A series of pyrido[2,3-b[pyrazin-3(4 H)-one derivatives as aldose reductase inhibitors with antioxidant activity

Article abstract of DOI:10.1016/j.ejmech.2016.05.036

A series of pyrido[2,3-b]pyrazin-3(4H)-one based derivatives were designed as inhibitors of aldose reductase (ALR2), the enzyme which plays a key role in the development of diabetes complications as well as in the oxidative stress processes associated with diabetes and other pathologies. Most of the derivatives, having a substituted C2 aromatic group and a N4 acetic acid group on the core structure, were found to be potent and selective aldose reductase inhibitors with submicromolar IC₅₀ values, and 9c was the most active with IC₅₀ value 0.009 μM. Particularly, a number of the designed compounds bearing phenolic hydroxyl substituted C2-styryl side chain showed excellent not only in ALR2 inhibition but also in antioxidant, and among these 11i was proved to be the top one with an antioxidant ability even comparable to that of the well-known antioxidant Trolox. Structure-activity relationship and molecular docking studies highlighted the importance of phenolic hydroxyl substituents and vinyl spacer in C2 side chain of the scaffold for the construction of efficient and multifunctional ALR2 inhibitors.

Full text of DOI:10.1016/j.ejmech.2016.05.036

Accepted Manuscript
A series of pyrido[2,3-b]pyrazin-3(4H)-one derivatives as aldose reductase inhibitors
with antioxidant activity
Zhongfei Han, Xin Hao, Bing Ma, Changjin Zhu
PII:
S0223-5234(16)30434-2
10.1016/j.ejmech.2016.05.036
EJMECH 8628
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 February 2016
Revised Date: 16 May 2016
Accepted Date: 18 May 2016
Please cite this article as: Z. Han, X. Hao, B. Ma, C. Zhu, A series of pyrido[2,3-b]pyrazin-3(4H)-one
derivatives as aldose reductase inhibitors with antioxidant activity, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.05.036.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
A series of pyrido[2,3-b]pyrazin-3(4H)-one derivatives as aldose reductase
inhibitors with antioxidant activity
Zhongfei Han, Xin Hao, Bing Ma, Changjin Zhu*
Department of Applied Chemistry, Beijing Institute of Technology, No.5,
Zhongguancun South Street, 100081, Beijing, China
*Corresponding author. Tel.: +86-10-68918506; E-mail: zcj@bit.edu.cn
Abstract
A series of pyrido[2,3-b]pyrazin-3(4H)-one based derivatives were designed as
inhibitors of aldose reductase (ALR2), the enzyme which plays a key role in the
development of diabetes complications as well as in the oxidative stress processes
associated with diabetes and other pathologies. Most of the derivatives, having a
substituted C2 aromatic group and a N4 acetic acid group on the core structure, were
found to be potent and selective aldose reductase inhibitors with submicromolar IC50
values, and 9c was the most active with IC50 value 0.009 ꢀM. Particularly, a number
of the designed compounds bearing phenolic hydroxyl substituted C2-styryl side
chain showed excellent not only in ALR2 inhibition but also in antioxidant, and
among these 11i was proved to be the top one with an antioxidant ability even
comparable to that of the well-known antioxidant Trolox. Structure-activity
relationship and molecular docking studies highlighted the importance of phenolic
hydroxyl substituents and vinyl spacer in C2 side chain of the scaffold for the
construction of efficient and multifunctional ALR2 inhibitors.
Keywords
：
Aldose
reductase
inhibitor;
Antioxidant
activity;
pyrido[2,3-b]pyrazin-3(4H)-one; Structure-activity relationship
1. Introduction
Diabetes mellitus (DM), companied with cancer and cardiovascular disease, are
recognized as the three most leading cause to human death. All forms of DM, include
type I and type II, are characterized by hyperglycaemia and the development of
chronic diabetic complications including neuropathy, nephropathy, cataracts and

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retinopathy.[1-3] Heart attack is also a major complication in diabetic patients
accounting for more than 50% of all fatalities. The International Diabetes Federations
predicts that almost 600 million adults worldwide will have diabetes by the year
2035.[4] Particularly, diabetic patients in China have reached 92 million in 2007, and
this number rocketed to 114 million in 2010.[5] More and more evidence has
established that all forms of diabetes are vulnerable to chronic diabetic complications,
and those diseases are the major menace to diabetic patients.[6-9]
In past several decades, increasing studies[10-12] have shown that ALR2 plays a
crucial role in the development of chronic diabetic complications. The ALR2 is a
cytosol enzyme presented in the cytoplasm of most human cells, and catalyzes the
reduction of a series of carbonyl compounds, especially glucose, accompanied with
NADPH as coenzyme.[8,13] More significantly, this enzyme is a member of the
aldo-keto reductase(AKR) superfamily, together with sorbitol dehydrogenase form
the polyol pathway of glucose metabolism (Fig. 1), [2,9,11,14-18] which is
considered to be the leading pathogenesis of the diabetic complications. In this
glucose metabolism pathway, the enzyme ALR2 firstly catalyses the reduction of
glucose to sorbitol with NADPH as the rate-limiting step of this metabolism pathway,
and then segmental sorbitol dehydrogenase converts sorbitol into fructose with
reduction of NAD+. Under euglycemia circumstances, the ALR2 has little activity to
the polyol pathway, and the most glucose is predominantly converted to
glucose-6-phosphate by hexokinase and then implements the glycolytic
pathway.[2,6,19] However, in hyperglycemia, the activity of ALR2 is stimulated and
about one third of the total glucose is metabolized through the polyol pathway in
tissues demonstrating insulin-independent uptake of glucose, such as lens, kidney,
retina, and peripheral nerves.[2,6,19,20] Sorbitol is endowed with stronger polarity
and hard to penetrate through cell membrane, therefore, the abnormal accumulation of
sorbitol through the polyol pathway results in osmotic imbalance, cell swelling, and
membrane
permeability changes,
and
further
grows
into
diabetic
complications.[10,13,21-25] Concurrently, the alteration in cellular redox potentials
and the activity of enzymes such as nitric oxide synthase (NOS) and glutathione
reductase caused by the decrease of NADPH and NAD+ would further lead to the
cellular oxidative stress, as a consequence of the imbalance between increased
production of radical oxygen species (ROS) and reduced intracellular antioxidant
defense.[7,10,26-28] In addition, the increased AGEs caused by abnormal enhance of
the levels of glycating agents could in return give rise to other pathological change in

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the functions of intracellular proteins and result in further accumulation of
ROS.[29,30]
All those abnormal changes rooted in the polyol pathway regulated by ALR2
were responsible for multiple pathogenic mechanisms of the diabetic complications.
Therefore, inhibiting the activity of ALR2 and further reducing the polyol pathway of
glucose metabolism is supposed to be an efficient therapy to remedy and delay the
development of diabetic complications.
Fig. 1. Polyol pathway of glucose metabolism
In recent decades, a number of structurally different ARIs have been developed
and some of them are endowed with excellent inhibitory activity(Fig. 2.),
[6,10,17,18,31-38] but epalrestat is the only ARI available for the therapy in Japan
and more recently in China and India. Most of those ARIs that appeared to be
promising have not yet succeeded in the clinical trials mainly due to low in vivo
efficacies or pharmacokinetic drawbacks.[20,34,38,39] Although at present the
underlying mechanism of the low efficacy is not clear,[10,37] indeed, the
pathogenesis of the chronic diabetes complications and oxidative stress always
promote to each other. Thus, the design and synthesis of multifunctional ARIs to
inhibit the activity of ALR2 and simultaneously to reduce the progress of oxidative
stress process may be a new efficient therapy strategy.
We have developed several groups of ARIs in recent years,[15,16,19] and herein
describe
a
new
series
of
multifunctional
ARIs
based
on
pyrido[2,3-b]pyrazin-3(4H)-one and discuss their activities of ALR2 inhibition and
antioxidant.

