9-Benzoyl 9-deazaguanines as potent xanthine oxidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.12.006

A novel potent xanthine oxidase inhibitor, 3-nitrobenzoyl 9-deazaguanine (LSPN451), was selected from a series of 10 synthetic derivatives. The enzymatic assays were carried out using an on-flow bidimensional liquid chromatography (2D LC) system, which allowed the screening the measurement of the kinetic inhibition constant and the characterization of the inhibition mode. This compound showed a non-competitive inhibition mechanism with more affinity for the enzyme-substrate complex than for the free enzyme, and inhibition constant of 55.1 ± 9.80 nM, about thirty times more potent than allopurinol. Further details of synthesis and enzymatic studies are presented herein.

Full text of DOI:10.1016/j.bmc.2015.12.006

Bioorganic & Medicinal Chemistry 24 (2016) 226–231
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
9-Benzoyl 9-deazaguanines as potent xanthine oxidase inhibitors
Marili V. N. Rodrigues a,b, Alexandre F. Barbosa c, Júlia F. da Silva c, Deborah A. dos Santos c,
b,
Kenia L. Vanzolini b, Marcela C. de Moraes b,d, Arlene G. Corrêa c, , Quezia B. Cass
⇑
⇑
a Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas, Universidade Estadual de Campinas, 13148-218 Paulínia, SP, Brazil
b Separare—Nucleo de Pesquisa em Cromatografia, Departamento de Química, Universidade Federal de São Carlos, 13565-905 São Carlos, SP, Brazil
c Laboratório de Síntese de Produtos Naturais—LSPN, Departamento de Química, Universidade Federal de São Carlos, 13565-905 São Carlos, SP, Brazil
d Departamento de Química Orgânica, Instituto de Química, Universidade Federal Fluminense, 24020-141 Niterói, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel potent xanthine oxidase inhibitor, 3-nitrobenzoyl 9-deazaguanine (LSPN451), was selected from
a series of 10 synthetic derivatives. The enzymatic assays were carried out using an on-flow bidimen-
sional liquid chromatography (2D LC) system, which allowed the screeningß the measurement of the
kinetic inhibition constant and the characterization of the inhibition mode. This compound showed a
non-competitive inhibition mechanism with more affinity for the enzyme–substrate complex than for
the free enzyme, and inhibition constant of 55.1 9.80 nM, about thirty times more potent than allopuri-
nol. Further details of synthesis and enzymatic studies are presented herein.
Received 17 September 2015
Revised 23 November 2015
Accepted 5 December 2015
Available online 8 December 2015
Keywords:
Xanthine oxidase inhibitor
9-Deazaguanine
Ó 2015 Elsevier Ltd. All rights reserved.
On-flow bidimensional liquid
chromatography
Non-competitive inhibition
Allopurinol
Green synthesis
Microwave
1. Introduction
Additionally, XO is frequently used in coupled enzymatic assay
to screen purine nucleoside phosphorylase (PNP) inhibitors, as
The screening of potential enzymatic inhibitors in synthetic or
natural libraries is an important step in drug discovery and devel-
opment process. Besides its inhibitory potency, to require lower
doses to achieve the desired effect, these compounds should exhi-
bit a high degree of selectivity toward the target enzyme.
described elsewhere by Kalckar.4 PNP enzyme is also associated
with purine metabolism and catalyzes the cleavage of (deoxy)ri-
bonucleosides, like inosine, in the presence of inorganic phosphate
(Pi) to the corresponding purine bases and ribose(deoxyribose)-1-
phosphate. Human PNP has been considered an important target to
the development of new drugs to treat severe T-cell mediated dis-
orders. In this assay, XO is used to oxidize the product of PNP catal-
One of the target enzymes of considerable interest in human
therapy is xanthine oxidase (XO). This enzyme participates in the
purines metabolism and catalyzes the uric acid production using
xanthine and hypoxanthine as substrates. In parallel, the reactive
species of oxygen (ROS) are produced, which has been associated
with some pathological conditions such as post-ischemic reperfu-
sion injury, diabetes and chronic heart failure.1 Therefore, the
selective inhibition of XO may result in broad-spectrum
chemotherapeutic for gout, cancer, inflammation and oxidative
damage. Allopurinol is the first clinically used XO inhibitor
although causes many side effects such as renal toxicity due to
impairment of pyrimidine metabolism.2 Thus there is a great inter-
est for the development of novel XO inhibitors.3
ysis
(hypoxanthine)
producing
uric
acid
that
is
spectrophotometrically monitored at 293 nm. For PNP, false posi-
tive results may be obtained by this approach, thus the recognition
of which enzyme is inhibited is high-priority.5
To overcome this problem, new models of inhibitors screening
assays are necessary and, we have been working on this sub-
ject.6–8 The use of enzymatic bioreactors on a 2D LC system fur-
nishes advantage over other approaches as it allows the
screening and characterization of ligands in automatized sys-
tems.5–7,9
The deazapurines show a great variety of biological activities,
among them, 7-deazapurine nucleoside analogues have demon-
strated potent antiviral,10 and antibacterial activities.11 Rosemeyer
et al.12 have described a 7-deazaxanthine derivative as an irre-
versible inhibitor of xanthine oxidase.
⇑
Corresponding authors. Tel.: +55 16 33518281; fax: +55 16 33518350.
