Discovery of Potent and Orally Bioavailable GPR40 Full Agonists

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Discovery of Potent and Orally Bioavailable GPR40 Full Agonists Bearing Thiophen-2-ylpropanoic Acid Scaffold He Li, Qi Huang, Cheng Chen, Bin Xu, Heyao Wang, and Ya-Qiu Long J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01357 • Publication Date (Web): 09 Mar 2017 Downloaded from http://pubs.acs.org on March 10, 2017

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Journal of Medicinal Chemistry

Discovery of Potent and Orally Bioavailable GPR40 Full Agonists Bearing Thiophen-2-ylpropanoic Acid Scaffold He Li,1,2,# Qi Huang,1,2,# Cheng Chen,1,3 Bin Xu,3 He-Yao Wang,1,* Ya-Qiu Long1,* 1

CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,

Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China; 2

University of Chinese Academy of Sciences, Beijing 100049, China; 3Department of

Chemistry, Shanghai University, 99 Shangda Road, Shanghai 200444, China

KEY WORDS: GPR40, full agonist, glucose-stimulated insulin secretion, type 2 diabetes mellitus, thiophen-2-ylpropanoic acid

ACS Paragon Plus Environment


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ABSTRACT The free fatty acid receptor GPR40 is predominantly expressed in pancreatic β-cells and enhances insulin secretion in a glucose dependent manner. Therefore, GPR40 agonists are possible novel insulin secretagogues with reduced or no risk of hypoglycemia for the treatment of type 2 diabetes mellitus (T2DM). Chemically and structurally diverse GPR40 agonists with high safety are pursued for the clinical development of GPR40-based pharmacotherapeutics. Herein we report our design and discovery of a new chemotype of GPR40 agonists free of the typical phenylpropanoic acid scaffold. The thiophen-2-ylpropanoic acid containing GPR40 modulators functioned as full agonists with high-efficacy response (Emax) and reduced lipophilicity. Significantly, the lead compound in this series, (R)-7k exhibited more potent in vitro glucose-stimulated insulin secretion and in vivo glucose-lowering effects (10 mg/kg, p.o.) than the GPR40 partial agonist TAK-875 which was once in phase III clinical trials, and high selectivity over the relevant receptors GPR120 and PPARγ.


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INTRODUCTION Type 2 diabetes mellitus (T2DM) is a serious metabolic disorder characterized by insulin resistance and insufficient insulin secretion from the pancreas. GPR40 (also referred to as free fatty acid receptor 1, FFA1) belongs to the class A family of G-protein coupled receptors (GPCR) and is mainly expressed in pancreatic β-cells1 and enteroendocrine cells.2 Activation of GPR40 by its endogenous ligands elicits the enhancement of glucose-stimulated insulin secretion (GSIS) in a glucose dependent manner.1 GPR40 agonists stimulate insulin secretion only in the presence of elevated glucose levels. On the basis of this mechanism, GPR40 has emerged as a promising anti-diabetic drug target posing little or no risk of hypoglycemia.

Figure 1 Representative GPR40 partial agonists and full agonists In the past decade, a number of potent synthetic GPR40 agonists have been developed3-5 and the most advanced compound was 1 (TAK-875)6 from Takeda, which was once under clinical trial phase III and provided clinical proof-of-concept for the GPR40 agonist as an effective treatment for T2DM. Based on the mode of action, the GPR40 agonists can be divided into partial agonists and full agonists (Figure 1), and most of the current GPR40 agonists published in the literature belong to partial agonists [e.g. 17, 2 (AMG 837)8]. The termination of 1 in phase III clinical trials for the hepatotoxicity issue threw doubt over the long-term safety of targeting GPR40.



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Structurally diverse and new mechanism GPR40 agonists with high safety are needed to address these concerns and give new insights into the receptor’s basic biology and physiological roles. The recently reported GPR40 full agonists as potential anti-diabetic drugs by Amgen stimulated our intensive interest. GPR40 full agonists endow attractive therapeutic characteristics, exemplified by 3 (AM 1638)9 and AM 526210 (for the structures, see Figure 1), showing superior in vivo efficacy to the partial agonist (i.e. 2), as well as the significant advantage of stimulating incretin secretion (GLP-1 and GIP) simultaneously.10-12 Moreover, the full agonists may bind to a different site from that of the partial agonists, offering the potential of positive functional cooperativity to deliver synergistic therapeutic benefits.13 Therefore, GPR40 full agonists might afford access to a robust mechanism for maintaining glycemic control and clinical potential for the treatment of type 2 diabetic patients. However, 3 suffered from the issue of high lipophilicity which might result in lipotoxicity14 and off-target effects in CNS.15 We therefore aimed to develop novel GPR40 full agonists with improved physicochemical properties. Initially, by analyzing the structures of various synthetic GPR40 agonists reported, we concluded the typical pharmacophore of GPR40 agonists, which have also been explored by various groups,5 comprising phenylpropanoic acid core, linker and aromatic fragment (Figure 2). According to the co-crystal structure of GPR40 in complex with 1,16-17 the carboxylic acid head interacts with the key residues, Arg183, Arg258, Tyr240 and Tyr91 of the receptor, while the linker and the aromatic moiety both mimic the hydrophobic tail of the endogenous ligands. For the few GPR40 full agonists reported by Amgen,5,

18

they bear the similar modular structures to the

GPR40 partial agonists, yet they function differently and bind to distinct binding sites, probably due to the different spatial arrangement. The comparison of the partial and


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full agonists in the modular structure framework indicated that the modification of the phenylpropanoic core might lead to the agonist type transformation. For example, changing the middle ether linkage from the para to the meta-position of the phenyl core along with switching the stereochemistry at the -position and relocating the biphenyl linkage from the 3- to 4-position of 2 led to the identification of 3 (Figure 1), a potent GPR40 full agonist.9

Figure 2. The typical pharmacophore model for GPR40 agonist and our design of new structure GPR40 full agonist Therefore, we were intrigued to design and discover a new structure series of GPR40 full agonists with low lipophilicity, focused on the variation of the phenylpropanoic core. Starting from the template compound 4,6 the precursor of 1, replacement of the phenyl core with polar heterocycle along with further chemical modifications on the aromatic tail and the propanoic acid head delivered a new class of GPR40 agonists free of phenylpropanoic acid (Figure 2), with the lead compound being demonstrated as full agonist by plasmid titration experiments.11 Notably, the choice of the five-membered heteroaromatic ring was based on the notion that 2,5-disubstituted heteroaromatic structure could mimic the meta-substitution pattern of the phenyl core of Amgen’s full agonists (Figure 1) with respect to the spatial



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orientation.

Significantly,

after

a

global

structural

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modification,

the

thiophen-2-ylpropanoic acid-based GPR40 full agonist exhibited more potent in vitro GSIS and in vivo glucose-lowering activity than 1, and high selectivity over GPR120 and PPARγ, holding promise for further clinical development.

CHEMISTRY Starting from a simple model GPR40 agonist 4, a mechanism-based drug design and systematic SAR study was undertaken to furnish new structure GPR40 full agonists (Figure 3). The first round of structural modification was focused on the central aromatic ring. As mentioned in the introduction section, a polar five-membered heteroaromatic ring was incorporated to mimic the meta-substitution pattern of the phenylpropanoic acid core of Amgen’s full agonists with reduced lipophilicity. Thus, triazole- and thiophene-based various analogs were synthesized and screened. Based on the outcome of the first round of SAR study, the thiophen-2-ylpropanoic acid scaffold was chosen for further structural modification on the aromatic tail and the β-position to the carboxylic acid to improve the activity and PK property (Figure 3).

Figure 3. The structural exploration of the new structure GPR40 full agonists starting from a model compound 4. The 1H-1,2,3-triazole-based compounds 5a-c were constructed by employing CuAAC (copper-catalyzed azide-alkyne cycloaddition) and Suzuki coupling as the key

reactions

(Scheme

1).

For

example,

the


preparation

of

the

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3-(1/4-((2',6'-dimethylbiphenyl-3-yloxy)methyl)-1H-1,2,3-triazol-4-yl)propanoic acid (5a/5b) was commenced with 3-bromophenol which was converted to the azide/alkyne (9/11) followed by a sequential CuAAC with the corresponding alkyne/azide and a Suzuki coupling with 2,6-dimethylphenylboronic acid. For the 3-(4-((2',6'-dimethylbiphenyl-3-yl)methoxy)-1H-1,2,3-triazol-1-yl)propanoic acid (5c), convergent synthesis was employed by the alkylation of two building blocks 14 and 16 which were prepared by Suzuki coupling and CuAAC, respectively. Scheme 1

Reagents and conditions: (a) NaH, NaI, (chloromethyl)(methyl)sulfane, DMF, 0℃-r.t., 72%; (b) SO2Cl2, DCM, -78℃; (c) NaN3, DMSO, r.t.; (d) pent-4-ynoic acid, CuSO4.5H2O, sodium ascorbate, tBuOH/H2O=1:1, M.W. 80℃, 75%; (e) 2,6-dimethylphenylboronic acid, Pd(PPh3)4, Na2CO3, dioxane/H2O=3:1, 100℃; (f) 3-bromoprop-1-yne, K2CO3, acetone, 60℃, 78%; (g) methyl 3-azidopropanoate, CuSO4.5H2O, sodium ascorbate, tBuOH/H2O=1:1, M.W. 80℃, 84% for 12, 28% for 15; (h) PBr3, DCM, 0℃ (i) BBr3, DCM, r.t., 94%; (j) Cs2CO3, acetone, 60℃, 73%; (k) LiOH.H2O, MeOH/THF/H2O=2:2:1, r.t., 78%.

The thiophen-2-ylpropanoic acid-based analogs (5d-f, 5i-l, 6a-h) were furnished from the formylation of the substituted thiophene with nBuLi/DMF or LDA/DMF followed by Wittig reaction to introduce the propanoic acid moiety and the Suzuki coupling to install the aromatic fragment (Scheme 2 for compounds 5 series, Scheme



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3 for compounds 6 series). The substituted thiophene (17d, 17f, 17i) was readily prepared from the coupling of bromobenzyl alcohol with iodothiophene.19 Notably, the preparation of the 5-(oxymethyl)thiophen-2-ylpropanoic acid (5e) was different from that of the 5-(methyloxy)thiophen-2-ylpropanoic acid (5d, 5f, 5i, 6a-h) in the reaction sequence. Specifically, the formylation occurred at the 5-position of the thiophene ring with Vilsmeier reagent to provide a handle for further introduction of aromatic tail. The resulting aldehyde (21e) was reduced with NaBH4 followed by the chlorination with SOCl2 and subsequent alkylation of 2',6'-dimethylbiphenyl-3-ol to give 5e. It was worth noting that the synthetic route toward 5d, 5f and 5i could achieve higher yield than that toward 6a-h with diversification on the terminal aryl ring. However, for the synthesis of the compounds collection with variation on the aromatic substitution, it is an efficient strategy to diversify at late stage from a common intermediate 26. Scheme 2

Reagents

and

conditions:

(a)

CuI,

3,4,7,8-tetramethyl-1,10-phenanthroline,


Cs2CO3,

80℃;

(b)

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2,6-dimethylphenylboronic acid, Pd(PPh3)4, Na2CO3, 1,4-dioxane/H2O=3:1, 100℃; (c) nBuLi, DMF, THF, -78℃; (d) methyl (triphenylphosphoranylidene) acetate, DMF, 60℃; (e) CoCl2.6H2O, NaBH4, MeOH/THF=1:2, r.t.; (f) LiOH.H2O, THF/MeOH/H2O=2:2:1, r.t.; (g) POCl3, DMF, 100℃, 43%; (h) NaBH4, MeOH/toluene=1:1, 0℃-r.t., 72%; (i) SOCl2, DCM, 0-50℃; (j) 2',6'-dimethylbiphenyl-3-ol, NaI, K2CO3, acetone, 60℃, 34% yield over 3 steps of i, j and f; (k) I2, PhI(O2CCF3)2, CCl4, r.t., 62%; (l) Na2PdCl4, CuI, 2-(di-tert-butylphosphino)-1-phenylindole, TMEDA/H2O=9:1,

80℃,

65%;

(m)

triethoxymethane,

NH4Cl,

EtOH,

60℃,

90%;

(n)

2,2-dimethyl-1,3-dioxane-4,6-dione, DMAP, THF, r.t., 64%; (o) 1-propynylmagnesium bromide, THF, -10℃-r.t.; (p) HCl (aq), r.t.; (q) DMF/H2O=10:1, 90℃, 52% over 3 steps; (r) spiro[indene-1,4'-piperidine], NaB(OAc)3H, HOAc, DCE, r.t., 25%; (s) thiazolidine-2,4-dione, piperidine, EtOH, 120℃, 48%; (t) Mg, MeOH, r.t., 36%.

Several thiophen-2-ylpropanoic acid derivatives incorporated with privileged aromatic fragment, favorable β-substituent or carboxylic acid bioisosteres (5j-l) were synthesized to compare the SAR of thiophen-2-ylpropanoic acid-based GPR40 agonists with the phenylpropanoic acid prototype. The synthesis was outlined in Scheme 2 along with other thiophene series, by employing the similar synthetic procedures to the known phenylpropanoic acid counterparts. Sonogashira coupling, condensation with Meldrum’s acid and 1,4-addtion of Grignard reagent served as the key reactions. For the synthetic details of compound 5j-l, please refer to the Supporting Information. Scheme 3

Reagents and conditions: (a) LDA, DMF, THF, -78℃, 48%; (b) ethyl (triphenylphosphoranylidene) acetate, DMF, 60℃, 82%; (c) CoCl2.6H2O, NaBH4, MeOH/THF=1:2, r.t., 73%; (d) R-B(OH)2, Pd(PPh3)4, Na2CO3, 1,4-dioxane/H2O=3:1, 100℃.

The synthetic route toward thieno[3,2-b]thiophene derivatives with different length of carboxylic acid chains 5g-h were depicted in Scheme 4. Starting from



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thieno[3,2-b]thiophene, the introduction of formyl group through Vilsmeier reaction followed by sequential oxidation and esterification or sequential Wittig reaction and reduction gave the key intermediate 3-(thieno[3,2-b]thiophen-2-yl)propanoate 30g or thieno[3,2-b]thiophene-2-carboxylate 30h. Then iodination and coupling between iodothieno[3,2-b]thiophene and (2',6'-dimethylbiphenyl-3-yl)methanol 13 followed by hydrolysis produced 5g and 5h, respectively. Scheme 4

Reagents and conditions: (a) POCl3, DMF, DCE, 90℃, 59%; (b) methyl (triphenylphoranylidene)acetate, THF/H2O=2:1, r.t., 72%; (c) NaClO2, NaH2PO4, 2-methyl-2-butene, tBuOH, 77%; (d) CoCl2.6H2O, NaBH4, THF/MeOH=2:1, r.t., 83%; (e) EDCI, HOBt, MeOH, DIEA, DCM; (f) PIFA, I2, CCl4, r.t.; (g) 13, CuI, Me4Phen, Cs2CO3, toluene, 80℃; (h) 0.5 M NaOH in MeOH/THF/H2O=2:2:1, r.t.

The derivatization on the β-position to the carboxylic acid was illustrated in Scheme 5. The diversified propanoic acid chain was synthesized from a common precursor 19d. Condensation with Meldrum’s acid yielded the key intermediate 32, from which were generated 7a-e and 33 directly through 1,4-addition by corresponding Grignard reagents and decarboxylation. For raceme 7h, the intermediate 32 was treated with KCN, followed by decarboxylation under the microwave-assisted FeCl3.6H2O-catalysis conditions.20 Further conversion was


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performed on 33 through sequential esterification, click reaction and hydrolysis to give 7i. On the other direction, the nucleophilic addition of an organozinc reagent to 19d followed by Ag2O-promoted alkylation and hydrolysis delivered the -alkoxy substituted derivatives 7g-h. The synthesis of 7j was accomplished according to the reported method,21 through Henry reaction between 19d and nitromethane, Michael addition of (S)-3-acetyl-4-benzyl-2-oxazolidinone, formation of isoxazole under (Boc)2O/vinyl

bromide/DMAP/Et3N

conditions

and

final

removal

of

the

oxazolidinone. Scheme 5

Reagents and conditions: (a) Meldrum's acid, DMAP, THF, 45℃, 72%; (b) RMgX, THF, -10℃; (c) DMF/H2O=10:1, 90℃; (d) KCN, MeOH, r.t., 89%; (e) FeCl3.6H2O, MeNO2/DMF=3:2, H2O, M.W. 110℃, 46%; (f) MeOH, EDCI, HOBt, Et3N, DCM, r.t., 75%; (g) TMSN3, CuSO4.5H2O, sodium ascorbate, tBuOH/H2O=1:1, M.W. 80℃, 39%; (h) LiOH.H2O, MeOH/THF/H2O=2:2:1, r.t.; (i) (2-ethoxy-2-oxoethyl)zinc(II) bromide, THF, 0℃, 52%; (j) RX, Ag2O, toluene, 80℃; (k) MeNO2, NH4OAc, THF, 70℃, 44%; (l) (S)-3-acetyl-4-benzyloxazolidin-2-one, TiCl4, DIEA, DCM, -78℃, 40%; (m) vinyl bromide, (Boc)2O, DMAP, Et3N, THF, r.t., 46%; (n) LiOH.H2O, H2O2, THF/H2O, 0℃, 25%.

The chiral (R)-7g and (S)-7g were enantioselectively synthesized via Ru-catalyzed asymmetric reduction of carbonyl group, as shown in Scheme 6. After the construction

of

ethyl

3-(5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)-3-oxopropanoate 38, the Ru-catalyzed

asymmetric

hydrogenation

furnished


the

(R)-34

in

good


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enantioselectivity (98% ee).22 Alkylation of the hydroxyl and hydrolysis gave (R)-7g. And the enantiomer (S)-7g was obtained according to the same procedure just by utilizing RuCl[(S,S)-Tsdpen](mesitylene) as the catalyst instead.22 Scheme 6

Reagents and conditions: (a) nBuLi, CO2 (g), THF, -78℃, 84%; (b) CDI, THF (c) potassium ethyl malonate, MgCl2, Et3N, MeCN, 52% over 2 steps; (d) HCO2H, Et3N, RuCl[(R,R)-Tsdpen](mesitylene), 38℃, 90%, 98% ee; (e) EtI, Ag2O, toluene, 80℃; (f) LiOH.H2O, MeOH/THF/H2O=2:2:1, r.t.

