Discovery of phenylsulfonyl acetic acid derivatives with improved efficacy and safety as potent free fatty acid receptor 1 agonists for the treatment of type 2 diabetes

Article abstract of DOI:10.1016/j.ejmech.2017.07.001

The free fatty acid receptor 1 (FFA1) has emerged as an attractive anti-diabetic target that mediates glucose-stimulated insulin secretion. Several FFA1 agonists have been reported, but many of them possessed somewhat high lipophilicity and/or molecular weight. Herein, we describe the identification of sulfone-carboxylic acid moiety with the multiple advantages of reducing lipophilicity, cytotoxicity and β-oxidation associated with compound 2. Further structure-activity relationship study based on the previleged scaffolds led to the discovery of 2-{(4-[(2’-chloro-[1,1’-biphenyl]-3-yl)methoxy]phenyl)sulfonyl}acetic acid (compound 20), which showed a better balance than compound 2 in terms of physicochemical properties, cytotoxicity profiles and pharmacokinetic properties. Subsequent in vivo studies demonstrated that compound 20 robustly improves the glucose tolerance both in normal and type 2 diabetic models without the risk of hypoglycemia. Compared to the high risk of TAK-875 induced liver toxicity, there was no significant adverse effects such as hepatic and renal toxicity were observed in the chronic toxicity studies of compound 20 even at the higher dose.

Full text of DOI:10.1016/j.ejmech.2017.07.001

Accepted Manuscript
Discovery of phenylsulfonyl acetic acid derivatives with improved efficacy and safety
as potent free fatty acid receptor 1 agonists for the treatment of type 2 diabetes
Zheng Li, Chunxia Liu, Xue Xu, Qianqian Qiu, Xin Su, Yuxuan Dai, Jianyong Yang,
Huilan Li, Wei Shi, Chen Liao, Miaobo Pan, Wenlong Huang, Hai Qian
PII:
S0223-5234(17)30523-8
10.1016/j.ejmech.2017.07.001
EJMECH 9564
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 April 2017
Revised Date: 15 June 2017
Accepted Date: 2 July 2017
Please cite this article as: Z. Li, C. Liu, X. Xu, Q. Qiu, X. Su, Y. Dai, J. Yang, H. Li, W. Shi, C. Liao, M.
Pan, W. Huang, H. Qian, Discovery of phenylsulfonyl acetic acid derivatives with improved efficacy
and safety as potent free fatty acid receptor 1 agonists for the treatment of type 2 diabetes, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.001.
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ACCEPTED MANUSCRIPT
Discovery of Phenylsulfonyl Acetic Acid Derivatives with Improved Efficacy and Safety as
Potent Free Fatty Acid Receptor 1 Agonists for the Treatment of Type 2 Diabetes
a
a
a
a
a
a
a
Zheng Li , Chunxia Liu , Xue Xu , Qianqian Qiu , Xin Su , Yuxuan Dai , Jianyong Yang , Huilan
a
a
a
a
a, b*
a, b*
Li , Wei Shi , Chen Liao , Miaobo Pan , Wenlong Huang , Hai Qian
a
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China.
b
Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
Graphical Abstract
Aiming to develop potent agonist with improved efficacy and safety, we have identified series of
FFA1 agonists bearing sulfone-carboxylic acid moiety, exemplified by the potent and orally
bioavailable agonist 20.
∗
Co-corresponding authors: Wenlong Huang and Hai Qian, Centre of Drug Discovery, State Key Laboratory of
Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China.
Tel: +86-25-83271050; Fax: +86-25-83271050.
E-mail: ydhuangwenlong@126.com (W.l. Huang), qianhai24@163.com (H. Qian).

ACCEPTED MANUSCRIPT
Discovery of Phenylsulfonyl Acetic Acid Derivatives with Improved Efficacy and Safety as
Potent Free Fatty Acid Receptor 1 Agonists for the Treatment of Type 2 Diabetes
a
a
a
a
a
a
a
Zheng Li , Chunxia Liu , Xue Xu , Qianqian Qiu , Xin Su , Yuxuan Dai , Jianyong Yang , Huilan
a
a
a
a
a, b*
a, b*
Li , Wei Shi , Chen Liao , Miaobo Pan , Wenlong Huang , Hai Qian
a
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China.
b
Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
ABSTRACT: The free fatty acid receptor 1 (FFA1) has emerged as an attractive anti-diabetic
target that mediates glucose-stimulated insulin secretion. Several FFA1 agonists have been
reported, but many of them possessed somewhat high lipophilicity and/or molecular weight.
Herein, we describe the identification of sulfone-carboxylic acid moiety with the multiple
advantages of reducing lipophilicity, cytotoxicity and β-oxidation associated with compound 2.
Further structure-activity relationship study based on the previleged scaffolds led to the discovery
∗
Co-corresponding authors: Wenlong Huang and Hai Qian, Centre of Drug Discovery, State Key Laboratory of
Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China.
Tel: +86-25-83271050; Fax: +86-25-83271050.
E-mail: ydhuangwenlong@126.com (W.l. Huang), qianhai24@163.com (H. Qian).
1