E-mail addresses: agcorrea@ufscar.br (A.G. Corrêa), quezia@pq.cnpq.br
(Q.B. Cass).
http://dx.doi.org/10.1016/j.bmc.2015.12.006
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

M. V. N. Rodrigues et al. / Bioorg. Med. Chem. 24 (2016) 226–231
227
Table 1
9-Substituted 9-deazaguanines have been described as purine
nucleoside phosphorylase (PNP) inhibitors.13–15 The isosteric
replacement of a heterocyclic nitrogen by a carbon unit is a well-
established procedure for the study of the interactions of purines
with their biological targets.16,17
Synthesis of 2-amino-4(1H)-pyrimidinone (1)
Entry Solvent Heating
Temperature
(°C)
Time
(min)
Yieldb
(%)
1
2
3
4
5
6
7
8
9
10
11
12
13
MeOH
EtOH
Conventional Reflux
Conventional Reflux
1080
1080
480
5
65
81
66
27
32
43
65
65
25
38
45
33
55
In this study, a series of 9-benzoyl 9-deazaguanine derivatives
was screened for XO, and since they are structurally correlated
with inosine, the PNPs substrate, their inhibitory specificity was
also investigated by screening the compounds for human (Hs)
MeOHa Conventional 45
—
—
—
—
MW
MW
MW
MW
MW
MW
MW
MW
MW
MW
80
100
120
120
120
65
65
65
75
75
5
5
and Schistosoma mansoni (Sm) PNPs. From these assays,
a
10
15
15
30
45
15
30
—
3-nitrobenzoyl-derivative was identified as a potent and specific
XO inhibitor. Furthermore, its inhibition constant and mechanism
modality were determined.
MeOH
MeOH
MeOH
EtOH
EtOH
2. Results and discussion
2.1. Synthesis
a
MeONa was employed as base.
Isolated yield.
b
Few methods are reported in the literature for the synthesis of
pyrrolo[3,2-d]pyrimidine (9-deazapurines). One of the strategies
involves the formation of the pyrimidine ring onto a preformed
3-aminopyrrole intermediate.18–20 However, the most generally
used approach to pyrrolo[3,2-d]pyrimidines has so far involved
elaboration of the pyrrole ring onto a preformed pyrimidine bear-
ing reactive functionalities at C-4 and C-5.21–23
In this work, we have synthesized 9-benzoyl 9-deazapurines
based on the work described by Shih et al.24 (Scheme 1). Several
procedures are reported for the preparation of the starting mate-
rial, 2-amino-4(1H)-pyrimidinone (1), using guanidine, ethyl ace-
toacetate, with or without base, under reflux of ethanol or
methanol for several hours.25,26 We have tested some of the
reported conditions and using reflux of ethanol for 18 h, compound
1 was obtained in 81% yield.
alkylation using N,N-dimethylformamide dimethyl acetal furnish-
ing compound 3.30 Then, this compound was treated with sodium
dithionite in water under reflux for 2 h affording 2-amino-3,5-
dihydro-pyrrolo[3,2-d]pyrimidin-4-one (5) in only 61% yield. We
tested this reaction under MW irradiation at 90 °C and after
30 min we obtained a mixture of compound 5 and a by-product,
the imidamide 4,31 in 38% and 43% yield, respectively. This imi-
damide was hydrolyzed under basic conditions to furnish the
desired product 5.
Based on this result and following the procedure described by
Taylor and Young,32 treatment of compound 3 with sodium
dithionite in a 2:1 mixture of THF/H2O at rt for 2 h, afforded the
imidamide 4 which was then submitted to hydrolysis, using NaOH
in water under MW irradiation at 90 °C for 15 min, furnishing com-
pound 5 in 91% yield, after 2 steps. Finally, Friedel–Crafts reaction
using 12 different acyl chlorides and trifluoromethane sulfonic acid
as catalyst,24 followed by hydrolysis lead to the 9-benzoyl 9-deaza-
purine series (LSPN440–452), in moderate to good yields (Table 2).
Schmink et al.27 described a microwave (MW) assisted synthe-
sis of 6-methylthiouracil using KOH in ethanol. In an ongoing pro-
gram toward the development of greener procedures for the
preparation of heterocycles,28,29 we have evaluated different reac-
tion conditions to prepare pyrimidinone 1, including MW irradia-
tion (Table 1). The best result was obtained without base and in
neat conditions under MW irradiation for 10 min at 120 °C afford-
ing 1 in 65% yield.
2.2. Screening assay
The products of XO and PNP catalyzes (uric acid and hypoxan-
thine, respectively) were used to determine the enzymatic
The synthesis of 9-deazapurines was carried out by nitration of
1 furnishing 2-amino-5-nitropyrimidin-4(1H)-one (2), followed by
O
O
O
guanidine,
O
O
NO2
NO2
30W, 120oC
HNO3, H2SO4
rt, 3h, 69%
N
DMF.DMA, THF
100oC, 24h, 96%
N
N
OEt
10 min., 65%
N
N
H
H2N
N
H
1
H2N
N
H
2
3
N
N
O
N
O
O
H
N
H
N
H
N
NaOH, 40W, 90oC,
15 min., 95%
HN
ArCOCl, triflic acid
HN
Na2S2O4, THF/H2O,
rt, 1h, 94%
HN
120oC, 48h;
N
NaOH, 60oC, 2h
H2N
N
H2N
N
4
O
5
N
LSPN440-452
R
Scheme 1. Synthesis of 9-benzoyl 9-deazapurines.

