Design, synthesis, and molecular docking studies of N-(9,10-anthraquinone-2-carbonyl)amino acid derivatives as xanthine oxidase inhibitors

Article abstract of DOI:10.1111/cbdd.13156

A series of N-(9,10-anthraquinone-2-carbonyl)amino acid derivatives (1a–j) was designed and synthesized as novel xanthine oxidase inhibitors. Among them, the L/D-phenylalanine derivatives (1d and 1i) and the L/D-tryptophan derivatives (1e and 1j) were effective with micromolar level potency. In particular, the L-phenylalanine derivative 1d (IC₅₀?=?3.0?μm) and the D-phenylalanine derivative 1i (IC₅₀?=?2.9?μm) presented the highest potency and were both more potent than the positive control allopurinol (IC₅₀?=?8.1?μm). Preliminary SAR analysis pointed that an aromatic amino acid fragment, for example, phenylalanine or tryptophan, was essential for the inhibition; the D-amino acid derivative presented equal or greater potency compared to its L-enantiomer; and the 9,10-anthraquinone moiety was welcome for the inhibition. Molecular simulations provided rational binding models for compounds 1d and 1i in the xanthine oxidase active pocket. As a result, compounds 1d and 1i could be promising lead compounds for further investigation.

Full text of DOI:10.1111/cbdd.13156

PROFESSOR FAN-HAO MENG (Orcid ID : 0000-0002-1331-2164)
Article type : Research Article
Design, synthesis and molecular docking studies of N-(9,10-anthraquinone-2-carbonyl)amino
acid derivatives as xanthine oxidase inhibitors
Ting-jian Zhang a, Song-ye Li a, Wei-yan Yuan, Yi Zhang, Fan-hao Meng*
School of Pharmacy, China Medical University, 77 Puhe Road, North New Area, Shenyang 110122,
China
* Corresponding author.
E-mail address: fhmeng@cmu.edu.cn (F-h. Meng).
a These two authors contributed equally to this work.
Abstract
A series of N-(9,10-anthraquinone-2-carbonyl)amino acid derivatives (1a-j) was designed and
synthesized as novel XO inhibitors. Among them, the L/D-phenylalanine derivatives (1d and 1i) and
the L/D-tryptophan derivatives (1e and 1j) were effective with micromolar level potency. In
particular, the L-phenylalanine derivative 1d (IC50 = 3.0 µM) and the D-phenylalanine derivative 1i
(IC50 = 2.9 µM) presented the highest potency, and were both more potent than the positive control
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doi: 10.1111/cbdd.13156
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allopurinol (IC50 = 8.1 µM). Preliminary SAR analysis pointed that an aromatic amino acid fragment,
e.g., phenylalanine or tryptophan, was essential for the inhibition; the D-amino acid derivative
presented equal or greater potency compared to its L-enantiomer; and the 9,10-anthraquinone moiety
was welcome for the inhibition. Molecular simulations provided rational binding models for
compounds 1d and 1i in the XO active pocket. As a result, compounds 1d and 1i could be promising
lead compounds for further investigation.
Keywords: Anthraquinone; Synthesis; Xanthine oxidase inhibitor; Hyperuricemia
Introduction
Xanthine oxidase (XO) is a key rate-limited enzyme in uric acid production in humans which
oxidizes hypoxanthine from nucleic acid metabolites into xanthine, and xanthine into uric acid.[1, 2]
The over-production of uric acid can lead to hyperuricemia, which is a major cause of gout. And
hyperuricemia and gout are associated with chronic diseases such as metabolic syndrome, renal and
cardiovascular disorders.[3, 4] Besides, reactive oxygen species (ROS) overproduction may be also
caused by the enhancement of XO expression and activity. This hypothesis is supported by clear
evidence showing that XO is involved in various forms of ischemic and vascular injuries,
inflammatory diseases, and chronic heart failure.[5] Therefore, inhibition of XO not only is one of the
most promising methods for the treatment for hyperuricemia and gout, but also has widely therapeutic
potential for series XO-associated diseases.
Allopurinol (Fig. 1) is the first XO inhibitor approved in the 1960s and has been the main therapy
for the management of gout and conditions associated with hyperuricemia for several decades.