The synthetic routes of raceme 7k-m with modification on the terminal phenyl ring portion were shown in Scheme 7. Starting from compound 17d, the boronic acid 39 was prepared via lithium-bromide exchange and boronation. Suzuki coupling with 4-bromo-3,5-dimethylphenol followed by protecting the phenolic hydroxyl with TBSCl provided compound 40. Then formylation and condensation with Meldrum’s acid generated compound 42. The key intermediate 43 was produced through Michael addition, decarboxylation, esterification and removal of the TBS protective group. Various hydrophilic side chains were coupled to 44 by substitution or Mitsunobu reactions followed by hydrolysis, affording the target compounds 7k-m. Scheme 7


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Reagents and conditions: (a) nBuLi, B(OiPr)3, THF, -78℃, 54%; (b) 4-bromo-3,5-dimethylphenol, Pd(PPh3)4, Na2CO3, dioxane/H2O=3:1, 100℃, 68%; (c) TBSCl, Et3N, DCM, r.t., 89%; (d) nBuLi, DMF, THF, -78℃, 77%; (e) Meldrum's acid, DMAP, THF, 45℃, 86%; (f) 1-propynyl magnesium bromide, THF, -10℃; (g) DMF/H2O=10:1, 90℃; (h) MeOH, EDCI, HOBt, Et3N, DCM, r.t. 64% over 3 steps; (i) TBAF, THF, r.t., 98%; (j) 3-(methylsulfonyl)propyl

4-methylbenzenesulfonate,

K3PO4,

DMF,

90℃,

88%;

(k)

3-(chloromethyl)-3-methyloxetane, KI, K2CO3, DMF, 80℃, 69%; (l) 1-methylpiperidin-4-ol, DEAD, PPh3, THF, 0℃-r.t., 54%; (m) LiOH.H2O, MeOH/THF/H2O=2:2:1, r.t.; (n) (S)-2-methylbutan-1-ol, EDCI, HOBt, Et3N, DCM, r.t., 56%; (o) resolution on chiral AD-H column; (p) 0.25 M NaOH in MeOH/THF/H2O=2:2:1, r.t.

The direct resolution of raceme 7k on the normal-phase chiral column was inefficient due to its high polarity. So the raceme 7k was condensed with (S)-2-methyl-1-butanol to produce the separable diastereoisomer. Resolution on chiral AD-H column and hydrolysis with sodium hydroxide gave the optical pure enantiomer (R)-7k and (S)-7k, respectively.

RESULTS AND DISCUSSION The efforts to pursue new structure GPR40 full agonists with reduced lipophilicity was commenced with the lead compound 4, from which was derived the drug candidate 1.6 A systematic structural exploration was undertaken based on the pharmacophore model for the GPR40 agonists (Figure 2), with respect to the phenyl



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core, the aromatic tail and the -substitution on the propanoic acid portion. Identification

of

2,5-disubstituted

thiophenylpropanoic

acid

with

2',6'-dimethylbiphenyl-3-yl tail being an optimal scaffold. First round of structural modification was focused on the phenyl ring of the phenylproponoic acid head. Considering that 5-membered heterocycle might induce a similar conformation to the meta-substituted phenyl core of Amgen’s full agonists9 as well as lowering the lipophilicity, various 5-membered heteroaromatic rings were screened as the surrogate of the phenyl core in compound 4. As shown in Table 1, the polar triazole was not tolerated (compound 5a-c) regardless of the substitution pattern and the ether position, with an activity loss by at least 3 orders of magnitude. Then, we turned to the less polar thiophene ring. To our delight, the thiophene derivative 5d almost maintained the GPR40 agonistic activity of the phenyl parent compound 4, and significantly the maximal efficacy increased to 140% relative to compound 4. As it was commonly observed with the classic phenylpropanoic acid-based GPR40 agonists, the inverted central linker remarkably affected the GPR40 agonistic activity, with the alkoxy/amino-substituted

phenylpropanoic

acid

being

superior

to

the

oxy/aminoalkyl-substituted counterpart. In the case of thiophen-2-ylpropanoic acid–based GPR40 agonists, the alkoxy substituent on the thiophene core was indeed beneficial for the interaction with GPR40 displaying more than an order of magnitude more potent than the corresponding oxyalkyl thiophenylpropanoic acid (compound 5d vs 5e). The specific effect of the central linkers might be due to hydrogen bond interactions, conformational effects, or overall effects on the dipolar momentum. Even on the inferior triazole scaffold series (compound 5a-c), the alkoxy linker exerted a boosting effect on the maximal efficacy of the GPR40 agonism compared to the oxyalkyl linker counterpart (compound 5c vs 5b), though both EC50 values were


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mediocre. Considering the fused-ring alkanoic acid conferred an improved PK profile of 1 and yet had slight effect on the activity,23-25 we further investigated the thieno[3,2-b]thiophene derivatives (compound 5g-h) with an enlarged aromatic ring which might gain more π-π interaction with the receptor.17, 23 The length of the acidic chain was also evaluated. However, the large rigid aromatic ring turned out disfavored within the 2,5-disubstituted thiophenylpropanoic acid framework. Furthermore, the favorable location of the biphenyl linkage at 4-position of 3 proved to be adverse for the

2,5-disubstituted

thiophenylpropanoic

acid

scaffold.

The

switch

of

2,6-dimethylphenyl from meta- to para-position led to a complete loss of the activity (compound 5i). In addition, we tested whether the thiophene replacement for the phenyl core could be applied to other structure type of potent GPR40 agonists reported. However, the corresponding thiophene derivatives 5j and 5k were much less potent than the phenyl counterpart TUG-48826 and LY2881835,27 indicating the importance of the compatibility of lipophilic tail and aryl alkanoic acid head. However, the carboxylic acid function could be replaced by a bioisostere, e.g. 2,4-thiazolidinedione with GPR40 agonistic activity almost remaining (compound 5l). Table 1. Agonistic activity of 5-membered heteroaryl derivatives

Compound

Heteroaryl core

-Y-X-


EC50 (μM)

Emax (%)a


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Page 16 of 89

0.0270.003

90.9

4

-CH2-O-

0.0080.001

100

5a

-O-CH2-

32.25.21

95.6

5b

-O-CH2-

14.70.9

79.9

5c

-CH2-O-

14.81.1

97.9

5d

-CH2-O-

0.0320.006

140

5e

-O-CH2-

2.420.046

136

5f

-CH2-O-

2.320.095

135

5g

-CH2-O-

NAb

NTc

5h

NAb

NTc

5i

NAb

NTc

5j

0.6400.039

NTc

5k

NAb

NTc


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a

NTc

0.0820.014

5l

Maximal efficacy (Emax) was determined compared to compound 4.

b

NA: No activity was

observed at the concentration of 20 M. c NT: Not tested. Data are expressed as mean values with standard deviation from three independent experiments.

Fixing the 2,5-disubstitued thiophenylpropanoic acid as an optimal scaffold, further structure-activity relationship study was focused on the terminal aryl fragment. A range of aromatic rings with various substituents were screened. However, none of these analogues exhibited satisfactory activity (Table 2). All results indicated that the orthogonal conformation of the biaryl rings was an important factor for the agonistic function, which was in agreement with Takeda’s previous report.23 The less hindered ortho-monosubstituted derivatives exhibited lowered activity (compound 6a-b, 6h) and some even lost the activity (compound 6c-e). The analogues lacking the ortho-substituent were completely inactive (compound 6f-g). Table 2. Exploration of the terminal aryl ring

EC50 (μM)

Compound

5d

0.0320.006

6e

NAa

6a

0.580.034

6f

NAa

6b

2.710.31

6g

NAa

Compound

Ar


Ar

EC50 (μM)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a

6c

NAa

6d

NAa

Page 18 of 89

2.010.154

6h

NA = No agonism activity was observed at the initial concentration of 10 μM. Data are expressed

as mean values with standard deviation from three independent experiments.

Optimal combination of chiral β-substituent and the terminal polar chain affording potent thiophenylpropanoic acid-based GPR40 full agonist. After identification of the 2,6-dimethylbiphenyl-3-yl moiety as the favored aromatic fragment, further structural modification was switched to the substitution at the β-position to the carboxylic acid with the notion that introduction of small residues could reduce the potential of β-oxidation.28 As shown in Table 3, propynyl (raceme 7e) was preferred to the small alkyl (raceme 7a-c) or cyclopropyl (raceme 7d) group at R2. But a further increase in the size of R2 to 5-membered heteroaromatic rings (raceme 7i-j) resulted in a complete loss in activity. However, the introduction of hydrogen bond acceptor with suitable size (raceme 7g-h) led to additional gain in activity relative to the propynyl counterpart (7e). The chirality of the β-substituent was reported to be critical for the GPR40 interaction. So we continued to obtain the two enantiomers of raceme 7g separately by asymmetric synthesis. Indeed, an increased activity was observed for the (R)-7g isomer, while the (S)-7g was almost inactive. Interestingly, the configuration of the more active isomer is opposite to that of the literature-reported GPR40 agonists.29 Unfortunately, although the ethoxy or cyano substituted racemes were more potent, both of them were not chemically stable with elimination or hydrolysis liability. So our further structural optimization was focused on the propynyl substituted prototype. Based on the previous SAR study6,

14, 24-25, 30

and the co-crystal structure of


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hGPR40 and 1,17 the 4-position of the 2,6-dimethylphenyl moiety could tolerate various modification of different length and polarity, to improve the physicochemical property and PK profile of the whole compound. Therefore, we assayed distinct types of polar chains at R1 to reduce lipophilicity (raceme 7k-m). Gratifyingly, the agonistic activity could be well retained with the mesylalkoxy substituted raceme 7k. Then, further chiral separation of the raceme 7k confirmed that (R)-7k isomer was superior to the (S)-7k in terms of the GPR40 agonistic potency. More significantly, even though the EC50 values of thiophenylpropanoic acid-based GPR40 agonists 7k and (R)-7k were higher (less potent) than the phenyl parent compound 4, their maximal efficacy Emax values were improved by a factor of 1.5-2 fold relative to compound 4, which suggested a possible different mechanism and in vivo pharmacological behavior. Furthermore, compared to the Amgen’s developed GPR40 full agonists (Figure 1), our compounds generally exhibited much lower lipophilicity (smaller cLogP values). Table

3.

The

GPR40

agonistic

activity

3-(5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)propanoic

of

the acid

derivatives with variation on the β-position and the polar side chain

Compound

R1

R2

Configuration

EC50 (μM)

CLogPd

7a

H

Me

racemic

NAa

6.046

7b

H

Et

racemic

NAa

6.576

7c

H

n

racemic

NAa

6.974

7d

H

racemic

3.920.102

6.490

7e

H

racemic

0.210.016

6.096

Pr



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a

Page 20 of 89

7f

H

OMe

racemic

0.210.045

5.219

7g

H

OEt

racemic

0.0570.022

5.608

(R)-7g

H

(R)

0.0500.007b

5.608

(S)-7g

H

(S)

40.15.31

5.608

7h

H

racemic

0.110.023

4.708

7i

H

racemic

NAa

4.758

7j

H

(R)

NAa

5.064

7k

racemic

0.240.025

5.100

(R)-7k

(R)

0.0810.007c

5.100

(S)-7k

(S)

0.410.064

5.100

7l

racemic

0.450.021

5.668

7m

racemic

NAa

3.634

CN

NA = No agonism activity was observed at the initial concentration of 10 μM. b Emax was 147%

compared to compound 4.

c

Emax was 244% compared to compound 4.

d

CLogP values were

calculated by the BioByte’s algorithm as implemented in ChemBioDraw Ultra 11.0 (the “CLogP” option). Data are expressed as mean values with standard deviation from three independent experiments.

As expected, preliminary mechanism study revealed that compound (R)-7k with high Emax value functioned as GPR40 full agonist by plasmid titration experiments.11 HEK293T cells transfected with different quantities of GPR40 expression plasmids (5 μg and 0.05 μg) were used. When 5 μg plasmids were transfected, DHA (docosahexaenoic acid, endogenous ligand), 1 and compound (R)-7k displayed similar Emax (Figure 4A). Under the 0.05 μg plasmid transfected condition, compound (R)-7k still showed comparable Emax to DHA while the maximal responses of 1


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substantially decreased (Figure 4B). Definitely, the thiophenylpropanoic acid-derived lead compound (R)-7k acted as GPR40 full agonist. Though the hit in our thiophenyl series (compound 5d) was also disclosed by Toda N. et al in 2014,31 they did not pay further attention to its potential of intrinsic efficacy and structural derivatization due to its lowered EC50 value compared to the phenylpropanoic acid prototype, thus missing the discovery of its derived full agonists through the global optimization with the thiophenyl core, aromatic moiety and the β-substitution on the propanoic acid chain.

(A)

(B)

Figure 4. Plasmid titration experiments. (A) Effect of DHA, 1 and compound (R)-7k in HEK293T cells transfected with 5 μg of GPR40 expression plasmid. (B) Effect of DHA, 1 and compound (R)-7k in HEK293T cells transfected with 0.05 μg of GPR40 expression plasmid. Data are expressed as mean values with standard deviation from three independent experiments.

Thiophen-2-ylpropanoic acid-based GPR40 full agonists displaying potent in vitro glucose-stimulated insulin secretion and in vivo glucose-lowering effects. The GPR40 full agonist nature of the thiophen-2-ylpropanoic acid chemotype encouraged us to proceed with further biological evaluation. The glucose-stimulated insulin secretion of compound 5d, (R)-7g and (R)-7k were examined in MIN6 cell line. Compound (R)-7k showed higher enhancement of glucose-stimulated insulin secretion than 1 while compound 5d and (R)-7g were less potent at the concentration



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

of 10 μM in the presence of 25 mM glucose (Figure 5A). As we mentioned, the unstability of (R)-7g might lead to the compromised cellular activity. Then the stimulation of insulin release under gradient concentrations of compound (R)-7k and 1 was tested, and compound (R)-7k turned out much more potent than 1 at the same concentration of 300 nM (Figure 5B). On the other hand, compound (R)-7k produced no obvious increase in insulin secretion in the presence of 2 mM glucose compared to 2 mM glucose alone (Figure 5B), confirming that compound (R)-7k as a GPR40 agonist just induced a glucose-dependent insulin secretion.

(A)

(B)

Figure 5. (A) Activity of compound 5d, (R)-7g, (R)-7k and 1 in glucose-stimulated insulin secretion (GSIS) in MIN6 cell line. (B) The dose-GSIS activity relationship of 1 and (R)-7k at different concentrations of glucose ∗, p < 0.05; ∗∗, p < 0.01, compared to DMSO; #, p < 0.05, compared to 0.3 μM 1 in the presence of 25 mM glucose. Data are expressed as mean values with standard deviation from four independent experiments (n = 4).

Before the in vivo efficacy evaluation, we determined the PK properties of lead compound (R)-7k in SD rats. Encouragingly, the thiophenylpropanoic acid derivative displayed acceptable metabolic stability with a half-life of 3.9 ±0.5 h. The blood concentration, the AUC, and MRT values are satisfactory, but the oral bioavailability (F) was just 13.3%, which needed improving by further structural optimization (Table 4). Table 4. PK parameters for compound (R)-7k in SD ratsa


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Cmax ±sd

AUC0-t ±sd

MRTlast

PK properties

t1/2 (h)

Tmax (h)

F (%)

(ng/mL)

(h*ng/mL)

(h)

iv 1 mg/kg

2181.2 ±534.6

3322.9 ±783.5

3.8 ±0.6

/

3.2 ±0.5

po 10 mg/kg

834.0 ±278

4423.7 ±1051.8

3.9 ±0.5

1.0

5.0 ±0.3

13.3

a

Administered at a dose of 1 mg/kg, iv; 10 mg/kg, po, in SD rats. The values for Cmax and AUC

were expressed as equivalent of anhydrous (R)-7k. Data are expressed as mean values with standard deviation from four independent experiments (n = 4).

Since the lead compound (R)-7k possessed good PK properties, the in vivo efficacy of the thiophenylpropanoic acid-based GPR40 full agonist was demonstrated by an oral glucose tolerance test (OGTT) in ICR mice. The compounds were orally administered 30 min prior to a glucose challenge (2.5 g/kg). Compound (R)-7k significantly lowered the blood glucose at both tested doses (10 mg/kg and 20 mg/kg) and showed higher potency than 1 at the same dose (10 mg/kg) (Figure 6), even though the oral bioavailability of (R)-7k (F:13.3% in rat) was much lower than that of 1 (F: 76% in rat).6 The superior efficacy data proved the advantage of the full agonist over the partial agonist with respect to the in vivo overall glucose-lowering effect and glycemic control.

(A)

(B)

Figure 6 In vivo efficacy of compound (R)-7k during an OGTT in ICR mice. (A) Time-dependent changes of glucose levels. (B) Area under the curve (AUC0-120 min) of plasma glucose. ∗∗, p < 0.01, ∗∗∗, p < 0.001, compared to Vehicle. Data are expressed as mean values with standard deviation



from ten independent experiments (n = 10).

