methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl-3-(4-
methoxyphenyl)-2-methyl-4-oxo-3,4-dihydroquinazoline-6-
carboxylate
4.1.102. 3-(2-Fluorophenyl)-6-(5-hydroxy-1,3-dimethyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (2d)
White solid, mp 193–195 °C. Yield: 29.4%. 1H NMR (600 MHz,
CDCl3) d 8.54 (s, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 9.0 Hz,
1H), 7.55 (t, J = 6.0 Hz, 1H), 7.35 (m, 3H), 3.65 (s, 3H), 2.35 (s,
3H), 2.08 (s, 3H).13C NMR (101 MHz, CDCl3) d 190.87, 161.01,
160.23, 158.63, 156.14, 155.88, 149.47, 146.71, 136.34, 133.73,
131.66, 129.77, 127.61, 127.33, 125.38, 119.85, 117.18, 116.98,
102.26, 32.51, 23.69, 15.81. MS (EI) m/z: 392.03(M)+. Anal. Calcd
for C21H17FN4O3: C, 64.28; H, 4.37; N, 14.28. Found: C, 64.13; H,
4.35; N, 13.99.
White solid, yield: 60%. Mp 162–164 °C. 1H NMR (600 MHz,
CDCl3) d 9.06 (s, 1H), 8.47 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.4 Hz,
1H), 7.19 (d, J = 8.4 Hz, 2H), 7.09 (d, J = 8.4 Hz, 2H), 6.60 (s, 1H),
3.90 (s, 3H), 3.88 (s, 3H), 2.34 (s, 3H). MS (EI) m/z: 458.80(M)+.
4.1.97. 1,3-Dimethyl-1H-pyrazol-5-yl-3-(4-chloro-2-
methylphenyl)-2-methyl-4-oxo-3,4-dihydroquinazoline-6-
carboxylate
White solid, yield: 54%. Mp 220–222 °C. 1H NMR (600 MHz,
CDCl3) d 9.08 (s, 1H), 8.50 (d, J = 9.0 Hz, 1H), 7.84 (d, J = 7.8 Hz,
1H), 7.45 (s, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H),
6.11 (s, 1H), 3.79 (s, 3H), 2.31 (s, 3H), 2.27 (s, 3H), 2.14 (s, 3H).
MS (EI) m/z: 422.28(M)+.
4.1.103. 6-(5-Hydroxy-1,3-dimethyl-1H-pyrazole-4-carbonyl)-2-
methyl-3-m-tolylquinazolin-4(3H)-one (2e)
White solid, mp 222–223 °C. Yield: 37.8%. 1H NMR (600 MHz,
CDCl3) d 8.54 (s, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 9.0 Hz,
1H), 7.46 (t, J = 7.8 Hz, 1H), 7.33 (d, J = 7.8 Hz, 1H), 7.08 (d,
J = 12.0 Hz, 2H), 3.65 (s, 3H), 2.44 (s, 3H), 2.30 (s, 3H), 2.08 (s,
3H). 13C NMR (100 MHz, CDCl3) d 190.85, 161.59, 160.14, 156.37,
149.44, 146.72, 140.22, 137.04, 136.07, 133.47, 130.24, 129.81,
128.17, 127.59, 127.16, 124.66, 120.03, 102.26, 32.47, 24.40,
21.26, 15.79. MS (EI) m/z: 388.03(M)+. Anal. Calcd for
4.1.98. General procedure for title compounds 2a–t and 3a–y
The mixture of intermediate 10 (1 mmol) dissolved in acetoni-
trile (15 mL), triethylamine (0.32 mL), and acetone cyanohydrin
(0.01 mL) was stirred at room temperature for 3–8 h. When the
reaction was completed, as monitored by TLC detection, the sol-
vent was removed under reduced pressure. After adding 30 mL
dichloromethane, the solution was washed sequentially with 1 N
hydrochloric acid (30 mL), water and saturated brine, and dried
with anhydrous sodium sulfate. The solvent was removed under
reduced pressure. Finally, the desired products were recrystallized
from ethyl acetate/petroleum ether (1:10) after solvent evapora-
tion under reduced pressure.
C22H20N4O3: C, 68.03; H, 5.19; N, 14.42. Found: C, 67.79; H, 5.33;
N, 14.13.
4.1.104. 3-(3-Chlorophenyl)-6-(5-hydroxy-1,3-dimethyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (2f)
White solid, mp 216–219 °C. Yield: 51%. 1H NMR (600 MHz,
CDCl3) d 8.53 (s, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.4 Hz,
1H), 7.52 (s, 2H), 7.33 (s, 1H), 7.21 (s, 1H), 3.65 (s, 3H), 2.31 (s,
3H), 2.07 (s, 3H). 13C NMR (100 MHz, CDCl3) d 190.87, 161.38,
160.25, 155.62, 149.28, 146.66, 138.19, 136.41, 135.67, 133.75,
131.05, 129.91, 128.32, 127.54, 127.32, 126.28, 119.91, 102.27,
32.53, 24.41, 15.83. MS (EI) m/z: 408.05(M)+. Anal. Calcd for C21H17
ClN4O3: C, 61.69; H, 4.19; N, 13.70. Found: C, 61.73; H, 4.31; N,
13.62.
4.1.99. 3-(2-Chlorophenyl)-6-(5-hydroxy-1,3-dimethyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (2a)
White solid, mp 187–189 °C. Yield: 31%. 1H NMR (600 MHz,
CDCl3)
d 8.56 (s, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.89 (d,
J = 7.2 Hz, 1H), 7.65 (d, J = 6.6 Hz, 1H), 7.52 (d, J = 3.6 Hz, 2H),
7.39 (d, J = 6.6 Hz, 1H), 3.66 (s, 3H), 2.33 (s, 3H), 2.08 (s, 3H).
13C NMR (100 MHz, CDCl3) d 190.88, 160.79, 160.27, 155.82,
149.45, 146.70, 136.33, 134.86, 133.75, 132.31, 131.03, 130.83,
129.66, 128.48, 127.66, 127.26, 119.96, 102.25, 32.53, 23.61,
15.81. MS (EI) m/z: 408.05(M)+. Anal. Calcd for C21H17ClN4O3:
C, 61.69; H, 4.19; N, 13.70. Found: C, 61.36; H, 4.37; N,
13.47.
4.1.105. 3-(3-Bromophenyl)-6-(5-hydroxy-1,3-dimethyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (2g)
White solid, mp 226–228 °C. Yield: 37.8%. 1H NMR (600 MHz,
CDCl3) d 8.53 (s, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.4 Hz,

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Y.-L. Xu et al. / Bioorg. Med. Chem. 22 (2014) 5194–5211
1H), 7.68 (d, J = 7.8 Hz, 1H), 7.48 (s, 1H), 7.46 (d, J = 7.8 Hz, 1H),
3.65 (s, 3H), 2.31 (s, 3H), 2.07 (s, 3H). 13C NMR (100 MHz, CDCl3)
d 190.79, 161.19, 160.29, 156.04, 148.66, 146.69, 138.10, 136.65,
133.89, 132.94, 131.34, 131.12, 127.56, 126.95,126.75, 123.46,
119.85, 102.32, 32.55, 24.20, 15.80. MS (EI) m/z: 452.04(M)+. Anal.
Calcd for C21H17BrN4O3: C, 55.64; H, 3.78; N, 12.36. Found: C,
55.70; H, 3.99; N, 12.65.
4.1.111. 3-(4-Chlorophenyl)-6-(5-hydroxy-1,3-dimethyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (2m)
White solid, mp 131–134 °C. Yield: 31%. 1H NMR (400 MHz,
CDCl3)
d 8.52 (s, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.78 (d,
J = 8.4 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H),
3.65 (s, 3H), 2.29 (s, 3H), 2.06 (s, 3H). 13C NMR (100 MHz,
CDCl3)
d 190.93, 161.54, 160.29, 155.78, 149.40, 146.70,
136.40, 135.65, 133.71, 130.39, 129.31, 127.57, 127.35, 119.98,
102.29, 32.55, 24.46, 15.82. MS (EI) m/z: 408.13(M)+. Anal. Calcd
for C21H17N4O3: C, 61.69; H, 4.19; N, 13.70. Found: C, 61.46; H,
3.88; N, 13.92.
