In silico design and synthesis of N-arylalkanyl 2-naphthamides as a new class of non-purine xanthine oxidase inhibitors

Article abstract of DOI:10.1002/ddr.21782

A series of N-arylalkanyl 2-naphthamides (Xa~e), which were predicted from virtual molecular docking on a built xanthine oxidase template as potential inhibitors, were synthesized. Their inhibitory activity against xanthine oxidase was assayed. Among these prepared, compounds Xb (IC₅₀ 13.6?μM), Xc (IC₅₀ 13.1?μM), and Xd (IC₅₀ 12.5?μM) showed comparable inhibitory activity to allopurinol (IC₅₀ 22.1?μM). The in vitro assay result correlated well with molecular docking scores, ΔG?=??16.99, ?17.66, and ?17.13 Kcal/mol, respectively. On the potassium oxonate-induced hyperuricemic mice model, oral administration of Xc-Ac (40 mg/ Kg), the per-O-acetylated Xc, could reduce the blood uric acid level by 60% in comparison to the normal control group and is statistically significant (p <.01) while compared with the hyperuricemic mice group.

Full text of DOI:10.1002/ddr.21782

Received: 31 May 2020
DOI: 10.1002/ddr.21782
Revised: 8 December 2020
Accepted: 26 December 2020
R E S E A R C H A R T I C L E
In silico design and synthesis of N-arylalkanyl 2-naphthamides
as a new class of non-purine xanthine oxidase inhibitors
Sheau Ling Ho1† | Ching-Ting Lin2† | Shoei-Sheng Lee2
1Department of Chemical & Materials
Abstract
Engineering, Chinese Culture University,
Taipei, Taiwan
A series of N-arylalkanyl 2-naphthamides (Xaꢀe), which were predicted from virtual
molecular docking on a built xanthine oxidase template as potential inhibitors, were
2School of Pharmacy, College of Medicine,
National Taiwan University, Taipei, Taiwan
synthesized. Their inhibitory activity against xanthine oxidase was assayed. Among
Correspondence
these prepared, compounds Xb (IC50 13.6 μM), Xc (IC50 13.1 μM), and Xd (IC50
Shoei-Sheng Lee, School of Pharmacy, College
of Medicine, National Taiwan University,
Taipei 10050, Taiwan.
12.5 μM) showed comparable inhibitory activity to allopurinol (IC50 22.1 μM). The in
vitro assay result correlated well with molecular docking scores, ΔG = −16.99,
−17.66, and −17.13 Kcal/mol, respectively. On the potassium oxonate-induced hyp-
eruricemic mice model, oral administration of Xc-Ac (40 mg/ Kg), the per-O-
acetylated Xc, could reduce the blood uric acid level by 60% in comparison to the
normal control group and is statistically significant (p < .01) while compared with the
hyperuricemic mice group.
Email: shoeilee@ntu.edu.tw
Funding information
Ministry of Science and Technology, Taiwan,
Grant/Award Number: MOST 103-2320-B-
002-011-MY3
K E Y W O R D S
in vitro and in vivo assay, molecular docking, N-arylalkanyl naphthamides, synthesis, xanthine
oxidase inhibitor
1
|
INTRODUCTION
(Robinson & Dalbeth, 2015). Therefore, exploration of potent XOIs
void of side effects is in urgent need. Febuxostat, the first non-purine
Xanthine oxidase (XO), a molybdenum-containing hydroxylase, is an
important enzyme which catalyzes oxidative hydroxylation of purine
substrates (hypoxanthine and xanthine) to produce uric acid and reac-
tive oxygen species (ROS) (Hille, 2006). The excessive production or
insufficient excretion of uric acid results in hyperuricemia, which is
associated with gout and the risk factor of chronic renal disease, met-
abolic syndrome, diabetes, and cardiovascular disease (Rock,
Kataoka, & Lai, 2013). XO inhibitors (XOIs) block the formation of uric
acid from purine, which mainly occurs in intestine and liver, leading to
lowering blood uric acid level (Pacher, Nivorozhkin, & Szabo, 2006).
Thus, the development of XOIs has become one of the therapeutic
approaches for treating hyperuricemia.
XOI with higher potency than allopurinol, was approved in 2009 for
gout treatment (Kumar, Darpan, & Singh, 2011). Many research
groups have synthesized various non-purine XOIs, such as 2-phenyl-
1H-imidazoles (Chen et al., 2015; Zhang et al., 2018), 5-arylazo-
tropolones (Saito, Kisen, Kumagai,
& Ohta, 2018), and N-(9,-
10-anthraquinone-2-carbonyl)amino acids (Zhang, Li, Yuan, Zhang, &
Meng, 2018) (Figure 1). Additionally, many natural products like flavo-
noids, coumarins, and stilbenes (Cao, Pauff, & Hille, 2014; Cos
et al., 1998; Lin et al., 2008; Pauff & Hille, 2009; Silva, Mira, Lima, &
Manso, 1996) (Figure 1) also displayed XO inhibitory activity.
Five 1,4-benzodioxane type neolignans and one styrenyl caffeate
(Figure 2), isolated from Hyptis rhomboids, had been reported as active
XOIs (IC50 0.6–39.5 μM) in our previous study (Tsai & Lee, 2014).
These active natural products are too minor to proceed further stud-
ies, such as in vivo experiments. Thus, alternative approaches to sup-
ply such analogs are required.
Allopurinol, an XOI, is the most commonly used anti-gout drug
(Pacher et al., 2006). However, it might cause several serious side
effects, including hypersensitivity reactions, Steven's Johnson syn-
drome, hepatitis, nephropathy, and 6-mercaptopurine toxicity
To gain insights into the binding mode of these neolignans,
molecular docking of these compounds into the built XO template
†
Equal contribution.
Drug Dev Res. 2021;1–13.
wileyonlinelibrary.com/journal/ddr
© 2021 Wiley Periodicals, LLC.
1

2
HO ET AL.
FIGURE 1 Structures of some synthetic and natural occurring xanthine oxidase inhibitors
FIGURE 2 Structures of five 1,4-benzodioxane type neolignans and one styrenyl caffeate from Hyptis rhomboids and dereplication of VII
using MOE software was undertaken. The results showed that the
docking scores of compounds V, VII, and IV with ΔG = −21.84,
−21.46, −20.89 Kcal/mol, respectively, correlated well with the
in vitro inhibition assays with the respective IC50 value of
0.6 0.3 μM, 2.0 0.1 μM, and 5.2 0.5 μM, validating the built XO
template (Ho, Tsai, Lin, Kim, & Lee, 2020).
Dereplication of the scaffold of epihyprhombin B (VII) (IC50
2.0 μM) led to two parts, a 6-phenyl-benzo[b]-1,4-dioxane moiety