However, in some cases, severe life-threatening side effects have been reported such as fulminant
hepatitis, renal failure, and Stevense Johnson syndrome.[6] Febuxostat[7] and topiroxostat[8] (Fig. 1) are
both non-purine XO inhibitors which possess excellent XO inhibitory activities and have been
introduced into market in 2009 and 2013, respectively. Their approval greatly promoted the research
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of non-purine XO inhibitors and a number of compounds based on the scaffolds of febuxostat and
topiroxostat have been recently reported such as Y-700,[9] isoxazoles,[10] imidazoles,[11] 1,2,3-
triazoles,[12] selenazoles,[13] 2-(indol-5-yl)thiazoles,[14] isocytosines,[15-18] phenyl-1,2,4-triazoles,[19] 4-
(pyridin-4-yl)-1,2,3-triazoles[20] and isonicotinamides.[21]
9,10-Anthraquinone is a class of functionally diverse aromatic compound which exists widely in
traditional Chinese medicines such as aloe-emodin, rhein, emodin, chrysophanol and physcion.[22] Up
to now, various medicinal values of 9,10-anthraquinone were discovered, including anticancer,
antibacterial, anti-inflammatory, antioxidant, antidiabetic, antiviral, hepatoprotection and purgation.[23]
Shi et al [24] reported a series of aloe-enodin derivatives which presented well XO inhibitory potency.
Our previous studies discovered a series of XO inhibitors based on a (1H-1,2,3-triazol-4-
yl)methoxybenzaldehyde scaffold and contained an 9,10-anthraquinone moiety.[25] Structure-activity
relationship (SAR) analysis and molecular modeling studies demonstrated that the 9,10-anthraquinone
moiety located at the outer region of XO active pocket and performed a role of the lipophilic fragment
as in the case of the isobutoxy group of febuxostat.[25]
In this context, we used a N-acylamino acid to mimic an open-ring fragment of the thiazole-5-
carboxylic acid moiety of febuxostat and adopted an 9,10-anthraquinone moiety to play a combined
role of benzonitrile moiety and the lipophilic tail to design a series of N-(9,10-anthraquinone-2-
carbonyl)amino acid derivatives (Fig. 2), expecting to identify a new class of novel XO inhibitors
based on the endogenous amino acids and 9,10-anthraquinone moiety. Additionally, molecular
modeling simulations were carried out to explore the action mode of the representative compounds.
Materials and methods
Chemistry
Unless otherwise indicated, reagents and solvents were purchased from commercial sources and
used without further purification. All reactions were monitored by TLC using silica gel aluminum
cards (0.2 mm thickness) with a fluorescent indicator at 254 nm. The column chromatography was
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performed using silica gel (200-300 mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong,
China). 1H NMR and 13C NMR spectra were recorded on a Bruker 600 MHz spectrometer. Chemical
shifts were expressed in parts per million using DMSO-d6 as the solvent.
General procedure for the preparation of N-(9,10-anthraquinone-2-formyl)amino acids (1a-j)
A mixture of 7 (10 mmol), 10 M NaOH (5 mL), methanol (1 mL), and tetrahydrofuran (1 mL)
was stirred at 50 °C for 4 h. Then the reaction mixture was diluted with water and adjusted pH to 3
with dilute sulfuric acid. The formed precipitate was collected and purified by recrystallization to
provide pure 1a-j.
N-(9,10-anthraquinone-2-formyl)glycine (1a): A yellow solid, yield 85%; MS: m/z 308.1 [M-H]-1; 1H
NMR (600 MHz, DMSO-d6): δ = 12.71 (s, 1H, COOH), 9.36 (t, 1H, J = 5.7 Hz, Ar-H), 8.66 (d, 1H, J
= 1.4 Hz, Ar-H), 8.28 (m, 4H, Ar-H), 7.94 (d, 2H, J = 5.7 Hz, Ar-H), 4.00 (d, 2H, J = 5.8 Hz, CH2);
13C NMR (150 MHz, DMSO-d6): δ = 182.54, 182.50, 171.42, 165.41, 139.01, 135.13, 135.06, 135.00,
133.54, 133.47, 133.43, 133.20, 127.57, 127.24, 127.20, 125.91, 41.76, 40.29, 40.15, 40.01, 39.88,
39.74, 39.60, 39.46.