4.1.106. 3-(3-Fluorophenyl)-6-(5-hydroxy-1,3-dimethyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (2h)
White solid, mp 183–184 °C. Yield: 27%. 1H NMR (600 MHz,
CDCl3) d 8.53 (s, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 8.4 Hz,
1H), 7.58 (dd, J = 14.4, 7.8 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 7.12 (d,
J = 7.8 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 3.66 (s, 3H), 2.37 (s, 3H),
2.06 (s, 3H). 13C NMR (100 MHz, CDCl3) d 190.56, 164.27, 161.78,
161.25, 159.88, 155.62, 149.13, 146.64, 138.35, 138.25, 136.30,
133.64, 131.28, 131.19, 127.42, 127.11, 123.79, 119.81, 116.81,
116.60, 115.76, 115.53, 102.21, 32.39, 24.14, 15.62. MS (EI) m/z:
392.41(M)+. Anal. Calcd for C21H17FN4O3: C, 64.28; H, 4.37; N,
14.28. Found: C, 63.94; H, 4.41; N, 14.63.
4.1.112. 3-(2,4-Dimethylphenyl)-6-(5-hydroxy-1,3-dimethyl-
1H-pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (2n)
White solid, mp 146–148 °C. Yield: 51%. 1H NMR (600 MHz,
CDCl3) d 8.57 (s, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 8.4 Hz,
1H), 7.23 (s, 1H), 7.19 (d, J = 7.8 Hz, 1H), 7.06 (d, J = 7.8 Hz, 1H),
3.65 (s, 3H), 2.41 (s, 3H), 2.25 (s, 3H), 2.09 (s, 3H), 2.08 (s, 3H).
13C NMR (100 MHz, CDCl3) d 190.90, 161.07, 160.18, 156.67,
149.60, 146.70, 139.73, 136.09, 134.59, 133.58, 133.43, 132.25,
128.34, 127.65, 127.35, 127.08, 120.07, 102.25, 32.47, 23.89,
21.08, 17.21, 15.73. MS (EI) m/z: 402.20(M)+. Anal. Calcd for
C23H22N4O3: C, 68.64; H, 5.51; N, 13.92. Found: C, 68.37; H, 5.88;
N, 13.64.
4.1.107. 3-(4-Fluorophenyl)-6-(5-hydroxy-1,3-dimethyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (2i)
White solid, mp 185–187 °C. Yield: 31%. 1H NMR (600 MHz,
cdcl3) d 8.53 (s, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.3 Hz,
1H), 7.28 (d, J = 6.5 Hz, 4H), 3.65 (s, 3H), 2.30 (s, 3H), 2.07 (s, 3H).
13C NMR (100 MHz, CDCl3) d 190.71, 164.00, 161.51, 160.07,
156.20, 149.04, 146.68, 136.38, 133.68, 132.92, 129.75, 129.67,
127.54, 127.06, 119.90, 117.27, 117.04, 102.26, 32.49, 24.32,
15.72. MS (EI) m/z: 392.10(M)+. Anal. Calcd for C21H17FN4O3: C,
64.28; H, 4.37; N, 14.28. Found: C, 64.49; H, 4.54; N, 14.52.
4.1.113. 3-(2-Fluoro-4-methylphenyl)-6-(5-hydroxy-1,3-
dimethyl-1H-pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-
one (2o)
White solid, mp 200–201 °C. Yield: 50%. 1H NMR (600 MHz,
CDCl3) d 8.54 (d, J = 2.4 Hz, 1H), 8.03 (dd, J = 8.4, 1.2 Hz, 1H), 7.79
(d, J = 8.4 Hz, 1H), 7.20 (t, J = 8.4 Hz, 1H), 7.15 (t, J = 7.8 Hz, 2H),
3.65 (s, 3H), 2.46 (s, 3H), 2.34 (s, 3H), 2.08 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) d 187.69, 160.74, 157.91, 155.85, 155.45,
149.19, 148.24, 142.31, 142.23, 137.24, 134.77, 130.08, 127.70,
126.44, 126.11, 122.05, 121.91, 119.23,117.00, 116.81, 103.09,
31.79, 23.54, 20.80, 14.08. MS (EI) m/z: 406.34(M)+. Anal. Calcd
for C22H19FN4O3: C, 65.02; H, 4.71; N, 13.79. Found: C, 65.26; H,
4.43; N, 13.58.
4.1.108. 6-(5-Hydroxy-1,3-dimethyl-1H-pyrazole-4-carbonyl)-3-
(4-methoxyphenyl)-2-methylquinazolin-4(3H)-one (2j)
White solid, mp 125–128 °C. Yield: 41%. 1H NMR (400 MHz,
CDCl3) d 8.54 (s, 1H), 8.03 (d, J = 7.6 Hz, 1H), 7.88 (s, 1H), 7.20 (d,
J = 8.8 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 3.89 (s, 3H), 3.65 (s, 3H),
2.37 (s, 3H), 2.07 (s, 3H).13C NMR (150 MHz, CDCl3) d 190.89,
161.83, 160.16, 160.03, 156.89, 149.38, 146.79, 136.08, 133.48,
129.55, 128.75, 127.63, 127.13, 120.03, 115.2, 102.25, 55.48,
32.51, 24.45, 15.78. MS (EI) m/z: 404.08(M)+. Anal. Calcd for
4.1.114. 3-(4-Fluoro-2-methylphenyl)-6-(5-hydroxy-1,3-
dimethyl-1H-pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-
one (2p)
C22H20N4O4: C, 65.34; H, 4.98; N, 13.85. Found: C, 65.07; H, 4.99;
N, 13.93.
White solid, mp 156–158 °C. Yield: 30%. 1H NMR (600 MHz,
CDCl3)
d 8.55 (s, 1H), 8.04 (d, J = 8.3 Hz, 1H), 7.81 (d,
4.1.109. 3-(4-Bromophenyl)-6-(5-hydroxy-1,3-dimethyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (2k)
White solid, mp 160–162 °C. Yield: 45%. 1H NMR (600 MHz,
CDCl3) d 8.53 (s, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 9.0 Hz,
1H), 7.73 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 7.8 Hz, 2H), 3.66 (s, 3H),
2.32 (s, 3H), 2.06 (s, 3H). 13C NMR (100 MHz, CDCl3) d 190.89,
161.43, 160.24, 155.78, 149.23, 146.71, 136.44, 136.11, 133.73,
133.38, 129.59, 127.56, 127.24, 123.74, 119.93, 102.30, 32.54,
24.40, 15.78. MS (EI) m/z: 452.08(M)+. Anal. Calcd for C21H17BrN4-
O3: C, 55.64; H, 3.78; N, 12.36. Found: C, 55.87; H, 3.94 N, 12.48.
J = 8.3 Hz, 1H), 7.21–7.13 (m, 2H), 7.10 (t, J = 8.3 Hz, 1H), 3.66
(s, 3H), 2.25 (s, 3H), 2.14 (s, 3H), 2.07 (s, 3H). 13C NMR
(100 MHz, CDCl3)
d 190.82, 164.01, 161.53, 160.91, 160.26,
156.37, 149.20, 146.63, 137.87, 137.79, 136.46, 133.71, 132.03,
129.52, 127.63, 127.05, 119.95, 118.46, 118.26, 114.85, 114.62,
102.26, 32.52, 23.83, 17.56, 15.76. MS (EI) m/z: 406.36(M)+.
Anal. Calcd for C22H19FN4O3: C, 65.02; H, 4.71; N, 13.79. Found:
C, 65.33; H, 4.79; N, 13.51.
