Design, synthesis and biological evaluation of a novel class of potent TGR5 agonists based on a 4-phenyl pyridine scaffold

European Journal of Medicinal Chemistry 69 (2013) 55e68

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Original article

Design, synthesis and biological evaluation of a novel class of potent

TGR5 agonists based on a 4-phenyl pyridine scaffold

,

Junjie Zhu ^{a 1}, Mengmeng Ning ^{a 1}, Cen Guo ^b, Lina Zhang ^a, Guoyu Pan ^b, Ying Leng ^a

**

*

,

Jianhua Shen ^a

,

^aState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China

^bCenter for Drug Safety Evaluation & Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

a r t i c l e i n f o

a b s t r a c t

Article history:

TGR5, a GPCR, is involved in energy and glucose homeostasis, and as such, is a target for the treatment of

diabetes, obesity and other metabolic syndromes. A new class of TGR5 agonists based on a 4-phenyl

pyridine scaffold was designed, synthesized and evaluated in vitro and in vivo. Extensive structureeac-

tivity relationship studies are reported herein. The most potent compounds 15a, 18b and 18c showed

comparable activity with the lead compound 2. 15a had the best potency in vitro but displayed an un-

favorable pharmacokinetic proﬁle and was found to be ineffective during an oral glucose tolerance test in

imprinting control region mice at a dose of 50 mg/kg.

Received 4 April 2013

Received in revised form

26 July 2013

Accepted 30 July 2013

Available online 13 August 2013

Keywords:

TGR5

GPCR

Diabetes

4-Phenyl pyridine

1. Introduction

murine enteroendocrine L cells (STC-1), is increased signiﬁcantly

via the stimulation of TGR5 by various agonists [10]. Thus, TGR5 is

TGR5 (Takeda G-protein-coupled receptor 5), also known as

M-BAR (membrane-type receptor for bile acids), GPBAR1 (G-pro-

tein coupled bile acid receptor 1) or BG37 [1e3], is a Gs-coupled

GPCR identiﬁed in 2002 and is expressed in various tissues,

particularly in the intestine, gallbladder, spleen, placenta and ovary

[2e4]. As one of the bile acid (BA) receptors, which are recognized

as complex metabolic integrators and signaling factors with para-

crine and endocrine functions [5,6], TGR5 mediates many physio-

logical activities. Through the BA-TGR5-cAMP-D2 signaling

pathway [7], BAs can increase energy expenditure in brown adipose

tissue by enhancing mitochondrial activity, the decrease of which is

considered to be one of the early hallmarks of type 2 diabetes

mellitus [8]. In addition, secretion of glucagon-like peptide-1 (GLP-

1), which has a wide variety of physiological effects [9] including

stimulating pancreatic insulin secretion, inhibiting the release of

glucagon, reducing appetite and delaying gastric emptying from

becoming of interest as a potential target to treat diabetes, obesity

and other metabolic syndromes.

The Pellicciari group has reported their research into the search

for potent and selective TGR5 agonists based on natural BAs.

Among them, INT-777 (1, Fig. 1) exhibited good properties in vitro

and in vivo and was considered a promising anti-diabetic drug

candidate, and has been used as the reference compound in our lab

[11,12]. Takeda as one of the earliest companies entering this ﬁeld

has also reported their work in a series of patents [13e16], among

which compounds 2 and 3 (Fig. 1) containing an oxazepine ring

showed the best activity in vitro. Meanwhile, 2-phenoxy-nicotin-

amide 4 (EC₅₀¼ 10 nM) developed by Roche represented a class of

very potent TGR5 agonists but suffered from the disadvantage of

high metabolic clearance [17]. It can be noted that 2 is structurally

similar to 4 to some degree: the 3,5-ditriﬂuoromethylphenyl ring of

2 and tetrahydroquinoline ring of 4, and the 4-(2-ﬂuorophenyl)

pyridine moiety of 2 and 2-(2,5-dichlorophenoxy)pyridine part of 4

may play the same role when binding to TGR5 (Fig. 2) [18]. On this

basis, our group has identiﬁed a class of TGR5 agonists by changing

the position of the nitrogen atom at the pyridine ring of compound

4 [19]. Although very potent analogs in vitro and in vivo were ob-

tained, Roche holds the patent [20] and serious gallbladder side

* Corresponding author. Tel.: þ86 21 50806059.

** Corresponding author. Tel.: þ86 21 20231969.

E-mail addresses: yleng@mail.shcnc.ac.cn (Y. Leng), jhshen@mail.shcnc.ac.cn

(J. Shen).

1

These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.ejmech.2013.07.050

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J. Zhu et al. / European Journal of Medicinal Chemistry 69 (2013) 55e68

2. Results and discussion

2.1. Chemistry

The synthesis of the initial amide derivatives was carried out as

shown in Scheme 1. Ester 5 was converted to the corresponding

ethyl 4-arylnicotinates 6aec by Suzuki coupling with excellent

yields. The catalytic system for 6c was Pd₂(dba)₃/t-Bu₃P [22], while

Pd(PPh₃)₄was used as the catalyst for 6a and 6b [23]. Aldehydes

7a,b were synthesized from 6a,b via reduction with DIBAL-H [24]

followed by oxidation with iodobenzene diacetate (DIB) [25].

Reductive amination of 7a,b with 3,5-bis(triﬂuoromethyl)benzyl-

amine followed by acylation with acyl chloride or acid gave amides

9aek. Azides 10aec were prepared from 6aec through reduction

with DIBAL-H followed by Thompson’s reaction [26]. Staudinger

reaction [27] of 10aec gave the key intermediate amines 11aec in

high yields. A stepwise reductive amination between 11a and the

appropriate ketones followed by acylation with 2-furoyl chloride

using N-ethylmorpholine as the base yielded amides 12a,b [28].

Modiﬁcation at the

in Scheme 2. Similar to the preparation of 12a,b, amines 13a,b were

synthesized from 11b by a stepwise reductive amination. -Amino

a-position of the amino of 8b was described

Fig. 1. Selection of previously published TGR5 agonists.

a

nitriles 15aec were prepared from amines 11aec through dehy-

dration with 3,5-bis(triﬂuoromethyl)benzaldehyde catalyzed by

Ti(i-PrO)₄followed by reaction with TMSCN [29]. Condensation of

11b with 3,5-bis(triﬂuoromethyl)benzoic acid afforded amide 14,

while sulfonylation of 11b,c with 3,5-bis(triﬂuoromethyl)benze-

nesulfonyl chloride produced sulfonamides 16a,b. Reductive ami-

nation of 15a with aldehydes afforded the alkylated products 17a,b.

The N-substituted derivatives 18aed were obtained by reaction

with benzyl bromide or acyl chlorides after deprotonation of 16a

with NaH in excellent yields [30].

effects of these derivatives were observed in our assay. Hence, there

is still a need to search for a proprietary scaffold that would yield

potent TGR5 agonists with lessened gallbladder toxicities.

Following our previous work [19], we focused on the 4-phenyl

pyridine scaffold of compounds 2 and 3. We believed that the

role of oxazepine ring of these compounds was to ﬁx the confor-

mation of the three aromatic cycles in a well-deﬁned geometric

orientation. In addition, although compound 4 does not contain a

cycle like oxazepine ring connecting to the pyridine part, it also

displayed excellent activity. Based on such logic, by opening the

oxazepine ring and introducing additional groups to maintain the

potency (A, Fig. 2), a class of TGR5 agonists possessing dissimilar

chemotypes was developed. Ligand efﬁciency (LE), deﬁned as the

free energy of binding of a ligand for a speciﬁc protein averaged for

each “heavy atom” (or non-hydrogen atoms), is now widely used as

a governing parameter to avoid undue increases in molecular size

during hit optimization [21]. Therefore, LEs were also monitored

throughout the optimization process. Herein, we report the syn-

thesis, biological evaluation and extensive structureeactivity rela-

tionship (SAR) studies of this novel series of TGR5 agonists.

Ester 20 was obtained from commercially available 2⁰-ﬂuo-

roacetophenone 19 following the procedure reported by Ishichi

[31]. Using the same method as for 11aec, 20 was successfully

converted to amine 23. Sulfonylation of 23 afforded sulfonamide

24. Initially, we tried to use 24 as the substrate for quick derivati-

zation at position 2 of the pyridine ring by Suzuki coupling or

Ullmann reaction, but the reactions proved to be very sluggish and

afforded side products that might resulted from the weak acid of

the sulfonamide group. Intermediate 25 was transformed into the

2-phenyl substituted compound 26 via Suzuki coupling with phe-

nylboronic acid, or was converted to 28 by nucleophilic substitution

with sodium ethoxide under reﬂux in EtOH. Treatment of 26 and 28

with 4 N HCl/EtOAc followed by sulfonylation produced the ﬁnal

sulfonamides 27 and 29, respectively (Scheme 3).

2.2. Human TGR5 (hTGR5) agonism activity and SAR studies

All the ﬁnal compounds and certain intermediates were evalu-

ated for their hTGR5 agonism ability using a cAMP-responsive

element (CRE)-driven luciferase assay in HEK293 cells similar to

that reported in the literature [11,12]. Compounds 1 and 2 showed

an EC₅₀of 1.23 mM and 19 nM toward hTGR5 in our assay, respec-

tively. The results are summarized in Tables 1 and 2.

Our initial strategy was to break the oxazepine ring of com-

pound 2 and use the amide group to introduce an extra substituent

as compensation for the loss of binding energy owning to the

change of the overall conformation. Fortunately, the ﬁrst acetamide

9a (EC₅₀¼ 0.91 M, effect ¼ 117%) possessed moderate potency,

m

although a slight decrease in LE (0.26) was observed (Table 1).

However, when the methyl was changed to other aliphatic groups,

such as ethyl (9b) or cyclohexyl (9c), the results were not satis-

factory. Many derivatives had EC₅₀s of 1

mM or less, and the larger

Fig. 2. Design strategy of novel scaffold of TGR5 agonists.

the aliphatic ring, the poorer the activity. Replacement of the

J. Zhu et al. / European Journal of Medicinal Chemistry 69 (2013) 55e68

57

Scheme 1. Reagents and conditions: (a) for 6a,b, PhB(OH)₂, K₂CO₃, Pd(PPh₃)₄, DME/H₂O, 85 ^ꢀC, 75e81%; for 6c, 2,6-di-F-PhB(OH)₂, K₃PO₄, Pd₂(dba)₃, t-Bu₃P, toluene, reﬂux, 73%; (b)

(i) DIBAL-H, CH₂Cl₂, ꢁ78 ^ꢀC; (ii) TEMPO, DIB, CH₂Cl₂, rt; 52e54% for the two steps; (c) 3,5-bis(triﬂuoromethyl)benzylamine, NaBH(OAc)₃, CH₂Cl₂, rt, 51e55%; (d) acyl chloride, Et₃N,

CH₂Cl₂, rt; or acid, HATU, DIEA, CH₃CN, reﬂux; 33e87%; (e) (i) DIBAL-H, CH₂Cl₂, ꢁ78 ^ꢀC; (ii) DPPA, DBU, THF, reﬂux; 41e88% for the two steps; (f) PPh₃, THF/H₂O, rt, 77e86%; (g) (i)

ꢀ

Et₃N, ketone, 4 A molecular sieves, CH₂Cl₂, reﬂux; (ii) NaBH₄, MeOH, rt; (iii) 2-furoyl chloride, N-ethylmorpholine, DMAP, THF, rt; 9e42% for the three steps.

cyclohexyl with phenyl yielded 9d (EC₅₀¼ 0.35

a compound possessing a signiﬁcant increase in potency, which

might indicate the existence of a interaction between the

phenyl ring and the receptor. Therefore, systematic variation of the

phenyl ring became the focus of subsequent modiﬁcations.

Introduction at different positions with either electron with-

drawing substituents (such as halogens) or electron donating sub-

stituents (such as methyl groups) caused a large drop inpotency (data

notshown)and9eisshownas anexample. Switchingthephenylwith

a pyridyl also gave a disappointing result (9f). This may indicate that

the binding pocket in TGR5 that recognizes this phenyl region is

strongly hydrophobic and relatively narrow, and thus it does not

tolerate a hydrophilic nitrogen atom or any substituents on the ring.

Hence, ﬁve-membered aromaticheterocycles withsmaller sizes were

synthesized. Pleasingly, all the compounds (9gek) displayed

acceptable activity. Among them, thienyl was better than furanyl (9g

m

M, effect ¼ 116%),

introduction of additional groups proved to be futile (data not

shown). The activity of intermediates 8a (EC₅₀¼ 580 nM) and 8b

(EC₅₀¼ 458 nM) were surprising (Table 2). Both displayed similar

potency to 9d but possessed higher LE values in the absence of

the amide group, which revealed that the amide was not indis-

pensable. Coexistence of 4-phenyl pyridine and 3,5-

bis(triﬂuoromethyl)benzene was essential since complete loss

of potency was observed for only the 4-phenyl pyridine part

pep

(11b). By comparing 12a and 12b with 9g, a small space at the a-

position of the amino group was identiﬁed. Thus, we next turned

our attention to modify this region using 8b as the starting point.

