Discovery of N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin- 2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline (EW-7197): A highly potent, selective, and orally bioavailable inhibitor of TGF-β type I receptor kinase as cancer immunotherapeutic/antifibrotic agent

Article

pubs.acs.org/jmc

Discovery of N‑((4-([1,2,4]Triazolo[1,5‑a]pyridin-6-yl)-5-

(6-methylpyridin-2-yl)‑1H‑imidazol-2-yl)methyl)-2-ﬂuoroaniline

(EW-7197): A Highly Potent, Selective, and Orally Bioavailable

Inhibitor of TGF‑β Type I Receptor Kinase as Cancer

Immunotherapeutic/Antiﬁbrotic Agent

Cheng Hua Jin,^†Maddeboina Krishnaiah,^†Domalapally Sreenu,^†,#Vura B. Subrahmanyam,^†,∥

Kota S. Rao,^†,⊥Hwa Jeong Lee,^†So-Jung Park,^‡Hyun-Ju Park,^‡Kiho Lee,^§,∇Yhun Yhong Sheen,^†

and Dae-Kee Kim*^,†

^†Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-gu,

Seoul 120-750, Korea

^‡School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea

^§Bioevaluation Center, Korea Research Institute of Bioscience and Biotechnology, Cheongwon, Chungbuk 363-883, Korea

S

* Supporting Information

ABSTRACT: A series of 2-substituted-4-([1,2,4]triazolo[1,5-a]-

pyridin-6-yl)-5-(6-methylpyridin-2-yl)imidazoles was synthesized

and evaluated to optimize a prototype inhibitor of TGF-β type

I receptor kinase (ALK5), 6. Combination of replacement of a

quinoxalin-6-yl moiety of 6 with a [1,2,4]triazolo[1,5-a]pyridin-

6-yl moiety, insertion of a methyleneamino linker, and a o-F

substituent in the phenyl ring markedly increased ALK5

inhibitory activity, kinase selectivity, and oral bioavailability. The 12b (EW-7197) inhibited ALK5 with IC₅₀value of 0.013 μM in

a kinase assay and with IC₅₀values of 0.0165 and 0.0121 μM in HaCaT (3TP-luc) stable cells and 4T1 (3TP-luc) stable cells,

respectively, in a luciferase assay. Selectivity proﬁling of 12b using a panel of 320 protein kinases revealed that it is a highly selective

ALK5/ALK4 inhibitor. Pharmacokinetic study with 12b·HCl in rats showed an oral bioavailability of 51% with high systemic exposure

(AUC) of 1426 ng × h/mL and maximum plasma concentration (C_max) of 1620 ng/mL. Rational optimization of 6 has led to the

identiﬁcation of a highly potent, selective, and orally bioavailable ALK5 inhibitor 12b.

diﬀerentiation, proliferation, apoptosis, migration, and extrac-

ellular matrix production.¹⁻³

INTRODUCTION

■

Transforming growth factor-β (TGF-β) belongs to the TGF-β

superfamily, which consists of TGF-β1, TGF-β2, TGF-β3,

activins, inhibins, and bone morphogenetic proteins. TGF-β is

involved in many cellular processes, including cell proliferation,

cell migration, invasion, epithelial−mesenchymal transition

(EMT), extracellular matrix production, and immune suppres-

sion. TGF-β and its receptors are often chronically overex-

pressed in various human diseases, including cancer, tissue

ﬁbrosis, inﬂammation, and autoimmunity. Therefore, blockade

of TGF-β signaling pathway is an attractive target for drug

development. TGF-β signals via two related transmembrane

type I and type II serine/threonine kinase receptors. Following

TGF-β binding to the constitutively active type II receptor,

the type I receptor (activin receptor-like kinase 5 (ALK5)) is

phosphorylated and creates a binding site for Smad2/Smad3

proteins, which are further phosphorylated. Phosphorylated

Smad2/Smad3 proteins form a heteromeric complex with

Smad4, which translocates into the nucleus, assembles with

speciﬁc DNA-binding cofactors and comodulators, and binds to

the promoters of TGF-β target genes involved in cell

Small molecule ATP-competitive ALK5 inhibitors such as 1

(SB-505124),⁴2 (SB-525334),⁵3 (GW6604),⁶4 (SD-208),^7,8

and 5 (LY-2157299)⁹(Figure 1) speciﬁcally inhibited the Smad

pathway by occupying the ATP binding site of the ALK5 kinase

domain, which is essential for the phosphorylation of its

substrates, Smad2/Smad3 proteins. The ALK5 inhibitors 4 and

5 showed promise in blocking TGF-β-mediated tumor

progression, metastasis, and suppression of antitumor immun-

ity, and 2, 3, and 4 eﬀectively retarded progressive tissue

ﬁbrosis in animal models. Among them, 5 has progressed to

phase II trials for glioma, glioblastoma, hepatocellular

carcinoma, and pancreatic cancer.³We have previously reported

an imidazole-based ALK5 inhibitor, IN-1130 (6) (Figure 1).¹⁰

The inhibitor 6 eﬀectively prevented tumor progression and

enhanced immune response in mice bearing prostate cancer.¹¹

It showed its remarkable activity as a suppressor of ﬁbrogenic

Received: January 22, 2014

Published: May 2, 2014

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Figure 1. ALK5 inhibitors under development.

process in models of unilateral ureteral obstruction (UUO) in

adult mice¹²and rats,¹³ameliorated experimental autoimmune

encephalomyelitis in mice,¹⁴and signiﬁcantly reduced tunical

ﬁbrosis and corrected penile curvature in rats.¹⁵Although 6

demonstrated its pronounced eﬃcacy in various animal models

of cancer and ﬁbrosis, there were two drawbacks to develop it

as a clinical candidate: (1) pharmacokinetic studies with 6

showed good oral bioavailability in dog (85%) and monkey

(34%) but much lower in mouse (9%) and rat (11%), and (2)

the kinase selectivity index of 6 against p38α having one of the

most homologous kinase domains to that of ALK5 is <35.

Therefore, in this report, we attempted further structural

optimization of 6 to get a clinical candidate possessing favorable

pharmacologic, pharmacokinetic, and toxicologic proﬁles.

Our strategy of structural optimization of 6 consists of replace-

ment of a quinoxalin-6-yl moiety of 6 with a [1,2,4]triazolo[1,5-a]-

pyridin-6-yl moiety,¹⁶insertion of a suitable linker with a

heteroatom between a central imidazole ring and a phenyl ring,

and reﬁnement of phenyl ring substituent with various functional

groups.

inhibitory activity.¹⁹To further optimize the linker, we

compared the potential of methyleneoxy or methylenethio

group with that of methyleneamino group. It was one of our

concerns whether an incorporated heteroatom such as O, S, or

N into those three linkers might form another binding

interaction with ALK5.

RESULTS AND DISCUSSION

■

As our starting point, we designed three target molecules,

6-(5-(6-methylpyridin-2-yl)-2-(phenoxymethyl)-1H-imidazol-4-yl)-

[1,2,4]triazolo[1,5-a]pyridine (9a), 6-(5-(6-methylpyridin-2-yl)-2-

((phenylthio)methyl)-1H-imidazol-4-yl)-[1,2,4]triazolo[1,5-a]-

pyridine (9b), and N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-

(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)aniline (12a), to

examine the potential of the three diﬀerent linker moieties,

methyleneoxy, methylenethio, and methyleneamino in ALK5

inhibition and kinase selectivity against p38α.

The 9a and 9b were prepared as shown in Scheme 1. The 1-

([1,2,4]triazolo[1,5-a]pyridin-6-yl)-2-(6-methylpyridin-2-yl)-

a

In pharmacokinetic studies with orally administered 6 in

rodents, 3-((4-(2-hydroxyquinoxalin-6-yl)-5-(6-methylpyridin-

2-yl)-1H-imidazol-2-yl)methyl)benzamide or 3-((4-(3-hydrox-

yquinoxalin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)-

methyl)benzamide (7) was isolated as a major metabolite in the

systemic circulation of rat and mouse, showing that extensive

oxidation occurred in the quinoxaline ring (Figure 2).^17,18

Scheme 1

a

Reagents and conditions: (a) 2-phenoxyacetaldehyde (for 9a) or 2-

(phenylthio)acetaldehyde (for 9b), NH₄OAc, t-BuOMe/MeOH, rt,

70−180 min.

ethane-1,2-dione (8)²²was condensed with either 2-phenoxy-

acetaldehyde²³or 2-(phenylthio)acetaldehyde²⁴and NH₄OAc

in a mixture of t-BuOMe and MeOH at room temperature to

produce the 2-(phenoxymethyl)imidazole 9a or the 2-((phenylthio)-

methyl)imidazole 9b in 56% or 38% yield, respectively.

The 12a was prepared as shown in Scheme 2. Condensation

of the diketone 8 with 2,2-dimethoxyacetaldehyde and

NH₄OAc in a mixture of t-BuOMe and MeOH aﬀorded the

2-(dimethoxymethyl)imidazole 10 in 83% yield. The acetal

protecting group of the 10 was removed in 1 N HCl solution to

give the imidazole-2-carbaldehyde 11 in 96% yield. Coupling of

the 11 with aniline in the presence of AcOH in 1,2-dichloro-

ethane followed by reduction of the resulting imine with

NaBH₄gave the 2-((phenylamino)methyl)imidazole 12a in

87% yield.

Figure 2. Structure of a major metabolite of 6 in rat and mouse.

On the basis of this ﬁnding, we envisioned that replacement

of a quinoxalin-6-yl moiety of 6 with a [1,2,4]triazolo[1,5-a]-

pyridin-6-yl moiety could increase its oral bioavailability

because metabolic oxidation of 2-position of the latter moiety

is assumed to be diﬃcult due to the presence of two adjacent

nitrogen atoms.