Moreover, the selectivity of the thiophenylpropanoic acid derivatives was examined. Compound 5d, (R)-7g and (R)-7k exhibited very high selectivity over both GPR120 and PPARγ (EC50>10 μM). Though the molecular basis of hepatotoxicity caused by 1 was unclear, recently Chen X.Y. et al speculated that the hepatobiliary transporter inhibition and long half-life in diabetic patients’ systemic circulation of 1 might contribute to cholestatic hepatotoxicity.32 So the selectivity of our compound (R)-7k over the hepatobiliary transporters was assessed. Similar to 1, compound (R)-7k displayed moderate hepatobiliary transporter inhibition (Figure 7A). And inhibitory effect of compound (R)-7k on biliary excretion index (BEI) of d8-TCA was stronger than 1 (Figure 7B). The decreased BEI of d8-TCA demonstrated the inhibition of bile acids efflux into bile. The off-target inhibition might result from the alkanoic acid moiety which is similar to the bile acid in structure. As the carboxylic acid head is an essential pharmacophore of GPR40 agonist, an alternative to reduce the hepatotoxicity risk might be accomplished by increasing the therapeutic window through developing potent full agonists and decreasing the over long half-life. 6

60

Ca2+ (+) Ca2+ (-) BEI (%)

4

2

0

40

20

M Tr 1 og lit az on e

25

10

M

R)-7k (M)

25

C

20

20

(A)

1

on tr ol

M Tr 1 og lit az on e

25

R)-7k (M)

25

10

1

0

M

C

on tr ol

Concentration of d8-TCA (ng/mg protein)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 89

(B)

Figure 7 The inhibitory effects of compound (R)-7k on hepatobiliary transporters. (A) Effect on


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hepatobiliary transporters. (B) Effect on BEI of d8-TCA. Data are expressed as mean values with standard deviation from three independent experiments (n = 3).

CONCLUSIONS In conclusion, we discovered a novel class of GPR40 agonists free of phenylpropanoic acid based on the pharmacophore model and protein structure. The modification on the key pharmacophore of 1 successfully transformed the GPR40 partial agonist to low lipophilic full agonist. Besides robust glucose-stimulated insulin secretion effect in the cellular settings, the lead compound (R)-7k exhibited more potent in vivo glucose-lowering activity than 1 at the same dose (10 mg/kg, p.o.), and high selectivity over the relevant receptors GPR120 and PPARγ. Our work highlighted the therapeutic advantage of GPR40 full agonist over the partial agonist and provided a new chemotype lead compound for the further development of safe and effective treatment for T2DM.

EXPERIMENTAL SECTION Chemistry. Unless otherwise specified, all reactions were carried out in oven-dried glassware with magnetic stirring. All reagents were weighed and handled in air at room temperature. Column chromatography was performed on silica gel (200-300 mesh). All new compounds were characterized by 1H NMR,

13

C NMR and low/high resolution

mass spectroscopy. NMR spectra were recorded on Brucker AVANCE 300 NMR spectrometer or Brucker AVANCE III 400 NMR spectrometer. Chemical shifts for proton magnetic resonance spectra (1H NMR) were quoted in parts per million (ppm) referenced to the signals of residual chloroform (7.26 ppm), dimethyl sulfoxide (2.50 ppm) or methanol (3.30 ppm). All

13

C NMR spectra are reported in ppm relative to

deuterochloroform (77.23 ppm), dimethyl sulfoxide (39.52 ppm) or methanol (49.00



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ppm). The following abbreviations were used to describe peak splitting patterns when appropriate: br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multriplet, dd = doublet of doublet. Mass spectra were recorded using an ESI ion source unless stated otherwise. Some products were isolated by semi-preparative HPLC on a EasySepTM-1010 with XBridgeTM Prep C18 reversed-phase column (19 ×150 mm) in the solvent systems of solvent A: 0.1% TFA in water, solvent B: 0.1% TFA in acetonitrile, flow rate, 8 mL/min, UV detector 254 nm. All melting points were measured using a BÜCHI 510 melting point apparatus. The yields in this paper refer to isolated yields of compounds estimated to be ≥95% pure as determined by 1H NMR or HPLC on Waters 1525EF with Denali C18 reversed-phase column (4.6 × 250 mm) in the solvent systems of solvent A: 0.1% TFA in water, solvent B: 0.1% TFA in acetonitrile with the gradient 10-90% B over 8 min, 90% B over 20 min, flow rate 1 mL/min, UV detector 254 nm. General procedure A for azide-alkyne click reactions Azide (1.1 equiv), alkyne (1 equiv), CuSO4.5H2O (0.1 equiv) and sodium ascorbate (0.5 equiv) were dissolved in tBuOH/H2O=1:1 (0.1-0.2 M) and heated at 80 ℃ under microwave for 2 h. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate. Usual work-up and purification by silica gel column chromatography afforded the product. General procedure B for Suzuki coupling reactions Aryl halide (1 equiv), aryl boronic acid (1.2 equiv), Na2CO3 (3 equiv) and Pd(PPh3)4 (0.05 equiv) were protected by N2. 1,4-Dioaxne/H2O=3:1 (0.1 M) was added by syringe and the solution was stirred at 100 ℃ overnight. After cooling to room temperature, the reaction mixture was acidified by diluted HCl (aq) and extracted with ethyl acetate for 3 times. Usual work-up and purification by silica gel column chromatography or


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HPLC afforded the product. General procedure C for hydrolysis of methyl esters To the solution of methyl ester (1 equiv) in MeOH/THF/H2O=2:2:1 (0.1 M) was added LiOH.H2O (3 equiv) and the mixture was stirred at room temperature for 4 h. The reaction mixture was acidified by diluted HCl (aq) and extracted with ethyl acetate for 3 times. Usual work-up and purification by silica gel column chromatography or HPLC afforded the product. General procedure D for CuI/Me4Phen catalyzed coupling reactions CuI (0.1 equiv), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me4Phen, 0.2 equiv) and Cs2CO3 (1.5 equiv) were protected by N2. Iodothiophene (1 equiv) and (3-bromophenyl)methanol (2 M) were added by syringe and the slurry was heated at 80 ℃ overnight. After cooling to room temperature, the resulting mixture was filtered through a plug of silica gel and further eluted with additional ethyl acetate. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel chromatography to afford the product. General procedure E for formylation via nBuLi/DMF Thiophene derivative (1 equiv) was protected by N2 and anhydrous THF (0.1 M) was added by syringe. The solution was cooled to -78 ℃ and nBuLi (2.5 M in hexanes, 1 equiv) was added dropwise. After stirring at -78 ℃ for 2 h, DMF (1.1 equiv) in anhydrous THF was added dropwise and the resulting mixture was stirred at -78 ℃ overnight. The reaction was quenched by aqueous NH4Cl and warmed to room temperature. The reaction mixture was extracted with ethyl acetate and the organic layer was dried over anhydrous Na2SO4. After removal of the drying agent, the filtrate was concentrated under reduced pressure. The residue was crystallized from DCM/PE or purified by silica gel column chromatography to afford the product.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

General procedure F for Wittig reaction To the solution of aldehyde (1 equiv) in DMF (0.1 M) was added methyl (triphenylphosphoranylidene) acetate (1.5 equiv) and the reaction mixture was stirred at 60 ℃ overnight. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate. Usual work-up and purification by silica gel column chromatography afforded the product. General procedure G for reduction of aryl acrylates Aryl acrylate (1 equiv) and CoCl2.6H2O (0.1 equiv) were dissolved in MeOH/THF=1:2 (0.1 M). NaBH4 (2.5-4 equiv) was added in portions at room temperature. After the starting material was completely consumed, aqueous NH4Cl was added. Usual work-up and purification by silica gel column chromatography afforded the product. General procedure H for iodination via I2/PIFA Thiophene derivative (1 equiv) was dissolved in CCl4 (0.5 M). I2 (0.5 equiv) and PhI(O2CCF3)2 (PIFA,0.55 equiv) were added in portions and the mixture was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate. The resulting mixture was washed with Na2SO3 (aq) and brine for 3 times respectively and the organic layer was dried over anhydrous Na2SO4. After removal of the drying agent, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to afford the product. General procedure I for condensation of aldehydes and Meldrum’s acid To the solution of aldehyde (1 equiv) in THF (0.2 M) was added Meldrum’s acid (1.3 equiv) and DMAP (0.1 equiv). The reaction mixture was stirred at 45 ℃ until the starting material was completely consumed. The resulting mixture was diluted with ethyl acetate and washed with diluted HCl (aq) and brine. The organic layer was dried over anhydrous Na2SO4. After removal of the drying agent, the filtrate was


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concentrated under reduced pressure. The residue was purified by slurrying in acetone/water. The product was isolated by filtration and dried under vacuum. General procedure J for 1,4-addition and decarboxylation Substrate (1 equiv) was protected by N2 and anhydrous THF (0.1 M) was added by syringe. The solution was cooled to -10 ℃ and Grignard reagent (2 equiv) was added dropwise. After the starting material was completely consumed, the reaction was quenched by diluted HCl and warmed to room temperature. The reaction mixture was extracted with ethyl acetate and the organic layer was dried over anhydrous Na2SO4. After removal of the drying agent, the filtrate was concentrated under reduced pressure. The residue was dissolved in DMF/H2O=10:1 and the solution was stirred at 90℃ for 2 h. After cooling to room temperature, the mixture was acidified by diluted HCl (aq) and extracted with ethyl acetate. Usual work-up and purification by silica gel column chromatography afforded the product. General procedure K for alkylation of secondary alcohol with iodoalkane To the solution of secondary alcohol (1 equiv) in toluene (0.1 M) was added iodoalkane (5 equiv) and Ag2O (5 equiv) and the reaction mixture was stirred at 80 ℃ for 3 h. Then the mixture was cooled to room temperature and iodoalkane (5 equiv) and Ag2O (5 equiv) were added. The reaction mixture was stirred at 80 ℃ for additional 3 h. After cooling to room temperature, the resulting mixture was filtered through a plug of silica gel and further eluted with ethyl acetate. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel chromatography to afford the product. General procedure L for esterification To the solution of acid (1 equiv) in DCM (0.1 M) was added EDCI (1.2 equiv), HOBt (1.5 equiv) and Et3N (2 equiv) and the mixture was stirred at room temperature for



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Page 30 of 89

additional 30 min. Alcohol (1.2 equiv) was added to the active ester and the reaction mixture was stirred overnight. Usual work-up and purification by silica gel column chromatography afforded the product. 3-(1-((2',6'-Dimethylbiphenyl-3-yloxy)methyl)-1H-1,2,3-triazol-4-yl)propanoic acid (5a): Following general procedure B, target product 5a was obtained from intermediate 10 as white solid, m.p. 81-85 ℃, yield: 67%, purity 96.2% (tR=13.9 min). 1

H NMR (300 MHz, CDCl3) δ 7.60 (s, 1H), 7.34 (t, J = 7.9 Hz, 1H), 7.20 – 6.98 (m, 4H),

6.84 (d, J = 7.6 Hz, 1H), 6.79 (dd, J = 2.4, 1.4 Hz, 1H), 3.05 (t, J = 7.2 Hz, 2H), 2.78 (t, J = 7.2 Hz, 2H), 1.98 (s, 6H). ESI-MS m/z: 352.1 ([M+H]+), 350.0 ([M-H]-). 13C NMR (125 MHz, CDCl3) δ 177.2, 156.1, 147.0, 143.0, 140.9, 135.9, 130.0, 127.4, 127.3, 124.2, 121.4, 117.1, 114.5, 76.4, 33.2, 20.7, 20.6. 3-(4-((2',6'-Dimethylbiphenyl-3-yloxy)methyl)-1H-1,2,3-triazol-1-yl)propanoic acid (5b): Following general procedure B, target product 5b was obtained from intermediate 12 as colorless oil, yield: 71%, purity 97.0% (tR=14.8 min). 1H NMR (300 MHz, CDCl3) δ 8.38 (s, 1H), 7.76 (s, 1H), 7.38 – 7.29 (m, 1H), 7.19 – 7.05 (m, 3H), 6.95 (ddd, J = 8.3, 2.5, 1.1 Hz, 1H), 6.79 – 6.72 (m, 2H), 5.19 (s, 2H), 4.64 (t, J = 6.4 Hz, 2H), 3.01 (t, J = 6.4 Hz, 2H), 2.02 (s, 6H). ESI-MS m/z: 352.1 ([M+H]+), 350.0 ([M-H]-).

13

C NMR (125 MHz, CDCl3) δ 175.4, 158.5, 144.1, 142.8, 141.6, 136.1,

129.8, 127.5, 127.3, 124.1, 122.4, 115.5, 113.1, 61.8, 46.2, 35.2, 20.9. 3-(4-((2',6'-Dimethylbiphenyl-3-yl)methoxy)-1H-1,2,3-triazol-1-yl)propanoic acid (5c): To the solution of 14 (15.7 mg, 0.057 mmol) and 16 (6.5 mg, 0.038 mmol) in 0.35 mL acetone was added Cs2CO3 (25 mg, 0.076 mol) and the reaction mixture was stirred at 60 ℃ overnight. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate. Usual work-up and purification by silica gel column chromatography

afforded


methyl

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3-(4-((2',6'-dimethylbiphenyl-3-yl)methoxy)-1H-1,2,3-triazol-1-yl)propanoate

as

colorless oil, yield: 73%. 1H NMR (300 MHz, CDCl3) δ 7.47 – 7.38 (m, 2H), 7.22 (s, 1H), 7.19 – 7.06 (m, 5H), 5.33 (s, 2H), 4.55 (d, J = 6.3 Hz, 2H), 3.70 (s, 3H), 2.94 (d, J = 6.3 Hz, 2H), 2.01 (s, 6H). Following general procedure C, 5c was obtained as white solid, m.p. 122-126 ℃, yield: 78%, purity 95.3% (tR=14.1 min). 1H NMR (400 MHz, CDCl3) δ 7.46 – 7.37 (m, 2H), 7.21 (s, 1H), 7.18 – 7.07 (m, 5H), 5.31 (s, 2H), 4.54 (t, J = 6.4 Hz, 2H), 2.97 (t, J = 6.4 Hz, 2H), 2.00 (s, 6H). ESI-MS m/z: 352.1 ([M+H]+), 350.0 ([M-H]-).

13

C NMR (125 MHz, CDCl3) δ 174.5, 160.4, 141.4, 136.5, 136.0,

129.0, 128.7, 128.5, 127.3, 127.2, 126.2, 107.7, 72.2, 46.2, 34.4 29.7, 20.9. 3-(5-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)propanoic acid (5d): Following

general

procedure

G,

methyl

3-(5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)propanoate was obtained from the intermediate 20d as colorless oil, yield: 87%. 1H NMR (300 MHz, CDCl3) δ 7.44 (t, J = 7.4 Hz, 1H), 7.38 (d, J = 7.6 Hz, 1H), 7.21 – 7.04 (m, 5H), 6.39 (d, J = 3.9 Hz, 1H), 6.05 (d, J = 3.7 Hz, 1H), 5.07 (s, 2H), 3.68 (s, 3H), 2.99 (t, J = 7.8 Hz, 2H), 2.61 (t, J = 7.6 Hz, 2H), 2.01 (s, 6H). Following general procedure C, 5d was obtained as white solid, m.p. 73-77 ℃, yield: 71%, purity 99.2% (tR=14.9 min). 1H NMR (400 MHz, CDCl3) δ 7.48 – 7.35 (m, 2H), 7.22 – 7.07 (m, 5H), 6.42 (d, J = 3.7 Hz, 1H), 6.06 (d, J = 3.7 Hz, 1H), 5.07 (s, 2H), 3.00 (t, J = 7.5 Hz, 2H), 2.67 (t, J = 7.5 Hz, 2H), 2.02 (s, 6H). ESI-MS m/z: 366.9 ([M+H]+), 364.9 ([M-H]-).

13

C NMR (125 MHz, CDCl3) δ

178.3, 163.2, 141.6, 136.5, 136.2, 129.8, 129.3, 128.9, 128.8, 127.5, 127.4, 126.4, 121.9, 105.8, 75.9, 35.8, 25.5, 21.1. 3-(5-((2',6'-Dimethylbiphenyl-3-yloxy)methyl)thiophen-2-yl)propanoic acid (5e): Following general procedure C, 5e was obtained from intermediate 23e as colorless oil,



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yield: 55%, purity 96.3% (tR=14.9 min). 1H NMR (300 MHz, CDCl3) δ 7.32 (t, J = 7.4 Hz, 1H), 7.17 – 7.07 (m, 3H), 6.94 (d, J = 8.4 Hz, 1H), 6.89 (d, J = 3.2 Hz, 1H), 6.76 – 6.70 (m, 3H), 5.12 (s, 2H), 3.13 (t, J = 7.4 Hz, 2H), 2.72 (t, J = 7.5 Hz, 2H), 2.02 (s, 6H). ESI-MS m/z: 364.9 ([M-H]-). 13C NMR (125 MHz, CDCl3) δ 178.3, 158.6, 144.4, 142.7, 141.7, 137.5, 136.2, 129.7, 127.4, 127.3, 127.1, 124.6, 122.2, 115.7, 113.7, 65.3, 36.1, 25.4, 20.9. 3-(4-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)propanoic acid (5f): Following

general

procedure

G,

methyl

3-(4-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)propanoic

acid

was

obtained from the intermediate 20f as colorless oil, yield: 91%. 1H NMR (300 MHz, CDCl3) δ 7.44 (t, J = 7.2 Hz, 1H), 7.38 (d, J = 7.7 Hz, 1H), 7.21 – 7.06 (m, 5H), 6.57 (s, 1H), 6.10 (s, 1H), 5.02 (s, 2H), 3.69 (s, 3H), 3.06 (t, J = 7.7 Hz, 2H), 2.66 (t, J = 7.6 Hz, 2H), 2.02 (s, 6H). Following general procedure C, 5f was obtained as colorless oil, yield: 58%, purity 95.1% (tR=14.7 min). 1H NMR (300 MHz, CDCl3) δ 7.48 – 7.34 (m, 2H), 7.19 – 7.09 (m, 5H), 6.59 (s, 1H), 6.10 (s, 1H), 5.02 (s, 2H), 3.06 (t, J = 7.4 Hz, 2H), 2.71 (t, J = 7.6 Hz, 2H), 2.02 (s, 6H). ESI-MS m/z: 365.1 ([M-H]-). 13C NMR (125 MHz, CDCl3) δ 177.2, 156.7, 141.9, 141.7, 141.5, 137.2, 136.2, 128.9, 128.9, 128.4, 127.5, 127.3, 126.0, 118.0, 96.3, 72.0, 35.4, 25.6, 21.1. 3-(5-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thieno[3,2-b]thiophen-2-yl)propanoic acid (5g): Following general procedure D, starting from the intermediate 31g,

the

benzyl alcohol was specified to (2',6'-dimethylbiphenyl-3-yl)methanol (1.5 equiv) in toluene (2M), affording the mixture of target product and coupling product with ester functionality.

