Discovery of 2H-Chromen-2-one Derivatives as G Protein-Coupled Receptor-35 Agonists

Article abstract of DOI:10.1021/acs.jmedchem.6b01431

A family of 2H-chromen-2-one derivatives were identified as G protein-coupled receptor-35 (GPR35) agonists using dynamic mass redistribution assays in HT-29 cells. The compounds with 1H-tetrazol-5-yl in 3-substituted position displayed higher potency than the corresponding carboxyl analogs, and the hydroxyl group in the 7-position also played an important role in GPR35 agonistic activity. 6-Bromo-7-hydroxy-8-nitro-3-(1H-tetrazol-5-yl)-2H-chromen-2-one (50) was found to be the most potent GPR35 agonist with an EC₅₀ of 5.8 nM. Calculating the physicochemical properties of compounds with moderate to high potency suggested that compounds 30, 50, and 51 showed good druggability. This study provides a novel series of GPR35 agonists, and compound 50 may be a powerful tool to study GPR35.

Full text of DOI:10.1021/acs.jmedchem.6b01431

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Article
Discovery of 2H-chromen-2-one Derivatives
as G Protein-Coupled Receptor-35 Agonists
Lai Wei, Jixia Wang, Xiuli Zhang, Ping Wang, Yaopeng Zhao, Jiaqi
Li, Tao Hou, Lala Qu, Liying Shi, Xinmiao Liang, and Ye Fang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01431 • Publication Date (Web): 15 Dec 2016
Downloaded from http://pubs.acs.org on December 15, 2016
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Discovery of 2H-chromen-2-one Derivatives as G
Protein-Coupled Receptor-35 Agonists
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Lai Wei a, Jixia Wang a, Xiuli Zhang *a,c, Ping Wanga, Yaopeng Zhao a, Jiaqi Li a, Tao
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Hou a, Lala Qu a, Liying Shi a, Xinmiao Liang *a,c and Ye Fang *b
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Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of
Chemical Physics, Chinese Academy of Sciences, Dalian, China
b
Biochemical Technologies, Science and Technology Division, Corning, New York
14831, United States
c Coꢀinnovation Center of Neuroregeneration, Nantong University, Nantong, 226019,
China
*Corresponding authors: Xiuli Zhang, Xinmiao Liang, Ye Fang
Tel.: +86 411 84379519; fax: +86 411 84379539.
Eꢀmail addresses: zhangxiuli@dicp.ac.cn, liangxm@dicp.ac.cn,
fangy2@corning.com.
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ABSTRACT
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A family of 2Hꢀchromenꢀ2ꢀone derivatives was identified as G proteinꢀcoupled
receptorꢀ35 (GPR35) agonists using dynamic mass redistribution assays in HTꢀ29
cells. The compounds with 1Hꢀtetrazolꢀ5ꢀyl in 3ꢀsubstituted position displayed higher
potency than the corresponding carboxyl analogs, and the hydroxyl group on
7ꢀposition also played an important role in GPR35 agonistic activity.
6ꢀBromoꢀ7ꢀhydroxyꢀ8ꢀnitroꢀ3ꢀ(1Hꢀtetrazolꢀ5ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone (50) was found
to be the most potent GPR35 agonist with an EC50 of 5.8 nM. Calculating the
physicochemical property of compounds with moderate to high potency suggests that
compounds 30, 50 and 51 showed good druggability. This study provides a novel
series of GPR35 agonists and compound 50 may be a powerful tool to study GPR35..
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INTRODUCTION
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Historically, G proteinꢀcoupled receptors (GPCRs) are proven to be the largest
family of druggable targets, as 30ꢀ40% of all approved small molecule drugs directly
target this receptor family.1 GPCRs still remain to be one of the most active target
classes in drug discovery programs today, as these receptors play critical roles in a
great number of (patho)physiological processes. Given the historical success of
GPCRs as drug targets, there are strong interests in discovering lead compounds for
orphan GPCRs whose natural agonists remain to be unidentified.2 The orphan G
proteinꢀcoupled receptorꢀ35 (GPR35), first described in 1998,3 has been implicated in
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a number of diseases such as coronary artery disease,4 pain,5, cancers,7 and
hypertension.8
Kynurenic acid (1), an endogenous tryptophan metabolite, was the first GPR35
agonist described in literature.9 However, the weak potency of this compound in the
high micromolar range challenges the notion that kynurenic acid is the endogenous
agonist for GPR35.5, 10ꢀ12 Several other ligands were also postulated to be the natural
agonists for GPR35, including 2ꢀacyl lysophosphatidic acid,13 guanosineꢀ3′,5′ꢀcyclic
monophosphate,14 multiple tyrosine metabolites,13ꢀ15 and the mucosal chemokine
CXCL1716. Although the true natural agonist for GPR35 remains to be controversial,
several families of synthetic agonists have been identified including zaprinast (2)
which has become the most widely used reference agonist for GPR35.17, 18 Several
approved drugs were also found to be GPR35 agonists; for instance, the anticoagulant
dicoumarol (6),3 and the antiallergic drugs cromolyn (3), lodoxamide (4), amlexanox
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(5), and bufrolin (7) (Figure 1).14, 19ꢀ21 Pamoic acid (9), a salt component in several
pharmaceutical products, was also found to be a partial GPR35 agonist.3, 5 Recently,
several new families of GPR35 agonists, such as YE210 (8), were identified through
high throughput screening.22 However, there are a limited number of structure activity
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relationship
(SAR)
studies
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chromenꢀ4ꢀoneꢀ2ꢀcarboxylic acid derivatives (10). 23, 24
Here we report the synthesis and characterization of a series of coumarin
(2Hꢀchromenꢀ2ꢀone) derivatives as GPR35 agonists using labelꢀfree dynamic mass
redistribution (DMR) assays. The detailed SARs study exhibited that 3ꢀcarboxyl or
tetrazol coumarin derivatives could be novel potent GPR35 agonists.
RESULTS AND DISCUSSION
Structure Considerations
The natural products ellagic acid and dicoumarol were reported to activate the
GPR35 with moderate potency.3, 25 Several studies also suggested the importance of
carboxylic acid group or a phenolic structure for the agonistic activity of a compound
at the receptor.22 Thus, we designed and synthesized a series of derivatives depending
on the coumarin scaffold. By introducing an acidic group and a phenolic hydroxyl
group in the molecule, we finally obtained three subclasses of 3ꢀsubstituted coumarin
derivatives.