We have also reported that insertion of a small linker such as

methylene, ethylene, n-propylene, methyleneamino, amino-

methylene, thioamido, methyleneamido, or methylenethioami-

do between two pharmacophores, the central ﬁve-membered

heterocyclic ring and a phenyl ring signiﬁcantly increased ALK5

inhibitory activity and selectivity.^10,19−21Among them, the

methyleneamino linker was the most beneﬁcial in ALK5

The ALK5 inhibitory activity of 9a possessing a methyl-

eneoxy linker (IC₅₀= 0.013 μM) was comparable to that of

12a possessing a methyleneamino linker (IC₅₀= 0.013 μM),

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a

Scheme 2

a

Reagents and conditions: (a) 60% 2,2-dimethoxyacetaldehyde in H₂O, NH₄OAc, t-BuOMe/MeOH, rt, 4 h; (b) 1 N HCl, 70 °C, 4 h; (c) AcOH,

1,2-dichloroethane, reﬂux (for 12a−h, 12j−l, 12n−an, 12ap, 12ar−az, and 12bb−bh), rt (for 12i, 12ao, and 12bi (without AcOH)), or 120 °C (for

12ba, in sealed tube), then (AcO)₃BHNa (for 12c, 12e−h, 12j, 12k, 12q, 12s−x, 12ab−af, 12ah, 12aj−an, 12ap, 12ar, 12as, 12au, 12ax, 12az,

12bd, 12be, and 12bg−bi) or NaBH₄(12a, 12b, 12d, 12i, 12m, 12n−p, 12r, 12y, 12z, 12aa, 12ag, 12ai, 12ao, 12at, 12av, 12aw, 12ay, 12ba−bc,

and 12bf), THF, MeOH, rt; (d) 34.5% H₂O₂, 1 N NaOH, EtOH, rt, 2−3 h (for 12m and 12aq).

whereas that of 9b possessing a methylenethio linker (IC₅₀

=

our attention to the reﬁnement of phenyl ring substituent to

examine whether the substituent could increase the ALK5

inhibitory activity and oral bioavailability of 12a. Thus, a series

of the 60 2-((phenylamino)methyl)imidazoles possessing

various substituents such as F, Cl, Br, Me, Et, i-Pr, OMe,

OCF_3,SMe, CHCH₂, CN, CONH₂, CH₂CN, COMe,

CO₂Me, NHSO₂Me, and NHCOMe in the phenyl ring,

12b−bi, were prepared (Scheme 2). Most of the requisite

anilines are commercially available, and the commercially

unavailable anilines such as 2-(3-aminophenyl)acetonitrile,²⁶

N-(2-aminophenyl)methanesulfonamide,²⁷N-(4-aminophenyl)-

methanesulfonamide,²⁸and 3-aminophthalonitrile²⁹were pre-

pared according to the known procedures in the literatures.

Coupling of 11 with appropriately substituted anilines in the

presence of AcOH in 1,2-dichloroethane followed by reduction

of the resulting imines with either (AcO)₃BHNa or NaBH₄in a

mixture of THF and MeOH gave the target compounds 12b−l,

12n−ap, and 12ar−bi in various yields (14−92%). Trans-

formation of the carbonitrile group of 12l and 12ap to the

corresponding carboxamide group was accomplished by

treatment with 34.5% H₂O₂and 1 N NaOH in EtOH at

room temperature to provide the 12m and 12aq in 84% and

63% yields, respectively.

0.021 μM) was somewhat lower than that of 12a in a kinase

assay (Table 1). As we hoped, introduction of a [1,2,4]triazolo-

[1,5-a]pyridin-6-yl moiety signiﬁcantly inﬂuenced selectivity

against p38α. Regardless of the linker moiety, all the three

compounds showed high selectivity against p38α, thus

exhibiting selectivity indices with >176. Because 9a, 9b, and

12a showed the similar level of potency in ALK5 inhibition in a

kinase assay, a cell-based luciferase assay was performed using

HaCaT cells permanently transfected with p3TP-luciferase

reporter construct²⁵at a lower concentration, 0.03 μM, to

discriminate their ALK5 inhibitory activity (Table 2). Contrary

to the kinase assay, 9a (11% inhibition) and 9b (8% inhibition)

were much less inhibitory than 12a (52% inhibition) in a cell-

based luciferase assay, suggesting that a methyleneoxy or methyl-

enethio linker contributes to the lower cellular permeability

compared to a methyleneamino linker.

We next determined the solubility of 9a, 9b, and 12a in pH

1.2 and 6.8 aqueous buﬀers (Table 2). Indeed, although 9a, 9b,

and 12a were almost insoluble in pH 6.8 aqueous buﬀer

(<0.001 mg), 12a (38.5 mg/mL) was 4.0- and 2.2-fold more

soluble in pH 1.2 aqueous buﬀer than 9a (9.6 mg/mL) and 9b

(17.6 mg/mL), respectively.

Because a methyleneamino linker was proved to be the most

beneﬁcial to the ALK5 inhibition in both assays, we next turned

The results indicated that introduction of a halogen atom

such as F, Cl, or Br at ortho-position in the phenyl ring had

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Table 1. ALK5 and p38α Inhibitory Activity of 2-(Phenoxymethyl)imidazole 9a, 2-((Phenylthio)methyl)imidazole 9b, and

2-((Phenylamino)methyl)imidazoles 12a−bi in Kinase Assay

IC₅₀(μM)

selectivity

a

b

c

compd

X

R

ALK5

p38α

index

12ab

12ac

12ad

12ae

12af

12ag

12ah

12ai

12aj

12ak

12al

12am

12an

12ao

12ap

12aq

12ar

12as

12at

12au

12av

12aw

12ax

12ay

12az

12ba

12bb

12bc

12bd

12be

12bf

12bg

12bh

12bi

1

NH

3-CH₂CN

3-COMe

3-CO₂Me

3-NHSO₂Me

3-NHCOMe

4-F

0.011

0.013

0.017

0.015

0.017

0.011

0.017

0.016

0.046

0.048

0.060

0.040

0.030

0.032

0.055

0.025

0.059

0.078

0.047

0.039

0.009

0.012

0.018

0.058

0.021

0.010

0.008

0.017

0.044

0.006

0.015

0.014

0.054

0.017

1.41

1.28

128

98

1.39

82

2.27

151

3.98

265

IC₅₀(μM)

3.93

231

selectivity

a

b

c

4-Cl

9.30

845

compd

X

R

ALK5

p38α

index

4-Br

>10.00

7.81

>588

>625

>217

>208

>166

>250

>333

244

9a

O

H

0.013

0.021

0.013

0.007

0.009

0.007

0.014

0.011

0.014

0.012

0.010

0.014

0.020

0.030

0.015

0.040

0.009

0.006

0.005

0.009

0.008

0.016

0.014

0.023

0.007

0.012

0.005

0.009

4.89

6.24

2.29

1.72

>10.00

1.98

1.94

1.53

1.10

3.68

2.56

1.30

2.05

3.64

3.94

1.63

2.83

0.82

0.73

0.87

1.31

1.18

1.21

2.43

1.86

0.70

1.46

1.50

1.54

376

297

176

246

>1111

283

139

79

4-Me

9b

S

H

4-i-Pr

12a

12b

12c

12d

12e

12f

NH

H

4-OMe

2-F

2-Cl

2-Br

2-Me

2-Et

2-i-Pr

4-OCF₃

4-SMe

4-CHCH₂

4-CN

4-CONH₂

4-CH₂CN

4-COMe

4-CO₂Me

4-NHSO₂Me

4-NHCOMe

2,3-F₂

>10.00

2.54

>181

>400

>169

>128

>212

>256

282

12g

12h

12i

2-OMe

2-OCF₃

2-SMe

263

213

130

146

182

131

109

71

12j

12k

12l

2-CHCH₂

2-CN

12m

12n

12o

12p

12q

12r

12s

12t

12u

12v

12w

12x

12y

12z

12aa

2-CONH₂

2-NHSO₂Me

2-NHCOMe

3-F

2,3-Cl₂

8.64

720

2,3-Me₂

2,3-(OMe)₂

2,3-(CN)₂

3,4-F₂

3.49

194

>10.00

1.25

>172

>476

125

91

3-Cl

122

174

146

148

76

3-Br

3,4-Cl₂

3.89

486

3-Me

3,4-Me₂

3,4-(OMe)₂

3,5-F₂

>10.00

0.50

>588

>227

83

3-Et

3-i-Pr

3-OMe

3-OCF₃

3-SMe

174

81

3,5-Cl₂

1.07

71

3,5-Me₂

3,5-(OMe)₂

3.00

214

100

122

300

171

2.85

204

3-CHCH₂

0.59

11

3-CN

6

0.48

28

3-CONH₂

a

ALK5 was expressed in Sf9 insect cells as human recombinant GST-fusion protein by means of the vaculovirus expression system. A proprietary

radioisotopic protein kinase assay (³³PanQinase activity assay) was performed at ProQinase GmbH (Freiburg, Germany) using casein as a

b

substrate. p38α MAP kinase was expressed as untagged human recombinant protein in E. coli. The enzyme was puriﬁed by Ni-NTH-agarose

(Qiagen). A proprietary radioisotopic protein kinase assay (³³PanQinase activity assay) was performed at ProQinase GmbH (Freiburg, Germany)

c

using ATF2 as a substrate. IC₅₀of p38α/IC₅₀of ALK5.

impact on ALK5 inhibitory activity, thus, 12b (R = 2-F, IC₅₀

=

be not accommodated favorably into the ATP binding pocket

of ALK5. In the 13 disubstituted compounds, di-F substituted

compounds 12aw (R = 2,3-F₂, IC₅₀= 0.009 μM), 12bb

0.007 μM), 12c (R = 2-Cl, IC₅₀= 0.009 μM), and 12d (R = 2-Br,

IC₅₀= 0.007 μM) displayed higher ALK5 inhibitory activity

than the unsubstituted 12a (IC₅₀= 0.013 μM) in a kinase assay

(Table 1). At meta-position, halogen atoms, small alkyl groups

such as Me and Et, and small functional groups, SMe and CN,

were beneﬁcial to the ALK5 inhibitory activity. Hence, 12p

(R = 3-F, IC₅₀= 0.009 μM), 12q (R = 3-Cl, IC₅₀= 0.006 μM),

12r (R = 3-Br, IC₅₀= 0.005 μM), 12s (R = 3-Me, IC₅₀= 0.009

(R = 3,4-F₂, IC₅₀= 0.010 μM), and 12bf (R = 3,5-F₂, IC₅₀

=

0.006 μM) and di-Cl substituted compounds 12ax (R = 2,3-Cl₂,

IC₅₀= 0.012 μM) and 12bc (R = 3,4-Cl₂, IC₅₀= 0.008 μM)

were more inhibitory than 12a.