N-(9,10-anthraquinone-2-formyl)-L-alanine (1b): A yellow solid, yield 76%; MS: m/z 322.1 [M-H]-1;
1H NMR (600 MHz, DMSO-d6): δ = 12.67 (s, 1H, COOH), 9.21 (s, 1H, NH), 8.67 (s, 1H, Ar-H), 8.34
(d, 1H, J = 7.8 Hz, Ar-H), 8.22 (d, 3H, J = 5.7 Hz, Ar-H), 7.93 (s, 2H, Ar-H), 4.47 (q, 1H, J = 7.5 Hz,
CH), 1.45 (d, 3H, J = 7.2 Hz, CH3); 13C NMR (150 MHz, DMSO-d6): δ = 182.57, 182.51, 174.32,
165.03, 139.14, 139.11, 135.12, 135.04, 133.46, 133.41, 127.43, 127.39, 127.22, 127.19, 126.14,
126.10, 48.82, 40.29, 40.15, 40.01, 39.87, 39.73, 39.60, 39.46, 17.16.
N-(9,10-anthraquinone-2-formyl)-L-valine (1c): A yellow solid, yield 83%; MS: m/z 350.2 [M-H]-1;
1H NMR (600 MHz, DMSO-d6): δ = 12.73 (s, 1H, COOH), 9.04 (d, 1H, J = 8 Hz, Ar-H), 8.68 (s, 1H,
NH), 8.36 (d, 1H, J = 8 Hz, Ar-H), 8.26 (m, 3H, Ar-H), 7.96 (m, 2H, Ar-H), 4.35 (t, 1H, J = 4.5 Hz,
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CH), 2.24 (dd, 1H, J = 13.2 Hz, J = 6.9 Hz, CH), 1.01 (t, 6H, J = 5.7 Hz, CH3); 13C NMR (150 MHz,
DMSO-d6): δ = 182.54, 182.53, 173.25, 165.96, 139.47, 135.06, 135.04, 133.64, 133.50, 133.45,
133.36, 127.33, 127.23, 127.21, 126.47, 126.44, 59.03, 29.86, 19.69, 19.23.
N-(9,10-anthraquinone-2-formyl)-L-phenylalanine (1d): A yellow solid , yield 78% ; MS: m/z 398.1
[M-H]-1; 1H NMR (600 MHz, DMSO-d6): δ = 12.85 (s, 1H, COOH), 9.26 (s, 1H, NH), 8.62 (s, 1H,
Ar-H), 8.23 (m, 4H, Ar-H), 7.95 (m, 2H, Ar-H), 7.25 (m, 5H, Ar-H), 4.69 (t, 1H, J = 2.4 Hz, CH),
3.22 (m, 2H, CH2); 13C NMR (150 MHz, DMSO-d6): δ = 182.54, 182.49, 173.35, 165.20, 139.21,
138.56, 135.07, 135.03, 133.47, 133.42, 133.24, 129.52, 129.45, 129.07, 128.75, 128.57, 128.47,
127.45, 127.22, 127.18, 126.74, 126.02, 54.94, 40.30, 40.16, 40.02, 39.88, 39.74, 39.61, 39.47, 36.65.
N-(9, 10-anthraquinone-2-formyl)-L-tryptophan (1e): A purple solid, yield 83%; MS: m/z 437.1 [M-
H]-1; 1H NMR (600 MHz, DMSO-d6): δ = 10.86 (s, 1H, NH), 9.27 (s, 1H, NH), 8.66 (s, 1H, Ar-H),
8.25 (m, 4H, Ar-H), 7.95 (m, 2H, Ar-H), 7.63 (d, 1H, J = 7.7 Hz, Ar-H), 7.17 (m, 3H, Ar-H), 4.72 (t,
1H, J = 4.5 Hz, CH), 3.33 (m, 2H, CH2); 13C NMR (150 MHz, DMSO-d6): δ = 182.51, 182.47,
173.65, 165.31, 139.18, 136.50, 135.11, 135.08, 135.05, 133.48, 133.46, 133.43, 133.35, 127.51,
127.41, 127.23, 127.19, 126.12, 123.91, 121.35, 118.80, 118.52, 111.82, 110.76, 54.34, 40.30, 40.16,
40.02, 39.88, 39.75, 39.61, 39.47, 26.95.
