Structure-activity relationships of benzothiazole GPR35 antagonists

Article abstract of DOI:10.1016/j.bmcl.2016.12.012

The first structure-activity relationships for a benzothiazole scaffold acting as an antagonist at GPR35 is presented. Analogues were designed based on a lead compound that was previously determined to have selective activity as a GPR35 antagonist. The synthetic route was modular in nature to independently explore the role of the middle and both ends of the scaffold. The activities of the analogues illustrate the importance of all three segments of the compound.

Full text of DOI:10.1016/j.bmcl.2016.12.012

Accepted Manuscript
Structure-Activity Relationships of Benzothiazole GPR35 Antagonists
Manahil M. Abdalhameed, Pingwei Zhao, Dow P. Hurst, Patricia H. Reggio,
Mary E. Abood, Mitchell P. Croatt
PII:
S0960-894X(16)31279-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.12.012
BMCL 24501
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 August 2016
1 December 2016
2 December 2016
Please cite this article as: Abdalhameed, M.M., Zhao, P., Hurst, D.P., Reggio, P.H., Abood, M.E., Croatt, M.P.,
Structure-Activity Relationships of Benzothiazole GPR35 Antagonists, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.12.012
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Structure-Activity Relationships of
Benzothiazole GPR35 Antagonists
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Manahil M. Abdalhameeda, Pingwei Zhaob, Dow P. Hursta, Patricia H. Reggioa ,*, Mary E. Aboodb,*, and
Mitchell P. Croatta ,*

Bioorganic & Medicinal Chemistry Letters
Structure-Activity Relationships of Benzothiazole GPR35 Antagonists
Manahil M. Abdalhameeda, Pingwei Zhaob, Dow P. Hursta, Patricia H. Reggioa,*, Mary E. Aboodb,*, and
Mitchell P. Croatta,*
a
b
* Corresponding authors: Tel.: +1 336 334 3785.
E-mail addresses: mpcroatt@uncg.edu (M.P. Croatt), mabood@temple.edu (M.E. Abood), phreggio@uncg.edu (P.H. Reggio).
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The first structure-activity relationships for a benzothiazole scaffold acting as an
antagonist at GPR35 is presented. Analogues were designed based on a lead
compound that was previously determined to have selective activity as a GPR35
antagonist. The synthetic route was modular in nature to independently explore the
role of the middle and both ends of the scaffold. The activities of the analogues
illustrate the importance of all three segments of the compound.
Keywords:
GPR35
2009 Elsevier Ltd. All rights reserved.
Antagonist
G Protein-Coupled Receptor
Benzothiazole
SAR
* Corresponding author: Tel.: +1 336 334 3785
E-mail address: mpcroatt@uncg.edu
GPR35, a G protein-coupled receptor (GPCR), was discovered
and classified as an orphan GPCR in 19981 and deorphanized in
2006 by the discovery of kynurenic acid as the endogenous
agonist.2 Since its discovery, limited references on the GPR35
receptor have appeared, due in part to a scarcity of endogenous or
exogenous ligands that regulate GPR35 with sufficient potency
and selectivity. The deorphanization of GPR35 receptor has even
been debated due to the poor potency of kynurenic acid (IC50 of
39 µM) and it has recently been proposed that GPR35 should be
renamed to CXCR8 due to a reported binding to CXCL17.3
GPR35 has gained special consideration as a result of its
association with many diseases including type-2 diabetes,4
nociceptive pain,5 inflammation,1 mild mental retardation
syndrome,6 metabolic disorders,7 and gastric cancer.8 Based on
the association of GPR35 with these major diseases and
disorders, there is a current and urgent need for compounds to
regulate the GPR35 receptor and serve as research tools in
molecular characterization of GPR35 receptor. Herein, we report
a structure-activity relationship (SAR) study of benzothiazoles as
antagonists of GPR35. This research sets the foundation for the
design of future potent and selective ligands.
antagonism activity at GPR35 (IC50 = 20.1 nM), however, this
compound has a rhodanine ring. Compounds with a rhodanine
ring have been recommended to be excluded from libraries
screening results due to the potentially misleading results, as
described as Pan Assay Interference Compounds (PAINS).10
Compound 13 is also a potent GPR35 antagonist (IC50 = 160
nM), but the thiosemicarbazone functionality could also react in a
nonselective manner, similar to the rhodanine ring.10 Although
benzothiazole 1 was not as potent as the other potential leads
(IC50 = 0.55 µM), it was selected because it could be
synthetically modified and had a greater opportunity for
selectivity since it was inherently less reactive for covalent
attachment.
An image-based high-throughput, high-content primary screen
using the Molecular Libraries Probe Production Initiative
evaluated ~300,000 compounds.9 This screen identified a few
relatively potent and selective GPR35 antagonists: benzothiazole
1 (CID1231538), rhodanine 12 (CID2286812), and pyrazole 13
(CID2745684; Figure 1). Compound 12 displayed high
Figure 1. GPR35 Antagonists
The route to synthesize compounds 1-3 and 8-10 initiated with
ring-opening of phthalic anhydride 14 using 6-amino-2-
mercaptobenzothiazole 15 to form acid 16 in 96% yield or with
5-amino-2-mercaptobenzoimidazole to form compound 17, also