Considering the IC₅₀of ALK5 inhibition and selectivity index

against p38α, the 12b−d, 12p−s, 12x, 12z, 12aa, 12ai, 12aj,

12aw, and 12bf were chosen and their ALK5 inhibitory activity

was compared at a concentration of 0.03 μM in a luciferase

assay (Table 3).

μM), 12t (R = 3-Et, IC₅₀= 0.008 μM), 12x (R = 3-SMe, IC₅₀

=

0.007 μM), and 12z (R = 3-CN, IC₅₀= 0.005 μM) were

more inhibitory than 12a. In contrast with the ortho- and

meta-substituted compounds, 15 out of 16 para-substituted

compounds displayed lower ALK5 inhibitory activity than 12a,

suggesting that a para-substituent in the phenyl ring seemed to

All the tested compounds except 12aa (2% inhibition)

showed higher inhibitory activity (37−71% inhibition) than the

competitor 1 (26% inhibition) in this luciferase assay. In the

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less soluble in pH 6.8 aqueous buﬀer (<0.001−0.042 mg/mL)

(Table 3). Nevertheless, it should be pointed out that

introduction of a F atom at ortho- or meta-position in the

phenyl ring fairly increased solubility in pH 6.8 aqueous buﬀer,

thus 12b (0.042 mg/mL) and 12p (0.029 mg/mL) were at

least 42- and 29-fold more soluble than the other compounds

(<0.001 mg/mL). Interestingly, the di-F substituted 12aw and

12bf (<0.001 mg/mL) were less soluble than 12b and 12p.

At this stage, we speculated that introduction of an additional

tert-amino group such as dimethylamino, (dimethylamino)-

methyl, pyrrolidin-1-ylmethyl, or morpholinomethyl in the

phenyl ring might increase the compound’s oral bioavailability

by either increasing its aqueous solubility or allowing it to form

a water-soluble acid salt with an acid. Thus, for this purpose,

12b was selected as one of candidates in the 3 ortho-substituted

compounds because it showed slightly higher ALK5 inhibitory

activity in luciferase assay and much higher solubility in pH 6.8

aqueous buﬀer than the others. The 12z was chosen as another

candidate due to its the highest ALK5 inhibitory activity

(IC₅₀= 0.005 μM) in a kinase assay and selectivity index against

p38α (300) among the seven meta-substituted compounds.

The di-F substituted 12aw and 12bf were not considered as a

candidate because they did not show any advantages over 12b.

In the cellular assays, 12a, 12b, and 12z eﬃciently inhibited

TGF-β1-induced Smad signaling, cell migration, invasion, and

epithelial−mesenchymal transition (EMT) in mammary

epithelial cells, MCF10A, NMuMG, and 4T1 cells.³⁰⁻³²

On the basis of our ﬁnding that a para-substituent in

the phenyl ring mostly decreased ALK5 inhibitory activity, a

tert-amino group was preferentially introduced at the ortho- or

meta-position rather than the para-position.

Among the required anilines for the synthesis of the target

compounds 14a−p, the known 4-ﬂuoro-N¹,N¹-dimethylbenzene-

1,3-diamine (13a)³³was prepared according to the reported

procedure, and the novel ﬂuoroanilines or cyanoanilines possessing

a (dimethylamino)methyl, pyrrolidin-1-ylmethyl, or morpholino-

methyl group, 13b−p, were prepared by new synthetic routes

developed by us (Supporting Information).

With these anilines in hand, another series of 2-substituted-

4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

imidazoles 14a−p possessing a tert-amino group in the phenyl

ring were prepared as shown in Scheme 3. Coupling of 11 with

13a−p in the presence of AcOH in 1,2-dichloroethane followed

by reduction of the resulting imines with either (AcO)₃BHNa

or NaBH₄in a mixture of MeOH and THF gave 14a−p in

42−79% yields.

Table 2. ALK5 Inhibitory Activity in Luciferase Assay and

Solubility of 9a, 9b, and 12a

solubility in aqueous

buﬀer (mg/mL)

a,b

luciferase activity (% control)

c

d

e

compd

p3TP-luc

pH 1.2

pH 6.8

mock

TGF-β

9a

4

100

89

9.6

17.6

38.5

<0.001

9b

92

<0.001

12a

1

48

78

a

Activity is given as the mean of three independent experiments run in

triplicate relative to control incubations with DMSO vehicle.

b

Luciferase activity was determined at a concentration of 0.03 μM

c

d

of inhibitor. HaCaT (3TP-luc) stable cells were used. The buﬀer

solution was prepared by mixing of 50 mL of 0.2 M KCl and 85.0 mL

e

of 0.2 M HCl. The buﬀer solution was prepared by mixing of 100 mL

of 0.1 M KH₂PO₄and 44.8 mL of 0.1 M NaOH.

Table 3. ALK5 Inhibitory Activity of 12b−d, 12p−s, 12x,

12z, 12aa, 12ai, 12aj, 12aw, and 12bf in Luciferase Assay and

Solubility of 12b−d, 12p−s, 12x, 12z, 12ai, 12aj, 12aw, and

12bf

solubility in aqueous

buﬀer (mg/mL)

a,b

luciferase activity (% control)

c

d

e

compd

p3TP-luc

pH 1.2

pH 6.8

mock

TGF-β

12b

12c

4

100

41

54

47

38

34

29

46

40

48

98

52

63

58

36

74

31.4

38.0

36.9

44.6

21.0

16.8

54.1

33.2

29.5

0.042

<0.001

0.029

12d

12p

12q

12r

<0.001

12s

12x

12z

12aa

12ai

12aj

12aw

12bf

1

12.6

25.6

18.6

9.7

<0.001

a

Activity is given as the mean of three independent experiments run in

triplicate relative to control incubations with DMSO vehicle.

b

Luciferase activity was determined at a concentration of 0.03 μM

The ALK5 and p38α inhibitory activity of 14a−p in a kinase

assay is shown in Table 4. Introduction of a dimethylamino group

in 12b (IC₅₀= 0.0008 μM) led to 3.6-fold decrease in potency

(14a, IC₅₀= 0.0029 μM), and the bulkier analogues 14b−e were

8.5−27.6-fold less potent than 12b. Similarly, although a

dimethylamino analogue of 12z, 14m (IC₅₀= 0.0013 μM)

c

d

of inhibitor. HaCaT (3TP-luc) stable cells were used. The buﬀer

solution was prepared by mixing of 50 mL of 0.2 M KCl and 85.0 mL

e

of 0.2 M HCl. The buﬀer solution was prepared by mixing of 100 mL

of 0.1 M KH₂PO₄and 44.8 mL of 0.1 M NaOH.

ortho-substituted compounds, the 2-F substituted 12b (59%

inhibition) was slightly more inhibitory than the 2-Cl

substituted 12c (46% inhibition) and 2-Br substituted 12d

(53% inhibition). Among the meta-substituted compounds,

3-Br substituted 12r (71% inhibition) and 3-Cl substituted 12q

(66% inhibition) were the most inhibitory, and the rest of

compounds showed the similar level of inhibition (52−62%

inhibition). The 3,5-F₂substituted 12bf (64% inhibition)

displayed much higher inhibition than the 2,3-F₂substituted

12aw (42% inhibition).

showed comparable level of inhibition to that of 12z (IC₅₀

=

0.0008 μM), the rest of analogues 14f−l and 14n−p were 3.0−

22.2-fold less potent than 12z. It is worth noting that introduction

of a tert-amino group in 12b and 12z signiﬁcantly aﬀected the

potency, but not the selectivity index, showing the variation less

than 2-fold compared with that of 12b and 12z.

To investigate whether these analogues could increase their

oral bioavailability, the most potent two analogues of 12z, 14m

and 14f (IC₅₀= 0.0024 μM), were chosen, and their

pharmacokinetic proﬁle was evaluated and compared with

that of 12z along with 12b. Pharmacokinetic studies with their

Like the unsubstituted 12a, all the compounds were suﬃciently

soluble in pH 1.2 aqueous buﬀer (9.7−54.1 mg/mL) but much

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a

12b, thus showing the residual activity of 4, 7, 31, and 52% of

control, respectively. At the lowest concentration of 0.01 μM,

the residual activity of ALK5 and ALK4 only were ≤50%.

Overall, it was found that 12b is a highly selective ALK5/ALK4

inhibitor in this selectivity proﬁling assay.

Scheme 3

Activin signaling and overexpression of activin are associated

with pathological disorders that involve extracellular matrix

accumulation and ﬁbrosis. Ichida and his colleagues reported

that activin A, the ligand of ALK4, was overexpressed in rat

cirrhotic and ﬁbrotic livers, and activin A and TGF-β could act

synergistically to induce extracellular matrix production.³⁴

Matsuse et al. observed the abundant expression of activin A

in bleomycin-treated murine lungs compared with in normal

lungs, suggesting that activin A may play a role in the

pathogenesis of pulmonary ﬁbrosis.³⁵It was also reported that

intimal activin A expression was markedly upregulated in

balloon-injured rat carotid arteries, implying that it may play an

important role in the response to vascular injury and accompanying

neointimal formation.³⁶Werner and his colleagues demonstrated a

strong induction of activin in granulation tissue after skin injury in

mice³⁷and a marked increase in the amount of granulation tissue

in the wounds of the transgenic mice³⁸overexpressing the activin

βA chain in the epidermis after skin injury. They also observed a

severe delay in wound healing and a signiﬁcantly reduced

granulation tissue formation in the transgenic mice overexpressing

the activin antagonist follistatin in the epidermis after skin injury,

eventually leading to smaller scar area than in control animals after

healing.³⁹These reports strongly support that dual inhibition of

ALK5/ALK4 with 12b may further potentiate its therapeutic

eﬀects on a number of ﬁbrotic diseases.

a

Reagents and conditions: (a) AcOH, 1,2-dichloroethane, reﬂux, then

(AcO)₃BHNa (for 14a−e and 14j−l) or NaBH₄(for 14f−i and

14m−p), THF, MeOH, rt.