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Fig. 2. Structures of ARIs
2. Chemistry
The designed compounds with a carboxylate substituent at N4 position and a variety
of aromatic substituents at C2 position of the pyrido[2,3-b]pyrazin-3(4H)-one
framework were obtained by the syntheses starting from the crucial intermediate
2-chloropyrido[2,3-b]pyrazine-3(4H)-one (4), which was prepared by synthetic
method depicted in Scheme 1. The synthetic route of the compound 4 using various
substituted pyridine-2,3-diamine (1a-c) as substrate is similar to our previous
work.[40]
0
Scheme 1. i: CH3CH2OOCCOOCH2CH3, 120 C; ii:POCl3, 100℃; iii: LiOH, THF/H2O(5:2), 65
0C.
As shown in Scheme 2, compounds 4a-c were alkylated at the N4 position with
methyl bromoacetate to form methyl esters 5a-c as key intermediates to obtain various
final compounds. Different phenyl groups were then attached to the C2 position of
5a-c forming compounds 6a-e and 8a-e in high yields, and then hydrolysis of 8a-e

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with saturated aqueous lithium hydroxide gave compounds 9a-e. Then, demethylation
of partial or all the methoxyl at 6b-e with AlCl3 or BBr3 followed by hydrolysis of the
esters with hydroxide gave compounds 7a-i. On the other hand, different styryl groups
were also attached to the C2 position of compound 5a-c by the Heck coupling
reaction in the presence of Pd(OAc)2 forming the intermediates 10a-i, which were
followed by demethylation and hydrolysis to form compounds 11a-l. In addition, the
reduction reaction was applied to 10c and 10e to provide compounds 12a-b, and then
compounds 13a-b obtained by the hydrolysis of 12a-b.

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Scheme 2 a: BrCH2COOCH3, K2CO3, CH3CN, 65°C; b: polyphenol, AlCl3, CH2ClCH2Cl, 80°C
;
c: LiOH, H2O, THF, rt, then 0.1N HCl; d: Substituted-C6H5B(OH)2, CsCO3, Pd(OAc)2, Ph3P,
dioxane, H2O, 90°C, N2; e: i) AlCl3 or BBr3, Dry Dichloromethane; ii) c; f: Substituted-styrene,
DMF, Et3N, (C6H4CH3)3P, 100°c; g: H2, 10% Pd/C, CH3OH.
3. Results and discussion
In addition to the essential groups of N4-acetic acid and C2-benzyl on the core
structure for excellent ARIs, the C7-halogen substituent could obviously enhance the
inhibitory activity of ARIs.[1] Thus, in the present study, the phenolic hydroxyl that
often shows antioxidant activity was further introduced to the core structure in order
to attach an antioxidant property to the ARIs.
3.1. Inhibition of Enzymes
The prepared compounds were tested for their inhibitory activity against ALR2
extracted from rat lenses, and the selectivity for the ALR2 inhibition by the
identification of inhibitory activity against aldehyde reductase (ALR1) isolated from
rat kidneys. ALR1 is closely related to ALR2 and plays a pivotal role in detoxification
process by metabolizing toxic aldehydes such as methylglyoxal, 3-deoxyglucosone,
and hydroxynonenal (HNE).[41,42] Thus, ALR1 inhibition could give rise to some
undesirable side effects. IC50 and the percentage of enzyme inhibition (%) were
applied to express the test results of ARL2 and ARL1, respectively, as summarized in
Table 1.
Most of compounds 7 and 9 showed inhibitory activity against ALR2 with IC50
values at submicromolar level. It was found that the phenolic hydroxyl group played
an important role in the ALR2 inhibition determined by comparing the activity of 7b
and 7a. Regarding the position and number of phenolic hydroxyl, the phenolic
dihydroxyl compounds (7d, 7f and 9a) and the phenolic trihydroxyl compounds (7i
and 9b) showed an obviously level enhancement in the ALR2 inhibition comparing
with the single phenolic hydroxyl compound 7b and the single-phenolic like
compounds (7c, 7e and 7g). Among the phenolic dihydroxyl compounds,
2,4-dihydroxyl (9a) was more active than 3,5-dihydroxyl (7d) and3,4-dihydroxyl (7f).
The activity of 2,4-dihydroxyl compound could be significantly reinforced by
introduction of chloro and then bromo substitution to the C7-position of the core
structure producing 7-Cl 9c and 7-Br 9d, and 9c was the most active ARI with IC50
value of 0.009 ꢀM in the present study. The structure-activity relationship (SAR)

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studies manifest that the number of the phenolic hydroxyl is a more crucial factor in
the inhibitory activity than the position of phenolic hydroxyl, and C7-halogen
substitution is essential to obtain excellent ARIs.
Insertion of a vinyl group as the spacer into the C2 side chain provided series
11a-l (IC50 = 0.013-1.515 ꢀM). Almost compounds in the series except only one (11d)
showed inhibitory activity against ARL2 at submicromolar level of IC50. Among this
series, the 4-vinylphenol compound 11g was found to be the most active with IC50
value of 0.013ꢀM. Obviously, the extended spacer of the C2 side chain by the vinyl
insertion had a great enhancement effect on the ARL2 inhibition by comparing 11a,
11c, and 11f with their counterparts 7a, 7b, and 7f, respectively. The substituent
groups in C2 side chain also had an effect on the potency of ALR2 inhibition with the
rank order of 4-OH > 3,4-(OH)2 > 3-OCH3,4-OH > 4-OCH3.
In order to further identify the function of the vinyl spacer, the double bond in
10c and 10e was reduced to produce 13a-b, respectively. Comparing to their
precursors, the reduction products declined greatly in the inhibitory activity.
All the synthetic compounds showed inhibitory activity against ALR1 with
inhibition percentages no more than 40% at the concentration of 10 ꢀM, indicating
well selectivity for ALR2. In particularly, 11h-i were potent in the ALR2 inhibition
but very less active in the ALR1 inhibition. 9c-e produced not only top ALR2
inhibition but also higher percentage in the ALR1 inhibition in the present study, and
this implies that the C7-halogen substitution may result in a decrease in the
selectivity of compounds 7 and 9.
Table 1. Biological activity of target compounds.
substituent
R2
IC50(µM)
Inhib(%)
ALR1
35.7
DPPH sca. %
Compd.
7a
ALR2
100µM
50µM
10µM
R1
X
-
H
H
9.942(8.900-10.638)
1.032(0.908-1.156)
5.207(4.946-5.468)
0.296(0.270-0.321)
2.207(2.026-2.388)
0.831(0.757-0.905)
0.578(0.557-0.599)
0.319(0.276-0.362)
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
7b
H
H
H
H
H
H
H
4-OH
-
28.1
6.5
7c
3-OH, 5-OCH3
3,5-(OH)2
-
21.3
*
7d
-
22.8
30.3
32.1
50.5
20.5
45.6
7e
3-OH, 4-OCH3
3,4-(OH)2
-
10.9
7f
-
21.8
7g
3-OH, 4,5-(OCH3)2
3,5-(OH)2, 4-OCH3
-
5.6
7h
10.3

