Synthesis and agonistic activity at the GPR35 of 5,6-dihydroxyindole-2- carboxylic acid analogues

Article abstract of DOI:10.1021/ml300076u

5,6-Dihydroxyindole-2-carboxylic acid (DHICA), an intermediate of melanin synthesis and an eumelanin building block, was recently discovered to be a GPR35 agonist with moderate potency. Here, we report the synthesis and pharmacological characterization of a series of DHICA analogues against GPR35 using both label-free dynamic mass redistribution and Tango β-arrestin translocation assays. This led to identification of novel GPR35 agonists with improved potency and/or having biased agonism.

Full text of DOI:10.1021/ml300076u

Letter
pubs.acs.org/acsmedchemlett
Synthesis and Agonistic Activity at the GPR35 of 5,6-
Dihydroxyindole-2-carboxylic Acid Analogues
Huayun Deng and Ye Fang*
Biochemical Technologies, Science and Technology Division, Corning Inc., Corning, New York 14831, United States
S
* Supporting Information
ABSTRACT: 5,6-Dihydroxyindole-2-carboxylic acid (DHICA), an intermediate
of melanin synthesis and an eumelanin building block, was recently discovered to
be a GPR35 agonist with moderate potency. Here, we report the synthesis and
pharmacological characterization of a series of DHICA analogues against GPR35
using both label-free dynamic mass redistribution and Tango β-arrestin
translocation assays. This led to identification of novel GPR35 agonists with
improved potency and/or having biased agonism.
KEYWORDS: GPR35, 5,6-dihydroxyindole-2-carboxylic acid, biased agonism, melanin synthesis
GPR35, an orphan G protein-coupled receptor (GPCR), is
implicated in inflammation, pain, cardiovascular diseases, and
metabolic disorders.1 Recent pharmacological screens have
identified several clinically used drugs to be GPR35 agonists;
these drugs include the antiasthma drugs cromolyn and
dicumarol,2 the loop diuretics bumetanide and furosemide,3
the catechol-O-methyl transferase inhibitor drug entacapone
used for the treatment of Parkinson’s disease,4 and the
antinociception niflumic acid.5,6 Certain abundant natural
phytochemicals including myricetin, morin, and ellagic acid,6
and gallic acid7 also have been found to be GPR35 agonists
with moderate potency. Coupled with up-regulation of GPR35
in several types of inflamed cells,1,2,8 these findings provide
rationales for proposing activation of GPR35 as a therapeutic
mechanism with opportunity for development in certain
diseases, including inflammatory disorders. Therefore, we
have been interested in discovering GPR35 agonists as
potential therapeutics.
The natural agonists for GPR35 remain controversial. The
possible natural agonists include kynurenic acid,8 2-acyl
lysophosphatidic acid,9 and more recently, 5,6-dihydroxyin-
dole-2-carboxylic acid (DHICA, 3).10 DHICA is a building
block of eumelanin, the black to brown pigment produced by
melanin and found in human skin, hair, and eyes. To expand
pharmacological tools for GPR35 as well as to understand the
structural basis of DHICA to activate GPR35, we synthesized a
series of DHICA analogues and characterized them using both
label-free dynamic mass redistribution (DMR) and Tango β-
arrestin translocation assays. The DMR assay is based on whole
cell phenotypic responses,11,12 while Tango is an end point
measurement of gene reporter activity linked to the GPR35
activation-mediated β-arrestin translocation.4,6
β-arrestin translocation signal in an engineered U2OS cell line
stably expressing a C-terminal modified GPR35 (U2OS-
GPR35-bla).10 DHICA presents several positions for structure
activity relationship (SAR) modifications (Figure 1). We and
Figure 1. Structures of DHICA analogues. DHICA: X = N, R1 = R2 =
R3 = H, R4 = OH.
others have shown that the carboxylic acid group of several
different classes of GPR35 agonists identified to date is critical
to activate GPR35.5,13 Thus, we decided to focus on analogues
bearing modifications at other positions of DHICA.
The synthesis of all DHICA analogues is outlined in Scheme
1. We first synthesized N-alkylated derivatives of DHICA from
2-bromo-4,5-dimethoxybenzaldehyde (6−12) (Scheme 1A).