We next compared the inhibitory pattern of 12b with those

of potential competitors 1, 5, and 6 using the most sensitive

kinases to 12b, ALK5, ALK4, RIPK2, and VEGF-R2. The IC₅₀

values and selectivity indices of the compounds for these

kinases are summarized in Table 8. 1, 5, and 6 strongly

inhibited ALK5, ALK4, and RIPK2. This proﬁle of potent and

nonselective inhibition is similar to that of 12b. A diﬀerential

inhibitory pattern was observed on VEGF-R2 that was strongly

inhibited by 12b only.

hydrochloride salts of the four compounds were carried out in

Sprague−Dawley rats at an intravenous dose of 2 mg/kg (as a

free base) and oral dose of 10 mg/kg (Table 5). As we

anticipated, a dimethylamino analogue 14m (27%) and a

(dimethylamino)methyl analogue 14f (24%) were 3.0-fold and

2.7-fold more bioavailable than 12z (9%), respectively.

Gratifyingly, after oral administration, 12b showed a superior

pharmacokinetic proﬁle over 12z, 14m, and 14f, with higher

systemic exposure (AUC) of 1426 ng × h/mL and maximum

plasma concentration (C_max) of 1620 ng/mL, which lead to an

oral bioavailability of 51%.

With 12b showing a favorable pharmacokinetic proﬁle, we

evaluated the selectivity of this compound on ALK family

kinases, ALK1, ALK2, ALK3, ALK4, ALK5, and ALK6. The

IC₅₀values of 12b along with staurosporine and dorsomor-

phine as positive controls are summarized in Table 6. The 12b

selectively inhibited ALK5 and ALK4 with IC₅₀values of 0.011

and 0.013 μM, respectively, but did not inhibit ALK1, ALK2,

ALK3, and ALK6 (IC₅₀>10 μM). Although ALK7 was not

included in this assay, we expect that 12b would inhibit this

family member, also, which is very highly related to ALK5 in its

kinase domain.⁴

After conﬁrming the selectivity proﬁle of 12b on ALK family

kinases, we expanded the selectivity proﬁling of this compound

using a panel of 320 protein kinases. The residual activity of

each kinase was measured at four diﬀerent concentrations of

0.01, 0.1, 1, and 10 μM. The 12b reduced the residual activity

of 45 protein kinases to ≤50% and 11 protein kinases to ≤10%

of control at a concentration of 10 μM (Table 7). The residual

activity of these 11 protein kinases were ≤50% of control at

1 μM. Of these, at a lower concentration of 0.1 μM, ALK5,

ALK4, RIPK2, and VEGF-R2 were signiﬁcantly inhibited by

In this kinase assay, 12b and 6 (IC₅₀= 0.013 μM) showed

the same level of potency on ALK5 and were 3.6-fold and

6.6-fold more potent than 1 (IC₅₀= 0.047 μM) and 5 (IC₅₀

0.086 μM), respectively.

=

The potency of the four compounds was also compared in a

luciferase assay using HaCaT (3TP-luc) and 4T1 (3TP-luc)

stable cells. Luciferase activity of HaCaT (3TP-luc) stable cells

and 4T1 (3TP-luc) stable cells was increased by 65-fold and

46-fold after treatment of TGF-β1 (2 ng/mL), respectively, and

12b displayed the most potent inhibition on the TGF-β1-induced

luciferase reporter activity with IC₅₀values of 0.0165 and

0.0121 μM in HaCaT (3TP-luc) stable cells and 4T1 (3TP-luc)

stable cells, respectively (Table 9).

To determine whether 12b is competitive with respect to

ATP against ALK5 and to obtain a K_ivalue of 12b, kinase ATP

competition study was performed (Reaction Biology Corpo-

ration, Malvern, PA). The progress curves for ALK5 reactions

were not linear with time in the absence and presence of 12b

for 120 min (Figure 3A). Because progress curves were

nonlinear, the slopes obtained from the linear portion of the

progress curves (15−60 min) were plotted against ATP

concentrations for Michaelis−Menten plot (Figure 3B) and

double-reciprocal plot (Figure 3C) for 12b at concentrations of

41.2, 123, and 370 nM against ALK5. As shown in Figure 3B

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Table 4. Inhibitory Activity of 2-Substituted-4-([1,2,4]triazolo[1,5-a]pyridine-6-yl)-5-(6-methylpyridin-2-yl)imidazoles 14a−p

on ALK5

a

ALK5 was expressed in Sf9 insect cells as human recombinant GST-fusion protein by means of the vaculovirus expression system. A proprietary

radioisotopic protein kinase assay (³³PanQinase activity assay) was performed at ProQinase GmbH (Freiburg, Germany) using casein as a substrate.

b

p38α MAP kinase was expressed as untagged human recombinant protein in E. coli. The enzyme was puriﬁed by Ni-NTH-agarose (Qiagen). A

Proprietary radioisotopic protein kinase assay (³³PanQinase activity assay) was performed at ProQinase GmbH (Freiburg, Germany) using ATF2 as

a substrate. IC₅₀of p38α/IC₅₀of ALK5.

c

a

Table 5. Rat PK Data of Selected Compounds

b,

c

b,c

intravenous dose, 2 mg/kg

oral dose, 10 mg/kg

d

e

f

h

i

j

compd

CL (L/h/kg)

V_ss(L/kg)

AUC (ng × h/mL)

C_max(ng/mL)

t_1/2(h)

AUC (ng × h/mL)

F (%)

12b·HCl

12z·HCl

14f·HCl

14m·HCl

3.6

5.5

7.0

3.5

1.3

2.4

2.3

1.0

558

368

293

1620

139

510

880

2.5

2.3

0.4

1426

168

51

9

353

24

g

k

g

l

562

nd

768

27

a

Pharmacokinetic parameters were estimated by a noncompartmental analysis of the mean plasma concentration (n = >3) versus time proﬁle after

oral and intravenous administration. The amount of dose is as a free base. Dosed as a solution in 0.9% saline solution. Clearance. Volume of

distribution at steady state. AUC (area under the curve) was extrapolated to inﬁnity. AUC_last(AUC from zero hour to last quantiﬁable time point).

Maximum concentration observed. Half-life. Oral bioavailability. Not determined. Terminal phase could not be deﬁned. As a result, t_1/2could not

b

c

d

e

f

g

h

i

j

k

l

be determined. Oral bioavailability was estimated using AUC_last

.

and Table 10, the apparent K_mvalues for ATP are increased

when 12b concentration is increased in Michaelis−Menten plot

and all lines are converged on the Y-axis in the double-

reciprocal plot (Figure 3C), suggesting that 12b is competitive

with respect to ATP against ALK5. Although the K_ivalue can

be obtained by traditional graphical method, global ﬁt is more

accurate, therefore, we performed global ﬁt using GraFit

software. The global ﬁt of Michaelis−Menten plot is shown

in Figure 3D, and its results are listed in Table 11. If the

compound is purely noncompetitive, the K_i(inhibitor aﬃnity

for enzyme) and K_i′ (inhibitor aﬃnity for enzyme/ATP complex)

values become the same. The very high K_i′ value (almost inﬁnity)

means the compound is competitive with respect with ATP.

Therefore, it should be concluded that 12b is purely competitive

with respect to ATP with the K_ivalue of 20.59 nM.

Melanoma is a prototypical immunogenic tumor expressing a

number of cell surface antigens and melanosomal antigens,

which are targeted by cytotoxic T-lymphocytes (CTLs).⁴⁰

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a

Table 6. ALK Family Kinase Proﬁling of 12b

b

IC₅₀(μM)

compd

ALK1

>10

0.850

ALK2

>10

10.005

ALK3

>10

4.288

ALK4

ALK5

ALK6

>10

c

12b

staurosporine

0.013

9.118

0.011

d

>18.500

d

dorsomorphine

0.467

a

b

A radioisotopic protein kinase assay (HotSpot assay) was performed at Reaction Biology Corporation (Malvern, PA). Mean of four independent

c

d

assays. Compound was tested in 10-dose IC₅₀mode with 3-fold serial dilution starting at 10 μM. Control compound was tested in 10-dose IC₅₀

mode with 3-fold serial dilution starting at 20 μM.

Table 7. Selectivity Proﬁling of 12b Using Selected Protein

Kinases

Table 9. ALK5 Inhibitory Activity of 12b, 1, 5, and 6 in

Luciferase Assay

a

b

a

residual activity (%)

IC₅₀for p3TP-luc (μM)

b

c

protein kinase

ALK5

0.01 μM

0.1 μM

1 μM

10 μM

compd

HaCaT

4T1

35

39

4

7

1

6

0

1

12b

1

0.0165

0.0438

0.0121

0.0766

ALK4

MAP4K4

MINK1

92

83

76

74

93

31

84

82

52

75

41

21

43

47

7

4

5

>0.1

0.0196

>0.1

0.0442

104

90

4

6

a

b

c

PDGFR-α V561D

PDGFR-α wt

RIPK2

−7

9

Mean of triplicates. HaCaT (3TP-luc) stable cells were used. 4T1

(3TP-luc) stable cells were used.

93

81

3

of perforin and granzyme B in the draining lymph node CD8⁺

T cells, thus enhancing CTL activity. The T cell-speciﬁc

deﬁciency of Smad4 suppressed the progression of primary

melanomas and lymph node metastasis by derepressing

eomesodermin, a T-box transcription factor regulating CTL

function. Notably, a daily dose of 2.5 mg/kg (QD) of 12b was

more eﬃcacious than a daily dose of 150 mg/kg (75 mg/kg,

BID) of 5 in suppressing the growth of primary tumors (tumor

weight, 76% inhibition for 12b, 61% inhibition for 5; tumor

volume, 97% inhibition for 12b, 81% inhibition for 5) and lymph

node metastasis (green ﬂuorescent protein (GFP)-positive B16

cells in the draining lymph nodes: 78% inhibition for 12b, 64%

inhibition for 5) compared with the vehicle control.⁴⁴

TGFβR2

87

47

38

11

28

8

VEGF-R1

VEGF-R2

VEGF-R3

100

87

6

1

98

3

a

A proprietary radioisotopic protein kinase assay (³³PanQinase activity

assay) was performed at ProQinase GmbH (Freiburg, Germany).