HO ET AL.
3
(VII-A) and a styryl phenylpropenoate moiety (II). Molecular docking
of VII-A and II on XO template showed good docking score for both
compounds (ΔG = −19.32 Kcal/mol, both). The 1,4-benzodioxane
type neolignans such as VII-A have been synthesized via biomimetic
approach through horseradish peroxidase catalyzed oxidative coupling
of catechol containing phenylpropenoids, such as caffeic acid
(Takahashi, Matsumoto, Ueda, Miyake, & Fukuyama, 2002). Besides
the low yield, this approach gave isomeric products which raised puri-
fication issue, thus hampering the scale-up of the desired product.
Consequently, the availability of such analogs from either synthesis or
the nature is not practical. Netpetoidin B (II) is a relatively good XOI
(IC50 11.7 μM; cf 5.3 μM, allopurinol) (Tsai & Lee, 2014) but the styryl
ester and conjugated double bond are potentially labile under stress
(acid, base, light, air). Based on in silico molecular docking to XO tem-
plate, a series of N-arylalkyl-2-naphthamides (Xa−e) was designed to
overcome such structure drawback. The following describes the
design, synthesis, and anti-XO activity of these target compounds.
The compounds were docked into the same binding site, and then
the initial model was loaded into MOE working environment ignor-
ing water molecules and heteroatoms. The structure with all the
atoms shown was put in generalized born implicit solvated envi-
ronment using the default setting. The dock scoring was
implemented using London ΔG scoring function and enhanced by
the Affinity ΔG refinement method. Refined poses were updated
to satisfy the 3D spatial restraints of the specified conformations.
The rotatable bonds were allowed and then the best 20 poses
were retained to analyze their binding scores. Energy minimization
was conducted through MMFF94x forcefield optimization by using
a gradient cut-off value of 0.05 kcal/mol/Å for determining low
energy conformations with the lowest energy geometry
(Halgren, 1996). From the final list of these 20 docked conforma-
tions, the nondistorted one with the best docking score was cho-
sen for further analysis.
2.2
|
Chemistry
2
|
EXPERIMENTAL SECTION
| Molecular docking studies
MP: MEL-TEMP; UV: U-2001 spectrophotometer; FT-IR: JASCO
FT/IR-410; 1H and 13C NMR: Bruker AV 400 or Bruker AV-III
600 NMR spectrometer (CDCl3, δH 7.24 and δC 77.0 ppm; methanol-
d4, δH 3.30 and δC 49.0 ppm; pyridine-d5, δH 8.71 and δC 149.9 ppm;
J in Hz); ESIMS: Bruker Esquire 200 ESI-ion trap spectrometer; HR-
ESI-MS: Bruker micrOTOF orthogonal ESI-TOF mass spectrometer.
2.1
2.1.1
|
Data collection and preparation
The crystal structure of xanthine oxidase (XO) from bovine milk
source was retrieved from the Protein Data Bank (PDB ID: 1FIQ).
Xanthine oxidase is a homodimeric enzyme and each monomer con-
sists of four components, including a molybdopterin cofactor (Mo-pt)
center, a pair of spinach ferredoxin-like clusters [2Fe–2S], and one fla-
vin adenine dinucleotide (FAD) cofactor (Enroth et al., 2000). Since
the other three components are far from the active site, only the
active site component, Mo-pt, was kept for molecular docking study.
The active site of XO was constructed using the co-crystal structure
of salicylate−xanthine oxidase complex as a reference.
2.2.1
|
Preparation of N-(3-[3,4-dimethoxyphenyl]
propyl)-6,7-dimethoxy-2-naphthamide (28), a general
procedure for preparing 26–30
The reaction mixture of the carboxylic acid 4 (250.3 mg, 1.08 mmol),
the amine HCl salt 15 (284.2 mg, 1.23 mmol), triethylamine (166 μL),
EDC (240.6 mg), and HOBt (164.0 mg) in CH2Cl2 − DMF (11 mL:
1 mL) was stirred at rt for 16 hr, then quenched with H2O (120 mL).
The resultant suspension was extracted with CHCl3 (3 × 120 mL). The
combined CHCl3 layers were dried over anhy. Na2SO4 and concen-
trated under reduced pressure to give a residue, which was redis-
solved in CHCl3 (60 mL) and the solution was washed sequentially
with 5% citric acid (aq) (3 × 40 mL), sat. NaHCO3 (aq) (3 × 60 mL), and
brine (2 × 20 mL), dried over anhy. Na2SO4, and concentrated under
reduced pressure to give a residue, which upon purification over a sil-
ica gel column (230–400 mesh, CHCl3) yielded 28 (357.0 mg, 81%) as
white amorphous solid.
The 2D structures of selected ligands were constructed using
ChemBioDraw and converted into 3D structure using ChemBio3D
software where energy minimization and molecular dynamics were
undertaken.
2.1.2
|
Docking simulation
All simulations were performed using Molecular Operating Environ-
ment (MOE), 2010.10 (Chemical Computing Group Inc., 1010
Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7,
2010). The partial atomic charges were assigned to XO using
MMFF94x forcefield in MOE. The triangle matcher algorithm of the
MOE software packages was selected to dock the identified hit com-
pounds into the Mo-pt active site. Docking calculations were carried
out using standard default variables. The RMSD was computed in
terms of all the atoms in a protein backbone and the value was less
than 0.6 Å, which is indicative of considerable structural similarity.
UV (MeCN): λmax (log ε) 332 (3.23), 282 (4.03), 244 nm (4.76).
IR (KBr): 3369, 3058, 2999, 2936, 2833, 1626, 1538, 1511, 1464,
1417, 1258, 1161, 1140, 1028, 1007, 858, 764, 735 cm−1
1H NMR (400 MHz, CDCl3):
7.96 (br s, 1H), 7.65 (d,
.
δ
J = 8.5 Hz, 1H), 7.57 (dd, J = 8.5, 1.7 Hz, 1H), 7.11 and 7.08 (each
s, 2H), 6.77–6.71 (m, 3H), 6.32 (br t, 1H, D2O exchangeable), 3.97
(s, 3H), 3.95 (s, 3H), 3.83 (s, 3H), 3.79 (s, 3H), 3.52 (q, J = 6.7 Hz,
2H), 2.67 (t, J = 7.4 Hz, 2H), 1.95 (quin, J = 7.0 Hz, 2H).