The route commences with copper-catalyzed domino four-
component coupling and cyclization of 2-bromo-4,5-dimethox-
ybenzaldehyde with ethyl 2-isocyanoacetate using CuI to
produce the ethyl ester 1, N-alkylation to produce the
intermediate 4, hydrolysis to produce the carboxylic acid
intermediate 5, and then demethylation to give 1-alkylated
analogues 6−12 in good yield. Next, we synthesized 3-
substituted DHICA analogues 15 and 18−27, whose syntheses
were also outlined in Scheme 1A. Intermediate 1 was initially
reacted with N-bromosuccinimide (NBS) to produce inter-
mediate 13, then hydrolyzed to produce 14, and demethylated
to produce 3-bromo-DHICA 15. For targets 18−27, the route
begins with Suzuki cross-coupling of the intermediate 13 with
DHICA is an indolic intermediate of melanin synthesis.
Recently, we found that DHICA is a GPR35 agonist.10 It
triggers a robust DMR in HT-29, a native colon cancer cell line
endogenously expressing GPR35,4 with an EC50 of 23.6 μM;
and it exhibits comparable potency (EC50, 23.2 μM) to cause a
Received: April 1, 2012
Accepted: June 6, 2012
Published: June 6, 2012
© 2012 American Chemical Society
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Letter
Scheme 1. Synthesis of DHICA Analogues
Reagents and conditions: (A) (a) CuI, Cs2CO3, ethyl 2-isocyanoacetate in DMSO, 80 °C, 60%; (b) NaOH, 1:1 EtOH/H2O, rt, 75%; (c) BBr3,
CH2Cl2, dropwise/78 °C, rt, 43%; (d) CH3I, CH3CH2I, benzyl bromide, 4-methylbenzyl bromide, or methyl bromoacetate for 6, 7, 8, 9, or 12,
respectively, NaH/THF, rt; or 2-bromo-1-phenylethanone, or 2-bromo-1-p-tolylethanone for 10 or 11, respectively; DMF, 95 °C, 65−85%; (e)
LiOH/EtOH, 50 °C, 86−93%; (f) BBr3, CH2Cl2, dropwise/−15 °C, rt, 65−91%; (g) NBS, DMF, 0 °C, 76%; (h) LiOH, 1:1 EtOH/H2O, rt, 79%;
(i) BBr3, CH2Cl2, rt, 19%; (j) phenylboronic acid, 3-chlorophenylboronic acid, pyridin-3-ylboronic acid, thiophen-3-ylboronic acid, 3-
(methyloxycarbonyl)phenylboronic acid, 4-(methoxycarbonyl)phenylboronic acid, 3,5-dichlorophenylboronic acid, 3,5-difluorophenylboronic acid,
4-chlorophenylboronic acid, or 4-(trifluoromethyl)phenylboronic acid for 18 to 27, respectively, Na2CO3, [1,1′-bis(diphenylphosphino)ferrocene]-
dichloropalladium(II)/CH2Cl2 (1:1), dioxane/water, 100 °C, 63−81%; (k) LiOH, EtOH/H2O, 78−92%, rt to 50 °C; (l) BBr3, CH2Cl2, rt, 4−31%.
(B) (m) BF3Et2O, Ac2O, 90 °C, 17%; (n) methyl 2-bromoacetate, K2CO3, 100 °C, 81%; (o) Na/MeOH, 60 °C, 88%; (p) NaOH, EtOH/H2O, rt,
82%; (q) BBr3, CH2Cl2, rt, 33%. (C) (r) ethyl 2-mercaptoacetate, K2CO3, DMF, 18-crown-6 (catalytic amount), 80 °C, 73%; (s) NaOH, EtOH/
H2O, rt, 56%; (t) BBr3, CH2Cl2, rt, 34%; (u) methylmagnesium bromide, THF, −78 °C, N2, 67%; (v) pyridinium chlorochromate, rt, 95%; (w) ethyl
2-mercaptoacetate, K2CO3, DMF, 80 °C, 72%; (x) Na, EtOH/H2O, rt, 89%; (y) BBr3, CH2Cl2, rt, 34%. (D) (z) methylmagnesium bromide, −78
°C, N2, 88%; (aa) pyridinium chlorochromate, CH2Cl2, rt, 95%; (ab) ethyl 2-mercaptoacetate, catalytic amount of 18-crown-6, K2CO3, DMF, 80 °C,
63%; (ac) BBr3, CH2Cl2, rt, 21%; (ad) Br2, HOAc, rt, 30%; (ae) LiOH, EtOH/H2O, rt, 29%.
boronic acid to produce 3-substituted ethyl ester 16, then
hydrolysis to produce 17, and demethylation to produce 3-
substituted DHICA analogues 18−27. All compounds with
purity greater than 95% were obtained from the contract
research organization BioDuro Co. (Beijing, China).