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7i
9a
H
H
3,4,5-(OH)3
2,4-(OH)2
2,4,6-(OH)3
2,4-(OH)2
2,4-(OH)2
2,4,6-(OH)3
H
-
0.179(0.154-0.204)
0.185(0.149-0.221)
0.068(0.054-0.082)
0.009(0.007-0.011)
0.024(0.007-0.040)
0.018(0.007-0.029)
0.895(0.725-1.065)
0.126(0.099-0.153)
0.035(0.012-0.058)
1.515(1.345-1.685)
0.307(0.236-0.337)
0.165(0.137-0.193)
0.013(0.006-0.021)
0.087(0.064-0.110)
0.036(0.027-0.045)
0.029(0.025-0.033)
0.389(0.347-0.431)
0.108(0.080-0.136)
0.918(0.901-0.934)
0.689(0.659-0.719)
0.091(0.078-0.104)
19.5
18.6
12.4
38.6
36.6
39.5
24.1
8.2
70.7
37.7
83.5
41.5
43.5
85.2
78.9
42.7
57.6
52.8
85.5
89.1
66.2
94.9
97.1
61.2
90.1
95.8
35.2
40.1
62.4
*
34.6
*
-
9b
H
-
70.89
*
40.23
*
9c
Cl
Br
Cl
H
-
9d
-
*
*
9e
-
69.8
64.1
*
41.3
35.5
*
11a
11b
11c
11d
11e
11f
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH2-CH2
CH2-CH2
H
F
H
4-OH
37.6
14.3
18.6
16.5
39.2
17.6
13.7
12.9
23.4
18.4
27.6
23.7
50.3
44.5
39.1
75.6
78.4
47.5
81.8
87.4
43.8
75.4
82.4
*
*
H
4-OCH3
*
H
3-OCH3, 4-OH
3,4-(OH)2
4-OH
48.6
55.1
*
H
11g
11h
11i
Cl
Cl
Cl
Br
Br
Br
H
3-OCH3, 4-OH
3,4-(OH)2
4-OH
62.3
74.7
*
11j
11k
11l
3-OCH3, 4-OH
3,4-(OH)2
4-OH
59.6
70.8
*
13a
13b
epalrestat
Trolox
H
3-OCH3, 4-OH
25.8
*
96.2
89.6
78.2
a IC50 values represent the concentration required to produce 50% enzyme inhibition.
b The inhibitory effect was estimated at a concentration of 10 µM.
* Not tested.
3.2. DPPH Radical Scavenging Activity
The scavenging of stable free radicals of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was
used to determine potential antioxidant properties of the synthesized compounds, and
6-hydroxy-2,5,7,8-chroman-2-carboxylic acid (Trolox) was employed as a positive
control (Table 1).[43] Ten compounds revealed obvious antioxidant activity with
DPPH radical scavenging rates ranging from 34.6 to 74.7% at the concentration of 10
ꢀM. Of them, 11i and 11l containing phenolic 3,4-dihydroxyl groups in the C2-styryl
side chain were the most excellent and even comparable to Trolox indicating that the
combination of 3,4-dihydroxyl groups may be important to the antioxidant activity.
This effect of the combined 3,4-dihydroxyl groups could also be observed in the
comparison of 7f with 7d. Notably, the single 4-hydroxyl group that had an impact
both on inhibitions against ALR2 and ALR1 was insignificant and even negative for
the development of antioxidant activity (11c vs 11d and 11a), in addition, top three
antioxidant compounds in series 7 and 9 all have three phenolic hydroxyl groups in

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the C2-styryl side chain. Moreover, the vinyl spacer of the C2-styryl played a crucial
role in the antioxidant activity by comparing the antioxidant activity of compounds
13a and 11c, 13b and 11e, respectively. This C2 vinyl-dependence was also supported
by the comparison of 11f with 7f.
4. Molecular modeling
The compound 11i endowed with excellent activity of the ALR2 inhibition and
antioxidant activity was studied by molecular docking performed on the human
ALR2/NADP+/IDD594 complex (PDB entry code 1Z3N).[44,45] The result shows
that the compound fitted well to the active site of ALR2 (Fig. 3), in which the
carboxylate group formed tight hydrogen bonds with Tyr48 (2.62 Å), Trp111 (3.12 Å)
and His110 (2.49 Å and 2.87 Å). On the other hand, the 4-hydroxyl oxygen atom of
the C2-side chain formed an additional hydrogen bond with the side chain of Thr113
(3.09 Å) explaining the key role of the hydroxyl in the enhancement of the ALR2
inhibition. Moreover, the phenyl ring of C2 side chain was well paralleled to the
indole ring of Trp 111 forming a stacking interaction. Molecular modeling of typical
acetic acid inhibitor IDD594 was also compared with that of compound 11i, and
indicated that compound 11i endowed with a more intense hydrogen-bond interaction
and lower binding energy. This further demonstrates that 11i is a more potent aldose
reductase inhibitor (Supplementary material).
Fig. 3. Docking of 11i into the active site of ALR2. (a) The protein structure is shown in ribbon and
tube representation with selected residues labelled and shown in line representation, ligand 11i is shown as
stick models. The docked pose of 11i is shown in cyan (C), red (O), blue (N), and green (Cl). Hydrogen
bonds are shown as blue dashed lines. (b) Protein residues are in surface representation.

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5. Conclusion
Our design and synthetic efforts based on pyrido[2,3-b]pyrazin-3(4H)-one resulted in
a group of multifunctional ARIs, in which compounds 11 except 11d were found to be
potent in ALR2 inhibition with IC50 values at submicromolar level, and less active in
ALR1 inhibition with inhibition percentages no more than 40% at concentration of 10
ꢀM. In addition, DPPH radical scavenging potency was also tested to investigate the
antioxidant activity of the designed compounds. Most of compounds 11 displayed
excellent antioxidant activity, and 11i containing 3,4-dihydroxyl groups in the
C2-styryl was identified as a distinguished antioxidant even comparable with Trolox.
11i was also excellent in the ALR2 inhibition but less active in the ALR1 inhibition,
and thus suggested a potent, selective, and difunctional ARI. SAR studies on the
substituents except the carboxylate head group on the framework indicated that the
phenolic hydroxyl-substituted C2-styryl side chain is the important structure for the
potent and selective ARIs based on pyrido[2,3-b]pyrazin-3(4H)-one, and the
combination of 3,4-dihydroxyl groups in the C2-styryl is particularly the key for the
selectivity. In addition, the vinyl spacer of the C2-styryl played a pivotal role in the
development of antioxidant ability of the ARIs besides its significant enhancement
effect on the ALR2 inhibition.
6. Experimental section
6.1. Chemistry
Melting points were recorded on an X-4 microscopic melting point apparatus and are
uncorrected. All reactions were checked by TLC on silica gel Merck 60F254. The 1H
and 13C NMR spectra were recorded on a Bruker Avance 400 spectrometer (400 MHz,
Bruker (Beijing) Technologies and Services Co., Ltd.). Chemical shifts are given in δ
units (ppm) relative to internal standard TMS and refer to CDCl3 or DMSO-d6
solutions. The following HPLC methods were used to determine the purity of acetic
acid derivatives using a Hitachi D-2000 Elite HPLC system. All target acetic acid
derivatives tested in biological assays were >95% pure with the following method:
Inertsil ODS-2 250 mm ×10 mm, 5 mm column; mobile phase: CH3CN (0.1%
TFA)/CH3OH = 75/25, for 8min; room temperature; flow rate: 1 mL min−1; detection
at λ 254 nm.
6.1.1. General Procedure for Synthesis of 2-chloropyrido[2,3-b]pyrazin-3(4H)-one (4)