4
HO ET AL.
13C NMR (100 MHz, CDCl3): δ 167.6 (C), 150.7 (C), 149.9 (C),
UV (MeCN): λmax (log ε) 334 (3.23), 282 (4.08), 244 nm (4.82).
148.9 (C), 147.3 (C), 134.1 (C), 130.8 (C), 130.1 (C), 128.3 (C), 126.5
(CH), 125.6 (CH), 121.8 (CH), 120.2 (CH), 111.6 (CH), 111.3 (CH),
106.9 (CH), 105.9 (CH), 55.8 (CH3), 55.8 (CH3), 55.8 (CH3), 39.9 (CH2),
33.2 (CH2), 31.2 (CH2).
IR (KBr): 3390, 3000, 2934, 2855, 2834, 1627, 1537, 1510,
1463, 1438, 1417, 1257, 1247, 1161, 1140, 1028, 1007,
860, 772 cm−1
.
1H NMR (400 MHz, CDCl3): δ 8.11 (br s, 1H), 7.69 (d, J = 8.5 Hz,
1H), 7.63 (dd, J = 8.5, 1.7 Hz, 1H), 7.14 and 7.11 (each s, 2H), 6.77 (d,
J = 8.2 Hz, 1H), 6.71 (dd, J = 8.2, 1.8 Hz, 1H), 6.69 (d, J = 1.8 Hz, 1H),
6.24 (br t, 1H, D2O exchangeable), 3.99 (s, 3H), 3.97 (s, 3H), 3.84 (s,
3H), 3.83 (s, 3H), 3.50 (q, J = 6.6 Hz, 2H), 2.60 (t, J = 7.2 Hz, 2H),
1.73–1.66 (m, 4H).
13C NMR (100 MHz, CDCl3): δ 167.7 (C), 150.8 (C), 150.0 (C),
148.8 (C), 147.1 (C), 134.7 (C), 130.8 (C), 130.2 (C), 128.4 (C), 126.6
(CH), 125.8 (CH), 121.8 (CH), 120.1 (CH), 111.6 (CH), 111.1 (CH),
106.9 (CH), 106.0 (CH), 55.9 (CH3), 55.9 (CH3), 55.9 (CH3), 55.8 (CH3),
39.9 (CH2), 35.1 (CH2), 29.2 (CH2), 28.9 (CH2).
MS (ESI): m/z 431.9 [M+Na]+, 409.9 [M+H]+.
HRMS (ESI) m/z: [M+Na]+ calcd. for C24H27NNaO5 432.1781,
found 432.1784.
N-(3,4-Dimethoxybenzyl)-6,7-dimethoxy-2-naphthamide (26)
Following the general procedure as described above, reacting
4 (47.5 mg, 0.20 mmol) with 7 (46.8 mg, 0.23 mmol) yielded 26
(67.6 mg, 87%) as yellowish amorphous solid.
UV (MeCN): λmax (log ε) 334 (3.20), 284 (4.05), 244 nm (4.74).
IR (KBr): 3456, 1639, 1510, 1488, 1463, 1418, 1259, 1161, 1139,
750 cm−1
.
MS (ESI): m/z 445.9 [M+Na]+, 423.9 [M+H]+.
1H NMR (400 MHz, CDCl3): δ 8.15 (br s, 1H), 7.67 (s, 2H), 7.11
and 7.09 (each s, 2H), 6.91–6.89 (m, 2H), 6.81 (d, J = 8.6 Hz, 1H), 6.58
(br t, 1H, D2O exchangeable), 4.59 (d, J = 5.3 Hz, 2H), 3.98 (s, 3H),
3.94 (s, 3H), 3.84 (s, 3H), 3.84 (s, 3H).
13C NMR (100 MHz, CDCl3): δ 167.5 (C), 150.8 (C), 150.0 (C),
149.1 (C), 148.4 (C), 130.9 (C), 130.8 (C), 129.8 (C), 128.3 (C), 126.6
(CH), 125.9 (CH), 121.9 (CH), 120.2 (CH), 111.3 (CH), 111.1 (CH),
106.9 (CH), 105.9 (CH), 55.8 (CH3), 55.8 (CH3), 44.0 (CH2).
MS (ESI): m/z 403.9 [M+Na]+, 381.9 [M+H]+.
HRMS (ESI) m/z: [M+Na]+ calcd. for C25H29NNaO5 446.1938,
found 446.1930.
N-(5-[3,4-Dimethoxyphenyl]pentyl)-6,7-dimethoxy-
2-naphthamide (30)
Following the general procedure as described above, reacting
4 (18.3 mg, 78 μmol) with 25 (22.4 mg, 86 μmol) yielded 30 (12.8 mg,
37%) as viscous liquid.
UV (MeCN): λmax (log ε) 332 (3.15), 282 (3.93), 242 nm (4.69).
IR (KBr): 3380, 3000, 2933, 2854, 1627, 1540, 1508, 1463, 1417,
HRMS (ESI) m/z: [M+Na]+ calcd. for C22H23NNaO5 404.1468,
found 404.1464.
1258, 1248, 1161, 1140, 1028, 1007, 857, 771 cm−1
.
1H NMR (400 MHz, CDCl3): δ 8.12 (br s, 1H), 7.70 (d, J = 8.4 Hz,
1H), 7.62 (d, J = 8.4 Hz, 1H), 7.15 and 7.11 (each s, 2H), 6.75 (d,
J = 8.6 Hz, 1H), 6.70–6.68 (m, 2H), 6.22 (br. s, 1H, D2O exchangeable),
4.00 (s, 3H), 3.98 (s, 3H), 3.84 (s, 3H), 3.81 (s, 3H), 3.48 (m, 2H), 2.56
(t, J = 7.7 Hz, 2H), 1.67–1.63 (m, 4H), 1.47–1.39 (m, 2H).
13C NMR (100 MHz, CDCl3): δ 167.7 (C), 150.8 (C), 150.1 (C),
148.7 (C), 147.0 (C), 135.0 (C), 130.8 (C), 130.2 (C), 128.4 (C), 126.6
(CH), 125.8 (CH), 121.8 (CH), 120.1 (CH), 111.7 (CH), 111.1 (CH),
107.0 (CH), 106.0 (CH), 55.9 (CH3), 55.9 (CH3), 55.8 (CH3), 40.1 (CH2),
35.3 (CH2), 31.2, (CH2), 29.6 (CH2), 26.5 (CH2).
N-(3,4-Dimethoxyphenethyl)-6,7-dimethoxy-2-naphthamide (27)
Following the general procedure as described above, reacting
4 (51.4 mg, 0.2 mmol) with 8 (54.3 mg, 42 μL; 0.3 mmol) yielded 27
(78.4 mg, 90%) as white amorphous solid.
UV (MeCN): λmax (log ε) 334 (3.14), 282 (4.00), 244 nm (4.72).
IR (KBr): 3561, 2959, 2835, 1639, 1509, 1489, 1464, 1418, 1259,
1160, 1141, 1028, 1006, 862, 764, 752 cm−1
.
1H NMR (400 MHz, CDCl3): δ 8.08 (br s, 1H), 7.68 (d, J = 8.5 Hz,
1H), 7.57 (dd, J = 8.5, 1.7 Hz, 1H), 7.12 and 7.10 (each s, 2H), 6.82 (d,
J = 8.0 Hz, 1H), 6.78 (dd, J = 8.0, 1.6 Hz, 1H), 6.76 (d, J = 1.6 Hz, 1H),
6.26 (br t, 1H, D2O exchangeable), 3.99 (s, 3H), 3.98 (s, 3H), 3.85 (s,
3H), 3.82 (s, 3H), 3.72 (q, J = 6.7 Hz, 2H), 2.89 (t, J = 6.8 Hz, 2H).
13C NMR (100 MHz, CDCl3): δ 167.7 (C), 150.8 (C), 150.1 (C),
149.0 (C), 147.7 (C), 131.5 (C), 130.9 (C), 130.0 (C), 128.4 (C), 126.6
(CH), 125.9 (CH), 121.7 (CH), 120.7 (CH), 111.9 (CH), 111.4 (CH),
106.9 (CH), 106.0 (CH), 55.9 (CH3), 55.8 (CH3), 41.3 (CH2), 35.3 (CH2).
MS (ESI): m/z 417.9 [M+Na]+, 395.9 [M+H]+ .
MS (ESI): m/z 459.9 [M+Na]+, 437.9 [M+H]+.
HRMS (ESI) m/z: [M+Na]+ calcd. for C26H31NNaO5 460.2094,
found 460.2089.
2.2.2
|
Preparation of N-(3-[3,4-Dihydroxyphenyl]
propyl)-6,7-dihydroxy-2-naphthamide (Xc), a general
procedure for preparing N-arylalkanyl 2-naphthamides
(Xa−Xe)
HRMS (ESI) m/z: [M+Na]+ calcd. for C23H25NNaO5 418.1625,
found 418.1622.
To a solution of 28 (151.7 mg) in CH2Cl2 (16 mL) was added a solution
of boron tribromide in CH2Cl2 (1.0 M, 3 mL) dropwise at 0 ꢁC. The
reaction mixture was stirred at 0 ꢁC for 1 hr, then quenched by adding
MeOH (20 mL) dropwise and stirred for 1 hr, concentrated under
reduced pressure to give a residue, which was added H2O (5 mL) and
the insoluble was collected by filtration. The filtrate was passed
N-(4-[3,4-Dimethoxyphenyl]butyl)-6,7-dimethoxy-
2-naphthamide (29)
Following the general procedure as described above, reacting
4 (17.0 mg, 73 μmol) with 19 (20.0 mg, 78 μmol) yielded 29 (23.5 mg,
85%) as viscous liquid.

