Discovery of 2-phenylthiazole-4-carboxylic acid, a novel and potent scaffold as xanthine oxidase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2019.01.005

The xanthine oxidase (XO) plays an important role in producing uric acid, and therefore XO inhibitors are considered as one of the promising therapies for hyperuricemia and gout. We have previously reported a series of XO inhibitors with pyrazole scaffold to extend the chemical space of current XO inhibitors. Herein, we describe further structural optimization to explore the optimal heterocycle by replacing the thiazole ring of Febuxostat with 5 heterocycle scaffolds unexplored in this field. All of these efforts resulted in the identification of compound 8, a potent XO inhibitor (IC₅₀ = 48.6 nM) with novel 2-phenylthiazole-4-carboxylic acid scaffold. Moreover, lead compound 8 exhibited hypouricemic effect in potassium oxonate-hypoxanthine-induced hyperuricemic mice. These results promote the understanding of ligand-receptor interaction and might help to design more promising XO inhibitors.

Full text of DOI:10.1016/j.bmcl.2019.01.005

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 2-phenylthiazole-4-carboxylic acid, a novel and potent scaffold
as xanthine oxidase inhibitors
⁎
⁎
Xue Xua, Liming Dengb, Lu Niec, Yueming Chenb,d,e, Yanzhi Liub, Rongrong Xieb, , Zheng Lib,d,
a Guangzhou General Pharmaceutical Research Institute Co., Ltd., Guangzhou 510240, PR China
b School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China
c Jiujiang Vocational University, 88 Lian Xi Road, Jiujiang, Jiangxi 332000, PR China
d Key Laboratory of New Drug Discovery and Evaluation of Ordinary Universities of Guangdong Province, Guangdong Pharmaceutical University, Guangzhou 510006, PR
China
e Guangzhou Key Laboratory of Construction and Application of New Drug Screening Model Systems, Guangdong Pharmaceutical University, Guangzhou 510006, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The xanthine oxidase (XO) plays an important role in producing uric acid, and therefore XO inhibitors are
considered as one of the promising therapies for hyperuricemia and gout. We have previously reported a series of
XO inhibitors with pyrazole scaffold to extend the chemical space of current XO inhibitors. Herein, we describe
further structural optimization to explore the optimal heterocycle by replacing the thiazole ring of Febuxostat
with 5 heterocycle scaffolds unexplored in this field. All of these efforts resulted in the identification of com-
pound 8, a potent XO inhibitor (IC50 = 48.6 nM) with novel 2-phenylthiazole-4-carboxylic acid scaffold.
Moreover, lead compound 8 exhibited hypouricemic effect in potassium oxonate-hypoxanthine-induced hy-
peruricemic mice. These results promote the understanding of ligand-receptor interaction and might help to
design more promising XO inhibitors.
Gout
Xanthine oxidase
Hyperuricemia
Hypouricemic effect
XO inhibitor
In the purine metabolic pathway, xanthine oxidase (XO) is a key
enzyme to produce the uric acid, while the hyperuricemia is an un-
derlying cause of gout. Gout is an inflammatory disease resulting from
the urate crystal deposition in tissues and joints, which is often related
to hypertension, diabetes, and cardiovascular disease.1 Since gout can
be treated by decreasing the levels of uric acid, the uricosuric or XO
inhibitors are considered as promising therapies for the treatment of
hyperuricemia and gout.2,3
The synthetic routes of designed compounds 1–8 are summarized in
Scheme 1. Treated ethyl 2-chloroacetoacetate with 1a or 3a–b formed
oxazole 2a or thiazole 4a–b.10 Thiazole 5a–b was generated from ring-
closure reaction of ethyl bromopyruvate and 3a–b, which were ob-
tained by treating benzamide with Lawesson’s reagent.11 The basic
hydrolysis of intermediates 2a, 4a–b, and 5a–b in the presence of li-
thium hydroxide, afforded target compounds 1–3, 7, and 8. Cyclization
of 6a with 7a provided thiazole ester 8a. The synthesis of 11a was
obtained from ring-closure reaction of 9a and 10a.12 While isoxazole
14a was prepared by cyclization using hydroxylamine hydrochloride
and 13a, which was obtained by Claisen condensation.13,14 These ob-
tained esters 8a, 11a, and 14a were subsequently converted to target
compounds 4–6 in the presence of lithium hydroxide.