At ﬁrst, 13a and 13b was synthesized, both of which were slightly

more potent than 8b. The methyl derivative was still better than

ethyl, in accordance with the previous outcome. Then, the sub-

stituents were changed to hydrophilic groups. However,

replacement of the ethyl with a carboxylic acid group resulted in

vs. 9i, 9h vs. 9j); 3-thienyl 9j (EC₅₀¼ 0.12

m

M) in particular was about

a loss in potency (16% at 10 mM, see Supporting information). On

3-fold more potent than 9d. Moreover, an additional ﬂuorine atom

did not result in an obvious change of activity (9g vs. 9k). As the

methyl group of compound 3 was tolerated, an alkyl group was also

introduced to the same position in our compounds. Compared with

compound 9g, the methyl derivative (12a) slightly enhanced the

potency while maintained the LE (0.24). Replacement of the methyl

with a slightly larger group such as ethyl (12b) caused signiﬁcant loss

this basis, we speculated that the binding pocket of TGR5 that

recognizes this region does not contain a large space or positively

charged residues, but may be endowed with a narrow neutral

charged site that can form hydrogen bonds with ligands. Hence,

smaller and more polar nitrile group as a hydroxyl surrogate was

tested [32]. Surprisingly, compound 15a (effect ¼ 101%) not only

exhibited an EC₅₀of 35 nM, but also showed a higher LE value

(0.32) than 2. To verify our hypothesis about the structure of the

binding pocket, another two groups (14 and 16a) possessing a

much smaller size than the nitrile were tested. Amide 14

completely lost the activity, whereas sulfonamide 16a

(EC₅₀¼ 63 nM, effect ¼ 102%, LE ¼ 0.31) retained most of the

potency. These results indicated that keeping the 3,5-

bis(triﬂuoromethyl)phenyl moiety ﬂexible was important, as

of both potency (EC₅₀¼ 1.23

mM) and LE (0.21).

Although 9j was much more potent than INT-777, it was still a

weaker TGR5 agonist compared with compound 2. Furthermore,

a minimum LE of 0.3 is required in an ideal lead [21], but all the

compounds of this series suffered from lower LE values. How-

ever, further optimization of the thienyl group was limited since

either replacement with other ﬁve-membered cycles or

58

J. Zhu et al. / European Journal of Medicinal Chemistry 69 (2013) 55e68

ꢀ

Scheme 2. Reagents and conditions: (a) (i) Et₃N, ketones, 4 A molecular sieves, CH₂Cl₂, reﬂux; (ii) NaBH₄, MeOH, rt; 7e40% for the two steps; (b) 3,5-bis(triﬂuoromethyl)benzoic

acid, Et₃N, EDCI, HOBt, CH₂Cl₂, rt, 93%; (c) Et₃N, 3,5-bis(triﬂuoromethyl)benzaldehyde, Ti(i-PrO)₄, TMSCN, CH₂Cl₂, rt, 18e79%; (d) 3,5-bis(triﬂuoromethyl)benzenesulfonyl chloride,

Et₃N, CH₂Cl₂, rt, 52e70%; (e) aldehyde, NaBH(OAc)₃, HOAc, 1,2-dichloroethane, rt, 6e44%; (f) NaH, benzyl bromide or acyl chloride, DMF, 0 ^ꢀCert, 22e60%.

well as having an atom that could form a hydrogen bond with the

receptor. Furthermore, removal (15b) or addition (15c and 16b) of

the ﬂuorine atom did not cause any obvious changes in activity

and LE. However, unfortunately, asymmetric synthesis using a

literature method [33] and chiral separation of 15a were both

unsuccessful. This may be because racemization occurred at a

higher temperature (about 50 ^ꢀC), although follow-up work

revealed that the (R)-enantiomer of a similar structure possessed

a 2.5-fold increase in potency compared with the racemate [18].

As a result of the optimization of the two different regions,

2.3. Mouse TGR5 (mTGR5) agonism activity

Several potent hTGR5 agonists were selected for testing

against mTGR5 using a similar method to that used for hTGR5 in

an effort to select a compound that was potent against both

species for further evaluation in vivo. The results summarized in

Table 3 demonstrated that the potencies of the tested com-

pounds toward mTGR5 were inconsistent with those obtained for

hTGR5. Compounds 1 and 2 had EC₅₀s of 0.36 mM and 0.13 mM

toward mTGR5 in our assay, respectively. As for our own com-

pounds, compared with hTGR5 agonism activity, some were

similar (9i), some were slightly weaker (9j and 15c) and most

were much weaker (12a, 13a, 15a,b, 16a,b and 18bed). These

differences in potencies may be attributed to the relatively weak

sequence homology (83%) between the two species [3]. Thus, the

sulfonamide scaffold had the largest differences in potencies

we identiﬁed the potent compounds amide 9j,

a-amino nitrile

15a and sulfonamide 16a. Next, the derivation of the sub-

stituents of the amino group of 15a and 16a was explored

(Table 2). In the beginning, the failure of acylation of the sec-

ondary amino group of compound 15a with benzoyl chloride led

us to synthesize the alkylated products 17a,b. However, both had

much lower potencies, which forced us to turn to the sulfon-

amide scaffold. Except for the alkylated compound 18a, all the

acylation products gave good results, notably 18b (EC₅₀¼ 31 nM,

effect ¼ 109%) and 18c (EC₅₀¼ 32 nM, effect ¼ 112%), which

showed the best potency but at the expense of diminishing LE

(both below 0.3). Finally, some hydrophobic groups were intro-

duced at position 2 of the pyridine ring to take the place of the

furan ring of compound 18c and increase the LE values. However,

the results were disappointing. All compounds showed

decreased potencies, and some (27 and 29) even lost activity

entirely. Thus, upon binding, the substituents at position 2 of the

pyridine ring and furan ring of 18c might not be oriented in the

same pocket of TGR5.

between hTGR5 and mTGR5, whereas the a-amino nitrile scaffold

possessed the relative best potencies against the two species and

the highest LE values. Compound 15a (hTGR5 EC₅₀¼ 35 nM,

mTGR5 EC₅₀¼ 150 nM) was selected for further evaluation

in vivo.

2.4. In vivo pharmacokinetic (PK) study

The PK parameters of compound 15a, which possessed the

best potency in vitro, were evaluated in rats after p.o. (5 mg/kg)

administration. As shown in Table 4, compound 15a had a short

half-life (2.3 h), high clearance (91.2 L/h/kg) and rather low

plasma exposure (AUC_0et

¼

49.1 ng h/ml). However, to

J. Zhu et al. / European Journal of Medicinal Chemistry 69 (2013) 55e68

59

Scheme 3. Reagents and conditions: (a) (i) CNCH₂COOEt, NH₄OAc, HOAc, toluene, reﬂux, 63%; (ii) DMFDMA, 0 ^ꢀCert, 90%; (iii) 4 N HCl/EtOAc, rt, 78%; (b) DIBAL-H, CH₂Cl₂, ꢁ78 ^ꢀC,

89%; (c) DPPA, DBU, THF, reﬂux, 87%; (d) PPh₃, THF/H₂O, rt, 63%; (e) 3,5-bis(triﬂuoromethyl)benzenesulfonyl chloride, Et₃N, CH₂Cl₂, rt, 95%; (f) Boc₂O, Et₃N, CH₂Cl₂, rt, 90%; (g)

PhB(OH)₂, K₂CO₃, Pd(PPh₃)₄, DME/H₂O, 85 ^ꢀC, 72%; (h) 4 N HCl/EtOAc, rt; (i) EtONa, EtOH, reﬂux, 40%.

compounds 18b and 18c, none of the parent compounds and

possible metabolic products could be detected in rat plasma at

the same oral dose to that of 15a.

comparable to the lead compound 2. 15a with the best potency

in vitro displayed an unfavorable PK proﬁle and was found to be

ineffective during an OGTT in ICR mice at a dose of 50 mg/kg.

Further optimization of these structures, particularly the sulfon-

amides and

a-amino nitriles, is ongoing. In addition, chronic

2.5. Anti-diabetic evaluation in vivo

pharmacodynamic evaluation of 15a in mouse and further inves-

tigation of 18b and 18c in other animal model that share a higher

percentage of sequence homology with human TGR5 than mouse

are planned.

An oral glucose tolerance test (OGTT) was carried out in

imprinting control region (ICR) mice to examine the acute effect of

15a on blood glucose. Unfortunately, a single oral administration of

15a at a dose of 50 mg/kg did not cause a signiﬁcant reduction in

blood glucose AUC_0e120min(area under the curve) compared with

the vehicle control group (Fig. 3A). This outcome may be related to

the unfavorable PK proﬁle of 15a along with its relative weak

mTGR5 agonism activity. Therefore, more focused structural mod-

iﬁcations to optimize the PK properties as well as the mTGR5 po-

tency are needed.

The effect of dosing 15a on the mouse gallbladder was also

investigated after the OGTT. At the 50 mg/kg dose, the bile weights

from the gallbladders of the two groups were not signiﬁcantly

different (Fig. 3B). This lack of apparent toxicity to the gallbladder

might indicate the possibility of identifying a TGR5 agonist tool

compound that is safe in vivo despite the marginal effects of 15a on

blood glucose.

4. Experimental section

4.1. Chemistry

All reagents were purchased from commercial suppliers and

used without further puriﬁcation unless otherwise stated. Yields

were not optimized. Analytical thin-layer chromatography was

performed on HSGF 254 (0.15e0.2 mm thickness, Yantai Jiangyou

Company, Yantai, Shandong, China). Column chromatography was

carried out on silica gel (200e300 mesh) or using prepacked silica

cartridges (from 4 to 40 g) from Bonna-Agela Technologies Inc.

(Tianjin, China) and eluted using Teledyne Isco CombiFlash Rf ﬂash

chromatography system. ¹H NMR and ¹³C NMR spectra were

recorded on a Varian Mercury 300 or 400 spectrometer using tet-

ramethylsilane (TMS) or solvent peaks as an internal reference.

3. Conclusions

Chemical shifts (d) are reported in parts per million (ppm), and the

In summary, we have developed a novel class of amides, sul-

signals are described as brs (broad singlet), d (doublet), dd (doublet

of doublet), m (multiple), q (quarter), s (singlet), t (triplet) and td

(triplet doublet) and coupling constants (J) values are given in hertz

(Hz). Low resolution mass (LRMS) spectra were determined on

Waters ZQ2000a or Agilent liquid chromatography-mass

fonamides and

a-amino nitriles containing a 4-phenyl pyridine

scaffold as potent TGR5 agonists. Extensive SAR studies, PK studies

and anti-diabetic evaluation in vivo of the selected compounds are

disclosed herein. The most potent exemplars 15a,18b and 18c, were

60

J. Zhu et al. / European Journal of Medicinal Chemistry 69 (2013) 55e68

spectrometer system (LCMS) that consisted of an Agilent 1260 in-

ﬁnity LC coupled to Agilent 6120 Quadrupole mass spectrometer

(electrospray positive ionization, ESI) using an Agilent ZORBAX

4.1.2. Ethyl 4-(2-ﬂuorophenyl)nicotinate (6b)

Compound 6b (18.5 g, 75%) was prepared as a colorless oil from

5 (22 g, 100 mmol) following a procedure similar to that described

1.8

mm SB-C18 column (2.1 ꢂ 50 mm) with aqueous CH₃CN (10e

for the preparation of 6a. ¹H NMR (400 MHz, CDCl₃)

d 9.15 (s, 1H),

90%) containing 0.05% formic acid monitored at 240 nm. High

resolution mass spectra (HRMS) were recorded on a Q-Tof Ultima

Globe mass spectrometer (Micromass, Manchester, UK). Prepara-

tive HPLC was performed on an Unimicro Easysep-1010 series LC

8.78 (d, J ¼ 5.1 Hz, 1H), 7.42 (m, 1H), 7.34e7.21 (m, 3H), 7.16e7.09

(m, 1H), 4.19 (q, J ¼ 7.1 Hz, 2H), 1.12 (t, J ¼ 7.1 Hz, 3H); LCMS (ESI^þ)

m/z 246.1 (M þ H)^þ.

system (Unimicro ChemStation, Agilent Prep-C18, 10

m

m,

4.1.3. Ethyl 4-(2,6-diﬂuorophenyl)nicotinate (6c)

21.2 ꢂ 250 mm, 30 ^ꢀC, UV 240 nm, 10 ml/min) with aqueous MeOH.

HPLC analysis for all compounds tested in biological systems were

performed on an Agilent 1200 series LC system (Agilent Chem-

A ﬂask was charged with ethyl 4-chloronicotinate hydrochlo-

ride

5 (2.2 g, 10 mmol), Et₃N (1.5 ml, 11 mmol), 2,6-

diﬂuorophenylboronic acid (2.0 g, 13 mmol), K₃PO₄(4.2 g,

20 mmol), Pd₂(dba)₃(732 mg, 0.8 mmol), t-Bu₃P (2.4 g, 0.12 mmol,

10% in hexanes) and toluene (30 ml) under nitrogen. The mixture

was reﬂuxed for 36 h, then cooled to room temperature and

ﬁltered. The aqueous layer was extracted with EtOAc. The com-

bined organic phases were washed with brine, dried over MgSO₄

and concentrated. The residue was puriﬁed by ﬂash column

chromatography to give 6c as colorless oil (1.93 g, 73%). ¹H NMR

Station, Agilent Eclipse XDB-C18, 5

254 nm, 1.0 ml/min) with aqueous CH₃CN (50e90%) containing

m

m, 4.6 ꢂ 150 mm, 30 ^ꢀC, UV

0.05% formic acid for 30 min.