The residual activity was measured at four concentrations in singlicate

using 320 protein kinases.

b

CTLs lyse the target tumor cells with the cytolytic molecules

such as perforin, granzymes A and B, and FasL.⁴¹Intensive

eﬀorts have been made to develop immunotherapies and tumor

vaccines that activate antimelanoma T-cell responses.⁴²

However, resistance of melanoma cells to the antiproliferative

activity of TGF-β limits the success of immunotherapies and

tumor vaccines by rendering the host immune response

tolerant to tumor-associated antigens.⁴³TGF-β is one of the

most potent immunosuppressive cytokines, therefore, it was of

our particular interest whether 12b could enhance antimelano-

ma CTL responses in a melanoma-bearing animal model. We

have recently reported the therapeutic eﬃcacy and the cellular

and molecular mechanisms of 12b in B16 melanoma-bearing

mice.⁴⁴

Oral administration of 12b (2.5 mg/kg, daily) for 18 days not

only blocked Smad2/Smad3 phosphorylation but also induced

ubiquitin-mediated degradation of Smad4 mainly in CD8⁺T

cells in melanoma-bearing mice.⁴⁴Treatment with 12b

signiﬁcantly increased the proportions and numbers of CD8⁺

T cells in the draining lymph nodes and the protein expression

The antiﬁbrotic eﬀect of 12b was investigated in the CCl₄-

induced liver ﬁbrosis model which is the best characterized

toxin-based experimental model for the induction of ﬁbrosis.⁴⁵

After intraperitoneal injection of CCl₄(1 mL/kg) to mice twice

a week for 4 weeks, the protein levels of α-smooth muscle actin

and collagen type 1, common hallmarks of ﬁbrotic diseases, in

CCl₄-induced mice liver were elevated by 2.3-fold and 4.1-fold,

respectively, compared to those in control mice liver. Oral

administration of 12b (0.625, 1.25, 2.5, or 5 mg/kg, daily)

5 times a week for 4 weeks reduced signiﬁcantly the elevated

level of α-smooth muscle actin at doses of 1.25, 2.5, and

5 mg/kg and of collagen type 1 at all dose levels tested.⁴⁶

A

signiﬁcant increase (2.1-fold) in serum level of AST was observed

in CCl₄-induced mice compared to that in control mice. The 12b

decreased signiﬁcantly the elevated serum level of AST at all dose

levels, reﬂecting improvement of liver function.⁴⁶

a

Table 8. Selectivity Proﬁling of 12b, 1, 5, and 6 Using Four Protein Kinases

ALK5

selectivity index

ALK4

selectivity index

RIPK2

selectivity index

VEGF-R2

selectivity index

b

c

compd

IC₅₀(μM)

12b

1

0.013

0.047

0.086

0.013

1

0.017

0.070

0.199

0.023

1.3

1.5

2.3

1.8

0.054

0.685

0.044

0.111

4

0.097

>10

7

>213

28

15

5

0.5

2.440

0.442

6

9

34

a

b

A proprietary radioisotopic protein kinase assay (³³PanQinase activity assay) was performed at ProQinase GmbH (Freiburg, Germany). Mean of

c

duplicate. IC₅₀of each kinase/IC₅₀of ALK5.

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Figure 3. (A) Progress curves for ALK5. (B) Michaelis−Menten plot for ALK5/12b. (C) Lineweaver−Burk plot for ALK5/12b. (D) Global ﬁt of

Michaelis−Menten plot by GraFit software for ALK5/12b.

Table 10. V_maxand K_mValues on Diﬀerent Concentrations of

a

12b

concentration (nM)

b

0

41.2

123

370

V_max

K_m

0.2226

71.99

0.2643

0.5844

1.067 × 10¹¹

8.673 × 10¹⁴

341.3

1648

a

A radioisotopic protein kinase assay (HotSpot assay) was performed

b

at Reaction Biology Corporation (Malvern, PA). DMSO 0.1% as

vehicle control.

Table 11. Kinetic Parameters of 12b Obtained from Global

Fit of Michaelis−Menten Plot

kinetic parameter

value

0.2278

V_max(μM/min)

K_m(μM)

78.1764

K_i(nM)

20.5937

6.29600 × 10²³

K_i′ (nM)

Figure 4. X-ray crystallographic structure of 12b·MeOH.

The 12b was crystallized from MeOH, and a single crystal

was obtained as a 12b·MeOH solvate. A single-crystal X-ray

analysis was conducted on 12b·MeOH to determine its solid-

state conformation.⁴⁷The X-ray crystallographic structure and

crystal data of 12b·MeOH are shown in Figure 4 and Table 12,

respectively. Because the crystal structure of 12b bound to

ALK5 is not available to us, we carried out a docking study to

examine docking poses of 12b along with 12a, 9a, and 9b in the

active site of ALK5 using ﬂexible molecular docking program

Surﬂex-Dock. After docking, we compared the docking pose of

12b with those of 12a, 9a, and 9b. As shown in Figure 5, all

ligands docked into the acive site cavity of ALK5 well with

similar docking pose. The [1,2,4]triazolo[1,5-a]pyridin-6-yl

moiety occupies the pocket for adenine ring of ATP and

commonly forms a hydrogen bonding with NH of His283 in

the backbone of hinge region of ALK5. Similar to LY580276

having 6-methylpyridine in the X-ray structure (PDB: 1RW8),⁴⁸

the methyl group of 6-methylpyridine forms hydrophobic

interaction with the aromatic ring of Tyr249 and the nitrogen

atom forms a water-mediated hydrogen bond network with the

side chain of Tyr249 and Glu245 and the backbone of Asp351.

Additionally, the imidazole ring NH of our compounds is also

involved in this hydrogen bond network, which may contribute

to tighter ﬁt into the active site. The amino group linked to the

imidazole 2-position in 12b forms hydrogen bonds with both

backbone Lys337 and side chain Asn338, while that in 12a forms

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in 9a and the sulfur atom in 9b did not interact with ALK5. Our

docking results demonstrate that the most active compound 12b

forms the most favorable intermolecular interactions in the ATP-

binding pocket of ALK5.

IC₅₀values of 12b against the ﬁve most common drug

metabolizing CYP450 isoforms (CYP1A2, CYP2C9, CYP2C19,

CYP2D6, and CYP3A4) in human liver microsomes were deter-

mined by N-in-1 approach⁴⁹with speciﬁc CYP450 substrates.

All IC₅₀values of 12b against the ﬁve CYP450 isoforms were

>10 μM (Table 13), indicating that 12b has low potential for

CYP-mediated drug/drug interactions.

Table 12. Crystal Data of 12b·MeOH

empirical formula

formula weight

crystal system

space group

C₂₂H₁₈FN₇·CH₃OH

431.46

monoclinic

P2(1)/n

a, b, c (Å)

7.9179(2), 10.3592(3), 25.6193(6)

α, β, γ (deg)

volume (Å³)

temperature (K)

Z

90.00, 91.643(10), 90.00

2100.51(9)

100(1)

4

goodness-of-ﬁt on F²

1.069

ﬁnal R indices [5212 data; I > 2σ(I)] R₁= 0.0465, wR₂= 0.1266

R indices [all data] R₁= 0.0490, wR₂= 0.1288

Table 13. CYP450 Inhibition of 12b in Human Liver

Microsomes

a

CYP450

IC₅₀(μM)

% inhibition at 10 μM

1A2

2C9

2C19

2D6

3A4

>10

23.3 3.9

30.1 3.3

30.7 4.0

14.6 1.6

44.8 5.3

a

Mean SD (n = 3).

The safety pharmacology studies of 12b were conducted to

assess its eﬀect on the cardiovascular system, central nervous

system, and respiratory system.

Inhibition of hERG channels is associated with potential risk

of QT prolongation. The whole cell patch-clamp technique was

used to record inward rectifying potassium currents (I_Kr) from

single HEK293 cells stably expressing the potassium hERG

channel (MPI Research, Inc. Mattawan, MI).⁵⁰Superfusion of

12b at concentrations of 1, 10, 30, and 100 μM produced mean

concentration-dependent inhibition of 1.79%, 22.13%, 50.84%,

and 76.60%, respectively, with an IC₅₀value of 31.04 μM,

indicating low cardiac toxicity risk (Table 14).

Table 14. Eﬀect of 12b on Cloned hERG Channels

Expressed in Human Embryonic Kidney (HEK293) Cells

a

concentration

hERG current

inhibition (%)

IC₅₀

(μM)

compd

(μM)

b

c

vehicle

0

1

1.18 0.343

1.79 0.457

22.13 3.579

50.84 0.219

76.60 2.135

83.60 0.977

12b

31.04

12b

10

12b

30

12b

100

0.1

d

cisapride

a

Assay was performed at MPI Research, Inc. (Mattawan, MI).

b

c

Physiological salt solution (PSS) with 0.1% DMSO. Mean

SE

d

(n = 3). Positive control compound.

The potential cardiovascular eﬀect of 12b was evaluated in

conscious freely moving beagle dogs instrumented with

radiotelemetry transmitters measuring electrocardiogram

(ECG), heart rate, and blood pressure (MPI Research, Inc.

Mattawan, MI). Oral administration of 12b at dose levels of 10,

30, and 100 mg/kg was without eﬀect on clinical observations,

body weight, body temperature, ECG, heart rate, and blood

pressure, establishing a no-observed-eﬀect level (NOEL) of at

least 100 mg/kg.

Figure 5. Docking pose of candidate compounds in the active site of

ALK5 (PDB: 1RW8). (A) Docking pose of 12b (magenta capped sticks).

(B) 12a (cyan capped sticks). (C) 9a (yellow capped sticks). (D) 9b

(green capped sticks). Gray capped sticks represent key amino acid

residues within the binding site and ribbon is backbone of ALK5. Ball and

stick is water molecule. Red dashed lines are hydrogen bonding

interactions (<2.8 Å). (E) Docking pose of 12b (magenta carbon).

The active site is shown as MOLCAD lipophilic potential surface map.

Lipophilicity increases from blue (hydrophilic) to brown (lipophilic).

a hydrogen bond with side chain Asn338. The 2-ﬂuorophenyl

ring of 12b is stretched to the backside hydrophobic pocket

consisting of Ile339 and Leu340 (Figure 5E). The oxygen atom

The potential acute neurobehavioral toxicity of 12b and the

potential eﬀect of 12b on respiratory function were evaluated

in SD rats (MPI Research, Inc. Mattawan, MI). Acute oral

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administration of 12b at 1000 mg/kg produced mortality (3 out

of 10 animals) and was associated with various clinical signs and

eﬀects on measured functional observational battery (FOB)

and respiratory parameters. Speciﬁcally, these animals experi-

enced decreased activity, low carriage, partially or completely

closed eyelids, salivation, diﬃculty breathing, and lacrimation

between 1 and 6 h postdose. Various eﬀects observed on FOB

end points were consistent with the general toxicity and/or

morbidity observed in animals at the same dose level. There

were some slight decrease in respiratory rate and increase in

tidal volume and minute volume. These changes in respiration

are considered to be related to 12b but are not considered to be

adverse.