Recently, a lot of XO inhibitors have been reported in the literatures
(Fig. 1). Febuxostat, a classical XO inhibitor, has approved by FDA for
the treatment of chronic gout.4 Besides the thiazole ring in Febuxostat,
other heterocycle scaffolds were also explored in this field, such as
pyrazoles5, isoxazoles6 and imidazoles.7 Moreover, Topiroxostat, a XO
inhibitor with triazole scaffold, has also been approved in Japan for the
management of hyperuricemia.8 In our previous study, the chemical
space of XO inhibitor with pyrazole scaffold has also been system-
atically explored.9 Herein, another 5 heterocycle scaffolds unexplored
in this field were designed and synthesized to explore the optimal
heterocycle for further structure-activity relationship (SAR) studies.
To obtain an optimal heterocycle for extending the chemical space
of XO inhibitors, five heterocycles were introduced to replace the
thiazole ring of Febuxostat. The XO inhibitory activities were in-
vestigated at the concentration of 10 μM. As shown in Table 1, the un-
substituted derivative of Febuxostat (compound 1) revealed potent in-
hibitory activity against XO despite it is inferior to that of Febuxostat.
Abbreviations: XO, xanthine oxidase; FDA, the Food and Drug Administration; SAR, structure-activity relationship; THF, tetrahydrofuran; NMR, nuclear magnetic
resonance; PBS, phosphate buffer saline; SD, standard deviation
⁎ Corresponding authors at: School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.
E-mail addresses: happyyunyun_1985@126.com (R. Xie), li.zheng.sky@163.com (Z. Li).
https://doi.org/10.1016/j.bmcl.2019.01.005
Received 30 November 2018; Received in revised form 3 January 2019; Accepted 5 January 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Xu,X.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2019.01.005

X. Xu et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Fig. 1. The structures of representative non-
purine XO inhibitors.
Scheme 1. Reagents and conditions: (a) ethyl 2-chloroacetoacetate, EtOH, reflux, 16 h; (b) LiOH·H2O, THF/MeOH/H2O, r.t., 4 h; (c) Lawesson’s reagent, THF, reflux,
4 h; (d) ethyl bromopyruvate, EtOH, reflux, 4 h; (e) EtOH, reflux, 6 h; (f) EtOH, reflux, 2 h; (g) diethyl oxalate, NaOEt, 0-rt, 18 h; (h) hydroxylamine hydrochloride,
EtOH, reflux, 2 h.
Although there was slightly difference between heterocycles, the ox-
azole analog 2 indicated a decreased inhibitory activity compared to
compound 1. A possible explanation is that the oxazole ring is a more
hydrophilic heterocycle compared to that of thiazole ring, which de-
creased the hydrophobic interaction with the receptor. Interestingly,
the inhibitory activity of compound 3 was recovered by switching the
location of sulphur and nitrogen in compound 1, which further verified
our hypothesis above. However, position exchange of sulphur atom in
compound 3 to afford compound 4 appeared to diminish the inhibitory
activity, might attributing to the strict interaction in the binding pocket
of XO. Compound 5, the methyl analog of Y-700, resulted in a sig-
nificant loss in potency compared with compounds 1 and 3. The po-
tency losses might be related to the change in conformation between
phenyl group and pyrazole ring. Meanwhile, the isoxazole scaffold
(compound 6) was also not appropriate for the building of XO inhibitor,
which might be attributed to the decreased hydrophobic interaction in
presence of hydrophilic isoxazole ring.