Chemistry
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A series of 3ꢀsubstituted coumarin derivatives were synthesized. All these
compounds were prepared from salicylaldehyde derivatives through Knoevenagel
condensation.26 The salicylaldehyde derivatives were reacted with bromine to obtain
compounds 11a and 11b (1:1 ratio),27, 28 or 12a and 12b (1:2.5 ratio) (Scheme 1).29
The nitrosalicylaldehyde derivatives 13a and 13b were obtained from 12a and 12b,
respectively, through nitration at 85oC.
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The 2ꢀoxoꢀ2Hꢀchromeneꢀ3ꢀcarboxylic acid derivatives 21-30 were prepared with
a good yield from 84% to 93% through the Knoevenagel condensation reaction of
salicylaldehyde derivatives and Meldrum’s acid (Scheme 2).26
Scheme 3 shows the synthesis route of 3ꢀtetrazolcoumarin compounds. First, 3ꢀ
cyanoꢀcoumarins 31ꢀ40 were prepared in oneꢀpot method as reported in literature.30
The 3ꢀ(1Hꢀtetrazolꢀ5ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone derivatives 41, 42, 44-49 were
synthesized from 3ꢀcyanoꢀcooumarins via the [3+2] cycloaddition of nitriles and
sodium azide catalyzed by aluminium chloride at 90oC with excellent yields
(85~97%).31 The compound 43 was obtained in a good yield of 94% by demethylation
of 44. To introduce nitro group into 3ꢀtetrazolcoumarin to synthesize compounds 50
and 51, the phenolic hydrxoy group of 35 was first reacted with choromethyl methyl
ether, followed by [3+2] cycloaddition of nitriles and sodium azide to form compound
49, which was subject to nitration to form compound 50 (1:1 with nitric acid) and 51
(1:3 with nitric acid), respectively. The purity of all these compounds was found to be
greater than 95% by analysis of high performance liquid chromatography (HPLC)
coupled to electrospray ionization mass spectrometry (LC/ESIꢀMS).
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Pharmacological Evaluation
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DMR assays afforded by labelꢀfree resonant waveguide grating biosensors were
applied to profile compound activity at the GPR35 endogenously expressed in
human colorectal adenocarcinoma cell line HTꢀ29.32 In previous studies, zaprinast
was used as a full agonist to perform DMR desensitization assay.32 As expected,
zaprinast triggered a robust DMR in HTꢀ29 cells with an EC50 of 0.34±0.025 ꢁM
(n=4). Furthermore, all 3ꢀsubstituted coumarin derivatives not only gave rise to a
doseꢀdependent DMR in HTꢀ29, but also desensitized the cells responding to 1 µM
zaprinast. For all 3ꢀsubstituted coumarins, the potency to trigger DMR was found to
be almost equivalent that to desensitize the zaprinast response (Table 1), suggesting
that these compounds acted as GPR35 agonists.
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DMR antagonist assay using the known GPR35 antagonist compound 11
(MLꢀ145) (Figure.1) further showed that compound 11 doseꢀdependently and
completely blocked the DMR arising from all 3ꢀsubstituted coumarins, each at its
respective EC80 to EC100 dose.33 The potency of compound 11 to block the
coumarinꢀinduced DMR was mostly similar (Table 1), suggesting that the DMR of
these cormarin compounds were specific to the activation of GPR35.
Structure-Activity Relationship analysis
Given the importance of introducing acidic group in determining the GPR35
agonistic activity,22, 34ꢀ36 we synthesized a series of 3ꢀsubstituted coumarin derivatives.
These compounds can be divided into three families according to the substituent
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group in R4, namely 3ꢀcarboxylic acidꢀcoumarins, 3ꢀcyanoꢀcoumarins, and
3ꢀ1Hꢀtetrazolꢀ5ꢀylꢀcoumarins (Table 1). For the parent compounds of each family,
compound 31 was inactive, while compound 41 had the highest potency (EC50 of 6.76
ꢁM), and compound 21 had the relatively low potency (EC50 of 92.26 ꢁM) (Figure 2).
The tetrazole and carboxy are isosteres in drug design. Since the carboxyl group has
certain stimulative activity in stomach, it is considered to be not a good druggable
group. In contrast, tetrazolyl has much lower stimulative activity than carboxy acid,
although it is also an acidic group because of its ionizable hydrogen ions.37 Because
of this, tetrazole has been used in antihypertensive,38 antiallergic,39 antiactrial40 and
antispasmodic drugs41.
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For 3ꢀcarboxylꢀcoumarin derivatives, consistent with a previous study23 was that
introducing halogen atoms (chlorine or bromine) in 6 or 6/8 positions of coumarins, as
indicated by compounds 22, 23, 24, 26, led to increased potency to activate the
GPR35. The potency rank order was found as follows for 3ꢀcarboxyl coumarin
derivatives: 6,8ꢀdibromo (26, EC50 of 1.75 ꢁM)＞6,8ꢀdichloro (24, 2.02 ꢁM)＞
6ꢀbromo (23, 10.76 ꢁM)＞6ꢀchloro (22, 13.52 ꢁM). This suggests that a lipophilic
pocket may exist in GPR35 and can be fitted with halogen atom(s).
Furthermore, introducing a hydroxyl group in 7ꢀposition of coumarins also had a
clear impact on their GPR35 agonistic activity, as indicated by the 10ꢀfold increase in
potency after introducing a hydroxyl group to the 7ꢀposition of compound 26 (27,
EC50 of 0.15 ꢁM). In contrast, replacing the hydroxyl group of compound 27 with the
methoxyl group led to decreased potency (28, EC50 of 0.89 ꢁM). Removal the
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8ꢀbromo of compound 27 also reduced its potency (25, EC50 of 4.34 ꢁM).
Furthermore, replacing 8ꢀbromine of compound 26 with nitro group also decreased its
potency (29, EC50 of 4.27 ꢁM). Unexpectedly, introducing hydroxyl group in
7ꢀposition of compound 29 greatly increased its potency (30, EC50 of 0.051 ꢁM),
which was much higher than compound 27.
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For 3ꢀcyanoꢀcoumarin derivatives, a different potency rank order was obtained
for halogenꢀmodified compounds: 6,8ꢀdichloro (33, EC50 of 8.61 ꢁM)＞6,8ꢀdibromo
(34, EC50 of 17.70 ꢁM)＞6ꢀchloro (32, EC50 of 21.43 ꢁM). Furthermore, unlike
3ꢀcarboxylꢀcoumarins, replacing the bromo group with nitro group at 8ꢀposition of
compound 34 significantly increased its potency by about 25ꢀfold (38, EC50 of 0.70
ꢁM). However, similar to 3ꢀcarboxylꢀcoumarin compounds, a potency increase was
found for introducing hydroxyl group in 7ꢀposition of compound 34 (36, EC50 of 2.33
ꢁM), while a potency decrease was found when the bromo group at 8ꢀposition of
compound 36 was removed to yield compound 35, or the hydroxyl group was
replaced with methoxy group at 7ꢀposition to yield compound 37.