Then

the

mixture

was

dissolved

in

0.5

M

NaOH

in

MeOH/THF/H2O=2:2:1 and stirred until the carboxylate was completely hydrolyzed. The reaction mixture was acidified by diluted HCl (aq) and extracted with ethyl


Page 33 of 89

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acetate for three times. Usual work-up and purification by silica gel column chromatography afforded the product 5g as light yellow solid, yield over 2 steps: 3%, m.p. 136-139 ℃, purity 95.1% (tR=15.5 min). 1H NMR (300 MHz, CDCl3) δ 7.48 – 7.35 (m, 2H), 7.19 (s, 1H), 7.16 – 7.04 (m, 4H), 6.82 (s, 1H), 6.43 (s, 1H), 5.13 (s, 2H), 3.17 (t, J = 7.4 Hz, 2H), 2.74 (t, J = 7.4 Hz, 2H), 1.99 (s, 6H). ESI-MS m/z: 445.1 ([M+Na]+), 421.1 ([M-H]-). 13C NMR (125 MHz, CDCl3) δ 177.6, 165.1, 141.7, 141.5, 141.3, 136.2, 136.1, 134.0, 129.4, 129.0, 128.9, 127.5, 127.4, 126.6, 126.5, 117.4, 100.3, 76.4, 36.3, 29.9, 26.1, 21.1. 5-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thieno[3,2-b]thiophene-2-carboxylic acid (5h): Following the procedure for preparation of 5g, 5h was obtained from the intermediate 31h as light yellow solid, yield over 2 steps: 7%. 1H NMR (300 MHz, CDCl3+CD3OD) δ 7.69 (s, 1H), 7.37 – 7.27 (m, 2H), 7.09 (s, 1H), 7.06 – 6.95 (m, 4H), 6.39 (s, 1H), 5.10 (s, 2H), 1.87 (s, 6H).

13

C NMR (125 MHz, CDCl3) δ 170.6, 167.8,

143.9, 141.9, 141.3, 136.2, 135.4, 129.7, 129.3, 129.2, 128.9, 128.2, 127.6, 127.5, 127.2, 126.4, 99.4, 76.0, 21.1. 3-(5-((2',6'-Dimethylbiphenyl-4-yl)methoxy)thiophen-2-yl)propanoic acid (5i): Following general procedure G, methyl 3-(5-((2',6'-dimethylbiphenyl-4-yl)methoxy)thiophen-2-yl)propanoate was obtained from the intermediate 20i as colorless oil, yield: 72%. 1H NMR (300 MHz, CDCl3) δ 7.47 (d, J = 7.8 Hz, 2H), 7.21 – 7.06 (m, 5H), 6.43 (d, J = 3.1 Hz, 1H), 6.11 (d, J = 3.3 Hz, 1H), 5.08 (s, 3H), 3.70 (s, 3H), 3.02 (t, J = 7.4 Hz, 2H), 2.64 (t, J = 7.6 Hz, 2H), 2.03 (s, 6H). Following general procedure C, 5i was obtained as white solid, m.p. 98-102 ℃, yield: 85%, purity 99.2% (tR=14.9 min). 1H NMR (300 MHz, CDCl3) δ 7.47 (d, J = 7.8 Hz, 2H), 7.21 – 7.05 (m, 5H), 6.45 (d, J = 3.2 Hz, 1H), 6.12 (d, J = 3.6



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Page 34 of 89

Hz, 1H), 5.08 (s, 2H), 3.03 (t, J = 7.3 Hz, 2H), 2.70 (t, J = 7.5 Hz, 2H), 2.03 (s, 6H). ESI-MS m/z: 364.9 ([M-H]-). 13C NMR (125 MHz, CDCl3) δ 178.4, 163.5, 141.54, 141.48, 136.2, 134.5, 129.7, 129.5, 128.3, 127.5, 127.4, 122.0, 105.4, 75.8, 35.9, 25.6, 21.1. 3-(5-((2'-Fluoro-5'-methoxybiphenyl-3-yl)methoxy)thiophen-2-yl)propanoic acid (6a): Following general procedure B, 6a was obtained from the intermediate 26 as colorless oil, yield: 26%, purity 99.5% (tR=13.8 min). 1H NMR (400 MHz, CDCl3) δ 7.57 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.49 – 7.40 (m, 2H), 7.07 (t, J = 9.5 Hz, 1H), 6.94 (dd, J = 6.2, 3.2 Hz, 1H), 6.84 (dt, J = 8.8, 3.4 Hz, 1H), 6.43 (d, J = 3.7 Hz, 1H), 6.08 (d, J = 3.7 Hz, 1H), 5.09 (s, 2H), 3.83 (s, 3H), 3.01 (t, J = 7.5 Hz, 2H), 2.68 (t, J = 7.5 Hz, 2H).ESI-MS m/z: 385.1 ([M-H]-).

C NMR (125 MHz, CDCl3) δ 177.9, 163.1, 155.8

13

(d, J = 1.8 Hz), 154.2 (d, J = 240.9 Hz), 136.3, 136.2, 129.6, 129.2 (d, J = 15.1 Hz), 129.0 (d, J = 2.6 Hz), 128.7, 128.5 (d, J = 2.6 Hz), 127.3, 121.8, 116.7 (d, J = 24.8 Hz), 115.5 (d, J = 3.0 Hz), 114.1 (d, J = 8.1 Hz), 105.3, 75.5, 55.9, 35.6, 25.3. 3-(5-((2'-(Trifluoromethyl)biphenyl-3-yl)methoxy)thiophen-2-yl)propanoic

acid

(6b): Following general procedure B, 6b was obtained from the intermediate 26 as white solid, m.p. 125-129 ℃, yield: 13%, purity 98.4% (tR=14.4 min). 1H NMR (300 MHz, CDCl3) δ 7.75 (d, J = 8.2 Hz, 1H), 7.60 – 7.27 (m, 7H), 6.42 (d, J = 3.7 Hz, 1H), 6.06 (d, J = 3.8 Hz, 1H), 5.07 (s, 2H), 3.01 (t, J = 7.6 Hz, 2H), 2.68 (t, J = 7.5 Hz, 2H). ESI-MS m/z: 405.1 ([M-H]-). 13C NMR (125 MHz, CDCl3) δ 176.8, 163.3, 141.1, 140.3, 135.9, 132.2, 131.5, 129.8, 129.2, 128.7 (q, J = 30.1 Hz), 128.7, 128.2, 127.7, 127.3, 126.3 (q, J = 5.3 Hz), 124.3 (d, J = 274.4 Hz), 121.9, 105.6, 75.7, 35.6, 25.5.


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3-(5-((2'-Cyanobiphenyl-3-yl)methoxy)thiophen-2-yl)propanoic

acid

(6c):

Following general procedure B, 6c was obtained from the intermediate 26 as white solid, m.p. 131-134 ℃, yield: 9%, purity 96.1% (tR=13.3 min). 1H NMR (300 MHz, CDCl3) δ 7.77 (d, J = 7.7 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.60 – 7.40 (m, 6H), 6.43 (d, J = 3.6 Hz, 1H), 6.09 (d, J = 3.7 Hz, 1H), 5.11 (s, 2H), 3.00 (t, J = 7.4 Hz, 2H), 2.67 (t, J = 7.6 Hz, 2H). ESI-MS m/z: 362.0 ([M-H]-).

13

C NMR (125 MHz, CDCl3) δ 178.2,

163.2, 145.3, 138.7, 136.9, 134.0, 133.1, 130.3, 129.8, 129.2, 129.0, 128.4, 128.3, 128.0, 122.0, 118.8, 111.5, 105.6, 75.4, 35.8, 25.5. 3-(5-((2'-Carbamoylbiphenyl-3-yl)methoxy)thiophen-2-yl)propanoic acid (6d): Following general procedure B (2-cyanophenylboronic acid was used as starting material), 6d was obtained from the intermediate 26 as white solid, m.p. 155-158 ℃, yield: 15%, purity 95.3% (tR=11.9 min). 1H NMR (300 MHz, DMSO) δ 7.67 (s, 1H), 7.53 – 7.27 (m, 9H), 6.46 (d, J = 3.4 Hz, 1H), 6.19 (d, J = 3.7 Hz, 1H), 5.09 (s, 2H), 2.85 (t, J = 7.3 Hz, 2H), 2.50 (t, J = 7.3 Hz, 2H). ESI-MS m/z: 380.1 ([M-H]-).

13

C NMR

(125 MHz, DMSO) δ 173.4, 171.0, 162.2, 140.7, 138.5, 137.3, 136.0, 129.9, 129.7, 129.3, 128.3, 128.3, 127.9, 127.6, 127.1, 126.8, 121.8, 104.9, 74.6, 35.4, 25.1. 3-(5-((2'-Methoxybiphenyl-3-yl)methoxy)thiophen-2-yl)propanoic

acid

(6e):

Following general procedure B, 6e was obtained from the intermediate 26 as colorless oil, yield: 26%, purity 96.4% (tR=13.9 min). 1H NMR (300 MHz, CDCl3) δ 7.57 (s, 1H), 7.51 (dt, J = 7.6, 1.5 Hz, 1H), 7.47 – 7.28 (m, 4H), 7.05 (d, J = 7.4 Hz, 1H), 6.99 (d, J = 8.6 Hz, 1H), 6.43 (d, J = 3.7 Hz, 1H), 6.08 (d, J = 3.8 Hz, 1H), 5.08 (s, 2H), 3.81 (s, 3H), 3.01 (t, J = 7.5 Hz, 2H), 2.68 (t, J = 7.5 Hz, 2H). ESI-MS m/z: 368.8 ([M+H]+), 366.9 ([M-H]-).

13

C NMR (125 MHz, CDCl3) δ 179.4, 157.6, 156.6, 139.0, 136.9,

132.7, 131.1, 130.6, 129.4, 129.4, 128.9, 128.9, 128.4, 126.2, 121.0, 115.1, 111.4, 70.3, 55.7, 36.1, 29.9.



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Page 36 of 89

3-(5-((4'-(Trifluoromethyl)biphenyl-3-yl)methoxy)thiophen-2-yl)propanoic

acid

(6f): Following general procedure B, 6f was obtained from the intermediate 26 as white solid, m.p. 117-120 ℃, yield: 24%, purity 96.6% (tR=14.8 min). 1H NMR (300 MHz, CDCl3) δ 7.70 (s, 4H), 7.63 (s, 1H), 7.60 – 7.54 (m, 1H), 7.52 – 7.40 (m, 2H), 6.43 (d, J = 3.7 Hz, 1H), 6.09 (d, J = 3.8 Hz, 1H), 5.10 (s, 2H), 3.01 (t, J = 7.5 Hz, 2H), 2.68 (t, J = 7.5 Hz, 2H). ESI-MS m/z: 405.0 ([M-H]-).

13

C NMR (125 MHz, CDCl3) δ 178.1,

163.2, 144.5, 140.4, 137.1, 129.9, 129.8 (q, J = 32.4 Hz), 129.5, 127.8, 127.7, 127.5, 126.9, 126.0 (q, J = 3.7 Hz), 124.5 (q, J = 272.7 Hz), 122.0, 105.6, 75.6, 35.8, 25.5. 3-(5-(3-(2,6-Dichloropyridin-4-yl)benzyloxy)thiophen-2-yl)propanoic acid (6g): Following general procedure B, 6g was obtained from the intermediate 26 as light yellow oil, yield: 19%, purity 97.3% (tR=14.4 min). 1H NMR (300 MHz, CDCl3) δ 7.62 (s, 1H), 7.59 – 7.50 (m, 3H), 7.47 (s, 2H), 6.43 (d, J = 3.7 Hz, 1H), 6.09 (d, J = 3.7 Hz, 1H), 5.10 (s, 2H), 3.01 (t, J = 7.4 Hz, 2H), 2.68 (t, J = 7.4 Hz, 2H). ESI-MS m/z: 405.9 ([M-H]-), 408.0 ([M+2-H]-). 13C NMR (125 MHz, CDCl3) δ 178.6, 157.3, 153.9, 151.3, 138.8, 136.3, 133.2, 129.9, 129.6, 129.4, 126.9, 126.2, 121.1, 115.1, 69.7, 36.0, 30.0. 3-(5-(3-(3-Chloropyridin-4-yl)benzyloxy)thiophen-2-yl)propanoic

acid

(6h):

Following general procedure B, 6h was obtained from the intermediate 26 as white solid, m.p. 115-117 ℃, yield: 11%, purity 98.9% (tR=12.5 min). 1H NMR (300 MHz, CDCl3) δ 8.69 (s, 1H), 8.54 (d, J = 4.9 Hz, 1H), 7.55 – 7.41 (m, 4H), 7.30 (d, J = 5.0 Hz, 1H), 6.43 (d, J = 3.8 Hz, 1H), 6.08 (d, J = 3.4 Hz, 1H), 5.10 (s, 2H), 3.02 (t, J = 7.5 Hz, 2H), 2.68 (t, J = 7.5 Hz, 2H). ESI-MS m/z: 371.9 ([M-H]-), 373.9 ([M+2-H]-).

13

C

NMR (125 MHz, CDCl3) δ 176.7, 163.0, 150.1, 147.8, 147.7, 137.0, 136.8, 130.5, 130.2, 129.1, 129.0, 128.6, 128.5, 125.7, 121.9, 105.7, 75.4, 35.7, 25.7.


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3-(5-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)butanoic

acid

(7a):

Following general procedure J, 7a was obtained from the intermediate 32 as yellow oil, yield: 57%, purity 95.0% (tR=15.2 min). 1H NMR (300 MHz, CDCl3) δ 7.44 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.21 – 7.06 (m, 5H), 6.42 (d, J = 3.7 Hz, 1H), 6.04 (d, J = 3.8 Hz, 1H), 5.07 (s, 2H), 3.47 – 3.31 (m, 1H), 2.68 (dd, J = 15.8, 6.7 Hz, 1H), 2.52 (dd, J = 15.7, 7.7 Hz, 1H), 2.01 (s, 6H), 1.33 (d, J = 6.8 Hz, 3H). ESI-MS m/z: 379.0 ([M-H]-).

13

C NMR (125 MHz, CDCl3) δ 177.7, 162.9, 141.6, 141.6, 136.5, 136.5,

136.2, 129.2, 128.9, 128.8, 127.5, 127.4, 126.4, 120.0, 105.4, 75.8, 43.6, 32.3, 22.5, 21.1. 3-(5-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)pentanoic acid (7b): Following general procedure J, 7b was obtained from the intermediate 32 as yellow oil, yield: 55%, purity 95.1% (tR=15.9 min). 1H NMR (300 MHz, CDCl3) δ 7.44 (t, J = 7.5 Hz, 1H), 7.37 (d, J = 7.9 Hz, 1H), 7.20 – 7.06 (m, 5H), 6.41 (d, J = 3.8 Hz, 1H), 6.04 (d, J = 3.8 Hz, 1H), 5.07 (s, 2H), 3.20 – 3.05 (m, 1H), 2.60 (d, J = 7.9 Hz, 2H), 2.01 (s, 6H), 1.77 – 1.48 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). ESI-MS m/z: 392.9 ([M-H]-).

13

C NMR

(125 MHz, CDCl3) δ 177.7, 163.0, 141.60, 141.56, 136.5, 136.2, 134.5, 129.2, 128.9, 128.8, 127.5, 127.4, 126.4, 121.4, 105.3, 75.7, 42.1, 39.7, 30.0, 29.9, 21.1, 11.9. 3-(5-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)hexanoic

acid

(7c):

Following general procedure J, 7c was obtained from the intermediate 32 as yellow oil, yield: 55%, purity 95.9% (tR=16.3 min). 1H NMR (300 MHz, CDCl3) δ 7.43 (t, J = 7.5 Hz, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.21 – 7.04 (m, 5H), 6.40 (d, J = 3.7 Hz, 1H), 6.02 (d, J = 3.7 Hz, 1H), 5.06 (s, 2H), 3.28 – 3.12 (m, 1H), 2.58 (d, J = 7.3 Hz, 2H), 2.00 (s, 6H), 1.66 – 1.41 (m, 2H), 1.36 – 1.14 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). ESI-MS m/z: 408.9 ([M+H]+), 407.0 ([M-H]-). 13C NMR (125 MHz, CDCl3) δ 176.0, 163.0, 141.61, 141.55,



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136.5, 136.2, 134.7, 129.2, 128.9, 128.8, 127.5, 127.4, 126.4, 121.4, 105.3, 75.7, 42.1, 39.3, 37.9, 21.1, 20.5, 14.0. 3-Cyclopropyl-3-(5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)propan oic acid (7d): Following general procedure J, 7d was obtained from the intermediate 32 as colorless oil, yield: 52%, purity 95.7% (tR=17.5 min). 1H NMR (300 MHz, CDCl3) δ 7.47 – 7.32 (m, 2H), 7.20 – 7.03 (m, 5H), 6.43 (d, J = 3.8 Hz, 1H), 6.05 (d, J = 3.8 Hz, 1H), 5.06 (s, 2H), 2.75 (d, J = 7.2 Hz, 2H), 2.49 (dd, J = 16.6, 7.6 Hz, 1H), 2.00 (s, 6H), 1.04 – 0.89 (m, 1H), 0.58 – 0.51 (m, 2H), 0.33 – 0.21 (m, 2H). ESI-MS m/z: 405.0 ([M-H]-).

C NMR (125 MHz, CDCl3) δ 177.0, 163.2, 141.60, 141.56, 136.5,

13

136.2, 134.4, 129.2, 128.9, 128.8, 127.5, 127.4, 126.4, 120.6, 105.3, 75.7, 42.7, 41.6, 21.1, 18.2, 5.3, 5.0. 3-(5-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)hex-4-ynoic acid (7e): Following general procedure J, 7e was obtained from the intermediate 32 as light yellow oil, yield: 56%, purity 97.9% (tR=15.1 min). 1H NMR (300 MHz, CDCl3) δ 7.48 – 7.33 (m, 2H), 7.20 – 7.05 (m, 5H), 6.55 (d, J = 3.8 Hz, 1H), 6.04 (d, J = 3.8 Hz, 1H), 5.06 (s, 2H), 4.23 – 4.18 (m, 1H), 2.86 – 2.70 (m, 2H), 2.00 (s, 6H), 1.81 (s, 3H). ESI-MS m/z: 404.9 ([M+H]+), 402.8 ([M-H]-).