4.1.1. Ethyl 4-phenylnicotinate (6a)

A ﬂask was charged with ethyl 4-chloronicotinate hydrochloride

5 (11 g, 50 mmol), phenylboronic acid (7.3 g, 60 mmol), K₂CO₃(20 g,

150 mmol), H₂O (50 ml, 50 mmol), Pd(PPh₃)₄(3 g, 2.5 mmol) and

DME (200 ml) under nitrogen. The mixture was stirred at 85 ^ꢀC for

12 h, then cooled to room temperature and ﬁltered. The aqueous

layer was extracted with EtOAc. The combined organic phases were

washed with brine, dried over MgSO₄and concentrated. The res-

idue was puriﬁed by column chromatography to give 6a as colorless

(300 MHz, CDCl₃)

d

9.27 (s, 1H), 8.80 (d, J ¼ 5.1 Hz, 1H), 7.47e7.28

(m, 2H), 6.99 (t, J ¼ 7.8 Hz, 2H), 4.23 (q, J ¼ 7.1 Hz, 2H), 1.16 (t,

J ¼ 7.1 Hz, 3H); LCMS (ESI^þ) m/z 264.1 (M þ H)^þ.

4.1.4. 4-Phenylnicotinaldehyde (7a)

DIBAL-H (108 ml, 162 mmol, 1.5 M toluene solution) was added

dropwise to a stirred solution of 6a (9.21 g, 40.5 mmol) in dry

CH₂Cl₂at ꢁ78 ^ꢀC under nitrogen and the mixture was stirred for

1.5 h.1 M aqueous potassium sodium tartrate solution was added to

quench the reaction, and the mixture was extracted with CH₂Cl₂.

The combined organic phases were washed with brine, dried over

MgSO₄and concentrated to give the crude (4-phenylpyridin-3-yl)

methanol (6.6 g) that was used for the next step without further

puriﬁcation.

oil (9.2 g, 81%). ¹H NMR (400 MHz, CDCl₃)

d

9.02 (d, J ¼ 0.6 Hz, 1H),

8.72 (d, J ¼ 5.1 Hz, 1H), 7.46e7.39 (m, 3H), 7.32 (m, 3H), 4.16 (q,

J ¼ 7.1 Hz, 2H), 1.06 (t, J ¼ 7.1 Hz, 3H); LCMS (ESI^þ) m/z 228.1

(M þ H)^þ.

Table 1

SAR of amide analogs.^a

To a solution of the above crude alcohol (3.58 g, 19.3 mmol) in

dry CH₂Cl₂was added DIB (9.3 g, 29 mmol) and TEMPO (603 mg,

3.86 mmol). The reaction mixture was stirred at room temperature

overnight, and then the solvent was concentrated. The residue was

puriﬁed by column chromatography to give 7a as a light yellow

solid (2.18 g, 54%). ¹H NMR (400 MHz, CDCl₃)

d 10.07 (s, 1H), 9.14 (s,

1H), 8.80 (d, J ¼ 5.2 Hz, 1H), 7.55e7.48 (m, 3H), 7.43e7.36 (m, 3H);

MS (ESI^þ) m/z 184.00 (M þ H)^þ.

4.1.5. 4-(2-Fluorophenyl)nicotinaldehyde (7b)

Compound 7b (2.09 g, 52%) was prepared as a light yellow solid

from 6b (4.9 g, 20 mmol) following a procedure similar to that

described for the preparation of 7a. ¹H NMR (300 MHz, CDCl₃)

b

Cmpd

R₁

R₂

R₃

hTGR5 EC₅₀

(

m

M)

Max effect (%)

LE^c

9a

9b

9c

9d

9e

9f

9g

9h

9i

Me

Et

Cyclohexyl

Phe

4-ClePhe

4-Pyridyl

2-Furanyl

3-Furanyl

2-Thienyl

3-Thienyl

2-Furanyl

e

H

Me

Et

e

H

F

0.91 ꢃ 0.21

1.27 ꢃ 0.58

117

104

NT

116

72

100

88

87

88

86

95

91

0.26

0.24

e

d

10.01 (d, J ¼ 3.1 Hz,1H), 9.18 (s,1H), 8.85 (d, J ¼ 5.1 Hz,1H), 7.51 (m,

1H), 7.43e7.29 (m, 3H), 7.25e7.19 (m, 1H); MS (ESI^þ) m/z 202.04

19%@10 mM

0.35 ꢃ 0.19

7.43 ꢃ 0.23

4.08 ꢃ 1.05

0.47 ꢃ 0.00

0.32 ꢃ 0.04

0.26 ꢃ 0.05

0.12 ꢃ 0.02

0.50 ꢃ 0.02

0.31 ꢃ 0.07

1.23 ꢃ 0.09

1.23 ꢃ 0.36

0.24

0.18

0.20

0.24

0.25

0.26

0.23

0.24

0.21

0.25

0.31

(M þ H)^þ.

4.1.6. N-(3,5-Bis(triﬂuoromethyl)benzyl)-1-(4-phenylpyridin-3-yl)

methanamine (8a)

To the solution of 7a (1.75 g, 9.56 mmol) and 3,5-

bis(triﬂuoromethyl)benzylamine (2.44 g, 10 mmol) in dry

CH₂Cl₂was added NaBH(OAc)₃(4.05 g, 19.1 mmol). The mixture

was stirred at room temperature overnight and then quenched

with saturated aq NaHCO₃. The mixture was extracted with

CH₂Cl₂, and the combined organic phases were washed with

brine, dried over MgSO₄and concentrated. The residue was pu-

riﬁed by ﬂash column chromatography to give 8a as colorless oil

9j

9k

12a

12b

1

H

e

95

103

106

2

e

0.019 ꢃ 0.005

a

EC₅₀values were calculated using the CRE-Luciferase assay from the results of at

least two independent experiments with eight concentrations each and are

expressed as the mean ꢃ SD. Effect: using effect of DMSO vehicle control as 0%, effect

(2.03 g, 51%). ¹H NMR (300 MHz, DMSO)

d 8.72 (s, 1H), 8.49 (d,

of 20

m

M INT-777 as 100%. Max effect (cmpd)% ¼ [Data_(cmpd)ꢁ Data_{(DMSO control)}]/

J ¼ 5.0 Hz, 1H), 7.95 (s, 2H), 7.91 (s, 1H), 7.40 (s, 5H), 7.24 (d,

J ¼ 5.0 Hz, 1H), 3.82 (s, 2H), 3.66 (s, 2H); MS (ESI^þ) m/z 411.40

(M þ H)^þ; HRMS (ESI^þ) calcd for C₂₁H₁₆F₆N₂Na (M þ Na)^þ

411.1290, found 411.1298.

[Data₍₂₀

ꢁ Data_{(DMSO control)}] ꢂ 100%. “NT”, not tested.

m

M INT-777)

b

All the chiral compounds are racemates.

c

LEs were calculated by ꢁ

D

G ¼ RTlnK_D, presuming that EC₅₀zK_D. Values are

given in kcal/mol per non-hydrogen atom.

J. Zhu et al. / European Journal of Medicinal Chemistry 69 (2013) 55e68

61

Table 2

SAR of selected compounds.^a

Cmpd

X^b

R₁

R₂

R₃

hTGR5 EC₅₀(nM)

Max effect (%)

LE^c

8a

8b

eCH₂e

H

e

H

Pr

H

e

H

2-F

H

2,6-di-F

2-F

2,6-di-F

2-F

580 ꢃ 8.7

458 ꢃ 8.2

120

81

NT

90

91

0.29

e

11b

13a

13b

14

15a

15b

15c

16a

16b

17a

e

1%@10

m

M

eCH(Me)e

eCH(Et)e

eCOe

eCH(CN)e

eSO₂e

204 ꢃ 15

0.29

0.27

e

384 ꢃ 43

46%@10

35 ꢃ 5.7

61 ꢃ 2.8

51 ꢃ 17

63 ꢃ 15

62 ꢃ 11

m

M

NT

101

107

104

102

107

89

0.32

0.30

0.31

0.30

0.25

H

eSO₂e

eCH(CN)e

466 ꢃ 118

17b

eCH(CN)e

H

2-F

892 ꢃ 77

94

0.22

18a

18b

eSO₂e

Bn

Bz

H

2-F

2100 ꢃ 139

31 ꢃ 2.8

112

109

0.20

0.25

18c

18d

eSO₂e

H

2-F

32 ꢃ 2.4

112

101

0.26

0.25

53 ꢃ 12

24

27

29

eSO₂e

H

Cl

Ph

EtOe

2-F

406 ꢃ 46

87

NT

0.27

e

18%@10

m

M

e

a

EC₅₀values were calculated using the CRE-Luciferase assay from the results of at least two independent experiments with eight concentrations each and are expressed as

the mean ꢃ SD. Effect: using effect of DMSO vehicle control as 0%, effect of 20 m int-777 as 100%. Max effect (cmpd)% ¼ [Data_(cmpd)ꢁ Data_{(DMSO control)}]/[Data₍₂₀

m

M INT-

ꢁ Data_{(DMSO control)}] ꢂ 100%. “NT”, not tested.

777)

b

All the chiral compounds are racemates.

c

LEs were calculated by ꢁ G ¼ RTlnK_D, presuming that EC₅₀zK_D. Values are given in kcal/mol per non-hydrogen atom.

D

4.1.7. N-(3,5-Bis(triﬂuoromethyl)benzyl)-1-(4-(2-ﬂuorophenyl)

pyridin-3-yl)methanamine (8b)

Compound 8b (2.35 g, 55%) was prepared as a colorless oil from

7b (2.09 g,10 mmol) following a procedure similar to that described

for the preparation of 8a. ¹H NMR (400 MHz, CDCl₃)

d 8.75 (s, 1H),

8.58 (d, J ¼ 5.0 Hz, 1H), 7.72 (s, 1H), 7.66 (s, 2H), 7.46e7.34 (m, 1H),

7.25e7.20 (m, 1H), 7.18 (d, J ¼ 5.0 Hz, 1H), 7.13 (m, 1H), 3.76 (s, 4H);

LCMS (ESI^þ) m/z 429.1 (M þ H)^þ; HRMS (ESI^þ) calcd for C₂₁H₁₆F₇N₂

(M þ H)^þ429.1196, found 429.1208.

4.1.8. N-(3,5-Bis(triﬂuoromethyl)benzyl)-N-((4-phenylpyridin-3-yl)

methyl)acetamide (9a)

Table 3

mTGR5 activity of selected compounds.^a

To the solution of 8a (90 mg, 0.22 mmol) and Et₃N (61

0.44 mmol) in dry CH₂Cl₂was added acetyl chloride (19

m

l,

Cmpd

mTGR5

EC₅₀M)

Max effect

(%)

Cmpd

mTGR5

EC₅₀M)

Max

effect (%)

(

m

(

m

0.26 mmol). The reaction mixture was stirred at room temperature

overnight and then saturated aq. NaHCO₃was added. The mixture

was extracted with CH₂Cl₂, and the combined organic phases were

washed with brine, dried over MgSO₄and concentrated. The res-

idue was puriﬁed by ﬂash column chromatography to give 9a as

colorless oil (70 mg, 70%). ¹H NMR (400 MHz, CDCl₃, ca 2:3 mixture

9i

9j

0.30 ꢃ 0.07

0.23 ꢃ 0.02

1.18 ꢃ 0.26

2.04 ꢃ 0.26

0.15 ꢃ 0.03

0.37 ꢃ 0.12

0.10 ꢃ 0.01

127

111

119

117

121

120

123

16a

16b

18b

18c

18d

1

0.82 ꢃ 0.02

0.55 ꢃ 0.08

0.85 ꢃ 0.02

0.91 ꢃ 0.02

1.09 ꢃ 0.11

0.36 ꢃ 0.08

0.13 ꢃ 0.03

148

123

144

139

146

100

130

12a

13a

15a

15b

15c

2

of atropisomers)

d 8.68 (s, 0.37H), 8.64e8.56 (m, 1H), 8.52 (s,

a

EC₅₀values were calculated using the CRE-Luciferase assay from the results of at

least two independent experiments with eight concentrations each and are

expressed as the mean ꢃ SD. Effect: using effect of DMSO vehicle control as 0%, effect

0.60H), 7.73 (s, 0.36H), 7.71 (s, 0.57H), 7.43 (m, 3H), 7.35 (m, 1H),

7.27 (s, 1H), 7.18 (d, J ¼ 4.9 Hz, 1H), 7.17e7.10 (m, 2H), 4.70 (s, 0.7H),

4.53 (s, 1.16H), 4.47 (s, 1.16H), 4.29 (s, 0.7H), 2.13 (s, 1H), 2.05 (s, 2H);

of 20

[Data₍₂₀

m

M INT-777 as 100%. Max effect (cmpd)% ¼ [Data_(cmpd)ꢁ Data_{(DMSO control)}]/

ꢁ Data_{(DMSO control)}] ꢂ 100%.

¹³C NMR (100 MHz, CDCl₃, ca 2:3 mixture of atropisomers)

d 171.0

m

M INT-777)

62

J. Zhu et al. / European Journal of Medicinal Chemistry 69 (2013) 55e68

Table 4

PK properties of 15a after oral administration to rats.^a

Cmpd

T_max(h)

C_max(ng/ml)

16.3 ꢃ 4.4

AUC_0et(ng h/ml)

AUC_0eN(ng$h/ml)

55.0 ꢃ 3.8

MRT_0et(h)

MRT_0eN(h)

Cl (L/h/kg)

T_1/2(h)

15a

1.9 ꢃ 1.0

49.1 ꢃ 6.6

2.6 ꢃ 0.2

3.6 ꢃ 0.8

91.2 ꢃ 6.5

2.3 ꢃ 0.6

a

n ¼ 6 rats/group; male SD rats (BK, Shanghai, China); p.o., 5 mg/kg; formulated in 0.25% CMCeNa; data were presented as mean ꢃ SD and the PK parameters were

calculated by non-compartmental model analysis on Pheonix WinNonlin 6.0 (Pharsight, Mountain View, CA).