N-(9,10-anthraquinone-2-formyl)-L-aspartic acid (1f): A green solid , yield 83%; MS: m/z 366.1 [M-
H]-1; 1H NMR (600 MHz, DMSO-d6): δ = 12.73 (s, 2H, COOH), 9.31 (d, 1H, J = 7.7 Hz, Ar-H), 8.66
(d, 1H, J = 1.3 Hz, Ar-H), 8.28 (m, 4H, Ar-H), 7.95 (d, 2H, J = 5.8 Hz, Ar-H), 4.82 (q, 1H, J = 7.6
Hz, CH), 2.85 (m, 2H, CH2); 13C NMR (150 MHz, DMSO-d6): δ = 182.51, 182.47, 172.79, 172.23,
164.92, 139.07, 135.12, 135.05, 134.79, 133.49, 133.46, 133.41, 133.36, 127.52, 127.23, 127.19,
125.97, 50.02, 40.28, 40.14, 40.00, 39.86, 39.72, 39.59, 39.45.
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N-(9,10-anthraquinone-2-formyl)-L-glutamic acid (1g): A yellow solid , yield 81%; MS: m/z 380.1
[M-H]-1; 1H NMR (600 MHz, DMSO-d6): δ = 12.54 (s, 1H, COOH), 9.13 (s, 1H, NH), 8.69 (s, 1H,
Ar-H), 8.36 (d, 1H, J = 8.1 Hz, Ar-H), 8.26 (m, 3H, Ar-H), 7.95 (m, 2H, Ar-H), 4.47 (t, 1H, J = 4.5
Hz, CH), 2.40 (t, 2H, J = 7.2 Hz, CH2), 2.07 (m, 2H, CH2); 13C NMR (150 MHz, DMSO-d6): δ =
182.58, 182.53, 174.19, 173.50, 165.51, 139.10, 138.78, 135.22, 135.13, 135.06, 133.48, 133.46,
133.44, 127.46, 127.24, 127.21, 126.14, 52.58, 40.29, 40.15, 40.01, 39.88, 39.74, 39.60, 39.46, 30.78,
26.20.
N-(9,10-anthraquinone-2-formyl)-L-glutamine (1h): A green solid , yield 75%; MS: m/z 379.1 [M-H]-
1; 1H NMR (600 MHz, DMSO-d6): δ = 12.54 (s, 1H, COOH), 9.12 (d, 1H, J = 7.6 Hz, Ar-H), 8.68 (d,
1H, J = 1.7 Hz, Ar-H), 8.35 (dd, 1H, J = 8.1 Hz, J = 1.7 Hz, NH), 8.29 (d, 1H, J = 8.0 Hz, Ar-H), 8.23
(m, 2H, Ar-H), 7.94 (m, 2H, Ar-H), 4.47(m, 1H, CH), 2.39 (t, 2H J = 7.6 Hz, CH2), 2.14 (m, 1H,
CH2), 2.00 (m, 1H, CH2); 13C NMR (150 MHz, DMSO-d6): δ = 182.52, 182.47, 174.25, 173.57,
165.45, 139.14, 135.10, 135.05, 134.99, 133.47, 133.44, 133.39, 127.45, 127.23, 127.20, 126.17,
126.13, 52.68, 40.29, 40.15, 40.01, 39.87, 39.73, 39.59, 39.45, 30.86, 26.30.
N-(9,10-anthraquinone-2-formyl)-D-phenylalanine (1i): A yellow solid, yield 83%; MS: m/z 398.2
[M-H]-1; 1H NMR (600 MHz, DMSO-d6): δ = 12.85 (s, 1H, COOH), 9.26 (s, 1H, NH), 8.62 (s, 1H,
Ar-H), 8.23 (m, 4H, Ar-H), 7.95 (m, 2H, Ar-H), 7.25 (m, 5H, Ar-H), 4.69 (t, 1H, J = 2.4 Hz, CH),
3.22 (m, 2H, CH2); 13C NMR (150 MHz, DMSO-d6): δ = 182.57, 182.49, 173.35, 165.20, 139.21,
138.56, 135.07, 135.03, 133.47, 133.42, 133.24, 129.52, 129.45, 129.07, 128.75, 128.57, 127.45,
127.22, 127.18, 126.74, 126.25, 126.02, 54.94, 40.30, 40.16, 40.02, 39.88, 39.74, 39.61, 39.47, 36.65.
N-(9,10-anthraquinone-2-formyl)-D-tryptophan (1j): A brown solid, yield 83%; MS: m/z 437.1 [M-
H]-1; 1H NMR (600 MHz, DMSO-d6): δ = 10.86 (s, 1H, NH), 9.28 (s, 1H,NH), 8.66 (s, 1H), 8.25 (m,
4H), 7.95 (m, 2H), 7.63 (d, 1H, J = 7.6 Hz,), 7.16 (m, 3H), 4.86 – 4.65 (m, 1H), 3.32 (dd, 2H, J =
14.3, 7.1 Hz,); 13C NMR (150 MHz, DMSO-d6) δ = 182.56, 182.51, 173.65, 165.31, 139.18, 136.50,
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135.08, 135.05, 134.99, 133.48, 133.46, 133.43, 133.35, 127.51, 127.41, 127.23, 127.19, 126.12,
123.91, 121.35, 118.80, 118.52, 111.82, 110.76, 54.34, 40.30, 40.16, 40.02, 39.88, 39.75, 39.61,
39.47, 26.95.