C NMR (125 MHz, CDCl3) δ 177.0,

13

163.8, 141.6, 141.5, 136.4, 136.2, 131.1, 129.3, 128.9, 128.7, 127.5, 127.4, 126.4, 121.8, 105.6, 79.5, 78.6, 75.8, 43.4, 29.7, 21.0, 3.8. 3-(5-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)-3-methoxypropanoic acid

(7f):

Following

general

procedure

K,

ethyl

3-(5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)-3-methoxypropanoate was obtained from the intermediate 34 as colorless oil, yield: 70%. 1H NMR (300 MHz, CDCl3) δ 7.49 – 7.35 (m, 2H), 7.21 – 7.06 (m, 5H), 6.62 (d, J = 3.8 Hz, 1H), 6.10 (d, J = 3.8 Hz, 1H), 5.10 (s, 2H), 4.70 (dd, J = 8.7, 5.0 Hz, 1H), 4.15 (q, J = 7.1 Hz, 2H), 3.24


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(s, 3H), 2.84 (dd, J = 15.4, 8.8 Hz, 1H), 2.64 (dd, J = 15.2, 5.1 Hz, 1H), 2.01 (s, 6H), 1.24 (t, J = 7.1 Hz, 3H). Then following general procedure C, 7f was obtained as colorless oil, yield: 47%, purity 95.1% (tR=14.6 min). 1H NMR (300 MHz, CDCl3) δ 7.49 – 7.36 (m, 2H), 7.22 – 7.07 (m, 5H), 6.64 (d, J = 3.3 Hz, 1H), 6.10 (d, J = 3.8 Hz, 1H), 5.11 (s, 2H), 4.69 (dd, J = 9.1, 4.3 Hz, 1H), 3.27 (s, 3H), 2.89 (dd, J = 15.8, 9.0 Hz, 1H), 2.72 (dd, J = 15.7, 4.6 Hz, 1H), 2.01 (s, 6H). ESI-MS m/z: 394.9 ([M-H]-).

13

C

NMR (150 MHz, CDCl3) δ 176.1, 165.3, 141.6, 141.5, 136.20, 136.19, 130.2, 129.3, 129.0, 128.7, 127.5, 127.4, 126.4, 124.0, 105.1, 76.2, 75.7, 56.5, 43.5, 21.1. 3-(5-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)-3-ethoxypropanoic acid

(7g):

Following

general

procedure

K,

3-(5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)-3-ethoxypropanoate obtained from the intermediate 34 as colorless oil, yield: 76%.

1

ethyl was

H NMR (300 MHz,

CDCl3) δ 7.45 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 7.6 Hz, 1H), 7.20 – 7.07 (m, 5H), 6.59 (d, J = 3.8 Hz, 1H), 6.08 (d, J = 3.8 Hz, 1H), 5.09 (s, 2H), 4.81 (dd, J = 8.7, 5.2 Hz, 1H), 4.14 (q, J = 7.1 Hz, 2H), 3.56 – 3.31 (m, 2H), 2.84 (dd, J = 15.3, 8.7 Hz, 1H), 2.63 (dd, J = 15.3, 5.3 Hz, 1H), 2.01 (s, 6H), 1.24 (t, J = 7.1 Hz, 3H), 1.14 (t, J = 7.0 Hz, 3H). Then following general procedure C, 7g was obtained as colorless oil, yield: 43%, purity 96.5% (tR=17.0 min, mobile phase without additive was used). 1H NMR (300 MHz, CDCl3) δ 7.45 (t, J = 7.0 Hz, 1H), 7.38 (d, J = 7.3 Hz, 1H), 7.22 – 7.08 (m, 5H), 6.62 (d, J = 3.5 Hz, 1H), 6.09 (d, J = 3.4 Hz, 1H), 5.10 (s, 2H), 4.81 (dd, J = 8.7, 4.4 Hz, 1H), 3.57 – 3.33 (m, 2H), 2.89 (dd, J = 16.0, 8.1 Hz, 1H), 2.72 (dd, J = 15.8, 4.5 Hz, 1H), 2.01 (s, 6H), 1.16 (t, J = 7.0 Hz, 3H). ESI-MS m/z: 432.8 ([M+Na]+), 408.9 ([M-H]-). (R)-3-(5-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)-3-ethoxypropanoi c acid ((R)-7g): According to the same procedure of 7g, (R)-7g was obtained using (R)-34 as precursor, overall yield over 2 steps: 33%, 98% ee [HPLC condition:



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CHIRALPAK IB 4.6 mm × 250 mm column by isocratic elution: 0.1 % TFA in hexane/0.1% TFA in 2-propanol=90/10 (v/v) at a flow rate of 1.0 mL/min, with UV detection at 254 nm]. Retention time: minor product (S)-7g 8.0 min, major product (R)-7g 24.4 min. 1H NMR (300 MHz, CDCl3) δ 7.45 (t, J = 7.0 Hz, 1H), 7.38 (d, J = 7.3 Hz, 1H), 7.22 – 7.08 (m, 5H), 6.62 (d, J = 3.5 Hz, 1H), 6.09 (d, J = 3.4 Hz, 1H), 5.10 (s, 2H), 4.81 (dd, J = 8.7, 4.4 Hz, 1H), 3.57 – 3.33 (m, 2H), 2.89 (dd, J = 16.0, 8.1 Hz, 1H), 2.72 (dd, J = 15.8, 4.5 Hz, 1H), 2.01 (s, 6H), 1.16 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 175.3, 165.0, 141.6, 141.5, 136.2, 136.1, 130.9, 129.3, 128.9, 128.7, 127.5, 127.3, 126.3, 123.4, 115.1, 105.0, 75.6, 74.1, 64.3, 43.3, 21.0, 15.1. HRMS (ESI) calcd for C24H25O4S [M-H]-: 409.1474, found 409.1476. [α]D +28.4o (c 0.175, CH2Cl2). 3-Cyano-3-(5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)propanoic acid (7h): To the solution of 32 (19 mg, 0.042 mmol) in 0.8 mL MeOH was added KCN (3.3 mg, 0.051 mmol) and stirred at room temperature for 2 h. The reaction mixture was diluted with ethyl acetate and washed with brine. The organic layer was dried over anhydrous Na2SO4. After removal of the drying agent, the filtrate was concentrated

under

reduced

pressure

to

afford

2-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)-2-(5-((2',6'-dimethylbiphenyl-3-yl)metho xy)thiophen-2-yl)acetonitrile which could be used for next step without further purification, yield: 89%. 1H NMR (300 MHz, CDCl3) δ 7.36 – 7.26 (m, 2H), 7.08 – 6.92 (m, 5H), 6.54 (d, J = 3.2 Hz, 1H), 5.94 (d, J = 3.4 Hz, 1H), 5.21 (s, 1H), 5.01 – 4.93 (m, 1H), 4.92 (s, 2H), 1.88 (s, 6H), 1.52 (s, 6H). To the solution of 2-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)-2-(5-((2',6'-dimethylbiphenyl-3-yl)metho xy)thiophen-2-yl)acetonitrile (190 mg, 0.4 mmol) in 5 mL MeNO2/DMF=3:2 was added FeCl3.6H2O (0.54 mg, 0.002 mmol) and H2O (7.2 μL, 0.4 mmol). The reaction


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mixture was stirred at 110 ℃ under microwave for 5 min. After cooling to room temperature, the mixture was acidified by diluted HCl (aq) and extracted with ethyl acetate for 3 times. Usual work-up and purification by silica gel column chromatography afforded the product as light yellow oil, yield: 46%, purity 96.2% (tR=15.6 min). 1H NMR (300 MHz, CDCl3) δ 7.46 (t, J = 7.5 Hz, 1H), 7.37 (d, J = 7.7 Hz, 1H), 7.21 – 7.07 (m, 5H), 6.72 (d, J = 3.9 Hz, 1H), 6.11 (d, J = 3.9 Hz, 1H), 5.10 (s, 2H), 4.36 (t, J = 7.3 Hz, 1H), 3.07 (dd, J = 17.2, 7.8 Hz, 1H), 2.94 (dd, J = 17.2, 6.8 Hz, 1H), 2.01 (s, 6H). HRMS (ESI) calcd for C23H20NO3S [M-H]-: 390.1164, found 390.1175.

13

C NMR (150 MHz, CDCl3) δ 173.7, 165.3, 141.7, 141.4, 136.2, 135.8,

129.5, 129.1, 128.8, 127.6, 127.4, 126.4, 124.6, 122.3, 118.9, 105.9, 76.0, 39.6, 28.9, 21.1. 3-(5-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)-3-(1H-1,2,3-triazol-4yl)propanoic acid (7i): Following general procedure J and L in sequence, methyl 3-(5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)pent-4-ynoate was obtained as yellow oil, yield: 53%. 1H NMR (300 MHz, CDCl3) δ 7.49 – 7.34 (m, 2H), 7.19 – 7.09 (m, 5H), 6.60 (dd, J = 3.8, 0.8 Hz, 1H), 6.07 (d, J = 3.8 Hz, 1H), 5.08 (s, 2H), 4.30 – 4.24 (m, 1H), 3.70 (s, 3H), 2.89 – 2.73 (m, 2H), 2.30 (d, J = 3.1 Hz, 1H), 2.01 (s, 6H). Following general procedure A (the amount of TMSN3 was increased to 2 equiv and reaction

time

was

increased

to

24

h),

methyl

3-(5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)-3-(1H-1,2,3-triazol-4-yl)pr opanoate was obtained as yellow oil, yield: 39%. 1H NMR (300 MHz, CDCl3) δ 7.51 (s, 1H), 7.44 (t, J = 7.5 Hz, 1H), 7.36 (d, J = 7.1 Hz, 1H), 7.21 – 7.06 (m, 5H), 6.49 (d, J = 3.9 Hz, 1H), 6.06 (d, J = 3.8 Hz, 1H), 5.06 (s, 2H), 4.79 (t, J = 7.4 Hz, 1H), 3.65 (s, 3H), 3.17 (dd, J = 16.0, 8.4 Hz, 1H), 2.99 (dd, J = 16.0, 7.3 Hz, 1H), 2.00 (s, 6H). Following general procedure C, 7i was obtained as yellow oil, yield: 72%, purity 95.3% (tR=13.6



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Page 42 of 89

min). 1H NMR (300 MHz, CD3OD) δ 7.61 (s, 1H), 7.44 (t, J = 7.5 Hz, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.16 – 7.00 (m, 5H), 6.50 (d, J = 3.8 Hz, 1H), 6.09 (d, J = 3.8 Hz, 1H), 5.08 (s, 2H), 4.74 (t, J = 7.6 Hz, 1H), 3.10 (dd, J = 16.1, 7.9 Hz, 1H), 2.96 (dd, J = 16.0, 7.5 Hz, 1H), 1.93 (s, 6H). ESI-MS m/z: 431.9 ([M-H]-). (R)-3-(5-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)-3-(isoxazol-3-yl)p ropanoic acid (7j): To the solution of 36 (104 mg, 0.18 mmol) and DMAP (2.2 mg, 0.018 mmol) in vinyl bromide (1 M in THF, 1.8 mL, 1.8 mmol) was added (Boc)2O (28 mg, 0.27 mmol). The reaction mixture was stirred at room temperature overnight. Then vinyl bromide (1 M in THF, 1.8 mL, 1.8 mmol) and (Boc)2O (28 mg, 0.27 mmol) were added and the mixture was stirred for additional 24 h. The resulting mixture was diluted with ethyl acetate and washed with brine. Usual work-up and purification by silica

gel

column

chromatography

or

HPLC

afforded

(S)-4-benzyl-3-((R)-3-(5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)-3-(iso xazol-3-yl)propanoyl)oxazolidin-2-one as colorless oil, yield: 46%. 1H NMR (300 MHz, CDCl3) δ 8.30 (s, 1H), 7.44 (t, J = 7.5 Hz, 1H), 7.39 – 7.27 (m, 4H), 7.20 – 7.09 (m, 7H), 6.55 (d, J = 3.8 Hz, 1H), 6.26 (s, 1H), 6.08 (d, J = 3.8 Hz, 1H), 5.07 (s, 2H), 4.92 (t, J = 7.3 Hz, 1H), 4.65 (ddd, J = 13.1, 6.9, 3.2 Hz, 1H), 4.22 – 4.08 (m, 2H), 3.94 (dd, J = 17.5, 8.0 Hz, 1H), 3.60 (dd, J = 17.5, 6.6 Hz, 1H), 3.21 (dd, J = 13.4, 2.9 Hz, 1H), 2.75 (dd, J = 13.3, 9.4 Hz, 1H), 2.00 (s, 6H). To

the

solution

of

(S)-4-benzyl-3-((R)-3-(5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)-3-(iso xazol-3-yl)propanoyl)oxazolidin-2-one (45 mg, 0.076 mmol) in 1 mL THF and hydrogen peroxide (30%, 8.2 μL, 0.27 mmol) in ice bath was added LiOH.H2O (6.4 mg, 0.15 mmol) in 0.3 mL H2O. The reaction mixture was stirred at 0 ℃ for 1 h and quenched by aqueous Na2SO3. After warming to room temperature, the mixture was


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acidified by diluted HCl (aq) and extracted with ethyl acetate for 3 times. Usual work-up and purification by silica gel column chromatography afforded the product as colorless oil, yield: 25%, purity 96.7% (tR=14.6 min). 1H NMR (300 MHz, CDCl3) δ 8.29 (s, 1H), 7.44 (t, J = 7.5 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.20 – 7.06 (m, 5H), 6.52 (d, J = 3.5 Hz, 1H), 6.19 (s, 1H), 6.06 (d, J = 3.8 Hz, 1H), 5.06 (s, 2H), 4.72 (t, J = 7.4 Hz, 1H), 3.28 (dd, J = 16.6, 7.8 Hz, 1H), 3.01 (dd, J = 16.6, 7.4 Hz, 1H), 1.99 (s, 6H). ESI-MS m/z: 456.1 ([M+Na]+), 431.9 ([M-H]-).

C NMR (125 MHz, CDCl3) δ 176.1,

13

164.4, 163.9, 158.8, 141.6, 141.5, 136.2, 129.9, 129.3, 129.0, 128.7, 127.5, 127.4, 126.4, 122.9, 105.7, 104.1 75.8, 40.0, 35.5, 21.0. 3-(5-((2',6'-Dimethyl-4'-(3-(methylsulfonyl)propoxy)biphenyl-3-yl)methoxy)thiop hen-2-yl)hex-4-ynoic acid (7k): Following general procedure C, 7k was obtained from the intermediate 45k as colorless oil, yield: 61%, purity 98.1% (tR=13.7 min). 1H NMR (300 MHz, CDCl3) δ 7.43 (t, J = 7.5 Hz, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.15 (s, 1H), 7.10 (d, J = 7.3 Hz, 1H), 6.65 (s, 2H), 6.57 (d, J = 3.8 Hz, 1H), 6.05 (d, J = 3.8 Hz, 1H), 5.07 (s, 2H), 4.20 (t, J = 7.3 Hz, 1H), 4.13 (t, J = 5.7 Hz, 2H), 3.34 – 3.20 (m, 2H), 2.97 (s, 3H), 2.90 – 2.66 (m, 2H), 2.35 (td, J = 11.7, 5.8 Hz, 2H), 1.99 (s, 6H), 1.82 (d, J = 2.0 Hz, 3H). ESI-MS m/z: 563.0 ([M+Na]+), 539.0 ([M-H]-).

13

C NMR (125 MHz,

CDCl3) δ 175.9, 163.8, 157.3, 141.2, 137.8, 136.3, 134.9, 131.2, 129.8, 129.3, 128.9, 126.3, 121.8, 113.4, 105.5, 79.4, 78.7, 75.8, 65.5, 52.0, 43.3, 41.0, 22.9, 21.3, 3.8, 1.2. (R)-3-(5-((2',6'-dimethyl-4'-(3-(methylsulfonyl)propoxy)biphenyl-3-yl)methoxy)t hiophen-2-yl)hex-4-ynoic acid ((R)-7k): The diastereoisomer 46k was separated by HPLC using a CHIRALPAK AD-H semi-preparative column (10 mm × 250 mm) [Conditions: isocratic elution, hexane/EtOH/MeOH=35/26/39 (v/v/v) at a flow rate of 4.0 mL/min, with UV detection at 254 nm] to afford (R)-46k with a retention time of 23

minutes.

Compound

(R)-46k

was hydrolyzed in 0.25 M NaOH in



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MeOH/THF/H2O=2:2:1 to obtained (R)-7k. 1H NMR (300 MHz, CDCl3) δ 7.46 – 7.32 (m, 2H), 7.15 (s, 1H), 7.10 (d, J = 7.3 Hz, 1H), 6.65 (s, 2H), 6.57 (d, J = 3.8 Hz, 1H), 6.05 (d, J = 3.7 Hz, 1H), 5.07 (s, 2H), 4.20 (t, J = 7.3 Hz, 1H), 4.13 (t, J = 5.7 Hz, 2H), 3.34 – 3.20 (m, 2H), 2.97 (s, 3H), 2.89 – 2.68 (m, 2H), 2.35 (td, J = 11.3, 5.8 Hz, 2H), 1.99 (s, 6H), 1.82 (d, J = 2.3 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 176.1, 163.8, 157.3, 141.2, 137.8, 136.3, 134.9, 131.1, 129.8, 129.3, 128.9, 126.3, 121.8, 113.4, 105.5, 79.5, 78.7, 75.8, 65.5, 52.0, 43.3, 41.0, 29.7, 22.9, 21.3, 3.8. HRMS (ESI) calcd for C29H31O6S2 [M-H]-: 539.1562, found 539.1576. [α]D +4.2o (c 0.210, CH2Cl2). (S)-3-(5-((2',6'-dimethyl-4'-(3-(methylsulfonyl)propoxy)biphenyl-3-yl)methoxy)th iophen-2-yl)hex-4-ynoic acid ((S)-7k): The diastereoisomer 46k was separated by HPLC using a CHIRALPAK AD-H semi-preparative column (10 mm × 250 mm) [Conditions: isocratic elution, hexane/EtOH/MeOH=35/26/39 (v/v/v) at a flow rate of 4.0 mL/min, with UV detection at 254 nm] to afford (S)-46k with a retention time of 15 minutes.