228
M. V. N. Rodrigues et al. / Bioorg. Med. Chem. 24 (2016) 226–231
Table 2
Table 3
Yields obtained in the synthesis of 9-benzoyl 9-deazapurines
Inhibition selectivity of 9-benzoyl 9-deazapurine derivatives
Entry
Compound LSPN
R
Yielda (%)
Compound
Inhibition (%)
HsPNP-ICER⁄⁄
1
2
3
4
5
6
7
8
9
440
441
442
443
444
445
446
447
448
449
450
451
H
53
64
68
65
56
71
73
61
75
62
52
68
XO-ICER⁄
SmPNP-ICER⁄⁄
4-Fluoro
4-Chloro
4-Bromo
4-Iodo
3-Fluoro
3-Chloro
2-Chloro
3,4-Dichloro
3,5-Dichloro
2,4,6-Trichloro
3-Nitro
LSPN440
LSPN441
LSPN442
LSPN443
LSPN444
LSPN446
LSPN447
LSPN450
LSPN451
44.0
42.1
27.1
57.3
15.5
53.5
41.4
0.0
0.0
0.0
7.13
19.8
0.0
0.0
0.0
0.0
25.7
0.0
0.0
2.52
12.8
0.0
6.39
0.0
0.0
10
11
12
83.6
1.50
Inhibition (%) at 100
l
M (⁄) and 200 M (⁄⁄). Mean percent inhibition is reported
l
(n = 2)⁄⁄
.
a
Isolated yields after purification by column chromatography.
activities of XO-ICERs and PNP-ICERs. Figure
enzymatic routes for PNP and XO catalyzes.
1
shows the
Ten out of the twelve compounds synthetized were screened for
XO, SmPNP and HsPNP inhibition (Table 2). For that, previously on-
flow 2D LC assays were used.5–7 The screening assay was carried
out at high concentrations levels (100 and 200 lM) to select inhi-
bitors that have different potency. The screening was carried in a
single concentration level against a control sample. Table 3 fur-
nishes the percentage of inhibition for all compounds. In general,
the 9-benzoyl 9-deazapurine derivatives showed none or very
weak activity toward the PNPs, being significantly more potent
to XO. Due to the higher selectivity index of LSPN451 toward XO,
this compound was selected for kinetics evaluation and mechanis-
tic studies.
The IC50 value found for compound LSPN451 was 65 9.4 nM.
To determine the inhibition mechanism, a visual inspection of
Lineweaver–Burk (LB) plots, as well as an analysis of the statistical
significance in both slope and intercept replots33 was carried out.
The visual enquiries of LB (Fig. 2) with statistical analysis of
slope and y-intercept replot data were used to determine the mode
of inhibition of compound LSPN451. The LB plot showed that KM
and Vmax decreases curvilinear with increasing inhibitor concentra-
tion indicating a non-competitive inhibition with
a
< 1.34 That was
confirmed by statistical analysis of slope and y-intercept
replot data which showed a significantly non-zero slopes in the
Figure 2. Lineweaver–Burk plot (A) and slope/intercept replots (B and C, respec-
tively) of xanthine oxidase inhibition by compound LSPN451. Each point of the LB
plot represents the average of duplicate determinations.
regression line for both slope and intercept.33 These results denote
that the inhibitor has higher affinity for the enzyme–substrate
complex than for the free enzyme.
Afterward, the inhibition constant was calculated fitting the
data to the appropriate nonlinear regression model, which resulted
in a Ki = 55.1 9.8 nM with
a = 0.29 0.095 that was similar to the
obtained IC50 value (IC50 = 65.2 9.4 nM).
The determined Ki places LSPN451 as a hit to control uric
acid-related health problems. At the same assay conditions the
determined Ki value for allopurinol was of only 1.55 l
M.7
3. Experimental section
3.1. General experimental information
The enzyme xanthine oxidase from bovine milk (E.C. 1.17.3.2)
was purchased from Sigma–Aldrich Co. (St. Louis, MO, USA).
Hs- and SmPNP expression and purification were conducted as
reported elsewhere.35,36
Figure 1. Enzymatic route of purine nucleoside phosphorylase (PNP) and xanthine
oxidase (XO).

M. V. N. Rodrigues et al. / Bioorg. Med. Chem. 24 (2016) 226–231
229
Unless otherwise noted, all commercially available reagents
were purchased from Sigma–Aldrich Co. Reagents and solvents
were purified when necessary according to the usual procedures
described in the literature.
(25 mL) and DMF dimethyl acetal (7.5 mL, 70.0 mmol). The mix-
ture was stirred at 100 °C for 24 h and then cooled. The mixture
was dried under vacuum. Acetone (15 mL) was added and the
formed precipitate was filtered and washed with acetone (10 mL)
For analytical thin-layer chromatography a 0.25
l
m film of sil-
furnishing 3 in 96% yield (3.16 g) as an orange solid, mp:
ica gel containing fluorescent indicator UV254 supported on an
aluminum sheet (Sigma–Aldrich) were used. Silica gel (Kieselgel
60, 230–400 mesh, E. Merck) was used for flash column
chromatography.
276–278 °C. 1H NMR (400 MHz, CDCl3) d (ppm): 8.58 (s, 1H),
7.88 (d, J = 12.2 Hz, 1H), 5.60 (d, J = 12.2 Hz, 1H), 3.24 (s, 3H),
3.14 (s, 3H), 2.80–3.10 (m, 6H). 13C NMR (100 MHz, CDCl3) d
(ppm): 35.2, 41.5, 89.7, 124.4, 151.7, 156.2, 156.4, 157.5, 158.4.
1H and 13C NMR spectra were recorded on a Bruker ARX-400
(400 and 100 MHz, respectively). The IR spectra refer to films and
were measured on a Bomem M102 spectrometer.