N-benzoyl-L-phenylalanine (8a): A yellow solid, yield 86%; MS: m/z 268.2 [M-H]-1; 1H NMR (600
MHz, DMSO-d6) δ = 12.77 (s, 1H, OH), 8.70 (d, 1H, J = 8.2 Hz, NH), 7.81 – 7.76 (m, 2H, Ar-H),
7.51 (d, 1H, J = 7.4 Hz, Ar-H), 7.45 (d, 2H, J = 7.8 Hz, Ar-H), 7.32 (d, 2H, J = 7.3 Hz, Ar-H), 7.27 (t,
2H, J = 7.6 Hz, Ar-H), 7.18 (t, 1H, J = 7.3 Hz, Ar-H), 4.62 (m, 1H, CH2), 3.19 (m, 1H, CH2), 3.07 (m,
1H, CH); 13C NMR (150 MHz, DMSO-d6): δ = 173.70, 166.82, 138.69, 136.99, 134.38, 131.83,
129.77, 129.53, 129.14, 128.72, 128.65, 127.80, 127.35, 126.82, 54.70, 40.39, 40.25, 40.11, 39.97,
39.84, 39.70, 39.56, 36.71.
N-benzoyl-D-phenylalanine (8b): A brown solid, yield 89%; MS: m/z 268.1 [M-H]-1; 1H NMR (600
MHz, DMSO-d6): δ = 12.76 (s, 1H, OH), 8.70 (d, 1H, J = 8.1 Hz, NH), 7.81 – 7.77 (m, 2H, Ar-H),
7.52 (t, 1H, J = 7.3 Hz, Ar-H), 7.45 (t, 2H, J = 7.8 Hz, Ar-H), 7.32 (d, 2H, J = 7.5 Hz, Ar-H), 7.27 (t,
2H, J = 7.6 Hz, Ar-H), 7.18 (t, 1H, J = 7.3 Hz, Ar-H), 4.62 (m, 1H, CH2), 3.19 (m, 1H, CH2), 3.07 (m,
1H, CH); 13C NMR (150 MHz, DMSO-d6): δ = 173.70, 166.82, 138.69, 136.99, 134.38, 131.83,
129.77, 129.53, 129.14, 128.72, 128.65, 127.80, 127.35, 126.82, 54.70, 40.39, 40.25, 40.11, 39.97,
39.84, 39.70, 39.56, 36.71.
N-benzoyl-L-tryptophan (8c): A brown solid, yield 88%; MS: m/z 307.2 [M-H]-1; 1H NMR (600 MHz,
DMSO-d6): δ = 12.69 (s, 1H, OH), 10.81 (s, 1H, NH), 8.63 (s, 1H, NH), 7.82 (d, 1H, J = 7.1 Hz, Ar-
H), 7.60 (d, 1H, J = 7.9 Hz, Ar-H), 7.52 (t, 1H, J = 7.4 Hz, Ar-H), 7.44 (t, 2H, J = 7.6 Hz, Ar-H), 7.32
(d, 1H, J = 8.1 Hz, Ar-H), 7.21 (d, 1H, J = 2.2 Hz, Ar-H), 7.08 – 7.03 (m, 2H, Ar-H), 6.98 (t, 1H, J =
7.4 Hz, Ar-H), 4.66 (m, 1H, CH2), 3.30 (d, 1H, J = 4.4 Hz, CH2), 3.22 (m, 1H, CH); 13C NMR (150
MHz, DMSO-d6): δ = 173.70, 166.83, 138.68, 136.99, 134.37, 131.84, 129.77, 129.53, 129.14,
128.72, 128.66, 127.81, 127.35, 126.83, 54.69, 40.38, 40.25, 40.11, 39.97, 39.83, 39.69, 39.55, 36.71.