(0.6C), 170.7 (0.4C), 150.7 (0.4C), 149.8 (0.6C), 149.6 (0.6C), 149.5

(0.4C), 148.8 (1C), 139.7 (0.6C), 138.7 (0.4C), 137.4 (0.4C), 137.1

(0.6C), 131.8 (m, 2C), 129.5 (1C), 128.9 (1.2C), 128.8 (0.8C), 128.5

(0.8C), 128.4 (0.8C), 128.3 (1.2C), 127.9 (1.2C), 126.2 (1C), 124.6

(0.6C), 124.2 (0.4C), 123.0 (m, 2C), 121.7 (0.4C), 121.4 (0.6C), 50.3

(0.4C), 47.5 (0.6C), 47.4 (0.6C), 42.8 (0.4C), 21.3 (0.4C), 21.2 (0.6C);

HRMS (ESI^þ) calcd for C₂₃H₁₉F₆N₂O (M þ H)^þ453.1402, found

453.1397.

2:3 mixture of atropisomers) d 176.6, 150.0 (0.4C), 149.7 (0.6C),

149.6 (0.4C), 149.3 (0.6C), 148.9 (0.6C), 148.5 (0.4C), 140.0 (0.6C),

139.1 (0.4C), 137.4 (0.4C), 137.1 (0.6C), 131.8 (m, 2C), 129.8 (0.4C),

129.0 (0.6C), 128.8 (1.2C), 128.7 (0.8C), 128.5 (0.6C), 128.4 (0.4C),

128.3 (0.8C), 128.0 (1.2C), 127.9 (1.2C), 126.3 (0.8C), 124.6 (0.6C),

124.2 (0.4C), 123.0 (q, J ¼ 216.8 Hz, 2C), 121.7 (0.4C), 121.3 (0.6C),

49.3 (0.4C), 47.4 (0.6C), 46.1 (0.6C), 42.9 (0.4C), 40.8 (0.4C), 40.6

(0.6C), 29.5 (0.8C), 29.3 (1.2C), 25.6 (1.8C), 25.5 (1.2C); HRMS (ESI^þ)

calcd for C₂₈H₂₇F₆N₂O (M þ H)^þ521.2028, found 521.2030.

4.1.9. N-(3,5-Bis(triﬂuoromethyl)benzyl)-N-((4-phenylpyridin-3-yl)

methyl)propionamide (9b)

4.1.11. N-(3,5-Bis(triﬂuoromethyl)benzyl)-N-((4-phenylpyridin-3-

yl)methyl)benzamide (9d)

To the solution of 8a (82 mg, 0.2 mmol) and propionic acid

(18

m

l, 0.24 mmol) in acetonitrile was added HATU (99 mg,

Compound 9d (55 mg, 59%) was prepared as a white solid from

8a (75 mg, 0.18 mmol) following a procedure similar to that of

0.26 mmol) and DIEA (67

ml, 0.4 mmol). The reaction mixture was

reﬂuxed under nitrogen overnight and then concentrated. The

residue was diluted with EtOAc and washed with brine, then dried

over MgSO₄and concentrated. The residue was puriﬁed by ﬂash

column chromatography to give 9b as colorless oil (60 mg, 64%). ¹H

preparation of 9a. ¹H NMR (400 MHz, CDCl₃)

d

8.64 (m, 2H), 7.73 (s,

1H), 7.40 (m, 9H), 7.11 (m, 4H), 4.93e4.09 (m, 4H); ¹³C NMR

(100 MHz, CDCl₃) 172.2,150.8,149.6, 149.2 (2C),139.2,137.2, 134.9,

d

131.9 (q, J ¼ 26.8 Hz, 2C), 130.2 (2C), 129.0, 128.7 (3C), 128.6, 128.2

(2C), 126.6 (3C), 124.5, 122.9 (q, J ¼ 216.6 Hz, 2C), 121.6, 51.0 (0.5C),

47.9 (0.5C), 49.0 (0.5C), 42.4 (0.5C); HRMS (ESI^þ) calcd for

NMR (300 MHz, CDCl₃, ca 2:3 mixture of atropisomers)

d 8.62 (m,

1.4H), 8.49 (s, 0.6H), 7.71 (s, 1H), 7.44 (m, 3H), 7.31 (m, 2H), 7.22e

7.06 (m, 3H), 4.72 (s, 0.76H), 4.55 (s, 1.12H), 4.46 (s, 1.13H), 4.30 (s,

0.77H); ¹³C NMR (100 MHz, CDCl₃, ca 2:3 mixture of atropisomers)

C

₂₈H₂₀F₆N₂ONa (M þ Na)^þ537.1378, found 537.1389.

d

174.1 (0.6C), 173.9 (0.4C), 150.6 (0.4C), 149.7, 149.4 (0.6C), 148.8

4.1.12. N-(3,5-Bis(triﬂuoromethyl)benzyl)-4-chloro-N-((4-

phenylpyridin-3-yl)methyl)benzamide (9e)

(0.4C), 148.7 (0.6C), 139.8 (0.6C), 138.9 (0.4C), 137.5 (0.4C), 137.1

(0.6C), 132.0 (m, 2C), 129.6 (0.4C), 128.8 (1.2C), 128.7 (0.8C), 128.6

(0.6C), 128.5 (0.6C), 128.4 (0.4C), 128.3 (0.8C), 128.2 (1.2C), 127.9

(1.2C), 126.1 (0.8C), 124.6 (0.6C), 124.2 (0.4C), 123.0 (m, 2C), 121.7

(0.4C), 121.4 (0.6C), 49.4 (0.4C), 47.7 (0.6C), 46.5 (0.6C), 43.1 (0.4C),

26.3 (0.4C), 26.1 (0.6C), 9.3; HRMS (ESI^þ) calcd for C₂₄H₂₁F₆N₂O

(M þ H)^þ467.1558, found 467.1563.

Compound 9e (90 mg, 75%) was prepared as a white solid from

8a (90 mg, 0.22 mmol) following a procedure similar to that

described for the preparation of 9a. ¹H NMR (300 MHz, DMSO)

d

8.54 (m, 2H), 7.93 (s, 1H), 7.85e6.88 (m, 12H), 4.67 (m, 4H); ¹³C

NMR (100 MHz, CDCl₃) d 171.1, 149.8, 149.2 (2C), 138.9, 137.0, 136.4,

133.1, 132.0 (q, J ¼ 27.6 Hz, 2C), 131.2, 129.0 (2C), 128.8 (2C), 128.7

(2C),128.1 (4C),126.7,124.6,123.9 (2C),121.7, 50.8, 47.4, 42.4; HRMS

(ESI^þ) calcd for C₂₈H₁₉ClF₆N₂ONa (M þ Na)^þ571.0988, found

571.0978.

4.1.10. N-(3,5-Bis(triﬂuoromethyl)benzyl)-N-((4-phenylpyridin-3-

yl)methyl)cyclohexanecarboxamide (9c)

Compound 9c (65 mg, 62%) was prepared as a colorless oil from

8a (84 mg, 0.2 mmol) following a procedure similar to that

described for the preparation of 9b. ¹H NMR (400 MHz, CDCl₃, ca

4.1.13. N-(3,5-Bis(triﬂuoromethyl)benzyl)-N-((4-phenylpyridin-3-

yl)methyl)isonicotinamide (9f)

2:3 mixture of atropisomers)

d

8.69e8.48 (m, 2H), 7.76 (s, 0.38H),

Compound 9f (70 mg, 62%) was prepared as a white solid from

8a (90 mg, 0.22 mmol) following a procedure similar to that

described for the preparation of 9a. ¹H NMR (300 MHz, CD₃OD, ca

7.72 (s, 0.60H), 7.45 (m, 3H), 7.34 (m, 2H), 7.20 (m, 3H), 4.69 (s,

0.67H), 4.55 (s, 1.19H), 4.49 (s, 1.17H), 4.38 (s, 0.69H), 2.32 (m, 1H),

1.86e1.50 (m, 6H), 1.37e1.12 (m, 4H); ¹³C NMR (100 MHz, CDCl₃, ca

2:3 mixture of atropisomers)

d 8.70e8.42 (m, 4H), 7.34 (m, 11H),

A

B

25

8

20

15

10

5

6

4

2

0

Control

15a

Control

15a

Fig. 3. Effect of 15a during OGTT in ICR mice: (A) blood glucose AUC_0e120min; (B) weight of the bile. 50 mg/kg 15a or 0.25% CMC (control) was orally administered to ICR mice

(n ¼ 7e8 animals/group). After the OGTT experiment, the fasting mice were re-fed for 2 h. Then, the gallbladders were ligated and removed, and the weight of the bile was

calculated by subtracting the net weight of the gallbladder from the gross weight of the gallbladder with bile. There was no signiﬁcant difference between the two groups. The error

bar indicates the SEM.

J. Zhu et al. / European Journal of Medicinal Chemistry 69 (2013) 55e68

63

4.90 (m, 1.4H), 4.82e4.64 (m, 2H), 4.33 (brs, 0.6H); ¹³C NMR

(100 MHz, CDCl₃) 169.6,151.0 (0.4C), 150.4 (2C), 149.7 (0.6C), 149.4

(0.4C), 149.0 (0.6C), 142.4, 138.8 (0.6C), 137.8 (0.4C), 137.3 (0.4C),

136.7 (0.6C), 132.1 (q, J ¼ 26.5 Hz, 2C), 128.9, 128.7 (2C), 128.4, 128.2,

127.8 (2C), 126.5, 124.7 (0.6C), 124.5 (0.4C), 124.0, 121.8, 120.7 (1.2C),

120.5 (0.8C), 50.6 (0.4C), 47.9 (0.6C), 47.1 (0.6C), 42.3 (0.4C); HRMS

(ESI^þ) calcd for C₂₇H₂₀F₆N₃O (M þ H)^þ516.1511, found 516.1516.

(s, 1H), 7.35 (m, 1H), 7.24e7.10 (m, 4H), 7.05 (m, 1H), 6.50 (dd,

d

J ¼ 3.5, 1.7 Hz, 1H), 4.62 (m, 4H); ¹³C NMR (100 MHz, CDCl₃)

d 160.4,

158.6 (d, J ¼ 196.1 Hz), 149.1 (2C), 147.0, 144.5, 143.5, 139.2, 131.8 (q,

J ¼ 26.5 Hz, 2C), 130.9, 130.3, 130.1, 128.4, 124.7 (4C), 123.0 (q,

J ¼ 216.9 Hz, 2C), 121.6, 118.2, 115.9 (d, J ¼ 17.3 Hz), 111.8, 49.1, 47.0;

HRMS (ESI^þ) calcd for C₂₆H₁₇F₇N₂O₂Na (M þ Na)^þ545.1070, found

545.1072.

4.1.14. N-(3,5-Bis(triﬂuoromethyl)benzyl)-N-((4-phenylpyridin-3-

yl)methyl)furan-2-carboxamide (9g)

Compound 9g (95 mg, 86%) was prepared as a white solid from

8a (90 mg, 0.22 mmol) following a procedure similar to that

described for the preparation of 9a. ¹H NMR (400 MHz, CDCl₃)

4.1.19. 3-(Azidomethyl)-4-phenyl pyridine (10a)

Following the similar procedure to that described for the

preparation of 7a, 6a was reduced to the corresponding alcohol

with DIBAL-H. To the solution of the crude alcohol (970 mg,

5.2 mmol) in anhydrous THF was added DBU (1.95 ml, 13.1 mmol)

and DPPA (2.26 ml, 10.5 mmol) successively. The reaction mixture

was stirred under reﬂux for 5 h under nitrogen until consumption

of the alcohol monitored by TLC, then mixture was cooled to room

temperature and the solvent was concentrated. The residue was

diluted with water and extracted with EtOAc. The combined

organic phases were washed with brine, dried over MgSO₄and

concentrated. The residue was puriﬁed by ﬂash column chroma-

tography to give 10a as colorless oil (980 mg, 88%). ¹H NMR

d

8.72 (brs, 1H), 8.60 (d, J ¼ 4.9 Hz, 1H), 7.70 (s, 1H), 7.60e7.30 (m,

6H), 7.24e7.09 (m, 4H), 6.51 (dd, J ¼ 3.5, 1.6 Hz, 1H), 4.82 (brs, 2H),

4.61 (s, 2H); ¹³C NMR (100 MHz, CDCl₃)

160.4, 149.7, 148.9, 147.2,

d

144.5 (2C), 139.2, 137.2, 131.8 (q, J ¼ 26.5 Hz, 2C), 129.1, 128.7 (3C),

128.6, 128.3 (2C), 124.3 (3C), 123.0 (q, J ¼ 216.9 Hz, 2C), 118.4, 111.9,

48.8, 43.8; HRMS (ESI^þ) calcd for C₂₆H₁₈F₆N₂O₂Na (M þ Na)^þ

527.1170, found 527.1177.