IR (m
max, film, cmꢁ1): 3429, 2929, 1666, 1608, 1542, 1477, 1388,
865, 752, 740.
Gas chromatography was performed in a Shimadzu GC-17A
with H2 as carrier and using a DB-5 column (30 m ꢀ 0.25 mm ID,
3.5. Synthesis of 2-amino-3,5-dihydro-pyrrolo[3,2-d]pyrimidin-
4-one (5)28
0.25 lm film thicknesses, J and W Scientific). Melting point was
obtained on a MQAPF-301 apparatus. Reactions were carried out
In a 500 mL round-bottom flask were combined 3 (4.6 g,
16.4 mmol), sodium dithionite (12.0 g, 69 mmol) and THF/H2O
(2:1, 300 mL). The mixture was stirred at room temperature for
1 h. The solvent was removed under reduced pressure, then H2O
(50 mL) was added and the solid formed was filtered and washed
with water (2 ꢀ 100 mL). The product was purified by silica gel
chromatography using 2% MeOH in CH2Cl2 as eluent. Compound
4 was obtained in 94% yield (3.16 g) as a white solid. 1H NMR
(200 MHz, DMSO-d6) d (ppm): 11.58 (s, 1H), 8.51 (s, 1H), 7.19 (t,
J = 5.9 Hz, 1H), 6.09 (t, J = 4.7 Hz, 1H), 3.13 (s, 3H), 3.03 (s, 3H).
13C NMR (100 MHz, DMSO-d6) d (ppm): 39.7, 41.3, 103.3, 115.6,
in a CEM DiscoveryÒ focused microwave oven.
The exact mass measurement was carried out using a Shimadzu
Nexera X2 UHPLC system coupled to a Bruker Impact HD QqTOF
mass spectrometer (FAPESP PROEM 2014/50244-6). A BetasilÒ
C18 (20 ꢀ 2.1 mm, 5
lm) pre-column was used for injection.
3.2. Synthesis of 2-amino-6-methylpyrimidin-4(3H)-one (1)37
Guanidine (0.2 g, 3.38 mmol) and ethyl acetoacetate (0.45 g,
3.45 mmol) (and eventually 3 mL of solvent) were placed in a glass
tube, sealed and irradiated during 20–60 min in a microwave oven,
as shown in Table 1. The crude mixture was cooled in an ice bath
and a precipitate was observed. The solid was filtered and washed
thoroughly with distilled water (5 mL). The dry product was
obtained as a white solid. The solvent-free reaction afforded the
desired product 1 in 65% yield (274 mg) as a white solid, mp:
296–298 °C (lit. mp >300 °C). 1H NMR (400 MHz, DMSO-d6) d
(ppm): 1.97 (s, 3H), 5.37–5.39 (m, 1H), 6.50–6.80 (m, 2H). 13C
NMR (100 MHz, DMSO-d6) d (ppm): 23.2, 100.2, 155.5, 156.0,
129.2, 145.2, 154.4, 156.7, 157.3. IR (m
max, film, cmꢁ1): 3431,
3166, 1685, 1620, 1492, 1400, 1336, 1108, 1093, 784.
In the sequence, in a microwave tube was added 4 (200 mg,
0.97 mmol) and NaOH (195.0 mg, 4.84 mmol) in H2O (5 mL). The
set conditions were 95 °C for 15 min with an average power of
40 W. This procedure was repeated four times and then the crude
products were mixed. The solvent was evaporated under vacuum
and then water (5 mL) was added. The mixture was acidified to
pH 5.0 with glacial acetic acid. The precipitate was filtered and
then recrystallized in ethanol/H2O (3:1) affording the desired pro-
duct 5 in 96% yield (701.0 mg) as a white solid, mp: degraded at
300 °C. 1H NMR (400 MHz, DMSO-d6) d (ppm): 7.43 (d, J = 5.9 Hz,
1H), 6.44 (s, 2H), 5.87 (d, J = 4.9 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6) d (ppm): 100.1, 112.0, 127.4, 144.8, 151.2, 153.0. IR
165.1. IR (m
max, film, cmꢁ1): 771, 829, 1018, 1047, 1504, 1666,
2943, 3074, 3332.
3.3. Synthesis of 2-amino-6-methyl-5-nitropyrimidin-4(3H)-
one (2)38,39
(m
max, film, cmꢁ1): 3450, 3207, 3134, 1647.
In a two-neck round-bottom flask containing precooled sulfuric
acid (3 mL) was added 2-amino-4-hydroxy-6-methylpyrimidine
(1) (100 mg, 0.8 mmol). An effective temperature control is
required to the nitration occur properly. Nitric acid (0.2 mL,
3.0 mmol) was added to the solution dropwise maintaining the
temperature below 6 °C using an ice bath. The mixture was
warmed to room temperature and stirred for 3 h. The reaction mix-
ture was added in cold ethyl ether (10 mL), remaining the temper-
ature below 10 °C. The precipitate was filtered and dissolved in
boiling 1 mol/L NaOH solution. Acetic acid was added to the solu-
tion for precipitation of the product (pH among 6–8). The solid was
filtered under vacuum, washed with water (2 ꢀ 1 mL) and dried in
vacuum for 8 h. The pyrimidinone 2 was obtained in 69% yield
(117 mg) as a pale yellow solid, mp: >300 °C (lit. mp = 300 °C). 1H
NMR (400 MHz, DMSO-d6) d (ppm): 2.24 (s, 3H), 11.62 (s, 1H).