For 3ꢀtetrazyl coumarin derivatives, the potency rank order among compounds
with varying substituents in the 6, 7, 8ꢀpostisions was: 6ꢀdromoꢀ8ꢀnitroꢀ7ꢀhydroxy (50,
EC50 of 0.0058 ꢁM)＞6,8ꢀdinitroꢀ7ꢀhydroxy (51, 0.014 ꢁM)＞6,8ꢀdibromoꢀ7ꢀhydroxy
(43, 0.068 ꢁM)＞6ꢀdibromoꢀ7ꢀhydroxy (49, 0.71 ꢁM) ≈ 6,8ꢀdibromo (42, 0.93 ꢁM) ≈
6,8ꢀdibromoꢀ7ꢀmethoxy (44, 1.41 ꢁM)＞6ꢀdromoꢀ8ꢀnitroꢀ7ꢀmethoxy (46, 2.66 ꢁM)＞
6ꢀdibromoꢀ7ꢀmethoxy (47, 14.76 ꢁM). The trend in potency for 6, 7, 8 position
substitutions was almost identical to those found for 3ꢀcyanoꢀcoumarin derivatives.
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β-Arrestin Translocation Induced by Selected Compounds
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Next, Tango GPR35 βꢀarrestin assay was used to examine the agonistic activity of
selected compounds (27, 30, 42, 43, 44, 50 and 51) to cause βꢀarrestin translocation
via GPR35. This assay uses Tango GPR35ꢀbla U2OS cells to detect GPR35
agonistꢀinduced recruitment of TEV protease tagged βꢀarrestin molecules to the
GPR35ꢀGal4ꢀVP16 transcription factor fusion protein linked by a TEV protease
cleavage site. Results showed that all these selected compounds resulted in a
doseꢀdependent response with a rightꢀshifted potency, compared to DMR assay results
(Figure 4, Table 2). Compounds 27, 30, 43, 50 and 51 all showed higher potency than
zaprinast in this translocation assay. In addition, the potency rank order (in EC50) was
50 (0.20ꢁM) ＞ 51 (0.29ꢁM) ＞ 30 (0.48ꢁM) > 43 (1.54 ꢁM) > 27 (3.63 ꢁM) > 42
(7.05 ꢁM) > 44 (14.06 ꢁM), which is to large degree the same as the potency rank
order obtained using DMR assays. These results suggested that these compounds were
agonists in the βꢀarrestin translocation assay.
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Receptor Selecivity
To assess the selectivity of these new GPR35 agonists versus other GPCRs, we
tested the representative compound 50 using DMR assays on other cell lines. The
results showed that 50 did not display activity on histamine H1 receptor, GPR109a,
β2ꢀadrenergic receptor, angiotensin AT1 receptor and bradykinin B2 receptor (Figure
S1 and Figure S2). Therefore, 50 had selectivity agonistic activity on GPR35.
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Physicochemical Properties of Selected Compounds
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Physicochemical parameters have been proposed to predict drugꢀlike properties
including the partition coefficient (clogP), the ligand efficiency (LE) and the
ligandꢀlipophilicity efficiency (LLE).42ꢀ44 We calculated these parameters for
compounds with relatively high potency, including compounds 26, 27, 28, 30, 36, 38,
42, 43, 44, 45, 50 and 51 (Table 3). Lipinski’s rule of five considered that the clogP
value should be lower than 5 if a compound is druggable.45, 46 The clogP values of all
these selected compounds were within this range. For perorally administered drugs,
the clogP value is often between 2 and 3. Most of these selected compounds exhibited
a clogP value between 2 and 3 or closed to this range.
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The LE is calculated as follows: LE = pEC50/N (N= nonꢀhydrogen atoms). This
parameter represents the binding force between per atom and receptors. For all of
these selected compounds except compound 44, a LE value is lower than 0.3. Another
useful parameter LLE which combines the potency and lipophilicity is also calculated
(LLE = pEC50ꢀclogP). Among these compounds, 30, 50 and 51 showed a good ligand
lipophilicity efficiency with a LLE value of 5.009, 6.495 and 6.935, respectively. For
the suitable drug candidates, LE＞0.3 and LLE＞5 could be optimal. Combining all
of these parameters, the highly potent compounds 30, 50 and 51 showed very good
clogP value, and suitable LE and LLE values.
Conclusion
In this article, a series of coumarin derivatives were synthesized based on the
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structure characteristics of GPR35 agonists reported previously. SAR analysis showed
that 6ꢀbromo substituent, 8ꢀnitro substituent, 7ꢀhydroxyl substituent and 3ꢀtetrazyl
substituent were found to significantly increase the potency of coumarin compounds
to activate the GPR35. Combining these substitutions resulted in compound 50 with
the highest potency found within this class of compounds (EC50, 5.8nM). Compound
50 also exhibited good physicochemical properties. Together, this study provides a
probe ligand to elucidate the biology and functions of GPR35.
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EXPERIMENTAL SECTION
General
All experimental reagents and solvents were obtained from various providers and
used without any additional purification or drying except for tetrahydrofuran, which
was distilled over calcium. The purity of all coumarin compounds was ≥ 95%. The
reactions were monitored by thin layer chromatography (TLC). If necessary, the
products were purified with column chromatography. NMR data were collected on a
Bruker Ascend 600 MHz NMR spectrometer at 600 MHz (1H) or 151 MHz (13C). The
chemical shifts were reported in parts per million (ppm) relative to the deuterated
1
solvent DMSOꢀd6; that is: δ H, 2.49 ppm; 13C, 39.7 ppm. Highꢀresolution mass
spectra (HRMS) were performed on an Agilent 1290 Infinity LC instrument (Agilent,
USA) coupled to an Agilent 6540 series QTOFꢀMS (Agilent, USA) equipped with an
ESI source, a diodeꢀarray detector (DAD), an automatic sample injector, a degasser
and a column thermostat.
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The purity of all final compounds analyzed by highꢀpressure liquid chromatography
(HPLC) was ＞ 95%. The determination of purity was conducted on a Waters
ACQUITY UPLCTM system (Waters Corp., Milford, MA, USA) with ACQUITY
UPLC® HSS T3 column (2.1×100 mm,1.8 ꢁm). Elution was performed with a
gradient of water/acetonitrile (containing 0.1% formic acid) from 95/5 to 5/95 for 10
min and maintained 5/95 for another 10min. The flow rate was 200ꢁL/min. Peaks
were detected at 290nm or 254nm.