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N-benzamido-D-tryptophan (8d): A brown solid ,yield 85%; MS: m/z 307.2 [M-H]-1; 1H NMR (600
MHz, DMSO-d6): δ = 12.69 (s, 1H, OH), 10.81 (s, 1H, NH), 8.63 (s, 1H, NH), 7.83 – 7.81 (m, 2H,
Ar-H), 7.60 (d, 1H, J = 7.9 Hz, Ar-H), 7.52 (t, 1H, J = 7.4 Hz, Ar-H), 7.44 (t, 2H, J = 7.6 Hz, Ar-H),
7.32 (d, 1H, J = 8.1 Hz, Ar-H), 7.21 (d, 1H, J = 2.0 Hz, Ar-H), 7.06 (t, 1H, J = 7.2 Hz, Ar-H), 6.98
(t, 1H, J = 7.2 Hz, Ar-H), 4.66 (m, 1H, CH2), 3.30 (d, 1H, J = 4.4 Hz, CH2), 3.22 (dd, 1H, J = 14.7,
10.0 Hz, CH); 13C NMR (150 MHz, DMSO-d6): δ = 174.08, 166.82, 136.57, 134.41, 132.00, 131.82,
129.14, 128.69, 127.84, 127.61, 124.04, 121.42, 118.85, 118.61, 111.90, 110.92, 54.15, 40.38, 40.24,
40.10, 39.96, 39.82, 39.68, 39.54, 27.10.
XO inhibitory activity
Bovine in vitro XO inhibitory potency was assayed spectrophotometrically by measuring the
production of uric acid at 294 nm at 25°C. The reactive mixture contained 0.1 M sodium
pyrophosphate buffer (pH 8.3), 0.3 mM Na2EDTA, 100 μM xanthine, 25 U/L XO (Sigma, X1875)
and the tested compound. The inhibition of XO was evaluated by the reduction of the uric acid
formation. The enzyme was pre-incubated for 10 min with the tested compound at 25℃, and the
reaction was started by an addition of xanthine. All tests were performed in triplicate. Compounds
presenting inhibitory effects over 50% at a concentration of 66 μM were further tested at a wide range
of concentrations to calculate their IC50 values using SPSS 20.0 software.
Molecular modeling
Molecular modeling studies were performed with MOE (Molecular Operating Environment,
version 2016.08, Chemical Computing Group Inc., and Canada) software. The crystal structure of
bovine XO in complex with febuxostat (PDB code: 1N5X) was adopted as docking receptor.[7] The
structure was protonated, polar hydrogens were added and energy minimization was carried out
(RMSD gradient = 0.1 kcal/mol, AMBER10: EHT field).[28] The binding site was designated by the
original ligand atoms and other parameters were maintained as the defaults.[29] The standard protocol
implemented in MOE was used in docking calculations. The 3-D binding modes were analyzed by
Surfaces and Maps tool of the MOE software.
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Results and discussion
Chemistry
The synthesis of compounds 1a-j is outlined in Scheme 1. Commercially available phthalic
anhydride reacted with toluene via a Friedel-Craft reaction to yield 2-(4-methylbenzoyl)benzoic acid
(2), which underwent an intramolecular cyclization in conc. H2SO4 conditions, leading to 2-methyl-
9.10-anthraquinone (3).[25] The oxidation of 3 with chromium trioxide in an acetic acid solution
obtained 9,10-anthraquinone-2-carboxylic acid (4),[26] which was then treated with thionyl chloride to
get 9,10-anthraquinone-2-carbonyl chloride (5). A solution of amino acid in methanol was treated with
thionyl chloride to provide its methyl ester (6).[27] Acylation of 6 with 5 produced methyl N-(9,10-
anthraquinone-2-carbonyl)amino carboxylates (7, as shown in the Supporting Information), which
were hydrolyzed with sodium hydroxide to provide target compounds 1a-j. Compounds 9a-d were
synthesized by a similar procedure for the preparation of compounds 1a-j as shown in Scheme 2. The
structures of the synthesized compounds were elucidated by MS, 1H NMR and 13C NMR. All spectral
data were in accordance with assumed structures.
Biological activity
In vitro bovine XO inhibitory potency was spectrophotometrically measured by determining the
uric acid formation at 294 nm. The testing method has been described in our previous study.[12]
Allopurinol was included as a reference compound. Compounds presenting inhibitory effects higher
than 50% at the concentration of 60 μM were further tested at a wide range of concentrations to
calculate associated IC50 values. The results are shown in Table 1.