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A mixture of substituted pyridine-2,3-diamine 1 (20mmol) and diethyl oxalate(10mL)
was stirred at 100℃ for 12 hours and cooled to room temperature subsequently.
Ethoxyethane was added to obtain solid. The formed solid was then centrifuged and
dried to obtain brown solid compound of pyrido[2,3-b]pyrazine-2,3(1H,4H)-dione 2.
The mixture of pyrido[2,3-b]pyrazine-2,3(1H,4H)-dione and POCl3(20ml) was stirred
at 130 oC and then cooled to room temperature. The mixture was poured into the ice
water. The formed solid was filter and then evaporated solvent in vacuo to obtain
unpurified 2,3-dichloropyrido[2,3-b]pyrazine 3. Then the unpurified was mixture with
LiOH (10mmol) in THF/H2O (5:2, 35 mL). The mixture was stirred at 65 oC for 12 h.
The resulting mixture was cooled to room temperature, and then brine (10 mL) was
added to mixture. After extraction with THF (3×50ml), the combined organic layers
were washed with brine (30 mL), dried over anhydrous MgSO4, and filtered,
followed by evaporation of the solvent in vacuo to obtain the unpurified
2-chloropyrido[2,3-b]pyrazin-3(4H)-one 4a-c. The crude products were purified by
column chromatography on silica gel using petroleum ether /EtOAc (3:1) as eluent to
provide the desired product 2-chloropyrido[2,3-b]pyrazin-3(4H)-one derivatives4a-c.
6.1.2. General Procedure for Synthesis of methyl 2-(2-chloro-3-oxopyrido[2,3-b]pyrazin
-4(3H)-yl)acetate (5)
A mixture of substituted 2-chloropyrido[2,3-b]pyrazin-3(4H)-one 4 (10 mmol),
K2CO3 (30 mmol), and methyl bromoacetate (10.5 mmol) dissolved in CH3CN (50
mL) was stirred at 65°C for 2 h. After the reaction, the resulting mixture was cooled
to room temperature, filtered and solvent evaporated in vacuo to obtain a crude
mixture. The residue was recrystallized from EtOAc to give desired
2-(2-chloroo-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetate 5.
6.1.3. General Procedure for Synthesis of 2-(3-oxo-2-phenylpyrido[2,3-b]pyrazin
-4(3H)-yl)acetic acid Derivatives (7)
A mixture of the appropriate 5 (1 mmol), the substituted phenylboronic acid (1.1
mmol), Cs2CO3 (2mmol), Pd(OAc)2 (0.05 mmol), and PPh3 (0.15mmol) were added
in H2O /dioxane (1:10, 11mL) charged in round-bottom flask. The mixture was stirred
at 100°C for 18 h with a reflux condenser and under an atmosphere of nitrogen. After
the reaction, the resulting mixture was cooled to room temperature, filtered and
solvent evaporated in vacuo to obtain a crude mixture, and then the crude residue
purified by silica gel chromatography to obtain compounds 6a-e. Subsequently, the
compounds 6b-e were mixture with BBr3 (1mmol) dissolved in dried dichloromethane
to get rid of partial or all methyl in N3 position of compound 6. The mixture was
stirred at room temperature for 12h and poured into ice water. And then filtered and

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solvent evaporated in vacuo to obtain a crude mixture, the solid was purified through
recrystallization and then added in saturated aq LiOH (5 mL) in THF (4 mL) was
stirred for 2 h at room temperature. Upon completion, the alkaline suspension was
acidified with 0.1 N HCl to pH 2-3. The resulting precipitate was collected by
filtration, washed with H2O, dried in vacuo, and recrystallized from CH3OH to give
desired final product 2-(3-oxo-2-phenylpyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid
Derivatives 7.
6.1.3.1. 2-(3-oxo-2-phenylpyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid (7a)
1
Yellow solid (0.19 g, 71%); purity: 95.21%; mp: 239-240 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.82 (m, 1H), 8.01-7.98(m, 2H), 7.66-7.40 (m, 6H), 4.15 (s, 2H); 13C NMR (100 MHz,
[D6]DMSO) δ= 175.6, 155.4, 154.1, 152.2, 146.6, 132.6, 131.4, 129.1, 129.0, 128.1 128.5,
113.1,112.3, 110.7, 53.7 ppm.
6.1.3.2. 2-(2-(4-hydroxyphenyl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid (7b)
1
Yellow solid (0.20 g, 68%); purity: 95.17%; mp: 261-263 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.82 (m, 1H), 8.01-7.98(m, 2H), 7.66-7.40 (m, 5H),5.65 (s, 1H), 4.15 (s, 2H); 13C NMR (100
MHz, [D6]DMSO) δ= 175.6, 155.4, 154.1, 152.2, 146.6, 132.6, 131.4, 129.1, 129.0, 128.1 128.5,
113.1,112.3, 110.7, 53.7 ppm.
6.1.3.3. 2-(2-(3-hydroxy-5-methoxyphenyl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid
(7c)
1
Yellow solid (0.23 g, 73%); purity: 96.32%; mp: 259-260 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.82 (m, 1H), 8.05-7.87(m, 2H), 7.61-7.39 (m, 3H),5.65 (s, 1H), 4.15 (s, 2H), 3.65 (s, 3H);
13C NMR (100 MHz, [D6]DMSO) δ= 175.6, 155.4, 154.1, 152.2, 146.6, 132.6, 131.4, 129.1, 129.0,
128.1 128.5, 113.1,112.3, 110.7, 55.8, 53.7 ppm.
6.1.3.4. 2-(2-(3,5-dihydroxyphenyl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid (7d)
1
Yellow solid (0.21 g, 65%); purity: 98.75%; mp: 271-273 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.82 (m, 1H), 8.01-7.98(m, 2H), 7.66-7.40 (m, 5H),5.65 (s, 2H), 4.15 (s, 2H); 13C NMR (100
MHz, [D6]DMSO) δ= 175.6, 155.4, 154.1, 152.2, 146.6, 132.6, 131.4, 129.1, 129.0, 128.1 128.5,
113.1,112.3, 110.7, 53.7 ppm.
6.1.3.5 2-(2-(3-hydroxy-4-methoxyphenyl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid
(7e)
1
Yellow solid (0.19 g, 61%); purity: 96.34%; mp: 284-285 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.82 (m, 1H), 8.05-7.87(m, 2H), 7.61-7.39 (m, 3H),5.65 (s, 1H), 4.15 (s, 2H), 3.65 (s, 3H);
13C NMR (100 MHz, [D6]DMSO) δ= 175.6, 155.4, 154.1, 152.2, 146.6, 132.6, 131.4, 129.1, 129.0,
128.1 128.5, 113.1,112.3, 110.7, 55.8, 53.7 ppm.
6.1.3.6. 2-(2-(3,4-dihydroxyphenyl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid (7f)
1
Yellow solid (0.18 g, 58%); purity: 97.15%; mp: 281-283 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.82 (m, 1H), 8.01-7.98(m, 2H), 7.66-7.40 (m, 5H),5.65 (s, 2H), 4.15 (s, 2H); 13C NMR (100
MHz, [D6]DMSO) δ= 175.6, 155.4, 154.1, 152.2, 146.6, 132.6, 131.4, 129.1, 129.0, 128.1 128.5,
113.1,112.3, 110.7, 53.7 ppm.
6.1.3.7
2-(2-(3-hydroxy-4,5-dimethoxyphenyl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic
acid (7g)
1
Yellow solid (0.16 g, 45%); purity: 96.25%; mp: 278-279 oC; H NMR (400 MHz, [D6]DMSO):