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Synthesis of compounds 11a,11b, 12a, 12b, 13a and 13b
Compounds 11a, 11b, 12a and 12b were synthesized as described in literature.47
The filtrate was purified with column chromatography (8:2 DCM/PE).
5-Bromo-2-hydroxy-4-hydroxy-3-nitrobenzaldehyde
(13a)
and
5-bromo-2-hydroxy-4-methoxy-3-nitrobenzaldehyde (13b). To synthesize 13a and
13b, nitric acid (65%) (1.0 equiv) was added dropwise to a solution of
5ꢀbromoꢀ2,4ꢀdihydroxybenzaldehyde or 5ꢀbromoꢀ2ꢀhydroxyꢀ4ꢀmethoxybenzaldehyde
(1.0 equiv), respectively, in acetic acid at 85 oC for 3h. Catalytic amount of sulfuric
was added. The resulting mixture was poured into ice water. The precipitated product
was filtered, washed with water, and dried under vacuum. The crude solids were
recrystallized from methyl alcohol. The products were filtered off, and dried under
vacuum at 50 oC. The NMR data of all these benzaldehyde derivatives was shown in
support information.
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General procedures of method A for the synthesis of compounds 21ꢀ30
The appropriate salicylaldehyde derivatives (1.0 equiv) and Meldrum's acid (1.5
equiv) were mixed in 20ꢀ40 mL of water at room temperature. Catalytic amount of
ammonium acetate were added, and the reaction mixture was stirred under the room
temperature for 2ꢀ3h. Hydrochloric acid solution (2M) was added to adjust pH to 4ꢀ5.
The resulted solid was filtered, washed three times with 5 ml of methanol, and dried
under vacuum at 50 oC.
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General procedure of method B for the synthesis of compounds 31ꢀ40
The appropriate salicylaldehyde derivatives (1.0 equiv) and malononitrile (1.5
equiv) were mixed in 20ꢀ40 mL of water at room temperature. Catalytic amount of
ammonium acetate were added, and the reaction mixture was stirred under the room
temperature for 2ꢀ3h. The resulted solid was filtered and washed three times with 10
mL of water. Next, the products were added in 20 mL 3M hydrochloric acid solution,
and the reaction mixture was stirred at 75 oC for 30 min. After reaction, the material
was cooled to room temperature. The resulted solid was filtered, and washed three
times with 5 mL of methanol. If necessary, the filtrate was separated by column
chromatography (40:1 DCM/MeOH) to yield the pure compound and dried under
vacuum at 50 oC.
General procedure of method C for the synthesis of compounds 41ꢀ47
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Aluminum chloride (3.0 equiv) was added to 20 mL of anhydrous tetrahydrofuran.
Subsequently, sodium azide (6.0 equiv) and 3ꢀcyano coumarin derivatives (1.0 equiv)
were added in order. The mixure was refluxed for 5 hours with agitation under an
argon atmosphere. After the completion of the reaction, the reaction mixture was
extracted three times with excessive diluted hydrochloric acid to remove Al3+. The
organic phase was dried with anhydrous Na2SO4 and was concentrated under reduced
pressure until the product started to crystallize and cooled in iceꢀbath to complete the
crystallization. The product was filtered off, washed with modicum methyl alcohol
(MeOH), and dried under vacuum at 50 oC.
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2-oxo-2H-chromene-3-carboxylic acid (21).
21 was obtained from
2ꢀhydroxybenzaldehyde and Meldrum's acid as described for method A. 86% yield as
a white solid; 1H NMR (600 MHz, DMSOꢀd6) δ 13.27 (s, 1H), 8.76 (s, 1H), 7.92 (d, J
= 7.5 Hz, 1H), 7.74 (t, J = 7.5 Hz, 1H), 7.56 – 7.32 (m, 2H). 13C NMR (151 MHz,
DMSOꢀd6) δ 164.43, 157.16, 154.92, 148.81, 134.72, 130.64, 125.26, 118.80, 118.44,
116.57. Purity: 99.30%. High resolution MS (HRMS) for C10H6O4: calcd, 190.0281;
found, 189.0208 [MꢀH]ꢀ, 145.0317 [MꢀCOOH]ꢀ.
6-chloro-2-oxo-2H-chromene-3-carboxylic acid (22). 22 was obtained from
6ꢀchloroꢀ2ꢀhydroxybenzaldehyde and Meldrum's acid as described for method A. 91%
yield as a white solid; 1H NMR (600 MHz, DMSOꢀd6) 1H NMR (600 MHz, DMSO) δ
13.39 (s, 1H), 8.70 (s, 1H), 8.04 (s, 1H), 7.76 (d, J = 8.6 Hz, 1H), 7.48 (d, J = 8.6 Hz,
1H). 13C NMR (151 MHz, DMSOꢀd6) δ 164.22, 156.58, 153.59, 147.47, 134.09,
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129.46, 128.89, 120.06, 119.84, 118.65. Purity: 98.64%. HRMS for C10H5ClO4: calcd,
223.9882; found, 222.9809 [MꢀH]ꢀ, 224.9775 [MꢀH+2]ꢀ, 178.9915 [MꢀCOOH]ꢀ,
180.9876 [MꢀCOOH+2]ꢀ.
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6-bromo-2-oxo-2H-chromene-3-carboxylic acid (23). 23 was obtained from
6ꢀbromoꢀ2ꢀhydroxybenzaldehyde and Meldrum's acid as described for method A. 84%
yield as a white solid; 1H NMR (600 MHz, DMSOꢀd6) δ 8.66 (s, 1H), 8.16 (d, J = 2.4
Hz, 1H), 7.86 (dd, J = 8.8, 2.4 Hz, 1H), 7.41 (d, J = 8.8 Hz, 1H).13C NMR (151 MHz,
DMSOꢀd6) δ 164.29, 156.61, 153.93, 146.93, 136.70, 132.38, 120.41, 118.87, 116.66.
Purity: 99.01%. HRMS for C10H5BrO4: calcd, 267.9379; found, 266.9306 [MꢀH]ꢀ,
268.9286 [MꢀH+2]ꢀ, 222.9408 [MꢀCOOH]ꢀ, 224.9388 [MꢀCOOH+2]ꢀ.
6,8-dichloro-2-oxo-2H-chromene-3-carboxylic acid (24). 24 was obtained from
6,8ꢀdichloroꢀ2ꢀhydroxybenzaldehyde and Meldrum's acid as described for method A.