Compound

(S)-46k

was

hydrolyzed

in

0.25

M

NaOH

in

MeOH/THF/H2O=2:2:1 to give (S)-7k. [α]D -5.1o (c 0.145, CH2Cl2). 3-(5-((2'-Methyl-4'-((3-methyloxetan-3-yl)methoxy)biphenyl-3-yl)methoxy)thioph en-2-yl)hex-4-ynoic acid (7l): Following general procedure C, 7l was obtained from the intermediate 45l as light yellow oil, yield: 82%, purity 96.4% (tR=14.9 min). 1H NMR (300 MHz, CDCl3) δ 7.46 – 7.33 (m, 2H), 7.16 (s, 1H), 7.11 (d, J = 7.3 Hz, 1H), 6.71 (s, 2H), 6.57 (d, J = 3.8 Hz, 1H), 6.05 (dd, J = 3.7, 1.4 Hz, 1H), 5.07 (s, 2H), 4.65 (d, J = 5.9 Hz, 2H), 4.48 (d, J = 5.9 Hz, 2H), 4.20 (t, J = 6.8 Hz, 1H), 4.04 (s, 2H), 2.89 – 2.66 (m, 2H), 2.00 (s, 6H), 1.82 (d, J = 2.2 Hz, 3H), 1.45 (s, 3H). ESI-MS m/z: 527.0 ([M+Na]+), 502.9 ([M-H]-). 13C NMR (125 MHz, CDCl3) δ 174.9, 163.8, 158.1, 141.3, 137.8, 136.3, 134.6, 131.1, 129.9, 129.4, 128.9, 126.3, 121.9, 113.4, 105.6, 80.2, 79.5, 78.7, 75.9, 72.9, 43.1, 39.9, 29.8, 21.5, 21.3, 3.8.


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3-(5-((2',6'-Dimethyl-4'-(1-methylpiperidin-4-yloxy)biphenyl-3-yl)methoxy)thiop hen-2-yl)hex-4-ynoic acid (7m): Following general procedure C, 7m was obtained from the intermediate 45m as white solid, m.p. 89-93 ℃, yield: 65%, purity 97.6% (tR=12.3 min). 1H NMR (300 MHz, CDCl3) δ 7.44 – 7.28 (m, 2H), 7.13 (s, 1H), 7.07 (d, J = 7.3 Hz, 1H), 6.62 (s, 2H), 6.58 (d, J = 3.8 Hz, 1H), 6.02 (d, J = 3.8 Hz, 1H), 5.04 (s, 2H), 4.48 (s, 1H), 4.24 (dd, J = 8.6, 6.4 Hz, 1H), 2.93 (s, 4H), 2.78 – 2.60 (m, 2H), 2.54 (s, 3H), 2.23 – 2.08 (m, 2H), 2.07 – 1.98 (m, 2H), 1.95 (s, 6H), 1.80 (d, J = 2.0 Hz, 3H). ESI-MS m/z: 518.1 ([M+H]+), 516.0 ([M-H]-).

C NMR (125 MHz, CDCl3) δ 176.7,

13

163.3, 155.7, 141.1, 137.9, 136.6, 135.0, 133.1, 129.7, 129.3, 128.9, 126.3, 121.2, 114.8, 105.7, 105.5, 80.4, 78.1, 75.8, 50.3, 45.6, 44.2, 30.8, 28.4, 21.3, 4.0. ((3-Bromophenoxy)methyl)(methyl)sulfane (8): 3-Bromophenol (613 μL, 5.8 mmol) and NaI (870 mg, 5.8 mmol) were dissolved in 50 mL DMF and cooled in ice bath. NaH (55%, 506 mg, 11.6 mmol) was added in small portions and the reaction mixture was stirred for additional 30 min. (Chloromethyl)(methyl)sulfane (538 μL, 6.4 mmol) was added dropwise. Then the mixture was warmed to room temperature and stirred overnight. The reaction was quenched by ice water and extracted with Et2O. The organic layer was dried over anhydrous Na2SO4. After removal of the drying agent, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to afford the product as colorless oil, yield: 72%. 1H NMR (300 MHz, CDCl3) δ 7.20 – 7.09 (m, 3H), 6.89 (dt, J = 7.2, 2.3 Hz, 1H), 5.13 (s, 2H), 2.25 (d, J = 1.4 Hz, 3H). 1-(Azidomethoxy)-3-bromobenzene (9): Compound 8 (1.0 g, 4.3 mmol) was dissolved in 15 mL anhydrous DCM and cooled to -78 ℃. SO2Cl2 (350 μL, 4.3 mmol) in 5 mL anhydrous DCM was added dropwise by syringe. The reaction mixture was



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Page 46 of 89

stirred at -78 ℃ for additional 1 h. The mixture was concentrated under reduced pressure. The residue was dissolved in 20 mL DMSO and NaN3 (559 mg, 8.6 mmol) was added. The reaction mixture was stirred at room temperature overnight. The mixture was diluted with Et2O and washed with aqueous NaHCO3 and brine. The organic layer was dried over anhydrous Na2SO4. After removal of the drying agent, the filtrate was concentrated under reduced pressure to afford the crude 9 which could be used for next step without further purification. 1H NMR (300 MHz, CDCl3) δ 7.23 – 7.16 (m, 3H), 6.94 (dd, J = 7.6, 4.0 Hz, 1H), 5.15 (s, 2H). 3-(1-((3-Bromophenoxy)methyl)-1H-1,2,3-triazol-4-yl)propanoic

acid

(10):

Following general procedure A, 10 was obtained as white solid, yield: 75%. 1H NMR (300 MHz, DMSO) δ 8.10 (s, 1H), 7.40 (s, 1H), 7.34 – 7.20 (m, 2H), 7.12 (d, J = 7.1 Hz, 1H), 6.36 (s, 2H), 2.86 (t, J = 7.4 Hz, 2H), 2.59 (t, J = 7.3 Hz, 2H). 1-Bromo-3-(prop-2-ynyloxy)benzene (11): 3-Bromophenol (613 μL, 5.8 mmol) was dissolved in 10 mL acetone and K2CO3 (1.6 g, 11.6 mmol) was added. The reaction mixture was stirred at 60 ℃ for 1.5 h. After cooling to room temperature, the resulting mixture was diluted with Et2O. The mixture was washed with diluted NaOH (aq) and brine respectively and the organic layer was dried over anhydrous Na2SO4. Usual work-up and purification by silica gel column chromatography afforded the product as colorless oil, yield: 78%.1H NMR (300 MHz, CDCl3) δ 7.22 – 7.10 (m, 3H), 6.92 (d, J = 7.5 Hz, 1H), 4.68 (d, J = 2.2 Hz, 2H), 2.55 (t, J = 2.1 Hz, 1H). Methyl 3-(4-((3-bromophenoxy)methyl)-1H-1,2,3-triazol-1-yl)propanoate (12): Following general procedure A, 12 was obtained as white solid, yield: 84%. 1H NMR (300 MHz, CDCl3) δ 7.71 (s, 1H), 7.19 – 7.06 (m, 3H), 6.92 (d, J = 7.8 Hz, 1H), 5.17 (s, 2H), 4.66 (t, J = 6.3 Hz, 2H), 3.70 (s, 3H), 2.99 (t, J = 6.3 Hz, 2H).


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(2',6'-Dimethyl- biphenyl-3-yl)methanol (13): Following general procedure B, 13 was obtained as colorless oil, yield: 76%. 1H NMR (300 MHz, CDCl3) δ 7.48 – 7.30 (m, 2H), 7.21 – 7.04 (m, 5H), 4.74 (s, 2H), 2.03 (s, 6H). 3'-(Bromomethyl)-2,6-dimethylbiphenyl (14): Compound 13 (70 mg, 0.33 mmol) was dissolved in 2 mL DCM and cooled in ice bath. PBr3 (31 μL, 0.33 mmol) was added to the solution, then the reaction mixture was warmed to room temperature. After the starting material was completely consumed, the reaction was quenched by ice water and extracted with DCM. The organic layer was dried over anhydrous Na2SO4. After removal of the drying agent, the filtrate was concentrated under reduced pressure to afford the crude 14 which could be used for next step without further purification. Methyl 3-(4-ethoxy-1H-1,2,3-triazol-1-yl)propanoate (15): Following general procedure A, 15 was obtained as light yellow oil, yield: 28%. 1H NMR (300 MHz, CDCl3) δ 7.05 (s, 1H), 4.54 (t, J = 6.5 Hz, 2H), 4.25 (q, J = 7.0 Hz, 2H), 3.69 (s, 3H), 2.93 (t, J = 6.5 Hz, 2H), 1.38 (t, J = 7.0 Hz, 3H). Methyl 3-(4-hydroxy-1H-1,2,3-triazol-1-yl)propanoate (16): Compound 15 (56 mg, 0.28 mmol) was dissolved in 3 mL DCM and cooled in ice bath. BBr3 (4 M in DCM, 0.7 mL, 2.8 mmol) was added dropwise, then the reaction mixture was warmed to room temperature. After the starting material was completely consumed, the reaction was quenched by ice water. Usual work-up and purification by silica gel column chromatography afforded the product as light yellow oil, yield: 94%. 1H NMR (300 MHz, CDCl3) δ 7.05 (s, 1H), 5.56 (s, 1H), 4.54 (t, J = 6.5 Hz, 2H), 3.71 (s, 3H), 2.94 (t, J = 6.5 Hz, 2H). 2-(3-Bromobenzyloxy)thiophene (17d): Following general procedure D, 17d was obtained as colorless oil, yield: 61%. 1H NMR (300 MHz, CDCl3) δ 7.53 (s, 1H), 7.46 –



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7.38 (m, 1H), 7.33 – 7.26 (m, 1H), 7.21 (dd, J = 8.1, 7.4 Hz, 1H), 6.66 (dd, J = 5.8, 3.7 Hz, 1H), 6.56 – 6.50 (m, 1H), 6.22 (dt, J = 3.7, 1.9 Hz, 1H), 4.99 (s, 2H). 3-(3-Bromobenzyloxy)thiophene (17f): Following general procedure D, 17f was obtained as colorless oil, yield: 61%. 1H NMR (300 MHz, CDCl3) δ 7.58 (s, 1H), 7.45 (d, J = 7.9 Hz, 1H), 7.33 (d, J = 7.7 Hz, 1H), 7.24 (s, 1H), 7.22 – 7.15 (m, 1H), 6.80 (dd, J = 5.3, 1.5 Hz, 1H), 6.29 (dd, J = 3.0, 1.5 Hz, 1H), 4.97 (s, 2H). 2-(4-Bromobenzyloxy)thiophene (17i): Following general procedure D, the (4-bromophenyl)methanol was dissolved in small amount of toluene. 17i was obtained as colorless solid, yield: 65%. 1H NMR (300 MHz, CDCl3) δ 7.50 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.2 Hz, 2H), 6.74 – 6.64 (m, 1H), 6.62 – 6.52 (m, 1H), 6.28 – 6.20 (m, 1H), 5.01 (s, 2H). 2-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thiophene

(18d):

Following

general

procedure B, 18d was obtained as colorless oil, yield: 84%. 1H NMR (300 MHz, CDCl3) δ 7.49 – 7.35 (m, 2H), 7.19 – 7.09 (m, 5H), 6.69 (dd, J = 5.8, 3.8 Hz, 1H), 6.55 (dd, J = 5.8, 1.4 Hz, 1H), 6.26 (dd, J = 3.7, 1.4 Hz, 1H), 5.11 (s, 2H), 2.01 (s, 6H). 3-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thiophene

(18f):

Following

general

procedure B, 18f was obtained as colorless oil, yield: 85%. 1H NMR (300 MHz, CDCl3) δ 7.50 – 7.37 (m, 2H), 7.24 – 7.07 (m, 6H), 6.82 (ddd, J = 5.2, 1.5, 0.5 Hz, 1H), 6.32 (dd, J = 3.0, 1.5 Hz, 1H), 5.08 (s, 2H), 2.03 (s, 6H). 2-((2',6'-Dimethylbiphenyl-4-yl)methoxy)thiophene

(18i):

Following

general

procedure B, 18i was obtained as colorless solid, yield: 63%. 1H NMR (300 MHz, CDCl3) δ 7.49 (d, J = 7.8 Hz, 2H), 7.22 – 7.08 (m, 5H), 6.75 (dd, J = 5.4, 3.8 Hz, 1H), 6.59 (d, J = 5.5 Hz, 1H), 6.33 (d, J = 3.7 Hz, 1H), 5.13 (s, 2H), 2.04 (s, 6H). 5-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thiophene-2-carbaldehyde

(19d):

Following general procedure E, 19d was obtained as white solid, yield: 86%. 1H NMR


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(300 MHz, CDCl3) δ 9.65 (s, 1H), 7.52 – 7.35 (m, 3H), 7.20-7.09 (m, 5H), 6.40 (d, J = 4.3 Hz, 1H), 5.21 (s, 2H), 2.00 (s, 6H). 4-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thiophene-2-carbaldehyde

(19f):

Following general procedure E, 19f was obtained as white solid, yield: 23%. 1H NMR (300 MHz, CDCl3) δ 9.82 (s, 1H), 7.50 – 7.35 (m, 3H), 7.22 – 7.07 (m, 5H), 6.80 (s, 1H), 5.12 (s, 2H), 2.01 (s, 6H). 5-((2',6'-Dimethylbiphenyl-4-yl)methoxy)thiophene-2-carbaldehyde

(19i):

Following general procedure E, 19i was obtained as white solid, yield: 84%. 1H NMR (300 MHz, CDCl3) δ 9.69 (s, 1H), 7.54 (d, J = 4.2 Hz, 1H), 7.49 (d, J = 7.8 Hz, 2H), 7.24 – 7.07 (m, 5H), 6.46 (d, J = 4.3 Hz, 1H), 5.23 (s, 2H), 2.03 (s, 6H). (E)-Methyl

3-(5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)acrylate

(20d): Following general procedure F, 20d was obtained as white solid, yield: 82%. 1H NMR (300 MHz, CDCl3) δ 7.63 (d, J = 15.6 Hz, 1H), 7.46 (t, J = 7.7 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.22 – 7.07 (m, 5H), 6.92 (d, J = 3.8 Hz, 1H), 6.22 (d, J = 4.1 Hz, 1H), 5.93 (d, J = 15.6 Hz, 1H), 5.16 (s, 2H), 3.76 (s, 3H), 2.01 (s, 6H). (E)-Methyl


(20f): Following general procedure F, 20f was obtained as white solid, yield: 90%. 1H NMR (300 MHz, CDCl3) δ 7.65 (d, J = 15.7 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 7.7 Hz, 1H), 7.19 – 7.09 (m, 5H), 6.97 (d, J = 1.6 Hz, 1H), 6.38 (d, J = 1.5 Hz, 1H), 6.21 (d, J = 15.7 Hz, 1H), 5.07 (s, 2H), 3.79 (s, 3H), 2.01 (s, 6H). (E)-Methyl


(20i): Following general procedure F, 20i was obtained as white solid, yield: 91%. 1H NMR (400 MHz, CDCl3) δ 7.66 (dd, J = 15.5, 0.5 Hz, 1H), 7.48 (d, J = 8.3 Hz, 2H), 7.22 – 7.14 (m, 3H), 7.11 (d, J = 7.5 Hz, 2H), 6.96 (d, J = 4.0 Hz, 1H), 6.27 (d, J = 4.0 Hz, 1H), 5.96 (d, J = 15.5 Hz, 1H), 5.17 (s, 2H), 3.77 (s, 3H), 2.03 (s, 6H).