ACCEPTED MANUSCRIPT
of 2-{(4-[(2'-chloro-[1,1'-biphenyl]-3-yl)methoxy]phenyl)sulfonyl}acetic acid (compound 20),
which showed a better balance than compound 2 in terms of physicochemical properties,
cytotoxicity profiles and pharmacokinetic properties. Subsequent in vivo studies demonstrated that
compound 20 robustly improves the glucose tolerance both in normal and type 2 diabetic models
without the risk of hypoglycemia. Compared to the high risk of TAK-875 induced liver toxicity,
there was no significant adverse effects such as hepatic and renal toxicity were observed in the
chronic toxicity studies of compound 20 even at the higher dose.
Keywords: Free fatty acid; FFA1; Insulin secretion; Type 2 diabetes mellitus
1
. Introduction
Type 2 diabetes mellitus (T2DM) is a progressive chronic disease characterized by impaired
insulin secretion and increased insulin resistance in different tissues, resulting in chronic
hyperglycemia.[1, 2] Persistent hyperglycemia is considered a high risk factor for macro- and
microvascular complications including nephropathy, coronary heart disease and atherosclerosis.[3]
Thus, it is crucially important for T2DM patients to control glucose homeostasis throughout their
lives. At present, there are many inspiring drugs for T2DM, mainly including insulin, insulin
secretagogue and sensitizer, glucose absorption and reabsorption inhibitors.[4] However, it is still
difficult for most of T2DM patients to maintain perfect glycemic control due to limited efficacy of
current hypoglycemic drugs and associated some adverse effects (e.g., hypoglycemia, body weight
gain and gastric symptoms).[5-8] Hence, a novel drug that have increased durability and safety
would be advantageous for T2DM management.[9, 10] The free fatty acid receptor 1 (FFA1,
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previously designated as GPR40), a new antidiabetic target highly expressed in pancreatic β-cells,
plays a key role in enhancing insulin secretion only in the state of hyperglycemia.[11-14] FFA1
mainly couples with the G protein α-subunit of G family (G ) until activated by agonist, the
q
αq
dissociated Gαq protein activates phospholipase C, leading to the increased level of inositol
triphosphate and diacylglycerol. The inositol triphosphate enhances the release of intracellular
2+
2+
Ca , whereas diacylglycerol, alone or combine with intracellular Ca , activates the protein kinase
C and D1. All of these pathways are related to improved insulin secretion in a glucose
concentration-dependent manner.[15-17] Therefore, the glucose-dependent insulinotropic effect of
FFA1 agonists provides enormous potentiality for reducing blood glucose level without the risk of
hypoglycemia.
As summarized in our recent review [18], several series of FFA1 agonists have already been
reported by many drug discovery teams (representative examples in Figure 1),[19-30] and the
candidates TAK-875 and AMG-837 have reached in clinical trials. More recently, Phase II clinical
trials have provided clinical proof-of-concepts for the glucose control in T2DM. The 12 weeks
oral treatment of TAK-875 in T2DM leads to a reduction in HbA1c superior to that of DPP4
inhibitors and equivalent to that of glimepiride with a lower risk of hypoglycemia.[31, 32]
However, Takeda terminated the Phase III clinical trial of TAK-875 due to the risk of liver
toxicity.[33, 34] It is widely speculated that the hepatotoxicity is most likely to be
compound-related since the expression of FFA1 in the liver is quite low.[12, 35-37] Indeed, some
FFA1 agonists have somewhat high lipophilicity and/or molecular weight even beyond the
“
Rule-of-Five” (red mark in the Figure 1), which were considered negative properties for
candidates as it is correlated with metabolic instability, poor absorption, off-target toxicity and
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thereby higher risk of attrition.[38-42] To counteract this, the concepts of ligand efficiency (LE)
and ligand lipophilicity efficiency (LLE) have been extensively implemented to avoid highly
lipophilic and oversized compounds in the optimization process.[43, 44] Considering these factors,
our previous study explored several series of polar building blocks including thiazole,[45]
pyrrole,[46] triazole,[47] oxime ether[48] and phenoxyacetamide linker[49]. In this study, the
Takeda’s compound 2 (Figure 1) was chosen as lead structure, because it has robust efficacy and
simple scaffold to avoid excessive increased molecular size in the optimization process. However,
compound 2 has been suffering from relatively high lipophilicity (ClogP = 5.876) and poor
pharmacokinetic (PK) profiles as a result of β-oxidation in phenylpropanoic acid moiety.[21] As
shown in Figure 2, the process of β-oxidation proceeds through the CoA ester which is
dehydrogenated to afford the cinnamic acid A1 and ultimately lead to the production of benzoic
acid A2,[21, 50] and the reaction process can be interrupted by several classic strategies including
α- and/or β-substitution or the incorporation of a heteroatom to replace the β-carbon.[51, 52]
Moreover, the researchers of Takeda have shown that the cytotoxicity appears to be roughly
related to the lipophilicity of compound 2 series, and the LogD value below 2.8 would be
preferable to minimize the potential risk of cytotoxicity.[23, 53] With the dual purposes of
improving lipophilicity and β-oxidation of compound 2, a practical strategy was developed by
introducing a sulfone group to block the β-oxidation of phenylpropionic acid (Figure 2).
Herein, we report our research efforts toward the identification of 2-(phenylsulfonyl)acetic
acid derivatives and the subsequent structure-activity relationship (SAR) study based on the
previleged scaffolds of current FFA1 agonists. These efforts ultimately led to the discovery of
2
-{(4-[(2'-chloro-[1,1'-biphenyl]-3-yl)methoxy]phenyl)sulfonyl}acetic acid (compound 20) with
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a >29-fold higher plasma exposure than compound 2. Additionally, compound 20 successfully
overcame the cytotoxicity issues in human hepatocyte HepG2 cells associated with compound 2.
The in vivo efficacy and safety including the risk of hypoglycemia and chronic toxicity studies
were also assessed in rodent models.
2
. Results and Discussion
2
.1. Chemistry
The synthetic routes of biphenyl derivatives 3-5 and 11-32 are summarized in Scheme 1. The key
intermediates 2a-2r, 5a and 5b were synthesized by Suzuki coupling reactions of commercially
available (3-formylphenyl)boronic acid or (4-formylphenyl)boronic acid with appropriate
bromobenzene (1a-1r, 4a or 4b) in the presence of Pd(PPh ) . Reduction of the aldehyde group in
3
4
intermediates 2a-2r, 5a and 5b with sodium borohydride followed by treating with thionyl
chloride provide the chlorinated intermediates 3a-3r, 6a and 6b. The commercially starting
material 4-methoxybenzenethiol 7a was condensed with methyl chloroacetate to afford the
intermediate 8a, which was further converted to 9a by demethylation with boron tribromide. The
intermediate 9a was condensed with 3a by Williamson ether synthesis, followed by basic
hydrolysis of ester, afforded the target compound 3. Oxidation of the thioether 8a with oxone,
gave sulfone 10a, which was treated with boron tribromide to give the desired phenol 11a.
Subsequently, condensing the chlorinated intermediates 3a-3r with phenol 11a in the presence of
K CO , followed by hydrolysis, furnished the desired carboxylic acids 4, 5 and 11-26. The
2
3
synthesis of target compounds 27-30 were accomplished via alkylation of the methyl ester 12a and
subsequently basic hydrolysis. The 3-methoxybenzenethiol 13a was converted to the desired
compounds 31 and 32 by the identical reaction sequence as compound 4.
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The synthesis of compounds 6 to 10 are depicted in Scheme 2. The commercially starting
material 3-methoxybenzenethiol 13a was condensed with methyl 4-chloroacetoacetate to afford
the intermediate 16a, which was converted to compound 17a by intramolecular cyclisation,
reduction and demethylation. Condensation of 17a with chlorinated intermediate 3b afforded the
desired ester 18a, which was further converted to sulfone 19a by oxidation with m-CPBA. Basic
hydrolysis of 18a and 19a with lithium hydroxide gave the target compounds 6 and 7 in high yield.
The starting material 20a was treated with ethylacetoacetate in the presence of catalytic amount of
piperidine for 48 h at room temperature, followed by acidic hydrolysis with KOH in 90% ethanol
to provide the diacid 21a. Compound 21a was treated by intramolecular Friedeld-Crafts
cyclization with polyphosphoric acid, followed by demethylation to afford the ketonic product 22a.
Condensing the chlorinated intermediate 3b with 22a furnished the common intermediate 23a,
which was treated with lithium hydroxide to get compound 8. Reduction of the carbonyl group in
compound 23a with sodium borohydride followed by esterolysis gave compound 9. The simple
mixing of 23a and methoxyamine hydrochloride in DMSO followed by basic hydrolysis provided
compound 10.
The target compounds 33-46 were synthesized as outlined in Scheme 3. The substituted
thiobenzamides 24a-f was condensed with ethyl 2-chloroacetoacetate to form the substituted
thiazole 25a-f.[54] The ester 25a-f and 30a were reduced with sodium borohydride in methanol,
followed by treating with thionyl chloride to generate the chlorinated intermediates 26a-f and 31a.
The starting material 2-bromoacetophenone 27a was treated with methoxyamine hydrochloride to
afford the intermediate 28a, which was further condensed with 4-hydroxybenzyl alcohol and
converted to chlorinated intermediate 29a. The pyrrole 33a was obtained by Paal-Knorr pyrrole
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synthesis with methyl 3-aminobenzoate and acetonylacetone. The ester 33a was reduced with
sodium borohydride, followed by treating with PBr in pyridine to generate the intermediate 34a.
3
The key intermediates 37a-e were prepared by Suzuki coupling reactions of commercially
available 35a with substituted bromobenzene (36a-e). Reduction of the aldehydes 37a-e with
sodium borohydride followed by treating with thionyl chloride provides the chlorinated
intermediates 38a-e. Condensing the intermediates (26a-f, 29a, 31a, 34a and 38a-e) with phenol
1
1a in the presence of K CO , followed by hydrolysis, provide the desired carboxylic acids 33-46.
2
3
2
.2. FFA1 agonistic activity and SAR study
2
+
The agonistic activities of target compounds were investigated by monitoring Ca signal using a
fluorometric imaging plate reader (FLIPR) assay. Besides agonistic activity, the LE and LLE, as
well as LogD7.4 were taken advantage of in the optimization process. Moreover, the cytotoxicity of
selected compounds was also evaluated by detecting the caspase-3/7 activity, a biomarker of cell
apoptosis and the larger value represented a higher risk of apoptotic cytotoxicity.
To suppress the β-oxidation of compound 2, we firstly focused on blocking β-position of
phenylpropanoic acid moiety by either replacing the β-carbon (compounds 3-5, Table 1) or
constructing fused-ring (compounds 6-10). Introduction of sulphur in the β-position of the acidic
portion remarkably reduced the potency (compound 2 vs 3). Interestingly, an approximately 3-fold
increase in agonistic activity over compound 3 was provided only by switching the sulphur to
sulphone in compound 4, which had lipophilicity decreased by more than one log unit and thereby
a higher LLE value than compound 2. Besides, methoxy substitution at the para-position of
terminal dimethylbiphenyl ring (compound 5) was shown to slightly improve the agonistic activity
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for human FFA1. It should be noted that the introduction of sulphone moiety, as seen in
compounds 4 and 5, provided the advantage of markedly decreasing the cytotoxicity that was
likely due to the reduced lipophilicity (LogD : 2.49 and 2.45, respectively). The sulphone
7
.4
derivative 7, however, turned out a dramatic loss of potency compared to the parent compound 6,
a bioisostere of TAK-875. One possible explanation is that the limited space in the ligand-binding
pocket around the sulphone position of compound 7. Indeed, the flexible hydroxy (compound 9)
and bulkier oxime ether (compound 10) gave a pronounced drop in activity, whereas smaller
ketone (compound 8) was tolerated in this position.
Having identified a favorable sulfone-carboxylic acid moiety with improved lipophilicity and
modest potency, we next shifted our focus on comprehensive exploration of SAR in this series. As
shown in Table 2, the agonistic activity of 5 (Me) > 11 (Et) ＞ 12 (Pr) ＞ 14 (c-PrCH ) ＞ 13
2
(
i-Bu) ＞ 15 (MeOC H ) > 16 (MsOC H ) suggested that the steric effect in the biphenyl
2 4 3 6
para-position might influence potency for FFA1, and the previous SAR of TAK-875 series is not
directly transferrable to the present series.[23] Gratifyingly, the monomethyl analog 17, compared
to the parent compound 4, resulted in a 1.4-fold increased potency and a marked improvement in
terms of lipophilicity and LE value. The positive result encouraged us to attach varies of
substituents at the biphenyl 2-position to further investigate the SAR of present series.
Introduction of smaller 2-hydrogen (18) or 2-fluoro group (19) drastically decreased the potency
compared with 2-methyl derivative 17, while incorporation of a bulkier group such as 2-chloro
(20), 2-trifluoromethyl (21) or 2-isopropyl (22) maintained the potent activity. Exceptionally, the
steric substitution 2-methoxy (23) or 2-nitrile (24) turned out a dramatic drop in potency despite a
distinct advantage in terms of lipophilicity. Moreover, in terms of bulkier group, as the size of
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group increases the activity decrease (compound 20 > 17 > 21 > 4 > 22), indicating that the
modest dihedral angle between biphenyl was also very important for agonistic activity. Next, we
evaluated the additional substituents at the biphenyl para-position of the most potent derivative 20.
Introducing a para-methyl substituent (25) maintained the agonistic activity but unfortunately
exhibited cytotoxicity at 30 µM in the caspase assay. Incorporation of ethyoxyl substituent (26) led
to a decrease of activity back to the level of compound 11 and a drop in LLE as a result of
increased lipophilicity. Consistent with the previous findings,[23, 53] the selected compounds (17,
1
8 and 20) with low LogD7.4 value (< 2.8) tend to have a lower risk of activating caspase-3/7.
Further incorporation of alkyl group in α-position of acid moiety afforded compounds 27-30,
while none of them showed the desired potency, likely suggesting that the bulkier alkyl group
moves the carboxylic acid into an unfavorable conformation. Inspired by the discovery process of
full agonists AM-1638,[26] we also relocated the orientation of ether spacer from 4- to 3-position
and the connection of biphenyl from 3’- to 4’-position, but the obtained 31 and 32 almost lost the
agonistic activity for FFA1.
To further decrease the lipophilicity, the previous reported thiazole scaffold was introduced as
bioisostere of middle phenyl (Table 3).[45, 55] As expected, an approximately one log unit
decrease of lipophilicity was observed on the replacement of the middle phenyl by thiazole (18 vs
3
3) but unfortunately also markedly decreased potency (4 vs 33). From the results of fluoro scan
in the terminal phenyl (compound 34-36), the 4- and 3-position seemed to be better than the
-position to retain agonistic activity, which shared a similar SAR with our previous reported
2
thiazole series.[45] Furthermore, several previous optimal substituents were also incorporated in
compound 37 and 38. Although a significantly reduced lipophilicity and relatively high LLE value,
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none of these derivatives explored (33-38) exhibited the desired activity. To expand the SAR study,
series of previleged scaffolds reported in current agonists were introduced to replace the terminal
phenyl (compounds 39-42, Table 4). The oxime ether 39 and pyrrole 41, two optimal scaffolds in
our previous studies,[46, 48] only revealed moderate agonistic activity for human FFA1.
Compound 40, a hybrid structure combining sulfone moiety and GW9508,[55] exhibited the best
activity in the terminal hydrophobic scaffolds (compounds 39-42). In terms of lipophilicity and
LLE value, we ultimately selected the isoxazole derivative 42 as our starting point despite a lower
potency compared to compound 40. Based on the reported SAR of isoxazole series,[56] the
preferred methyl and chlorine substituents were located to the favorable 2-, 4- and 6-position of
middle phenyl (compound 43-46). Although all these analogs exhibited desired lipophilicity and
LLE value, the agonistic activities were found to be rather low (hEC50 > 200 nM). One possible
rational explanation is that the need for lipophilicity to enter the binding pocket of FFA1 via the
lipid bilayer,[57] which preclude the entrance of agonists with low lipophilicity.
2
.3. Molecular modeling study
To better understand the binding mode of optimal compound 20, a molecular docking study based
on the reported co-crystal complex of FFA1 (PDB code: 4PHU) was developed by using MOE.[57]
As shown in Figure 3A, compound 20 fit very tightly into the same binding pocket as TAK-875
despite the presence of the different acidic head in our series, and the model can also explain some
SAR reasonably. As with TAK-875, the acidic moiety of compound 20 formed anchor point with
residues Arg183 and Arg2258 by strong H-bond interactions. Moreover, Trp174 is oriented
roughly perpendicular to the plane of benzene sulfone where an edge-on interaction was formed
1
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(Figure 3A). These essential interactions render the SAR of present series very sensitive to even
small structural changes (compound 27-30). Interestingly, the chloro of biphenyl moiety may form
halogen bonding with carbonyl oxygen of Pro80 (d = 3.4 Å, Figure 3B)[58, 59], consistent with
the better activity of chloro substituent compared to other kinds of substituents in this series.
2
.4. Pharmacokinetic evaluation of compound 20
Next, compound 20 was further evaluated for oral PK parameters in rats (Table 5). As we
expected, compound 20 (10 mg/kg) presented superior PK profiles, in particular, a higher
maximum concentration (Cmax = 2563.52 µg/L), lower clearance (CL = 0.154 L/h/kg) and longer
plasma half-life (T1/2 = 5.57 h), resulting in a > 29-fold higher exposure (AUC0−24h = 30204.43
µg/L·h) than compound 2 (10 mg/kg, AUC0−24h = 1024.23 µg/L·h). Indeed, the β-oxidation
metabolites of compound 2 such as cinnamic acid A1 and benzoic acid A2 were detected as major
components in rat plasma as previous reported.[21, 50] However, the β-oxidation metabolite was
not observed in rat plasma after oral administration of compound 20 (10 mg/kg), and the main
component in rat plasma was unchanged compound 20. In addition, a few of biphenylylcarboxylic
acid B1 was detected in plasma as a first-pass metabolite derived from cytochrome P450 oxidation
(Figure 4). The taurine conjugate B2 and acyl glucuronide B3 were presumed to be major
metabolites of compound 20 in the rat plasma (Figure 4). The more detailed metabolic analysis of
compound 20 will be presented elsewhere. These results indicated that our strategy to replace the
β-carbon with sulphone indeed decreased β-oxidation of phenylpropanoic acid moiety, resulting in
an improved PK profile. Moreover, the superior PK parameters noted with sulphone derivative 20
may also be attributed to the decreased first-pass drug metabolism due to the lower lipophilicity of
1
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compound 20.
2
.5. Dose-response relationship of compound 20
Based on its excellent FFA1 agonistic activity, PK properties and minimal risk of cytotoxicity,
compound 20 was selected and evaluated for its potential to improve the glucose tolerance in
Sprague−Dawley (SD) rats. Single oral administration of 20 robustly reduced the plasma glucose
excursion (Figure 5A) and enhanced insulin secretion (Figure 5B) during an oral glucose
tolerance test (OGTT) in a dose-dependent manner from 1 to 10 mg/kg when compound 20 was
dosed 60 min prior to the oral glucose challenge. The area under the curve of blood glucose
(AUC0−120min) and blood insulin (AUC0−120min) revealed that the minimum effective dose of
compound 20 was 3 mg/kg (Figure 5C and D, respectively).
2
.6. Glucose-lowering effects of compound 20 explored in type 2 diabetic mice
Moreover, the in vivo efficacy of compound 20 was also assessed by the OGTT in male HF/STZ
mice, a high-fat fed and low-dose streptozotocin-treated type 2 diabetic model with insulin
resistance and impaired insulin secretion.[60] As shown in Figure 6, the hyperglycemia state was
markedly improved in compound 20 (20 mg/kg) treated group, and the plasma glucose
AUC0−120min was better than that of TAK-875 (20 mg/kg) and comparable with Liraglutide (12
µg/kg), a well-known long-lasting glucagon-like peptide 1 agonist.
2
.7. Effects of compound 20 on the risk of hypoglycemia
Obtaining these promising pharmacological profiles, the risk of hypoglycemia was also evaluated
in the fasting SD rats by oral administrating a high dose of compound 20 (30 mg/kg) in
1
2