1
86% yield as a white solid; H NMR (600 MHz, DMSOꢀd6) δ 8.71 (s, 1H), 8.07 –
8.02 (m, 2H). 13C NMR (151MHz, DMSOꢀd6): 163.91, 155.56, 149.43, 147.29,
133.36, 128.73, 128.58, 121.20, 120.92, 120.69. Purity: 100%. HRMS for C10H4Cl2O4:
calcd, 257.9497; found, 256.9425 [MꢀH]ꢀ, 258.9392 [MꢀH+2]ꢀ, 212.9542 [MꢀCOOH]ꢀ,
214.9502 [MꢀCOOH+2]ꢀ.
6-bromo-7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid (25). 25 was obtained
from 11a and Meldrum's acid as described for method A. 84% yield as a white solid;
1H NMR (600 MHz, DMSOꢀd6) δ 13.02 (s, 1H), 11.92 (s, 1H), 8.65 (d, J = 3.6 Hz,
1H), 8.14 (d, J = 8.2 Hz, 1H), 6.88 (d, J = 3.6 Hz, 1H).13C NMR (151 MHz,
DMSOꢀd6) δ 164.48, 160.04, 157.28, 156.16, 148.71, 134.37, 114.36, 112.34, 107.11,
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102.94. Purity: 99.46%. HRMS for C10H5BrO5: calcd, 283.9320; found, 282.9347
[MꢀH]ꢀ, 284.9327 [MꢀH+2]ꢀ.
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8
9
6,8-dibromo-2-oxo-2H-chromene-3-carboxylic acid (26). 26 was obtained from
6,8ꢀdibromoꢀ2ꢀhydroxybenzaldehyde and Meldrum's acid as described for method A.
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89% yield as a white solid; H NMR(600 MHz, DMSOꢀd6) δ 13.52(s, 1H), 8.65(s,
1H), 8.36 – 8.08(m, 2H). 13C NMR (151 MHz, DMSOꢀd6) δ 163.93, 155.79, 150.87,
147.01, 138.59, 132.08, 121.40, 120.90, 116.67, 110.50. Purity: 99.17%. HRMS for
C10H4Br2O4: calcd, 345.8501; found, 344.8419 [MꢀH]ꢀ, 346.8411 [MꢀH+2]ꢀ, 348.8380
[MꢀH+4]ꢀ.
6,8-dibromo-7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid (27). 27 was
obtained from 12a and Meldrum's acid as described for method A. 93% yield as a
1
white solid; H NMR (600 MHz, DMSOꢀd6) δ 13.05 (s, 1H), 8.66 (s, 1H), 8.19 (s,
1H). 13C NMR (151 MHz, DMSOꢀd6) δ 164.23, 157.00, 156.64, 153.09, 148.52,
132.89, 114.91, 113.18, 108.19, 99.13. Purity: 96.25%. HRMS for C10H4Br2O5: calcd,
361.8460; found, 360.8374 [MꢀH]ꢀ, 362.8373 [MꢀH+2]ꢀ, 364.8335 [MꢀH+4]ꢀ.
6,8-dibromo-7-methoxy-2-oxo-2H-chromene-3-carboxylic acid (28). 28 was
obtained from 12b and Meldrum's acid as described for method A: 87% yield as a
white solid; 1H NMR (600 MHz, DMSOꢀd6) δ 8.69 (s, 1H), 8.30 (s, 1H), 3.91 (s, 3H).
13C NMR (151 MHz, DMSOꢀd6) δ 163.93, 158.12, 155.84, 152.56, 147.56, 133.15,
118.57, 117.51, 112.93, 106.25, 61.51. Purity: 99.20%. HRMS for C11H6Br2O5: calcd,
375.8582; found, 374.8516 [MꢀH]ꢀ, 376.8502 [MꢀH+2]ꢀ, 378.8477 [MꢀH+4]ꢀ,
330.8630[MꢀCOOH]ꢀ, 332.8629 [MꢀCOOH+2]ꢀ, 334.8590 [MꢀCOOH+4]ꢀ.
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6-bromo-8-nitro-2-oxo-2H-chromene-3-carboxylic acid (29). 29 was obtained from
5ꢀbromoꢀ2ꢀhydroxyꢀ3ꢀnitrobenzaldehyde and Meldrum's acid as described for method
A. 89% yield as a wihite solid; 1H NMR (600 MHz, DMSOꢀd6) δ 13.65 (s, 1H), 8.75
(s, 1H), 8.56 (d, J = 2.3 Hz, 1H), 8.51 (d, J = 2.3 Hz, 1H). 13C NMR (151MHz,
DMSOꢀd6): 163.63, 154.50, 146.81, 146.46, 137.64, 137.45, 131.36, 122.32, 120.91,
115.28. Purity: 100%. HRMS for C10H4BrNO6: calcd, 312.9234; found, 311.9161
[MꢀH]ꢀ, 313.9142 [MꢀH+2]ꢀ, 267.9342[MꢀCOOH]ꢀ, 269.9321 [MꢀCOOH+2]ꢀ.
6-bromo-7-hydroxy-8-nitro-2-oxo-2H-chromene-3-carboxylic acid (30). 30 was
obtained from 13a and Meldrum's acid as described for method A. 84% yield as a
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yellow solid; H NMR (600 MHz, DMSOꢀd6) δ 8.59 (s, 1H), 8.15 (s, 1H). 13C NMR
(151 MHz, DMSOꢀd6) δ 168.85, 164.55, 158.06, 156.91, 149.01, 148.94, 134.24,
129.89, 113.01, 108.78, 107.37. Purity: 99.51%. HRMS for C10H4BrNO7: calcd,
328.9171; found, 327.9241 [MꢀH]ꢀ, 329.9222 [MꢀH+2]ꢀ .
2-oxo-2H-chromene-3-carbonitrile
(31).
31
was
obtained
from
2ꢀhydroxybenzaldehyde and malononitrile as described for method B. 58% yield as a
yellow solid; 1H NMR (600 MHz, DMSOꢀd6) δ 8.95 (s, 1H), 7.84 – 7.78 (m, 2H),
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7.53 – 7.45 (m, 2H). C NMR (151 MHz, DMSOꢀd6) δ 157.35, 154.53, 153.93,
135.92, 130.45, 125.95, 117.98, 117.27, 115.07, 102.63. Purity: 98.69%. HRMS for
C10H5NO2: calcd, 171.0320; found, 172.0393 [M+H]+.