HO ET AL.
5
through an Amberlite XAD-II column (20–60 mesh, pore radius 90 Å),
eluted by H2O and MeOH in sequence. The insoluble part and the
residue of the MeOH eluate after evaporation were combined and
then separated over a Sephadex LH-20 column (57 cm × 1.5 cm,
MeOH-CHCl3 7:3) to give Xc (129.7 mg, 99%) as colorless amorphous
solid.
13C NMR (100 MHz, CD3OD): δ 170.8 (C), 149.7 (C), 148.6 (C),
146.3 (C), 144.8 (C), 132.6 (C), 132.3 (C), 130.2 (C), 129.9 (C), 127.0
(CH), 126.8 (CH), 122.2 (CH), 121.1 (CH), 117.0 (CH), 116.4 (CH),
111.4 (CH), 110.3 (CH), 43.1 (CH2), 36.1 (CH2).
MS (ESI): m/z 337.8 [M−H]−.
HRMS (ESI) m/z: [M−H]− calcd. for C19H16NO5 338.1034, found
UV (MeOH): λmax (log ε) 288.0 (4.03), 248.0 nm (4.74).
338.1030.
IR (KBr): 3402, 1620, 1602, 1525, 1431, 1362, 1319, 1251, 1202,
1116, 864, 764, 639 cm−1
.
N-(4-[3,4-Dihydroxyphenyl]butyl)-6,7-dihydroxy-2-naphthamide (Xd)
Following the general procedure as described above, Xd (7.5 mg,
60%) was yielded by reacting 29 (14.5 mg) with BBr3 in CH2Cl2
(1.0 M, 0.3 mL) as amorphous solid.
1H NMR (400 MHz, CD3OD): δ 8.02 (br s, 1H), 7.60 (d, J = 8.6 Hz,
1H), 7.57 (dd, J = 8.6, 1.6 Hz, 1H), 7.20 and 7.13 (each s, 2H), 6.67 (d,
J = 8.1 Hz, 1H), 6.66 (d, J = 2.0 Hz, 1H), 6.54 (dd, J = 8.1, 2.0 Hz, 1H),
3.40 (t, J = 7.1 Hz, 2H), 2.56 (t, J = 7.4 Hz, 2H), 1.89 (quintet,
J = 7.5 Hz, 2H).
UV (MeOH): λmax (log ε) 288.0 (4.00), 248.0 nm (4.70).
IR (KBr): 3363, 2937, 1618, 1602, 1523, 1430, 1359, 1321, 1250,
13C NMR (100 MHz, CD3OD): δ 170.8 (C), 149.6 (C), 148.5 (C),
146.2 (C), 144.3 (C), 134.7 (C), 132.6 (C), 130.2 (C), 129.8 (C), 127.0
(CH), 126.8 (CH), 122.2 (CH), 120.7 (CH), 116.5 (CH), 116.3 (CH),
111.5 (CH), 110.3 (CH), 40.9 (CH2), 33.8 (CH2), 32.5 (CH2).
MS (ESI): m/z 351.7 [M−H]−.
1202, 1116, 863, 758, 611 cm−1
.
1H NMR (400 MHz, CD3OD): δ 8.05 (br s, 1H), 7.59–7.58 (m, 2H),
7.19 and 7.13 (each s, 2H), 6.65 (d, J = 8.0 Hz, 1H), 6.62 (d, J = 2.0 Hz,
1H), 6.50 (dd, J = 8.0, 2.0 Hz, 1H), 3.40 (t, J = 6.6 Hz, 2H), 2.51 (t,
J = 6.8 Hz, 2H), 1.64 (m, 4H).
HRMS (ESI) m/z: [M−H]− calcd. for C20H18NO5 352.1190, found
13C NMR (100 MHz, CD3OD): δ 170.8 (C), 149.6 (C), 148.6 (C),
146.1 (C), 144.2 (C), 135.3 (C), 132.6 (C), 130.2 (C), 129.9 (C), 127.0
(CH), 126.8 (CH), 122.2 (CH), 120.7 (CH), 116.5 (CH), 116.2 (CH),
111.4 (CH), 110.3 (CH), 40.9 (CH2), 35.9 (CH2), 30.3 (CH2), 30.1 (CH2).
MS (ESI): m/z 365.8 [M−H]−.
352.1204.
N-(3,4-Dihydroxybenzyl)-6,7-dihydroxy-2-naphthamide (Xa)
Following the general procedure as described above, Xa (29.7 mg,
98%) was obtained by reacting 26 (35.6 mg) with BBr3 in CH2Cl2
(1.0 M, 0.8 mL) as white amorphous solid.
HRMS (ESI) m/z: [M−H]− calcd. for C21H20NO5 366.1347, found
366.1342.
UV (MeOH): λmax (log ε) 288.0 (4.09), 248.0 nm (4.78).
IR (KBr): 3485, 3413, 3341, 3293, 3069, 1593, 1578, 1519, 1491,
1428, 1366, 1288, 1265, 1206, 1156, 1115, 996, 903, 869, 825,
N-(5-[3,4-Dihydroxyphenyl]pentyl)-6,7-dihydroxy-
2-naphthamide (Xe)
766, 638, 597, 515, 476 cm−1
.
Following the general procedure as described above, Xe (3.0 mg, 51%)
was obtained by reacting 30 (6.8 mg) with BBr3 in CH2Cl2 (1.0 M,
0.15 mL) as amorphous solid.
1H NMR (400 MHz, CD3OD): δ 8.10 (d, J = 0.9 Hz, 1H), 7.64 (dd,
J = 8.5, 1.7 Hz, 1H), 7.60 (d, J = 8.5 Hz, 1H), 7.19 and 7.13 (each s,
2H), 6.82 (d, J = 1.8 Hz, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.69 (dd, J = 8.1,
1.8 Hz, 1H), 4.45 (s, 2H).
UV (MeOH): λmax (log ε) 290.0 (3.85), 248.0 nm (4.47).
IR (KBr): 3375, 2934, 2857, 1618, 1524, 1430, 1363, 1317, 1251,
13C NMR (100 MHz, CD3OD): δ 170.6 (C), 149.7 (C), 148.6 (C),
146.4 (C), 145.5 (C), 132.6 (C), 131.9 (C), 130.1 (C), 129.9 (C), 127.1
(CH), 127.0 (CH), 122.3 (CH), 120.1 (CH), 116.3 (CH), 115.8 (CH),
111.5 (CH), 110.3 (CH), 44.2 (CH2).
1202, 1116, 1058, 1016, 864, 766, 639 cm−1
.
1H NMR (400 MHz, CD3OD): δ 8.04 (br s, 1H), 7.59–7.58 (m, 2H),
7.19 and 7.13 (each s, 2H), 6.63 (d, J = 8.0 Hz, 1H), 6.60 (d, J = 2.0 Hz,
1H), 6.48 (dd, J = 8.0, 2.0 Hz, 1H), 3.38 (t, J = 7.2 Hz, 2H), 2.48 (t,
J = 7.4 Hz, 2H), 1.69–1.58 (m, 4H), 1.44–1.37 (m, 2H).
13C NMR (100 MHz, CD3OD): δ 170.9 (C), 149.6 (C), 148.6 (C), 146.0
(C), 144.1 (C), 135.5 (C), 132.6 (C), 130.3 (C), 129.9 (C), 127.1 (CH), 126.8
(CH), 122.2 (CH), 120.7 (CH), 116.5 (CH), 116.2 (CH), 111.5 (CH), 110.3
(CH), 41.0 (CH2), 36.1 (CH2), 32.6 (CH2), 30.4 (CH2), 27.6 (CH2).
MS (ESI): m/z 379.8 [M−H]−.
MS (ESI): m/z 323.8 [M−H]−.
HRMS (ESI) m/z: [M−H]− calcd. for C18H14NO5 324.0877, found
324.0869.
N-(3,4-Dihydroxyphenethyl)-6,7-dihydroxy-2-naphthamide (Xb)
Following the general procedure as described above, Xb (13.9 mg,
84%) was obtained by reacting 27 (19.2 mg) with BBr3 in CH2Cl2
(1.0 M, 0.4 mL) as amorphous solid.
HRMS (ESI) m/z: [M−H]− calcd. for C22H22NO5 380.1503, found
380.1509.
UV (MeOH): λmax (log ε) 288.0 (3.99), 248.0 nm (4.67).
IR (KBr): 3332, 1620, 1526, 1455, 1422, 1360, 1320, 1249, 1198,
1117, 1058, 861, 806, 762, 611 cm−1
.
2.2.3
|
N-(3-[3,4-Diacetoxyphenyl]propyl)-
1H NMR (400 MHz, CD3OD): δ 8.03 (br s, 1H), 7.60 (d, J = 8.5 Hz,
1H), 7.57 (dd, J = 8.5, 1.6 Hz, 1H), 7.18 and 7.13 (each s, 2H), 6.70 (d,
J = 2.0 Hz, 1H), 6.69 (d, J = 8.0 Hz, 1H), 6.58 (dd, J = 8.0, 2.0 Hz, 1H),
3.54 (t, J = 7.7 Hz, 2H), 2.77 (t, J = 7.7 Hz, 2H).
6,7-diacetoxy-2-naphthamide (31)
The solution of Xc (99.5 mg) in pyridine (3 mL) and acetic anhydride
(1.5 mL) was stirred at rt for 16 hr, quenched with abs. EtOH (3 mL),