ACCEPTED MANUSCRIPT
δ= 11.82 (m, 1H), 8.05-7.87(m, 2H), 7.61-7.39 (m, 3H),5.65 (s, 1H), 4.15 (s, 2H), 3.65 (s, 6H);
13C NMR (100 MHz, [D6]DMSO) δ= 175.6, 155.4, 154.1, 152.2, 146.6, 132.6, 131.4, 129.1, 129.0,
128.1 128.5, 113.1,112.3, 110.7, 55.8, 55.6, 53.7 ppm.
6.1.3.8.
2-(2-(3,5-dihydroxy-4-methoxyphenyl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic
acid (7h)
1
Yellow solid (0.13 g, 39%); purity: 96.24%; mp: 285-286 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.82 (m, 1H), 8.01-7.98(m, 2H), 7.66-7.40 (m, 5H),5.65 (s, 2H), 4.15 (s, 2H), 3.65 (s, 3H);
13C NMR (100 MHz, [D6]DMSO) δ= 175.6, 155.4, 154.1, 152.2, 146.6, 132.6, 131.4, 129.1, 129.0,
128.1 128.5, 113.1,112.3, 110.7, 55.8, 53.7 ppm.
6.1.3.9. 2-(3-oxo-2-(3,4,5-trihydroxyphenyl)pyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid (7i)
1
Yellow solid (0.13 g, 41%); purity: 95.67%; mp: 291-292 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.82 (m, 1H), 8.01-7.98(m, 2H), 7.66-7.40 (m, 5H),5.65 (s, 3H), 4.15 (s, 2H); 13C NMR (100
MHz, [D6]DMSO) δ= 175.6, 155.4, 154.1, 152.2, 146.6, 132.6, 131.4, 129.1, 129.0, 128.1 128.5,
113.1,112.3, 110.7, 53.7 ppm.
6.1.4. General Procedure for Synthesis of 2-(3-oxo-2-phenylpyrido[2,3-b]pyrazin
-4(3H)-yl)acetic acid Derivatives (9)
A mixture of compound 5 (1 mmol), resorcinol or other substituted phenol groups (1
mmol), and AlCl3 (1.1 mmol) in dichloroethane (5 mL) was stirred at 80°C for 2 h
under an atmosphere of argon. After the reaction, the mixture was poured into ice
water (15 mL), stirred for 15 min, and then extracted with ethyl acetate (3×20 mL).
The organic layers were collected, then dried over anhydrous Mg2SO4, and
concentrated under vacuum to obtain the residue to form unpurified methyl
2-(3-oxo-2-phenylpyrido[2,3-b]pyrazin-4(3H)-yl)acetate derivatives 8. The solid
purified through column chromatography. Subsequently, the compounds 8 were added
in saturated aq LiOH (5 mL) in THF (4 mL) and stirred for 2 h at room temperature.
Upon completion, the alkaline suspension was acidified with 0.1 N HCl to pH 2-3.
The resulting precipitate was collected by filtration, washed with H2O, dried in vacuo,
and
recrystallized
from
CH3OH
to
give
desired
final
product
2-(3-oxo-2-phenylpyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid Derivatives 9.
6.1.4.1. 2-(2-(2,4-dihydroxyphenyl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid (9a)
1
Yellow solid (0.26 g, 84%); purity: 97.51%; mp: 283-284 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.82 (m, 1H), 8.01-7.98(m, 2H), 7.66-7.40 (m, 5H),5.65 (s, 2H), 4.15 (s, 2H); 13C NMR (100
MHz, [D6]DMSO) δ= 175.6, 155.4, 154.1, 152.2, 146.6, 132.6, 131.4, 129.1, 129.0, 128.1 128.5,
113.1,112.3, 110.7, 53.7 ppm.
6.1.4.2. 2-(3-oxo-2-(2,4,6-trihydroxyphenyl)pyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid (9b)
1
Yellow solid (0.29 g, 89%); purity: 96.45%; mp: 291-292 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.82 (m, 1H), 8.01-7.98(m, 2H), 7.66-7.40 (m, 5H),5.65 (s, 3H), 4.15 (s, 2H); 13C NMR (100
MHz, [D6]DMSO) δ= 175.6, 155.4, 154.1, 152.2, 146.6, 132.6, 131.4, 129.1, 129.0, 128.1 128.5,
113.1,112.3, 110.7, 53.7 ppm.
6.1.4.3. 2-(7-chloro-2-(2,4-dihydroxyphenyl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid
(9c)
1
Yellow solid (0.33 g, 94%); purity: 97.54%; mp: 294-295 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.82 (m, 1H), 8.01-7.98(m, 2H), 7.66-7.40 (m, 5H),5.65 (s, 2H), 4.15 (s, 2H); 13C NMR (100

ACCEPTED MANUSCRIPT
MHz, [D6]DMSO) δ= 175.6, 155.4, 154.1, 152.2, 146.6, 132.6, 131.4, 129.1, 129.0, 128.1 128.5,
113.1,112.3, 110.7, 53.7 ppm.
6.1.4.4. 2-(7-bromo-2-(2,4-dihydroxyphenyl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid
(9d)
1
Yellow solid (0.33 g, 83%); purity: 98.57%; mp: 278-279 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.82 (m, 1H), 8.01-7.98(m, 2H), 7.66-7.40 (m, 5H),5.65 (s, 2H), 4.15 (s, 2H); 13C NMR (100
MHz, [D6]DMSO) δ= 175.6, 155.4, 154.1, 152.2, 146.6, 132.6, 131.4, 129.1, 129.0, 128.1 128.5,
113.1,112.3, 110.7, 53.7 ppm.
6.1.4.5.
2-(7-chloro-3-oxo-2-(2,4,6-trihydroxyphenyl)pyrido[2,3-b]pyrazin-4(3H)-yl)acetic
acid (9e)
1
Yellow solid (0.32 g, 87%); purity: 96.42%; mp: 275-276 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.82 (m, 1H), 8.01-7.98(m, 2H), 7.66-7.40 (m, 5H),5.65 (s, 3H), 4.15 (s, 2H); 13C NMR (100
MHz, [D6]DMSO) δ= 175.6, 155.4, 154.1, 152.2, 146.6, 132.6, 131.4, 129.1, 129.0, 128.1 128.5,
113.1,112.3, 110.7, 53.7 ppm.
6.1.5. General Procedure for Synthesis of 2-(3-oxo-2-styrylpyrido[2,3-b]pyrazin
-4(3H)-yl)acetic acid Derivatives (11)
A mixture of compounds 5 (1 mmol), Pd(OAc)2 (0.03 mmol), and P(o-tolyl)
(0.07mmol) was stirred at room temperature under argon for 10 min. Add alkene (1.5
mmol), dry Et3N (3 mmol), and DMF(1 mL). The reaction mixture was heated at
100°C for 18 h. After the completion of the reaction, the resulting mixture was cooled
to room temperature poured in water, filtered, and solvent evaporated in vacuo to
obtain
a
crude
mixture,
which
was
purified
by
column
chromatography(ethylacetate–hexane; 1:3) to provide the product 10a-i, Subsequently,
the compounds 10d-i were mixture with BBr3 (1mmol) dissolved in dried
dichloromethane to get rid of partial or all methyl in C3 position of compounds 10,
and after the hydrolysis reaction, the desired products 11a-l were obtained
.
6.1.5.1. (E)-2-(3-oxo-2-styrylpyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid (11a)
white solid (0.14 g, 45%); purity: 98.11%; mp: 279-280 oC; 1H NMR (400 MHz, [D6]DMSO): δ=
11.7 (s, 1H), 8.07-7.33 (m, 7H), 6.95-5.67 (m, 3H), 3.95 (s, 2H); 13C NMR (100 MHz, [D6]DMSO)
δ= 171.12, 157.14, 142.21, 135.82, 134.37, 133.19, 132.16, 131.32, 129.28, 128.54, 127.13,
119.34, 118.25, 115.51, 105.23, 103.21, 45.21 ppm.
6.1.5.2. (E)-2-(2-(4-fluorostyryl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid (11b)
1
Yellow solid (0.14 g, 44%); purity: 97.47%; mp: 281-282 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.7 (s, 1H), 8.07-7.33 (m, 7H), 6.95-5.67 (m, 2H), 3.95 (s, 2H); 13C NMR (100 MHz,
[D6]DMSO) δ= 171.12, 157.14, 142.21, 135.82, 134.37, 133.19, 132.16, 131.32, 129.28, 128.54,
127.13, 119.34, 118.25, 115.51, 105.23, 103.21, 45.21 ppm.
6.1.5.3. (E)-2-(2-(4-hydroxystyryl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid (11c)
1
Yellow solid (0.13 g, 41%); purity: 95.51%; mp: 283-284 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.7 (s, 1H), 8.07-7.33 (m, 7H), 6.95-5.67 (m, 3H), 3.95 (s, 2H); 13C NMR (100 MHz,
[D6]DMSO) δ= 171.12, 157.14, 142.21, 135.82, 134.37, 133.19, 132.16, 131.32, 129.28, 128.54,
127.13, 119.34, 118.25, 115.51, 105.23, 103.21, 45.21 ppm.
6.1.5.4. (E)-2-(2-(4-methoxystyryl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid (11d)

