Design and synthesis of novel 2-(indol-5-yl)thiazole derivatives as xanthine oxidase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.01.055

Xanthine oxidase (XO) inhibitors have been widely used for the treatment of gout. Indole rings are frequently used as active scaffold in designing inhibitors for enzymes. Herein, we describe the structure-activity relationship for novel xanthine oxidase inhibitors based on indole scaffold. A series of novel tri-substituted 2-(indol-5-yl)thiazole derivatives were synthesized, and their in vitro inhibitory activities against xanthine oxidase and in vivo efficacy lowering uric acid level in blood were measured. Among them, 2-(3-cyano-2-isopropylindol-5-yl)-4-methylthiazole-5-carboxylic acid exhibits the most potent XO inhibitory activity (IC₅₀ value: 3.5 nM) and the excellent plasma uric acid lowering activity. Study of structure activity relationship indicated that hydrophobic moiety (e.g., isopropyl) at 1-position and electron withdrawing group (e.g., CN) at 3-position of indole ring and small hydrophobic group (CH₃) at 4-position of the thiazole ring enhanced the XO inhibitory activity. Hydrophobic substitution such as isopropyl at 1-position of the indole moiety without any substitution at 2-position has an essential role for enhancing bioavailability and therefore for high in vivo efficacy.

Full text of DOI:10.1016/j.bmcl.2015.01.055

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of novel 2-(indol-5-yl)thiazole derivatives
as xanthine oxidase inhibitors
Jeong Uk Song a,b, Sung Pil Choi b, Tae Hun Kim b, Cheol-Kyu Jung b, Joo-Youn Lee b, Sang-Hun Jung a,
,
⇑
Geun Tae Kim b,
⇑
a College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea
b Research & Development, LG Life Sciences Ltd, 188 Munji-ro, Yuseong-gu, Daejeon 305-380, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Xanthine oxidase (XO) inhibitors have been widely used for the treatment of gout. Indole rings are
frequently used as active scaffold in designing inhibitors for enzymes. Herein, we describe the struc-
ture–activity relationship for novel xanthine oxidase inhibitors based on indole scaffold. A series of novel
tri-substituted 2-(indol-5-yl)thiazole derivatives were synthesized, and their in vitro inhibitory activities
against xanthine oxidase and in vivo efficacy lowering uric acid level in blood were measured. Among
them, 2-(3-cyano-2-isopropylindol-5-yl)-4-methylthiazole-5-carboxylic acid exhibits the most potent
XO inhibitory activity (IC50 value: 3.5 nM) and the excellent plasma uric acid lowering activity. Study
of structure activity relationship indicated that hydrophobic moiety (e.g., isopropyl) at 1-position and
electron withdrawing group (e.g., CN) at 3-position of indole ring and small hydrophobic group (CH3)
at 4-position of the thiazole ring enhanced the XO inhibitory activity. Hydrophobic substitution such
as isopropyl at 1-position of the indole moiety without any substitution at 2-position has an essential role
for enhancing bioavailability and therefore for high in vivo efficacy.
Received 6 July 2014
Revised 11 January 2015
Accepted 22 January 2015
Available online xxxx
Keywords:
Gout
Hyperuricemia
Xanthine oxidase inhibitors
2-(Indol-5-yl)thiazole
Ó 2015 Elsevier Ltd. All rights reserved.
Gout is commonly recognized as recurrent pains of acute
inflammatory arthritis. High level of uric acid in blood, or hyperuri-
cemia causes such symptoms by inducing the precipitation of
monosodium urate crystals.1 The disease occurs by overproduction
of uric acid and/or impaired renal excretion of uric acid.1–3 Uric
acid is a product of purine metabolism, which involves xanthine
oxidase (XO) in the two final steps (from hypoxanthine to xan-
thine, then to uric acid). Therefore inhibition of this enzyme has
been an obvious target to control uric acid level in blood and
eventually to cure gout. In this regard, allopurinol (Fig. 1) has been
clinically used to treat hyperuricemia for more than forty years.
