Design, synthesis and biological evaluation of 2-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acid derivatives as novel xanthine oxidase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.07.061

In our previous study, we reported a series of 1-hydroxy-2-phenyl-1H-imidazole-5-carboxylic acid derivatives that presented excellent in vitro xanthine oxidase (XO) inhibitory potency. To further investigate the structure-activity relationships of these compounds, the imidazole ring was transformed to a pyrimidine ring to design 2-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acids (8a-8j), 2-(4-alkoxy-3-cyano)phenyl-4-methyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acids (9c, 9e, 9j, 9l) and 2-(4-alkoxy-3-cyano)phenyl-6-imino-1,6-dihydropyrimidine-5-carboxylic acids (10c, 10e, 10j, 10l). These compounds exhibited remarkable in vitro XO inhibitory potency with IC₅₀ values ranging from 0.0181 μM to 0.5677 μM. Specifically, compounds 10c and 10e, with IC₅₀ values of 0.0240 μM and 0.0181 μM, respectively, emerged as the most potent XO inhibitors, and their potencies were comparable to that of febuxostat. Structure-activity relationship analysis revealed that the methyl group at 4-position of pyrimidine ring could damage the potency, and the XO inhibitory potency was maintained when carbonyl group was changed to an imino group. Lineweaver-Burk plot analysis revealed that the representative compound 10c acted as a mixed-type inhibitor. A potassium oxonate induced hyperuricemia model in rats was chosen to further confirm the hypouricemic effect of compound 10c, and the results showed that compound 10c (5 mg/kg) was able to significantly lower the serum uric acid level. Furthermore, in acute oral toxicity study, no sign of toxicity was observed when the mice were administered with a single 2000 mg/kg oral dose of compound 10c. These results suggested that compound 10c was a potent and promising uric acid-lowing agent for the treatment of hyperuricemia.

Full text of DOI:10.1016/j.ejmech.2019.07.061

Accepted Manuscript
Design, synthesis and biological evaluation of 2-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-
dihydropyrimidine-5-carboxylic acid derivatives as novel xanthine oxidase inhibitors
Qing Mao, Xiwen Dai, Gaoyang Xu, Yu Su, Bing Zhang, Dan Liu, Shaojie Wang
PII:
S0223-5234(19)30682-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.061
EJMECH 11558
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 May 2019
Revised Date: 13 July 2019
Accepted Date: 21 July 2019
Please cite this article as: Q. Mao, X. Dai, G. Xu, Y. Su, B. Zhang, D. Liu, S. Wang, Design, synthesis
and biological evaluation of 2-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acid
derivatives as novel xanthine oxidase inhibitors, European Journal of Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.ejmech.2019.07.061.
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ACCEPTED MANUSCRIPT
Design,
synthesis
and
biological
evaluation
of
2
-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic
acid
derivatives as novel xanthine oxidase inhibitors
a
a
b
b
a
a
a,
Qing Mao , Xiwen Dai , Gaoyang Xu , Yu Su , Bing Zhang , Dan Liu , Shaojie Wang
*
a
Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, School of Pharmaceutical
Engineering, Shenyang Pharmaceutical University, 103 Culture Road, Shenhe District, Shenyang 110016, China
b
Department of Pharmacology, Shenyang Pharmaceutical University, No.103 103 Culture Road, Shenhe District,
Shenyang, Liaoning110016, China
*Corresponding authors.
Tel/Fax: +86-24-43520230, E-mail: sjwang_99@163.com (S. J. Wang);
1

ACCEPTED MANUSCRIPT
Abstract
In our previous study, we reported a series of 1-hydroxy-2-phenyl-1H-imidazole-5-carboxylic
acid derivatives that presented excellent in vitro xanthine oxidase (XO) inhibitory potency. To
further investigate the structure-activity relationships of these compounds, the imidazole ring was
transformed
to
a
pyrimidine
ring
to
design
2
2
9
1
-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic
acids
(8a-8j),
-(4-alkoxy-3-cyano)phenyl-4-methyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acids (9c, 9e, 9j,
l) and 2-(4-alkoxy-3-cyano)phenyl-6-imino-1,6-dihydropyrimidine-5-carboxylic acids (10c, 10e,
0j, 10l). These compounds exhibited remarkable in vitro XO inhibitory potency with IC values
5
0
ranging from 0.0181 µM to 0.5677 µM. Specifically, compounds 10c and 10e, with IC values of
5
0
0
.0240 µM and 0.0181 µM, respectively, emerged as the most potent XO inhibitors, and their
potencies were comparable to that of febuxostat. Structure-activity relationship analysis revealed
that the methyl group at 4-position of pyrimidine ring could damage the potency, and the XO
inhibitory potency was maintained when carbonyl group was changed to an imino group.
Lineweaver-Burk plot analysis revealed that the representative compound 10c acted as a mixed-type
inhibitor. A potassium oxonate induced hyperuricemia model in rats was chosen to further confirm
the hypouricemic effect of compound 10c, and the results showed that compound 10c (5 mg/kg)
was able to significantly lower the serum uric acid level. Furthermore, in acute oral toxicity study,
no sign of toxicity was observed when the mice were administered with a single 2000 mg/kg oral
dose of compound 10c. These results suggested that compound 10c was a potent and promising uric
acid-lowing agent for the treatment of hyperuricemia.
2

ACCEPTED MANUSCRIPT
Keywords: 1,6-Dihydropyrimidine-5-carboxylic acid; Xanthine oxidase inhibitor; Synthesis;
Biological evaluation.
1
. Introduction
Xanthine oxidase (XO) is a key enzyme in the purine metabolic pathway that catalyzes the
oxidation of hypoxanthine to xanthine and eventually to the final product uric acid [1]. The
overproduction of uric acid could lead to hyperuricemia, which has been established as the major
etiologic factor in gout [2]. The human body accumulates purines externally through the intake of
food and internally through the degradation of DNA and RNA molecules. Specifically, modern
humans have dramatically increased the consumption of purine-rich foods, alcohol, and soft drinks
sweetened with fructose, which can cause overproduction of hypoxanthine and eventually lead to
overproduction of uric acid [3-4]. Moreover, the high expression of XO is associated with
overproduction of uric acid [4]. Many evidences established that inhibition of XO can effectively
reduce the level of uric acid, and XO inhibitors are used as uric acid-lowering drugs for the
treatment of hyperuricemia and gout [5-8].
Allopurinol, a classical XO inhibitor, has been used to treat hyperuricemia for several decades.
However, as a purine analog, allopurinol affects the metabolism of purines and leads to severe
life-threatening side effects, including fulminant hepatitis, renal failure, and Stevens-Johnson
syndrome [4], in some cases. Hence, there is a considerable need for developing nonpurine XO
inhibitors with potent XO inhibitory activity and with fewer side effects to replace allopurinol. As a
result, the discovery of novel nonpurine XO inhibitors has drawn extensive attention from the
3