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comparison with glibenclamide (10 mg/kg), a widely used sulfonylurea insulin secretagogue. As
shown in Figure 7, no statistical difference was found in the fasting glucose levels between
vehicle and compound 20-treated groups despite the dramatic hypoglycemic side effects exist in
the glibenclamide-treated group. Thus, these results suggested that compound 20 showed a low
risk of hypoglycemia, a common adverse effect of sulfonylureas.
2
.8. Effects of compound 20 on the risk of chronic toxicity
To assess the liver and renal toxicity, a 30-days chronic toxicity study was subsequently evaluated
in normal ICR mice. At the end of chronic toxicity study, several parameters indicative of liver
and renal function including ALT (alanine aminotransferase), AST (aspartate transaminase), TBIL
(total bilirubin), BUN (blood urea nitrogen) and Scr (serum creatinine) in serum were measured by
using automatic biochemical analyzer. As shown in Table 6, the serum levels of ALT and AST
were significantly increased in TAK-875 (20 mg/kg) treated group despite the liver enzyme levels
has not reached three times of normal reference, likely indicating a high risk of TAK-875-induced
liver toxicity. Gratifyingly, no significant differences were found in these parameters between
vehicle and compound 20-treated groups even at the higher dose of 30 mg/kg, preliminary
indicating the hypotoxicity of compound 20 to the liver and kidney. Furthermore, no particular
adverse effects were observed in ICR mice after 30-days of daily oral administration, even at the
dose of 30 mg/kg.
3
. Conclusion
Aiming to develop potent agonists with decreased lipophilicity and molecular size, we have
identified series of FFA1 agonists bearing sulfone-carboxylic acid moiety, exemplified by the
1
3