Eight natural amino acid derivatives (1a-h) were initially synthesized as shown in Table 1. Only
two aromatic amino acid derivatives (1d and 1e) were effective, whereas the rest were inactive (i.e.,
possessing less 50% inhibition at the concentration of 60 µM). Among them, the L-tryptophan
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derivative 1e (IC50 = 35.6 µM), bearing an (indol-3-yl)methyl for R1 group, possessed a weak
potency; and the L-phenylalanine derivative 1d (IC50 = 3.0 µM) with a benzyl for R1 group,
fortunately presented a promising potency and was 2.7-fold more effective than the positive control
allopurinol (IC50 = 8.1 µM). Removal of the R1 group produced inactive glycine derivative (1a),
meaning that the R1 group is requisite for the potency; moreover, replacement of the aromatic R1
group with alkyl (1b and 1c) or further introduction of a carboxyl (1f and 1g) or an amide fragment
(1h) in R1 group resulted in vanished potency, suggesting that the aromatic R1 group played a crucial
role for the potency.
Furthermore, a D-phenylalanine derivative (1i, IC50 = 2.9 µM) and a D-tryptophan derivative (1j,
IC50 = 11.8 µM) were synthesized to explore the influence of three-dimensional configuration.
Interestingly, compound 1i showed comparable potency with its L-enantiomer 1d and compound 1j
exhibited 3-fold higher effects compared with its L-counterpart 1e. This result demonstrated that the
potency of D-amino acid derivative was equal to its L-enantiomer or better.
In addition, to investigate the role of the 9,10-anthraquinone moiety, four structure simplified
derivatives (9a-d) were involved by replacing the 9,10-anthraquinone moiety of 1d, 1e, 1i and 1j by a
phenyl group, respectively. Expectedly, only compound 9c (IC50 = 50.5 µM) exhibited a weak
potency, whereas the rest were totally useless (Table 2), showing that the 9,10-anthraquinone moiety
obviously benefits the potency of compounds 1d, 1e, 1i and 1j.
Molecular modeling
Molecular modeling simulations were performed to further understand the action modes of
compounds 1d and 1i in the XO binding pocket with MOE (Molecular Operating Environment,
version 2016.08, Chemical Computing Group Inc., and Canada) software. The crystal structure of
bovine XO/febuxostat complex (PDB code 1N5X) was adopted as the protein template.[7] The small
molecules were built and minimized with MOE software. The carboxyl group of 1d and 1i was
calculated in its dissociated form. The docking modes are illustrated in Fig. 3.
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As presented in Fig. 3, compound 1d appeared as a flat conformation in the active pocket and
partially overlaid with the original ligand febuxostat.[7] The benzyl moiety inserted into the long-
narrow active pocket at a position of 2.75 Å from Glu1261 and 3.50 Å from molybdenum atom and
the phenyl ring formed a strong Pi-Pi stack interaction with Phe914. The presence of the benzyl
moiety made the ligand draw back from the cavity and therefore led the carboxyl group far away from
Arg880. However, the carboxyl group was till a powerful pharmacophore which strongly interacted
with Thr1010 and Val1011 by H-bonds. Additionally, the flat 9,10-anthraquinone ring occupied the
outer region of the active pocket and stacked with isobutoxyphenyl moiety of febuxostat and a
carbonyl group of 9,10-anthraquinone formed an H-bond with Lys771, meaning that the 9,10-
anthraquinone played a well combined role of the benzonitrile moietyand the lipophilic tail of
febuxostat as expected. The binding model of 1i in XO binding pocket was as similar as that of 1d.
The differences are that 1i was more close to Glu1261 (2.57 Å) and absented the H-bond interaction
with Lys771. However, the H-bond interactions between carboxylate and the Thr1010 were stronger
than that of 1d. As a result, compounds 1d and 1i exhibited the equal potency. The above interactions
provided rational action modes of 1d and 1i in XO binding pocket and well interpreted their XO
inhibitory potency.
Conclusion
Herein, we report a series of N-(9,10-anthraquinone-2-carbonyl)amino acid derivatives (1a-j ) as
novel XO inhibitors. Among them, the L/D-phenylalanine derivatives (1d and 1i) and L/D-tryptophan
derivatives (1e and 1j) were effective with micromolar level potency. Particularly, 1d and 1i
presented the highest potency, which were better than that of allopurinol (IC50 = 8.1 µM). Preliminary
SAR analysis exhibited that an aromatic amino acid fragment, e.g., phenylalanine or tryptophan, was
essential for the inhibition; the D-amino acid derivative presented equal or greater effects compared
with its L-enantiomer; and the 9,10-anthraquinone moiety was welcome for the inhibitory potency.