4.1.15. N-(3,5-Bis(triﬂuoromethyl)benzyl)-N-((4-phenylpyridin-3-

yl)methyl)furan-3-carboxamide (9h)

Compound 9h (37 mg, 33%) was prepared as a white solid from

8a (90 mg, 0.22 mmol) following a procedure similar to that

described for the preparation of 9b. ¹H NMR (300 MHz, CDCl₃)

(300 MHz, CDCl₃)

d

8.68 (s, 1H), 8.63 (d, J ¼ 5.0 Hz, 1H), 7.54e7.43

(m, 3H), 7.41e7.33 (m, 2H), 7.26 (m, 1H), 4.35 (s, 2H); LCMS (ESI^þ)

m/z 211.1 (M þ H)^þ.

4.1.20. 2-(Azidomethyl)-2⁰-ﬂuorobiphenyl (10b)

d

8.79e8.51 (m, 2H), 7.73 (s, 1H), 7.66 (brs, 1H), 7.39 (m, 5H), 7.20 (d,

J ¼ 4.8 Hz, 1H), 7.13 (m, 2H), 6.50 (brs, 1H), 4.71 (s, 2H), 4.56 (brs,

2H); ¹³C NMR (100 MHz, CDCl₃)

165.4, 149.6, 149.3, 143.9, 143.4

Compound 10b (10.8 g, 88%) was prepared as colorless oil from

6b (13 g, 53 mmol) following a procedure similar to that of prep-

d

aration of 10a. ¹H NMR (300 MHz, CDCl₃)

d 8.72 (s, 1H), 8.65 (d,

(2C), 139.0, 137.1, 132.0 (q, J ¼ 28.1 Hz, 2C), 128.7 (3C), 128.6 (2C),

128.0 (2C), 124.5 (2C), 122.9 (q, J ¼ 217.1 Hz, 2C), 121.7, 120.2, 109.8,

50.2, 47.5; HRMS (ESI^þ) calcd for C₂₆H₁₉F₆N₂O₂(M þ H)^þ505.1351,

found 505.1355.

J ¼ 5.0 Hz, 1H), 7.51e7.40 (m, 1H), 7.33e7.16 (m, 4H), 4.33 (s, 2H);

LCMS (ESI^þ) m/z 229.1 (M þ H)^þ.

4.1.21. 3-(Azidomethyl)-4-(2,6-diﬂuorophenyl)pyridine (10c)

Compound 10c (730 mg, 41%) was prepared as a colorless oil

from 6c (1.93 g, 7.3 mmol) following a procedure similar to that

described for the preparation of 10a. ¹H NMR (300 MHz, CDCl₃)

4.1.16. N-(3,5-Bis(triﬂuoromethyl)benzyl)-N-((4-phenylpyridin-3-

yl)methyl)thiophene-2-carboxamide (9i)

Compound 9i (100 mg, 87%) was prepared as a white solid from

8a (90 mg, 0.22 mmol) following a procedure similar to that

described for the preparation of 9a. ¹H NMR (400 MHz, CDCl₃)

d

8.76 (s,1H), 8.68 (d, J ¼ 5.0 Hz,1H), 7.44 (tt, J ¼ 8.5, 6.4 Hz,1H), 7.26

(m, 2H), 7.13e6.95 (m, 2H), 4.30 (s, 2H); LCMS (ESI^þ) m/z 247.1

(M þ H)^þ.

d

8.74 (s, 1H), 8.63 (d, J ¼ 5.0 Hz, 1H), 7.72 (s, 1H), 7.53e7.40 (m, 3H),

7.39e7.30 (m, 3H), 7.26e7.23 (m, 1H), 7.21 (d, J ¼ 4.9 Hz, 1H), 7.12

4.1.22. (4-Phenylpyridin-3-yl)methanamine hydrochloride (11a)

To a stirred solution of azide 10a (970 mg, 4.61 mmol) in THF/

H₂O was added PPh₃(2.42 g, 9.22 mmol) portionwise. The mixture

was stirred at room temperature overnight and then was acidiﬁed

with concentrated HCl. The solvent was concentrated and the

residue was dissolved in EtOAc and extracted with water. The

combined aqueous phases were washed with EtOAc ﬁve times

until most of non-saliﬁed side products were removed. Then the

aqueous solution was neutralized with solid NaOH and extracted

with EtOAc. The combined organic phases were washed with

brine, dried over MgSO₄. After ﬁltration, the ﬁltrate was acidiﬁed

with 4 N HCl/EtOAc solution. The resulting mixture was ﬁltered to

give 11a (910 mg, 77%) as a white solid. ¹H NMR (300 MHz, CDCl₃)

(m, 2H), 7.09e6.92 (m, 1H), 4.79 (s, 2H), 4.61 (s, 2H); ¹³C NMR

(100 MHz, CDCl₃)

d 165.1, 149.7, 149.2 (2C), 138.9, 137.0, 136.1, 131.9

(q, J ¼ 26.9 Hz, 2C), 130.0, 129.1, 128.7 (3C), 128.6, 128.1 (2C), 127.7,

127.1 (2C), 124.5, 122.9 (q, J ¼ 216.9 Hz, 2C), 121.7, 49.5; HRMS (ESI^þ)

calcd for C₂₆H₁₈F₆N₂OSNa (M þ Na)^þ543.0924, found 543.0936.

4.1.17. N-(3,5-Bis(triﬂuoromethyl)benzyl)-N-((4-phenylpyridin-3-

yl)methyl)thiophene-3-carboxamide (9j)

Compound 9j (55 mg, 48%) was prepared as a colorless gum

from 8a (90 mg, 0.22 mmol) following a procedure similar to that

described for the preparation of 9b. ¹H NMR (300 MHz, CDCl₃)

d

8.66 (m, 2H), 7.72 (s, 1H), 7.36 (m, 7H), 7.24e6.98 (m, 4H), 4.62 (m,

4H); ¹³C NMR (100 MHz, CDCl₃)

d

167.5, 149.8, 149.3 (2C), 139.1,

d

8.57 (s, 1H), 8.52 (d, J ¼ 5.0 Hz, 1H), 7.46e7.37 (m, 3H), 7.33 (m,

137.1, 135.2, 132.0 (q, J ¼ 25.4 Hz, 2C), 128.8 (3C), 128.7 (2C), 128.1

(2C), 127.0, 126.7, 126.6, 124.6 (2C), 123.0 (q, J ¼ 217.1 Hz, 2C), 121.7,

48.2, 41.2; HRMS (ESI^þ) calcd for C₂₆H₁₉F₆N₂OS (M þ H)^þ521.1122,

found 521.1118.

2H), 7.16 (d, J ¼ 5.0 Hz, 1H), 3.69 (s, 2H); MS (ESI^þ) m/z 185.07

(M þ H)^þ.

4.1.23. (4-(2-Fluorophenyl)pyridin-3-yl)methanamine

hydrochloride (11b)

4.1.18. N-(3,5-Bis(triﬂuoromethyl)benzyl)-N-((4-(2-ﬂuorophenyl)

pyridin-3-yl)methyl)furan-2-carboxamide (9k)

Compound 9k (135 mg, 60%) was prepared as a light yellow

solid from 8b (183 mg, 0.42 mmol) following a procedure similar to

that described for the preparation of 9a. ¹H NMR (400 MHz, CDCl₃)

Compound 11b (11 g, 85%) was prepared as a white solid from

10b (10.8 g, 47 mmol) following a procedure similar to that

described for the preparation of 11a. ¹H NMR (400 MHz, CDCl₃)

d

8.73 (s, 1H), 8.56 (d, J ¼ 4.9 Hz, 1H), 7.42 (m, 1H), 7.30e7.22 (m,

2H), 7.21e7.13 (m, 2H), 3.78 (s, 2H); LCMS (ESI^þ) m/z 203.1

d

8.72 (s, 1H), 8.64 (d, J ¼ 4.9 Hz, 1H), 7.72 (s, 1H), 7.54 (brs, 2H), 7.44

(M þ H)^þ.

64

J. Zhu et al. / European Journal of Medicinal Chemistry 69 (2013) 55e68

4.1.24. (4-(2,6-Diﬂuorophenyl)pyridin-3-yl)methanamine

hydrochloride (11c)

4.1.27. 1-(3,5-Bis(triﬂuoromethyl)phenyl)-N-((4-(2-ﬂuorophenyl)

pyridin-3-yl)methyl)ethanamine (13a)

Compound 11c (750 mg, 86%) was prepared as a white solid

from 10c (730 mg, 2.96 mmol) following a procedure similar to

that described for the preparation of 11a. ¹H NMR (400 MHz,

Compound 13a (120 mg, 40%) was prepared as colorless oil from

11b (242 mg, 0.88 mmol) following a procedure similar to that

described for the preparation of 1-(3,5-bis(triﬂuoromethyl)

phenyl)-N-((4-phenylpyridin-3-yl)methyl)ethanamine. ¹H NMR

DMSO)

d

9.11 (s, 1H), 8.77 (d, J ¼ 5.1 Hz, 1H), 8.69 (brs, 2H), 7.69 (m,

2H), 7.36 (t, J ¼ 8.1 Hz, 2H), 3.94 (m, 2H); LCMS (ESI^þ) m/z 221.1

(300 MHz, CDCl₃) d 8.63 (m, 2H), 7.71 (s, 1H), 7.66 (s, 2H), 7.39 (m,

(M þ H)^þ.

1H), 7.18 (m, 3H), 7.15e7.06 (m, 1H), 3.75 (q, J ¼ 6.5 Hz, 1H), 3.66e

3.47 (m, 2H), 1.25 (d, J ¼ 6.4 Hz, 3H); HRMS (ESI^þ) calcd for

4.1.25. N-(1-(3,5-Bis(triﬂuoromethyl)phenyl)ethyl)-N-((4-

phenylpyridin-3-yl)methyl)furan-2-carboxamide (12a)

To the solution of amine hydrochloride 11a (283 mg,

C

₂₂H₁₈F₇N₂(M þ H)^þ443.1353, found 443.1368.

4.1.28. 1-(3,5-Bis(triﬂuoromethyl)phenyl)-N-((4-(2-ﬂuorophenyl)

pyridin-3-yl)methyl)propan-1-amine (13b)

1.1 mmol) and Et₃N (319

ml, 2.3 mmol) in dry CH₂Cl₂under ni-

trogen was added 1-(3,5-bis(triﬂuoromethyl)phenyl)ethanone

(181 ml, 1 mmol) and 4 A molecular sieves. The reaction mixture

Compound 13b (15 mg, 7%) was prepared as colorless oil from

11b (138 mg, 0.5 mmol) following a procedure similar to that

described for the preparation of 13a. ¹H NMR (400 MHz, CDCl₃)

ꢀ

was reﬂuxed overnight and was then concentrated. The residue

was dissolved in methanol, then NaBH₄(57 mg, 1.5 mmol) was

added at 0 ^ꢀC. The reaction mixture was warmed to room tem-

perature and stirred overnight. Water was added to quench the

reaction and then the mixture was concentrated. The residue was

diluted with water and extracted with EtOAc. The combined

organic phases were washed with brine, dried over MgSO₄and

concentrated. The crude was puriﬁed by ﬂash column chroma-

tography to give 1-(3,5-bis(triﬂuoromethyl)phenyl)-N-((4-

phenylpyridin-3-yl)methyl)ethanamine (230 mg, 54%) as color-

d

8.65 (s, 1H), 8.56 (d, J ¼ 5.0 Hz, 1H), 7.71 (s, 1H), 7.63 (s, 2H), 7.38

(m, 1H), 7.15 (m, 4H), 3.51 (m, 3H), 1.61e1.44 (m, 2H), 0.73 (t,

J ¼ 7.4 Hz, 3H); HRMS (ESI^þ) calcd for C₂₃H₂₀F₇N₂(M þ H)^þ

457.1509, found 457.1512.

4.1.29. N-((4-(2-Fluorophenyl)pyridin-3-yl)methyl)-3,5-

bis(triﬂuoromethyl)benzamide (14)

To the suspension of 3,5-bis(triﬂuoromethyl)benzoic acid

(113 mg, 0.44 mmol), EDCI (139 mg, 0.73 mmol) and HOBt (98 mg,

0.73 mmol) in CH₂Cl₂was added 11b (100 mg, 0.36 mmol) and Et₃N

less oil. ¹H NMR (300 MHz, CDCl₃)

d 8.60 (s, 1H), 8.54 (d,

J ¼ 5.0 Hz, 1H), 7.72 (s, 1H), 7.68 (s, 2H), 7.47e7.36 (m, 3H), 7.36e

7.28 (m, 2H), 7.17 (d, J ¼ 5.0 Hz, 1H), 3.76 (q, J ¼ 6.6 Hz, 1H), 3.71e

3.57 (m, 2H), 1.25 (d, J ¼ 6.6 Hz, 3H); LCMS (ESI^þ) m/z 425.1

(M þ H)^þ.