ACCEPTED MANUSCRIPT
pharmaceutical industry worldwide in recent years, and many nonpurine XO inhibitors with
excellent potency, such as pyrazoles (e.g., Y-700) [5-6], selenazoles [7], isoxazoles [8], imidazoles
[9], triazoles [10-11], thiazoles [12-13] and some flavone analogs [18-25], have been reported. As a
result of these efforts, febuxostat was approved by the FDA in 2009 [26], and another nonpurine XO
inhibitor, topiroxostat, was subsequently approved in 2013 [27]. They both showed a more potent
and longer-lasting urate-lowering effect than allopurinol (Fig. 1). However, in 2019, the FDA added
a boxed warning for increased risk for heart-related death with febuxostat, and the mechanism of
cardiotoxicity caused by febuxostat is not clear [28-29]. Therefore, new nonpurine XO inhibitors
are still urgently needed to fulfill the clinical requirement of treatment of hyperuricemia and gout
[30].
Fig. 1. Chemical structures of allopurinol and nonpurine XO inhibitors.
Pyrimidine is an important scaffold of many natural and synthetic products and is widely used in
4

ACCEPTED MANUSCRIPT
medicinal chemistry [31-33]. In particular, Rao [34-36] and Narjes [37] have reported a series of
pyrimidine-moiety containing XO inhibitors, such as compounds 6 and 7 (Fig.2), displaying
excellent XO inhibitory potency. According to their docking research, the pyrimidine ring of
compounds 6 and 7 as a whole is sandwiched between Phe914 and Phe1009 (face-to-face with
Phe914 and face-to-edge with Phe1009) [34-37], and more interestingly, the same pi-stacking
interaction could also be found in allopurinol with XO [38]. Therefore, the pyrimidine moiety
could be used as a significant pharmacophore for XO inhibitor design. There are many successful
cases of enlarging the five-membered heterocycles to corresponding six-membered ones in drug
design [39-40], such as from fluvastatin to pitavastatin, in which enlargement of a pyrrole ring to a
pyridine ring led to a significant improvement in potency [39]. The abovementioned findings
encouraged
us
-hydroxy-4-methyl-2-phenyl-1H-imidazole-5-carboxylic acids to a pyrimidine ring to design the
-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acids as nonpurine XO
to
enlarge
the
imidazole
ring
of
1
2
inhibitors
(Fig.
2).
In
addition,
4-methyl
derivatives
acids
of
2
2
-(4-alkoxy-3-cyano)phenyl-4-methyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic
and
-(4-alkoxy-3-cyano)phenyl-6-imino-1,6-dihydropyrimidine-5-carboxylic acids were also
designed for comparison.
5

ACCEPTED MANUSCRIPT
Fig. 2. Design of compounds.
In this study, we described the synthesis, in vitro XO inhibitory evaluation and structure-activity
relationships (SARs) of 2-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic
acid derivatives, and molecular modeling studies and steady-state kinetic analysis were also
performed to investigate the inhibitory behavior of the representative compound 10c. Furthermore,
the hypouricemic effect of 10c was also evaluated using a potassium oxonate induced
hyperuricemia model in rats. Finally, acute oral toxicity study of compound 10c was performed
according to OECD guidelines 2001 [41].
2
. Results and discussion
.1 Chemistry
2
6

ACCEPTED MANUSCRIPT
CHO
CN
CN
i
ii
iii
HO
HO
HO
R O
1
CHO
CHO
CN
CN
1
1
12
13a-l
a, R = iso-Propyl
g, R = Benzyl
1
1
b, R = iso-Butyl
h, R = p-Fluorobenzyl
1
1
c, R = iso-Pentyl
i, R = p-Chlorobenzyl
1
1
d, R = Allyl
j, R = p-Bromobenzyl
1
k, R = p-tert-Butylbenzyl
1
1
NH
e, R = iso-Butenyl
1
f, R = iso-Pentenyl
l, R = p-Methylbenzyl
1
1
iv
NH2
O
O
R O
1
•HCl
COOH
COOEt
CN
4a-l
HN
HN
1
v
vi
N
R2
N
R2
O
O
R O
R O
1
1
CN
8a-l R = H
O
O
CN
5a-l R = H
1
O
R2
2
2
vii
16c,16e,16j,16l R = CH
9c,9e,9j,9l R = CH
2
3
2
3
1
2
9 R = H
0 R = CH
2
2
3
Cl
N
NH
NH
COOEt
COOEt
COOH
N
HN
HN
viii
vi
N
N
R O
R O
R O
1
1
1
CN
CN
18c,18e,18j,18l
CN
10c,10e,10j,10l
1
7c,17e,17j,17l
Scheme 1. Reagents and conditions: (i) a. hydrochloric acid, paraformaldehyde, POCl , rt, 8 h. b. HMTA, 50%
3
CH COOH, reflux, 1 h. (ii) Hydroxylamine hydrochloride, HCOONa, HCOOH, NMP, reflux, 6 h. (iii) various alkyl
3
chlorides or alkyl bromides, K CO , KI, DMF, 60°C, N . (iv) a. CH ONa, CH OH, rt, 24 h. b. NH Cl, 50°C, 4 h. (v)
2
3
2
3
3
4
CH CH ONa, CH CH OH, 80°C, 2 h. (vi) LiOH, H O, THF, CH CH OH, 50°C, 3 h. (vii) SOCl , DMF, rt, 2 h. (viii)
3
2
3
2
2
3
2
2
NH OH, THF, rt, 2 h.
4
The syntheses of the target compounds were performed as outlined in Scheme 1. The
commercially available salicylaldehyde was chloromethylated with paraformaldehyde in
hydrochloric acid to give 5-chloromethyl-2-hydroxybenzaldehyde, which was then transformed to
4
-hydroxyisophthalaldehyde (11) via a Sommelet reaction [42]. Next, 4-hydroxyisophthalaldehyde
(11) was treated with hydroxylamine hydrochloride to obtain the key intermediate
7

ACCEPTED MANUSCRIPT
4
-hydroxyisophthalonitrile (12) [43], which was alkylated with various alkyl halides to obtain the
corresponding 4-alkoxy-isophthalonitriles (13a-l) with good yields [13]. Subsequently,
-alkoxy-3-cyanobenzimidamide hydrochlorides (14a-l) were obtained through the alcoholysis of
4
intermediate 4-alkoxy-isophthalonitriles (13a-l) in the presence of sodium methoxide followed by
aminolysis with ammonium chloride [44]. Cyclization of diethyl ethoxymethylenemalonate (19)
with 4-alkoxyisophthalonitriles (14a-l) in the presence of sodium ethoxide led to ethyl
2
-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylates (15a-l) [45]. Finally, the
target compounds, 2-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acids
8a-l), were obtained by hydrolyzing ethyl
-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylates (15a-l) in an aqueous
solution of lithium hydroxide. similar method was used to synthesize
-(4-alkoxy-3-cyano)phenyl-4-methyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acids (9c, 9e, 9j,
l). The corresponding ethyl
-(4-alkoxy-3-cyano)phenyl-6-imino-1,6-dihydropyrimidine-5-carboxylates (18c, 18e, 18j, 18l)
obtained through SOCl2 chlorination of ethyl
-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylates (15c, 15e, 15j, 15l)
followed by ammonia aminolysis, and the
-(4-alkoxy-3-cyano)phenyl-6-imino-1,6-dihydropyrimidine-5-carboxylic acids (10c, 10e, 10j, 10l)
were synthesized by subsequently hydrolyzing the corresponding ethyl
-(4-alkoxy-3-cyano)phenyl-6-imino-1,6-dihydropyrimidine-5-carboxylates (17c, 17e, 17j, 17l) in
the aqueous solution of lithium hydroxide [45].
(
2
A
2
9
2
were
2
2
2
1
13
The target compounds were elucidated by HRMS, IR, H NMR and C NMR spectra. All
8