Recently, febuxostat (Fig. 1) has been introduced. Both of these
drugs target xanthine oxidase to inhibit its function. The XO inhib-
itors block the biosynthesis of uric acid through competitive inhi-
bition, leading to lower uric acid level in blood.4,5 Although both of
them are inhibitors of XO, their mechanism of action is diverged.
The purine analog, allopurinol is a mechanism-based inhibitor of
xanthine oxidase.5,6 Allopurinol itself is a weak XO inhibitor
in vitro. However, it is rapidly oxidized by XO in vivo to form an
active metabolite called oxypurinol, which is a potent XO inhibitor.
On the other hand, febuxostat is a 2-phenylthiazole analog and
displays higher inhibitory activity by strong non-competitive
binding to active site of XO. However, it has not been proved to
be clinically more beneficial compared with allopurinol.7–9
Another approach to treat gout is to use recombinant urate oxi-
dase, which directly metabolizes uric acid to allantoin. Pegloticase,
approved in 2010, is a pegylated urate oxidase that treats refrac-
tory chronic gout.3 Uric acid levels may also be lowered by uricosu-
rics, which increase the urinary excretion of uric acid by interfering
the urate absorption from kidney to blood.
Although the link between hyperuricemia and gout has been
known for more than hundred years, the intrinsic metabolic rela-
tionship between hyperuricemia and diabetes was recently estab-
lished.10,11 Since hyperuricemia becomes serious health problem, it
calls for more diverse treatment option. In the recent years, many
synthetic scaffolds comprising triazole,12 isocytosines,13,14 pyrimi-
done15 and imidazole16 have been reported to display XO inhibi-
tion in the literatures. In this context, we have made an effort to
discover novel XO inhibitors based on indolethiazole 6 as shown
in Figure 1. The indole rings are frequently utilized as active scaf-
fold in designing target molecules or inhibitors for enzymes and
receptors.17–19
⇑
Corresponding authors. Tel.: +82 42 821 5939; fax: +82 42 823 6566 (S.-H.J.);
tel.: +82 42 866 2247; fax: +82 42 861 2566 (G.T.K.).
Although little was known about the effect of indole scaffold on
XO inhibition, it can be found easily in promising anticancer,
E-mail addresses: jungshh@cnu.ac.kr (S.-H. Jung), gtakim@lgls.com (G.T. Kim).
http://dx.doi.org/10.1016/j.bmcl.2015.01.055
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Song, J. U.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.01.055

2
J. U. Song et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
R1
R2
OH
N
OH
N
HO
N
R4
O
N
N
S
N
N
N
S
N
N
O
R3
HO
N
H
N
H
O
O
Allopurinol
Oxypurinol
Febuxostat
Compound 6
Figure 1. Structures of allopurinol, oxypurinol, febuxostat and compound 6 as xanthine oxidase inhibitors.
antimicrobial, anti-inflammatory, analgesic, anticonvulsants, anti-
oxidant and antidiabetic agents,17–19 indicating that indole may
serve as a prospective scaffold in inhibiting xanthine oxidase.
In order to obtain an agent with better pharmacological profile
and in vivo efficacy for the treatment of gout, we incorporated
indole moiety into 6 as an isosteric replacement of phenyl moiety
of febuxostat as shown in Figure 1.
The general synthetic route to 6 is shown in Scheme 1. Amide 2
was synthesized from acid 1 using HBTU [2-(1H-benzotriazole-1-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate] and ammo-
nium chloride. Amide 2 was converted to thioamides 3 by refluxing
with Lawesson’s agent in THF. Compound 3 and ethyl 2-chloroace-
toacetate were reacted with a catalytic amount of pyridine by
refluxing in ethanol to afford indole-5-thiazole 4 in a reasonable
yield of 70–85%. Compound 4 was reacted with alkyl bromide
and sodium hydride in DMF at room temperature to furnish com-
pound 5, and this type of reaction allowed the modification of R3
group. Ester 5 was hydrolyzed with sodium hydroxide in aqueous
THF and methanol, and the final compound 6 was obtained in a
good yield.
The established synthesis led us to evaluate in vitro xanthine
oxidase inhibition of 6. Assay of in vitro XO inhibition activity
was performed by measuring the inhibitor concentration needed
for 50% inhibition.20 IC50 values of 6e, 6k, 6m and 6n showed the
similar levels to febuxostat. Tables 1–3 represent the inhibitory
activities against XO of the test compound.