6
HO ET AL.
concentrated under reduced pressure to give a residue, which was
purified over a silica gel column (230–400 mesh, CHCl3) to give 31
(143.2 mg, 98%) as colorless amorphous solid.
at 20ꢁC with a regular light/dark cycle, a standard fodder and water
ad libitum during the study. The study protocol was approved by the
Institutional Animal Care and Use Committee (IACUC), NTU
(Approved no. IACUC No.20130383. MOST 103-2320-B-
002-011-MY3).
UV (MeCN): λmax (log ε) 280 (3.87), 232 nm (4.79).
IR (KBr): 3424, 2936, 1768, 1643, 1540, 1505, 1471, 1427, 1371,
1210, 1110, 1012, 917, 764, 748 cm−1
.
1H NMR (600 MHz, CDCl3): δ 8.12 (br s, 1H), 7.80 (d, J = 8.6 Hz,
1H), 7.73 (dd, J = 8.6, 1.6 Hz, 1H), 7.71 and 7.66 (each s, 2H), 7.08
(dd, J = 8.2, 1.6 Hz, 1H), 7.07 (d, J = 8.2 Hz, 1H), 7.03 (d, J = 1.6 Hz,
1H), 6.25 (br. t, 1H, D2O exchangeable), 3.53 (q, J = 6.8 Hz, 2H), 2.72
(t, J = 7.5 Hz, 2H), 2.33 and 2.33 (each s, 6H), 2.26 and 2.25 (each s,
6H), 1.99 (quin, J = 7.1 Hz, 2H).
13C NMR (150 MHz, CDCl3): δ 168.4 (C), 168.4 (C), 168.3 (C),
168.3 (C), 167.1 (C), 142.3 (C), 141.9 (C), 141.6 (C), 140.3 (C), 140.2
(C), 132.9 (C), 132.3 (C), 130.8 (C), 128.0 (CH), 126.7 (CH), 126.5 (CH),
124.1 (CH), 123.3 (CH), 123.3 (CH), 121.9 (CH), 120.8 (CH), 39.8
(CH2), 32.9 (CH2), 30.8 (CH2), 20.7 (CH3), 20.7 (CH3), 20.6 (CH3),
20.6 (CH3).
2.4.2
|
Potassium oxonate-induced hyperuricemic
mice model (Kong, Yang, Ge, Wang, & Guo, 2004; Wu,
Ruan, Zhang, Wang, & Zhang, 2014)
Blood was collected from the mice orbital sinus 1 day before experi-
ments. Fodder, but not water, was withdrawn 1.5 hr prior to drug
administration. In order to induce hyperuricemic status, potassium
oxonate (250 mg/kg) was intraperitoneally injected 1 hr before admin-
istration of test compounds at 1st, 3rd, 5th, and 7th day. The test
compounds (28, 31) were suspended and allopurinol was dissolved in
15% Tween 80 (aq), then individually administered orally once per
day. Blood (0.1 mL per mouse) was collected from the mouse orbital
sinus 1 hr after final administration. All mice were sacrificed after the
final blood collection.
MS (ESI): m/z 543.8 [M+Na]+, 521.8 [M+H]+.
HRMS (ESI) m/z: [M+Na]+ calcd. for C28H27NNaO9 544.1578,
found 544.1581.
2.3
|
Xanthine oxidase inhibitory assay
2.4.3
|
Measurement of plasma uric acid
concentration
The assay method for the prepared target compounds against XO
activity was modified from a previous report (Khan, Ali, Gul, &
Choudhary, 2008). To the corresponding well in a 96-well plate was
added vehicle [10 μL, MeOH – H2O or DMSO – H2O 1:9 (vol/vol)] or
sample in vehicle [10 μL, MeOH – H2O (vol/vol) for netpetoidin B (II)
and Xa-e; DMSO – H2O 1:9 (vol/vol) for 28 and 31], and 2 mM xan-
thine (Sigma) solution (60 μL). The reaction started when xanthine oxi-
dase (30 μL, 0.2 U/mL) (EC 1.2.3.2, bovine milk, Sigma) was added.
The produced uric acid was determined by measuring the absorbance
at 290 nm on a Microplate spectrophotometer SPECTRAmax® PLUS
(Molecular Devices) at 2 min intervals. The inhibitory percentage (%)
of the test sample against XO was calculated by the following equa-
tion: inhibition (%) = [1 − (Asample/Acontrol)] × 100, where Acontrol and
Asample stand for the recorded absorbance value (A) of the blank and
the test sample. The IC50 value was determined by the dose–response
curve of five concentrations (0.5, 1, 5, 10, and 100 μg/mL) of each test
sample in triplicate. Allopurinol (Synmosa Biopharma Corporation)
whose IC50 values were found to be 22.1 0.7 μM was used as a pos-
itive control.
The uric acid concentration in mouse serum was measured by HPLC
with the conditions modified from a previous report (Ingebretsen,
Borgen, & Farstad, 1982). Each collected blood sample was allowed to
clot at room temperature, centrifuged at 7000 × g for 10 min. The
supernatant collected was treated with an equivalent volume of
MeCN, votexed for 10 s, centrifuged at 10,000 × g for 10 min. The
supernatant was filtered through 0.45 μm filter membrane. The filtrate
was analyzed on an Agilent (Waldbronn, Germany) series 1100 HPLC,
equipped with an analytical RP-18 column (Phenomenex Prodigy
ODS-3, 250 × 4.6 mm, 5 μm) connected to a UV detector (Bruker,
Rheinstetten, Germany). The delivery system [50 mM KH2PO4 (A),
MeCN (B)] was as follows: 5% B/A for 10 min, 5% to 50% B/A in
10 min, 50% to 5% B/A in 10 min, all linear gradient; injection volume,
10 μL; flow rate, 0.5 mL/min; detection at 290 nm.
3
|
RESULTS AND DISCUSSION
3.1
|
Design of N-arylalkanyl-2-naphthamides as
xanthine oxidase inhibitors
2.4
|
Evaluation of the in vivo efficacy
| Animal
Structure–activity relationship of the active neolignans suggests that
the presence of cinnamic acid moiety coupling with a cis-styryl moiety
is an important structural feature for the inhibitory potency. The sim-
plest approach to design target compounds derived from the styryl
caffeate II was to replace the caffeic acid and styryl alcohol moieties
by naphthoic acid and phenethylamine, respectively. Condensation of
2.4.1
Male ICR mice (4 weeks old) were purchased from the BioLASCO Tai-
wan, Co., Ltd (Taipei, Taiwan). Animals were allowed to adapt to the
environment at least 1 week before experiments. They were housed