ACCEPTED MANUSCRIPT
spectral data were in accordance with the assumed structures. In HRMS analysis, the target
-
compounds showed [M-H] ion peaks. The IR spectra of the target compounds displayed the cyano
-1
group stretching vibrations at 2239-2240 cm and amide peaks at approximately 3406, 1645 and
-1
1
1
566 cm . In H NMR spectra, the CH of the pyrimidine ring was observed as a singlet at
approximately 8.70 ppm for compounds 8a-l. The singlet disappeared in 4-methyl derivatives 9c, 9e,
9
j, and 9l.
2
.2. XO inhibitory activity
The in vitro bovine XO inhibitory potencies of target compounds were measured
spectrophotometrically by determining uric acid production at 295 nm [27]. Febuxostat and
allopurinol were included as positive controls. As shown in Table 1 and Table 2, all compounds
exhibited excellent inhibitory potency with IC values ranging from 0.0181 µM to 0.5677 µM. In
5
0
particular, the 6-imino derivatives 10c (IC = 0.0240 µM) and 10e (IC = 0.0181 µM), bearing an
5
0
50
iso-pentyl and iso-butenyl substituent at R , respectively, emerged as the most potent XO inhibitors
1
which were comparable to febuxostat (IC = 0.0236 µM), and their potencies were 316-fold and
5
0
4
19-fold higher than that of allopurinol, respectively.
Table 1
In vitro XO inhibitory potency of compounds 8a-l
9

ACCEPTED MANUSCRIPT
a
a
Compound
R1
IC50 (µM)
Compound R1
IC50 (µM)
8
8
8
8
8
8
a
b
c
d
e
f
iso-Propyl
iso-Butyl
iso-Pentyl
Allyl
0.0916 ± 0.0059
0.0609 ± 0.0106
0.0250 ± 0.0066
0.0811 ± 0.0004
0.0336 ± 0.0082
0.0388 ± 0.0064
7.5902 ± 0.2145
0.0236 ± 0.0023
8g
8h
8i
Benzyl
0.0387 ± 0.0151
0.0382 ± 0.0013
0.0499 ± 0.0098
0.0298 ± 0.0093
0.1970 ± 0.0465
0.0354 ± 0.0075
p-Fluorobenzyl
p-Chlorobenzyl
p-Bromobenzyl
p-tert-Butylbenzyl
p-Methylbenzyl
8j
8k
8l
iso-Butenyl
iso-Pentenyl
Allopurinol
Febuxostat
a
Values are means ± SD of three independent experiments
As
shown
in
Table
1,
the
XO
inhibitory
potencies
for
the
2
-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acids were influenced by
the size of the carbon chain at R . The IC of compound 8c (IC = 0.0250 µM), which bears an
1
50
50
iso-pentyl substituent, was 3.7-fold and 2.4-fold higher than those of the iso-propyl and iso-butyl
substituted compounds 8a (IC = 0.0916 µM) and 8b (IC = 0.0609 µM), respectively, which
5
0
50
suggested that shortening the carbon chains could damage the potency. The IC50 value of the
allyl-substituted compound 8d was 0.0811 µM. Introducing a methyl substituent into the double
bond of the allyl group led to compounds 8e (IC = 0.0336 µM) and 8f (IC = 0.0388 µM), and
5
0
50
their potencies were 2.4-fold and 2.1-fold higher than that of 8d, respectively. This finding indicated
that the introduction of a methyl substituent into the double bond of the allyl group could favor the
1
0

ACCEPTED MANUSCRIPT
XO inhibitory potency.
The benzyl substituted compound 8g (IC = 0.0387 µM) showed a slight decrease in XO
5
0
inhibitory potency compared with 8c. To explore the possibility of improving the inhibitory activity
of benzyl-substituted derivatives, a halogen (F, Cl and Br), methyl group and tert-butyl group were
introduced at the para-position of the benzyl moiety. When a methyl group or a fluorine atom was
linked at the para-position (8l, IC = 0.0354 µM and 8h, IC = 0.0382 µM, respectively), the
5
0
50
potency was maintained. Attachment of a bromine atom resulted in a slight increase in potency (8j,
IC = 0.0298 µM). However, employment of a tert-butyl group at the para-position (8k, IC =
50
50
0
.1970 µM) led to a significant decline in potency, and this decline is probably due to the steric
hindrance created by the tert-butyl group. The above observations indicated that the bromine atom
substituted at the para-position of the benzyl moiety was preferable for the potency.
Table 2
In vitro XO inhibitory potency of compounds 9c, 9e, 9j, 9l and 10c, 10e, 10j, 10l.
a
Compound
R1
R2
R3
O
IC50 (µM)
9
9
9
9
1
1
c
iso-Pentyl
iso-Butenyl
p-Bromobenzyl
p-Methylbenzyl
iso-Pentyl
iso-Butenyl
CH
CH
CH
CH
H
3
3
3
3
0.5400 ± 0.0134
0.5677 ± 0.0743
0.1854 ± 0.0492
0.1590 ± 0.0605
0.0240 ± 0.0032
0.0181 ± 0.0006
e
O
j
O
l
O
0c
0e
NH
NH
H
11

ACCEPTED MANUSCRIPT
1
1
0j
0l
p-Bromobenzyl
p-Methylbenzyl
H
H
NH
NH
0.0271 ± 0.0011
0.0339 ± 0.0017
7.5902 ± 0.2145
0.0236 ± 0.0023
Allopurinol
Febuxostat
a
Values are means ± SD of three independent experiments
Methyl groups play an important role in rational drug design [46]. A methyl group can
modulate the physicochemical, pharmacodynamic and pharmacokinetic properties by the ortho
effect [46]. Thus, we attempted to introduce a methyl group into the 4-position of the pyrimidine
ring of compounds 8c, 8e, 8l and 8j, as the most potent compounds of
2
-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acids, to obtain 4-methyl
derivatives 9c, 9e, 9j, and 9l for comparison. As shown in Table 2, the introduction of a methyl
group led to a 4.5-21.6-fold decrease in potency (9c, 9e, 9j, 9l vs 8c, 8e, 8j, 8l). This finding
suggested that the presence of a methyl group at the 4-position of the pyrimidine moiety could be
detrimental to the potency.
The hydroxyl group of imidazole XO inhibitors acted as a hydrogen bond donor and linked to
Thr1010 via a hydrogen bond [13]. Due to the amide-enamine tautomerism, the carbonyl group in
the 6-position of the pyrimidine ring could function as a hydrogen bond acceptor or donor. Thus, we
attempted to change the carbonyl group to an imino group, with the hope of maintaining the
6
2
1
-position
group
as
a
hydrogen
bond
donor.
Then,
-(4-alkoxy-3-cyano)phenyl-6-imino-1,6-dihydropyrimidine-5-carboxylic acids (10c, 10e, 10j and
0l) were synthesized. As shown in Table 2, the testing results showed that these compounds were
effective with IC50 values ranging from 0.0181 to 0.0339 µM, and the XO inhibitory potency
1
2