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potent and orally bioavailable agonist 20. The important aspects that result in the discovery of
compound 20 were as follows: (1) the introduction of sulfone in the β-position of phenylpropanoic
acid moiety not only maintained the agonistic activity but also attained the dual purposes of
reducing lipophilicity and β-oxidation, and thereby superior PK profiles compared to that of
parent compound 2; (2) systematic exploration of SAR and cytotoxicity study allowed us to
achieve a better balance between potency and drug-likeness. Further in vivo studies demonstrated
that compound 20 not only robustly improves glucose tolerance both in normal and type 2 diabetic
models, but also reveals a low risk of hypoglycemia in normal fasting rats even at the high dose.
Compared to the high risk of liver toxicity induced by TAK-875, there was no significant side
effects including the hepatic and renal toxicity were observed in a chronic toxicity study of
compound 20 even at the higher dose. Potent and safe FFA1 agonist, such as compound 20, is
expected as a promising candidate for further development of novel therapeutics for T2DM.
4
. Experimental section
4
.1. General chemistry
All starting materials, reagents and solvents were obtained from commercial sources and used
without further purification unless otherwise indicated. Purifications by column chromatography
were carried out over silica gel (200-300 mesh) and monitored by thin layer chromatography
performed on GF/UV 254 plates and were visualized using UV light at 254 and 365 nm. Melting
points were taken on a RY-1 melting-point apparatus and were uncorrected. NMR spectra were
1
13
recorded on a Bruker ACF-300Q instrument (300 MHz for H NMR and 75 MHz for C NMR
spectra), chemical shifts are expressed as values (ppm) relative to tetramethylsilane as internal
standard, and coupling constants (J values) were given in hertz (Hz). Abbreviations are used as
1
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follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublets of doublet, br
=
broad. LC/MS spectra were recorded on a Waters liquid chromatography-mass spectrometer
system (ESI). Analytical HPLC was performed on a Shimadzu LC-20AT instrument, equipped
with Shimadzu C18 reversed-phase column (5 µm, 150 mm × 4.6 mm) with a 20 min linear
gradient from 70/30 to 5/95 and subsequently with a 7 min isocratic elution 5/95 A/B, where A =
H O−0.05% TFA, B = CH CN, at a flow rate of 1 mL/min, with UV detection at 254 nm, at
2
3
column temperature of 25 °C. Elemental analyses were performed by the Heraeus CHN-O-Rapid
analyzer and were within 0.4% of the theoretical values unless otherwise noted. The purity of
compounds was assessed by elemental analysis or analytical HPLC (>95%). Liraglutide (Victoza)
was purchased from Novo Nordisk, TAK-875 and compound 2 were synthesized as previously
reported.[21, 22]
General synthetic procedure for compounds 3a-r, 6a and 6b. Step 1: The substituted
bromobenzene (1 equiv) and (3-formylphenyl)boronic acid or (4-formylphenyl) boronic acid (1
equiv) were dissolved in a mixture of 1 M sodium carbonate aqueous solution (15 mL), EtOH
(
5mL) and toluene (15 mL). After nitrogen substitution, Pd(PPh ) (0.05 equiv) was added. The
3 4
reaction mixture was stirred at 80 °C under nitrogen atmosphere for 12 h. The reaction mixture
was cooled, and water (15 mL) was added. The mixture was diluted with AcOEt (15 ml), and the
insoluble material was filtered off through Celite. The organic layer of the filtrate was washed
with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was
purified by silica gel column chromatography using a mixture of petroleum ether/ethyl acetate
(10:1, v/v) as eluent to afford the desired product 2a-r, 5a and 5b as a solid. Step 2: To a solution
of 2a-r, 5a or 5b (1 equiv) in MeOH (10 mL) and THF (20 mL) was added portionwise sodium
1
5

ACCEPTED MANUSCRIPT
borohydride (3 equiv) at 0 °C and the mixture was stirred at 0 °C for 1 h. The reaction mixture
was pouring into ice water (10 ml), and extracted with ethyl acetate (3 × 15 mL), the organic
fractions were combined, washed with saturated brine (2 × 15 ml) prior to drying over anhydrous
sodium sulfate. After filtration and concentrate using a rotary evaporator, the residue was used in
next step without further purification. To a solution of the obtained solid (1 equiv) in
dichloromethane (20 ml) was slowly added thionyl chloride (6 equiv) and a catalytic amount of
DMF at room temperature. After stirring at 40 °C for 4 h, the reaction was concentrated under
reduced pressure. The residue was purified by silica gel column chromatography using a mixture
of petroleum ether/ethyl acetate (20:1, v/v) as eluent to afford the desired product.
1
3
'-(chloromethyl)-2,6-dimethyl-1,1'-biphenyl (3a). Yield: 65%; H NMR (300 MHz,
CDCl ) δ: 7.44–7.39 (m, 1H), 7.34 (d, J = 7.9 Hz, 1H), 7.07–7.19 (m, 5H), 4.52 (s, 2H), 1.97 (s,
3
6
H).
1
3
'-(chloromethyl)-4-methoxy-2,6-dimethyl-1,1'-biphenyl (3b). Yield: 54%; H NMR (300
MHz, CDCl ) δ: 7.43–7.38 (m, 1H), 7.34 (d, J = 7.6 Hz, 1H), 7.12 (s, 1H), 7.06–7.10 (m, 1H),
3
6
.82 (s, 2H), 4.52 (s, 2H), 3.75 (s, 3H), 1.96 (s, 6H).
'-(chloromethyl)-4-ethoxy-2,6-dimethyl-1,1'-biphenyl (3c). Yield: 59%; H NMR (300
MHz, CDCl ) δ: 7.44–7.39 (m, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7.14 (s, 1H), 7.05–7.10 (m, 1H),
1
3
3
6
.82 (s, 2H), 4.52 (s, 2H), 4.02 (q, J = 6.7 Hz, 2H), 1.96 (s, 6H), 1.31 (t, J = 6.7 Hz, 3H).
1
3
'-(chloromethyl)-2,6-dimethyl-4-propoxy-1,1'-biphenyl (3d). Yield: 53%; H NMR (300
MHz, CDCl ) δ: 7.44–7.39 (m, 1H), 7.34 (d, J = 7.9 Hz, 1H), 7.14 (s, 1H), 7.05–7.10 (m, 1H),
3
6
.82 (s, 2H), 4.52 (s, 2H), 3.93 (t, J = 6.5 Hz, 2H), 1.92 (s, 6H), 1.73–1.69 (m, 2H), 0.99 (t, J = 7.3
Hz, 3H).
1
6