HO ET AL.
7
TABLE 1 In vitro XO inhibition assay and computer assisted docking results of N-arylalkanyl 2-naphthamides, netpetoidin B (II), and
allopurinol
Number of ligand-receptor interaction
H-bond
n–π
π–π
Ionic
Compd
ΔGcalc kcal/mol
−16.11
Log p
2.35
2.63
3.04
3.46
3.88
4.10
2.95
2.78
0.32
IC50a (μM)
25.6 0.4
13.6 1.0
13.1 0.4
12.5 1.9
18.2 0.7
Tb
3
3
4
4
2
2
1
4
3
Ac
1
2
2
2
1
2
1
2
3
T
3
4
3
3
2
1
2
1
1
A
3
1
3
3
1
0
1
1
1
T
1
1
1
1
1
1
0
1
1
A
1
1
1
1
1
1
0
1
1
T
0
0
0
0
0
0
0
0
0
A
0
0
0
0
0
0
0
0
0
Xa
Xb
−16.99
Xc
−17.66
Xd
−17.13
Xe
−16.11
d
28
−15.86
—
d
31
−10.59
—
II
−19.11
7.4 0.2
allopurinol
−11.18
22.1 0.7
aIC50: the concentration of inhibitor required to produce 50% inhibition of XO (mean SD; n = 3).
bTotal interaction number.
cInteraction number at the active site.
dVery poor solubility.
SCHEME 1 Preparation of 6,7-dimehtoxy-2-naphthoic acid (4): (a) Me2SO4, K2CO3, Me2CO, reflux; (b) AcCl/AlCl3, DCM, 0ꢁC; (c) (i) Br2,
NaOH (aq); (ii) 6 M HCl
the carboxylic acids and the amines to form N-arylalkanyl
2-naphthamides (Xa−e) (Figure 2), which should be more bio-stable
than the ester II. In silico molecular docking of Xa−e on XO template
showed the docking scores around −17 Kcal/mol, of which Xb-c
(n = 2–4; Figure 2) showed better binding affinity (Table 1).
acid (4) with 3,4-dimethoxyphenyl-alkylamines (7, 8, 15, 19, 25),
followed by per-O-demethylation.
The carboxylic acid 4 was prepared starting from 2,3-dihydroxy
naphthalene. Per-O-methylation of 2,3-dihydroxynaphthalene (1) with
dimethyl sulfate gave di-O-methylated product (2) (91% yield).
Friedel-Crafts acylation of 2 with acetyl chloride/ AlCl3 yielded
2-acetylated (3) (53% yield) and 1-acetylated (3a) (42% yield)
products, both being separated by silica gel CC. Reaction of 3 with
bromine in strong alkali, followed by acid workup, yielded
6,7-dimehtoxy-2-naphthoic acid (4) (82% yield) (Goksu, Kazaz,
Sutbeyaz, & Secen, 2003) (Scheme 1).
3.2
|
Synthesis of N-arylalkanyl-2-naphthamides
N-Arylalkanyl 2-naphthamides Xa-e were prepared as follows. The
target Xa-e were prepared by coupling of 6,7-dimethoxy-2-naphthoic