ACCEPTED MANUSCRIPT
increased compared with the corresponding carbonyl compounds (10c, 10e, 10j, 10l vs 8c, 8e, 8j,
8
l). In particular, compound 10c (IC of 0.0240 µM) and 10e (IC of 0.0181 µM) showed the best
50 50
XO inhibitory potency of all the target compounds. Furthermore, a Lineweaver-Burk plot (Fig. 3)
revealed that compound 10c acted as a mixed-type inhibitor of XO, which was similar to imidazole
XO inhibitors and febuxostat [13, 26].
Fig. 3. Lineweaver-Burk plot analysis of xanthine oxidase by compound 10c.
Furthermore, in order to obtain enzyme inhibition properties of compound 10c, a kinetic
analysis on the inhibition was performed using Lineweaver-Burk plot analysis. The Km and Vmax
-1
values were calculated as 75.72 µM and 0.0573 min without the present of inhibitors, respectively.
As show in Fig.3, in the presence of different concentrations of compound 10c (0.025, 0.05, 0.08
and 0.1 µM), the apparent Km of enzyme was increased and the Vmax of the enzyme was decreased,
thus compound 10c inhibited the activity of XO through a mixed-type inhibition [22]. In a
mixed-type inhibition, inhibitor can affect both the enzyme affinity for its substrate and the
maximal rate of catalysis. The values of K (equilibrium constant for binding with free enzyme)
i
1
3

ACCEPTED MANUSCRIPT
and K (equilibrium constant for binding with enzyme-substrate complex) were calculated to be
is
0
.0042 µM and 0.1270 µM respectively, which confirmed that compound 10c bound to the free
XO much more easily and firmly than the XO-xanthine complex [23].
Table 3
Comparison of pyrimidines and imidazoles in XO inhibitory potency
a
a
Pyrimidines
R1
RIP
Imidazoles
R1
RIP
8
8
8
a
b
c
j
iso-Propyl
iso-Butyl
iso-Pentyl
p-Methylbenzyl
3.85
2.56
1.06
1.49
5a
5b
5c
5j
iso-Propyl
iso-Butyl
iso-Pentyl
16.67
0.60
3.03
8
p-Methylbenzyl 11.11
a
Relative Inhibitory Potency (RIP) = IC50 compound /IC50 febuxostat.
To compare the XO inhibitory
-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acids (pyrimidine XO
synthesized in this paper with those of
-hydroxy-4-methyl-2-phenyl-1H-imidazole-5-carboxylic acids (imidazole XO inhibitors)
potencies
of
2
inhibitors)
1
synthesized before in our laboratory [13], the pyrimidines 8a, 8b, 8c and 8j together with the
corresponding imidazoles 5a, 5b, 5c and 5j with the same R groups were chosen. The relative
1
inhibitory potency value [30] (RIP; the IC50 of a selected compound relative to the potency of
febuxostat) was adopted as a comparison parameter. As shown in Table 3, the pyrimidine
compounds showed relatively lower RIP values than those of imidazole compounds except the
1
4

ACCEPTED MANUSCRIPT
iso-butyl
substituted
compound.
These
results
suggested
that
the
6
-oxo-1,6-dihydropyrimidine-5-carboxylic acid moiety was more favorable for the XO inhibitory
potency than the 1-hydroxy-4-methyl-1H-imidazole-5-carboxylic acid moiety, and ring
enlargement of the imidazole ring to a pyrimidine ring led to an increase in potency.
2
.3. Molecular modeling
To explore the probable interaction model of inhibitors and the enzyme active site, molecular
docking of the compounds 8c, 9c and 10c into the substrate binding pocket of XO was performed.
The docking experiment was carried out using the Glide XP docking protocol (2016, Schrodinger
Suite). Since molybdenum-pterion sites of both XO and xanthine dehydrogenase (XDH) are
structurally equivalent [47], the X-ray crystal structure of the complex XDH/febuxostat (PDB code
1
N5X) was adopted in the docking calculation [48]. The protein was prepared by removing all water
molecules and adding all hydrogen atoms using Protein Preparation Wizard (2016, Schrodinger
Suite). The carboxyl groups of the compounds 8c, 9c and 10c and febuxostat were calculated in
dissociated form using the LIGPREP module in Schrodinger Suite. The binding models of the
representative compounds 8c, 9c and 10c were illustrated by Discovery Studio Visualizer 2017.
1
5

ACCEPTED MANUSCRIPT
Fig. 4. Binding modes of compounds 8c, 9c and 10c within the XO binding pocket. Hydrogen bonds are shown as
green dashed lines, and π-π stacking interactions are shown as violet dashed lines. A) Overlay of compound 8c
(purple), 9c (yellow) and febuxostat (gray) at the binding site. B) Binding modes of compound 10c within the XO
binding pocket.
As shown in Fig. 4A, compounds 8c and 9c presented a set of interactions similar to febuxostat.
As expected, the pyrimidine ring as a whole was sandwiched between Phe914 and Phe1009 via
face-to-face and face-to-edge pi-stacking interactions, respectively. In addition, the carbonyl group
of the pyrimidine formed two hydrogen bonds with Thr1010, which was similar to the 1-hydroxyl
group of the imidazole compound 5b [13], the nitrogen atom of the pyrimidine forming a hydrogen
bond with Glu802 and the cyano group of the phenyl unit forming a hydrogen bond with Asn768
and Lys771, and several hydrophobic interactions with Leu648, Phe649, Leu873, Val1011 and
Leu1014 were also observed at the hydrophobic pocket. Moreover, we found that the methyl group
at the 4-position of the pyrimidine ring was located on a hydrophilic surface (Fig. 4A), and the
presence of a methyl group may adversely affect the activity. This observation explained the
1
6

ACCEPTED MANUSCRIPT
decrease in activities after adding the methyl group at the 4-position of the pyrimidine ring.
The pyrimidine moiety of compound 10c flipped 180 degrees (Fig. 4B), and the amino group
established a hydrogen bond with the carboxyl group of Glu802 instead of forming a hydrogen bond
with Thr1010, presenting a set of similar interactions to Rao’s compound 6 [38-40]. Taking the
potency comparison between the carbonyl series and imino series into consideration, we believe
that the hydrogen bond formed between the amino group and Glu802 was stronger than that
between the carbonyl group and Thr1010, and the interaction of the amino group with Glu802 could
be highlighted for the design of novel nonpurine XO inhibitors.
2.4. Hypouricemic effect in vivo
Fig. 5. Effect of compound 10c, febuxostat and allopurinol on the serum uric acid levels in the potassium oxonate
induced hyperuricemic rat model. (a) Time course changes in the serum uric acid levels after oral administration of
compound 10c, febuxostat and allopurinol in the potassium oxonate induced hyperuricemic rat model. (b) The sUA
levels 2 h after oral administration of febuxostat, allopurinol and 10c in the potassium oxonate induced
hyperuricemic rat model. (c) The AUC (uric acid, 2-8 h) after oral administration of 10c, febuxostat and allopurinol
in the potassium oxonate induced hyperuricemic rat model. Data are expressed as the mean ± SD ****P < 0.0001,
** P < 0.001 vs hyperuricemic rats (model).
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7