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Methyl 3-(5-formylthiophen-2-yl)propanoate (21e): To the solution of methyl 3-(thiophen-2-yl)propanoate (1.08 g, 7.14 mmol) in 30 mL DMF in bath ice was added POCl3 (1.16 mL, 12.7 mmol) dropwise. After warming to room temperature, the reaction mixture was stirred at 100℃ for 4 h. The mixture was cooled to room temperature and quenched by ice water. The resulting mixture was extracted with ethyl acetate and washed with aqueous NaHCO3 and brine. Usual work-up and purification by silica gel column chromatography afforded the product as colorless solid, yield: 43%. 1H NMR (300 MHz, CDCl3) δ 9.80 (s, 1H), 7.59 (d, J = 3.7 Hz, 1H), 6.94 (d, J = 3.6 Hz, 1H), 3.68 (s, 3H), 3.20 (t, J = 7.4 Hz, 2H), 2.71 (t, J = 7.4 Hz, 2H). Methyl 3-(5-(hydroxymethyl)thiophen-2-yl)propanoate (22e): To the solution of methyl 3-(5-formylthiophen-2-yl) propanoate (542 mg, 2.74 mmol) in 6 mL toluene/MeOH=1:1 in ice bath was added NaBH4 (125 mg, 3.28 mmol) in small portions. The reaction mixture was stirred in ice bath for additional 1 h and aqueous NH4Cl was added. Usual work-up and purification by silica gel column chromatography afforded the product as colorless oil, yield: 72%. 1H NMR (400 MHz, CDCl3) δ 6.83 (d, J = 3.4 Hz, 1H), 6.70 (d, J = 3.4 Hz, 1H), 4.77 (d, J = 5.7 Hz, 2H), 3.71 (s, 3H), 3.15 (t, J = 7.6 Hz, 2H), 2.70 (t, J = 7.6 Hz, 2H), 1.80 (t, J = 6.0 Hz, 1H). Methyl

3-(5-((2',6'-dimethylbiphenyl-3-yloxy)methyl)thiophen-2-yl)propanoate

(23e): To the solution of 22e (100 mg, 0.5 mmol) in 5 mL DCM in ice bath was added SOCl2 (363 μL, 5 mmol). After warming to room temperature, the reaction mixture was stirred at 50℃ overnight. The mixture was concentrated under reduced pressure. The residue was dissolved in 5 mL acetone and NaI (75 mg, 0.5 mmol), 2',6'-dimethylbiphenyl-3-ol (119 mg, 0.6 mmol) and K2CO3 (138 mg, 1 mmol) were added. The reaction mixture was stirred at 60℃ for 8 h. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate. Usual work-up and


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purification by silica gel column chromatography afforded the product as colorless oil, yield over 2 steps: 63%. 1H NMR (300 MHz, CDCl3) δ 7.33 (t, J = 8.0 Hz, 1H), 7.17 – 7.06 (m, 3H), 6.93 (ddd, J = 8.3, 2.6, 1.0 Hz, 1H), 6.89 (d, J = 3.4 Hz, 1H), 6.78 – 6.72 (m, 2H), 6.69 (d, J = 3.4 Hz, 1H), 5.12 (s, 2H), 3.68 (s, 3H), 3.12 (t, J = 7.6 Hz, 2H), 2.66 (t, J = 7.6 Hz, 2H), 2.02 (s, 6H). 5-(3-Bromobenzyloxy)thiophene-2-carbaldehyde

(24):

Following

general

procedure E, LDA (2 M in THF/heptane/ethylbenzene) was used instead of nBuLi. Compound 24 was obtained as dark yellow solid, yield: 48%. 1H NMR (300 MHz, CDCl3) δ 9.68 (s, 1H), 7.59 (s, 1H), 7.55 – 7.48 (m, 2H), 7.35 (d, J = 7.5 Hz, 1H), 7.31 – 7.26 (m, 1H), 6.41 (d, J = 4.2 Hz, 1H), 5.14 (s, 2H). (E)-Methyl

3-(5-(3-bromobenzyloxy)thiophen-2-yl)acrylate

(25):

Following

general procedure F, ethyl (triphenylphosphoranylidene)acetate was used as Wittig reagent. Compound 25 was obtained as white solid, yield: 82%. 1H NMR (300 MHz, CDCl3) δ 7.62 (d, J = 15.5 Hz, 1H), 7.57 (s, 1H), 7.50 (d, J = 7.4 Hz, 1H), 7.34 (d, J = 7.2 Hz, 1H), 7.27 (t, J = 7.7 Hz, 1H), 6.92 (d, J = 4.0 Hz, 1H), 6.21 (d, J = 3.9 Hz, 1H), 5.95 (d, J = 15.4 Hz, 1H), 5.08 (s, 2H), 4.22 (q, J = 6.9 Hz, 2H), 1.31 (t, J = 7.2 Hz, 3H). Methyl 3-(5-(3-bromobenzyloxy)thiophen-2-yl)propanoate (26): Following general procedure G, 26 was obtained as colorless oil, yield: 73%. 1H NMR (300 MHz, CDCl3) δ 7.56 (s, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.36 – 7.21 (m, 2H), 6.40 (d, J = 3.6 Hz, 1H), 6.05 (d, J = 3.6 Hz, 1H), 4.99 (s, 2H), 4.14 (q, J = 7.1 Hz, 2H), 2.99 (t, J = 7.6 Hz, 2H), 2.61 (t, J = 7.5 Hz, 2H), 1.25 (t, J = 7.0 Hz, 3H). Thieno[3,2-b]thiophene-2-carbaldehyde

(27):

To

the

solution

of

thieno[3,2-b]thiophene (1 g, 7.14 mmol) in 25 mL DCE in bath ice were added DMF (550 μL, 7.14 mmol) and POCl3 (2 mL, 21.4 mmol) dropwise. After warming to room temperature, the reaction mixture was stirred at 90 ℃ for 48 h. The mixture was



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cooled to room temperature and quenched by ice water. Usual work-up and purification by silica gel column chromatography afforded the product as white solid, yield: 59%. 1H NMR (300 MHz, CDCl3) δ 9.96 (s, 1H), 7.94 (s, 1H), 7.69 (d, J = 5.3 Hz, 1H), 7.32 (d, J = 5.3 Hz, 1H). (E)-Methyl

3-(thieno[3,2-b]thiophen-2-yl)acrylate

(28):

Following

general

procedure F at room temperature, THF/H2O=2:1 was used as solvent. Compound 28 was obtained as white solid, yield: 72%. 1H NMR (300 MHz, CDCl3) δ 7.81 (d, J = 15.6 Hz, 1H), 7.46 (d, J = 5.3 Hz, 1H), 7.39 (s, 1H), 7.21 (d, J = 5.3 Hz, 1H), 6.22 (d, J = 15.6 Hz, 1H), 3.79 (s, 3H). Thieno[3,2-b]thiophene-2-carboxylic acid (29): To the solution of 27 (168 mg, 5 mmol) in 50 mL tBuOH was added 2-methyl-2-butene (2 M in THF, 10 mL) and cooled in ice bath. The solution of NaClO2 (80%, 619 mg, 5.5 mmol) and NaH2PO4.2H2O (5.65 g) in 5 mL H2O was added dropwise and the reaction mixture was stirred at room temperature overnight. The resulting mixture was acidified with diluted HCl (aq) and extracted with ethyl acetate for three times. The organic layer was dried over anhydrous Na2SO4. After removal of the drying agent, the filtrate was concentrated under reduced pressure. The residue was crystallized from DCM/PE to afford the product as pale yellow solid, yield: 77%. 1H NMR (300 MHz, DMSO) δ 13.27 – 12.68 (m, 1H), 8.12 (s, 1H), 7.94 (d, J = 5.3 Hz, 1H), 7.52 (d, J = 5.3 Hz, 1H). Methyl

3-(thieno[3,2-b]thiophen-2-yl)propanoate

(30g):

Following

general

procedure G, 30g was obtained as white solid, yield: 83%. 1H NMR (300 MHz, CDCl3) δ 7.28 (d, J = 5.2 Hz, 1H), 7.16 (d, J = 5.2 Hz, 1H), 6.99 (s, 1H), 3.69 (s, 3H), 3.21 (t, J = 7.5 Hz, 2H), 2.72 (t, J = 7.5 Hz, 2H).


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Methyl thieno[3,2-b]thiophene-2-carboxylate (30h): Following general procedure L, 30h was obtained as light yellow solid, yield: 80%. 1H NMR (300 MHz, CDCl3) δ 7.98 (s, 1H), 7.58 (d, J = 5.3 Hz, 1H), 7.27 (d, J = 5.3 Hz, 1H), 3.91 (s, 3H). Methyl 3-(5-iodothieno[3,2-b]thiophen-2-yl)propanoate (31g): Following general procedure H, 31g was obtained as light yellow solid, yield: 49%. 1H NMR (300 MHz, CDCl3) δ 7.31 (s, 1H), 6.92 (s, 1H), 3.68 (s, 3H), 3.18 (t, J = 7.4 Hz, 2H), 2.70 (t, J = 7.4 Hz, 2H). Methyl 5-iodothieno[3,2-b]thiophene-2-carboxylate (31h): Following general procedure H, 31h was obtained as light yellow solid, yield: 71%. 1H NMR (300 MHz, DMSO) δ 8.13 (s, 1H), 7.82 (s, 1H), 3.86 (s, 3H). 5-((5-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)methylene)-2,2-dimet hyl-1,3-dioxane-4,6-dione (32): Following general procedure I, 32 was obtained as yellow solid, yield: 72%. 1H NMR (300 MHz, CDCl3) δ 8.46 (s, 1H), 7.68 (d, J = 4.4 Hz, 1H), 7.49 (t, J = 7.5 Hz, 1H), 7.41 (d, J = 7.3 Hz, 1H), 7.24 – 7.07 (m, 5H), 6.57 (d, J = 4.5 Hz, 1H), 5.35 (s, 2H), 2.01 (s, 6H), 1.75 (s, 6H). Ethyl 3-(5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)-3-hydroxypropanoate (34): The solution of 19d (595 mg, 1.85 mmol) in 20 mL anhydrous THF was cooled in ice bath and freshly prepared (2-ethoxy-2-oxoethyl)zinc(II) bromide (about 0.5 M in THF, 11 mL, 5.5 mmol) was added dropwise by syringe. The reaction mixture was warmed to room temperature and stirred for 3 h. Then the mixture was cooled in ice bath again and quenched by aqueous NH4Cl. Usual work-up and purification by silica gel column chromatography afforded the product as colorless oil, yield: 52%. 1H NMR (300 MHz, CDCl3) δ 7.45 (t, J = 7.1 Hz, 1H), 7.38 (d, J = 7.6 Hz, 1H), 7.20 – 7.07 (m, 5H), 6.58 (d, J = 3.9 Hz, 1H), 6.10 (d, J = 3.9 Hz, 1H), 5.23 – 5.13 (m, 1H), 5.09 (s, 2H),



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4.19 (q, J = 7.1 Hz, 2H), 3.24 (d, J = 4.1 Hz, 1H), 2.81 (d, J = 3.6 Hz, 1H), 2.79 (d, J = 0.7 Hz, 1H), 2.01 (s, 6H), 1.27 (t, J = 7.2 Hz, 3H). (R)-Ethyl 3-(5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)-3-hydroxypropanoate ((R)-34): Compound 38 (127 mg, 0.31 mmol) was protected by N2 and Et3N (216 μL, 1.6 mmol) was added. The mixture was stirred at 38 ℃ until the substrate was dissolved completely. RuCl[(R,R)-Tsdpen](mesitylene) (5 mg, 0.0078 mmol) in HCO2H (53 μL, 1.4 mmol) was added and the reaction mixture was stirred at 38 ℃ for additional 4.5 h. The mixture was diluted with diluted with ethyl acetate. The resulting mixture was washed with brine and the organic layer was dried over anhydrous Na2SO4. After removal of the drying agent, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to afford the product as colorless oil, yield: 90%, 98% ee [HPLC condition: CHIRALPAK AD-H 4.6 mm × 250 mm column by isocratic elution: 0.1 % TFA in hexane/0.1% TFA in EtOH=95/5 (v/v) at a flow rate of 1.0 mL/min, with UV detection at 254 nm]. Retention time: minor product (S) 18.6 min, major product (R)-34 (R) 20.7 min. [α]D +5.3o (c 1.195, CH2Cl2). (E)-2-((2',6'-Dimethylbiphenyl-3-yl)methoxy)-5-(2-nitrovinyl)thiophene (35): To the solution of 5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophene-2-carbaldehyde (322 mg, 1 mmol) in 2 mL THF was added 2 mL MeNO2 and NH4OAc (85 mg, 1.1 mmol) and stirred at 70 ℃ for 24 h. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate. Usual work-up and purification by silica gel column chromatography afforded the product as yellow oil, yield: 44%. 1H NMR (300 MHz, CDCl3) δ 8.01 (d, J = 12.7 Hz, 1H), 7.50 – 7.42 (m, 1H), 7.38 (d, J = 7.2 Hz, 1H), 7.22 – 7.12 (m, 4H), 7.10 (d, J = 7.9 Hz, 2H), 6.35 – 6.28 (m, 1H), 5.20 (s, 2H), 2.00 (s,


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6H). (S)-4-Benzyl-3-((R)-3-(5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)-4nitrobutanoyl)oxazolidin-2-one

(36):

The

solution

of

(S)-3-acetyl-4-benzyloxazolidin-2-one (96 mg, 0.44 mmol) in 2.5 mL anhydrous DCM was cooled at -78 ℃ and TiCl4 (1 M in DCM, 0.46 mL, 0.46 mmol) was added dropwise. Then DIEA (87 μL, 0.53 mmol) was added and the mixture was stirred at -78 ℃ for additional 45 min. The solution of 35 (160 mg, 0.44 mmol) in 0.5 mL anhydrous DCM was added. TiCl4 (1 M in DCM, 0.46 mL, 0.46 mmol) was then added dropwise to the reaction. The mixture was stirred at –78 ℃ for another 2.5 h before it was slowly warmed to -10 ℃ and quenched by aqueous NH4Cl. The resulting mixture was extracted with ethyl acetate and washed with brine. The organic layer was dried over anhydrous Na2SO4. After removal of the drying agent, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to afford the product as yellow oil, yield: 40%. 1H NMR (300 MHz, CDCl3) δ 7.44 (t, J = 7.2 Hz, 1H), 7.39 – 7.27 (m, 4H), 7.22 – 7.06 (m, 7H), 6.57 (d, J = 3.9 Hz, 1H), 6.08 (d, J = 3.9 Hz, 1H), 5.07 (s, 2H), 4.77 – 4.55 (m, 3H), 4.31 – 4.14 (m, 3H), 3.48 (dd, J = 17.7, 7.5 Hz, 1H), 3.38 – 3.21 (m, 2H), 2.75 (dd, J = 13.3, 9.7 Hz, 1H), 2.01 (s, 6H). 5-((2',6'-Dimethylbiphenyl-3-yl)methoxy)thiophene-2-carboxylic

acid

(37):

2-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophene (237 mg, 0.81 mmol) was protected by N2 and 8 mL anhydrous THF was added by syringe. The solution was cooled to -78 ℃ and nBuLi (2.5 M in hexanes, 0.35 mL, 0.88 mmol) was added dropwise. After stirring at -78 ℃ for 2 h, the N2 balloon was replaced with CO2 balloon and the reaction mixture was stirred at -78 ℃ overnight. The reaction was



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quenched by aqueous NH4Cl and warmed to room temperature. The resulting mixture was acidified by diluted HCl (aq) and extracted with ethyl acetate for 3 times. Usual work-up and purification by silica gel column chromatography afforded the products white solid, yield: 84%. 1H NMR (400 MHz, DMSO) δ 7.50 (t, J = 7.5 Hz, 1H), 7.48 – 7.43 (m, 2H), 7.22 (s, 1H), 7.19 – 7.09 (m, 4H), 6.49 (d, J = 4.2 Hz, 1H), 5.28 (s, 2H), 1.94 (s, 6H). Ethyl 3-(5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)-3-oxopropanoate (38): Carbonyldiimidazole (43

mg,

0.24

mmol) was added to a solution of

5-((2',6'-dimethylbiphenyl-3-yl)methoxy)thiophene-2-carboxylic acid (82 mg, 0.24 mmol) in 0.5 mL THF and stirred at room temperature for 12 h. A mixture of potassium ethyl malonate (82 mg, 0.48 mmol), MgCl2 (58 mg, 0.62 mmol) and Et3N (101 μL, 0.73 mmol) in 1 mL CH3CN was stirred for 4 h. Then to the suspension in ice bath was added the solution of the active ester dropwise. The mixture was stirred for additional 12 h at room temperature and then quenched by diluted HCl (aq) at 0℃. T Usual work-up and purification by silica gel column chromatography afforded the product as brown oil, yield: 52%. 1H NMR (400 MHz, CDCl3) δ 7.51 – 7.43 (m, 2H), 7.39 (d, J = 7.5 Hz, 1H), 7.22 – 7.08 (m, 5H), 6.33 (d, J = 4.3 Hz, 1H), 5.20 (s, 2H), 4.20 (q, J = 7.1 Hz, 2H), 3.81 (s, 2H), 2.01 (s, 6H), 1.26 (t, J = 7.1 Hz, 3H). 3-((Thiophen-2-yloxy)methyl)phenylboronic acid (39): To the solution of 2-(3-bromobenzyloxy)thiophene (3 g, 11 mmol) in 50 mL anhydrous THF under N2 at -78 ℃ was added nBuLi (2.5 M in hexanes, 5 mL, 12 mmol) dropwise. After stirring at -78 ℃ for 2 h, B(OiPr)3 (3.9 mL, 16.7 mmol) in 10 mL anhydrous THF was added dropwise and the resulting mixture was stirred at -78 ℃ overnight. The reaction was quenched by diluted HCl (aq) and warmed to room temperature. Usual work-up and


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purification by silica gel column chromatography afforded the product as white solid, yield: 54%. 1H NMR (300 MHz, CDCl3) δ 8.22 (d, J = 8.7 Hz, 2H), 7.68 (d, J = 7.8 Hz, 1H), 7.54 (t, J = 7.4 Hz, 1H), 6.72 (dd, J = 5.8, 3.8 Hz, 1H), 6.57 (dd, J = 5.8, 1.4 Hz, 1H), 6.32 (dd, J = 3.7, 1.4 Hz, 1H), 5.19 (s, 2H). tert-Butyl(2,6-dimethyl-3'-((thiophen-2-yloxy)methyl)biphenyl-4-yloxy)dimethyls ilane

(40):

Following

general

procedure

B,

2,6-dimethyl-3'-((thiophen-2-yloxy)methyl)biphenyl-4-ol was obtained as colorless oil, yield: 68%. 1H NMR (300 MHz, CDCl3) δ 7.46 – 7.35 (m, 2H), 7.17 (s, 1H), 7.11 (d, J = 7.1 Hz, 1H), 6.70 (dd, J = 5.8, 3.7 Hz, 1H), 6.62 – 6.52 (m, 3H), 6.27 (dd, J = 3.8, 1.5 Hz, 1H), 5.11 (s, 2H), 4.68 (s, 1H), 1.97 (s, 6H). To the solution of 2,6-dimethyl-3'-((thiophen-2-yloxy)methyl)biphenyl-4-ol (1.03 g, 3.3 mmol) in 30 mL DCM was added TBSCl (552 mg, 3.7 mmol) and Et3N (691 μL, 5 mmol) and the mixture was stirred at room temperature overnight. TBSCl (498 mg, 3.3 mmol) and Et3N (555 μL, 4 mmol) were added and the reaction was quenched by aqueous NH4Cl after the starting material was consumed completely. Usual work-up and purification by silica gel column chromatography afforded the product as colorless oil, yield: 89%. 1

H NMR (300 MHz, CDCl3) δ 7.47 – 7.33 (m, 2H), 7.19 (s, 1H), 7.12 (d, J = 7.2 Hz,

1H), 6.70 (dd, J = 5.7, 3.8 Hz, 1H), 6.61 – 6.52 (m, 3H), 6.27 (dd, J = 3.7, 1.4 Hz, 1H), 5.11 (s, 2H), 1.96 (s, 6H), 1.01 (s, 9H), 0.23 (s, 6H). 5-((4'-(tert-Butyldimethylsilyloxy)-2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen e-2-carbaldehyde (41): Following general procedure E, 41 was obtained as white solid, yield: 77%. 1H NMR (300 MHz, CDCl3) δ 9.65 (s, 1H), 7.52 – 7.32 (m, 3H), 7.18 (s, 1H), 7.14 (d, J = 7.2 Hz, 1H), 6.57 (s, 2H), 6.39 (d, J = 4.3 Hz, 1H), 5.20 (s, 2H), 1.93 (s, 6H), 0.99 (s, 9H), 0.22 (s, 6H).