13C NMR (100 MHz, DMSO-d6) d (ppm): 22.2, 128.5, 155.0, 155.4,
3.6. General procedure for 9-benzoyl 9-deazapurines LSPN440–
451
A mixture of 9-deazapurine 5 (150.0 mg, 1.0 mmol) and benzoyl
chloride (2.2 equiv) in trifluoromethanesulfonic acid (3.45 g,
23 mmol) was stirred at 80–120 °C for 48 h. After the mixture
was cooled and H2O (15 mL) was added. Then the reaction was
neutralized with NaOH 1.0 mol/L, the volume adjusted to approx-
imately 35 mL by adding H2O and then 60 equiv of NaOH were
added. The reaction was stirred for 2.5 h at 60 °C. The mixture
was neutralized with glacial acetic acid and the formed solid was
filtered under vacuum and washed several times with dichloro-
methane (10 mL), ethyl acetate (10 mL), acetone (5 mL) and
methanol (5 mL). The precipitate purity was monitored by CCD
and depending on the product, further purification was necessary
by recrystallization in methanol or flash chromatography using
CH2Cl2/MeOH (4:1) as eluent.
164.4. IR (
1658, 1708, 3053, 3498, 3575.
m
max, film, cmꢁ1): 717, 788, 852, 1093, 1402, 1550,
3.4.
Synthesis
of
N0-[4-[2-(dimethylamino)ethenyl]-1,6-
3.6.1. 2-Amino-3H,5H-7-benzoylpyrrolo[3,2-d]pyrimidin-4-one
(LSPN440)24
dihydro-5-nitro-6-oxo-2-pyrimidinyl]-N,N-dimethyl-
methanimidamide (3)22
White solid (135 mg), mp: degraded at 280 °C. 1H NMR
(400 MHz, DMSO-d6) d (ppm): 7.72–7.68 (m, 2H), 7.54–7.44 (m,
3H), 7.41 (s, 1H), 6.60 (s, 2H). 13C NMR (100 MHz, DMSO-d6) d
(ppm): 188.5, 156.1, 152.6, 146.5, 142.2, 141.6, 130.4, 128.6,
In a 100 mL two-neck round-bottom flask under N2 atmosphere
and molecular sieve 3 Å was added 2 (2.0 g, 11.7 mmol) in dry DMF

230
M. V. N. Rodrigues et al. / Bioorg. Med. Chem. 24 (2016) 226–231
128.0, 114.2, 100.1. IR (
1525, 1483, 1411, 798, 769.
m
max, film, cmꢁ1): 3427, 3114, 3083, 1703,
3.6.9. 2-Amino-3H,5H-7-(3,4-dichlorobenzoyl)pyrrolo[3,2-d]
pyrimidin-4-one (LSPN448)24
White solid (243 mg), mp: degraded at 300 °C. 1H NMR
(400 MHz, DMSO-d6) d (ppm): 7.79 (d, J = 2.0 Hz, 1H), 7.69 (d,
J = 8.3 Hz, 1H), 7.61 (dd, J = 2.0 and 8.7 Hz, 1H), 7.13 (s, 1H). 13C
NMR (100 MHz, DMSO-d6) d (ppm): 188.6, 154.9, 151.4, 140.1,
3.6.2. 2-Amino-3H,5H-7-(4-fluorbenzoyl)pyrrolo[3,2-d]
pyrimidin-4-one (LSPN441)
White solid (174 mg), mp: degraded at 294 °C. 1H NMR
(400 MHz, DMSO-d6) d (ppm): 7.78–7.74 (m, 2H), 7.41 (s, 1H),
7.27 (t, J = 17.7 Hz, 2H), 6.65 (s, 2H). 13C NMR (100 MHz, DMSO-
d6) d (ppm): 186.9, 164.5, 162.0, 156.4, 152.4, 146.6, 142.9, 138.2,
137.8, 135.8, 135.0, 133.5, 130.1, 126.4, 113.2, 99.1. IR (mmax, film,
cmꢁ1): 3417, 3126, 3095, 1697, 1529, 1487, 1463, 1211, 1189,
773, 761.
131.1, 121.3, 114.9, 114.7, 114.1. IR (m
max, film, cmꢁ1): 3406,
3083, 1703, 1525, 1485, 1415, 1228, 1153, 790, 775.
3.6.10. 2-Amino-3H,5H-7-(3,5-dichlorobenzoyl)pyrrolo[3,2-d]
pyrimidin-4-one (LSPN449)24
3.6.3. 2-Amino-3H,5H-7-(4-chlorobenzoyl)pyrrolo[3,2-d]
pyrimidin-4-one (LSPN442)
White solid (200 mg), mp: degraded at 300 °C. 1H NMR
(400 MHz, DMSO-d6) d (ppm): 12.49 (s, 1H), 7.68 (s, 1H), 7.50–
7.43 (m, 3H), 6.68 (s, 2H). 13C NMR (100 MHz, DMSO-d6) d
(ppm): 190.2, 153.4, 150.5, 138.0, 135.2, 134.3, 132.5, 131.9,
White solid (196 mg), mp: degraded at 280 °C. 1H NMR
(400 MHz, DMSO-d6) d (ppm): 7.70 (d, J = 8.3 Hz, 2H), 7.53 (d,
J = 8.3 Hz, 2H), 7.36 (s, 1H), 6.57 (s, 2H). 13C NMR (100 MHz,
DMSO-d6) d (ppm): 186.6, 156.7, 152.4, 147.1, 144.3, 140.7,
130.0, 126.8, 125.9, 102.5, 96.3. IR (m
max, film, cmꢁ1): 3431, 3087,
1701, 1529, 1487, 1436, 1413, 1168, 1120, 771.