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Compound 10c was further tested in vivo to verify its hypouricemic effect in the acute
hyperuricemic rat model compared to the standard inhibitors febuxostat and allopurinol by
examining uric acid levels in the serum (Fig. 5). As expected, subcutaneous injection of 300
mg/kg potassium oxonate into rats markedly increased serum uric acid (sUA) levels in the
hyperuricemia vehicle group compared to the blank group (P < 0.001 for Blank vs Model). At a
single oral dose of 5 mg/kg, compound 10c was able to significantly reduce the serum
concentration of uric acid compared with the hyperuricemia vehicle group 2 h after administration
in hyperuricemic rats (P< 0.001). In addition, the AUC of sUA levels from a 2 h to 8 h period of
the study also showed that compound 10c was able to significantly lower the sUA level compared
with the model group (P< 0.0001), and the hypouricemic potency was similar to allopurinol (10
mg/kg). These results of in vivo hypouricemic activity evaluation suggested that compound 10c was
a potent and promising uric acid-lowering agent for the treatment of hyperuricemia.
2
.5 Acute oral toxicity study
In order to explore the preliminary toxicity profile of the most potent compound 10c, acute
oral toxicity study was performed according to OECD guidelines 2001 [41], and the result showed
that no gross behavioral abnormality was found within 24 h after the administration of test
compound at a single dose of 2000 mg/kg, which suggested that LD50 value of compound 10c
might greater than 2000 mg/Kg, and it was about 400 times over the effective dose (5 mg/Kg). Thus,
we can confirm that compound 10c have good potential to be a safety and effective uric acid-lowing
agent for the treatment of hyperuricemia.
1
8

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3
. Conclusion
In
summary,
we
designed,
synthesized
and
identified
a
series
of
2
-(4-alkoxy-3-cyano)phenyl-1,6-dihydropyrimidine-5-carboxylic acid derivatives as novel XO
inhibitors, and these compounds showed excellent in vitro XO inhibitory potency. Specifically,
compounds 10c and 10e emerged as the most potent XO inhibitors and were comparable to
febuxostat. The potency comparison between the present pyrimidine compounds and the imidazole
compounds reported before indicated that the 6-oxo-1,6-dihydropyrimidine-5-carboxylic acid
moiety
was
more
favorable
for
XO
inhibitory
potency
than
the
1
-hydroxy-4-methyl-1H-imidazole-5-carboxylic acid moiety. SAR analysis revealed that the
methyl group at the 4-position of the pyrimidine moiety could damage the potency, and an
increasing trend could be observed when the carbonyl group was changed to an imino group.
Molecular docking studies rationalized the SARs observed and provided insight into the binding
mode of the target compounds with XO. Furthermore, the Lineweaver-Burk plot showed that the
representative compound 10c acted as a mixed-type XO inhibitor. The results of in vivo
hypouricemic activity evaluation suggested that compound 10c could considerably reduce the
serum uric acid. Moreover, it was found noteworthy that no sign of toxicity was observed when the
mice were administered with a single 2000 mg/kg oral dose of compound 10c. Thus, these results
suggested that compound 10c was a potent and promising uric acid-lowering agent for the treatment
of hyperuricemia.
1
9

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4
4
. Experimental protocols
.1 Chemistry
Unless otherwise indicated, reagents and solvents were purchased from commercial sources
and used without further purification. All reactions were monitored by TLC using silica gel
aluminum cards (0.2 mm thickness) with 254 nm and 365 nm fluorescent indicators. Melting points
1
13
were recorded on a YRT-3 melting apparatus and were uncorrected. H NMR and C NMR spectra
were recorded on a Bruker 400 MHz or 600 MHz spectrometer. Chemical shifts were expressed in
parts per million using tetramethylsilane as an internal reference and DMSO-d as the solvent. IR
6
spectra were determined as KBr pellets on a Bruker IFS-55 spectrometer and expressed as the
number of wavelengths per centimeter. ESI-MS data were gathered using an Agilent 1100
instrument. ESI-HRMS data were recorded on the Agilent 6540 Series Q-TOF-MS system.
4
.1.1. Synthesis of 4-hydroxyisophthalaldehyde (11)
A mixture of salicylaldehyde (60 g, 0.39 mol) and paraformaldehyde (24.2 g, 0.64 mol) in
concentrated hydrochloric acid (390 ml) was stirred at room temperature for 1 h and then POCl (20
3
ml) was added dropwise at 0 - 10 °C, large amounts of solids gradually precipitated. After
completion of addition, the mixture was stirred at room temperature for another 8 h. The solid was
separated by filtration as a white powder and dried under vacuum at 40 °C. The above product was
2
0

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added to a solution of HMTA (74.7 g, 0.53 mol), H O (140 mL), CH COOH (140 mL) and refluxed
2
3
for 1 h. After completion of the reaction, 140 ml of concentrated hydrochloric acid was added and
refluxed for 10 min. Then, the mixture was poured into ice water and stirred for 30 min. The solid
was separated by filtration as a yellow powder (66 g) in 89.5% yield which was used directly in the
next step.
4
.1.2. Synthesis of 4-hydroxyisophthalonitrile (12)
A solution of 11 (66 g，0.44 mol), hydroxylamine hydrochloride (91.7 g, 1.32 mol), HCOONa
(110.7 g, 1.63 mol), NMP (30 ml) in HCOOH (400 ml) was refluxed for 6 h. The reaction mixture
was cooled at room temperature and stirred at 10 °C for 30 min. Then the precipitate was filtered
and recrystallized from 50% ethanol to give 12 (34.5 g, 54.5%) as a gray white solid. mp: degraded
1
at 190 °C. H NMR (600 MHz, DMSO-d ) δ 12.42 (s, 1H), 8.27 (s, 1H), 7.93 (d, J = 8.8 Hz, 1H),
6
-
7
.15 (d, J = 8.8 Hz, 1H). ESI-MS: m/z 142.8 [M-H] .
4
.1.3. General procedure for the synthesis of 4-alkoxy-isophthalonitrile 13a-l
A solution of 12 (50 mmol), anhydrous potassium carbonate (65 mmol), potassium iodide (1
mmol), and alkyl halide (75 mmol) in DMF (40 mL) was stirred at 50 °C for 6 h under nitrogen
atmosphere. After the reaction was completed, the mixture was poured into water (200 mL). Then
the precipitate was filtered and recrystallized from ethanol to yield 4-alkoxy- isophthalonitrile
(13a-l).
4
.1.3.1. 4-iso-Propoxyisophthalonitrile(13a). A white solid, yield: 77.9%. mp: 147.9 °C – 148.5 °C.
2
1