ACCEPTED MANUSCRIPT
1
3
'-(chloromethyl)-4-isobutoxy-2,6-dimethyl-1,1'-biphenyl (3e). Yield: 46%; H NMR (300
MHz, CDCl ) δ: 7.42–7.37 (m, 1H), 7.35 (d, J = 7.9 Hz, 1H), 7.13 (s, 1H), 7.06–7.11 (m, 1H),
3
6
6
.83 (s, 2H), 4.53 (s, 2H), 3.74 (d, J = 6.2 Hz, 2H), 1.92 (s, 6H), 1.39–1.16 (m, 1H), 0.99 (d, J =
.4 Hz, 6H).
Methyl 2-((4-methoxyphenyl)thio)acetate (8a). To a solution of 4-methoxybenzenethiol (0.88
ml, 7.13 mmol) and methyl chloroacetate (1.0 g, 9.27 mmol) in acetone was added K CO (2.96 g,
2
3
2
1.4 mmol) at room temperature. The reaction mixture was heated to reflux for 2 h. Then the
reaction mixture was cooled to room temprature followed by filtration and the filtrate was
concentrated under vacuum. The residue was purified by silica gel column chromatography using
a mixture of petroleum ether/ethyl acetate (20:1, v/v) as eluent to afford 8a (1.35 g, 96%) as a
1
colorless oil. H NMR (300 MHz, DMSO-d ) δ: 7.35 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 8.5 Hz, 2H),
6
3
.78 (s, 3H), 3.65 (s, 2H), 3.57 (s, 3H).
Methyl 2-((4-hydroxyphenyl)thio)acetate (9a). To a solution of 8a (1.0 g, 4.71 mmol) in
dichloromethane (20 ml) was slowly added BBr (2.36 g, 9.42 mmol) through a dropping funnel at
3
-78 °C. After addition was complete, the solution was keeping the temperature at -78 °C for 2 h,
and then, the mixture was stirred at room temperature for 16 h. After quenching with saturated
NH Cl aqueous solution (20 ml), the mixture was extracted with dichloromethane (3 × 15 mL),
4
and the combined organic phases were washed with brine (2 × 15 mL), dried and filtered. The
filtrate was evaporated under vacuum and the residue was purified by column chromatography
using a mixture of petroleum ether/ethyl acetate (10:1, v/v) as eluent to afford 9a (0.82 g, 88%) as
1
7

ACCEPTED MANUSCRIPT
1
a white solid. H NMR (300 MHz, DMSO-d ) δ: 10.26 (s, 1H), 7.37 (d, J = 8.6 Hz, 2H), 7.03 (d, J
6
=
8.6 Hz, 2H), 3.65 (s, 2H), 3.57 (s, 3H).
2
-((4-((2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)thio)acetic acid (3). To a solution
of 9a (0.26 g, 1.31 mmol) and 3a (0.36 g, 1.57 mmol) in acetone was added K CO (0.36 g, 2.62
2
3
mmol) and a catalytic amount of KI at room temperature. The reaction mixture was heated to
reflux with stirring overnight. Then the reaction mixture was cooled to room temprature followed
by filtration and the filtrate was concentrated under vacuum. The residue was purified by silica gel
column chromatography using a mixture of petroleum ether/ethyl acetate (5:1, v/v) as eluent to
afford a white solid. To a solution of the obtained solid (0.43 g, 1.1 mmol ) in 2:3:1
THF/MeOH/H O (18 ml) was added LiOH·H O (0.01 g, 3.3 mmol). After stirring at room
2
2
temperature for 4 h, the volatiles were removed under reduced pressure. The residue was acidified
with 1N hydrochloric acid solution, and then filtered and the filter cake was washed with 5 mL of
water, dried in vacuum to afford a white powder. The white powder was purified by column
chromatography using a mixture of petroleum ether/ethyl acetate (4:1–3:2, v/v) as eluent to give
1
the desired compound 3 (0.33 g, 78%) as a white solid, HPLC purity: 99.7%; m.p. 93–95 °C. H
NMR (300 MHz, DMSO-d ) δ: 7.51–7.41 (m, 2H), 7.35 (d, J = 8.5 Hz, 2H), 7.21–7.08 (m, 5H),
6
1
3
7
1
1
.00 (d, J = 8.5 Hz, 2H), 5.16 (s, 2H), 3.65 (s, 2H), 1.94 (s, 6H). C NMR (75 MHz, DMSO-d ) δ:
6
70.71, 157.51, 141.04, 140.43, 137.16, 135.13, 131.78, 128.69, 128.24, 127.99, 127.26, 127.03,
-
26.04, 125.82, 115.69, 69.17, 37.10, 20.46. ESI-MS m/z: 377.1 [M-H] . Anal. calcd. For
C H O S: C, 72.99; H, 5.86; Found: C, 72.95; H, 5.87.
2
3
22
3
1
8

ACCEPTED MANUSCRIPT
Methyl 2-((4-methoxyphenyl)sulfonyl)acetate (10a). To a solution of 8a (5 g, 23.56 mmol) in
MeOH (200 mL) was added dropwise a solution of Oxone (29.0 g, 47.12 mmol) in water (150 mL)
at 0 °C, and the mixture was stirred at 0 °C to room temperature for 20 h. MeOH was evaporated.
The residue was extracted with ethyl acetate (4 × 45 mL), and the combined organic phases were
washed with brine (2 × 25 mL), dried and filtered. The filtrate was evaporated under vacuum and
the residue was purified by column chromatography using a mixture of petroleum ether/ethyl
1
acetate (4:1, v/v) as eluent to afford 10a (5.2 g, 90%) as a colorless oil. H NMR (300 MHz,
DMSO-d ) δ: 7.72 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 4.47 (s, 2H), 3.79 (s, 3H), 3.59 (s,
6
3
H).
Methyl 2-((4-hydroxyphenyl)sulfonyl)acetate (11a). To a solution of 10a (5.0 g, 20.5 mmol) in
dichloromethane (50 ml) was slowly added BBr (15.4 g, 61.5 mmol) through a dropping funnel at
3
-78 °C. After addition was complete, the solution was keeping the temperature at -78 °C for 2 h,
and then, the mixture was stirred at room temperature for 16 h. After quenching with saturated
NH Cl aqueous solution (40 ml), the mixture was extracted with dichloromethane (3 × 25 mL),
4
and the combined organic phases were washed with brine (2 × 20 mL), dried and filtered. The
filtrate was evaporated under vacuum and the residue was purified by column chromatography
using a mixture of petroleum ether/ethyl acetate (3:1, v/v) as eluent to afford 11a (4.1 g, 87%) as a
1
white solid. H NMR (300 MHz, DMSO-d ) δ: 10.67 (s, 1H), 7.72 (d, J = 8.7 Hz, 2H), 6.97 (d, J =
6
8
.7 Hz, 2H), 4.47 (s, 2H), 3.59 (s, 3H).
General synthetic procedure for target compounds 4, 5 and 11-26. To a solution of 11a (1
1
9