Doses of 10 and 100 mg/kg did not produce mortality and

were without eﬀects on clinical observations and respiratory

parameters. Potential FOB eﬀects at doses of 10 and 100 mg/kg

were limited to slight decrease in activity and arousal at 24 h

postdose, establishing a no-observed-adverse-eﬀect level

(NOAEL) of at least 100 mg/kg.

The genotoxic potential of 12b was assessed using a bacterial

reverse mutation assay, a mammalian chromosome aberration

assay, and an in vivo rat bone marrow erythrocyte micronucleus

test.

The mutagenic potential of 12b was evaluated by measuring

its ability to induce reverse mutations at selected loci of

Salmonella typhimurium strains TA98, TA100, TA1535, and

TA1537, and Escherichia coli strain WP2 uvrA in the presence

and absence of aroclor-induced rat liver S9 metabolic activation

system (BioReliance, Rockville, MD). No positive mutagenic

responses were observed with any of the strains in either the

presence or absence of S9 activation at dose levels of 50, 150,

500, 1500, and 5000 μg per plate.

To evaluate the clastogenic potential of 12b, a chromosome

aberration assay was performed using Chinese hamster ovary

(CHO) cells in both the absence and presence of aroclor-

induced rat liver S9 (BioReliance, Rockville, MD). No

signiﬁcant or dose-dependent increase in aberrant metaphases,

polyploid, or endoreduplicated cells were observed in treatment

groups with or without S9 at dose levels tested.

The clastogenic eﬀect and aneugenic eﬀect of 12b were

evaluated by measuring its ability to induce micronucleated

polychromatic erythrocytes in rat bone marrow (BioReliance,

Rockville, MD). A single oral administration of 12b at dose

levels of 500, 1000, and 2000 mg/kg did not induce a

signiﬁcant increase in the incidence of micronucleated

polychromatic erythrocytes in bone marrow of female SD

rats. The results of these three studies indicate that 12b is

negative for the induction of genotoxicity.

A 4-week toxicity study in rats with a 4-week recovery period

was conducted to evaluate the potential subchronic toxicity and

toxicokinetics after oral administration of 12b at dose levels of

10, 20, and 50 mg/kg/day once daily for 4 weeks and to

examine reversibility, progression, or delayed appearance of any

observed changes following a 4-week post dose observation

period (MPI Research, Inc. Mattawan, MI). The microscopic

ﬁndings related to 12b were present in the liver (panlobular

hypertrophy), kidney (tubular hypertrophy), spleen (extra-

medullary hematopoiesis), femur, and sternum (subphyseal

hyperostosis, growth plate thickening) in males and/or females

and in ovaries (ovarian atrophy) in females at terminal

necropsy. In general, females had higher incidence and/or

severity of microscopic ﬁndings at a given dose level. There

was a complete resolution of compound-related ﬁndings in all

tissues in males and in the liver, kidney, spleen, and ovaries in

females. There was a partial resolution of microscopic ﬁndings

in the femur and sternum in females. For males, there were no

adverse 12b-related eﬀects noted for any parameter evaluated.

In females at 50 mg/kg/day, ovarian atrophy observed

microscopically was considered an adverse 12b-related eﬀect.

As a result, the NOAEL following once daily oral admin-

istration of 12b for 4 weeks was 50 mg/kg/day for males and

20 mg/kg/day for females. Following 4 weeks of dosing,

systemic exposure (AUC_{0−6 h}) to 12b in males at 50 mg/kg/day

was 3500 ng × h/mL and C_maxwas 2240 ng/mL. Systemic

exposure in females at 20 mg/kg/day was 5350 ng × h/mL and

C_maxwas 3910 ng/mL.

Frazier and his colleagues observed the three major toxicities,

pulmonary hemorrhage, bone physeal abnormalities (hyper-

trophy of the physes or growth plates), and heart valve lesions,

during an ALK5 program at GSK.⁵¹Heier et al. reported that

small molecule ALK5 inhibitors induced histopathological heart

valve lesions and physeal dysplasia characterized by increased

growth plate thickness in the femoro-tibial joint in rats.⁵²In this

study, we also observed physeal dysplasia characterized by

subphyseal hyperostosis and growth plate thickening in femur,

a ﬁnding consistent with that observed by Frazier’s group⁵³and

Heier’s group with ALK5 inhibitors. Frazier’s group hypothe-

sized that physeal dysplasia observed in test animals might not

be problematic because adults, the target population for ALK5

inhibitors, have already closed growth plates.⁵¹

Heart valve lesions were observed at doses that also caused a

physeal dysplasia in rats.⁵²The hemorrhagic, degenerative, and

inﬂammatory lesions in heart valves can cause hemodynamic

overload to the ventricles, which eventually leads to myocardial

dysfunction and heart failure.⁵⁴However, clinical trials with 5

to date have not revealed medically signiﬁcant cardiotoxicities

or bone physeal abnormalities.⁵⁵Nevertheless, considering the

key role of heart valves in cardiac function, in this study, we

performed an extended evaluation of the heart for the control

and high dose groups at both terminal and recovery necropsies

to capture/represent as many heart valves as possible in the

plane of the section including the attachment of the aorta at the

heart base. It should be noted that there was no evidence of

inﬂammation or necrosis at the base of the aorta and/or within

the heart valves in either sex at 50 mg/kg/day at terminal and

recovery necropsies.

A 4-week toxicity study in dogs with a 2-week recovery

period was conducted to evaluate the potential subchronic

toxicity and toxicokinetics after oral administration of 12b at

dose levels of 3, 10, and 30 mg/kg/day once daily for 4 weeks

and to examine reversibility, progression, or delayed appearance

of any observed changes following a 2-week post dose

observation period (MPI Research, Inc. Mattawan, MI). The

only detailed clinical observations that could be related to 12b

were soft and watery feces at 30 mg/kg/day in both sexes.

There were no 12b-related changes in ECG, hematology, or

clinical chemistry and no 12b-related macroscopic and micro-

scopic ﬁndings at all dose levels. Consequently, the NOAEL was

considered to be 30 mg/kg/day in both sexes. AUC_{0−6 h}and

C_maxvalues on day 28 for 30 mg/kg/day, sexes combined, were

21500 ng × h/mL and 11400 ng/mL, respectively.

The development of potent, selective, and orally bioavailable

ALK5 inhibitors such as 12b may oﬀer many therapeutic

opportunities. A clinically useful antibody drug, ipilimumab exerts

its eﬀect on melanoma by binding to cytotoxic T-lympocyte

antigen 4 (CTLA-4), a negative regulator of T-cell activation,

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and blocking the interaction of CTLA-4 with ligand CD80/CD86,

thus enhancing T-cell mediated antitumor immune responses. In a

phase III clinical trial in 676 patients with previously treated,

unresectable or metastatic melanoma, median overall survival in

patients receiving ipilimumab plus a peptide vaccine, gp100, was

10.0 months, compared with 6.4 months in patients receiving

gp100 alone.⁵⁶It also increased the rates of survival at 12 months

(46% vs 25%) and 24 months (24% vs 14%) by a near doubling

compared with the peptide vaccine. Although ipilimumab achieved

a breakthrough in metastatic melanoma, long-time exposure of this

antibody drug frequently caused severe and fatal immune-

mediated adverse reactions such as enterocolitis, hepatitis,

dermatitis, neuropathies, and endocrinopathies by T-cell activation

and proliferation.⁵⁶

pharmacology studies of 12b in dogs and rats, it showed no

adverse eﬀect on the cardiovascular system, central nervous

system, and respiratory system up to an oral dose of 100 mg/kg.

The genotoxic potential of 12b was found to be negative in a

bacterial reverse mutation assay, a mammalian chromosome

aberration assay, and an in vivo rat bone marrow erythrocyte

micronucleus test. In a 4-week toxicity study in rats with a 4-week

recovery period, the NOAEL following once daily oral admin-

istration of 12b at dose levels of 10, 20, and 50 mg/kg/day was

50 mg/kg/day for males and 20 mg/kg/day for females. Unlike

the earlier ALK5 inhibitors developed by GSK⁵¹and

AstraZeneca,⁵²there was no evidence of 12b-related inﬂamma-

tion or necrosis at the base of the aorta and/or within the heart

valves in either sex at 50 mg/kg/day at terminal and recovery

necropsies. In a 4-week toxicity study in dogs with a 2-week

recovery period, the NOAEL following once daily oral

administration of 12b at dose levels of 3, 10, and 30 mg/kg/day

was 30 mg/kg/day in both sexes. Taken together, 12b holds great

promise as an anticancer/antiﬁbrotic agent.

Because 12b has a short half-life and a diﬀerent mechanism

of action to activate CTLs from that of ipilimumab, it might

have a high potential to be developed as another much safer

cancer immunotherapeutic agent.

The potential clinical applications of ALK5 inhibitors on

various ﬁbrotic diseases have recently been reviewed by Akhurst

and Hata.³They include radiation-induced ﬁbrosis, idiopathic

pulmonary ﬁbrosis, renal ﬁbrosis, cardiac ﬁbrosis, scleroderma,

restenosis following coronary artery bypass and angioplasty,

Marfan syndrome, and postoperative scarring in ocular conditions.

We have also demonstrated the therapeutic utility of ALK5

inhibitors with 6,¹²⁻¹⁵3-((4-(6-methylpyridin-2-yl)-5-(quinolin-

6-yl)-1H-imidazol-2-yl)methyl)benzamide (IN-1233),⁵⁷⁻⁶¹and

12b⁴⁶in animal models of renal ﬁbrosis, multiple sclerosis, penile

ﬁbrosis, airway ﬁbrosis, stent-induced tissue hyperplasia, pulmo-

nary arterial hypertension, and liver ﬁbrosis.