6-chloro-2-oxo-2H-chromene-3-carbonitrile (32). 32 was obtained from
6ꢀchloro2ꢀhydroxybenzaldehyde and malononitrile as described for method B. 64%
yield as a light yellow solid; 1H NMR (600 MHz, DMSOꢀd6) δ 8.86 (s, 1H), 7.92 (d, J
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= 2.6 Hz, 1H), 7.84 (dd, J = 8.9, 2.6 Hz, 1H), 7.57 (d, J = 8.9 Hz, 1H). 13C NMR
(151MHz, DMSOꢀd6): 156.92, 153.20, 152.56, 135.20, 129.56, 129.17, 119.33,
119.27, 114.80, 104.03. Purity: 98.99%. HRMS for C10H4ClNO2: calcd, 204.9931;
found, 238.0272 [M+CH3OH+H]+, 240.0239 [M+CH3OH+H+2]+.
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6,8-dichloro-2-oxo-2H-chromene-3-carbonitrile (33). 33 was obtained from
6,8ꢀdichloroꢀ2ꢀhydroxybenzaldehyde and malononitrile as described for method B. 71%
yield as a light yellow solid; 1H NMR (600 MHz, DMSOꢀd6) δ 8.87 (s, 1H), 8.17 (d, J
= 2.4 Hz, 1H), 7.90 (d, J = 2.4 Hz, 1H). 13C NMR (151 MHz, DMSOꢀd6) δ 156.13,
152.20, 149.02, 134.52, 129.48, 128.30, 121.89, 120.34, 114.47, 104.94. Purity:
96.28%. HRMS for C10H3Cl2NO2: calcd, 238.9541; found, 271.9897
[M+CH3OH+H]+, 273.9858 [M+CH3OH+H+2]+.
6,8-dibromo-2-oxo-2H-chromene-3-carbonitrile (34). 34 was obtained from
6,8ꢀdibromoꢀ2ꢀhydroxybenzaldehyde and malononitrile as described for method B. 65%
yield as a light yellow solid; 1H NMR (600 MHz, DMSOꢀd6) δ 8.86 (s, 1H), 7.92 (d, J
= 2.6 Hz, 1H), 7.84 (dd, J = 8.9, 2.6 Hz, 1H), 7.57 (d, J = 8.9 Hz, 1H). 13C NMR (151
MHz, DMSOꢀd6) δ 156.32, 152.17, 150.51, 139.84, 131.80, 120.77, 117.40, 114.48,
111.22, 104.79. Purity: 96.42%. HRMS for C10H3Br2NO2: calcd, 326.8531; found,
359.8871
[M+CH3OH+H]+,
361.8861
[M+CH3OH+H+2]+,
363.8831[M+CH3OH+H+4]+.
6-bromo-7-hydroxy-2-oxo-2H-chromene-3-carbonitrile (35). 35 was obtained from
6ꢀbromoꢀ2ꢀhydroxybenzaldehyde and malononitrile as described for method B. 58%
1
yield as a yellow solid; H NMR (600 MHz, DMSOꢀd6) δ 8.67 (s, 1H), 7.97 (s, 1H),
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6.87 (s, 1H). 13C NMR (151 MHz, DMSOꢀd6) δ 161.34, 157.60, 155.83, 152.58,
133.98, 115.43, 111.69, 108.11, 103.50, 97.56. Purity: 98.64%. HRMS for
C10H4BrNO3: calcd, 264.9393; found, 263.9320 [MꢀH]ꢀ, 265.9300 [MꢀH+2]ꢀ.
6,8-dibromo-7-hydroxy-2-oxo-2H-chromene-3-carbonitrile (36). 36 was obtained
from 12a and malononitrile as described for method B. 52% yield as a yellow solid;
1H NMR (600 MHz, DMSOꢀd6) δ 8.68 (s, 1H), 8.03 (s, 1H). (151 MHz, DMSOꢀd6) δ
158.81, 157.37, 152.96, 152.21, 132.45, 115.36, 112.26, 109.35, 99.82, 97.53. Purity:
95.19%. HRMS for C10H3Br2NO3: calcd, 342.8511; found, 341.8433 [MꢀH]ꢀ,
343.8420 [MꢀH+2]ꢀ, 345.8393 [MꢀH+4]ꢀ.
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6,8-dibromo-7-methoxy-2-oxo-2H-chromene-3-carbonitrile (37). 37 was obtained
from 12b and malononitrile as described for method B: 69% yield as a light yellow
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solid; H NMR (600MHz, DMSOꢀd6): δ 8.83(s, 1H), 8.19(d, 1H), 3.93(s, 3H). 13C
NMR (151MHz, DMSOꢀd6): 159.02, 156.30, 152.19, 152.11, 132.88, 117.08, 114.61,
113.67, 107.05, 102.83, 61.70. Purity: 98.81%. HRMS for C11H5Br2NO2: calcd,
356.8636; found, 389.8973 [M+CH3OH+H]+, 391.8958 [M+CH3OH+H+2]+,
393.8935 [M+CH3OH+H+4]+.
6-bromo-8-nitro-2-oxo-2H-chromene-3-carbonitrile (38). 38 was obtained from
5ꢀbromoꢀ2ꢀhydroxyꢀ3ꢀnitrobenzaldehyde and malononitrile as described for method B.
54% yield as a yellow solid; 1H NMR (600 MHz, DMSOꢀd6) δ 9.04 (s, 1H), 8.84 (d, J
= 2.5 Hz, 1H), 8.77 (d, J = 2.3 Hz, 1H).13C NMR (151 MHz, DMSOꢀd6) δ 155.93,
154.93, 152.30, 144.41, 132.30, 125.16, 119.23, 114.27, 110.86, 105.64. Purity:
97.94%. HRMS for C10H3BrN2O4: calcd, 293.9276; found, 326.9613
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[M+CH3OH+H]+, 328.9593 [M+CH3OH+H+2]+.
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6-bromo-7-methoxy-8-nitro-2-oxo-2H-chromene-3-carbonitrile (39). 39 was
obtained from 13b and malononitrile as described for method B. 58% yield as a
yellow solid; 1H NMR (600 MHz, DMSOꢀd6) δ 8.86 (s, 1H), 8.40 (s, 1H), 4.04 (s,
3H). 13C NMR (151 MHz, DMSOꢀd6) δ 155.14, 152.86, 151.49, 145.82, 135.94,
134.02, 116.62, 114.36, 112.75, 103.73, 63.97. Purity: 95.76%. HRMS for
C11H5BrN2O5: calcd, 323.9382; found, 356.9723 [M+CH3OH+H]+, 358.9704
[M+CH3OH+H+2]+.