ACCEPTED MANUSCRIPT
equiv) and intermediates 3a-r (0.8 equiv) in acetone was added K CO (2 equiv) and a catalytic
2
3
amount of KI at room temperature. The reaction mixture was heated to reflux with stirring
overnight. Then the reaction mixture was cooled to room temprature followed by filtration and the
filtrate was concentrated under vacuum. The residue was purified by silica gel column
chromatography using a mixture of petroleum ether/ethyl acetate (4:1, v/v) as eluent to afford a
white solid. To a solution of the obtained solid (1 equiv) in 2:3:1 THF/MeOH/H O (18 ml) was
2
added LiOH·H O (3 equiv). After stirring at room temperature for 4 h, the volatiles were removed
2
under reduced pressure. The residue was acidified with 1N hydrochloric acid solution, and then
filtered and the filter cake was washed with 5 mL of water, dried in vacuum to afford a white
powder. The white powder was purified by column chromatography using a mixture of petroleum
ether/ethyl acetate (2:1–1:2, v/v) as eluent to afford the target compounds as white solid.
2
-((4-((2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)sulfonyl)acetic acid (4). Yield 69%;
1
white solid, HPLC purity: 98.6%; m.p. 125–127 °C. H NMR (300 MHz, DMSO-d ) δ: 7.83 (d, J
6
13
=
8.4 Hz, 2H), 7.55–7.40 (m, 2H), 7.24–7.07 (m, 7H), 5.25 (s, 2H), 4.14 (s, 2H), 1.93 (s, 6H). C
NMR (75 MHz, DMSO-d ) δ: 164.95, 162.44, 141.43, 140.99, 137.03, 135.60, 132.43, 130.78,
6
1
29.26, 128.95, 128.61, 127.76, 127.55, 126.68, 115.36, 70.04, 62.56, 20.97. ESI-MS m/z: 365.1
-
[
M-45] . Anal. calcd. For C H O S: C, 67.30; H, 5.40; Found: C, 67.34; H, 5.41.
2
3
22
5
2
-((4-((4'-methoxy-2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)sulfonyl)acetic
acid
1
(
5). Yield 73%; white solid, HPLC purity: 99.3%; m.p. 111–112 °C. H NMR (300 MHz,
DMSO-d ) δ: 7.81 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 7.8 Hz, 2H), 7.25–7.04 (m, 4H), 6.70 (s, 2H),
6
1
3
5
.25 (s, 2H), 3.88 (s, 2H), 3.75 (s, 3H), 1.93 (s, 6H). C NMR (75 MHz, DMSO-d ) δ: 163.87,
6
2
0

ACCEPTED MANUSCRIPT
1
7
5
62.49, 158.41, 140.89, 136.99, 136.88, 134.00, 133.19, 129.58, 129.18, 126.46, 115.91, 113.13,
-
0.07, 55.36, 44.40, 21.19. ESI-MS m/z: 395.1 [M-45] . Anal. calcd. For C H O S: C, 65.44; H,
2
4
24
6
.49; Found: C, 65.41; H, 5.48.
2
-((4-((4'-ethoxy-2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)sulfonyl)acetic acid (11).
1
Yield 65%; white solid, HPLC purity: 98.5%; m.p. 107–109 °C. H NMR (300 MHz, DMSO-d ) δ:
6
7
.82 (d, J = 8.6 Hz, 2H), 7.56–7.37 (m, 2H), 7.34–7.02 (m, 4H), 6.68 (s, 2H), 5.27 (s, 2H), 4.20 (s,
1
3
2
H), 4.02 (q, J = 6.7 Hz, 2H), 1.92 (s, 6H), 1.31 (t, J = 6.7 Hz, 3H). C NMR (75 MHz, DMSO-d )
6
δ: 163.90, 162.08, 157.18, 140.43, 136.48, 136.38, 133.40, 131.63, 130.27, 129.10, 128.78, 128.66,
-
1
26.03, 115.43, 114.95, 113.14, 69.57, 62.71, 61.55, 20.72, 14.71. ESI-MS m/z: 409.1 [M-45] .
Anal. calcd. For C H O S: C, 66.06; H, 5.77; Found: C, 66.09; H, 5.76.
2
5
26
6
2
-((4-((2',6'-dimethyl-4'-propoxy-[1,1'-biphenyl]-3-yl)methoxy)phenyl)sulfonyl)acetic
acid
1
(
12). Yield 68%; white solid, HPLC purity: 99.5%; m.p. 93–94 °C. H NMR (300 MHz,
DMSO-d ) δ: 7.82 (d, J = 8.5 Hz, 2H), 7.56–7.37 (m, 2H), 7.26–7.04 (m, 4H), 6.68 (s, 2H), 5.27 (s,
6
2
3
1
H), 4.19 (s, 2H), 3.93 (t, J = 6.5 Hz, 2H), 1.92 (s, 6H), 1.73–1.69 (m, 2H), 0.99 (t, J = 7.3 Hz,
1
3
H). C NMR (75 MHz, DMSO-d ) δ: 164.57, 162.01, 157.36, 140.44, 136.48, 136.35, 133.41,
6
30.28, 129.11, 128.65, 126.02, 114.89, 113.18, 69.57, 68.68, 62.54, 22.08, 20.72, 10.41. ESI-MS
-
m/z: 423.1 [M-45] . Anal. calcd. For C H O S: C, 66.65; H, 6.02; Found: C, 66.68; H, 6.03.
2
6
28
6
2
-((4-((4'-isobutoxy-2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)sulfonyl)acetic acid
1
(
13). Yield 52%; white solid, HPLC purity: 98.2%; m.p. 101–102 °C. H NMR (300 MHz,
2
1

ACCEPTED MANUSCRIPT
DMSO-d ) δ: 7.82 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 7.7 Hz, 2H), 7.30–7.00 (m, 4H), 6.69 (s, 2H),
6
5
6
1
.27 (s, 2H), 4.08 (s, 2H), 3.74 (d, J = 6.2 Hz, 2H), 1.92 (s, 6H), 1.39–1.16 (m, 1H), 0.99 (d, J =
1
3
.4 Hz, 6H). C NMR (75 MHz, DMSO-d ) δ: 164.53, 162.01, 157.47, 140.44, 136.47, 136.38,
6
33.40, 132.69, 129.10, 128.69, 125.96, 115.42, 113.20, 73.52, 69.57, 43.90, 27.71, 20.66, 19.03.
-
ESI-MS m/z: 437.1 [M-45] . Anal. calcd. For C H O S: C, 67.20; H, 6.27; Found: C, 67.25; H,
2
7
30
6
6
.28.
2
-((4-((4'-(cyclopropylmethoxy)-2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)sulfonyl)
1
acetic acid (14). Yield 56%; white solid, HPLC purity: 97.8%; m.p. 97–99 °C. H NMR (300
MHz, DMSO-d ) δ: 7.82 (d, J = 7.7 Hz, 2H), 7.56–7.35 (m, 2H), 7.17–7.06 (m, 4H), 6.68 (s, 2H),
6
5
2
1
7
6
.25 (s, 2H), 4.11 (s, 2H), 3.79 (d, J = 5.9 Hz, 2H), 1.91 (s, 6H), 1.39–1.35 (m, 1H), 0.58–0.55 (m,
1
3
H), 0.32–0.28 (m, 2H). C NMR (75 MHz, DMSO-d ) δ: 164.59, 162.75, 156.12, 141.44, 140.93,
6
36.93, 132.76, 131.07, 130.76, 128.95, 127.84, 126.91, 126.34, 118.41, 115.30, 113.63, 72.22,
-
0.08, 61.20, 21.23, 10.69, 3.57. ESI-MS m/z: 435.1 [M-45] . Anal. calcd. For C H O S: C,
2
7
28
6
7.48; H, 5.87; Found: C, 67.43; H, 5.86.
General synthetic procedure for target compounds 4, 5 and 11-26. To a solution of 11a (1
equiv) and intermediates 3a-r (0.8 equiv) in acetone was added K CO (2 equiv) and a catalytic
2
3
amount of KI at room temperature. The reaction mixture was heated to reflux with stirring
overnight. Then the reaction mixture was cooled to room temprature followed by filtration and the
filtrate was concentrated under vacuum. The residue was purified by silica gel column
chromatography using a mixture of petroleum ether/ethyl acetate (4:1, v/v) as eluent to afford a
2
2