ACCEPTED MANUSCRIPT
1
Yellow solid (0.16 g, 48%); purity: 96.75%; mp: 282-283 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.7 (s, 1H), 8.07-7.33 (m, 6H), 6.95-5.67 (m, 3H), 3.95 (s, 2H), 3.65(s, 3H); 13C NMR (100
MHz, [D6]DMSO) δ= 171.12, 157.14, 142.21, 135.82, 134.37, 133.19, 132.16, 131.32, 129.28,
128.54, 127.13, 119.34, 118.25, 115.51, 105.23, 103.21, 55.8, 45.21 ppm.
6.1.5.5. (E)-2-(2-(4-hydroxy-3-methoxystyryl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid
(11e)
1
Yellow solid (0.16 g, 46%); purity: 96.18%; mp: 276-278 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.7 (s, 1H), 8.07-7.33 (m, 6H), 6.95-5.67 (m, 3H), 3.95 (s, 2H), 3.65(s, 3H); 13C NMR (100
MHz, [D6]DMSO) δ= 171.12, 157.14, 142.21, 135.82, 134.37, 133.19, 132.16, 131.32, 129.28,
128.54, 127.13, 119.34, 118.25, 115.51, 105.23, 103.21, 55.8, 45.21 ppm.
6.1.5.6. (E)-2-(2-(3,4-dihydroxystyryl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid (11f)
white solid (0.13 g, 38%); purity: 97.41%; mp: 288-289 oC; 1H NMR (400 MHz, [D6]DMSO): δ=
11.7 (s, 1H), 8.07-7.33 (m, 7H), 6.95-5.67 (m, 3H), 3.95 (s, 2H); 13C NMR (100 MHz, [D6]DMSO)
δ= 171.12, 157.14, 142.21, 135.82, 134.37, 133.19, 132.16, 131.32, 129.28, 128.54, 127.13,
119.34, 118.25, 115.51, 105.23, 103.21, 45.21 ppm.
6.1.5.7. (E)-2-(7-chloro-2-(4-hydroxystyryl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid
(11g)
white solid (0.14 g, 39%); purity: 98.26%; mp: 289-290 oC; 1H NMR (400 MHz, [D6]DMSO): δ=
11.7 (s, 1H), 8.07-7.33 (m, 6H), 6.95-5.67 (m, 3H), 3.95 (s, 2H); 13C NMR (100 MHz, [D6]DMSO)
δ= 171.12, 157.14, 142.21, 135.82, 134.37, 133.19, 132.16, 131.32, 129.28, 128.54, 127.13,
119.34, 118.25, 115.51, 105.23, 103.21, 45.21 ppm.
6.1.5.8. (E)-2-(7-chloro-2-(4-hydroxy-3-methoxysty-ryl)-3-oxopyri- do[2,3-b]pyraz-in-4(3H)
-yl)acet- ic acid (11h)
1
Yellow solid (0.17 g, 45%); purity: 98.54%; mp: 284-286 oC; H NMR (400 MHz, [D6]DMSO):
δ= 11.7 (s, 1H), 8.07-7.33 (m, 5H), 6.95-5.67 (m, 3H), 3.95 (s, 2H), 3.65(s, 3H); 13C NMR (100
MHz, [D6]DMSO) δ= 171.12, 157.14, 142.21, 135.82, 134.37, 133.19, 132.16, 131.32, 129.28,
128.54, 127.13, 119.34, 118.25, 115.51, 105.23, 103.21, 55.8, 45.21 ppm.
6.1.5.9.
(E)-2-(7-chloro-2-(3,4-dihydroxystyryl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic
acid (11i)
white solid (0.15 g, 41%); purity: 96.37%; mp: 287-288 oC; 1H NMR (400 MHz, [D6]DMSO): δ=
11.7 (s, 1H), 8.07-7.33 (m, 6H), 6.95-5.67 (m, 3H), 3.95 (s, 2H); 13C NMR (100 MHz, [D6]DMSO)
δ= 171.12, 157.14, 142.21, 135.82, 134.37, 133.19, 132.16, 131.32, 129.28, 128.54, 127.13,
119.34, 118.25, 115.51, 105.23, 103.21, 45.21 ppm.
6.1.5.10. (E)-2-(7-bromo-2-(4-hydroxystyryl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid
(11j)
white solid (0.17 g, 43%); purity: 97.31%; mp: 293-294 oC; 1H NMR (400 MHz, [D6]DMSO): δ=
11.7 (s, 1H), 8.07-7.33 (m, 6H), 6.95-5.67 (m, 3H), 3.95 (s, 2H); 13C NMR (100 MHz, [D6]DMSO)
δ= 171.12, 157.14, 142.21, 135.82, 134.37, 133.19, 132.16, 131.32, 129.28, 128.54, 127.13,
119.34, 118.25, 115.51, 105.23, 103.21, 45.21 ppm.
6.1.5.11.
(E)-2-(7-bromo-2-(4-hydroxy-3-methoxystyryl)-3-oxopyrido[2,3-b]pyraz-
in-4(3H)-yl)acetic acid (11k)
white solid (0.17 g, 39%); purity: 97.34%; mp: 287-288 oC; 1H NMR (400 MHz, [D6]DMSO): δ=
11.7 (s, 1H), 8.07-7.33 (m, 5H), 6.95-5.67 (m, 3H), 3.95 (s, 2H), 3.65(s, 3H); 13C NMR (100 MHz,
[D6]DMSO) δ= 171.12, 157.14, 142.21, 135.82, 134.37, 133.19, 132.16, 131.32, 129.28, 128.54,
127.13, 119.34, 118.25, 115.51, 105.23, 103.21, 55.8, 45.21 ppm.