8
HO ET AL.
SCHEME 2 Preparation of 3,4-dimethoxyphenylalkanamines (7, 15, 19, 25): (a) NH2OHꢂHCl, EtOH, rt; (b) H2 (1 atm), 10% Pd/C, EtOH, conc.
HCl, rt; (c) EtOH, H2SO4/reflux; (d) H2 (1 atm), 10% Pd/C/EA, rt; (e) LiALH4/THF, rt; (f) PCC, DCM, 0ꢁC; (g) H2 (1 atm), 10% Pd/C/MeOH, conc.
HCl, rt; (h) CH2(CO2H)2, py.-piperidine, reflux; (i) (i) NaN3, H2SO4, CHCl3; (ii) NaOH (aq); (j) 50% H2SO4 (aq)
3,4-Dimethoxybenzylamine hydrochloride (7) was prepared by
reacting veratraldehyde (5) with hydroxylamine hydrochloride in the
presence of sodium acetate (Soni et al., 2013) to yield oxime 6 (66%),
followed by catalytic hydrogenation under acidic conditions
(10% Pd/C-HCl) (97% yield) (Tsou et al., 2009). While
3-(3,4-dimethoxyphenyl)propylamine hydrochloride (15) was prepared
starting from 3,4-dimethoxycinnamic acid (9). Esterification of 9 with
ethanol/ H2SO4 (cat.) yielded the ethyl ester 10 (95% yield), which
gave the alcohol 12 upon catalytic hydrogenation (10% Pd/C) (quant.
yield) and subsequent LAH reduction (99% yield) (Lal, Ghosh, &

HO ET AL.
9
SCHEME 3 Preparation of N-arylalkanyl-2-naphthamides (Xa-e)
Salomon, 1987). PCC oxidation (Pedrosa, Andres, & Iglesias, 2001) of
12 yielded the aldehyde 13 (66% yield), δCHO 9.80, which upon reac-
tion with hydroxylamine hydrochloride gave a mixture of Z/E oximes
(14) (89% yield) with a 4:1 ratio according to the integration of the
olefinic proton signal (δ 6.65, 1H, t, J = 5.2 Hz; δ 7.35, 0.25H, t) in the
1H NMR spectrum. Catalytic hydrogenation of the Z-oxime product
(14a), obtained by recrystallization from isopropanol, under the same
condition as described for the preparation of 7, gave
3-(3,4-dimethoxyphenyl)propylamineꢂHCl (15) (86% yield) (Scheme 2).
4-(3,4-Dimethoxyphenyl)butylamine (19) was prepared starting
from the aryl propanal 13. Reaction of 13 with malonic acid in the
presence of pyridine and piperidine (Knoevenagel-Doebner reaction)
(Duarte et al., 2004) gave a mixture of β,γ- and α,β-unsaturated pen-
tenoic acids (16a/b) with a ratio 3:1, determined by integration of the
olefinic proton signals in the 1H NMR spectrum, δ 7.07 (dt, J = 15.6,
6.7 Hz, H-3) and 5.81 (br. d, J = 15.6 Hz, H-2) (16b); δ 5.75–5.56 (m,
2H, H-3,4) (16a). Catalytic hydrogenation of 16a/b afforded the pen-
tanoic acid 17 (96% yield over two-step reaction from 13), which
through Schmidt reaction (sodium azide/ sulfuric acid) (Werner &
Casanova, Werner & Casanova Jr., 1967) yielded the sulfonated deriv-
ative (18) of the desired 4-(3,4-dimethoxyphenyl)butylamine (19)
(47% yield). Structure of 18 was elucidated based on analysis of the
1H NMR spectrum, showing two singlets for the para-aryl protons (δ
7.49 & 6.84) and the HRMS (ESI) data, showing [M+H]+ at m/z
afforded the amides 26–29 (81–90% yield), and 30 (37% yield), which
were per-O-demethylated by boron tribromide to give the target N-
arylalkanyl 2-naphthamides Xa-e (51–99% yield). Acetylation of Xc with
acetic anhydride in pyridine yielded the peracetylated 31 (Scheme 3).
3.3
|
In vitro anti-xanthine oxidase activity of
target compounds and correlation with molecular
docking
The inhibitory activity against xanthine oxidase (XO) of the prepared
N-arylalkanyl 2-naphthamides (Xa-e) was assayed (Khan et al., 2008)
and the result was shown in Table 1. Among these, Xb-d showed bet-
ter inhibitory activity against XO (IC50 ꢀ 13 μM), comparable to the
positive control allopurinol (IC50 22.1 μM) but weaker than II (IC50
7.4 μM). Xa and Xe, however, showed weaker inhibitory activity. The
in vitro assay result indicated that the activity of N-arylalkanyl
2-naphthamides increased apparently as the length of the alkyl-linker
extends from n = 1 to n = 2, and roughly the same from n = 2 to n = 4,
but decreased as n = 5. This bioassay results correlated well with the
molecular docking scores as indicated above (Table 1), verifying the
applicability of the virtual screening on this established XO template.
312.0878 for the molecular formula
312.0876). Desulfonation of 18 with 50% sulfuric acid under reflux
(Ehrenfeld Puterbaugh, 1932) gave the sulfate salt of
C
12H19NNaO5S (calcd.
3.4
|
In silico evaluation of target compounds
against xanthine oxidase
&
4-(3,4-dimethoxyphenyl)butylamine (19) (91% yield). Esterification of
16a/b with ethanol/sulfuric acid (cat.) yielded the ethyl ester 20a/b
(95% yield), which under similar conditions for the preparation of the
amine 15 afforded 5-(3,4-dimethoxyphenyl)pentylamine ꢂ HCl (25)
(Scheme 2).
According to our previous research, the caffeoyl residue of
netpetoidin B (II) was inserted into the binding site of XO to form one
H-bond to Arg880, and hence the styrenyl catechol residue was ori-
ented to form one H-bond to Ser876. Additional two H-bonds were
formed between the styrenyl catechol residue to His875 and Glu879,
both of which were close to the binding site (Figure 3c)
(Ho et al., 2020). As compounds Xa-e were derived from II, further
investigation of the binding behavior of Xa-e to the XO active site
Coupling of the naphthoic acid 4 with various arylalkanamines (7, 8,
15, 19, and 25), catalyzed by 1-ethyl-3-(30-dimethylamino)carbodiimide
(EDC) and 1-hydroxybenzotriazole (HOB) (Takahashi & Miyazawa, 2011),

10
HO ET AL.
FIGURE 3 Molecular modeling of XO with compounds Xa,c&e and II in the presence of Mo cofactor. Docking results, depicted as two-
dimensional (Xa: a; xc: b; II: c; Xe: d) and three-dimensional conformation (Xa: a1; Xc: b1; II (magenta)/Xc (blue): c1; Xe: d1), are shown in the left
and right panel, respectively
was performed and the results were revealed in Figure 3 (Xa,c&e) and
Supporting Information (Xb&d), where the dihydroxynaphthyl moiety
was positioned in the molybdenum center.
The interactions of Xa-d to XO bore some similarities, including
one H-bond with Arg880, one π–π interaction with Phe914, and one
π–H interaction with Ala1079 in the 2,3-dihydroxynaphthyl moiety to