139.2, 134.6, 130.9, 130.4, 127.9, 127.1, 113.9. IR (mmax, film,
cmꢁ1): 3407, 3080, 1699, 1527, 1481, 1413, 1282, 1191, 777,
761. HRMS: calc. for C13H10ClN4O2: 289.70; exp. 289.0493.
3.6.11. 2-Amino-3H,5H-7-(2,4,6-trichloro)pyrrolo[3,2-d]
pyrimidin-4-one (LSPN450)
White solid (218 mg), mp: degraded at 296 °C. 1H NMR
(400 MHz, DMSO-d6) d (ppm): 6.29 (s, 2H), 6.84 (s, 1H), 7.64 (s,
2H). 13C NMR (100 MHz, DMSO-d6) d (ppm): 177.6, 161.5, 159.6,
3.6.4. 2-Amino-3H,5H-7-(4-bromobenzoyl)pyrrolo[3,2-d]
pyrimidin-4-one (LSPN443)
White solid (216 mg), mp: degraded at 288 °C. 1H NMR
(400 MHz, DMSO-d6) d (ppm): 7.86 (d, J = 8.3 Hz, 2H), 7.52–7.58
(m, 3H), 6.70 (s, 2H). 13C NMR (100 MHz, DMSO-d6) d (ppm):
187.7, 155.0, 152.8, 145.2, 139.8, 136.9, 135.9, 131.3, 130.8,
152.7, 142.5, 140.2, 132.3 (2C), 132.2 (2C), 120.9, 113.1. IR (mmax,
film, cmꢁ1): 3481, 3230, 3080, 1670, 1583, 1514, 1415, 1201, 781.
3.6.12. 2-Amino-3H,5H-7-(3-nitrobenzoyl)pyrrolo[3,2-d]
pyrimidin-4-one (LSPN451)24
114.1, 98.6. IR (
1413, 1147, 775.
m
max, film, cmꢁ1): 3425, 3116, 3083, 1703, 1525,
White solid (203 mg), mp: degraded at 300 °C. 1H NMR
(400 MHz, DMSO-d6) d (ppm): 8.47 (s, 1H), 8.35 (d, J = 9.3 Hz,
1H), 8.13 (d, J = 7.3 Hz, 1H), 7.76 (t, J = 15.8 Hz, 1H), 7.38 (s, 1H),
6.37 (s, 2H). 13C NMR (100 MHz, DMSO-d6) d (ppm): 184.9, 156.3,
153.6, 148.6, 137.3, 136.3, 135.3, 132.8, 128.0, 126.4, 124.7,
3.6.5. 2-Amino-3H,5H-7-(4-iodobenzoyl)pyrrolo[3,2-d]
pyrimidin-4-one (LSPN444)
White solid (213 mg), mp: degraded at 300 °C. 1H NMR
(400 MHz, DMSO-d6) d (ppm): 7.82 (d, J = 8.3 Hz, 2H), 7.46 (d,
J = 8.3 Hz, 2H), 7.29 (s, 1H), 6.51 (s, 2H). 13C NMR (100 MHz,
DMSO-d6) d (ppm): 186.7, 157.0, 152.3, 147.5, 145.5, 141.7,
107.6, 105.7. IR (m
max, film, cmꢁ1): 3485, 3323, 3195, 3085, 1706,
1529, 1433, 1215, 781, 723. HRMS: calc. for C13H10N5O4: 300.07;
exp. 300.0729.
136.7, 130.5, 123.8, 113.9, 96.9. IR (m
max, film, cmꢁ1): 3423, 3116,
3080, 1703, 1525, 1411, 1147, 775, 754.
3.7. Screening and inhibition studies
3.6.6. 2-Amino-3H,5H-7-(3-fluorobenzoyl)pyrrolo[3,2-d]
pyrimidin-4-one (LSPN445)
The protocols to prepare XO and PNP-ICERs, as well as the val-
idated LC 2D methods for quantifying the enzymatic reactions
products are described in previously works.3–5
Ten 9-benzoyl 9-deazaguanine derivatives were screened with
XO-ICER. Stock solutions of each compound were prepared in
methanol/H2O (1:1, v/v) at 2 mM. The compounds were evaluated
White solid (193 mg), mp: degraded at 300 °C. 1H NMR
(400 MHz, DMSO-d6) d (ppm): 7.53–7.50 (m, 2H), 7.38–7.30 (m,
3H), 6.52 (s, 2H). 13C NMR (100 MHz, DMSO-d6) d (ppm): 186.1,
162.9, 160.5, 157.0, 152.4, 144.7, 129.9, 124.5, 116.7, 116.5,
114.9, 114.7, 113.8. IR (
1238, 786, 723.
m
max, film, cmꢁ1): 3427, 3083, 1701, 1411,
at 100
The assay solutions for XO-ICER were prepared with the addi-
tion of 100 L of xanthine (200 M), 80 L water and 20 L of
methanol/H2O (1:1, v/v) solution, maintaining methanol concen-
tration fixed in all samples. Aliquots of 10 L were injected in
lM using XO-ICER and 200 lM for PNP-ICERs.
l
l
l
l
3.6.7. 2-Amino-3H,5H-7-(3-chlorobenzoyl)pyrrolo[3,2-d]
pyrimidin-4-one (LSPN446)
l
White solid (216 mg), mp: degraded at 270 °C. 1H NMR
(400 MHz, DMSO-d6) d: 12.43 (s, 1H), 7.73–7.71 (m, 2H), 7.63–
7.60 (m, 2H), 7.50 (t, J = 8.0 Hz, 1H), 6.65 (s, 1H). 13C NMR
(100 MHz, DMSO-d6) d: 187.2, 153.9, 152.9, 144.0, 141.5, 133.7,
the chromatographic system3,5 and, the enzymatic activity evalu-
ated by the quantification of uric acid produced by XO-ICERs at
294 nm. For each tested compound, a control sample was prepared
using a solution of methanol/H2O (1:1, v/v) to substitute the com-
pound solutions, measuring the impact of methanol in the enzyme
activity.