4.1.115. 3-(2-Bromo-4-methylphenyl)-6-(5-hydroxy-1,3-
dimethyl-1H-pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-
one (2q)
4.1.110. 6-(5-Hydroxy-1,3-dimethyl-1H-pyrazole-4-carbonyl)-2-
methyl-3-p-tolylquinazolin-4(3H)-one (2l)
White solid, mp 131–133 °C. Yield: 69%. 1H NMR (600 MHz,
CDCl3) d 8.56 (d, J = 1.8 Hz, 1H), 8.03 (dd, J = 8.4, 1.8 Hz, 1H),
7.81 (d, J = 8.4 Hz, 1H), 7.62 (s, 1H), 7.33 (d, J = 7.8 Hz, 1H),
7.25 (d, J = 8.4 Hz, 1H), 3.65 (s, 3H), 2.45 (s, 3H), 2.08 (s, 3H).
13C NMR (100 MHz, CDCl3) d 190.62, 160.68, 159.95, 156.06,
149.22, 146.69, 141.72, 136.18, 134.22, 133.61, 129.83, 129.02,
127.61, 127.01, 121.94, 119.91, 102.22, 32.40, 23.60, 20.85,
15.60. MS (EI) m/z: 466.30(M)+. Anal. Calcd for C22H19BrN4O3:
C, 56.54; H, 4.10; N, 11.99. Found: C, 56.26; H, 4.28; N,
11.68.
White solid, mp 134–136 °C. Yield: 32%. 1H NMR (600 MHz,
CDCl3) d 8.53 (s, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H),
7.37 (d, J = 7.2 Hz, 2H), 7.17 (d, J = 7.8 Hz, 2H), 3.64 (s, 3H), 2.45 (s,
3H), 2.29 (s, 3H), 2.08 (s, 3H). 13C NMR (100 MHz, CDCl3) d 190.84,
161.64, 160.01, 156.59, 149.36, 146.78, 139.56, 136.05, 134.39,
133.47, 130.68, 127.50, 127.13, 127.10, 120.01, 102.23, 32.48,
24.42, 21.17, 15.79. MS (EI) m/z: 388.44(M)+. Anal. Calcd for
C22H20N4O3: C, 68.03; H, 5.19; N, 14.42. Found: C, 67.77; H, 4.99;
N, 14.70.

Y.-L. Xu et al. / Bioorg. Med. Chem. 22 (2014) 5194–5211
5207
4.1.116. 3-(4-Chloro-2-methylphenyl)-6-(5-hydroxy-1,3-
dimethyl-1H-pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-
one (2r)
7.45 (t, J = 7.8 Hz, 1H), 7.39 (d, J = 7.2 Hz, 1H), 3.73 (s, 3H), 2.33
(s, 3H). 13C NMR (100 MHz, CDCl3) d 187.97, 160.74, 159.80,
156.16, 150.03, 138.42, 136.50, 135.25, 134.01, 131.19, 129.63,
129.20, 128.16, 128.12, 127.58, 122.57, 120.41, 102.12,
33.02, 23.75. ESI-HRMS m/z calcd for [M+(H)]+ 439.0406, found
439.0419.
Yellow solid, mp 191–192 °C. Yield: 72.3%. 1H NMR (600 MHz,
CDCl3) d 8.55 (d, J = 1.8 Hz, 1H), 8.04 (dd, J = 8.4, 1.8 Hz, 1H), 7.82
(d, J = 8.4 Hz, 1H), 7.44 (s, 1H), 7.39 (dd, J = 8.4, 1.8 Hz, 1H), 7.14
(d, J = 8.4 Hz, 1H), 3.66 (s, 3H), 2.26 (s, 3H), 2.13 (s, 3H), 2.07 (s,
3H). 13C NMR (100 MHz, CDCl3) d 190.78, 160.76, 160.23, 156.03,
149.18, 146.61, 137.17, 136.48, 135.52, 134.69, 133.74, 131.59,
129.09, 127.93, 127.60, 127.06, 119.90, 102.26, 32.50, 23.79,
17.31, 15.73. MS (EI) m/z: 422.45(M)+. Anal. Calcd for C22H19ClN4O3:
C, 62.49; H, 4.53; N, 13.25. Found: C, 62.52; H, 4.50; N, 12.97.
4.1.122. 6-(5-Hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-2-
methyl-3-o-tolylquinazolin-4(3H)-one (3e)
Yellow solid, mp 195–197 °C. Yield: 71.6%. 1H NMR (600 MHz,
CDCl3) d 8.86 (s, 1H), 8.31 (d, J = 9.0 Hz, 1H), 7.87 (s, 1H), 7.82 (d,
J = 8.4 Hz, 1H), 7.44 (s, 2H), 7.41 (d, J = 8.4 Hz, 1H), 7.18 (d,
J = 7.8 Hz, 1H), 3.72 (s, 3H), 2.24 (s, 3H), 2.16 (s, 3H). 13C NMR
4.1.117. 3-(2-Chloro-4-methylphenyl)-6-(5-hydroxy-1,3-
dimethyl-1H-pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-
one (2s)
(100 MHz, CDCl3) d 187.96, 160.98, 159.62, 156.66, 150.31,
138.36, 136.20, 135.04, 134.97, 133.65, 131.52, 129.70, 128.06,
127.64, 127.62, 127.49, 120.43, 102.05, 32.94, 23.93, 17.27. MS
(EI) m/z: 374.04(M)+. Anal. Calcd for C21H18N4O3: C, 67.37; H,
4.85; N, 14.96. Found: C, 67.13; H, 4.58; N, 14.75.
Yellow solid, mp 149–150 °C. Yield: 52.4%. 1H NMR (600 MHz,
CDCl3) d 8.55 (s, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.4 Hz,
1H), 7.45 (s, 1H), 7.29 (d, J = 7.8 Hz, 1H), 7.24 (d, J = 7.8 Hz, 1H),
3.65 (s, 3H), 2.45 (s, 3H), 2.28 (s, 3H), 2.08 (s, 3H). 13C NMR
4.1.123. 6-(5-Hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-3-(2-
methoxyphenyl)-2-methylquinazolin-4(3H)-one (3f)
(100 MHz, CDCl3)
d 190.70, 160.80, 160.05, 156.11, 149.33,
146.69, 141.64, 136.21, 133.62, 131.99, 131.66, 131.13, 129.17,
129.09, 127.64, 127.09, 119.90, 102.22, 32.44, 23.50, 20.98, 15.68.
MS (EI) m/z: 422.30(M)+. Anal. Calcd for C22H19ClN4O3: C, 62.49;
H, 4.53; N, 13.25. Found: C, 62.56; H, 4.75; N, 13.17.
Yellow solid, mp 202–204 °C. Yield: 36.4%. 1H NMR (600 MHz,
CDCl3) d 8.85 (s, 1H), 8.30 (d, J = 8.4 Hz, 1H), 7.88 (s, 1H), 7.81 (d,
J = 8.4 Hz, 1H), 7.51 (t, J = 7.2 Hz, 1H), 7.23 (d, J = 7.2 Hz, 1H),
7.15–7.11 (m, 2H), 3.82 (s, 3H), 3.72 (s, 3H), 2.28 (s, 3H). 13C
NMR (100 MHz, CDCl3) d 188.03, 161.25, 159.71, 157.58, 154.24,
150.15, 138.43, 134.83, 133.60, 131.12, 128.97, 128.16, 127.30,
125.44, 121.38, 120.49, 112.21, 102.07, 55.70, 32.95, 23.48. MS
(EI) m/z: 390.03(M)+. Anal. Calcd for C21H18N4O4: C, 64.61; H,
4.65; N, 14.35. Found: C, 64.29; H, 4.85; N, 14.50.