HO ET AL.
11
two catechol moieties in Xc might cause stability problem, peracetylation
was performed to give the acetylated prodrug Xc-Ac (31). The per-
methylated Xc (28) was also assayed in vivo to clarify the role of these
catechol moieties in anti-hyperuricemic efficacy. The change of serum uric
level after 7 days' drug treatment was shown in Figure 4. The serum uric
level was found 60% and 80% reduction for 31 treated group (0.25 mg/
dL) in comparison to the normal control group (0.58 mg/dL) and the
potassium oxonate induced hyperuricemia model group (1.24 mg/dL), the
latter comparison being statistically significant (p = .0014, <.01), The com-
pound 28 treated group showed that serum uric acid level was declined
to almost the level of normal group but without statistical significance.
However, both compounds were less effective than allopurinol. The dock-
ing score of 31 is much less than Xc and 28. However, in vivo study rev-
ealed that it was more potent than 28, indicating Xc-Ac (31) to be
hydrolyzed to the parent compound Xc to achieve the uric acid lowering
effect. The in vivo efficacy of 31, however, might not reflect that of Xc,
partly attributable to its poor solubility. The poor solubility of 28 also cau-
sed its IC50 value against XO unavailable. Nevertheless, molecular docking
result showed that the binding potential of 28 was lower than Xc, corre-
lating well with the in vivo result.
FIGURE 4 Effect of oral administration of 28 (30 mg/kg) and Xc-
Ac (31; 40 mg/kg) on serum uric acid after i.p. administration of
potassium oxonate (PO, 250 mg/kg) in male ICR mice (the y-axis
represent as serum uric acid change level after 7 days). Data are the
mean SEM for four animals; NC: normal control group, PO:
potassium oxonate induced hyperuricemia group; *p < .01 comparing
with PO; for PO, p = .059 comparing with NC
the binding site of XO, and one H-bond to the cofactor
Mo. Additional π–π interaction was observed from this moiety in Xc
and Xd to Phe1009. Xa with a shorter aliphatic linker (n = 1) let the
catechol moiety reach only the edge of salicylate binding pocket, thus
showing weaker binding affinity. Compounds with longer aliphatic
linkers like Xc (n = 3) and Xd (n = 4) made the catechol moiety pose
nearby the salicylate binding site pocket and showed one π-H interac-
tion with Leu1014 and one H-bond with Asn768, thus having better
binding affinity. An extra H-bond interaction of the catechol moiety in
Xc to Lys771, located nearby the binding site, stabilized the fitting
conformation, leading to slightly higher binding affinity score than Xd
(ΔG −17.66 vs. −17.13 Kcal/mol) (Table 1).
Kidney is the main route for uric acid excretion and therefore the
uricosuric agents are considered effective to lower serum uric acid
(SUA) level. However, to achieve such effect, their systemic absorp-
tion and transport to renal tubules are necessary and these might
cause systemic adverse effect including liver and kidney damage. The
approved XOIs also have similar disadvantage (Robinson
&
Dalbeth, 2015). Recently some evidences emphasize that intestinal
tract is an important organ for lowering SUA in rats. Highly expressed
XO on upper intestinal tract was contributed to the level of uric acid
in the intestine tissue and serum (Yun et al., 2017). Compound Xc in
this study blocks the uric acid synthesis at least in the intestine to
achieve SUA lowering effect. Whether the poor solubility of these
prepared target compounds might hamper their absorption by the
intestine, resulting less systemic side effects, requires further
investigation.
The interactions of Xe (n = 5) to XO, showing one H-bond to the
binding site and one H-bond to the cofactor Mo (Figure 3d/d1), were
quite different from those of Xa-d, thus showing weak bonding affin-
ity (Table 1).
The docking comparisons between II and Xc, the latter of which
showing the highest docking score among Xa-e to XO, indicated the
common interactions including two H-bonds with Arg880 and Ser876
in the binding site. In II, the cis-styryl catechol served as proton donor
to form H-bond with Ser876 (Figure 3c/c1), contributing ΔG − 3.4
Kcal/mol. Nevertheless, it is the naphthoyl amide carbon in Xc serving
as proton acceptor (Figure 3b/b1), contributing ΔG −1.3 Kcal/mol
only. This explained why II had better binding score to XO than Xc
(ΔG −19.32 vs. −17.66 Kcal/mol).
4
|
CONCLUSION
In the present study, an established XO molecular docking template
was applied to virtually screen N-arylalkanyl naphthamides designed
from 1,4-benzodioxane type neolignans. The scaffold of such target
compounds has been identified as new class of XO inhibitors form
both in vitro and in vivo assays. Compounds Xc and Xd showed most
potent XO inhibitory effects, comparable to allopurinol. The in vivo
efficacy of the Xc prodrug on hyperuricemia mice model showed the
reduction of blood uric acid level as compared with the vehicle con-
trol. N-Arylalkanyl 2-naphthamides could be thought as new lead for
further development of XO inhibitors.
3.5
|
Evaluation of the in vivo efficacy in
hyperuricemia mice model
Xc, one of the most active compounds against XO, was subjected to
evaluate anti-hyperuricemic efficacy in potassium oxonate induced
hyperuricemia mice model (Kong et al., 2004; Wu et al., 2014). As the
ACKNOWLEDGMENT
This work was supported by the Ministry of Science and Technology,
Taiwan under the grants MOST 103-2320-B-002-011-MY3.

12
HO ET AL.
Khan, W. N., Ali, I., Gul, R., & Choudhary, M. I. (2008). Xanthine oxidase
inhibiting compounds from Ranunculus repens. Chemistry of Natural
Compounds, 44, 95–97.
CONFLICT OF INTEREST
The authors declare no potential conflict of interests.
Kong, L. D., Yang, C., Ge, F., Wang, H. D., & Guo, Y. S. A. (2004). Chinese
herbal medicine Ermiao wan reduces serum uric acid level and inhibits
liver xanthine dehydrogenase and xanthine oxidase in mice. Journal of
Ethnopharmacology, 93, 325–−330.
AUTHOR CONTRIBUTIONS
Ching-Ting Lin was involved in synthesis and bioactivity analysis and
wrote the manuscript draft. Sheau Ling Ho was involved in XO tem-
plate production, target compound design, data collection and refine-
ment for structural studies, and wrote/edited manuscript. Shoei-
Sheng Lee co-designed target compounds, supervised the experi-
ments, conceived the project and integrated all data and completed
the manuscript.
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Data has been attached as Supporting Information and in the
manuscript.
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ORCID
Shoei-Sheng Lee
https://orcid.org/0000-0002-6628-2448
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of this article.
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synthesis, and molecular docking studies of N-(9,10-anthraquinone-
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(2018). Design, synthesis and biological evaluation of 1-hydroxy-
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How to cite this article: Ho SL, Lin C-T, Lee S-S. In silico
design and synthesis of N-arylalkanyl 2-naphthamides as a
new class of non-purine xanthine oxidase inhibitors. Drug Dev
Res. 2021;1–13. https://doi.org/10.1002/ddr.21782