The activities of indole 6a–g were initially investigated as
shown in Table 1. Compound 6a (IC50 = 110 nM) with hydrogen
for R1 and isobutyl group for R3 was found as a hit compound.
The previous investigation indicated that an electron-withdrawing
group such as nitro, chloro and cyano groups at 2-position in feb-
uxostat plays an important role on xanthine oxidase inhibition.23
Thus, chloro and nitro groups were introduced at 3-position of
6a. Chloro compounds 6b, 6c and 6d showed remarkably low
IC50 values of 5.7 nM, 7.3 nM and 16.0 nM, respectively. Unfortu-
nately, 6b and 6d were not metabolically stable (Table 1). Although
6c exhibited highly potent inhibitory activity (IC50 = 7.3 nM), it
displayed in vivo uric acid-lowering activity of only 18.9%. Com-
pound 6e with nitro group showed IC50 values of 4.2 nM. However,
6e was less metabolically stable than 6g (Table 1). Compound 6f
exhibited moderate inhibitory activity. Compound 6g with cyano
group showed an IC50 value of 5.5 nM which was ten times more
potent in activity than the original hit compound 6a. Thus, It was
confirmed that substitution with electron-withdrawing group at
3-position of the indole ring increased in vitro XO inhibitory
activity.
In order to examine the effect of the substitution at 3-position
of the thiazole ring, analogs 6h–k were prepared and their XO
inhibitory activities were tested as shown in Table 2. Based on
50% cytotoxic concentration (CC50) using primary rat hepatocytes,
6k (CC50 = 200 lM) was less cytotoxic than 6g (CC50 = 82 lM).
Therefore, isopropyl group was fixed at 1-position for comparison.
Compound 6i with trifluoromethyl group exhibited high IC50 value
of 895.0 nM, and 6j with methoxy group much increased activity
(IC50 = 90.0 nM). Compound 6h (R4 = H) and 6k (R4 = CH3)
displayed better inhibitory activities compared with 6i and 6j. Oral
exposure in rats with 6h and 6k revealed that 6k exhibited high
Cmax and AUC values as shown in Table 2. Therefore, we focused
on the modification of 6k comprising cyano group with various
substituents for R2 and R3.
The substitution effect on XO inhibition is summarized in
Table 3. Most of the compounds in Table 3 showed good XO inhib-
itory activity. Further selection was carried out on the basis of
in vivo uric acid reduction at 10 mg/kg.
In order to check whether the R2 substituent is worthwhile to
be varied, 6l (R2 = CH3) was prepared. It exhibited moderate inhib-
itory activity (IC50 = 10.7 nM) and low in vivo efficacy. Therefore,
our interest was focused on the R3 substituent. Introduction of flu-
oroisopropyl, hydroxyl isopropyl and methoxy isopropyl group
afforded 6m, 6n and 6o, showing potent IC50 values of 4.9 nM,
3.0 nM and 9.0 nM, respectively. Unfortunately, those compounds
displayed weak uric acid lowering activity compared to 6k. Com-
pounds 6p (R3 = methanesulfonylethyl) and 6q (R3 = acetylamino-
ethyl) exhibited moderate inhibitory activities of 9.0 nM and
R1
O
R1
S
R1
O
c
b
a
H2N
H2N
HO
R2
R2
R2
N
H
N
H
N
H
3
2
1
R1
R1
R1
R2
NH
R2
R3
R2
R3
R4
O
d
e
R4
O
N
S
R4
O
N
N
S
N
N
O
HO
S
O
6
4
5
Scheme 1. Reagents and conditions: (a) HBTU, NH4Cl, Et3N, DMF, 74–90%; (b) Lawesson’s agent, THF, reflux, 90–95%; (c) ethyl 2-chloroacetoacetate, pyridine, EtOH, reflux,
70–85%; (d) alkyl bromide, sodium hydride, DMF, 70–80%; (e) THF/MeOH = 1:1, 1 N NaOH, 90%.