ACCEPTED MANUSCRIPT
white solid. To a solution of the obtained solid (1 equiv) in 2:3:1 THF/MeOH/H O (18 ml) was
2
added LiOH·H O (3 equiv). After stirring at room temperature for 4 h, the volatiles were removed
2
under reduced pressure. The residue was acidified with 1N hydrochloric acid solution, and then
filtered and the filter cake was washed with 5 mL of water, dried in vacuum to afford a white
powder. The white powder was purified by column chromatography using a mixture of petroleum
ether/ethyl acetate (2:1–1:2, v/v) as eluent to afford the target compounds as white solid.
2
-((4-((4'-(2-methoxyethoxy)-2',6'-dimethyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)sulfonyl)ace
1
tic acid (15). Yield 56%; white solid, HPLC purity: 97.5%; m.p. 86-88 °C. H NMR (300 MHz,
DMSO-d ) δ: 7.82 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 7.9 Hz, 2H), 7.31-7.00 (m, 4H), 6.70 (s, 2H),
6
1
3
5
.24 (s, 2H), 4.16 (s, 2H), 4.07-4.02 (m, 4H), 3.64 (s, 3H), 1.91 (s, 6H). C NMR (75 MHz,
DMSO-d ) δ: 164.25, 162.33, 157.61, 140.87, 137.02, 134.67, 132.58, 130.73, 129.13, 127.53,
6
1
4
26.51, 126.03, 115.27, 114.99, 113.66, 70.89, 69.25, 67.13, 63.52, 58.61, 21.22. ESI-MS m/z:
-
39.1 [M-45] . Anal. calcd. For C H O S: C, 64.45; H, 5.82; Found: C, 64.41; H, 5.81.
2
6
28
7
2
-((4-((2',6'-dimethyl-4'-(3-(methylsulfonyl)propoxy)-[1,1'-biphenyl]-3-yl)methoxy)phenyl)su
1
lfonyl)acetic acid (16). Yield 67%; white solid, HPLC purity: 98.6%; m.p. 117-119 °C. H NMR
(
300 MHz, DMSO-d ) δ: 7.82 (d, J = 8.1 Hz, 2H), 7.56-7.40 (m, 2H), 7.36-7.03 (m, 4H), 6.71 (s,
6
2
H), 5.26 (s, 2H), 4.18 (s, 2H), 4.09 (t, J = 5.9 Hz, 2H), 3.37-3.20 (m, 2H), 3.03 (s, 3H), 2.27-2.09
1
3
(
m, 2H), 1.92 (s, 6H). C NMR (75 MHz, DMSO-d ) δ: 164.20, 162.53, 156.78, 140.81, 137.08,
6
1
34.26, 132.17, 130.78, 129.16, 128.72, 126.59, 115.40, 113.75, 70.04, 65.84, 62.06, 51.00, 40.67,
-
2
2.51, 21.22. ESI-MS m/z: 501.1 [M-45] . Anal. calcd. For C H O S : C, 59.32; H, 5.53; Found:
2
7
30
8 2
C, 59.36; H, 5.52.
2
3

ACCEPTED MANUSCRIPT
2
-((4-((2'-methyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)sulfonyl)acetic acid (17). Yield 75%;
1
white solid, HPLC purity: 99.3%; m.p. 115-117 °C. H NMR (300 MHz, DMSO-d ) δ: 7.84 (d, J
6
13
=
8.5 Hz, 2H), 7.55-7.42 (m, 3H), 7.39-7.16 (m, 7H), 5.29 (s, 2H), 4.34 (s, 2H), 2.21 (s, 3H). C
NMR (75 MHz, DMSO-d ) δ: 164.50, 162.78, 141.41, 140.92, 136.68, 135.29, 133.62, 130.86,
6
1
29.95, 129.15, 128.94, 127.89, 126.90, 126.44, 115.49, 70.08, 61.20, 20.59. ESI-MS m/z: 351.1
-
[
M-45] . Anal. calcd. For C H O S: C, 66.65; H, 5.09; Found: C, 66.61; H, 5.08.
2
2
20
5
2
-((4-([1,1'-biphenyl]-3-ylmethoxy)phenyl)sulfonyl)acetic acid (18). Yield 79%; white solid,
1
HPLC purity: 99.5%; m.p. 110-112 °C. H NMR (300 MHz, DMSO-d ) δ: 7.90-7.75 (m, 3H),
6
1
3
7
.71-7.66 (m, 3H), 7.50-7.46 (m, 4H), 7.42-7.23 (m, 3H), 5.30 (s, 2H), 4.37 (s, 2H). C NMR (75
MHz, DMSO-d ) δ: 178.88, 164.54, 137.43, 137.38, 130.94, 130.87, 129.71, 129.48, 128.12,
6
-
1
27.43, 127.18, 126.92, 126.72, 115.60, 70.18, 61.17. ESI-MS m/z: 337.1 [M-45] . Anal. calcd.
For C H O S: C, 65.95; H, 4.74; Found: C, 65.92; H, 4.73.
21
18
5
2
-((4-((2'-fluoro-[1,1'-biphenyl]-3-yl)methoxy)phenyl)sulfonyl)acetic acid (19). Yield 71%;
1
white solid, HPLC purity: 98.6%; m.p. 112-114 °C. H NMR (300 MHz, DMSO-d ) δ: 7.85 (d, J
6
=
7.7 Hz, 2H), 7.65 (s, 1H), 7.60-7.39 (m, 5H), 7.30 (d, J = 7.6 Hz, 2H), 7.21 (d, J = 7.7 Hz, 2H),
1
3
5
.28 (s, 2H), 4.06 (s, 2H). C NMR (75 MHz, DMSO-d ) δ: 164.51, 162.03, 136.67, 135.27,
6
1
32.76, 130.77, 129.67, 129.18, 128.85, 128.50, 128.13, 127.24, 124.94, 116.25, 115.94, 115.31,
-
6
9.50, 43.89. ESI-MS m/z: 355.1 [M-45] . Anal. calcd. For C H FO S: C, 62.99; H, 4.28; Found:
2
1
17
5
C, 62.93; H, 4.27.
2
4

ACCEPTED MANUSCRIPT
2
-((4-((2'-chloro-[1,1'-biphenyl]-3-yl)methoxy)phenyl)sulfonyl)acetic acid (20). Yield 68%;
1
white solid, HPLC purity: 99.9%; m.p. 99-100 °C. H NMR (300 MHz, DMSO-d ) δ: 13.03 (s,
6
1
H), 7.85 (d, J = 8.8 Hz, 2H), 7.58-7.51 (m, 4H), 7.45-7.40 (m, 4H), 7.27 (d, J = 8.8 Hz, 2H), 5.30
1
3
(
s, 2H), 4.40 (s, 2H). C NMR (75 MHz, DMSO-d ) δ: 164.60, 162.86, 139.89, 139.35, 136.71,
6
1
6
6
31.96, 131.51, 130.88, 130.32, 129.79, 129.45, 129.12, 128.95, 128.02, 127.71, 115.57, 70.01,
-
0.81. ESI-MS m/z: 371.0 [M-45] . Anal. calcd. For C H ClO S: C, 60.51; H, 4.11; Found: C,
2
1
17
5
0.46; H, 4.12.
2
-((4-((2'-(trifluoromethyl)-[1,1'-biphenyl]-3-yl)methoxy)phenyl)sulfonyl)acetic acid (21).
1
Yield 62%; white solid, HPLC purity: 98.1%; m.p. 119-120 °C. H NMR (300 MHz, DMSO-d ) δ:
6
7
.89-7.78 (m, 3H), 7.70-7.64 (m, 2H), 7.57-7.39 (m, 4H), 7.31 (s, 1H), 7.17 (d, J = 8.3 Hz, 2H),
1
3
5
.25 (s, 2H), 3.80 (s, 2H). C NMR (75 MHz, DMSO-d ) δ: 173.79, 164.41, 161.53, 139.45,
6
1
36.01, 132.30, 132.13, 131.78, 130.20, 129.14, 128.37, 128.15, 127.96, 127.18, 126.07, 115.33,
-
1
14.48, 69.45, 64.08, 43.90. ESI-MS m/z: 405.1 [M-45] . Anal. calcd. For C H F O S: C, 58.66;
2
2
17
3
5
H, 3.80; Found: C, 58.61; H, 3.81.
2
-((4-((2'-isopropyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)sulfonyl)acetic acid (22). Yield 52%;
1
white solid, HPLC purity: 97.6%; m.p. 81-83 °C. H NMR (300 MHz, DMSO-d ) δ: 7.85 (d, J =
6
8
2
1
.9 Hz, 2H), 7.48 (d, J = 4.4 Hz, 2H), 7.40-7.33 (m, 3H), 7.30-7.11 (m, 5H), 5.30 (s, 2H), 4.42 (s,
1
3
H), 2.98-2.85 (m, 1H), 1.08 (d, J = 6.8 Hz, 6H). C NMR (75 MHz, DMSO-d ) δ: 164.59,
6
62.37, 147.35, 141.23, 136.78, 132.05, 131.49, 130.89, 129.60, 128.67, 127.63, 126.78, 126.43,
2
5