4.1.118. 6-(5-Hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-2-
methyl-3-phenylquinazolin-4(3H)-one (3a)
Yellow solid; Mp 184–186 °C. Yield: 58%. 1H NMR (600 MHz,
CDCl3) d8.85 (s, 1H), 8.30 (d, J = 8.4 Hz, 1H), 7.86 (s, 1H), 7.81 (d,
J = 8.4 Hz, 1H), 7.60 (t, J = 7.2 Hz, 2H), 7.55 (d, J = 7.2 Hz, 1H), 7.29
(d, J = 7.2 Hz, 2H), 3.72 (s, 3H), 2.30 (s, 3H). 13C NMR (150 MHz,
CDCl3) d 188.03, 161.66, 159.70, 156.57, 150.21, 138.40, 137.18,
135.00, 133.68, 130.05, 129.48, 128.04, 127.76, 127.56, 120.50,
102.10, 32.97, 24.51. MS (EI) m/z: 360.31(M)+. Anal. Calcd for
4.1.124. 3-(3-Fluorophenyl)-6-(5-hydroxy-1-methyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (3g)
Yellow solid, mp 181–183 °C. Yield: 32.7%. 1H NMR (600 MHz,
CDCl3) d 8.83 (d, J = 1.8 Hz, 1H), 8.32 (dd, J = 8.4, 1.8 Hz, 1H), 7.86
(d, J = 8.4 Hz, 1H), 7.85 (s, 1H), 7.59(q, 1H), 7.29 (dd, J = 8.4,
2.4 Hz, 1H), 7.11 (d, J = 7.8 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 3.73
(s, 3H), 2.36 (s, 3H).13C NMR (100 MHz, CDCl3) d 187.98, 164.37,
161.88, 161.44, 159.64, 155.96, 150.00, 138.39, 135.19, 133.87,
131.39, 131.30, 127.96, 127.62, 123.77, 120.33, 116.95, 116.75,
115.80, 115.57, 102.09, 33.01, 24.37. MS (EI) m/z: 378.17(M)+. Anal.
Calcd for C20H15FN4O3: C, 63.49; H, 4.00; N, 14.81. Found: C, 63.28;
H, 4.19; N, 14.58.
C20H16N4O3: C, 66.66; H, 4.48; N, 15.55. Found: C, 66.55; H, 4.37;
N, 15.35.
4.1.119. 3-(2-Fluorophenyl)-6-(5-hydroxy-1-methyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (3b)
Yellow solid, mp 173–174 °C. Yield: 53%. 1H NMR (600 MHz,
CDCl3) d 8.84 (s, 1H), 8.32 (d, J = 8.4 Hz, 1H), 7.87 (s, 1H), 7.83 (d,
J = 8.4 Hz, 1H), 7.55 (s, 1H), 7.44–7.33 (m, 3H), 3.73 (s, 3H), 2.34
(s, 3H). 13C NMR (100 MHz, CDCl3) d 187.92, 161.01, 159.67,
158.63, 156.17, 150.14, 138.37, 135.17, 133.87, 131.58, 129.72,
128.04, 127.64, 125.33, 124.74, 120.24, 117.13, 116.92, 102.09,
32.96, 23.69. MS (EI) m/z: 378.30(M)+. Anal. Calcd for C20H15FN4O3:
C, 63.49; H, 4.00; N, 14.81. Found: C, 63.80; H, 4.61; N, 14.90.
4.1.125. 3-(3-Chlorophenyl)-6-(5-hydroxy-1-methyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (3h)
Yellow solid, mp 193–195 °C. Yield: 63.8%. 1H NMR (600 MHz,
CDCl3) d 8.82 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 8.4 Hz,
1H), 7.84 (s, 1H), 7.55 (d, J = 4.2 Hz, 2H), 7.34 (s, 1H), 7.22 (s, 1H),
3.73 (s, 3H), 2.36 (s, 3H). 13C NMR (100 MHz, CDCl3) d 187.83,
161.34, 159.50, 155.90, 149.83, 138.32, 138.08, 135.54, 135.11,
133.82, 130.99, 129.84, 128.20, 127.87, 127.49, 126.18, 120.20,
102.02, 32.96, 24.40. MS (EI) m/z: 394.13(M)+. Anal. Calcd for C20
H15ClN4O3: C, 60.84; H, 3.83; N, 14.19. Found: C, 60.51; H, 3.88;
N, 14.25.
4.1.120. 3-(2-Chlorophenyl)-6-(5-hydroxy-1-methyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (3c)
Yellow solid, mp 200–202 °C. Yield: 66%. 1H NMR (600 MHz,
CDCl3) d 8.85 (s, 1H), 8.33 (d, J = 8.4 Hz, 1H), 7.87 (s, 2H), 7.66
(dd, J = 7.2, 1.2 Hz, 1H), 7.52 (t, J = 1.2, Hz, 2H), 7.38 (dd, J = 7.2,
3.0 Hz, 1H), 3.73 (s, 3H), 2.31 (s, 3H). 13C NMR (100 MHz, CDCl3)
d 187.89, 160.79, 159.68, 156.11, 150.13, 138.35, 135.14, 134.82,
133.87, 132.26, 131.00, 130.74, 129.61, 128.43, 128.04, 127.58,
120.31, 102.07, 32.96, 23.58. MS (EI) m/z: 394.26(M)+. Anal. Calcd
for C20H15ClN4O3: C, 60.84; H, 3.83; N, 14.19. Found: C, 60.63; H,
3.50; N, 13.92.
4.1.126. 3-(3-Bromophenyl)-6-(5-hydroxy-1-methyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (3i)
Yellow solid, mp 194–196 °C. Yield: 63.8%. 1H NMR (600 MHz,
CDCl3) d 8.82 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 10.2 Hz,
2H), 7.70 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 13.2 Hz, 2H), 3.73 (s, 3H),
2.35 (s, 3H). 13C NMR (100 MHz, CDCl3) d 188.0, 161.47, 159.72,
155.88, 150.05, 138.41, 138.33, 135.25, 133.90, 132.81, 131.28,
131.10, 128.00, 127.70, 126.72, 123.39, 120.36, 102.12, 33.03,
24.52. MS (EI) m/z: 438.00(M)+. Anal. Calcd for C20H15BrN4O3: C,
54.69; H, 3.44; N, 12.75. Found: C, 54.38; H, 3.69; N, 12.51.
4.1.121. 3-(2-Bromophenyl)-6-(5-hydroxy-1-methyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (3d)
Yellow solid, mp 209–211 °C. Yield: 47%. 1H NMR (600 MHz,
CDCl3) d 8.86 (s, 1H), 8.33 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 8.4 Hz,
1H), 7.87 (s, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H),

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Y.-L. Xu et al. / Bioorg. Med. Chem. 22 (2014) 5194–5211
4.1.127. 6-(5-Hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-2-
methyl-3-m-tolylquinazolin-4(3H)-one (3j)
3.73 (s, 3H), 2.30 (s, 3H). 13C NMR (150 MHz, DMSO-d6) d 186.04,
161.12, 156.18, 155.55, 149.55, 139.36, 136.95, 136.13, 133.98,
132.70, 130.75, 127.10, 122.45, 120.08, 103.38, 33.11, 24.31. MS
(EI) m/z: 438.04(M)+. Anal. Calcd for C20H15BrN4O3: C, 54.69; H,
3.44; N, 12.75. Found: C, 54.52; H, 3.73; N, 12.48.
Yellow solid, mp 185–187 °C. Yield: 66.7%. 1H NMR (600 MHz,
CDCl3) d 8.84 (s, 1H), 8.30 (d, J = 8.4 Hz, 1H), 7.85 (s, 1H), 7.82 (d,
J = 9.0 Hz, 1H), 7.47 (t, J = 7.2 Hz, 1H), 7.35 (d, J = 7.2 Hz, 1H), 7.08
(d, J = 12.6 Hz, 2H), 3.72 (s, 3H), 2.45 (s, 3H), 2.32 (s, 3H). 13C
NMR (100 MHz, CDCl3) d 187.97, 161.57, 159.68, 156.85, 149.87,
140.27, 138.39, 136.96, 135.01, 133.68, 130.35, 130.07, 128.13,
128.05, 127.33, 124.61, 120.42, 102.08, 32.96, 24.34, 21.25. MS
(EI) m/z: 374.02(M)+. Anal. Calcd for C21H18N4O3: C, 67.37; H,
4.85; N, 14.96. Found: C, 67.55; H, 4.82; N, 14.68.