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1
H NMR (600 MHz, DMSO-d ) δ 8.37 (s, 1H), 8.11 (d, J = 8.7 Hz, 1H), 7.47 (s, 1H), 4.95 (m,
6
+
1
H), 1.35 (d, J = 5.9 Hz, 6H). ESI-MS: m/z 187.1 [M+H] .
4
.1.3.2. 4-iso-Butoxyisophthalonitrile(13b). A white solid, yield: 88.1%. mp: 129.6 °C – 130.1 °C.
1
H NMR (600 MHz, DMSO-d ) δ 8.36 (d, J = 2.1 Hz, 1H), 8.11 (dd, J = 8.9, 2.1 Hz, 1H), 7.42 (d, J
6
=
8.9 Hz, 1H), 4.02 (d, J = 6.4 Hz, 2H), 2.07 (m, 1H), 1.00 (d, J = 6.7 Hz, 6H).。ESI-MS: m/z 223.8
+
[
M + Na] .
4
1
7
6
.1.3.3. 4-iso-Pentyloxyisophthalonitrile(13c). A white solid, yield: 66.6%. mp: 133.8 °C –
1
34.1 °C. H NMR (600 MHz, DMSO-d ) δ 8.36 (d, J = 2.1 Hz, 1H), 8.13 (dd, J = 8.9, 2.1 Hz, 1H),
6
.45 (d, J = 8.9 Hz, 1H), 4.26 (t, J = 6.6 Hz, 2H), 1.79 (m, 1H), 1.66 (q, J = 6.7 Hz, 2H), 0.94 (d, J =
+
.7 Hz, 6H). ESI-MS: m/z 215.7 [M+H] .
1
4
.1.3.4. 4-Allyloxyisophthalonitrile(13d). A white solid, yield: 76.3%. mp: 200.4 °C – 201.1 °C. H
NMR (600 MHz, DMSO-d ) δ 8.39 (d, J = 2.1 Hz, 1H), 8.14 (dd, J = 8.9, 2.1 Hz, 1H), 7.43 (d, J =
6
8
.9 Hz, 1H), 6.06 (m, 1H), 5.47 (m, 1H), 5.34 (m, 1H), 4.86 (d, J = 5.2 Hz, 2H). ESI-MS: m/z 185.3
+
[
M+H] .
4
1
7
.1.3.5. 4-(2-Methylallyloxy)isophthalonitrile(13e). A white solid, yield: 73.3%. mp: 157.6 °C –
1
58.5 °C. H NMR (600 MHz, DMSO-d ) δ 8.41 (d, J = 2.2 Hz, 1H), 8.14 (dd, J = 8.9, 2.2 Hz, 1H),
6
.43 (d, J = 8.9 Hz, 1H), 5.13 – 5.09 (m, 1H), 5.04 (t, J = 1.5 Hz, 1H), 4.78 (s, 2H), 1.79 (s, 3H).
+
ESI-MS: m/z 199.1 [M+H] .
2
2

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4
1
2
.1.3.6. 4-[(3-
M
ethylbut-2-en-1-yl)oxy]isophthalonitrile(13f). A white solid, yield: 61.2%. mp:
1
33.8 °C – 134.1 °C. H NMR (600 MHz, DMSO-d ) δ 8.37 (d, J = 2.2 Hz, 1H), 8.14 (dd, J = 8.9,
6
.2 Hz, 1H), 7.44 (d, J = 8.9 Hz, 1H), 5.49 – 5.42 (m, 1H), 4.82 (d, J = 6.9 Hz, 2H), 1.76 (d, J = 15.9
+
Hz, 6H). ESI-MS: m/z 213.4 [M+H] .
4
.1.3.7. 4-Benzyloxyisophthalonitrile(13g). A white solid, yield: 80.1%. mp: 184.6 °C – 185.7 °C.
1
H NMR (400 MHz, DMSO-d ) δ 8.40 (d, J = 2.2 Hz, 1H), 8.15 (dd, J = 8.9, 2.2 Hz, 1H), 7.53 (d, J
6
+
=
8.9 Hz, 1H), 7.51 – 7.34 (m, 5H), 5.40 (s, 2H). ESI-MS: m/z 235.4 [M+H] .
4
2
7
2
.1.3.8. 4-(4-Fluorobenzyloxy)isophthalonitrile(13h). A white solid, yield: 43.8%. mp: 217.9 °C –
1
18.1 °C. H NMR (400 MHz, DMSO-d ) δ 8.41 (d, J = 2.1 Hz, 1H), 8.16 (dd, J = 8.8, 2.1 Hz, 1H),
6
.64 (d, J = 8.1 Hz, 2H), 7.51 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 8.1 Hz, 2H), 5.38 (s, 2H). ESI-MS: m/z
+
53.3 [M+Na] .
4
2
7
.1.3.9. 4-(4-Chlorobenzyloxy)isophthalonitrile(13i). A white solid, yield: 43.6%. mp: 217.3 °C –
1
18.8 °C. H NMR (400 MHz, DMSO-d ) δ 8.41 (d, J = 2.2 Hz, 1H), 8.16 (dd, J = 8.9, 2.2 Hz, 1H),
6
.72 – 7.60 (m, 2H), 7.51 (d, J = 8.9 Hz, 1H), 7.48 – 7.40 (m, 2H), 5.38 (s, 2H). ESI-MS: m/z 269.0
+
[
M+H] .
4
.1.3.10. 4-(4-Bromobenzyloxy)isophthalonitrile(13j). A white solid, yield: 86.2%. mp: 184.7 °C –
1
1
85.6 °C. H NMR (400 MHz, DMSO-d ) δ 8.41 (d, J = 2.2 Hz, 1H), 8.16 (dd, J = 8.9, 2.2 Hz, 1H),
6
2
3

ACCEPTED MANUSCRIPT
+
7
.64 (d, J = 8.0 Hz, 2H), 7.48 (m, 3H), 5.38 (s, 2H). ESI-MS: m/z 313.1 [M+H] .
4
1
2
1
.1.3.11. 4-[4-(tert-Butyl)benzyloxy]isophthalonitrile(13k). A white solid, yield: 77.4%. mp:
1
33.3 °C – 133.9 °C. H NMR (600 MHz, DMSO-d ) δ 8.40 (d, J = 2.1 Hz, 1H), 8.16 (dd, J = 8.9,
6
.1 Hz, 1H), 7.55 (d, J = 8.9 Hz, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 5.36 (s, 2H),
+
.29 (s, 9H). ESI-MS: m/z 291.4 [M+H] .
4
–
7
.1.3.12. 4-[(4-Methyl)benzyloxy]isophthalonitrile(13l). A white solid, yield: 76.4%. mp: 174.5 °C
1
175.9 °C. H NMR (600 MHz, DMSO-d ) δ 8.40 (d, J = 2.1 Hz, 1H), 8.15 (dd, J = 8.9, 2.1 Hz, 1H),
6
.52 (d, J = 8.9 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 5.35 (s, 2H), 2.32 (s, 3H).
+
ESI-MS: m/z 249.2 [M+H] .
4
.1.4. General procedure for synthesis of 3-cyano-4-alkoxybenzimidamide hydrochloride(14a-l)
A 500 mL flask was charged with anhydrous methanol (300 mL), compound 13a-l (17.40
mmol) and sodium methoxide (5.22 mmol). The complex was protected from moisture and stirred
for 36 h. Then, NH Cl (34.8 mmol) was added and stirring at 50°C for 6 h. Unreacted reactant was
4
filtered, and the reaction mixture was concentrated under reduced pressure. The crude residue was
refluxed with ethyl acetate and the precipitate was collected by filtration to give compounds 14a-l,
which was used for the next reaction without further purification.
4
.1.4.1. 3-Cyano-4-isopropoxybenzimidamide hydrochloride(14a). A white solid, yield: 66.9%.
1
mp: >250 °C. H NMR (400 MHz, DMSO-d ) δ 8.33 (d, J = 2.5 Hz, 1H), 8.20 (dd, J = 9.1, 2.5 Hz,
6
+
1
H), 7.48 (d, J = 9.1 Hz, 1H), 4.97 (m, 1H), 1.37 (d, J = 5.9 Hz, 6H). ESI-MS: m/z 204.4 [M+H] .
2
4