ACCEPTED MANUSCRIPT
6.1.5.12. (E)-2-(7-bromo-2-(3,4-dihydroxystyryl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic
acid (11l)
white solid (0.16 g, 38%); purity: 96.11%; mp: 289-290 oC; 1H NMR (400 MHz, [D6]DMSO): δ=
11.7 (s, 1H), 8.07-7.33 (m, 6H), 6.95-5.67 (m, 3H), 3.95 (s, 2H); 13C NMR (100 MHz, [D6]DMSO)
δ= 171.12, 157.14, 142.21, 135.82, 134.37, 133.19, 132.16, 131.32, 129.28, 128.54, 127.13,
119.34, 118.25, 115.51, 105.23, 103.21, 45.21 ppm.
6.1.6.
General
Procedure
for
Synthesis
of
2-(3-oxo-2-phenethylpyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid Derivatives (13)
A mixture of 10% palladium on carbon (0.36 g) and compounds 12 (1 mmol)
dissolved in methanol (10 mL). The mixture was stirred at 25°C for 36 h under a
hydrogen atmosphere. After the reaction, the resulting mixture filtered, and solvent
evaporated in vacuo to obtain a crude mixture, which was purified by column
chromatography(ethylacetate–hexane; 1:4) to provide the product 13a-b.
6.1.6.1. 2-(2-(4-hydroxyphenethyl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic acid (13a)
white solid (0.27 g, 84%); purity: 98.21%; mp: 295-296 oC; 1H NMR (400 MHz, [D6]DMSO): δ=
11.82 (s, 1H), 8.07-6.62 (m, 7H), 5.35 (s, 1H), 3.90 (s, 2H), 2.56 (m, 2H), 1.68 ppm (m, 2H); 13
C
NMR (100 MHz, [D6]DMSO) δ= 171.12, 157.14, 142.21, 135.82, 134.37, 133.19, 132.16, 131.32,
129.28, 128.54, 127.13, 126.58, 124.24, 123.18, 55.23, 30.21, 25.21 ppm.
6.1.6.2.
2-(2-(4-hydroxy-3-methoxyphenethyl)-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)acetic
acid (13b)
white solid (0.30 g, 86%); purity: 98.21%; mp: 291-292 oC; 1H NMR (400 MHz, [D6]DMSO): δ=
11.82 (s, 1H), 8.07-6.62 (m, 6H), 5.35 (s, 2H), 3.90 (s, 2H), 2.56 (m, 2H), 1.68 ppm (m, 2H); 13
C
NMR (100 MHz, [D6]DMSO) δ= 171.12, 157.14, 142.21, 135.82, 134.37, 133.19, 132.16, 131.32,
129.28, 128.54, 127.13, 126.58, 124.24, 123.18, 55.23, 30.21, 25.21 ppm.
6.2. Enzyme assays
The ALR2 inhibition activity was tested in a reaction mixture containing 0.25 mL
NADPH (0.10 mM), 0.25 mL sodium phosphate buffer (pH=6.2, 0.1 M), 0.1 mL
enzyme extract, 0.15 mL deionized water, and 0.25 mL D,L-glyceraldehyde (10 mM)
as substrate in a final volume of 1 mL. Before adding to D,L-glyceraldehyde, the
reaction mixture was incubated at 30oC for 10 min, then the substrate was added to
start the reaction, which was monitored for 5 min.
The ALR1 inhibition activity was performed at 36oC in a reaction mixture
containing 0.25 mL NADPH (0.12 mM), 0.1 mL enzyme extract, 0.25 mL sodium
phosphate buffer (pH=7.2, 0.1 M), 0.15 mL deionized water, and 0.25 mL sodium
D-glucuronate (20 mM) as substrate in a final volume of 1 mL. Before adding to
sodium D-glucuronate, the reaction mixture was incubated at 37oC for 10 min, then
the substrate was added to start the reaction, which was monitored for 5 min.

ACCEPTED MANUSCRIPT
The inhibitory activity of the newly synthesized compounds against ALR2 and
ALR1 was assayed by adding 5ꢀL of the inhibitor solution to the reaction mixture
described above. All compounds were dissolved in dimethyl sulfoxide (DMSO) and
diluted with deionized H2O. To correct for the nonenzymatic oxidation of NADPH,
the rate of NADPH oxidation in the presence of all of the reaction mixture
components except the substrate was subtracted from each experimental rate. The
inhibitory effect of the synthetic compounds was routinely estimated at 10-4M (the
concentration is referenced to that of the compound in the reaction mixture). The
compounds found to be active were tested at additional concentrations between 10-4
and 10-8 M, the log (dose)–response curves were then constructed from the inhibitory
data, and the IC50 values were calculated by least-square analysis of the linear portion
of the log (dose) versus response curves (0.912 <r2< 0.996).
6.3. DPPH assay
In order to identify the radical scavenging activity of target compounds in a
homogeneous system, a method based on the scavenging of the stable free radical
DPPH was employed. A 100 ꢀL of methanolic solution of various compounds with
different concentrations was added to 1 mL DPPH methanolic solution (2.5 × 10-5
g/mL) and 1.9 ml methanol solution to give final concentrations of 0.1, 0.05, 0.01 mM
for the tested compounds, respectively. After vortexing thoroughly and leaving for 30
min at room temperature, the optical density was measured at λ 517 nm using the
Shimadzu UV-1800 spectrophotometer. The tested compounds and the reference
(Trolox) were dissolved in methanol, and after 240 min (steady state), the percentage
of DPPH radical scavenging was determined by the equation as shown below. The
experiments were performed in triplicate.
A
sample − A




blank
percentage of scavenging % = 1−
×100%
(
)


A
control
A
= DPPH + methanol
control
A
= tested compound + DPPH + methanol
= tested compound + mathanol
sample
A
blank
6.4. Docking studies
Docking studies were performed using Molegro Virtual Docker, version 5.0. The
crystal structure of human aldose reductase with bound inhibitor IDD594 retrieved
from the RCSB Protein Data Bank (PDB code: 1Z3N). All solvent molecules within

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the protein structure were removed for the docking procedure. Four binding cavities
were detected to get the potential binding site in the protein. The cavity around the
anion binding site (volume of 141 Å3) was chosen using the grid-based Mol-Dock
score (GRID) function with a grid resolution of 0.30 Å. The best ligand pose was
chosen according to the Mol-Dock score and ReRank score.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (grant
no. 21272025 and grant no. 21572021), the Research Fund for the Doctoral Program
of Higher Education of China (grant no. 20111101110042).
References
1.
Chen, X., et al. (2010) Acetic Acid Derivatives of 3,4-Dihydro-2H-1,2,4-benzothiadiazine
1,1-Dioxide as a Novel Class of Potent Aldose Reductase Inhibitors. Journal of Medicinal
Chemistry 53: 8330-8344.
2.
3.
Brownlee, M. (2001) Biochemistry and molecular cell biology of diabetic complications.
Nature 414: 813-820.
Mahapatra, D.K., V. Asati, and S.K. Bharti. (2015) Chalcones and their therapeutic targets for
the management of diabetes: Structural and pharmacological perspectives. European Journal
of Medicinal Chemistry 92: 839-865.
4.
5.
Aguiree, F., et al. (2013) IDF diabetes atlas.
Han, Z., et al. (2015) Aromatic Heterobicyclics-Based Design of Aldose Reductase Inhibitors as
Drug Candidates for the Treatment of Diabetic Complications. Adv Tech Biol Med 3:
2379-1764.1000152.
6.
7.
Maccari, R., et al. (2014) Structure–activity relationships and molecular modelling of new
5-arylidene-4-thiazolidinone derivatives as aldose reductase inhibitors and potential
anti-inflammatory agents. European Journal of Medicinal Chemistry 81: 1-14.
Kotsampasakou, E. and V.J. Demopoulos. (2013) Synthesis of derivatives of the
keto-pyrrolyl-difluorophenol scaffold: some structural aspects for aldose reductase inhibitory
activity and selectivity. Bioorganic & Medicinal Chemistry 21: 869–873.
Ramana, K.V. and S.K. Srivastava. (2010) Aldose reductase: A novel therapeutic target for
inflammatory pathologies. International Journal of Biochemistry & Cell Biology 42: 17–20.
Han, Z., et al. (2016) Multifunctional aldose reductase inhibitors based on 2 H-benzothiazine
1, 1-dioxide. RSC Advances 6: 12761-12769.
8.
9.
10.
Alexiou, P. and V.J. Demopoulos. (2010) A Diverse Series of Substituted Benzenesulfonamides
as Aldose Reductase Inhibitors with Antioxidant Activity: Design, Synthesis, and in Vitro
Activity. Journal of Medicinal Chemistry 53: 7756-7766.
11.
Polyxeni, A., et al. (2009) Aldose reductase enzyme and its implication to major health