DHICA, its N-alkylated derivatives generally caused a decrease
in potency, while, except for 20, 22, and 23, most of its 3-
substituted analogues increased its potency. The most potent
analogue was 24 (3-(3,5-dichlorophenyl)-DHICA), whose
EC50 was 1.06
0.12 μM (two independent measurements,
We employed both DMR and Tango β-arrestin translocation
assays to characterize the pharmacological activity of DHICA
analogues at the GPR35. First, we recorded the dose responses
of all DHICA analogues in native HT-29 cells using DMR
agonist assays. Results showed that out of the 18 analogues only
compound 12 was inactive at a dose up to 1 mM. All other
analogues led to a clear dose-dependent DMR whose
characteristics were similar to those of the known GPR35
agonists, including zaprinast, kynurenic acid,4 and DHICA.10
Their dose responses were best fitted with a monophasic
sigmoidal nonlinear regression, leading to a single EC50 for each
compound (Table 1). SAR analysis showed that, compared to
each in duplicate, n = 4). Compared to DHICA, 3-bromo-
DHICA (15) exhibited a 6-fold increase in potency; similarly,
3-(3-chlorophenyl)-DHICA (19), 3-(3,5-difluorophenyl)-
DHICA (25), and 3-(4-chlorophenyl)-DHICA (26) also
displayed increased potency. However, the efficacy, based on
the maximal DMR signal amplitude, was found to be
compound-dependent (Table 1). Except for 6, 7, and 21, all
other DHICA analogues led to DMR signals that were smaller
than those for DHICA.
Second, we measured the ability of DHICA analogues to
cause β-arrestin translocation using the Tango assay in the
engineered U2OS-GPR35-bla cell line. This cell line stably
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dose-dependent responses. Compared to DMR assay results,
the potency observed was mostly right-shifted, except for
DHICA, 6, 7, and 23. Among all indole analogues tested, the
most potent agonist was 3-(3-carboxyphenyl)-DHICA (23),
with an EC50 of 10.8 μM. Furthermore, compared to DHICA,
only N-methyl-DHICA (6) and 3-(4-chlorophenyl)-DHICA
(26) led to an increase in efficacy (85% and 89% of the
zaprinast maximal response, respectively). Together with DMR
results, these results suggest that except for 12 all other DHICA
analogues are GPR35 agonists, but with distinct potency and
efficacy.
Previously, we had discovered that certain thieno[3,2-
b]thiophene-2-carboxylic acid derivatives are GPR35 agonists;
particularly, YE210 (6-bromo-3-methylthieno[3,2-b]thiophene-
2-carboxylic acid) displayed a potency better than that of
zaprinast, and an efficacy comparable to that of zaprinast.13
Together with the observations that N-alkylation and 3-
substitution of DHICA altered the potency and efficacy of
DHICA, we designed and characterized several benzofuran and
benzo[b]thiophene analogues of DHICA. 5,6-Dihydroxy-3-
methylbenzofuran-2-carboxylic acid (3-methyl-DHFCA, 32)
was synthesized according to Scheme 1B. The synthesis
began with acetylation of 3,4-dimethoxyphenol with acetic
anhydride and BF3·Et2O to produce 28, the alkylation with
methyl 2-bromoacetate in the presence of K2CO3 to produce
29, the ring closure with NaOCH3 to produce 30, and the
sequential hydrolysis and demethylation to produce the
product 32. 5,6-Dihydroxybenzo[b]thiophene-2-carboxylic
acid (DHTCA, 35) was synthesized according to Scheme 1C.