ACCEPTED MANUSCRIPT
4
.1.4.2. 3-Cyano-4-isobutoxybenzimidamide hydrochloride(14b). A white solid, yield: 42.2%.
1
mp: >250 °C. H NMR (400 MHz, DMSO-d ) δ 8.31 (d, J = 2.6 Hz, 1H), 8.18 (dd, J = 8.8, 2.6 Hz,
6
1
H), 7.48 (d, J = 8.8 Hz, 1H), 4.06 (d, J = 6.4 Hz, 2H), 2.10 (m, 1H), 1.03 (d, J = 6.6 Hz, 6H).
+
ESI-MS: m/z 218.1 [M+H] .
4
.1.4.3. 3-Cyano-4-isopentyloxybenzimidamide hydrochloride(14c). A white solid, yield: 67.2%.
1
mp: >250 °C. H NMR (400 MHz, DMSO-d ) δ 8.30 (d, J = 2.5 Hz, 1H), 8.17 (dd, J = 9.0, 2.5 Hz,
6
1
H), 7.51 (d, J = 9.0 Hz, 1H), 4.30 (t, J = 6.5 Hz, 2H), 1.83 (m, 1H), 1.70 (q, J = 6.6 Hz, 2H), 0.96 (d,
+
J = 6.6 Hz, 6H). ESI-MS: m/z 232.3 [M+H] .
4
.1.4.4. 4-Allyloxy-3-cyanobenzimidamide hydrochloride(14d). A white solid, yield: 72.1%.
1
mp: >250 °C. H NMR (400 MHz, DMSO-d ) δ 8.33 (d, J = 2.4 Hz, 1H), 8.19 (dd, J = 9.0, 2.4 Hz,
6
1
2
H), 7.49 (d, J = 9.0 Hz, 1H), 6.09 (m, 1H), 5.55 – 5.44 (m, 1H), 5.37 (m, 1H), 4.89 (d, J = 5.2 Hz,
+
H). ESI-MS: m/z 202.3 [M+H] .
4
.1.4.5. 3-Cyano-4-(2-methylallyloxy)benzimidamide hydrochloride(14e). A white solid, yield:
1
6
8.1%. mp: >250 °C. H NMR (600 MHz, DMSO-d ) δ 8.38 (s, 1H), 8.21 (d, J = 9.2 Hz, 1H), 7.48
6
(d, J = 9.2 Hz, 1H), 5.11 (s, 1H), 5.03 (s, 1H), 4.80 (s, 2H), 1.79 (s, 3H). ESI-MS: m/z 216.2 [M+H]
+
.
4
.1.4.6. 3-Cyano-4-[(3-methylbut-2-en-1-yl)oxy]benzimidamide hydrochloride(14f). A white solid,
1
yield: 41.1%. mp: >250 °C. H NMR (600 MHz, DMSO-d ) δ 8.34 (d, J = 2.4 Hz, 1H), 8.21 (dd, J =
6
2
5

ACCEPTED MANUSCRIPT
9
.0, 2.4 Hz, 1H), 7.48 (d, J = 9.0 Hz, 1H), 5.46 (t, J = 6.9 Hz, 1H), 4.82 (d, J = 6.8 Hz, 2H), 1.75 (dd,
+
J = 10.5, 1.4 Hz, 6H). ESI-MS: m/z 230.3 [M+H] .
4
.1.4.7. 4-Benzyloxy-3-cyanobenzimidamide hydrochloride(14g). A white solid, yield: 24.6%.
1
mp: >250 °C. H NMR (400 MHz, DMSO-d ) δ 8.36 (d, J = 2.2 Hz, 1H), 8.12 (dd, J = 8.9, 2.2 Hz,
6
+
1
H), 7.50 (d, J = 8.9 Hz, 1H), 7.41 (m, 5H), 5.37 (s, 2H). ESI-MS: m/z 252.4 [M+H] .
4
5
2
.1.4.8. 3-Cyano-4-(4-fluorobenzyloxy)benzimidamide hydrochloride(14h). A white solid, yield:
1
8.3%. mp: >250 °C. H NMR (400 MHz, DMSO-d ) δ 8.34 (d, J = 2.5 Hz, 1H), 8.20 (dd, J = 9.0,
6
+
.5 Hz, 1H), 7.65 – 7.52 (m, 3H), 7.28 (m, 2H), 5.41 (s, 2H). ESI-MS: m/z 270.2 [M+H] .
4
5
2
.1.4.9. 4-(4-Chlorobenzyloxy)-3-cyanobenzimidamide hydrochloride(14i). A white solid, yield:
1
4.2%. mp: >250 °C. H NMR (400 MHz, DMSO-d ) δ 8.35 (d, J = 2.4 Hz, 1H), 8.21 (dd, J = 9.0,
6
+
.4 Hz, 1H), 7.56 (m, 5H), 5.43 (s, 2H). ESI-MS: m/z 286.4 [M+H] .
4
7
2
.1.4.10. 4-(4-Bromobenzyloxy)-3-cyanobenzimidamide hydrochloride(14j). A white solid, yield:
1
3.9%. mp: >250 °C. H NMR (400 MHz, DMSO-d ) δ 8.32 (d, J = 2.4 Hz, 1H), 8.16 (dd, J = 9.0,
6
.4 Hz, 1H), 7.65 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 9.0 Hz, 1H), 7.47 (d, J = 8.4 Hz, 2H), 5.41 (s, 2H).
+
ESI-MS: m/z 332.1 [M+H] .
4
.1.4.11. 4-(4-tert-Butylbenzyloxy)-3-cyanobenzimidamide hydrochloride(14k). A white solid, yield:
1
5
3.1%. mp: >250 °C. H NMR (400 MHz, DMSO-d ) δ 8.31 (d, J = 2.5 Hz, 1H), 8.15 (dd, J = 9.0,
6
2
6

ACCEPTED MANUSCRIPT
2
.5 Hz, 1H), 7.59 (d, J = 9.0 Hz, 1H), 7.51 – 7.38 (m, 4H), 5.38 (s, 2H), 1.29 (s, 9H). ESI-MS: m/z
+
3
08.4 [M+H] .
4
5
2
2
.1.4.12. 3-Cyano-4-(4-methylbenzyloxy)benzimidamide hydrochloride(14l). A white solid, yield:
1
5.2%. mp: >250 °C. H NMR (400 MHz, DMSO-d ) δ 8.28 (d, J = 2.4 Hz, 1H), 8.12 (dd, J = 9.0,
6
.4 Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 7.39 (d, J = 8.0 Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H), 5.37 (s, 2H),
+
.32 (s, 3H). ESI-MS: m/z 266.4 [M+H] .
4
.1.5.
-(4-alkoxy-3-cyanophenyl)-6-oxo-1,6-dihydropyrimidine-5-carboxylate(15a-l)
Diethyl ethoxymethylenemalonate (4.6 mmol) were add to a solution of sodium hydride (9.7
mmol) and compound 14a-l (4.2 mmol) in ethanol (20 mL), the reaction mixture was stirred at
0 °C for 2h. After the reaction completed, the mixture was added 10 mL 6 M hydrochloric acid and
stirred for 0.5 h, then the precipitate was collected by filtration. The resulting residue was
recrystallized from ethanol to yield ethyl
-(4-alkoxyphenyl-3-cyano)-6-oxo-1,6-dihydropyrimidine-5-carboxylate(15a-l).
General
procedure
for
synthesis
of
ethyl
2
8
2
4
.1.5.1. Ethyl 2-(3-cyano-4-isopropoxyphenyl)-6-oxo-1,6-dihydropyrimidine-5-carboxylate (15a).
1
A white solid, yield: 43.1%. mp:199.8-201.1 °C. H NMR (600 MHz, DMSO-d ) δ 13.14 (s, 1H),
6
8
6
3
.61 (s, 1H), 8.51 (d, J = 2.4 Hz, 1H), 8.46 – 8.40 (m, 1H), 7.45 (d, J = 9.2 Hz, 1H), 4.94 (hept, J =
.0 Hz, 1H), 4.25 (q, J = 7.1 Hz, 2H), 1.36 (d, J = 6.0 Hz, 6H), 1.28 (t, J = 7.1 Hz, 3H). ESI-MS: m/z
+
50.7 [M+Na] .
2
7