in good yield (95%) (Scheme 1). Alkylation of thiol 16 with 4-
chlorobenzyl chloride, benzyl bromide, or 4-(chloromethyl)
pyridine hydrochloride successfully produced thioethers 18, 19,
and 20, respectively. These different aryl groups would test the
potential and type of aromatic stacking.
Scheme 2. Oxidation of the exocyclic sulfur. Reagents and conditions: (i)
mCPBA (1 eq), CH2Cl2, 0 °C to rt., 3 hr, 82%; (ii) mCPBA (2.5 eq), CH2Cl2,
0 °C to rt., 5 hr, 83%; (iii) (-) DET, Ti(Oi-Pr)4, C6H5C(CH3)2OOH, DIPEA,
toluene, 2 h, 59%; (iv) (+) DET, Ti(Oi-Pr)4, C6H5C(CH3)2OOH, DIPEA,
toluene, 2 h, 53%.
Compound 11 was synthesized in a racemic fashion with this
study and plans for an enantioselective synthesis could be
developed based on the activity of racemic 11. Synthesis of
compound 11 followed a slightly different synthetic route due to
the ineffectuality of the SN2 reaction on the secondary position
(Scheme 3). The synthesis began with nucleophilic addition of
the commercially available 1-phenylethyl mercaptan 26 to 2-
chloro-6-nitrobenzothiazole 27 to form nitrobenzothiazole 28 in
80% yield. The nitro group was then reduced to amine 29
through catalytic hydrogenation. The resulting aniline was used
to open phthalic anhydride to produce acid 30 in high yield
(98%). Formation of the phthalisoimide ring (31, 70% yield) and
opening by morpholine successfully produced analogue 11 in
76% yield.
Scheme 1. General synthetic route to benzothiazole analogues. Reagents and
conditions: (i) THF, 30 °C, 17 hr; (ii) NaOH, EtOH, 0 °C to rt, ArCH2Cl, 17
hr; (iii) (CH3CO)2O, Et3N, 1,4-dioxane, rt, 17 hr; (iv) R2NH, THF, 50-35°C,
15 hr. See Supplementary data for specific conditions and yields.
Formation of the phthalisoimide ring was accomplished using
acetic
anhydride
and
triethylamine.11
Phthalisoimide
The activities of the synthesized analogues as antagonists at
GPR35 were examined using U2OS cells permanently expressing
HA-GPR35a and arr2-GFP (UGPR35β) assay; 10 µM Zaprinast
was used as the agonist as in our previous publication.9
Concentration-effect curves for agonist-mediated receptor
activation were analyzed by nonlinear regression techniques
using GraphPad Prism 5.0 software (GraphPad) and data were
fitted to sigmoidal dose-response curves to obtain IC50 (Figure 2,
curves for inactive and less active compounds are not shown for
clarity).
Evaluation of the activities as antagonists at GPR35 for the
analogues provide considerable information (Table 1). One of
the first observations is that the parent compound is not as active
as was previously determined. After some more extensive
studying of this, it was determined that many of the
benzothiazoles were not sufficiently stable at room temperature
and decomposed within 15 hours when exposed to air and light.
To get reproducible data, the analogues were all frozen in DMSO
until they were analyzed for their activity. The activity of the
parent compound is still less than was previously determined.
This could be due to the initially screened compound being
partially decomposed into an unknown mixture of compounds.
Fortunately, the parent compound was still moderately active
when pure, which allowed for trends to be observed.
intermediates 22, 23, and 24 were produced in 91%, 96%, and
87% yields, respectively, and were opened using morpholine,
piperidine, or pyrrolidine to produce final analogues 1, 2, 3, 8,
and 9. The morpholine surrogates were chosen to evaluate if
there is a requirement for hydrogen bonding in that region of the
receptor. Analogue 10 was synthesized following the same
protocol, except using 5-amino-2-mercaptobenzoimidazole as
starting substrate. The benzimidazole group was selected for
synthesis to test the role of the endocyclic sulfur. Benzimidazole
10 may also provide a positive potential surface instead of
negative potential surface of the sulfur.
Oxidation of the exocyclic sulfur to a sulfoxide or sulfone
could potentially provide strong and directional hydrogen bonds.
Sulfoxide 4 and sulfone 5 were synthesized using different
amounts of meta-chloroperoxybenzoic acid (mCPBA) to improve
this interaction (Scheme 2). Due to the chiral nature of the
sulfoxide, enantiomers 6 and 7 were individually synthesized
following procedures similar to those used to synthesize Nexium
(esomeprazole).12 The individual sulfoxide enantiomers (6 and 7)
were synthesized using titanium tetraisopropoxide and the
respective enantiomers of diethyl tartrate and cumene
hydroperoxide as the oxidant. The absolute configuration of
sulfoxides 6 and 7 were inferred based on the close analogy to
the process for esomeprazole.12 It was decided that the
confirmation of the absolute configurations would be made if
either of the two enantiomers were significantly potent.