4.1.133. 6-(5-Hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-2-
methyl-3-p-tolylquinazolin-4(3H)-one (3p)
Yellow solid, mp 234–236 °C. Yield: 68.9%.1H NMR (400 MHz,
CDCl3) d 8.82 (s, 1H), 8.28 (d, J = 8.4 Hz, 1H), 7.86 (s, 1H), 7.79 (d,
J = 8.4 Hz, 1H), 7.38 (d, J = 7.6 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H),
3.72 (s, 3H), 2.46 (s, 3H), 2.30 (s, 3H). 13C NMR (150 MHz, CDCl3)
d: 188.16, 161.81, 159.79, 156.97, 150.22, 139.66, 138.47, 135.01,
134.50, 133.69, 130.75, 128.14, 127.53, 127.43, 120.56, 102.15,
33.02, 24.51, 21.23. MS (EI) m/z: 374.32 (M)+. Anal. Calcd for
4.1.128. 6-(5-Hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-3-(3-
methoxyphenyl)-2-methylquinazolin-4(3H)-one (3k)
Yellow solid, mp 158–160 °C. Yield: 51%. 1H NMR (600 MHz,
CDCl3) d 8.84 (s, 1H), 8.30 (d, J = 9.0 Hz, 1H), 7.86 (s, 1H), 7.84 (d,
J = 8.4 Hz, 1H), 7.50 (t, J = 8.4 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 6.87
(d, J = 7.8 Hz, 1H), 6.82 (s, 1H), 3.86 (s, 3H), 3.73 (s, 3H), 2.36 (s,
3H). 13C NMR (100 MHz, CDCl3) d 187.87, 161.20, 160.79, 159.60,
157.23, 148.98, 138.38, 137.74, 135.26, 133.89, 130.86, 128.03,
126.82, 120.26, 119.66, 115.31, 113.39, 102.08, 55.47, 33.00,
23.90. MS (EI) m/z: 390.03(M)+. Anal. Calcd for C21H18N4O4: C,
64.61; H, 4.65; N, 14.35. Found: C, 64.80; H, 4.67; N, 14.01.
C21H18N4O3: C, 67.37; H, 4.85; N, 14.96. Found: C, 67.24; H, 4.75;
N, 14.89.
4.1.134. 6-(5-Hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-3-(4-
methoxyphenyl)-2-methylquinazolin-4(3H)-one (3q)
Yellow solid, mp 188–190 °C. Yield: 31.6%. 1H NMR (600 MHz,
CDCl3) d 8.81 (s, 1H), 8.27 (d, J = 7.8 Hz, 1H), 7.86 (s, 1H), 7.77 (d,
J = 7.8 Hz, 1H), 7.20 (d, J = 9.0 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H),
3.89 (s, 3H), 3.72 (s, 3H), 2.31 (s, 3H). 13C NMR (150 MHz, CDCl3)
d 188.08, 161.94, 160.02, 159.68, 157.17, 150.18, 138.44, 134.94,
133.63, 129.60, 128.74, 128.09, 127.51, 120.48, 115.23, 102.10,
55.49, 32.98, 24.55. MS (EI) m/z: 390.39(M)+. Anal. Calcd for
4.1.129. 6-(5-Hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-2-
methyl-3-(3-(trifluoromethoxy)phenyl) quinazolin-4(3H)-one
(3l)
Yellow solid, mp 154–156 °C. Yield: 66%. 1H NMR (600 MHz,
CDCl3) d 8.82 (s, 1H), 8.31 (d, J = 9.0 Hz, 1H), 7.84 (d, J = 10.2 Hz,
2H), 7.65 (t, J = 7.8 Hz, 1H), 7.43 (d, J = 9.0 Hz, 1H), 7.28 (s, 1H),
7.21 (s, 1H), 3.73 (s, 3H), 2.32 (s, 3H). 13C NMR (100 MHz, CDCl3)
d 187.60, 161.15, 159.48, 155.85, 149.87, 149.57, 138.24, 135.14,
133.82, 131.16, 127.82, 127.30, 126.44, 124.00, 121.77, 121.42,
120.83, 120.14, 118.90, 119.29, 101.98, 77.33, 77.32, 76.84 (d,
J = 32.0 Hz), 32.84 (s), 24.15 (s). MS (EI) m/z: 444.30(M)+. Anal.
Calcd for C21H15F3N4O4: C, 56.76; H, 3.40; N, 12.61. Found: C,
56.48; H, 3.68 N, 12.95.
C21H18N4O4: C, 64.61; H, 4.65; N, 14.35. Found: C, 64.33; H, 4.60;
N, 14.51.
4.1.135. 6-(5-Hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-2-
methyl-3-(4-(trifluoromethoxy)phenyl) quinazolin-4(3H)-one
(3r)
White solid, mp 202–204 °C. Yield: 69%. 1H NMR (600 MHz,
CDCl3) d 8.82 (d, J = 1.8 Hz, 1H), 8.31 (dd, J = 8.4, 2.4 Hz, 1H), 7.84
(s, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 8.4 Hz, 2H), 7.35 (dd,
J = 7.2, 1.2 Hz, 1H), 3.73 (s, 3H), 2.32 (s, 3H). 13C NMR (100 MHz,
CDCl3) d 195.91, 170.96, 166.45,165.55, 158.86, 158.47, 149.16,
146.26, 146.20, 143.99, 140.60, 136.99, 136.56, 132.03, 131.23,
129.94, 128.68, 113.35, 42.98, 33.90. MS (EI) m/z: 444.32(M)+. Anal.
Calcd for C21H15F3N4O4: C, 56.76; H, 3.40; N, 12.61. Found: C,
56.67; H, 3.61 N, 12.41.
4.1.130. 3-(4-Fluorophenyl)-6-(5-hydroxy-1-methyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (3m)
Yellow solid, mp 219–221 °C Yield: 62%. 1H NMR (600 MHz,
CDCl3) d 8.83 (s, 1H), 8.31 (d, J = 9.0 Hz, 1H), 7.84 (d, J = 9.0 Hz,
2H), 7.29 (d, J = 6.6 Hz, 4H), 3.73 (s, 3H), 2.33 (s, 3H).13C NMR
(100 MHz, CDCl3)
d
188.02, 164.06, 161.71, 159.77, 156.40,
4.1.136. 3-(4-Chloro-2-methylphenyl)-6-(5-hydroxy-1-methyl-
1H-pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (3s)
Yellow solid, mp 188–190 °C. Yield: 39.1%. 1H NMR (600 MHz,
CDCl3) d 8.84 (d, J = 1.8 Hz, 1H), 8.32 (dd, J = 8.4, 1.8 Hz, 1H), 7.86
(s, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.44 (s, 1H), 7.39 (dd, J = 8.4,
2.4 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 3.73 (s, 3H), 2.26 (s, 3H), 2.14
(s, 3H). 13C NMR (100 MHz, CDCl3) d 187.96, 160.93, 159.67,
156.17, 150.24, 138.39, 137.18, 135.45, 135.20, 134.79, 133.86,
131.57, 129.05, 128.06, 127.90, 127.64, 120.33, 102.08, 33.01,
23.98, 17.31. MS (EI) m/z: 408.14(M)+. Anal. Calcd for C21H17ClN4
O3: C, 61.69; H, 4.19; N, 13.70. Found: C, 61.47; H, 4.16; N, 13.41.