The synthesis begun with a one-step ring closure of 2-bromo-
4,5-dimethoxybenzaldehyde with ethyl 2-mercaptoacetate in
the presence of K2CO3 to produce 33, then the sequential
hydrolysis and demethylation to produce the product 35. 5,6-
Dihydroxy-3-methylbenzo[b]thiophene-2-carboxylic acid (3-
methyl-DHTCA, 40) and its analogue 46 were synthesized
following a similar procedure (Scheme 1C and D).
Table 1. Compounds, and Their EC50 and Efficacy (That Is,
the Maximal Responses) as Measured Using DMR and
a
Tango Assays
EC50 (μM)
efficacy
DMR
(pm)
Tango (%
ZAP)
compd
DMR
Tango
Zaprinast
0.14 0.02
23.6 2.5
64.2 5.5
77.1 6.5
43.5 3.2
17.3 1.5
43.7 3.7
39.7 3.1
inactive
5.3 0.7
23.2 2.1
45.4 3.2
45.7 3.7
inactive
245
256
275
232
184
170
132
150
100
75
3 (DHICA)
6
85
7
52
8
9
>500
>25
10
11
12
15
18
19
20
21
22
23
24
25
26
27
32
35
40
46
inactive
inactive
inactive
4.10 0.31
20.2 1.9
2.48 0.20
84.0 7.5
10.3 0.7
24.9 1.9
22.6 1.8
1.06 0.12
4.36 0.31
2.54 0.37
12.9 1.1
16.2 1.3
7.35 0.51
6.17 0.44
0.16 0.02
166 12
inactive
188
138
162
150
261
210
172
102
175
137
194
214
216
218
197
48
19
34.4 3.4
inactive
61.2 5.1
75.7 6.4
10.8 1.9
24.2 1.9
18
17
40
25
48
89
109
7
387 24
inactive
133 11
520 41
90.6 7.4
46.5 3.2
27
86
109
94
a
The data represents mean
ments (n = 4). ZAP, zaprinast.
SD from two independent measure-
expresses two fusion proteins: human GPR35 linked to a Gal4-
VP16 transcription factor via a TEV protease site, and β-
arrestin/TEV protease fusion protein. The cell line also stably
expresses the β-lactamase reporter gene under the control of a
UAS response element. The activation of GPR35 by agonists
leads to the recruitment of β-arrestin-TEV protease fusion
protein to the activated GPR35, leading to cleavage of Gal4-
VP16 transcription factor from the receptor by the protease.
The transcription factor then translocates to the nucleus and
activates the expression of β-lactamase. Such a β-arrestin
translocation is often specific to the GPR35 activation, given
that the test compound is neither fluorescent nor toxic to cells.6
All Tango assay results were reported after being normalized to
the maximal response of zaprinast within the same plate.
Results showed that only a subset of analogues including 8, 10,
11, 12, 18, 20, and 27 were inactive, while the rest led to clear
DMR assays showed that, for compounds 32, 35, and 40,
their DMR characteristics were similar to those of the GPR35
full agonists including zaprinast and YE210 (Figure 2a).
However, the DMR characteristics of 46 were almost identical
to those of DHICA (Figure 2b). All four analogues displayed
higher potency than DHICA, and compound 46 gave rise to
the highest potency with an EC50 of 0.16 μM, which is
comparable to that of zaprinast.4 Interestingly, compared to
DHICA, all gave rise to slightly lower efficacy (Figure 2c, Table
1).
The Tango assay revealed a different pattern. First, all four
compounds resulted in lower potency than DHICA (Figure
2d). Second, except for 32, which exhibited lower efficacy than
DHICA, all the other three compounds gave rise to an efficacy
Figure 2. Dose responses of DHICA analogues. (a, b) Real time dose DMR responses of 35 and 46, respectively, in HT-29 cells. (c) DMR
amplitudes of 32, 35, 40, and 46 as a function of their doses. The DMR amplitudes at 10 min poststimulation in HT-29 cells were calculated for all.
(d) β-Arrestin translocation signals of 32, 35, 40, and 46 as a function of their doses. The Tango signals were normalized to the zaprinast maximal
response, which was set to be 100%. The data represents mean SD from two independent measurements, each in duplicate (n = 4).