basicity of the nitrogen of the pyridine ring produced an
unfavorable negative potential surface. The same scenario exists
when the cyclic sulfur is replaced with a nitrogen atom in
benzimidazole 10.
Table 1. Biological results
Entry
Structure
IC50 (CI)a
1
4.56 (1.8-12)
2
3
4
5
6
>30
>30
13 (4.0-45)
Scheme 3. Synthesis of compound 11. Reagents and conditions: (i) NaH,
DMF, 5 hr, 80%; (ii) H2, Pd/C, EtOH, 17 hr; (iii) phthalic anhydride, THF, 30
°C, 17 hr; (iv) (CH3CO)2O, Et3N, 1,4-dioxane, rt, 17 hr, (52% over 3 steps);
(v) morpholine, THF, 35 °C, 15 hr, 76%.
5.24 (2.2-13)
150
1
4
5
8
>30
>30
100
11
7
8
50
0
7.23 (2.3-23)
>30
-8
-6
-4
Log (M)
9
-50
Figure 2. Concentration-response curves for GPR35 antagonists that had
obtainable IC50 values
One of the most important findings was that piperidine 2 and
pyrrolidine 3 are devoid of activity as GPR35 antagonists. This
indicates that there is probably a strong interaction between the
oxygen of the morpholine ring and a residue in the binding site in
this region. Prior modeling studies placed the morpholine ring in
the region of the toggle switch,9, 13 so these data can be used to
further refine the model.
10
>30
11
17.0 (5.6-54)
Oxidation of the exocyclic sulfur to sulfoxides 4, 6 and 7 and
sulfone 5 decreased the activity as an antagonist. These data
indicate that this area of the molecule is either not amenable to
expansion or is in a non-polar environment. The electronics of
the benzothiazole are also modified by having the adjacent
electron-withdrawing group; this might also be detrimental. The
individual enantiomers (6 and 7) appear to be less active than the
racemate (4), however, the confidence intervals (CI) for the IC50
values are overlapping for sulfoxides 4, 6, and 7, which explains
this otherwise unexpected result. Pyridine 9 was found to be
inactive as a GPR35 antagonist, which is an indication that the
a CI= 95% Confidence Intervals
In conclusion, we report herein an initial structure-activity
relationships study for a family of benzothiazoles that act as
antagonists at GPR35. A major finding was that a seemingly
innocuous oxygen of the morpholine ring actually plays a major
role. This and other results, including the location and type of
heteroatom substitutions, could be used in concert with an
updated GPR35 model, to generate more potent GPR35
antagonists.