150.10, 138.40, 135.21, 133.83, 133.06, 129.77, 129.69, 128.03,
127.64, 120.43, 117.31, 117.08, 102.14, 33.01, 24.53. MS (EI) m/z:
378.13(M)+. Anal. Calcd for C20H15FN4O3: C, 63.49; H, 4.00; N,
14.81. Found: C, 63.26; H, 4.26; N, 14.60.
4.1.131. 3-(4-Chlorophenyl)-6-(5-hydroxy-1-methyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (3n)
Yellow solid, mp 235–237 °C. Yield: 65%. 1H NMR (400 MHz,
DMSO-d6) d8.46 (d, J = 1.6 Hz, 1H), 8.21 (dd, J = 8.4, 1.6 Hz, 1H),
7.77 (d, J = 8.4 Hz, 1H), 7.68 (s, 1H), 7.66 (d, J = 2.4 Hz, 2H), 7.55
(d, J = 8.4 Hz, 2H), 3.59 (s, 3H), 2.18 (s, 3H). 13C NMR (100 MHz,
CDCl3) d 187.95, 161.51, 159.68, 156.14, 149.94, 138.41, 135.64,
135.58 135.24, 133.87, 130.36, 129.26, 127.99, 127.56, 120.33,
102.12, 33.01, 24.44. MS (EI) m/z: 394.10(M)+. Anal. Calcd for
4.1.137. 3-(4-Fluoro-2-methylphenyl)-6-(5-hydroxy-1-methyl-
1H-pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (3t)
Yellow solid, mp 197–200 °C. Yield: 71.1%. 1H NMR (600 MHz,
CDCl3) d 8.85 (s, 1H), 8.32 (d, J = 9.0 Hz, 1H), 7.87 (s, 1H),7.84 (d,
J = 8.4 Hz, 1H), 7.15 (d, J = 9.0 Hz, 2H), 7.11 (d, J = 8.4 Hz, 1H),
3.73 (s, 3H), 2.26 (s, 3H), 2.15 (s, 3H).13C NMR (100 MHz, CDCl3)
d 187.95, 163.97, 161.49, 161.03, 159.65, 156.61, 150.07, 138.39,
137.85, 137.76, 135.20, 133.85, 132.08, 129.46, 129.37, 128.07,
127.50, 120.32, 118.44, 118.21, 114.82, 114.59, 102.09, 32.99,
23.91, 17.53. MS (EI) m/z: 392.33(M)+. Anal. Calcd for C21H17FN4O3:
C, 64.28; H, 4.37; N, 14.28. Found: C, 64.19; H, 4.35; N, 14.59.
C20H15ClN4O3: C, 60.84; H, 3.83; N, 14.19. Found: C, 60.64; H,
3.76; N, 14.16.
4.1.132. 3-(4-Bromophenyl)-6-(5-hydroxy-1-methyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (3o)
Yellow solid, mp 234–236 °C. Yield: 71.4%.1H NMR (600 MHz,
CDCl3) d8.82 (s, 1H), 8.30 (d, J = 9.0 Hz, 1H), 7.84 (s, 1H), 7.80 (d,
J = 9.0 Hz, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H),

Y.-L. Xu et al. / Bioorg. Med. Chem. 22 (2014) 5194–5211
5209
4.1.138. 3-(2-Bromo-4-methylphenyl)-6-(5-hydroxy-1-methyl-
1H-pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (3u)
Yellow solid, mp 216–219 °C. Yield: 58.3%. 1H NMR (600 MHz,
CDCl3) d 8.86 (d, J = 1.2 Hz, 1H), 8.32 (dd, J = 8.4, 1.8 Hz, 1H), 7.88
(s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.64 (s, 1H), 7.34 (d, J = 7.8 Hz,
1H), 7.24 (d, J = 7.8 Hz, 1H), 3.73 (s, 3H), 2.46 (s, 3H), 2.30 (s, 3H).
13C NMR (100 MHz, CDCl3) d 187.90, 160.81, 159.68, 156.42,
150.03, 141.80, 138.38, 135.09, 134.30, 133.83, 133.63, 129.89,
129.03, 128.11, 127.50, 122.00, 120.36, 102.06, 32.95, 23.72,
20.92. MS (EI) m/z: 452.33(M)+. Anal. Calcd for C21H17BrN4O3: C,
55.64; H, 3.78; N, 12.36. Found: C, 55.32; H, 3.50; N, 12.27.
heated at 110 °C for a further 3 h. After removing the reaction sol-
vent under reduced pressure, the intermediate 2-chloro-4-(meth-
ylsulfonyl)benzoyl chloride was obtained, it was used directly in
the next step without further purification.
A reaction mixture of 1,3-cyclohexanedione 1.44 g (12.8 mmol),
Et3N 1.9 g (19.2 mmol) in CH2Cl2 (25 mL) was pre-stirred at room
temperature for 4 h. Then the intermediate 2-chloro-4-(methylsul-
fonyl)-benzoyl chloride prepared previously was dissolved in CH2
Cl2 (25 mL), and was added dropwise to the reaction mixture at
0 °C in about 15 min. After the mixture was stirred at room tem-
perate for 1 h, Et3N 1.3 g (12.8 mmol) and acetone cyanohydrin
0.11 g (1.3 mmol) was added, and the obtained mixture was heated
at 40 °C for another hour. After the reaction was completed, as
monitored by TLC, the mixture was extracted twice by 2 mol Lꢀ1
HCl (30 mL), and the organic layer was dried by anhydrous Na2SO4.
After removal of the solvent by rotary vapor, the residue was
recrystallized from methanol to give the final produce NTBC in
good yield. White solid, yield, 73.8%, mp 137.5–138.1 °C. 1H NMR
(600 MHz, CDCl3): d 16.26 (s, 1H), 8.48 (s, 1H), 7.95 (d, J = 7.8 Hz,
1H), 7.38 (d, J = 7.8 Hz, 1H), 2.82 (t, J = 6.6 Hz, 2H), 2.38 (t,
J = 6.6 Hz, 2H), 2.05 (quintuplet, J = 6.6, 2H). 13C NMR (101 MHz,
CDCl3): d 196.19, 195.77, 194.11, 145.37, 139.61, 132.37, 132.02,
131.68, 130.99, 130.76, 127.61, 126.57, 123.86, 121.14, 121.01,
118.43, 112.54, 37.18, 31.53, 18.98. HRMS (ESI): calcd for
4.1.139. 3-(3,4-Dichlorophenyl)-6-(5-hydroxy-1-methyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (3v)
Yellow solid, mp 226–228 °C. Yield: 75.8%. 1H NMR (600 MHz,
CDCl3) d 8.81 (s, 1H), 8.31 (d, J = 7.8 Hz, 1H), 7.84 (s, 1H), 7.82 (d,
J = 8.4 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.44 (s, 1H), 7.18 (d,
J = 7.8 Hz, 1H), 3.73 (s, 3H), 2.34 (s, 3H). 13C NMR (100 MHz, CDCl3)
d 187.95, 161.38, 159.74, 155.56, 149.90, 138.39, 136.33, 135.42,
134.39, 134.10, 134.03, 131.77, 130.10, 127.94, 127.71, 127.43,
120.25, 102.15, 33.03, 24.46. MS (EI) m/z: 428.21(M)+. Anal. Calcd
for C20H14Cl2N4O3: C, 55.96; H, 3.29; N, 13.05. Found: C, 56.18;
H, 3.50; N, 13.27.
4.1.140. 3-(2-Fluoro-4-methylphenyl)-6-(5-hydroxy-1-methyl-
1H-pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (3w)
Yellow solid, mp 149–151 °C. Yield: 62%. 1H NMR (600 MHz,
CDCl3) d 8.84 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H), 7.87 (s, 1H), 7.81 (d,
J = 8.4 Hz, 1H), 7.20 (t, J = 7.2, 1H), 7.15 (t, J = 7.2, 2H), 3.73 (s,
3H), 2.47 (s, 3H), 2.34 (s, 3H). 13C NMR (100 MHz, DMSO-d6) d
186.13, 160.76, 157.99, 156.44, 156.39, 155.83, 155.52,
149.48,142.53, 142.49, 139.48, 136.52, 134.39, 130.11, 127.31,
126.24, 122.02,121.89, 119.74, 117.13, 116.93, 103.50, 33.15,
23.62, 20.89. MS (EI) m/z: 392.41(M)+. Anal. Calcd for C21H17FN4O3:
C, 64.28; H, 4.37; N, 14.28. Found: C, 64.36; H, 4.65; N, 14.57.