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4
.1.5.2. Ethyl 2-(3-cyano-4-isobutoxyphenyl)-6-oxo-1,6-dihydropyrimidine-5-carboxylate (15b). A
1
white solid, yield: 51.3%. mp: 207.5-209.8 ℃. H NMR (600 MHz, DMSO-d ) δ 13.16 (s, 1H),
6
8
.61 (s, 1H), 8.52 (d, J = 2.4 Hz, 1H), 8.44 (d, J = 7.9 Hz, 1H), 7.42 (d, J = 9.1 Hz, 1H), 4.25 (q, J =
7
.1 Hz, 2H), 4.03 (d, J = 6.6 Hz, 2H), 2.15 – 2.05 (m, J = 6.6 Hz, 1H), 1.29 (t, J = 7.1 Hz, 3H), 1.02
+
(
d, J = 6.6 Hz, 6H). ESI-MS: m/z 342.4 [M+H] .
4
.1.5.3. Ethyl 2-(3-cyano-4-isopentyloxyphenyl)-6-oxo-1,6-dihydropyrimidine-5-carboxylate (15c).
1
A white solid, yield: 35.0%. mp: 190.7-193.4 ℃. H NMR (600 MHz, DMSO-d ) δ 13.17 (s, 1H),
6
8
.63 (s, 1H), 8.53 (d, J = 2.4 Hz, 1H), 8.46 (d, J = 9.1 Hz, 1H), 7.47 (d, J = 9.1 Hz, 1H), 4.26 (M, 4H),
1
.82 (m, J = 6.6 Hz, 1H), 1.69 (q, J = 6.6 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H), 0.96 (d, J = 6.6 Hz, 6H).
+
ESI-MS: m/z 378.6 [M+Na] .
4
.1.5.4. Ethyl 2-(4-allyloxy-3-cyanophenyl)-6-oxo-1,6-dihydropyrimidine-5-carboxylate (15d). A
1
white solid, yield: 51.3%. mp: 209.3-210.7 ℃. H NMR (600 MHz, DMSO-d ) δ 13.18 (s, 1H),
6
8
5
.64 (s, 1H), 8.55 (d, J = 2.3 Hz, 1H), 8.46 (d, J = 9.1 Hz, 1H), 7.45 (d, J = 9.1 Hz, 1H), 6.09 (m, 1H),
.49 (m, 1H), 5.36 (m, 1H), 4.87 (d, J = 5.2 Hz, 2H), 4.26 (q, J = 7.1 Hz, 2H), 1.29 (t, J = 7.1 Hz, 3H).
+
ESI-MS: m/z 347.8 [M+Na] .
4
.1.5.5. Ethyl 2-[3-cyano-4-(2-methylallyloxy)phenyl]-6-oxo-1,6-dihydropyrimidine-5-carboxylate
1
(
15e). A white solid, yield: 62.5%. mp: 199.7-201.3 ℃. H NMR (600 MHz, DMSO-d ) δ 13.18 (s,
6
1
H), 8.63 (s, 1H), 8.55 (d, J = 2.3 Hz, 1H), 8.50 – 8.38 (m, 1H), 7.43 (d, J = 9.1 Hz, 1H), 5.12 (s, 1H),
2
8

ACCEPTED MANUSCRIPT
5
.04 (s, 1H), 4.77 (s, 2H), 4.26 (q, J = 7.1 Hz, 2H), 1.81 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H). ESI-MS:
+
m/z 340.4 [M+H] .
4
.1.5.6.
Ethyl
2
-[3-cyano-4-(3-methylbut-2-en-1-yl)oxyphenyl]-6-oxo-1,6-dihydropyrimidine-5-carboxylate
1
(
15f). A white solid, yield: 38.4%.mp: 204.3-205.9 ℃. H NMR (600 MHz, DMSO-d ) δ 13.17 (s,
6
1
1
H), 8.63 (s, 1H), 8.53 (d, J = 2.3 Hz, 1H), 8.49 – 8.42 (m, 1H), 7.46 (d, J = 9.0 Hz, 1H), 5.49 (m,
H), 4.82 (d, J = 6.8 Hz, 2H), 4.26 (q, J = 7.1 Hz, 2H), 1.77 (m, 6H), 1.29 (t, J = 7.1 Hz, 3H).
+
ESI-MS: m/z 376.3[M+Na] .
4
.1.5.7. Ethyl 2-(4-benzyloxy-3-cyanophenyl)-6-oxo-1,6-dihydropyrimidine-5-carboxylate (15g). A
1
white solid, yield: 80.0%. mp: 215.7-217.9 ℃. H NMR (600 MHz, DMSO-d ) δ 13.17 (s, 1H), 8.62
6
(s, 1H), 8.55 (d, J = 2.4 Hz, 1H), 8.45 (d, J = 9.0 Hz, 1H), 7.54 (d, J = 9.1 Hz, 1H), 7.50 (d, J = 6.8
Hz, 2H), 7.44 (t, J = 7.6 Hz, 2H), 7.38 (t, J = 7.3 Hz, 1H), 5.40 (s, 2H), 4.25 (q, J = 7.1 Hz, 2H), 1.28
+
(
t, J = 7.1 Hz, 3H). ESI-MS: m/z 376.3[M+Na] .
4
2
.1.5.8.
Ethyl
-[3-cyano-4-(4-fluorobenzyloxy)phenyl]-6-oxo-1,6-dihydropyrimidine-5-carboxylate (15h).
A
1
white solid, yield: 46.9%.mp: 207.9-211.3 ℃. H NMR (600 MHz, DMSO-d ) δ 13.19 (s, 1H), 8.63
6
(s, 1H), 8.55 (d, J = 2.4 Hz, 1H), 8.47 (d, J = 9.0 Hz, 1H), 7.62 – 7.48 (m, 3H), 7.28 (t, J = 8.9 Hz,
+
2
H), 5.39 (s, 2H), 4.26 (q, J = 7.1 Hz, 2H), 1.29 (t, J = 7.1 Hz, 3H). ESI-MS: m/z 394.2[M+H] .
2
9