Furthermore, molecular modeling simulations of 1d and 1i indicated that the phenyl moiety could
insert into the long-narrow pocket and form a Pi-Pi stack interaction with Phe914; and the carboxyl
group strongly interacted with Thr1010 and Val1011 by H-bonds. In a word two promising lead
compounds, 1d and 1i, were successfully discovered in this work and further investigation based on
1d and 1i is in progress.
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Author contributions
F. Meng and T. Zhang conceived and designed the experiments; S. Li and W. Yuan synthesized
the compounds and evaluated the activity; Y. Zhang collected the spectral data; T. Zhang performed
the molecular modeling and wrote the manuscript; F. Meng reviewed the manuscript. All authors have
read and approved the final manuscript.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grant Nos.
81274182 and 81573687), the Project of the Liaoning distinguished professor and the Liaoning
Province Basic Scientific Research Project in University (LQNK201739).
Conflict of interest
The authors have declared no conflict of interest.
Legends for Fig.1, Fig. 2, Fig. 3, Scheme 1, Scheme 2, Table 1 and Table 2.
Fig. 1 Chemical structures of allopurinol, febuxostat and topiroxostat.
Fig. 2 Design of the target compounds 1a-j.
Fig. 3 (A) Binding mode of compoud 1d (blue) overlaid with the original ligand febuxostat (orange) in XO binding pocket;
(B) binding mode of compoud 1i (cyan) overlaid with the original ligand febuxostat (orange) in XO binding pocket; (C)
interaction of compound 1d in XO binding pocket; (D) interaction of compound 1i in XO binding pocket.
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Scheme 1. Reagents and conditions: (i) toluene, AlCl3, 50 °C, 4 h; (ii) H2SO4, 100 °C, 1 h; (iii) CrO3,
AcOH, reflux, 1 h; (iv) SOCl2, DMF, DCM, 40 °C, overnight; (v) SOCl2, MeOH, 0 °C then rt,
overnight; (vi) Et3N, DCM, 0 °C then rt, overnight; (vii) NaOH, MeOH, THF, 50 °C, 4 h.
Scheme 2. Reagents and conditions: (i) SOCl2, DMF, DCM, 40 °C, overnight; (ii) SOCl2, MeOH, 0
°C then rt, overnight; (iii) Et3N, DCM, 0 °C then rt, overnight; (iv) NaOH, MeOH, THF, 50 °C, 4 h.
Table.1 in vitro XO inhibitory potency of designed compounds 1a-j.
Table.2 in vitro XO inhibitory potency of compounds 9a-d.
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SUPPORTING INFORMATION
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Table.1 in vitro XO inhibitory potency of designed compounds 1a-j.
Inhibition rate
Compound R1
R2
(L/D)
IC50 (µM)
at 60 µM (%)
1a
1b
1c
1d
H
glycine
/
29.3
n.a. a
CH3
alanine
L
L
L
20.8
n.a.
(CH3)2CH2
benzyl
valine
24.0
n.a.
3.0± 0.32 #
phenylalanine
97.5
35.6± 1.23 #
n.a.
1e
1f
(indol-3-yl)methyl
HOOCCH2
tryptophan
L
L
L
L
D
70.4
28.2
10.6
20.8
99.1
aspartic acid
glutamic acid
glutamine
1g
1h
1i
HOOCCH2CH2
H2NCOCH2CH2
benzyl
n.a.
n.a.
2.9± 0.26 #
phenylalanine
11.8± 0.84 #
8.1± 1.02
1j
(indol-3-yl)methyl
/
tryptophan
/
D
90.1
99.6
Allopurinol
/
a n.a.: not active (<50% inhibition at 60 µM).
# P＜0.05, versus allopurinol.
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Table.2 in vitro XO inhibitory potency of compounds 9a-d.
Inhibition rate
Compound R1
R2
(L/D)
IC50 (µM)
at 60 µM (%)
9a
9b
9c
9d
benzyl
phenylalanine
phenylalanine
tryptophan
L
17.9
n.a. a
50.5± 2.12
n.a.
benzyl
D
L
60.3
(indol-3-yl)methyl
(indol-3-yl)methyl
18.9
tryptophan
D
21.5
n.a.
a n.a.: not active (<50% inhibition at 60 µM).
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.