ACCEPTED MANUSCRIPT
problems of the 21(st) century. Current Medicinal Chemistry 16: 734-752.
Ramana, K. and S. Srivastava. (2006) Mediation of aldose reductase in lipopolysaccharide
–induced inflammatory signals in mouse peritoneal macrophages. Cytokine 36: 115-122.
Alexiou, P., et al. (2009) Aldose reductase enzyme and its implication to major health
problems of the 21st century. Current medicinal chemistry 16: 734-752.
Demopoulos, V.J. and E. Rekka, . (1995) Isomeric benzoylpyrroleacetic acids: some structural
aspects for aldose reductase inhibitory and anti-inflammatory activities. Journal of
Pharmaceutical Sciences 84: 79-82.
12.
13.
14.
15.
16.
17.
Zou, Y., et al. (2015) Phenolic 4-hydroxy and 3,5-dihydroxy derivatives of
3-phenoxyquinoxalin-2(1H)-one as potent aldose reductase inhibitors with antioxidant
activity. Bioorganic & Medicinal Chemistry Letters 25.
Hao, X., et al. (2015) Chiral resolution, determination of absolute configuration, and biological
evaluation of (1,2-benzothiazin-4-yl)acetic acid enantiomers as aldose reductase inhibitors.
Journal of Enzyme Inhibition and Medicinal Chemistry 30: 846-851.
Xin, C., et al. (2011) Design and synthesis of potent and selective aldose reductase inhibitors
based on pyridylthiadiazine scaffold. European Journal of Medicinal Chemistry 46:
1536–1544.
18.
19.
20.
21.
Xin, C., et al. (2011) 1,2-Benzothiazine 1,1-dioxide carboxylate derivatives as novel potent
inhibitors of aldose reductase. Bioorganic & Medicinal Chemistry 19: 7262–7269.
Qin, X., et al. (2015) Design and Synthesis of Potent and Multifunctional Aldose Reductase
Inhibitors Based on Quinoxalinones. Journal of Medicinal Chemistry 58: 1254-1267.
Zhu, C., E.A. ., and et al. (2011) ChemInform Abstract: Design and Synthesis of Potent and
Selective Aldose Reductase Inhibitors Based on Pyridylthiadiazine Scaffold. Cheminform 42.
Antonioli, L., et al. (2012) Adenosine deaminase in the modulation of immune system and its
potential as a novel target for treatment of inflammatory disorders. Current drug targets 13:
842-862.
22.
23.
24.
25.
26.
Ottanà, R., et al. (2011) Identification of 5-arylidene-4-thiazolidinone derivatives endowed
with dual activity as aldose reductase inhibitors and antioxidant agents for the treatment of
diabetic complications. European journal of medicinal chemistry 46: 2797-2806.
La Motta, C., et al. (2007) Pyrido [1, 2-a] pyrimidin-4-one derivatives as a novel class of
selective aldose reductase inhibitors exhibiting antioxidant activity. Journal of medicinal
chemistry 50: 4917-4927.
Srivastava, S.K., K.V. Ramana, and A. Bhatnagar. (2005) Role of aldose reductase and oxidative
damage in diabetes and the consequent potential for therapeutic options. Endocrine Reviews
26: 380-392.
Ramana, K.V., et al. (2005) Requirement of aldose reductase for the hyperglycemic activation
of protein kinase C and formation of diacylglycerol in vascular smooth muscle cells. Diabetes
54: 818-829.
Srivastava, S.K., K.V. Ramana, and B. Aruni. (2005) Role of aldose reductase and oxidative
damage in diabetes and the consequent potential for therapeutic options. Endocrine Reviews
26: 380-92.
27.
28.
Ramana, K.V., et al. (2004) Activation of nuclear factor-kappaB by hyperglycemia in vascular
smooth muscle cells is regulated by aldose reductase. Diabetes 53: 2910-20.
Nicolaou, I. and V.J. Demopoulos. (2003) Substituted pyrrol-1-ylacetic acids that combine

ACCEPTED MANUSCRIPT
aldose reductase enzyme inhibitory activity and ability to prevent the nonenzymatic
irreversible modification of proteins from monosaccharides. Journal of medicinal chemistry
46: 417-426.
29.
Barski, O.A., K.H. Gabbay, and K.M. Grimshaw CEBohren. (1995) MECHANISM OF HUMAN
ALDEHYDE REDUCTASE - CHARACTERIZATION OF THE ACTIVE SITE POCKET. Biochemistry 34:
11264-11275.
30.
31.
32.
Larson, E.R., C.A.L. And, and R. Sarges. (1988) Medicinal chemistry of aldose reductase
inhibitors. Medicinal Research Reviews 8: 159&ndash;186.
Settimo, F., Da, et al. (2001) [1,2,4]Triazino[4,3-a]benzimidazole acetic acid derivatives: a new
class of selective aldose reductase inhibitors. Journal of Medicinal Chemistry 44: 4359-4369.
La Motta, C., et al. (2008) Acetic acid aldose reductase inhibitors bearing a five-membered
heterocyclic core with potent topical activity in a visual impairment rat model. Journal of
Medicinal Chemistry 51: 3182-3193.
33.
Parveen, S., et al. (2014) Selective synthesis and comparative activity of olefinic isomers of
1,2-benzothiazine-1,1-dioxide carboxylates as aldose reductase inhibitors. Rsc Advances 4:
21134-21140.
34.
35.
Hussain, S., et al. (2014) Structure–activity relationships studies of quinoxalinone derivatives
as aldose reductase inhibitors. European Journal of Medicinal Chemistry 80: 383–392.
Wu, B., et al. (2013) Synthesis and Structure&ndash;Activity Relationship Studies of
Quinoxaline Derivatives as Aldose Reductase Inhibitors. Chemmedchem 8:
1913&ndash;1917.
36.
37.
Shuzhen, Z., et al. (2013) Effect of C7 modifications on benzothiadiazine-1,1-dioxide
derivatives on their inhibitory activity and selectivity toward aldose reductase.
Chemmedchem 8: 603-613.
Pegklidou, K., et al. (2010) Design and synthesis of novel series of pyrrole based chemotypes
and their evaluation as selective aldose reductase inhibitors. A case of bioisosterism between
a carboxylic acid moiety and that of a tetrazole. Bioorganic & Medicinal Chemistry 18:
2107–2114.
38.
39.
Hotta, N., et al. (2006) Long-Term Clinical Effects of Epalrestat, an Aldose Reductase Inhibitor,
on Diabetic Peripheral Neuropathy: The 3-year, multicenter, comparative Aldose Reductase
Inhibitor-Diabetes Complications Trial. Diabetes Care 29: 1538-1544.
Iqbal, Z., et al. (2015) Synthesis, characterization, hypoglycemic and aldose reductase
inhibition activity of arylsulfonylspiro[fluorene-9,5 ′ -imidazolidine]-2 ′ ,4 ′ -diones.
European Journal of Medicinal Chemistry 98: 127-138.
40.
41.
Yang, Y., et al. (2012) An Efficient Synthesis of Quinoxalinone Derivatives as Potent Inhibitors
of Aldose Reductase. Chemmedchem 7: 823-835.
Feather, M.S., et al. (1995) Catalysis of reduction of carbohydrate 2-oxoaldehydes (osones) by
mammalian aldose reductase and aldehyde reductase. Biochimica Et Biophysica Acta 1244:
10–16.
42.
43.
44.
Carper, D.A., et al. (1989) A superfamily of NADPH-dependent reductases in eukaryotes and
prokaryotes. Experimental Eye Research 49: 377-88.
Blois, M.S. (1958) Antioxidant Determinations by the Use of a Stable Free Radical. Nature 181:
1199-1200.
El-Kabbani, O., et al. (2005) Structure of Aldehyde Reductase Holoenzyme in Complex with

ACCEPTED MANUSCRIPT
the Potent Aldose Reductase Inhibitor Fidarestat:ꢀ Implications for Inhibitor Binding and
Selectivity. J.med.chem 48: 5536-5542.
45.
Kinoshita, T., et al. (2002) The structure of human recombinant aldose reductase complexed
with the potent inhibitor zenarestat. Acta Crystallographica Section D 58: 622-626.

ACCEPTED MANUSCRIPT
Highlights
ꢀ
A series of novel aldose reductase inhibitors were synthesized.
ꢀ
Compound 11i was proved to be a multifunctional ARI with antioxidant activity.
ꢀ Structure-activity relationship and molecular docking studies were conducted.