C
14H9F3NO5 [MꢀH]ꢀ 328.0433, found 328.0437.
4.2. Biology
4.2.1. Plasmid construction
cDNA of human HPPD (pDONR223-HPD, BC024287) was pur-
chased from Yingrun Biotechnologies Inc. With pDONR223-HPD
as template, the human HPPD was amplified by PCR using the for-
ward primer 50-GGAATTCCATATGACGACTTACAGTGACA-30 and the
reverse primer 50-CGGGATCCTTACATGCCGGGCACCACCCCATT-30 to
introduce 5-NdeI and 30-BamHI restriction sites (underlined),
respectively. PCR conditions were 30 cycles at 94 °C for 30 s,
52 °C for 25 s and 72 °C for 1.25 min. The resulting amplicons were
separated on a 1% agarose gel, followed by QG buffer dissolvation
and further extracted with QIAGEN Plasmid Mini Kit (QIAGEN).
The amplion was digested with NdeI and BamHI, ligated in-frame
into the expression vector pET-28a digested with the same restric-
tion enzymes. Then the ligation mixture was transformed into
Escherichia coli (E. coli) BL-21(DE3) cells. The DNA sequences of
the positive clones were confirmed by DNA sequencing with
Shanghai SANGON Company.
4.1.141. 3-(2-Chloro-4-methylphenyl)-6-(5-hydroxy-1-methyl-
1H-pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (3x)
Yellow solid, mp 206–208 °C. Yield: 55.1%. 1H NMR (600 MHz,
CDCl3) d 8.85 (d, J = 2.4 Hz, 1H), 8.32 (dd, J = 8.4, 1.8 Hz, 1H), 7.87
(s, 2H), 7.47 (s, 1H), 7.30 (d, J = 7.2 Hz, 1H), 7.24 (d, J = 8.4 Hz,
1H), 3.73 (s, 3H), 2.46 (s, 3H), 2.32 (s, 3H). 13C NMR (100 MHz,
CDCl3) d 188.01, 160.96, 159.74, 156.46, 150.20, 141.72, 138.44,
135.10, 133.84, 132.06, 131.72, 131.18, 129.23, 129.08, 128.12,
127.61, 120.36, 102.09, 32.99, 23.65, 21.07. MS (EI) m/z:
408.29(M)+. Anal. Calcd for C21H17ClN4O3: C, 61.69; H, 4.19; N,
13.70. Found: C, 61.30; H, 4.03; N, 13.42.
4.2.2. Protein expression, extraction and purification
Recombinant human HPPD and HGA dioxygenase were over-
expressed in E. coli BL-21(DE3) cells with pET-28a-HPPD plasmid.
The cells were grown at 37 °C in Luria Bertani broth supplemented
4.1.142. 3-(2,4-Dimethylphenyl)-6-(5-hydroxy-1-methyl-1H-
pyrazole-4-carbonyl)-2-methylquinazolin-4(3H)-one (3y)
Yellow solid, mp 195–196 °C. Yield: 39%. 1H NMR (400 MHz,
CDCl3) d 10.65 (s, 1H), 8.84 (s, 1H), 8.29 (d, J = 8.4 Hz, 1H), 7.87
(s, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.23 (s, 1H), 7.19 (d, J = 8.0 Hz,
1H), 7.07 (d, J = 7.6 Hz, 1H), 3.72 (s, 3H), 2.41 (s, 3H), 2.26 (s, 3H),
2.11 (s, 3H). 13C NMR (100 MHz, CDCl3) d 187.94, 160.97, 159.68,
157.19, 149.93, 139.80, 138.38, 135.04, 134.56, 133.69, 133.44,
132.26, 128.37, 128.15, 127.29, 120.40, 102.06, 32.95, 23.80,
21.08, 17.19. MS (EI) m/z: 388.08(M)+. Anal. Calcd for
with 50 l
g/ml of kanamycin according to previous publications.5,28
Expression of the plasmid was induced by 0.5 mM IPTG for HPPD
and 1 mM for HGA dioxygenase when bacterial growth was equiv-
alent to an A600 of 0.6. The cells for HPPD over-expression were
incubated for another 14 h at 23 °C and harvested by centrifuga-
tion (5000 g, 30 min), while the cells for HGA dioxygenase over-
expression were incubated for another 40 h at 15 °C and harvested
by centrifugation (5000 g, 30 min). The pellet was resuspended in
buffer (20 mM HEPES, 20 mM NaCl, pH = 7.0) and washed twice,
followed by sonication using a cell disruptor. A crude cell-free
supernatant was obtained by centrifugation at 20,000g for 30 min.
The purification of His6-tagged recombinant HPPD was carried
out using nickel nitrilotriacetic acid (Ni–NTA) protocol (Invitrogen).
In general, the crude cell-free supernatant was subjected to affinity
chromatography on Ni–NTA column and eluted with 250 mM imid-
azole, followed by desalination and buffer exchange with Millipore
C22H20N4O3: C, 68.03; H, 5.19; N, 14.42. Found: C, 67.75; H, 5.12;
N, 14.17.
4.1.143. General procedure for the synthesis of NTBC
To a suspension of 2-nitro-4-(trifluoromethyl)benzoic acid 3 g
(12.8 mmol), N,N-dimethylformamide (0.1 mL) in toluene
(25 mL), thionyl chloride 1.8 g (15.3 mmol) was added with stir-
ring at room temperature for 10 min, and the suspension was then

5210
Y.-L. Xu et al. / Bioorg. Med. Chem. 22 (2014) 5194–5211
denaturation at 95 °C for 5 min. The 5
lL prepared samples,
10 L HPPD polyclonal antibody, 10 L peroxidase-conjugated goat
l
l
anti-rabbit IgG (Simon-01-01) and chemiluminescent substrate
were then dispensed in microliter volumes into the wells of a
384-well assay plate. Simple Western assay buffers, nanovolume
capillaries and the 384-well assay plate were placed in a Simon
instrument, which carried out the entire assay steps automatically.
In general, proteins of each sample were separated in capillaries as
they migrate through a stacking and separation matrix. The sepa-
rated proteins were immobilized on the capillary wall via a pro-
prietary, photoactivated capture chemistry. Just as the traditional
western blot, the target proteins were specifically identified with
a primary antibody and subsequent immunodetection using a
horseradish peroxidase (HRP)-conjugated secondary antibody.
However, the signal was produced by the conversion of the added
chemiluminescent substrate catalyzed by the second antibody. The
whole assay procedure took about 5 h. Finally, the results of molec-
ular weight and signal for immunodetected proteins were auto-
matically reported by Compass software. It is worth noting that
this software reports molecular weight and signal to noise for each
protein detected in three formats: graph, image and lane that is
commonly used.
Figure 5. Validation of the over-expression of recombinant human HPPD. (a)
Electrophoresis of over-expressed HPPD in SDS–PAGE: marker (Lane 1), the crude
cell lysate (Lane 2), and the purified HPPD (Lane 3). (b) Detection of the over-
expressed HPPD crude enzyme by Western blot analysis: marker (Lane 1), 100
times dilution of crude HPPD (Lane 2), 400 times dilutions of crude HPPD (Lane 3).
Amicon ultra-30 filter. As indicted in Figure 5, the over-expression
and the relative molecular weight of recombinant human HPPD
(40ꢁ55 kDa) were validated by both sodium dodecyl sulfate poly-
acrylamide gel electrophoresis (SDS–PAGE) stained with Coomassie
Brilliant Blue-R250, After mixing with 50% glycerol, the purified
enzyme was aliquoted and kept at ꢀ80 °C for storage.