Please cite this article in press as: Song, J. U.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.01.055

J. U. Song et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 1
IC50 and metabolic stability of compound 6 with various R1, R2 and R3
R1
R2
R3
H3C
HO
N
S
N
O
a
Compound
R1
H
R2
H
R3
In vitro IC50 (nM)
Metabolic stability (half life, min)
6a
6b
110
5.7
27.5
6.2
Cl
H
6c
6d
6e
6f
Cl
H
7.3
96.0
6.6
Cl
CH3
H
16.0
4.2
NO2
NO2
CN
79.5
b
H
12.0
NT
O
6g
H
5.5
105.7
174.0
Febuxostat
3.1–5.5
a
Metabolic stability was studied in rat liver microsomes (RLM).
NT: not tested.
b
Table 2
The effect of R4 in compound 6 on xanthine oxidase inhibition
CN
H
R4
O
N
S
N
HO
Compound
R4
In vitro IC50 (nM)
Cmax
(
l
g/mL)a at 10 mg/kg
AUC0–last (l
g h/mL)b at 10 mg/kg
6h
6i
6j
6k
H
4.4
0.94
NTa
NT
3.98
16.37
5.39
NT
NT
47.31
61.51
CF3
OCH3
CH3
895.0
90.0
3.5
Febuxostat
3.1–5.5
a,bTest compound was administered orally at 10 mg/kg in Sprague-Dawley (SD) rats.
a
NT: not tested.
12.3 nM, respectively. Less than 0.02 g/mL of low plasma concen-
tration at 1 h was observed when 6p or 6q was orally adminis-
trated in rats. This result indicates low in vivo efficacy of 6p or
6q is caused by low plasma exposure. Compound 6r with sulfon-
amide group had good XO inhibitory activity of 6.0 nM, but did
not reduce in vivo uric acid level. Thus, hydrophilic substitution
for R3 has a harmful effect on in vivo inhibition efficacy. Finally,
we decided to install benzyl group for R3 and the hydrophobic
effect was investigated. Compounds 6s and 6t had good XO inhib-
itory activities of 3.9 nM and 8.6 nM, but exhibited weak uric acid
lowering activities of 10.0% and 16.0% at 10 mg/kg, respectively.
Low plasma exposure of 6s (0.29 g/mL) and 6t (0.11 g/mL) at 1 h
was observed as well when each compound was treated in rats.
Their weak in vivo efficacy is attributed to low plasma concentra-
tion and low bioavailability in rats.
To visualize the interaction of indole scaffold with XO enzyme,
we performed molecular modeling studies using the Schrodigner
Glide program with 6k. The proposed binding model of 6k is ori-
ented similarly to febuxostat in its complex with bovine milk XO
as shown in Figure 2 (PDB code 1N5X).24 Since the residues around
the binding pocket are highly conserved in bovine and human XO,
our findings are also applicable to human XO.7 The major interac-
tions show the hydrogen bonds among acid group of the inhibitor,
Arg880 and Thr1010. Cyano group forms hydrogen bond with
Asn768 as well. The thiazole ring that interacts with Glu802
appears to have
p–p stacking interaction with Phe914 and
Phe1009. Non-bonded interactions are also observed with amino
acid residues, Leu648, Phe649, Leu873, Val1011, Phe1013 and
Leu1014 in the active site of XO.
The proposed docking model provides us with a reasonable
explanation of modification in compounds. In Table 1, substituents
of chloro, nitro and cyano groups at R1 position led to more than
10-fold gain in potency. Based on the docking model, chloro, nitro
Among test compounds for XO inhibition, 6k with cyano group
for R1 was found to display well-balanced and excellent in vitro
activity and in vivo efficacy.
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4
J. U. Song et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 3
Table 4
The effect of R2 and R3 in compound 6 on xanthine oxidase inhibition
Pharmacokinetic data of 6k in SD ratsa
CN
R2
Pharmacokinetic parameters
Dose (mg/kg)
po
iv
2
10
H3C
N
Cmax
(l
g/mL)
g h/mL)
g h/mL)
3.98
47.31
52.87
6
N
R3
AUC0–last
(l
12.39
12.54
HO
S
AUC0–infinity
Tmax (h)
(l
O
T1/2 (h)
Clearance (mL/h/kg)
Vd (mL/kg)
7.95
4.37
161.47
634.99
Compound R2
R3
In vitro
IC50 (nM)
Uric acid inhibition (%)a
at 1 h, 10 mg/kg
Bioavailability (%)
84.31
6k
6l
H
3.5
10.7
4.9
3.0
9.0
60.0
a
n = 3.