Based on these positive results, two thiazole scaffolds of compound
1 and 3 were selected as our starting point for further modification
(Table 2). In this field of XO inhibitors, the nitro group was usually
2

X. Xu et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Table 1
In vitro xanthine oxidase inhibitory activities of six heteroaromatics.
Compd
Het
XO inhibitory activity (%)a
Febuxostat
98.5
37.6
1
2
12.5
3
4
5
32.8
18.6
13.9
6
4.2
a
Xanthine oxidase inhibitory activity at the concentration of 10 μM in three
independent experiments.
Table 2
The inhibitory activities of the derivatives 7 and 8.
a
Compd
R
Het
IC50 (nM)
4.8
Fig. 2. The aligned docking poses of compound 7 (silvery), 8 (purple brown)
and Febuxostat (light black) at the binding site of XO. Key residues are labeled
in white, and interactions are represented by dashed lines.
Febuxostat
CN
7
8
a
NO2
NO2
33.4
48.6
only formed hydrogen-bond network with Arg880 (Fig. 2). These ab-
sences of hydrogen bond interactions were reasonably explained that
the inhibitory activities of compounds 7 and 8 were inferior to that of
Febuxostat. Moreover, the nitrile group of Febuxostat forming hy-
drogen bond with Asn768, but there was no direct interaction for the
nitro group of compound 7. Interestingly, the nitro group of compound
8 formed two hydrogen bonds with Asn768 and Lys771.
Values are mean of three independent experiments.
introduced as a bioisosteres of cyan group.5 As expected, the nitro
analog of Febuxostat (compound 7) revealed a significant improvement
on inhibitory activity compared with compound 1 but, nevertheless,
result in nearly 7-fold decrease in potency compared to Febuxostat.
Consistent with the finding above, incorporation of nitro and isobutoxy
groups (compound 8) led to an increased inhibitory activity rise to the
level of compound 7. These results indicated that the 2-phenylthiazole-
4-carboxylic acid scaffold might be introduced as a bioisosteres of
thiazole ring in Febuxostat.
Based on their favorable inhibitory activities, compounds 7 and 8
were selected to evaluate hypouricemic effect in potassium oxonate-
hypoxanthine-induced hyperuricemic mice. As shown in Fig. 3, sub-
cutaneous injection of potassium oxonate (300 mg/kg) combines with
intraperitoneal injection of hypoxanthine (500 mg/kg) induced an
acute hyperuricemia in mice. As expected, the increases of serum uric
acid levels were significantly suppressed in the treated groups, despite
the hypouricemic effects of compounds 7 and 8 were inferior to that of
Febuxostat, the most advanced drug in this field.
With the aim of exploring potent XO inhibitor with optimal het-
erocycle, we have identified a new 2-phenylthiazole-4-carboxylic acid
scaffold by comprehensive evaluating 5 heterocycle scaffolds un-
explored in this field. The lead compounds 7 and 8 exhibited potent XO
inhibitory activity in vitro and hypouricemic effect in potassium ox-
onate-hypoxanthine-induced hyperuricemic mice. Moreover, com-
pounds 7 and 8 fitted very well to the same binding site of Febuxostat in
the induced-fit docking studies. All of these results indicated that the
new 2-phenylthiazole-4-carboxylic acid scaffold was meaningful for
To better clarify the binding mode of compounds 7 and 8, the in-
duced-fit docking studies were performed based on the X-ray structure
of XO (PDB code: 1VDV). As shown in Fig. 2, compounds 7 and 8
docked very well to the same binding site for Febuxostat. The car-
boxylic acid of Febuxostat formed hydrogen-bond network with re-
sidues Arg880 and Thr1010, and the thiazole ring formed interactions
with Phe914 and Thr1010 (Fig. 2). Similarly, the thiazole ring of
compounds 7 and 8 also revealed interactions with residues Glu802 and
Thr1010, respectively. However, the carboxylic acid moiety of them
3

X. Xu et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Guangdong province (2017KSYS002), Innovation and strong school
project of Guangdong Pharmaceutical University (2017KQNCX111),
and Guangdong Province Medical Science and Technology Research
Fund (Grant A2018273 and B2018053).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2019.01.005.