Acknowledgments
This research was supported by National Institutes of
Health Grants R21NS077347 and R01DA023204,
T32DA007237, P30DA013429. The authors thank Dr.
Franklin J. Moy (UNCG) for assisting with analysis of NMR
data and Dr. Brandie M. Ehrmann (UNCG) for acquisition of
the high resolution mass spectrometry data at the Triad Mass
Spectrometry Laboratory at the University of North Carolina
at Greensboro.
Supplementary Material
Supplementary material
(synthetic procedures
and
characterization of all compounds) associated with article can be
found, in the online version, at xxx.
References and notes
1. O'Dowd, B. F.; Nguyen, T.; Marchese, A.; Cheng, R.; Lynch, K. R.; Heng,
H. H. Q.; Kolakowski Jr, L. F.; George, S. R. Genomics 1998, 47, 310.
2. Wang, J.; Simonavicius, N.; Wu, X.; Swaminath, G.; Reagan, J.; Tian, H.;
Ling, L. Journal of Biological Chemistry 2006, 281, 22021.
3. Maravillas-Montero, J. L.; Burkhardt, A. M.; Hevezi, P. A.; Carnevale, C.
D.; Smit, M. J.; Zlotnik, A. The Journal of Immunology 2014.
4. Horikawa, Y.; Oda, N.; Cox, N. J.; Li, X.; Orho-Melander, M.; Hara, M.;
Hinokio, Y.; Lindner, T. H.; Mashima, H.; Schwarz, P. E. H.; del Bosque-
Plata, L.; Horikawa, Y.; Oda, Y.; Yoshiuchi, I.; Colilla, S.; Polonsky, K. S.;
Wei, S.; Concannon, P.; Iwasaki, N.; Schulze, J.; Baier, L. J.; Bogardus, C.;
Groop, L.; Boerwinkle, E.; Hanis, C. L.; Bell, G. I. Nat Genet 2000, 26, 163.
5. Ohshiro, H.; Tonai-Kachi, H.; Ichikawa, K. Biochemical and Biophysical
Research Communications 2008, 365, 344.
6. Shrimpton, A. E.; Braddock, B. R.; Thomson, L. L.; Stein, C. K.; Hoo, J. J.
Clinical Genetics 2004, 66, 537.
7. Leonard, J. N.; Chu, Z. L. GPR35 and modulators thereof for the treatment
of metabolic-related disorders. US 20070077602 A1, 2007.
8. Okumura, S.-i.; Baba, H.; Kumada, T.; Nanmoku, K.; Nakajima, H.;
Nakane, Y.; Hioki, K.; Ikenaka, K. Cancer Science 2004, 95, 131.
9. Zhao, P.; Sharir, H.; Kapur, A.; Cowan, A.; Geller, E. B.; Adler, M. W.;
Seltzman, H. H.; Reggio, P. H.; Heynen-Genel, S.; Sauer, M.; Chung, T. D.
Y.; Bai, Y.; Chen, W.; Caron, M. G.; Barak, L. S.; Abood, M. E. Molecular
Pharmacology 2010, 78, 560.
10.Baell, J. B.; Holloway, G. A. Journal of Medicinal Chemistry 2010, 53,
2719.
11.de Toranzo, E. G. D.; Brieux, J. A. Journal of Medicinal Chemistry 1967,
10, 982.
12.Cotton, H.; Elebring, T.; Larsson, M.; Li, L.; Sörensen, H.; von Unge, S.
Tetrahedron: Asymmetry 2000, 11, 3819.
13.Zhao, P.; Lane, T. R.; Gao, H. G. L.; Hurst, D. P.; Kotsikorou, E.; Le, L.;
Brailoiu, E.; Reggio, P. H.; Abood, M. E. Journal of Biological Chemistry
2014, 289, 3625.