On the basis of the proposed therapeutic applications of

ALK5 inhibitors and favorable pharmacologic, pharmacokinetic,

and toxicologic proﬁles of 12b (EW-7197), a ﬁrst-in-human

phase I dose-escalation study of 12b is scheduled to start soon

in U.S. (IND 119528).

EXPERIMENTAL SECTION

■

General Methods. All commercial chemicals and solvents are of

reagent grade and were used without further puriﬁcation unless

otherwise stated. All reactions were monitored for completion by TLC

using Merck silica gel 60 F₂₅₄glass plates (20 cm × 20 cm). Spots were

detected by viewing under UV light (254 and 365 nm) and colorizing

with charring after dipping in either p-anislaldehyde solution with

acetic acid, or ninhydrin solution with EtOH, or KMnO₄solution with

water. MPLC was performed on Merck silica gel 60 (230−400 mesh)

with an YFLC-540 ceramic pump (Yamagen).

¹H NMR spectra were recorded on a Varian Unity-Inova 400 MHz

instrument. The ¹H NMR data are reported as peak multiplicities: s for

singlet, d for doublet, dd for double of doublets, t for triplet, q for

quartet, br s for broad singlet, br d for broad double, br t for broad

triplet, and m for multiplet. The chemical shifts are reported in parts

per million (ppm), and coupling constants are reported in hertz (Hz).

1

For H NMR spectra, CDCl₃, CD₃OD, or DMSO-d₆was used as

CONCLUSION

solvent, and it served as the internal standard at δ 7.26, 3.31, or 2.50.

¹³C NMR spectra were recorded on a Varian Unity-Inova 100 or

125 MHz instrument. For ¹³C NMR spectra, CDCl₃, CDCl₃/CD₃OD,

CD₃OD, or DMSO-d₆was used as solvent, and it served as the internal

standard at δ 77.23 (CDCl₃, CDCl₃/CD₃OD), 49.05 (CD₃OD), or

39.50 (DMSO-d₆). ¹⁹F NMR spectra were recorded on Varian Unity-

Inova 376 MHz instrument.

High resolution mass spectra electrospray ionization (HRMS-ESI)

was obtained on an Agilent Technologies 6220 TOF LC/MS

spectrometer. Infrared spectra were recorded on a FT-infrared

spectrometer (FTS-135, Bio-Rad) using KBr discs with absorption

in cm⁻¹. All melting points were taken in Pyrex capillaries using an

■

We have synthesized a series of 2-substituted-4-([1,2,4]triazolo-

[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)imidazoles to opti-

mize a prototype ALK5 inhibitor 6. The structure−activity

relationships in this series of compounds have been established

and discussed. Combination of replacement of a quinoxalin-6-yl

moiety of 6 with a [1,2,4]triazolo[1,5-a]pyridin-6-yl moiety,

insertion of a methyleneamino linker, and a o-F substituent in

the phenyl ring markedly increased ALK5 inhibitory activity,

kinase selectivity, and oral bioavailability. The 12b inhibited

ALK5 with IC₅₀value of 0.013 μM in a kinase assay and with

IC₅₀values of 0.0165 μM and 0.0121 μM in HaCaT (3TP-luc)

stable cells and 4T1 (3TP-luc) stable cells, respectively, in a

luciferase assay. Selectivity proﬁling of 12b using a panel of 320

protein kinases, including ALK family kinases ALK1, ALK2,

ALK3, ALK4, ALK5, and ALK6, revealed that it is a highly

selective ALK5/ALK4 inhibitor. Pharmacokinetic study with

12b·HCl in rats showed an oral bioavailability of 51% with high

systemic exposure (AUC) of 1426 ng × h/mL and maximum

plasma concentration (C_max) of 1620 ng/mL. Kinase ATP

competition study indicated that 12b is purely competitive with

respect to ATP with the K_ivalue of 20.59 nM. Docking pose of

12b generated by ﬂexible molecular docking program with

ALK5:12b complex shows it ﬁts well into the active site cavity

of ALK5 by forming several hydrogen bond interactions. The

12b inhibited the potassium hERG channel with IC₅₀value of

31.04 μM, indicating low cardiac toxicity risk. In the safety

electrothermal digital melting apparatus (B-540, Buchi) and are not

̈

corrected.

The purity of compounds was conﬁrmed by following method. The

HPLC analyses were accomplished with an Agilent 1100 series HPLC

system (Agilent Technologies, Waldbronn, Germany) consisting of a

solvent degasser (G1379A degasser), a quaternary pump (G1311A

liquid chromatography), and an auto sampler injector (G1329A).

Separations were carried out using a reversed-phase C₁₈analytical

column (Capcell-pak C₁₈MG 5 μm, 3.0 mm × 250 mm) (Shiseido,

Tokyo, Japan) with a solvent system consisting of an isocratic solvent.

The solvent contained 15−35% acetonitrile in distilled water

containing 0.1% triﬂuoroacetic acid and was delivered at 0.5 mL/min

over 25 min. The column was maintained at room temperature with a

column oven (G1316A). All compounds were dissolved in MeOH or

MeOH and CH₂Cl₂cosolvent and detected at 230 nm with a G1314A

variable wavelength detector (VWD). The HPLC system was controlled

by a ChemStation Software (Agilent). The purity of the each compound

was calculated from a surface integral of detected peaks and is described

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as percent (%). All compounds were found to be ≥95% pure. Retention

times are given in minutes (min).

through a glass ﬁltered funnel, and the ﬁltered cake was washed

with water (100 mL) and ether (200 mL). The solid was dried in a

vacuum oven over P₂O₅at 60 °C for 12 h to give the titled compound

as a light-yellow solid. Yield 96%; mp 193.8−196.2 °C. ¹H NMR

(400 MHz, DMSO-d₆) δ 10.03 (dd, 1 H, J = 1.6, 0.8 Hz), 9.56 (s,

1 H), 8.42 (s, 1 H), 8.27 (dd, 1 H, J = 9.2, 1.6 Hz), 7.82 (br d, 1 H, J =

0.8 Hz), 7.72 (dd, 1 H, J = 9.2, 0.8 Hz), 7.65 (t, 1 H, J = 7.8 Hz), 7.05

(d, 1 H, J = 7.6 Hz), 2.46 (s, 3 H). IR (KBr) 1691, 1438 cm⁻¹. HRMS-

ESI m/z [M + H]⁺calcd for C₁₆H₁₃N₆O 305.1145, found 305.1158.

General Procedure for the Preparation of the 2-Substituted-

4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

imidazoles 12a−l, 12n−ap, 12ar−bi, and 14a−p. To a stirred

solution of 11 (13.14 mmol) in 1,2-dichloroethane (240 mL) were

added appropriate substituted anilines (19.71 mmol) and AcOH

(13.14 mmol), and the mixture was heated to reﬂux for 2 h under

nitrogen atmosphere. The reaction mixture was cooled to 0 °C, and

MeOH (60 mL) and THF (20 mL) were added. To it, (AcO)₃BHNa

(26.20 mmol) or NaBH₄(52.56 mmol) was added portionwise, and

then the reaction mixture was allowed to warm to room temperature

and stirred for an additional 3 h. The pH of the reaction mixture was

adjusted to 7−8 at 0 °C with 1 N HCl, and then the organic layer was

separated. The aqueous layer was extracted with CH₂Cl₂(2 × 300 mL).

The combined organic layers was washed with brine (300 mL) and

dried over anhydrous Na₂SO₄, ﬁltered, and evaporated to dryness under

reduced pressure. The residue was puriﬁed by MPLC on silica gel to

aﬀord the titled compounds as a solid.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)aniline (12a). The residue was puriﬁed by

MPLC on silica gel using CH₂Cl₂/MeOH (24:1) as eluent to give the

titled compound as a yellow solid. Yield 87%; mp 162.1−164.3 °C. ¹H

NMR (400 MHz, CDCl₃) δ 10.43 (br s, 1 H), 8.96 (s, 1 H), 8.37

(s, 1 H), 7.82 (dd, 1 H, J = 9.2, 1.6 Hz), 7.77 (dd, 1 H, J = 9.2, 0.8 Hz),

7.45 (t, 1 H, J = 7.6 Hz), 7.25−7.19 (m, 3 H), 7.01 (d, 1 H, J = 7.6

Hz), 6.80 (tt, 1 H, J = 8.0, 1.2 Hz), 6.74−6.72 (m, 2 H), 4.55 (s, 2 H),

2.53 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃) δ 158.72, 154.49, 149.94,

147.91, 147.84, 147.51, 137.51, 133.54, 131.45, 129.61 (2C), 128.41,

127.96, 122.24, 122.20, 118.97, 117.99, 116.31, 113.50 (2C), 42.67,

24.28. IR (KBr) 3399, 2924, 1602, 1503, 1316 cm⁻¹. HRMS-ESI m/z

[M + H]⁺calcd for C₂₂H₂₀N₇382.1775, found 382.1786. HPLC

purity: 100%.

General Procedure for the Preparation of the 6-(5-(6-Methyl-

pyridin-2-yl)-2-(phenoxymethyl)- or -2-((phenylthio)methyl)-1H-

imidazol-4-yl)-[1,2,4]triazolo[1,5-a]pyridine (9a and 9b). To a

stirred solution of 1-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-2-(6-methylpyridin-

2-yl)ethane-1,2-dione (8) (1.50 mmol) in a mixture of tert-butyl methyl

ether (4 mL) and MeOH (4 mL) were added 2-phenoxyacetaldehyde

(1.80 mmol) or 2-(phenylthio)acetaldehyde (2.25 mmol) and NH₄OAc

(4.51 mmol), and the mixture was stirred at room temperature for

70−180 min under nitrogen atmosphere. The pH of the reaction

mixture was adjusted to 8 with saturated aqueous NaHCO₃solution.

After removal of solvent, the mixture was extracted with EtOAc

(2 × 50 mL), and the combined EtOAc solution was washed with

water (20 mL) and brine (20 mL), dried over anhydrous Na₂SO₄,

ﬁltered, and then evaporated to dryness under reduced pressure. The

residue was puriﬁed by MPLC on silica gel to aﬀord the titled

compounds as a solid.