132.9, 131.3, 130.1, 128.5, 127.8, 114.4, 113.1. IR (mmax, film,
cmꢁ1): 3431, 3087, 1701, 1529, 1409, 1211, 1149, 773, 738.
To investigate the inhibition specificity, the same library was
screened for Sm- and HsPNP. Inhibitory activity of these analogues
3.6.8. 2-Amino-3H,5H-7-(2-chlorobenzoyl)pyrrolo[3,2-d]
pyrimidin-4-one (LSPN447)
against SmPNP was evaluated at 200
quots of a sample containing Ino 250
7.4) 400 M, and 200 M of the assayed compound. The screening
for HsPNPs was carried out at 200 M, by injecting 20 L of a sam-
ple containing 5 mM phosphate buffer pH 7.0 and 382.5 M Ino.
l
M, by injecting 15
lL ali-
White solid (176 mg), mp: degraded at 300 °C. 1H NMR
(400 MHz, DMSO-d6) d (ppm): 7.54–7.42 (m, 4H), 7.25 (s, 1H),
6.75 (s, 2H). 13C NMR (100 MHz, DMSO-d6) d (ppm): 187.1, 153.9,
153.4, 144.3, 140.3, 135.0, 130.6, 129.6, 129.5, 128.6, 126.9,
lM, phosphate buffer (pH
l
l
l
l
l
115.1, 113.7. IR (
m
max, film, cmꢁ1): 3448, 3130, 3091, 1691, 1529,
The percentage of inhibitions were calculated by the following
1417, 1149, 1068, 769, 717.
expression: % I = 100 ꢁ (Ci/C0 ꢀ 100), where Ci is the enzymatic

M. V. N. Rodrigues et al. / Bioorg. Med. Chem. 24 (2016) 226–231
231
6. de Moraes, M. C.; Cardoso, C. L.; Cass, Q. B. Anal. Bioanal. Chem. 2013, 405, 4871.
7. Rodrigues, M. V. N.; Correa, R. S.; Vanzolini, K. L.; Santos, D. S.; Batista, A. A.;
Cass, Q. B. RSC Adv. 2015, 5, 37533.
8. Vanzolini, K. L.; Jiang, Z.; Zhang, X.; Vieira, L. C. C.; Correa, A. G.; Cardoso, C. L.;
Cass, Q. B.; Moaddel, R. Talanta 2013, 116, 647.
9. Cardoso, C. L.; Lima, V. V.; Zottis, A.; Oliva, G.; Andricopulo, A. D.; Wainer, I. W.;
Moaddel, R.; Cass, Q. B. J. Pharm. Biomed. Anal. 2013, 73, 44.
10. Vittori, S.; Dal Ben, D.; Lambertucci, C.; Marucci, G.; Volpini, R.; Cristalli, G. Curr.
Med. Chem. 2006, 13, 3529.
11. Vitali, L. A.; Petrelli, D.; Lambertucci, C.; Prenna, M.; Volpini, R.; Cristalli, G. J.
Med. Microbiol. 2012, 61, 525.
12. Rosemeyer, H.; Kretschmer, U.; Seela, F. Helv. Chim. Acta 1985, 68, 2165.
13. Hikishima, S.; Hashimoto, M.; Magnowska, L.; Bzowska, A.; Yokomatsu, T. S.
Bioorg. Med. Chem. 2010, 18, 2275.
14. Breer, K.; Glavas-Obrovac, L.; Suver, M.; Hikishima, S.; Hashimoto, M.;
Yokomatsu, T.; Wielgus-Kutrowska, B.; Magnowska, L.; Bzowska, A. FEBS J.
2010, 277, 1747.
15. Castilho, M. S.; Postigo, M. P.; Pereira, H. M.; Oliva, G.; Andricopulo, A. D. Bioorg.
Med. Chem. 2010, 18, 1421.
16. Borrmann, T.; Abdelrahman, A.; Volpini, R.; Lambertucci, C.; Alksnis, E.;
Gorzalka, S.; Knospe, M.; Schiedel, A. C.; Cristalli, G.; Muller, C. E. J. Med.
Chem. 2009, 52, 5974.
product (Hypo or uric acid) concentration obtained in the presence
of the tested compounds, and C0 is the enzymatic product concen-
tration calculated in the absence of the evaluated compounds.
An inhibition curve was constructed for LSPN451 (0, 0.02, 0.05,
0.10, 0.25, 0.50, 2.50, 3.50, 4.00, 6.00, 8.00 and 10.00
lM) at fixed
concentration of substrate (25 M) by plotting the percentage of
l
inhibition versus the inhibitor concentration and, the IC50 value
was, thus, obtained. The modality of inhibition was determined
by visual inspection of Lineweaver–Burk plot and statistical analy-
sis of slope and y-intercept replot data. The slopes of replot were
analyzed by GraphPad Prism software to determine its significantly
nonzero slopes in the regression line using F-test feature.31
Then, based in the obtained inhibition mechanism, the appro-
priate nonlinear regression model was selected in the GraphPad
Prism software to obtain Ki and
a values.