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5-((5-((4'-(tert-Butyldimethylsilyloxy)-2',6'-dimethylbiphenyl-3-yl)methoxy)thiop hen-2-yl)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (42): Following general procedure I, 42 was obtained as yellow solid, yield: 86%. 1H NMR (300 MHz, CDCl3) δ 8.46 (s, 1H), 7.68 (d, J = 4.5 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.39 (d, J = 7.4 Hz, 1H), 7.21 (s, 1H), 7.17 (d, J = 7.0 Hz, 1H), 6.59 (s, 2H), 6.57 (d, J = 4.5 Hz, 1H), 5.34 (s, 2H), 1.95 (s, 6H), 1.75 (s, 6H), 1.00 (s, 9H), 0.23 (s, 6H). Methyl 3-(5-((4'-(tert-butyldimethylsilyloxy)-2',6'-dimethylbiphenyl-3-yl)methoxy)thioph en-2-yl)hex-4-ynoate (43): Following general procedure J and L in sequence, 43 was obtained as colorless oil, yield over 3 steps: 64%. 1H NMR (300 MHz, CDCl3) δ 7.45 – 7.31 (m, 2H), 7.16 (s, 1H), 7.13 – 7.07 (m, 1H), 6.57 (d, J = 5.8 Hz, 2H), 6.55 (dd, J = 3.8, 0.9 Hz, 1H), 6.05 (d, J = 3.9 Hz, 1H), 5.06 (s, 2H), 4.25 – 4.16 (m, 1H), 3.69 (s, 3H), 2.83 – 2.63 (m, 2H), 1.95 (s, 6H), 1.82 (d, J = 2.4 Hz, 3H), 1.00 (s, 9H), 0.23 (s, 6H). Methyl 3-(5-((4'-hydroxy-2',6'-dimethylbiphenyl-3-yl)methoxy)thiophen-2-yl)hex-4-ynoa te (44): To the solution of 43 (703 mg, 1.28 mmol) in 10 mL THF was added TBAF (1 M in THF, 1.4 mL, 1.4 mmol) and the reaction mixture was stirred at room temperature for 2 h. Usual work-up and purification by silica gel column chromatography afforded the product as colorless oil, yield: 98%. 1H NMR (300 MHz, CDCl3) δ 7.46 – 7.32 (m, 2H), 7.15 (s, 1H), 7.14 – 7.06 (m, 1H), 6.59 (s, 2H), 6.55 (d, J = 3.8 Hz, 1H), 6.05 (d, J = 3.8 Hz, 1H), 5.06 (s, 2H), 4.64 (s, 1H), 4.27 – 4.17 (m, 1H), 3.69 (s, 3H), 2.86 – 2.63 (m, 2H), 1.97 (s, 6H), 1.82 (d, J = 2.4 Hz, 3H). Methyl 3-(5-((2',6'-dimethyl-4'-(3-(methylsulfonyl)propoxy)biphenyl-3-yl)methoxy)thiop hen-2-yl)hex-4-ynoate (45k): To the solution of 44 (81 mg, 0.19 mmol) in 2 mL


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DMF was added 3-(methylsulfonyl)propyl 4-methylbenzenesulfonate (73 mg, 0.25 mmol) and K3PO4 (61 mg, 0.29 mmol) and the reaction mixture was stirred at 90 ℃ for 2.5 h. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate. Usual work-up and purification by silica gel column chromatography afforded the product as colorless oil, yield: 88%. 1H NMR (300 MHz, CDCl3) δ 7.42 (t, J = 7.3 Hz, 1H), 7.36 (d, J = 7.7 Hz, 1H), 7.15 (s, 1H), 7.09 (d, J = 7.3 Hz, 1H), 6.64 (s, 2H), 6.55 (dd, J = 3.8, 0.9 Hz, 1H), 6.05 (d, J = 3.8 Hz, 1H), 5.06 (s, 2H), 4.26 – 4.08 (m, 3H), 3.69 (s, 3H), 3.31 – 3.23 (m, 2H), 2.97 (s, 3H), 2.83 – 2.66 (m, 2H), 2.41 – 2.30 (m, 2H), 1.99 (s, 6H), 1.82 (d, J = 2.4 Hz, 3H). Methyl 3-(5-((2',6'-dimethyl-4'-((3-methyloxetan-3-yl)methoxy)biphenyl-3-yl)methoxy)th iophen-2-yl)hex-4-ynoate (45l): To the solution of 44 (83 mg, 0.2 mmol) in 2 mL DMF were added KI (36 mg, 0.22 mmol), K2CO3 (55 mg, 0.4 mmol) and 3-(chloromethyl)-3-methyloxetane (33 μL, 0.3 mmol). The reaction mixture was stirred at 70 ℃ for 45 h. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate. Usual work-up and purification by silica gel column chromatography afforded the product as colorless oil, yield: 69%. 1H NMR (300 MHz, CDCl3) δ 7.43 (t, J = 7.4 Hz, 1H), 7.36 (d, J = 7.9 Hz, 1H), 7.16 (s, 1H), 7.11 (d, J = 7.3 Hz, 1H), 6.71 (s, 2H), 6.55 (d, J = 3.8 Hz, 1H), 6.05 (d, J = 3.8 Hz, 1H), 5.07 (s, 2H), 4.65 (d, J = 5.8 Hz, 2H), 4.47 (d, J = 5.9 Hz, 2H), 4.21 (t, J = 8.7 Hz, 1H), 4.05 (s, 2H), 3.69 (s, 3H), 2.84 – 2.65 (m, 2H), 2.00 (s, 6H), 1.82 (d, J = 2.3 Hz, 3H), 1.45 (s, 3H). Methyl 3-(5-((2',6'-dimethyl-4'-(1-methylpiperidin-4-yloxy)biphenyl-3-yl)methoxy)thiop hen-2-yl)hex-4-ynoate (45m): The solution of 44 (90 mg, 0.21 mmol),



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1-methylpiperidin-4-ol (38 μL, 0.32 mmol) and PPh3 (90 mg, 0.34 mmol) in 2 mL anhydrous THF was cooled in ice bath and DEAD (54 μL, 0.34 mmol) was added dropwise. Then the reaction mixture was warmed to room temperature and stirred overnight. 1-Methylpiperidin-4-ol (38 μL, 0.32 mmol), PPh3 (90 mg, 0.34 mmol) and DEAD (54 μL, 0.34 mmol) were added to the mixture and stirred at room temperature for additional 24 h. The reaction was quenched by aqueous NH4Cl at 0 ℃ and extracted with ethyl acetate. Usual work-up and purification by silica gel column chromatography afforded the product as colorless oil, yield: 54%. 1H NMR (300 MHz, CDCl3) δ 7.42 (t, J = 7.5 Hz, 1H), 7.36 (s, 1H), 7.16 (s, 1H), 7.11 (d, J = 7.4 Hz, 1H), 6.66 (s, 2H), 6.55 (d, J = 3.8 Hz, 1H), 6.04 (d, J = 3.8 Hz, 1H), 5.06 (s, 2H), 4.40 – 4.14 (m, 2H), 3.69 (s, 3H), 2.96 – 2.63 (m, 4H), 2.39 – 2.24 (m, 5H), 2.09 – 1.94 (m, 8H), 1.94 – 1.72 (m, 5H). (S)-2-methylbutyl 3-(5-((2',6'-dimethyl-4'-(3-(methylsulfonyl)propoxy)biphenyl-3-yl)methoxy)thiop hen-2-yl)hex-4-ynoate (46k): Following general procedure L, 46k was obtained as colorless oil. 1H NMR (300 MHz, CDCl3) δ 7.42 (t, J = 7.5 Hz, 1H), 7.36 (d, J = 7.1 Hz, 1H), 7.15 (s, 1H), 7.09 (d, J = 6.5 Hz, 1H), 6.64 (s, 2H), 6.55 (d, J = 3.5 Hz, 1H), 6.05 (d, J = 3.6 Hz, 1H), 5.06 (s, 2H), 4.25 – 4.16 (m, 1H), 4.13 (t, J = 5.5 Hz, 2H), 3.98 (dd, J = 10.6, 6.5 Hz, 1H), 3.90 (dd, J = 11.0, 6.7 Hz, 1H), 3.32 – 3.21 (m, 2H), 2.97 (s, 3H), 2.83 – 2.65 (m, 2H), 2.42 – 2.28 (m, 2H), 1.99 (s, 6H), 1.81 (s, 3H), 1.77 – 1.61 (m, 1H), 1.49 – 1.37 (m, 1H), 1.25 – 1.11 (m, 1H), 0.95 (t, J = 6.9 Hz, 3H), 0.89 (t, J = 7.1 Hz, 3H). Calcium Influx Activity Assay


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For CHO cell lines stably expressing hGPR40 or hGPR120, cells were seeded in 96-well cell culture plates (Corning) and incubated overnight in 5% CO2 at 37 ℃. For plasmid titration experiments, HEK293T cells were transiently transfected with hGPR40 expression plasmids using X-tremeGENE HP DNA Transfection Reagent (Roche), followed by plating in poly-D-lysine-coated black clear 96-well plates (BD Falcon) and incubating for 24 hours. These cells were then incubated in Hank’s Balanced Salt Solution (HBSS) containing 0.1% BSA, calcium sensitive dye Fluo 4-AM (3 μM) and probenecid (2.5 mM) for 90 min. The cells were then washed three times using HBSS and then subjected to equilibrate for 10 min in HBSS containing probenecid before conducting the assay. Intracellular Ca2+ concentrations were measured as the difference between 585/525 ratios before and after addition of the test compounds using a Flexstation 3.0 plate reader (Molecular Devices). EC50 and Emax values for each curve were calculated by Prism 5.0 software (GraphPad Software). PPAR Transactivation Assay The activity of PPARγ was tested on HEK293T cells transfected with corresponding plasmids by X-tremeGENE HP DNA Transfection Reagent. Luciferase assay was conducted as described previously.33 MIN6 Insulin Secretion Assay MIN6 cells were pre-incubated for 2 h at 37 ℃ with Krebs-Ringer bicarbonate–HEPES buffer (116 mM NaCl, 4.7 mM KCl, 1.17 mM KH2PO4, 1.17 mM MgSO4, 25 mM NaHCO3, 2.52 mM CaCl2 and 24 mM HEPES) containing 0.1% BSA. After discarding the pre-incubation buffer, cells were stimulated with 2 mM glucose, 25 mM glucose



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alone or in the presence of indicated reagents and the plates were incubated for 1 h at 37 ℃. For insulin content assay, supernatants were collected after incubation and secreted insulin concentrations of each well were measured using ELISA kit (Millipore) according to the manufacturer's instruction. Oral Glucose Tolerance Test All experimental protocols on animals were approved by the Shanghai Institute of Materia Medica Experimental Animal Care and Use Committee. Six-week-old male ICR mice were purchased from Slac Laboratory Animal (Shanghai, China) and maintained under a 12 h light/dark cycle with a normal chow diet and free access to water. After fasted for 16 h, mice were randomly divided into four groups with 10 mice in each group and were orally administered 0.5% methylcellulose with 20% PEG400 (vehicle), 1 (10 mg/kg), compound (R)-7k (20 mg/kg or 10 mg/kg). 30 minutes later, all the animals were given an oral glucose load (2.5 g/kg) by gavage. Blood glucose in the tail vein were measured using ACCU-CHEK (Roche) before drug administration (time -30 min), before glucose load (time 0), and time 30, 60, 90 and 120 min after the glucose load. Pharmacokinetic Study Male Sprague-Dawley (SD) rats (200 ± 20 g) were obtained from the Shanghai Laboratory Animal Center Co, Ltd (Shanghai, China). The animals were housed in an air-conditioned room with a 12 h on and 12 h off light cycle, and they received powdered rodent chow and tap water by the bottle ad libitum. The rats were acclimated for 7 days before the experiment was conducted. Experiments were approved by the


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Institutional Animal Care and Use Committee of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Rats were randomly assigned to two groups and fasted overnight with free access to water before dosing. 8 rats were assigned to two groups (n = 4 for each group). 10 mg/kg Compound (R)-7k was administered to the rats in group A by oral gavage. 1 mg/kg Compound (R)-7k was administered intravenously (i.v.) via the tail vein in group B. The two formulation was prepared by dissolving compound (R)-7k in DMSO and then the stock solution was diluted into 1mg/mL by 5% Tween 80 (DMSO final concentration was less than 1%). Approximately 300 μL of blood sample was collected from the orbital vein at 0.25, 0.5, 1, 2, 4, 8, and 24 hours under light ether anesthesia. Food was withheld during this process, but water was freely provided. Plasma samples were obtained after centrifugation at 5,000 rpm for 15 minutes and stored at -80 ℃ until analysis. The compound concentrations were measured using a Shimadzu LCMS-8030 triple quadrupole system (Shimadzu Corp., Japan) equipped with electrospray ionization (ESI). Pharmacokinetic

parameters

of

compound

(R)-7k

were

calculated

by

a

non-compartmental analysis (NCA) model using Phoenix WinNonlin (version 6.3; Pharsight, Cary, NC). The oral absolute bioavailability (F) was calculated using the Eq. (1) below: F=[AUC(p.o)/Dose(p.o)]/[AUC(i.v)/Dose(i.v)] × 100%

(1)

Bile acid (d8-TCA) uptake and biliary excretion in sandwich-cultured rat hepatocytes (SCRHs).



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Rat hepatocytes were isolated from male SD rats according to the previously described method.34 A sandwich configuration model was established as previously described.35 The uptake and biliary excretion of d8-TCA was conducted in SCRHs.36 In brief, after incubation with compound (R)-7k, 1 or troglitazone in standard HBSS for 15 min, SCRHs were rinsed two times with warm standard HBSS or Ca2+-free HBSS and pre-incubated with the same buffer at 37 ℃ for 15 min. After removing the buffer, the hepatocytes were incubated with standard HBSS containing above compounds in the presence of 1 µM d8-TCA for another 15 min, respectively. After incubation, the solution was aspirated from the cells, uptake was terminated by washing three times with ice-cold PBS, and the samples were frozen until analysis. The d8-TCA concentration in hepatocytes was analyzed by liquid chromatography-mass spectrometry tandem mass spectrometry (LC-MS/MS).36 Data analysis The biliary excretion index (BEI) was calculated as follows: BEI=[AHBSS–AHBSS (Ca2+ free)]/AHBSS×100%, which was defined as the proportion of accumulated taurocholate excreted into bile canaliculi,37 where AHBSS and AHBSS (Ca2+-free) represent the amount of accumulated d8-TCA in the standard buffer treatment wells (hepatocytes+bile) and Ca2+-free buffer treatment wells (hepatocytes), respectively. Data was presented as the mean ± standard deviation (SD) of 3 independent experiments. Statistical analysis was performed using GraphPad Prism 5.03 software. To test the statistical significance among groups, one-way ANOVA was employed. The difference between two groups was compared using the unpaired Student t-test.


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Differences were considered significant at P < 0.05.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.xxxxxxx. Synthetic procedures and characterization data of target compounds 5k-l, (S)-7g (PDF) Molecular formula strings (CSV)

AUTHOR INFORMATION Corresponding Author *For Ya-Qiu Long: phone & fax, 86-21-50806876; e-mail, [email protected] *For He-Yao Wang: phone, +86-50805785; Fax: +86-21-50807088; e-mail, [email protected] Author Contributions #

These authors contributed equally.

Funding Sources National Natural Science Foundation of China Chinese Academy of Sciences

ACKNOWLEDGEMENTS The work was supported by the National Natural Science Foundation of China (81325020, 81361120410, 81321092, and 81473262) and the "Personalized



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Medicines---Molecular Signature-based Drug Discovery and Development", Strategic Priority Research Program of the Chinese Academy of Sciences (XDA12040311).

ABBREVIATIONS AcOH, acetic acid; ADMET, absorption, distribution, metabolism, excretion, and toxicity; AUC, area under curve; t-BuOK, Potassium tert-butoxide; CNS, central nervous system; CuAAC, copper-catalyzed azide-alkyne cycloaddition; DEAD, diethyl azodiformate; DCM, Dichloromethane; DHA, docosahexaenoic acid; DMAP, 4-dimethylaminopyridine; DMF, Dimethylformamide; DMPK, drug metabolism and pharmacokinetics; EDCI, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide; FFA1, free fatty acid receptor 1; GIP, gastric inhibitory polypeptide; GLP-1, glucagon-like peptide1; GPR40, G protein-coupled receptor 40; GSIS, glucose-stimulated insulin secretion; HOBt, N-Hydroxybenzotrizole; ICR mice, institute of cancer research mice; LA, linoleic acid; Me4Phen, 3,4,7,8-tetramethyl-1,10-phenanthroline; MRT, mean retention time; NBS, N-bromosuccinimide; N-STZ rats, N-streptozotocin rats; OGTT,

oral glucose tolerance test; PE, petroleum ether; PIFA, PhI(O2CCF3)2; PIntB, 2-(di-tert-butylphosphino)-1-phenylindole; PPAR, peroxisome proliferator-activated receptor; SAR, Structure activity relationship; T2DM, type 2 diabetes mellitus; TFA, trifluoroacetic acid; THF, Tetrahydrofuran; ZDF rats, zucker diabetic fatty rats.

REFERENCES 1. Itoh, Y.; Kawamata, Y.; Harada, M.; Kobayashi, M.; Fujii, R.; Fukusumi, S.; Ogi,


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A.;

Hashiguchi,

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Uematsu,

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Table of Contents graphic.


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Figure 1 ACS Paragon Plus Environment


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A

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Figure 4


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A

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Figure 6


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A

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Scheme 1 ACS Paragon Plus Environment

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Table of Contents graphic ACS Paragon Plus Environment

Discovery of Potent and Orally Bioavailable GPR40 Full Agonists

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