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Figure 3. Dose-dependent inhibition of the DMR of DHICA analogues by GPR35 antagonists. (a, b) Real time DMR signals of compounds 35 and
40, respectively, in HT-29 cells in the presence of SPB05142 at different doses (indicated in graph). (c) The DMR amplitudes of 32, 35, 40, and 46
as a function of SPB05142 doses. The compound dose was fixed and was 64 μM, 32 μM, 32 μM, and 1 μM for compounds 32, 35, 40, and 46,
respectively. The DMR amplitudes at 10 min poststimulation were calculated for all. (d) Tango signal of 100 μM 46 in the U2OS-GPR35-bla cell
line as a function of ML-145 doses. The data represents mean SD from two independent measurements, each in duplicate (n = 4).
that was higher than that of DHICA. Compound 40 displayed
the highest efficacy among all the DHICA analogues; and its
efficacy was even slightly higher than that of zaprinast (Table
1).
Such a cell line- and assay readout-dependent relative efficacy
and potency indicates biased agonism of these compounds at
the GPR35.16
In summary, we synthesized a series of DHICA analogues
and found that several analogues display improved potency and
efficacy, and many ligands displayed biased agonism. Further
exploration of SAR may lead to the discovery of β-arrestin
biased agonists for GPR35. Considering the importance of β-
arrestin in regulating a diverse array of cell signaling pathway
downstream GPCRs, such a class of GPR35 agonists may open
the possibility to further elucidate the biology and physiology of
these compounds at the GPR35.16
Next, we determined the specificity of the DMR signals of all
DHICA analogues in HT-29 to the activation of endogenous
GPR35. This DMR antagonist assay was performed using a
recently discovered GPR35 antagonist, SPB05142.13,14 Com-
pound 12 was excluded in this analysis, since it was inactive.
Results showed that SPB05142 alone up to 64 μM did not
trigger any obvious DMR. Furthermore, SPB05142 dose-
dependently blocked the DMR of all other DHICA analogues,
each at a fixed dose which was close to its EC80. The SPB05142
dose inhibition of the DMR induced by representative
compounds 32, 35, 40, and 46 was illustrated in Figure 3a−
c, giving rise to an apparent IC50 of 3.6 0.4 μM, 12.0 1.0
μM, 5.0 0.4 μM, and 3.6 0.3 μM, respectively (n = 4).
Similarly, ML-145, a potent GPR35 antagonist,15 also dose-
dependently blocked the β-arrestin translocation signal of
compound 46 in U2OS-GPR35-bla cell lines, yielding an IC50
of 0.17 0.01 μM (two independent measurements, n = 4)
(Figure 3d). These results suggest that these DHICA analogues
were GPR35 agonists.
ASSOCIATED CONTENT
* Supporting Information
■
S
Full names, structures, and NMR and mass spectroscopy
characterization of all compounds, as well as detailed synthesis
procedures of compounds 26 and 46. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 607-9747203. E-mail: fangy2@corning.com.
■
Next, we performed correlation analysis between the results
obtained using the two pharmacological assays. Results showed
that only DHICA, 6, 7, and 23 displayed comparable potency
in both assays; however, the rest compounds displayed lower
potency in the Tango assay than those in the DMR assay
(Figure 4a). Further, only compounds 26, 40, and 46 exhibited
higher efficacy in the Tango assay than those in the DMR assay;
in contrast, the remaining compounds exhibited lower efficacy
in the Tango assay than those in the DMR assay (Figure 4b).
Notes
The authors declare the following competing financial
interest(s):H.D. and Y.F. are employees and stockholders of
Corning Inc.
ABBREVIATIONS
■
DHICA, 5,6-dihydroxyindole-2-carboxylic acid; DHFCA, dihy-
droxybenzofuran-2-carboxylic acid; DHTCA, dihydroxybenzo-
[b]thiophene-2-carboxylic acid; DMR, dynamic mass redis-
tribution; GPCR, G protein-coupled receptor; GPR35, G
protein-coupled receptor-35; NBS, N-bromosuccinimide; SAR,
structure activity relationship; YE210, 6-bromo-3-methylthieno-
[3,2-b]thiophene-2-carboxylic acid
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NOTE ADDED AFTER ISSUE PUBLICATION
■
The footnote was missing in Table 1 in the version published
on July 12, 2012. The revised version was published on the
Web on May 21, 2014.
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