CH3
H
8.6
2.5
2.0
1.5
1.0
0.5
0.0
6m
6n
6o
22.3
8.5
F
H
OH
**
H
14.1
***
O
***
***
S
O
6p
6q
H
H
H
12.3
5.6
0.0
0.0
0.0
O
O
N
H
l
l
g
g
k
g
k
o
kg
r
ro
O
S
/k
g/
nt
Co
nt
O
o
mg
mg/
mg/
6r
6s
6.0
3.9
0
N
H
l
1
e C
t
6k 5m
a
at
t
6k 10
mpo
6k 20
d
n
rma
No
d
d
n
o
n
n
u
x
xos
H
H
10.0
16.0
u
u
O
-
bu
K
mpo
Co
Fe
mpo
Co
F
Co
F
6t
8.6
Figure 3. Plasma uric acid concentration (mg/dl) (⁄⁄P<0.01,⁄⁄⁄P<0.001: ANOVA and
post Dunnet’s test).
Febuxostat
3.1–5.5
a
Uric acid inhibition was studied in SD rats at 10 mg/kg by oral route.
thiazole acid and appears to loose hydrogen bond between
Arg880 and the acid. Thus, compound 6i shows much less IC50
compared to 6k.
Pharmacokinetic profile of 6k was investigated in Sprague-
Dawley (SD) rats at a single 10 mg/kg dose by oral route (po) over
24 h. Pharmacokinetic parameters of 6k were described in Table 4,
showing high Cmax of 3.98 lg/mL, AUC0–last of 47.31 lg h/mL and
long half life of 7.95 h. Compound 6k exhibited excellent oral bio-
availability and good in vivo efficacy.
In order to estimate the plasma uric acid-lowering ability,
experiments were carried out using oxonic acid-induced high-uric
acid model.21,22 Figure 3 represents plasma uric acid concentration.
The uric acid inhibition rate of 6k (10 mg/kg) at 1 h after oral
administration (putting the plasma uric acid level of normal group
as 100% inhibition, and plasma uric acid level of control group as
0% inhibition) was 60%, indicating a suppression rate similar to feb-
uxostat (10 mg/kg). As revealed by the experiment, 6k exerts an
excellent inhibitory effect on XO. Therefore, 6k can be used as an
agent for the treatment and prevention of the diseases associated
with human xanthine oxidase such as hyperuricemia and gout.
In summary, we identified a novel indole scaffold for xanthine
oxidase inhibition. Among a series of indole derivatives, 6k exhib-
ited good pharmacokinetic parameters and oral availability. Com-
pound 6k has excellent plasma uric acid-lowering in vivo
efficacy, which will be further developed for clinical studies.
Figure 2. Docking model of bovine XO bound to 6k (cyan) and febuxostat (gray).
The hydrogen bonds are shown in yellow dashed line. Docking results are visualized
using PyMol.19
and cyano groups form hydrogen bonds with Asn768, explaining a
significant increase in inhibition for 6b, 6e and 6g. In Table 2, the
electronegative substituent such as trifluoromethyl group at R4
position lowers electron density of the hydrogen bond acceptor,
Acknowledgment
We would like to thank Dr. Dongchul Lim for his continuous
support to this structure-guided drug discovery project.
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J. U. Song et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
19. Hwang, I. K.; Yoo, K. Y.; Li, H.; Park, O. K.; Lee, C. H.; Choi, J. H.; Won, M.-H. J.
Neurosci. Res. 2009, 87, 2126.
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compounds of various concentrations (10% DMSO phosphate buffer) were
added at 20 L/well, and pre-incubated for 3 min at room temperature.
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initial velocity of uric acid formation was determined at 293 nm using
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Please cite this article in press as: Song, J. U.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.01.055