(126 ml, 0.9 mmol). The mixture was stirred at room temperature

overnight and then saturated aq NaHCO₃was added. The mixture

was extracted with CH₂Cl₂. The combined organic phases were

washed with brine, dried over MgSO₄and concentrated. The crude

was puriﬁed by ﬂash column chromatography to afford 14 (150 mg,

To the solution of 1-(3,5-bis(triﬂuoromethyl)phenyl)-N-((4-

phenylpyridin-3-yl)methyl)ethanamine (85 mg, 0.2 mmol)

nd DMAP (12 mg, 0.1 mmol) in dry THF was added N-ethylm

orpholine (115 l, 0.9 mmol) and 2-furoyl chloride (60 l, 0.6 mmol)

a

93%) as a white solid. ¹H NMR (300 MHz, CDCl₃)

d 8.72 (s, 1H), 8.59

(d, J ¼ 5.0 Hz, 1H), 8.09 (s, 2H), 7.98 (s, 1H), 7.52e7.40 (m, 1H), 7.33e

m

7.14 (m, 4H), 6.68 (brs, 1H), 4.63 (d, J ¼ 5.5 Hz, 2H); ¹³C NMR

under nitrogen. The mixture was stirred at room temperature for 30 h

and then concentrated. The residue was diluted with water and

extracted with EtOAc. The combined organic phases were washed

with brine, dried over MgSO₄and concentrated. The residue was

puriﬁed by ﬂash column chromatography to give 12a (80 mg, 77%) as

(100 MHz, CDCl₃)

d

164.2, 158.9 (d, J ¼ 234.5 Hz), 150.0, 148.7, 144.0,

135.9, 132.0 (q, J ¼ 33.7 Hz, 2C), 131.8, 131.1 (d, J ¼ 8.0 Hz), 130.5 (d,

J ¼ 2.7 Hz), 127.4 (2C), 125.3, 125.2, 125.0, 124.9 (d, J ¼ 3.4 Hz), 122.8

(q, J ¼ 261.8 Hz, 2C), 116.0 (d, J ¼ 21.6 Hz), 40.2; HRMS (ESI^þ) calcd

for C₂₁H₁₄F₇N₂O (M þ H)^þ443.0989, found 443.0987.

a white solid. ¹H NMR (300 MHz, CDCl₃)

d 8.61 (s, 1H), 8.49 (d,

J ¼ 5.0 Hz, 1H), 7.67 (s, 1H), 7.55 (s, 2H), 7.45 (brs, 1H), 7.44e7.36 (m,

3H), 7.21 (d, J ¼ 3.5 Hz, 1H), 7.13 (m, 2H), 7.09 (d, J ¼ 5.0 Hz, 1H), 6.52

(dd, J ¼ 3.4,1.7 Hz,1H), 5.92 (q, J ¼ 7.1 Hz,1H), 4.76 (d, J ¼ 17.1 Hz, 1H),

4.53 (d, J ¼ 16.9 Hz, 1H), 1.48e1.39 (d, J ¼ 7.1 Hz, 3H); ¹³C NMR

4.1.30. 2-(3,5-Bis(triﬂuoromethyl)phenyl)-2-((4-(2-ﬂuorophenyl)

pyridin-3-yl)methylamino)acetonitrile (15a)

To the solution of amine hydrochloride 11b (2.75 g, 10 mmol) and

Et₃N (3.1 ml, 22.5 mmol) in dry CH₂Cl₂was added 3,5-

bis(triﬂuoromethyl)benzaldehyde (1.64 ml, 10 mmol) and Ti(i-PrO)₄

(3.6 ml, 12 mmol) under nitrogen. The mixture was stirred at room

temperature for 4 h, then, TMSCN (2 ml, 15 mmol) was added drop-

wise at 0 ^ꢀC. The mixture was warmed to room temperature and

stirred for 24 h. Then water was added to quench the reaction. The

mixture was ﬁltered and the ﬁltrate was extracted with CH₂Cl₂. The

combined organic phases were washed with brine, dried over MgSO₄

and concentrated. The crude was puriﬁed by ﬂash column chroma-

tography to afford 15a (3.6 g, 79%) as a white solid. ¹H NMR (300 MHz,

(100 MHz, CDCl₃) d 160.8, 149.2,148.3,148.1, 147.5,144.4,142.6,137.2,

131.6 (q, J ¼ 26.5 Hz, 2C),130.4,128.7 (2C),128.6,128.2 (2C),127.5 (2C),

123.7, 123.0 (q, J ¼ 217.0 Hz, 2C), 121.6, 118.3, 111.8, 53.7, 43.7, 17.2;

HRMS (ESI^þ) calcd for C₂₇H₂₀F₆N₂O₂Na (M þ Na)^þ541.1327, found

541.1316.

4.1.26. N-(1-(3,5-Bis(triﬂuoromethyl)phenyl)propyl)-N-((4-

phenylpyridin-3-yl)methyl)furan-2-carboxamide (12b)

Compound 12b (27 mg, 9% for the three steps) was prepared

as a white solid from 11a (170 mg, 0.66 mmol) following a pro-

cedure similar to that described for the preparation of 12a. ¹H

CDCl₃)

d

8.76 (s,1H), 8.63 (d, J ¼ 5.0 Hz, 1H), 7.86 (m, 3H), 7.45 (m,1H),

7.30e7.15 (m, 4H), 4.74 (d, J ¼ 10.3 Hz, 1H), 3.98 (m, 2H); ¹³C NMR

NMR (300 MHz, CDCl₃)

d

8.51 (s, 1H), 8.45 (d, J ¼ 5.0 Hz, 1H), 7.65

(100 MHz, CDCl₃)

d

159.0 (d, J ¼ 195.8 Hz), 150.7, 149.4, 144.2, 136.8,

(s, 1H), 7.54 (s, 2H), 7.44 (m, 4H), 7.20 (d, J ¼ 3.5 Hz, 1H), 7.15 (m,

2H), 7.07 (d, J ¼ 5.0 Hz, 1H), 6.53 (m, 1H), 5.57 (t, J ¼ 7.4 Hz, 1H),

4.79 (d, J ¼ 17.5 Hz, 2H), 4.58 (d, J ¼ 17.5 Hz, 1H), 1.85 (m, 3H),

132.4 (q, J¼ 26.9 Hz, 2C),131.3,130.8 (d, J ¼ 6.5 Hz),130.4(d,J ¼ 2.2 Hz),

127.5 (2C), 125.5 (d, J ¼ 12.9 Hz),125.3,124.6 (d, J ¼ 2.7 Hz), 123.2 (m),

122.8 (q, J ¼ 217.0 Hz, 2C),116.8,116.0 (d, J ¼ 17.4 Hz), 53.0, 47.0; HRMS

(ESI^þ) calcd for C₂₂H₁₅F₇N₃(M þ H)^þ454.1154, found 454.1151.

0.81 (t, J ¼ 7.3 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃)

d 161.2, 149.4,

148.3, 148.2, 147.7, 144.3, 141.5, 137.3, 131.6 (q, J ¼ 26.5 Hz, 2C),

130.1, 128.8 (2C), 128.7, 128.3 (3C), 123.8 (2C), 123.0 (q,

J ¼ 217.0 Hz, 2C), 121.7, 118.3, 111.9, 60.1, 29.7, 23.7, 11.0; HRMS

4.1.31. 2-(3,5-Bis(triﬂuoromethyl)phenyl)-2-((4-phenylpyridin-3-

yl)methylamino)acetonitrile (15b)

Compound 15b (28 mg, 29%) was prepared as white solid from

11a (57 mg, 0.22 mmol) following a procedure similar to that

(ESI^þ) calcd for C₂₈H₂₃F₆N₂O₂(M

533.1663.

þ

H)^þ533.1664, found

J. Zhu et al. / European Journal of Medicinal Chemistry 69 (2013) 55e68

65

described for the preparation of 15a. ¹H NMR (300 MHz, CDCl₃)

8.70 (s, 1H), 8.62 (d, J ¼ 5.0 Hz, 1H), 7.88 (s, 1H), 7.85 (s, 2H), 7.53e

7.42 (m, 3H), 7.38 (m, 2H), 7.24 (d, J ¼ 5.0 Hz,1H), 4.74 (brs,1H), 4.05

(m, 2H); ¹³C NMR (100 MHz, CDCl₃)

150.8, 150.1, 149.6, 137.9,

136.8, 132.4 (q, J ¼ 26.9 Hz, 2C), 130.2, 128.7 (2C), 128.6, 128.1 (2C),

127.5 (2C), 124.8, 123.2, 122.8 (q, J ¼ 217.0 Hz, 2C), 116.9, 53.1, 46.9;

HRMS (ESI^þ) calcd for C₂₂H₁₆F₆N₃(M þ H)^þ436.1248, found

436.1255.

NaHCO₃and extracted with CH₂Cl₂. The combined organic phases

were washed with brine, dried over MgSO₄and concentrated. The

crude was puriﬁed by ﬂash column chromatography and prepara-

tive HPLC using 77% MeOH in water as the eluent to give 17a

d

(65 mg, 44%) as a white solid. ¹H NMR (400 MHz, CDCl₃)

d 8.78 (s,

1H), 8.63 (d, J ¼ 5.0 Hz, 1H), 7.84 (s, 1H), 7.81 (s, 2H), 7.47 (m, 1H),

7.28 (m, 1H), 7.22 (m, 3H), 4.77 (s, 1H), 4.10 (d, J ¼ 13.9 Hz, 1H), 3.50

(d, J ¼ 13.8 Hz, 1H), 2.33 (m, 1H), 2.12 (m, 1H), 1.26 (m, 1H), 1.06 (m,

1H), 0.55 (t, J ¼ 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃)

d 158.9 (d,

4.1.32. 2-(3,5-Bis(triﬂuoromethyl)phenyl)-2-((4-(2,6-

J ¼ 195.5 Hz), 151.4, 149.7, 144.7, 136.7, 132.2 (q, J ¼ 26.9 Hz, 2C),

130.8 (d, J ¼ 6.4 Hz), 130.6, 130.3, 127.8 (2C), 125.6 (d, J ¼ 13.0 Hz),

125.5, 124.7 (d, J ¼ 2.7 Hz), 122.9, 122.9 (q, J ¼ 217.0 Hz, 2C), 116.1 (d,

J ¼ 17.3 Hz), 114.0, 57.5, 53.0, 51.6, 20.1, 11.3; HRMS (ESI^þ) calcd for

diﬂuorophenyl)pyridin-3-yl)methylamino)acetonitrile (15c)

Compound 15c (22 mg, 18%) was prepared as a white solid from

11c (80 mg, 0.27 mmol) following a procedure similar to that

described for the preparation of 15a. ¹H NMR (400 MHz, CDCl₃)

C

₂₅H₂₀F₇N₃Na (M þ Na)^þ518.1443, found 518.1443.

d

8.81 (s, 1H), 8.67 (d, J ¼ 5.0 Hz, 1H), 7.87 (s, 3H), 7.49e7.35 (m, 1H),

7.25 (d, J ¼ 5.0 Hz, 1H), 7.04 (m, 2H), 4.72 (d, J ¼ 10.8 Hz, 1H), 3.97

(m, 2H); ¹³C NMR (100 MHz, CDCl₃)

159.4 (m, 2C), 151.1, 149.6,

4.1.36. 2-(3,5-Bis(triﬂuoromethyl)phenyl)-2-(((4-(2-ﬂuorophenyl)

pyridin-3-yl)methyl)(furan-2-ylmethyl)amino)acetonitrile (17b)

Compound 17b (30 mg, 6%) was prepared as a white solid from

15a (450 mg, 1 mmol) following a procedure similar to that

described for the preparation of 17a. ¹H NMR (400 MHz, CDCl₃)

d

138.1, 136.7, 132.4 (q, J ¼ 26.9 Hz, 2C), 131.8, 130.8 (t, J ¼ 8.0 Hz),

127.5 (2C), 125.9, 123.2, 122.8 (q, J ¼ 217.0 Hz, 2C), 116.7, 114.7 (t,

J ¼ 16.5 Hz), 111.7 (m, 2C), 52.8, 47.2; HRMS (ESI^þ) calcd for

C

₂₂H₁₄F₈N₃(M þ H)^þ472.1060, found 472.1054.

d

8.78 (s, 1H), 8.60 (d, J ¼ 4.9 Hz, 1H), 7.84 (m, 3H), 7.54e7.37 (m,

1H), 7.27 (m, 1H), 7.23 (m, 1H), 7.17 (m, 3H), 6.22 (dd, J ¼ 3.1, 1.9 Hz,

1H), 6.05 (d, J ¼ 3.2 Hz, 1H), 4.84 (s, 1H), 4.02 (d, J ¼ 13.9 Hz, 1H),

3.68 (d, J ¼ 13.9 Hz, 1H), 3.51 (d, J ¼ 14.5 Hz, 1H), 3.41 (d, J ¼ 14.5 Hz,

4.1.33. N-((4-(2-Fluorophenyl)pyridin-3-yl)methyl)-3,5-

bis(triﬂuoromethyl)benzenesulfonamide (16a)

To the suspension of 11b (83 mg, 0.3 mmol) in dry CH₂Cl₂was

1H); ¹³C NMR (100 MHz, CDCl₃)

d

158.6 (d, J ¼ 245.0 Hz), 151.1,

added Et₃N (146

ml, 1.05 mmol) and 3,5-bis(triﬂuoromethyl)ben-

149.4, 149.1, 144.5, 142.9, 136.0, 132.2 (q, J ¼ 33.6 Hz, 2C), 130.7 (d,

J ¼ 8.0 Hz), 130.3,130.1, 127.8 (2C),125.4 (d, J ¼ 15.8 Hz),125.3,124.6

(d, J ¼ 3.5 Hz),122.9,122.8 (q, J ¼ 271.5 Hz, 2C), 116.0 (d, J ¼ 21.6 Hz),

113.8, 110.2, 110.0, 57.0, 51.6, 47.7; HRMS (ESI^þ) calcd for

zenesulfonyl chloride (104 mg, 0.33 mmol). The reaction mixture

was stirred at room temperature overnight and then saturated aq

NaHCO₃was added. The mixture was extracted with CH₂Cl₂. The

combined organic phases were washed with brine, dried over

MgSO₄and concentrated. The crude was puriﬁed by ﬂash column

chromatography to afford 16a (100 mg, 70%) as a white solid. ¹H

C

₂₇H₁₉F₇N₃O (M þ H)^þ534.1411, found 534.1413.