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3-(1H-tetrazol-5-yl)-2H-chromen-2-one (41). 41 was obtained from 31 as described
for method C. 87% yield as a white solid; 1H NMR (600 MHz, DMSOꢀd6) δ 9.09 (s,
1H), 8.06 (dd, J = 7.7, 1.0 Hz, 1H), 7.83 – 7.78 (m, 1H), 7.58 (d, J = 8.3 Hz, 1H), 7.54
– 7.48 (m, 1H); 13C NMR (151 MHz, DMSOꢀd6) δ 158.56, 154.11, 150.10, 144.82,
134.41, 130.47, 125.70, 118.90, 116.85, 112.85. Purity: 100%. HRMS for C10H6N4O2:
calcd, 214.0497; found, 213.0424 [MꢀH]ꢀ.
6,8-dibromo-3-(1H-tetrazol-5-yl)-2H-chromen-2-one (42). 42 was obtained from
1
34 as described for method C. 89% yield as a white solid; H NMR (600 MHz,
DMSOꢀd6) δ 8.86 (s, 1H), 7.92 (d, J = 2.6 Hz, 1H), 7.84 (dd, J = 8.9, 2.6 Hz, 1H),
13
7.57 (d, J = 8.9 Hz, 1H); C NMR (151 MHz, DMSOꢀd6) δ 157.49, 150.89, 149.90,
142.20, 138.00, 131.72, 121.94, 117.15, 116.03, 110.68. Purity: 99.44%. HRMS for
C10H4Br2N4O2: calcd, 369.8726; found, 368.8651 [MꢀH]ꢀ, 370.8634 [MꢀH+2]ꢀ,
372.8612 [MꢀH+4]ꢀ.
Preparation of 6,8ꢀdibromoꢀ7ꢀhydroxyꢀ3ꢀ(1Hꢀtetrazolꢀ5ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone
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(43).The compound 44 (1.0 equiv) was dissolved in 10mL DCM, cooled to ꢀ78 C;
BBr3(3.0 equiv) was added dropwise. The reaction mixture was stirred in room
temperature overnight. After the reaction, 25mL of water was added, and the layers
were separated. And then extracted with 2 × 20 mL DCM. The combined organic
layers were dried over Na2SO4 and was concentrated under reduced pressure. The
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product was washed with modicum MeOH, and dried in vacuum at 50 C. H NMR
(600 MHz, DMSOꢀd6) δ 8.75 (s, 1H), 8.10 (s, 1H); 13C NMR (151 MHz, DMSOꢀd6) δ
161.60, 158.80, 153.34, 150.52, 143.84, 131.99, 111.71, 110.12, 103.43, 99.43. Purity:
100%. HRMS for C10H4Br2N4O3: calcd, 385.8679; found, 384.8603 [MꢀH]ꢀ, 386.8587
[MꢀH+2]ꢀ, 388.8564 [MꢀH+4]ꢀ.
6,8-dibromo-7-methoxy-3-(1H-tetrazol-5-yl)-2H-chromen-2-one (44). 44 was
1
obtained from 37 as described for method C. 96% yield as a yellow solid; H NMR
(600 MHz, DMSOꢀd6) δ 8.92 (s, 1H), 8.37 (s, 1H), 3.92 (s, 3H); 13C NMR (151 MHz,
DMSOꢀd6) δ 157.64, 157.62, 151.49, 150.45, 142.58, 132.76, 118.05, 113.74, 113.40,
106.52, 61.56. Purity: 98.94%. HRMS for C11H6Br2N4O3: calcd, 399.8834; found,
398.8760 [MꢀH]ꢀ, 400.8741 [MꢀH+2]ꢀ, 402.8720 [MꢀH+4]ꢀ.
6-bromo-8-nitro-3-(1H-tetrazol-5-yl)-2H-chromen-2-one (45). 45 was obtained
from 38 as described for method C. 91% yield as a yellow solid; 1H NMR (600 MHz,
DMSOꢀd6) δ 9.06 (s, 1H), 8.65 – 8.59 (m, 2H); 13C NMR (151 MHz, DMSOꢀd6) δ
156.42, 145.43, 142.38, 137.73, 137.35, 131.01, 122.68, 115.92, 115.53. Purity:
96.18%. HRMS for C10H4BrN5O4: calcd, 336.9467; found, 335.9393 [MꢀH]ꢀ,
337.9375 [MꢀH+2]ꢀ.
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6-bromo-7-methoxy-8-nitro-3-(1H-tetrazol-5-yl)-2H-chromen-2-one (46). 46 was
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obtained from 39 as described for method C. 89% yield as a yellow solid; H NMR
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(600 MHz, DMSOꢀd6) δ 9.02 (s, 1H), 8.63 (s, 1H), 4.04 (s, 3H); 13C NMR (151 MHz,
DMSOꢀd6) δ 156.32, 151.74, 149.89, 145.22, 142.27, 135.95, 134.04, 117.77, 114.06,
112.58, 63.93. Purity: 97.62%. HRMS for C11H6BrN5O5: calcd, 366.9585; found,
365.9513 [MꢀH]ꢀ, 367.9493 [MꢀH+2]ꢀ.
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6-bromo-7-methoxy-3-(1H-tetrazol-5-yl)-2H-chromen-2-one (47). 47 was obtained
from 40 as described for method C. 87% yield as a white solid; 1H NMR (600 MHz,
DMSOꢀd6) δ 8.96 (s, 1H), 8.31 (s, 1H), 7.36 (s, 1H), 4.01 (s, 3H); 13C NMR (151
MHz, DMSOꢀd6) δ 160.11, 158.51, 155.53, 150.05, 143.87, 133.51, 113.52, 109.95,
107.97, 101.22, 57.94. Purity: 97.97%. HRMS for C11H7BrN4O3: calcd, 321.9723;
found, 320.9650 [MꢀH]ꢀ, 322.9630 [MꢀH+2]ꢀ.
6-bromo-7-hydroxy-3-(1H-tetrazol-5-yl)-2H-chromen-2-one (49). 49 was obtained
from 48 as described for method C. 93% yield as a yellow solid; 1H NMR (600 MHz,
DMSOꢀd6) δ 8.87 (s, 1H), 8.19 (s, 1H), 7.00 (s, 1H); 13C NMR (151 MHz, DMSOꢀd6)
δ 159.69, 158.57, 155.12, 150.15, 144.14, 133.99, 112.69, 108.78, 107.56, 103.25.
Purity: 100%. HRMS for C10H5BrN4O3: calcd, 307.9576; found, 306.9503 [MꢀH]ꢀ,
308.9484 [MꢀH+2]ꢀ.