4.5. Computational modeling
Based on the crystal structure of human HPPD, the GLOD 3.0
version docking program was applied to dock the inhibitor into
the active sites of human HPPD. The chemical structures of the
small inhibitors were constructed and optimized by using SYBYL
7.0. The GOLD program uses a genetic algorithm (GA) to explore
the full range of ligand conformational flexibility, and the radius
of active site was set to be 10 Å. All docking runs were carried
out using the default settings of program on a population size of
100 individuals with a selection pressure of 1.1. All the complex
structures derived from the above docking were used as starting
structures for further energy minimizations using the Sander mod-
ule of the Amber 8 program. The atomic charges used for these
inhibitors were AM1-BCC (Bond Charge Corrections), determined
with Antechamber module of Amber 8.
4.3. HPPD activity assay and inhibitory kinetics
Our coupled enzyme assays on the in vitro activity and
inhibition of HPPD were measured by a modification of methods
previously reported in the literature.29 Assays were performed in
96-well plates at 30 °C using a UV/visible plate reader to monitor
the formation of maleylacetoacetate at 318 nm
500 Mꢀ1 cmꢀ1).30 The reaction mixture in a total assay volume of
200 L contained appropriate amounts of HPPA, 100 M FeCl2,
(e330 = 13
l
l
2 mM sodium ascorbate, 50 mM HEPES buffer (pH = 7.0), HPPD
(25 nM) and HGD (150 nM). Before assays were conducted, all
reaction components were preequilibrated at 30 °C for at least
30 min. The amount of HGD activity was predetermined to be in
large excess of the HPPD activity to ensure that the reaction was
tightly coupled. Each experiment was carried out around 30 min
and repeated at least three times, and then the values were aver-
aged. HPPD inhibitors were dissolved in dimethyl sulfoxide
(DMSO) for stock solution and diluted to various concentrations
with reaction buffer just before using. The inhibition constant
(Ki), the indication of an inhibitor’s potency, was obtained from
the Dixon plot of plotting 1/v against concentration of inhibitor
(totally five concentrations, and the specific concentration was
determined according to the potency of inhibitor) at fixed concen-
Acknowledgement
We are grateful to the financial support for this work from the
National Key Technologies R&D Program of China (2011BAE06B05).
References and notes
1. Moran, G. R. Arch. Biochem. Biophys. 2005, 433, 117.
2. Yang, C.; Pflugrath, J. W.; Camper, D. L.; Foster, M. L.; Pernich, D. J.; Walsh, T. A.
Biochemistry 2004, 43, 10414.
3. Crouch, N. P.; Adlington, R. M.; Baldwin, J. E.; Lee, M. H.; MacKinnon, C. H.
Tetrahedron 1997, 53, 6993.
4. Kvittingen, E. A.; Scand, J. Clin. Lab Invest. Suppl. 1986, 184, 27.
5. Meazza, G.; Scheffler, B. E.; Tellez, M. R.; Rimando, A. M.; Romagni, J. G.; Duke,
S. O.; Nanayakkara, D.; Khan, I. A.; Abourashed, E. A.; Dayan, F. E.
Phytochemistry 2002, 60, 281.
6. Russo, P. A.; Mitchell, G. A.; Tanguay, R. M. Pediatr. Dev. Pathol. 2001, 4, 212.
7. Lindblad, B.; Lindstedt, S.; Steen, G. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 4641.
8. El-Shabrawi, M. H. Orphan Drugs Res. Rev. 2013, 3, 1.
9. Brownlee, J. M.; Heinz, B.; Bates, J.; Moran, G. R. Biochemistry 2010, 49, 7218.
10. Lindstedt, S.; Holme, E.; Lock, E. A.; Hjalmarson, O.; Strandvik, B. Lancet 1992,
340, 813.
trations of substrate (final concentrations of HPPA are 40
lM and
80 M, respectively). Bovine serum albumin is usually added up
l
to 0.5% of the total reaction volume to avoid the coating of the tar-
get enzyme during the incubation. In our assay, no obvious effect
from bovine serum albumin on the activity of the compounds have
been found, which indicates that the new compounds selectively
inhibited the target enzyme, but did not interact with bovine
serum albumin.
11. Holme, E.; Lindstedt, S. Lancet 1992, 340, 850.
12. Suwannarat, P.; O’Brien, K.; Perry, M.; Gerber, L.; Bernardini, I.; Gahl, W. A. Mol.
Genet. Metab. 2005, 84, 207.
13. Phornphutkul, C.; Introne, W. J.; Perry, M. B.; Bernardini, I.; Murphey, M. D.;
Fitzpatrick, D. L.; Anderson, P. D.; Huizing, M.; Anikster, Y.; Gerber, L. H.; Gahl,
W. A. N. Engl. J. Med. 2002, 347, 2111.
4.4. Western assay
The crude recombinant HPPD samples were mixed with Sample
Buffer of the Simple Western kit (Simon Master Kit, ProteinSimple)
14. Ahrens, H.; Lange, G.; Muller, T.; Rosinger, C.; Willms, L.; van Almsick, A. Angew.
Chem., Int. Ed. 2013, 52, 9388.
and standards to a final concentration of 1 lg l
Lꢀ1, followed by

Y.-L. Xu et al. / Bioorg. Med. Chem. 22 (2014) 5194–5211
5211
15. Beaudegnies, R.; Edmunds, A. J.; Fraser, T. E.; Hall, R. G.; Hawkes, T. R.; Mitchell,
G.; Schaetzer, J.; Wendeborn, S.; Wibley, J. Bioorg. Med. Chem. 2009, 17, 4134.
16. Yang, D. Y. Drug News Perspect. 2003, 16, 493.
23. Peng, D. Y.; Sun, Q.; Zhu, X. L.; Lin, H. Y.; Chen, Q.; Yu, N. X.; Yang, W. C.; Yang,
G. F. Bioorg. Med. Chem. 2012, 20, 6739.
24. Hao, G. F.; Wang, F.; Li, H.; Zhu, X. L.; Yang, W. C.; Huang, L. S.; Wu, J. W.; Berry,
E. A.; Yang, G. F. J. Am. Chem. Soc. 2012, 134, 11168.
17. Mitchell, G. A. Hum. Exp. Toxicol. 1996, 15, 179.
18. Ellis, M. K.; Whitfield, A. C.; Gowans, L. A.; Auton, T. R.; Provan, W. M.; Lock, E.
A.; Smith, L. L. Toxicol. Appl. Pharmacol. 1995, 133, 12.
25. Zhao, P. L.; Wang, L.; Zhu, X. L.; Huang, X.; Zhan, C. G.; Wu, J. W.; Yang, G. F. J.
Am. Chem. Soc. 2010, 132, 185.
19. Ishikawa, M.; Azuma, H.; Eguchi, Y.; Sugimoto, A.; Ito, S.; Takashima, Y.;
Ebisawa, H.; Moriguchi, S.; Kotoku, I.; Suzuki, H. Chem. Pharm. Bull. (Tokyo)
1982, 30, 744.
26. Xue, S.; McKenna, J.; Shieh, W. C.; Repic, O. J. Org. Chem. 2004, 69, 6474.
27. El-Faham, A.; Albericio, F. Chem. Rev. 2011, 111, 6557.
28. Ruetschi, U.; Odelhog, B.; Lindstedt, S.; Barros-Soderling, J.; Persson, B.;
Jornvall, H. Eur. J. Biochem. 1992, 205, 459.
20. Ishikawa, M.; Eguchi, Y. Heterocycles 1981, 16, 31.
21. Grover, G.; Kini, S. G. Eur. J. Med. Chem. 2006, 41, 256.
22. Avallone, A.; Di Gennaro, E.; Silvestro, L.; Iaffaioli, V. R.; Budillon, A. Expert Opin.
Drug Saf. 2014, 13, 113.
29. Purpero, V. M.; Moran, G. R. Biochemistry 2006, 45, 6044.
30. Schmidt, S. R.; Muller, C. R.; Kress, W. Eur. J. Biochem. 1995, 228, 425.