4.1.37. N-Benzyl-N-((4-(2-ﬂuorophenyl)pyridin-3-yl)methyl)-3,5-

bis(triﬂuoromethyl)benzenesulfonamide (18a)

NMR (300 MHz, CDCl₃)

d

8.59 (s, 1H), 8.57 (d, J ¼ 5.0 Hz, 1H), 8.09 (s,

2H), 8.02 (s, 1H), 7.46e7.32 (m, 1H), 7.21 (td, J ¼ 7.5, 1.1 Hz, 1H), 7.12

(d, J ¼ 5.0 Hz, 1H), 7.07 (m, 2H), 5.10 (t, J ¼ 5.8 Hz, 1H), 4.22 (d,

NaH (10 mg, 0.24 mmol) was added to the solution of 16a

(96 mg, 0.2 mmol) in dry DMF at 0 ^ꢀC under nitrogen. After 0.5 h, KI

J ¼ 5.8 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃)

d

158.6 (d, J ¼ 195.8 Hz),

(4 mg, 0.02 mmol) and benzyl bromide (29 ml, 0.24 mmol) were

150.3, 149.5, 143.7, 142.8, 132.8 (q, J ¼ 27.4 Hz, 2C), 131.1 (d,

J ¼ 6.4 Hz), 130.4, 129.6, 127.2 (2C), 126.2, 125.1, 124.9 (d, J ¼ 2.7 Hz),

124.5 (d, J ¼ 12.8 Hz), 122.4 (q, J ¼ 217.5 Hz, 2C), 116.0 (d,

J ¼ 17.4 Hz), 42.9 (d, J ¼ 2.5 Hz); HRMS (ESI^þ) calcd for

added to the mixture. The mixture was stirred at room temperature

for 5 h and then water was added. The suspension was extracted

with EtOAc and the combined organic phases were washed with

brine, dried over MgSO₄and concentrated. The crude was puriﬁed

by ﬂash column chromatography to give 18a (25 mg, 22%) as a light

C

₂₀H₁₄F₇N₂O₂S (M þ H)^þ479.0664, found 479.0662.

yellow solid. ¹H NMR (300 MHz, CDCl₃)

d 8.63 (s, 1H), 8.55 (d,

4.1.34. N-((4-(2,6-Diﬂuorophenyl)pyridin-3-yl)methyl)-3,5-

bis(triﬂuoromethyl)benzenesulfonamide (16b)

J ¼ 5.0 Hz, 1H), 7.99 (s, 1H), 7.96 (s, 2H), 7.45 (m, 1H), 7.26e7.08 (m,

6H), 6.96 (m, 3H), 4.38 (s, 2H), 4.17 (s, 2H); ¹³C NMR (100 MHz,

Compound 16b (70 mg, 52%) was prepared as a white solid from

11c (80 mg, 0.27 mmol) following a procedure similar to that

described for the preparation of 16a. ¹H NMR (400 MHz, CDCl₃)

CDCl₃)

d

158.8 (d, J ¼ 196.7 Hz),150.2,148.8,143.7,142.9,134.1,132.8

(q, J ¼ 27.4 Hz, 2C),131.1 (d, J ¼ 6.4 Hz),130.6,129.4,128.7 (2C),128.5

(2C), 128.3, 127.1 (2C), 126.0, 124.9, 124.8, 124.7, 122.3 (q,

J ¼ 217.5 Hz, 2C), 116.1 (d, J ¼ 17.3 Hz), 52.6, 47.2 (d, J ¼ 3.4 Hz);

HRMS (ESI^þ) calcd for C₂₇H₂₀F₇N₂O₂S (M þ H)^þ569.1134, found

569.1131.

d

8.60 (s, 1H), 8.58 (d, J ¼ 5.0 Hz, 1H), 8.11 (s, 2H), 8.01 (s, 1H), 7.38

(m, 1H), 7.17 (d, J ¼ 5.0 Hz, 1H), 7.03e6.89 (m, 2H), 5.16 (t, J ¼ 5.6 Hz,

1H), 4.18 (d, J ¼ 5.6 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃)

d 158.8 (d,

J ¼ 198.0 Hz), 158.7 (d, J ¼ 198.0 Hz), 149.9, 148.7, 142.4, 137.1, 132.4

(q, J ¼ 27.4 Hz, 2C), 130.8 (t, J ¼ 8.0 Hz), 129.9, 126.6 (2C), 125.7,

125.4, 121.9 (q, J ¼ 217.4 Hz, 2C), 113.1 (t, J ¼ 16.3 Hz), 111.4 (m, 2C),

42.4; HRMS (ESI^þ) calcd for C₂₀H₁₃F₈N₂O₂S (M þ H)^þ497.0570,

found 497.0576.

4.1.38. N-(3,5-Bis(triﬂuoromethyl)phenylsulfonyl)-N-((4-(2-

ﬂuorophenyl)pyridin-3-yl)methyl)benzamide (18b)

NaH (6 mg, 0.15 mmol) was added to the solution of 16a (48 mg,

0.1 mmol) in dry DMF at 0 ^ꢀC under nitrogen. After 0.5 h, benzoyl

chloride (35 ml, 0.3 mmol) was added to the mixture. The mixture

4.1.35. 2-(3,5-Bis(triﬂuoromethyl)phenyl)-2-(((4-(2-ﬂuorophenyl)

pyridin-3-yl)methyl)(propyl)amino)acetonitrile (17a)

was stirred at room temperature for 5 h and then water was added.

The suspension was extracted with EtOAc and the combined

organic phases were washed with brine, dried over MgSO₄and

concentrated. The crude was puriﬁed by ﬂash column chromatog-

raphy to give 18b (35 mg, 60%) as a white solid. ¹H NMR (300 MHz,

To the solution of 15a (136 mg, 0.3 mmol) and propionaldehyde

(110

(318 mg, 1.5 mmol) and HOAc (18

stirred at room temperature, and propionaldehyde (110

m

l, 1.5 mmol) in dry CH₂ClCH₂Cl was added NaBH(OAc)₃

l, 0.3 mmol). The mixture was

l,

m

CDCl₃)

7.61e7.51 (m, 1H), 7.50e7.35 (m, 5H), 7.17 (t, J ¼ 7.2 Hz, 1H), 7.07 (m,

2H), 6.77 (m, 1H), 5.07 (s, 2H); ¹³C NMR (100 MHz, CDCl₃)

170.7,

158.7 (d, J ¼ 245.7 Hz), 150.2, 149.4, 143.8, 141.4, 133.0, 132.5 (q,

d

8.61 (s,1H), 8.57 (d, J ¼ 4.6 Hz,1H), 8.04 (s,1H), 8.00 (s, 2H),

1.5 mmol) and NaBH(OAc)₃(318 mg, 1.5 mmol) were added every

12 h. After 5 days, most of the starting material was consumed.

Then the reaction mixture was quenched with saturated aq

d

66

J. Zhu et al. / European Journal of Medicinal Chemistry 69 (2013) 55e68

J ¼ 34.4 Hz, 2C), 132.3, 131.2 (d, J ¼ 8.2 Hz), 130.3, 129.2 (3C), 128.7

(2C), 128.6 (2C), 127.2, 125.1, 124.9 (d, J ¼ 3.3 Hz), 124.3 (d,

J ¼ 15.8 Hz), 122.2 (q, J ¼ 272.0 Hz, 2C), 116.3 (d, J ¼ 21.7 Hz), 48.4;

HRMS (ESI^þ) calcd for C₂₇H₁₇F₇N₂O₃SNa (M þ Na)^þ605.0746, found

605.0750.

(40.5 g, 140 mmol). The mixture was stirred at room temperature

for 36 h and then the solvent was concentrated. The residue was

diluted with water and extracted with EtOAc. The combined

organic phases were washed with brine, 0.5 N HCl, saturated aq

NaHCO₃and brine successively, dried over MgSO₄and concen-

trated. The crude was puriﬁed by column chromatography to afford

4.1.39. N-(3,5-Bis(triﬂuoromethyl)phenylsulfonyl)-N-((4-(2-

ﬂuorophenyl)pyridin-3-yl)methyl)furan-2-carboxamide (18c)

Compound 18c (65 mg, 57%) was prepared as a white solid from

16a (96 mg, 0.2 mmol) following a procedure similar to that

described for the preparation of 18b. ¹H NMR (300 MHz, CDCl₃)

20 (30.6 g, 78%) as a white solid. ¹H NMR (400 MHz, CDCl₃)

d 8.48 (d,

J ¼ 5.1 Hz, 1H), 7.43 (m, 1H), 7.34e7.27 (m, 2H), 7.24e7.12 (m, 2H),

4.18 (q, J ¼ 7.1 Hz, 2H), 1.08 (t, J ¼ 7.1 Hz, 3H); MS (ESI^þ) m/z 280.1

(M þ H)^þ.

d

8.70 (s,1H), 8.61 (d, J ¼ 4.9 Hz,1H), 8.11 (s, 2H), 8.05 (s,1H), 7.49 (d,

4.1.42. (2-Chloro-4-(2-ﬂuorophenyl)pyridin-3-yl)methanol (21)

DIBAL-H (1.5 M toluene solution, 218 ml) was added dropwise to

a stirred solution of 20 (30.6 g, 109 mmol) in dry CH₂Cl₂at ꢁ78 ^ꢀC

under nitrogen and the mixture was stirred for 1.5 h. 1 M aqueous

potassium sodium tartrate solution was added to quench the re-

action, and the mixture was extracted with CH₂Cl₂. The combined

organic phases were washed with brine, dried over MgSO₄and

concentrated to give 21 (22 g, 89%) as a yellow solid that was used

for the next step without further puriﬁcation. ¹H NMR (300 MHz,

J ¼ 0.8 Hz, 1H), 7.38 (m, 1H), 7.19e7.06 (m, 3H), 7.01 (t, J ¼ 8.9 Hz,

1H), 6.81 (td, J ¼ 7.5, 1.6 Hz, 1H), 6.52 (dd, J ¼ 3.6, 1.7 Hz, 1H), 5.31

(brs, 2H); ¹³C NMR (100 MHz, CDCl₃)

d

158.6 (d, J ¼ 196.4 Hz), 158.3,

150.2, 149.8, 146.5, 145.2, 143.4,141.5, 132.4 (q, J ¼ 27.5 Hz, 2C), 131.1

(d, J ¼ 6.5 Hz), 130.2, 129.4 (2C), 127.1, 124.9, 124.8 (d, J ¼ 2.7 Hz),

124.4 (d, J ¼ 12.9 Hz), 122.3 (q, J ¼ 217.6 Hz, 2C), 121.7, 116.0 (d,

J ¼ 17.3 Hz), 112.7, 47.6 (d, J ¼ 3.5 Hz); HRMS (ESI^þ) calcd for

C

₂₅H₁₆F₇N₂O₄S (M þ H)^þ573.0719, found 573.0732.

DMSO)

d

8.40 (d, J ¼ 5.0 Hz, 1H), 7.61e7.43 (m, 2H), 7.43e7.24 (m,

4.1.40. N-(3,5-Bis(triﬂuoromethyl)phenylsulfonyl)-N-((4-(2-

ﬂuorophenyl)pyridin-3-yl)methyl)thiophene-3-carboxamide (18d)

Compound 18d (65 mg, 55%) was prepared as a white solid from

16a (96 mg, 0.2 mmol) following a procedure similar to that

described for the preparation of 18b. ¹H NMR (300 MHz, CDCl₃)

3H), 4.40 (s, 2H); MS (ESI^þ) m/z 237.9 (M þ H)^þ.

4.1.43. 3-(Azidomethyl)-2-chloro-4-(2-ﬂuorophenyl)pyridine (22)

Compound 22 (19.3 g, 87%) was prepared as a colorless oil from

21 (20 g, 84 mmol) following a procedure similar to that described

d

8.68 (s, 1H), 8.60 (d, J ¼ 5.0 Hz, 1H), 8.03 (brs, 3H), 7.73 (dd, J ¼ 2.9,

for the preparation of 10a. ¹H NMR (300 MHz, CDCl₃)

d 8.43 (d,

1.2 Hz, 1H), 7.43 (m, 1H), 7.32 (dd, J ¼ 5.1, 3.0 Hz, 1H), 7.21e7.11 (m,

J ¼ 5.0 Hz, 1H), 7.57e7.07 (m, 5H), 4.40 (brs, 2H); MS (ESI^þ) m/z

2H), 7.05 (m, 2H), 6.77 (td, J ¼ 7.5, 1.7 Hz, 1H), 5.13 (s, 2H); ¹³C NMR

263.1 (M þ H)^þ.

(100 MHz, CDCl₃)

d

164.9, 158.7 (d, J ¼ 246.7 Hz), 150.5, 149.8, 143.6,

141.4, 133.9, 132.9, 132.4 (q, J ¼ 27.4 Hz, 2C), 131.2 (d, J ¼ 6.7 Hz),

130.3,129.1 (2C),129.0,127.5,127.1,126.7,125.0,124.9 (d, J ¼ 2.7 Hz),

124.3 (d, J ¼ 12.8 Hz), 122.2 (q, J ¼ 269.7 Hz, 2C), 116.2 (d,

J ¼ 17.4 Hz), 48.6; HRMS (ESI^þ) calcd for C₂₅H₁₅F₇N₂O₃S₂Na

(M þ Na)^þ611.0310, found 611.0309.