6-bromo-7-hydroxy-8-nitro-3-(1H-tetrazol-5-yl)-2H-chromen-2- one (50). Nitric
acid (65%) (1.0 equiv) was added dropwise to a solution of 49 (1.0 equiv) in acetic
acid at 85 oC for 1h. Catalytic amount of sulfuric was added. The resulting mixture
was concentrated under reduced pressure to give yellow solid. Then, the solid was
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collected by filtration and purified by recrystallization from petroleum ether/ethyl
acetate (1:2) to give title compounds as yellow solid and dried in vacuum at 50 oC. 1H
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NMR (600 MHz, DMSOꢀd6) δ 8.85 (s, 1H), 8.49 (s, 1H); C NMR (151 MHz,
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DMSOꢀd6) δ 160.20, 157.62, 150.38, 149.95, 146.00, 139.60, 133.58, 129.61, 102.38,
100.73. Purity: 97.99%. HRMS for C10H4BrN5O5: calcd, 352.9422; found, 351.9349
[MꢀH]ꢀ, 353.9330 [MꢀH+2]ꢀ.
7-hydroxy-6,8-dinitro-3-(1H-tetrazol-5-yl)-2H-chromen-2-one (51). Nitric acid
(65%) (1.0 equiv) was added dropwise to a solution of 50 (1.0 equiv) in acetic acid at
85 oC for 1h. Catalytic amount of sulfuric was added. The resulting mixture was
concentrated under reduced pressure to give yellow solid. Then, the solid was washed
with modicum petroleum ether to give the compound as yellow solid and dried in
vacuum at 50 oC. 1H NMR (600 MHz, DMSO) δ 9.02 (s, 1H), 8.75 (s, 1H); 13C NMR
(151 MHz, DMSO) δ 157.79, 155.49, 155.36, 149.95, 144.16, 135.73, 127.04, 110.91,
110.06, 101.82. Purity: 98.99%. HRMS for C10H4N6O7: calcd, 320.0141; found,
319.0107 [MꢀH]ꢀ.
Materials and Cell Culture
Zaprinast was obtained from Sigmaꢀaldrich. MLꢀ145 was obtained from Tocris. Epic®
384ꢀwell biosensor microplates were obtained from Corning Incorporated (Corning,
NY). HTꢀ29 cells were cultured at 37 oC, 5% CO2 in McCoy’s 5A Medium modified
with 10% FBS, 50 ꢁg/mL penicillin and 100 ꢁg/mL streptomycin.
DMR Assays Using Epic BT System
All DMR assays were performed using Epic BT system (Corning Incorporated). Epic
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is a swept wavelength interrogation reader system tailored for resonant waveguide
grating biosensors in microtiter plates48. Cells were directly seeded in Epic plates and
cultured overnight to form a confluent monolayer in the cell culture medium. After
being washed, the cells were maintained with Hank’s Balanced Salt Solution and
further incubated inside the system for 1 h. For agonist profiling, a 2 min baseline was
then established. After the compound addition, the cellular responses were recorded
immediately. For desensitization assays, cells were initially treated with compounds
for 1 h, followed by stimulation with zaprinast at 1ꢁM. The cellular responses were
recorded throughout the assays. All EC50 or IC50 described in the main text were
calculated based on the amplitudes of DMR signals at 8 min postꢀstimulation. All
GPR35 agonists led to a sustained positiveꢀDMR signal. The data represents mean ±
sd from two independent measurement, each with four replicates (n=8).
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Tango β-Arrestin Translocation Gene Reporter Assays
Tango GPR35ꢀbla U2OS cells was used. This cell line stably expresses two fusion
proteins: human GPR35 linked to a TEV protease site and a Gal4ꢀVP16 transcription
factor and βꢀarrestin/TEV protease fusion protein. The cell line also stably expresses
the βꢀlactamase reporter gene under the control of a UAS response element. The
activation of GPR35 by agonists leads to the recruitment of βꢀarrestin/TEV protease
fusion proteins to the activated GPR35. As a result, the protease cleaves the
Gal4ꢀVP16 transcription factor from the receptor, which then translocates to the
nucleus and activates the expression of βꢀlactamase. Briefly, 10000 cells per well
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were seeded in 384ꢀwell, blackꢀwall, clear bottom assay plates with low fluorescence
background (Corning) and cultured in Dulbecco’s Modified Eagle Medium
(Invitrogen, 10569_010) supplemented with 10% dialyzed fetal bovine serum, 0.1
ꢁM nonessential amino acids, 25 ꢁM Hepes (pH 7.3), 100 ꢁg/mL penicillin, and
100 ꢁg/mL streptomycin. After overnight culture, the cells were stimulated with
ligands for 5 h in a humidified 37 oC/5% CO2 and then loaded with the cell permeable
LiveBLAzer FRET B/G substrate. After the 2 h incubation, the coumarin:fluorescein
ratio was measured using Tecan Safire II microplate reader (Männedorf, Switzerland).
In the absence of βꢀlactamase expression (i.e., no GPR35 activation), cells generated
green fluorescence. In the presence of βꢀlactamase expression upon receptor
activation, the substrate was cleaved and the cells generated blue fluorescence. The
coumarin: fluorescein ratio was used as a normalized reporter response.
ASSOCIATED COTENT
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Supporting Information
The Supporting Information is available free of charge on the ACS Publications
website.
Synthetic procedures and 1H and 13C NMR spectral data for compounds 11a, 11b, 12a,
12b, 13a and 13b were shown in supporting information.
Molecular formula strings
AUTHOR INFORMATION
Corresponding Author
*For Xiuli Zhang: phone, +86 411 84379519; Eꢀmail, zhangxiuli@dicp.ac.cn.
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*For Xinmiao Liang: phone, +86 411 84379519; Eꢀmail, liangxm@dicp.ac.cn,
*For Ye Fang: phone, +1ꢀ607ꢀ9747203; Eꢀmail, fangy2@corning.com.
ABBREVIATIONS USED
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GPR35, G proteinꢀcoupled receptor 35; GPCR, G proteinꢀcoupled receptor; SARs,
structure activity relationships; DMR, dynamic mass redistribution; HPLC, high
performance liquid chromatography; THF, tetrahydrofuran; LE, ligand efficiency;
LLE, ligandꢀlipophilicity efficiency; HRMS, highꢀresolution mass spectra.
ACKNOWLEDGMENT
This work was supported by “Project of National Science Foundation of China (81473
436) and Project of International Cooperation Plan from Ministry of Science and Tec
hnology of China (2015DFG32260). We also thank the supporting by the State Key Pr
ogram of National Natural Science of China (Grant No.U1508221 ) and Strategic Prio
rity Research Program of the Chinese Academy of Sciences, Personalized Medicines
——Molecular Signatureꢀbased Drug Discovery and Development (XDA12040314).
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