ACCEPTED MANUSCRIPT
-
1
25.66, 115.36, 69.73, 62.54, 28.92, 23.57. ESI-MS m/z: 379.1 [M-45] . Anal. calcd. For
C H O S: C, 67.90; H, 5.70; Found: C, 67.95; H, 5.71.
2
4
24
5
2
-((4-((2'-methoxy-[1,1'-biphenyl]-3-yl)methoxy)phenyl)sulfonyl)acetic acid (23). Yield 63%;
1
white solid, HPLC purity: 98.1%; m.p. 101-102 °C. H NMR (300 MHz, DMSO-d ) δ: 7.85 (d, J =
6
8
.8 Hz, 2H), 7.57 (s, 1H), 7.50-7.38 (m, 3H), 7.38-7.19 (m, 4H), 7.15-6.97 (m, 2H), 5.27 (s, 2H),
1
3
4
.38 (s, 2H), 3.74 (s, 3H). C NMR (75 MHz, DMSO-d ) δ: 164.53, 162.71, 157.72, 141.45,
6
1
40.91, 136.64, 132.82, 131.67, 129.91, 129.12, 128.96, 127.83, 126.92, 126.43, 116.70, 115.49,
-
7
0.08, 61.20, 56.13. ESI-MS m/z: 367.1 [M-45] . Anal. calcd. For C H O S: C, 64.07; H, 4.89;
2
2
20
6
Found: C, 64.01; H, 4.88.
2
-((4-((2'-cyano-[1,1'-biphenyl]-3-yl)methoxy)phenyl)sulfonyl)acetic acid (24). Yield 57%;
1
white solid, HPLC purity: 96.8%; m.p. 137-139 °C. H NMR (300 MHz, DMSO-d ) δ: 7.96 (d, J
6
=
6.8 Hz, 1H), 7.88-7.76 (m, 3H), 7.72-7.52 (m, 6H), 7.24 (d, J = 7.6 Hz, 2H), 5.31 (s, 2H), 4.16
1
3
(
s, 2H). C NMR (75 MHz, DMSO-d ) δ: 164.31, 162.39, 142.35, 138.50, 137.45, 134.32, 134.05,
6
1
30.85, 130.61, 129.69, 129.45, 128.81, 128.63, 115.80, 114.98, 110.65, 69.85, 61.23. ESI-MS
-
m/z: 362.1 [M-45] . Anal. calcd. For C H NO S: C, 64.85; H, 4.21; N, 3.44; Found: C, 64.81; H,
22
17
5
4
.21; N, 3.43.
2
-((4-((2'-chloro-4'-methyl-[1,1'-biphenyl]-3-yl)methoxy)phenyl)sulfonyl)acetic acid (25).
1
Yield 65%; white solid, HPLC purity: 99.3%; m.p. 123-125 °C. H NMR (300 MHz, DMSO-d ) δ:
6
7
.86 (d, J = 7.0 Hz, 2H), 7.56 – 7.45 (m, 3H), 7.44 – 7.34 (m, 2H), 7.33 – 7.17 (m, 4H), 5.29 (s,
2
6

ACCEPTED MANUSCRIPT
1
3
2
H), 4.41 (s, 2H), 2.35 (s, 3H). C NMR (75 MHz, DMSO-d ) δ: 164.01, 162.42, 139.15, 138.86,
6
1
36.47, 136.19, 131.17, 130.38, 130.08, 128.97, 128.62, 128.40, 128.20, 126.99, 115.11, 69.60,
-
6
0.42, 20.24. ESI-MS m/z: 385.1 [M-45] . Anal. calcd. For C H ClO S: C, 61.32; H, 4.44;
2
2
19
5
Found: C, 61.36; H, 4.43.
2
-((4-((2'-chloro-4'-ethoxy-[1,1'-biphenyl]-3-yl)methoxy)phenyl)sulfonyl)acetic acid (26).
1
Yield 68%; white solid, HPLC purity: 99.1%; m.p. 73-75 °C. H NMR (300 MHz, DMSO-d ) δ:
6
7
.85 (d, J = 6.8 Hz, 2H), 7.61 – 7.43 (m, 3H), 7.41 – 7.18 (m, 4H), 7.20 – 6.93 (m, 2H), 5.29 (s,
1
3
2
H), 4.41 (s, 2H), 4.09 (q, J = 6.7 Hz, 2H), 1.30 (t, J = 6.7 Hz, 3H). C NMR (75 MHz, DMSO-d )
6
δ: 164.01, 162.43, 138.71, 136.15, 132.06, 131.85, 131.08, 130.38, 129.07, 128.73, 128.38, 126.77,
-
1
15.45, 115.11, 114.03, 69.61, 63.68, 60.39, 14.46. ESI-MS m/z: 415.1 [M-45] . Anal. calcd. For
C H ClO S: C, 59.93; H, 4.59; Found: C, 59.90; H, 4.58.
2
3
21
6
2
-((3-([1,1'-biphenyl]-4-ylmethoxy)phenyl)sulfonyl)acetic acid (31). The title compound was
prepared by a method similar to that described for compound 4. Yield 73%; white solid, HPLC
1
purity: 98.2%; m.p. 63-65 °C. H NMR (300 MHz, DMSO-d ) δ: 7.73-7.66 (m, 4H), 7.61-7.37 (m,
6
1
3
9
H), 5.25 (s, 2H), 4.56 (s, 2H). C NMR (75 MHz, DMSO-d ) δ: 163.90, 158.43, 140.56, 139.89,
6
1
39.70, 135.48, 130.45, 128.94, 128.46, 128.12, 127.53, 126.79, 126.66, 120.59, 120.18, 113.72,
-
6
9.48, 59.78. ESI-MS m/z: 337.1 [M-45] . Anal. calcd. For C H O S: C, 65.95; H, 4.74; Found:
2
1
18
5
C, 65.91; H, 4.73.
2
7

ACCEPTED MANUSCRIPT
2
-((3-((2'-methyl-[1,1'-biphenyl]-4-yl)methoxy)phenyl)sulfonyl)acetic acid (32). The title
compound was prepared by a method similar to that described for compound 4. Yield 65%; white
1
solid, HPLC purity: 97.6%; m.p. 72-74 °C. H NMR (300 MHz, DMSO-d ) δ: 7.72-7.50 (m, 5H),
6
1
3
7
.47-7.35 (m, 3H), 7.33-7.13 (m, 4H), 5.25 (s, 2H), 4.53 (s, 2H), 2.24 (s, 3H). C NMR (75 MHz,
DMSO-d ) δ: 163.94, 158.52, 141.05, 140.84, 140.59, 134.89, 134.68, 130.45, 130.33, 129.47,
6
1
29.08, 127.79, 127.36, 125.94, 120.58, 120.20, 113.64, 69.63, 59.87, 20.13. ESI-MS m/z: 351.1
-
[
M-45] . Anal. calcd. For C H O S: C, 66.65; H, 5.09; Found: C, 66.60; H, 5.08.
2
2
20
5
General synthetic procedure for target compounds 27, 28 and 30. To a solution of 12a (1 equiv)
and alkyl halide (1.2 equiv) in acetone was added K CO (3 equiv) at room temperature. The
2
3
reaction mixture was heated to reflux with stirring overnight. Then the reaction mixture was
cooled to room temprature followed by filtration and the filtrate was concentrated under vacuum.
The residue was purified by silica gel column chromatography using a mixture of petroleum
ether/ethyl acetate (4:1, v/v) as eluent to afford a white solid. To a solution of the obtained solid (1
equiv) in 2:3:1 THF/MeOH/H O (18 ml) was added LiOH·H O (3 equiv). After stirring at room
2
2
temperature for 4 h, the volatiles were removed under reduced pressure. The residue was acidified
with 1N hydrochloric acid solution, and then filtered and the filter cake was washed with 5 mL of
water, dried in vacuum to afford a white powder. The white powder was purified by column
chromatography using a mixture of petroleum ether/ethyl acetate (2:1–1:2, v/v) as eluent to afford
the target compounds as white solid.
2
8