4.1.44. (2-Chloro-4-(2-ﬂuorophenyl)pyridin-3-yl)methanamine

hydrochloride (23)

Compound 23 (14.4 g, 63%) was prepared as a light yellow solid

from 22 (19.3 g, 74 mmol) following a procedure similar to that

described for the preparation of 11a. ¹H NMR (400 MHz, CDCl₃)

d

8.33 (d, J ¼ 4.9 Hz, 1H), 7.50e7.38 (m, 1H), 7.25 (m, 2H), 7.19 (m,

4.1.41. Ethyl 2-chloro-4-(2-ﬂuorophenyl)nicotinate (20)

1H), 7.13 (d, J ¼ 4.9 Hz, 1H), 3.75 (s, 2H); MS (ESI^þ) m/z 237.1

A mixture of 2⁰-ﬂuoroacetophenone (30.3 ml, 250 mmol), ethyl

cyanoacetate (26.7 ml, 250 mmol), ammonium acetate (5.78 g,

75 mmol) and acetic acid (12.8 ml, 225 mmol) was reﬂuxed in

toluene for 10 h, while water was removed azeotropically using

DeaneStark apparatus. Then the mixture was cooled to room

temperature, diluted with water and extracted with Et₂O. The

combined extracts were washed with water, 0.5 N HCl, saturated aq

NaHCO₃and brine successively. The organic layer was dried over

MgSO₄and concentrated. The residue was puriﬁed by column

chromatography to afford ethyl 2-cyano-3-(2-ﬂuorophenyl)but-2-

enoate (36.8 g, 63%) as a light yellow oil. ¹H NMR (300 MHz,

(M þ H)^þ.

4.1.45. N-((2-Chloro-4-(2-ﬂuorophenyl)pyridin-3-yl)methyl)-3,5-

bis(triﬂuoromethyl)benzenesulfonamide (24)

Compound 24 (1.42 g, 95%) was prepared as a white solid from

23 (900 mg, 2.9 mmol) following a procedure similar to that

described for the preparation of 16a. ¹H NMR (400 MHz, CDCl₃)

d

8.34 (d, J ¼ 5.0 Hz, 1H), 8.08 (s, 2H), 8.02 (s, 1H), 7.55e7.42 (m,

1H), 7.30e7.24 (m, 1H), 7.21e7.06 (m, 3H), 5.26 (t, J ¼ 6.1 Hz, 1H),

4.23 (d, J ¼ 6.1 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃)

d 158.6 (d,

J ¼ 196.4 Hz), 152.1, 149.1, 147.4, 142.4, 132.8 (q, J ¼ 27.4 Hz, 2C),

131.6 (d, J ¼ 6.5 Hz), 130.6 (d, J ¼ 1.7 Hz), 128.2, 127.2 (2C), 126.3,

124.9 (d, J ¼ 2.7 Hz), 124.8, 124.1 (d, J ¼ 2.4 Hz), 122.3 (q,

J ¼ 217.4 Hz, 2C), 116.0 (d, J ¼ 17.2 Hz), 42.1 (d, J ¼ 1.7 Hz); HRMS

(ESI^þ) calcd for C₂₀H₁₃ClF₇N₂O₂S (M þ H)^þ513.0269, found

513.0267.

CDCl₃)

d

7.43 (m, 1H), 7.36e7.10 (m, 3H), 4.35 (q, J ¼ 7.1 Hz, 2H), 2.67

(s, 3H), 1.38 (t, J ¼ 7.1 Hz, 3H); MS (ESI^þ) m/z 234.0 (M þ H)^þ.

N,N-Dimethylformamide dimethyl acetal (26.2 ml, 197 mmol)

was added dropwise to ethyl 2-cyano-3-(2-ﬂuorophenyl)but-2-

enoate (36.6 g, 157 mmol) at 0 ^ꢀC. The mixture was stirred at

room temperature for 2 h until it was turned to the purple residue.

Then diluted with petroleum ether and ﬁltered to give ethyl 2-

cyano-5-(dimethylamino)-3-(2-ﬂuorophenyl)penta-2,4-dienoate

(40.5 g, 90%) as a purple solid. The crude was used for the next step

without further puriﬁcation. ¹H NMR (400 MHz, CDCl₃, ca. 3:7

4.1.46. tert-Butyl (2-chloro-4-(2-ﬂuorophenyl)pyridin-3-yl)

methylcarbamate (25)

To the solution of 23 (2.64 g, 8.52 mmol) and Et₃N (4.7 ml,

34 mmol) in dry CH₂Cl₂was added Boc₂O (3 ml, 12.8 mmol). The

reaction mixture was stirred at room temperature overnight and

then washed with brine. The combined organic phases were dried

over MgSO₄and concentrated. The residue was puriﬁed by column

chromatography to afford 25 (2.58 g, 90%) as a white solid. ¹H NMR

mixture of cis and trans isomers) d 7.46e7.34 (m,1H), 7.28e6.99 (m,

4H), 6.52 (d, J ¼ 12.5 Hz, 0.67H), 6.45 (d, J ¼ 12.5 Hz, 0.34H), 5.95 (d,

J ¼ 12.5 Hz, 0.34H), 4.26 (q, J ¼ 7.1 Hz,1.3H), 4.03 (q, J ¼ 7.1 Hz, 0.7H),

3.03e2.97 (m, 6H), 1.33 (t, J ¼ 7.1 Hz, 2H), 1.15 (t, J ¼ 7.1 Hz, 1H); MS

(ESI^þ) m/z 289.1 (M þ H)^þ.

(300 MHz, CDCl₃)

d

8.37 (d, J ¼ 4.9 Hz, 1H), 7.50e7.39 (m, 1H), 7.23

To the solution of 4 N HCl in EtOAc (500 ml) was added ethyl 2-

cyano-5-(dimethylamino)-3-(2-ﬂuorophenyl)penta-2,4-dienoate

(m, 3H), 7.14 (d, J ¼ 4.9 Hz, 1H), 4.78 (brs, 1H), 4.31 (brs, 2H), 1.38 (s,

9H); LCMS (ESI^þ) m/z 337.1 (M þ H)^þ.

J. Zhu et al. / European Journal of Medicinal Chemistry 69 (2013) 55e68

67

4.1.47. tert-Butyl (4-(2-ﬂuorophenyl)-2-phenylpyridin-3-yl)

methylcarbamate (26)

Compound 26 (160 mg, 72%) was prepared as a white solid from

25 (200 mg, 0.59 mmol) following a procedure similar to that

described for the preparation of 6a. ¹H NMR (300 MHz, CDCl₃)

Luciferase activity in cell lysate was determined using the Steady-

Glo^ÒLuciferase Assay System (Promega, Madison, WI) according to

the manufacturer’s instructions.

4.3. OGTT and bile weight measurement in ICR mice

d

8.66 (d, J ¼ 4.9 Hz, 1H), 7.50 (m, 6H), 7.36e7.26 (m, 2H), 7.20 (m,

2H), 4.26 (m, 3H), 1.27 (s, 9H); LCMS (ESI^þ) m/z 379.2 (M þ H)^þ.

Male ICR mice were purchased from the Shanghai SLAC Labo-

ratory Animal Co. Ltd (Shanghai, China). The animals were main-

tained under a 12 h lightedark cycle with free access to water and

food. Animal experiments were approved by the Animal Care and

Use Committee, Shanghai Institute of Materia Medica, Chinese

Academy of Sciences.

4.1.48. N-((4-(2-Fluorophenyl)-2-phenylpyridin-3-yl)methyl)-3,5-

bis(triﬂuoromethyl)benzenesulfonamide (27)

Compound 26 (150 mg, 0.4 mmol) was dissolved in 10 ml 4 N HCl/

EtOAc and the mixture was stirred at room temperature for 2 h. The

suspensionwas ﬁltered to give (4-(2-ﬂuorophenyl)-2-phenylpyridin-

3-yl)methanamine hydrochloride (85 mg, 61%) as a white solid.

Compound 27 (120 mg, 89%) was prepared as a white solid from (4-

(2-ﬂuorophenyl)-2-phenylpyridin-3-yl)methanamine hydrochloride

(85 mg, 0.24 mmol) following a procedure similar to that described

50 mg/kg of 15a or vehicle (0.25% CMC) were administered

orally to overnight fasting ICR mice (n ¼ 7e8 mice/group) 30 min

prior to the oral glucose load (4 g/kg). Blood glucose levels were

measured via blood drops obtained by clipping the tail of the mice

using an ACCU-CHEK Advantage II Glucose Monitor (Roche, Indi-

anapolis, IN) before compound dosing and 0, 15, 30, 60 and 120 min

after the glucose load. The area under the concentrationetime

curve from 0 to 120 min (AUC_{0e120min, Glu}) of blood glucose after the

glucose load was calculated by the trapezoidal rule (using the

0 time glucose level for each animal as that animal’s baseline). After

the OGTTexperiment, the fasting mice were re-fed for 2 h. Then, the

gallbladders were ligated and removed. The size of the gallbladders

was measured using vernier caliper. The weight of the bile was

calculated by subtracting net weight of gallbladder from gross

weight of gallbladder with bile.

for the preparation of 16a. ¹H NMR (400 MHz, CDCl₃)

d 8.64 (d,

J ¼ 4.9 Hz, 1H), 7.99 (s, 1H), 7.85 (s, 2H), 7.46e7.37 (m, 6H), 7.26e7.22

(m, 2H), 7.16 (d, J ¼ 4.9 Hz, 1H), 7.14e7.07 (m, 1H), 4.34 (t, J ¼ 5.8 Hz,

1H), 4.25 (d, J ¼ 6.0 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃)

d 159.9, 158.7

(d, J ¼ 242.1 Hz), 149.0, 145.4, 142.0, 139.1, 132.6 (q, J ¼ 34.2 Hz, 2C),

131.2 (d, J ¼ 8.1 Hz), 130.6 (d, J ¼ 2.4 Hz), 129.1, 128.8 (2C), 128.4 (2C),

127.1, 127.0 (2C),126.2, 125.2 (d, J ¼ 16.0 Hz), 124.8 (d, J ¼ 8.4 Hz),124.1,

122.3 (q, J ¼ 271.8 Hz, 2C), 115.9 (d, J ¼ 21.6 Hz), 42.3; HRMS (ESI^þ)

calcd for C₂₆H₁₇F₇N₂O₂SNa (M þ Na)^þ577.0797, found 577.0801.

4.1.49. tert-Butyl (2-ethoxy-4-(2-ﬂuorophenyl)pyridin-3-yl)

methylcarbamate (28)

All data were expressed as the mean ꢃ S.E.M. The statistical

analysis between two groups was performed using unpaired Stu-

dent’s t test. P < 0.05 was considered to be statistically signiﬁcant.

To the solution of 25 (300 mg, 0.89 mmol) in ethanol was added

NaOEt (prepared from Na (200 mg, 8.9 mmol) and EtOH). The mixture

was reﬂuxed overnight and then cooled to room temperature. The

solvent was concentrated and the residue was diluted with water and

then extracted with EtOAc. The combined organic phases were

washed with brine, dried over MgSO₄and concentrated. The residue

was puriﬁed by column chromatography to give the crude product 28

(125 mg, 40%) as a yellow oil. LCMS (ESI^þ) m/z 347.2 (M þ H)^þ.

Acknowledgments

This work was ﬁnancially supported by grant from “National

Science and Technology Major Project-Key New Drug Creation

and Manufacturing Program, China” (2012ZX09103101-049) and

National Nature Science Foundation of China (Grant 81202571). P.G.

and G.C. thank National Science and Technology Major Project

(2012ZX09301001-006).

4.1.50. N-((2-Ethoxy-4-(2-ﬂuorophenyl)pyridin-3-yl)methyl)-3,5-

bis(triﬂuoromethyl)benzenesulfonamide (29)

Compound 29 (45 mg, 24%) was prepared as a white solid from

28 (125 mg, 0.36 mmol) following a procedure similar to that

described for the preparation of 27. ¹H NMR (400 MHz, CDCl₃)

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ejmech.2013.07.050.

d

8.02 (m, 3H), 7.97 (s, 1H), 7.47e7.38 (m, 1H), 7.23 (td, J ¼ 7.6, 1.1 Hz,

1H), 7.17e7.07 (m, 2H), 6.73 (d, J ¼ 5.2 Hz, 1H), 5.47 (t, J ¼ 6.4 Hz,

1H), 4.34 (q, J ¼ 7.1 Hz, 2H), 4.13 (d, J ¼ 6.2 Hz, 2H), 1.38 (t, J ¼ 7.1 Hz,

References

3H); ¹³C NMR (100 MHz, CDCl₃)

d

161.5, 158.8 (d, J ¼ 196.3 Hz),

146.3, 145.4, 143.0, 132.6 (q, J ¼ 27.4 Hz, 2C), 131.0 (d, J ¼ 6.5 Hz),

130.6 (d, J ¼ 2.0 Hz), 127.0 (2C), 126.0,124.8 (d, J ¼ 12.5 Hz), 124.6 (d,

J ¼ 2.8 Hz), 122.4 (q, J ¼ 217.4 Hz, 2C), 118.7, 116.1, 115.9 (d,

J ¼ 17.4 Hz), 62.4, 40.5 (d, J ¼ 2.4 Hz), 14.6; HRMS (ESI^þ) calcd for

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Article Doi

Design, synthesis and biological evaluation of a novel class of potent TGR5 agonists based on a 4-phenyl pyridine scaffold

DOI: 10.1016/j.ejmech.2013.07.050

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