4. Conclusions
17. Lougiakis, N.; Marakos, P.; Pouli, N.; Fragopoulou, E.; Tenta, R. Chem. Pharm.
Bull. 2015, 63, 134.
A series of new 9-benzoyl 9-deazaguanine derivatives were effi-
ciently synthesized and ten compounds were investigated for their
inhibition against XO and Sm- and HsPNPs. The 2D LC assay sys-
tem, with the XO-ICER and PNP-ICERs at the first dimension, has
once again showed to be a valuable tool, not only for selectively
identifying the inhibitors, but also for mechanistically characteriz-
ing them. LSPN451 emerged as a potent selective XO noncompet-
itive inhibitor with higher affinity for the enzyme–xanthine
complex.
18. Salaheldin, A. M.; Oliveira-Campos, A. M. F.; Rodrigues, L. M. ARKIVOC 2008, xiv,
180.
19. Montgomery, J. A.; Niwas, S.; Rose, J. D.; Secrist, J. A.; Babu, Y. S.; Bugg, C. E.;
Erion, M. D.; Guida, W. C.; Ealick, S. E. J. Med. Chem. 1993, 36, 55.
20. He, P.; Yan, Y.-M.; Ding, M.-W. J. Heterocycl. Chem. 2014, 51, E93.
21. Evans, G. B.; Furneaux, R. H.; Hutchison, T. L.; Kezar, H. S.; Morris, P. E.;
Schramm, V. L.; Tyler, P. C. J. Org. Chem. 2001, 66, 5723.
22. Otmar, M.; Masojidkova, I. M.; Votruba, I.; Holy, A. Bioorg. Med. Chem. 2004, 12,
3187.
23. Amarnath, V.; Madhav, R. Synthesis 1974, 837.
24. Shih, H.; Carson, H. B.; Carson, D. A. Chem. Pharm. Bull. 2002, 50, 364.
25. Foster, H.; Snyder, H. Org. Synth. Coll. 1963, 4, 638.
26. Zhou, J. P.; Ding, Y. W.; Zhang, H. B.; Xu, L.; Dai, Y. Chin. Chem. Lett. 2008, 19,
669.
27. Schmink, J. R.; Kormos, C. M.; Devine, W. G.; Leadbeater, N. E. Org. Process Res.
Dev. 2010, 14, 205.
28. Duarte, P. D.; Paixao, M. W.; Correa, A. G. Green Process. Synth. 2013, 2, 19.
29. Sangi, D. P.; Monteiro, J. L.; Vanzolini, K. L.; Cass, Q. B.; Paixão, M. W.; Corrêa, A.
G. J. Braz. Chem. Soc. 2014, 25, 887.
30. Furneaux, R. H.; Tyler, P. C. J. Org. Chem. 1999, 64, 8411.
31. Filla, S. A.; Mathes, B. M.; Johnson, K. W.; Phebus, L. A.; Cohen, M. L.; Nelson, D.
L.; Zgombick, J. M.; Erickson, J. A.; Schenck, K. W.; Wainscott, D. B.; Branchek, T.
A.; Schaus, J. M. J. Med. Chem. 2003, 46, 3060.
Acknowledgments
The authors are grateful to CAPES, FAPESP (research grants
2013/01710-1,
2013/07600-3
and 2013/50680-8),
CNPq
(150954/2013-1 and 471093/2013-0), and GSK Trust in Science
Program for financial support and fellowships.
Supplementary data
32. Taylor, E. C.; Young, W. B. J. Org. Chem. 1995, 60, 7947.
33. Barr, J. T.; Jones, J. P. Drug Metab. Dispos. 2011, 39, 2381.
34. Copeland, R. A. Reversible Modes of Inhibition Interactions with Enzymes,
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.12.006.
Evaluation of Enzyme Inhibitors in Drug Discovery:
A
Guide for Medicinal
Chemists and Pharmacologists; John Wiley
& Sons: Hoboken, New Jersey,
2005; pp 48–81.
35. Silva, R. G.; Carvalho, L. P. S.; Oliveira, J. S.; Pinto, C. A.; Mendes, M. A.; Palma, M.
S.; Basso, L. A.; Santos, D. S. Protein Expr. Purif. 2003, 27, 158.
36. Pereira, H. M.; Cleasby, A.; Pena, S. D. J.; Franco, G. R.; Garratt, R. C. Acta
Crystallogr., Sect. D Biol. Crystallogr. 2003, 59, 1096.
37. Erkin, A. V.; Krutikov, V. I. Russ. J. Gen. Chem. 2011, 81, 1699.
38. De la Cuesta, E.; Avendaño, C. J. Heterocycl. Chem. 1985, 22, 337.
39. Bailey, S.; Ayling, J. Biochemistry 1983, 22, 1790.
References and notes
1. Pacher, P.; Nivorozhkin, A.; Szabo, C. Pharm. Rev. 2006, 58, 87.
2. Horiuchi, H.; Ota, M.; Nishimura, S.; Kaneko, H.; Kasahara, Y.; Ohta, T.;
Komoriya, K. Life Sci. 2000, 66, 2051.
3. Chauhan, M.; Kumar, R. Med. Chem. Res. 2015, 24, 2259.
4. Kalckar, H. M. J. Biol. Chem. 1947, 167, 429.
5. de Moraes, M. C.; Ducati, R. G.; Donato, A. J.; Basso, L. A.; Santos, D. S.; Cardoso,
C. L.; Cass, Q. B. J. Chromatogr., A 2012, 1232, 110.