References
1. Karis E, Crittenden DB, Pillinger MH. Hyperuricemia, gout, and related comorbid-
ities: cause and effect on a two-way street. South Med J. 2014;107:235–241.
2. Borges F, Fernandes E, Roleira F. Progress towards the discovery of xanthine oxidase
inhibitors. Curr Med Chem. 2002;9:195–217.
3. Roddy E, Doherty M. Treatment of hyperuricaemia and gout. Clin Med.
2013;13:400–403.
4. Song JU, Choi SP, Kim TH, et al. Design and synthesis of novel 2-(indol-5-yl)thiazole
derivatives as xanthine oxidase inhibitors. Bioorg Med Chem Lett.
2015;25:1254–1258.
5. Ishibuchi S, Morimoto H, Oe T, et al. Synthesis and structure-activity relationships of
1-phenylpyrazoles as xanthine oxidase inhibitors. Bioorg Med Chem Lett.
2001;11:879–882.
Fig. 3. Mice were treated with the potassium oxonate and hypoxanthine 1 h
before drug administration, and the serum uric acid levels was measured at 1 h
after drug administration. Results are mean
SD (n = 6 per group).
6. Wang S, Yan J, Wang J, et al. Synthesis of some 5-phenylisoxazole-3-carboxylic acid
derivatives as potent xanthine oxidase inhibitors. Eur J Med Chem.
2010;45:2663–2670.
*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 compared to control group by Student’s
t test.
7. Chen S, Zhang T, Wang J, et al. Synthesis and evaluation of 1-hydroxy/methoxy-4-
methyl-2-phenyl-1H-imidazole-5-carboxylic acid derivatives as non-purine xanthine
oxidase inhibitors. Eur J Med Chem. 2015;103:343–353.
further evaluation, and the information obtained from this work might
help to design more promising XO inhibitors that are structurally re-
lated.
8. Matsumoto K, Okamoto K, Ashizawa N, Nishino T. FYX-051: a novel and potent
hybrid-type inhibitor of xanthine oxidoreductase. J Pharmacol Exp Ther.
2011;336:95–103.
9. Li J, Wu F, Liu X, et al. Synthesis and bioevaluation of 1-phenyl-pyrazole-4-carboxylic
acid derivatives as potent xanthine oxidoreductase inhibitors. Eur J Med Chem.
2017;140:20–30.
Notes
10. Nicolaou K, Zak M, Safina BS, Estrada AA, Lee SH, Nevalainen M. Total synthesis of
thiostrepton. assembly of key building blocks and completion of the synthesis. J Am
Chem Soc. 2005;127:11176–11183.
The authors confirm that this article content has no conflict of in-
terest.
11. Hwang JY, Attia RR, Zhu F, et al. Synthesis and evaluation of sulfonylni-
trophenylthiazoles (SNPTs) as thyroid hormone receptor–coactivator interaction
inhibitors. J Med Chem. 2012;55:2301–2310.
Acknowledgements
12. Schmidt A, Habeck T, Kindermann MK, Nieger M. New pyrazolium-carboxylates as
structural analogues of the pseudo-cross-conjugated betainic alkaloid Nigellicine. J
Org Chem. 2003;68:5977–5982.
This study was supported by the National Natural Science
Foundation of China (Grant 81803341), the Natural Science Foundation
of Guangdong Province, China (Grant 2018A030313445), the projects
of Guangzhou key laboratory of construction and application of new
drug screening model systems (201805010006) and Key Laboratory of
New Drug Discovery and Evaluation of ordinary universities of
13. Hauser CR, Swamer FW, Adams JT. The Acylation of Ketones to Form β-Diketones or
β-Keto Aldehydes. Org React. 1954;8:59–196.
14. Royals EE. The use of sodium methoxide in the Claisen reaction. J Am Chem Soc.
1945;67:1508–1509.
4