6-(5-(6-Methylpyridin-2-yl)-2-(phenoxymethyl)-1H-imidazol-4-yl)-

[1,2,4]triazolo[1,5-a]pyridine (9a). The residue was puriﬁed by MPLC

on silica gel using CHCl₃/MeOH (97:3) as eluent to give the titled

compound as a yellow solid. Yield 56%; mp 203.2−205.2 °C. ¹H NMR

(400 MHz, CDCl₃) δ 8.94 (s, 1 H), 8.37 (s, 1 H), 7.77−7.72 (m, 2 H),

7.51 (t, 1 H, J = 7.6 Hz), 7.32−7.25 (m, 3 H), 7.06 (d, 1 H, J = 7.6

Hz), 7.00 (t, 1 H, overlapped, J = 7.6 Hz), 6.97 (d, 2 H, overlapped,

J = 8.0 Hz), 5.22 (s, 2 H), 2.54 (s, 3 H). ¹³C NMR (125 MHz, DMSO-d₆)

δ 158.10, 157.16, 154.32, 149.81, 149.06, 144.29, 137.52, 131.81, 129.61

(2 C), 129.39, 128.01, 121.79, 121.14, 120.65, 118.56, 115.25, 114.88,

114.76 (2 C), 62.62, 23.73. IR (KBr) 3071, 2930, 1588, 1496, 1237 cm⁻¹.

HRMS-ESI m/z [M + H]⁺calcd for C₂₂H₁₉N₆O 383.1615, found

383.1631. HPLC purity: 98.08%.

6-(5-(6-Methylpyridin-2-yl)-2-((phenylthio)methyl)-1H-imidazol-

4-yl)-[1,2,4]triazolo[1,5-a]pyridine (9b). The residue was puriﬁed by

MPLC on silica gel using CH₂Cl₂/MeOH (40:1) as eluent to give the

1

titled compound as a white solid. Yield 38%; mp 168.0−170.0 °C. H

NMR (400 MHz, CDCl₃) δ 8.89 (t, 1 H, J = 1.4 Hz), 8.35 (s, 1 H),

7.76−7.19 (m, 2 H), 7.45 (t, 1 H, J = 7.8 Hz), 7.39−7.36 (m, 2 H),

7.30−7.26 (m, 2 H), 7.23−7.19 (m, 2 H), 7.01 (d, 1 H, J = 7.6 Hz),

4.26 (s, 2 H), 2.49 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃) δ 158.41,

154.60, 150.08, 147.16, 146.31, 138.17, 134.78, 131.50, 130.21 (2C),

129.50 (2C), 128.16, 127.36, 122.36, 117.81, 116.55, 31.68, 24.04

(3 peaks were not observed). IR (KBr) 3068, 2927, 1577, 1466,

1316 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₂H₁₉N₆S 399.1386,

found 399.1402. HPLC purity: 97.61%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-2-ﬂuoroaniline (12b). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as

eluent to give the titled compound as a pale-yellow solid. Yield 92%;

1

mp 129.5−131.5 °C. H NMR(400 MHz, CD₃OD) δ 9.13 (s, 1 H),

Synthesis of 6-(2-(Dimethoxymethyl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-4-yl)-[1,2,4]triazolo[1,5-a]pyridine (10). To a stirred

solution of 8 (13.50 g, 50.70 mmol) in a mixture of tert-butyl methyl

ether (80 mL) and MeOH (40 mL) were added 60% 2,2-dimethoxy-

acetaldehyde in H₂O (17.7 mL, 101.40 mmol) and NH₄OAc (9.77 g,

126.75 mmol). The mixture was stirred at room temperature for 4 h,

and the solvent was removed under reduced pressure. The pH of the

reaction mixture was adjusted to 8 with saturated aqueous NaHCO₃

solution and extracted with CH₂Cl₂(2 × 50 mL). The combined

CH₂Cl₂solution was washed with brine (30 mL), dried over

anhydrous Na₂SO₄, ﬁltered, and evaporated to dryness under reduced

pressure. The residue was puriﬁed by MPLC on silica gel using

CH₂Cl₂/MeOH (20:1) as eluent to give the titled compound as a

8.41 (s, 1 H), 7.84 (d, 1 H, J = 8.8 Hz), 7.74 (d, 1 H, J = 8.8 Hz), 7.65

(t, 1 H, J = 7.6 Hz), 7.36 (br s, 1 H), 7.17 (d, 1 H, J = 7.6 Hz), 6.99−

6.93 (m, 2 H), 6.84−6.79 (m, 1 H), 6.66−6.60 (m, 1 H), 4.55 (s, 2 H),

2.49 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃) δ 158.68, 154.40, 151.80

(d, J = 237.6 Hz), 149.87, 148.10, 147.56, 137.44, 135.88 (d, J = 11.6

Hz), 133.71, 131.38, 128.58, 127.75, 124.92 (d, J = 3.9 Hz), 122.34,

122.19, 118.32, 118.26, 116.18, 114.76 (d, J = 17.8 Hz), 112.71 (d, J =

3.1 Hz), 42.26, 23.91. ¹⁹F NMR (376 MHz, CDCl₃) −135.50. IR

(KBr) 3070, 2962, 1518, 749 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd

for C₂₂H₁₉FN₇400.1680, found 400.1689. HPLC purity: 100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-2-chloroaniline (12c). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as eluent

to give the titled compound as a yellow solid. Yield 67%; mp 181.4−

1

white solid. Yield 83%; mp 149.1−151.1 °C. H NMR (400 MHz,

CDCl₃) δ 10.54 (s, 1 H), 8.96 (s, 1 H), 8.36 (s, 1 H), 7.82 (dd, 1 H,

J = 9.2, 1.6 Hz), 7.77 (d, 1 H, J = 9.2, 0.8 Hz), 7.47 (t, 1 H, J = 7.8 Hz),

7.23 (d, 1 H, J = 7.6 Hz), 7.04 (d, 1 H, J = 8.0 Hz), 5.57 (s, 1 H), 3.48

(s, 6 H), 2.58 (s, 3 H). IR (KBr) 3079, 2960, 1576, 1502, 1191, 1115,

1067 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₁₈H₁₉N₆O₂

351.1564, found 351.1573.

1

183.4 °C. H NMR (400 MHz, CDCl₃) δ 8.97 (br s, 1 H), 8.37 (s, 1

H), 7.82 (dd, 1 H, J = 9.2, 1.6 Hz), 7.77 (dd, 1 H, J = 9.2, 1.0 Hz), 7.46

(t, 1 H, J = 7.6 Hz), 7.29 (dd, 1 H, J = 7.6, 1.6 Hz), 7.23 (br d, 1 H, J =

7.6 Hz), 7.14 (td, 1 H, J = 8.4, 1.6 Hz), 7.01 (dd, 1 H, J = 7.6, 0.4 Hz),

6.75 (dd, 1 H, J = 8.8, 1.4 Hz), 6.71 (td, 1 H, J = 8.4, 1.6 Hz), 5.01 (br

s, 1 H), 4.60 (br s, 2 H), 2.50 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃)

δ 158.68, 154.48, 149.95, 147.86, 147.34, 143.47, 137.63, 133.78,

131.45, 129.48, 128.48, 128.26, 127.93, 122.32, 122.21, 119.92, 118.95,

118.16, 116.31, 112.03, 42.47, 24.15. IR (KBr) 3413, 3083, 2927, 1597,

1507, 795 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₂H₁₉ClN₇

416.1385, found 416.1398. HPLC purity: 100%.

Synthesis of 4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methyl-

pyridin-2-yl)-1H-imidazole-2-carbaldehyde (11). Compound 10

(12.30 g, 35.11 mmol) was dissolved in 1 N HCl (105 mL), and the

mixture was heated at 70 °C for 4 h. The reaction mixture was allowed

to cool to 0 °C, and then it was neutralized with saturated aqueous

NaHCO₃solution. The light-yellow precipitates were collected

4225

dx.doi.org/10.1021/jm500115w | J. Med. Chem. 2014, 57, 4213−4238

Journal of Medicinal Chemistry

Article

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-2-bromoaniline (12d). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as eluent

to give the titled compound as a yellow solid. Yield 78%; mp 188.3−

190.3 °C. ¹H NMR (400 MHz, CDCl₃) δ 10.94 (br s, 1 H), 8.97 (br s,

1 H), 8.36 (s, 1 H), 7.82 (d, 1 H, J = 9.2 Hz), 7.75 (d, 1 H, J = 9.2 Hz),

7.45 (t, 1 H, overlapped, J = 7.8 Hz), 7.43 (dd, 1 H, J = 8.0, 1.6 Hz),

7.21 (d, 1 H, J = 8.0 Hz), 7.16 (td, 1 H, J = 8.4, 1.2 Hz), 6.99 (d, 1 H,

J = 7.6 Hz), 6.71 (dd, 1 H, J = 8.4, 1.2 Hz), 6.63 (td, 1 H, J = 8.0, 1.6

Hz), 4.99 (t, 1 H, J = 5.6 Hz), 4.57 (d, 2 H, J = 5.6 Hz), 2.44 (s, 3 H).

¹³C NMR (100 MHz, CDCl₃) δ 158.64, 154.49, 149.96, 147.81,

147.31, 144.46, 137.70, 133.83, 132.76, 131.44, 128.95, 128.42, 127.96,

122.34, 122.19, 119.50, 118.16, 116.34, 112.14, 110.38, 42.67, 24.15.

IR (KBr) 3393, 3072, 3003, 1593, 1505, 1460, 1315 cm⁻¹. HRMS-ESI

m/z [M + H]⁺calcd for C₂₂H₁₉BrN₇460.0880, found 460.0896.

HPLC purity: 100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-2-methylaniline (12e). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as eluent

to give the titled compound as a yellow solid. Yield 53%; mp 164.6−

166.6 °C. ¹H NMR(400 MHz, CDCl₃) δ 8.97 (s, 1 H), 8.37 (s, 1 H),

7.83 (dd, 1 H, J = 9.2, 1.6 Hz), 7.78 (dd, 1 H, J = 9.2, 0.8 Hz), 7.46

(t, 1 H, J = 7.8 Hz), 7.23 (br d, 1 H, J = 7.6 Hz), 7.14 (t, 1 H, overlapped,

J = 8.0 Hz), 7.12 (d, 1 H, overlapped, J = 7.6 Hz), 7.01 (d, 1 H, J =

8.0 Hz), 6.76 (td, 1 H, J = 7.6, 0.8 Hz), 6.69 (d, 1 H, J = 7.6 Hz), 4.61

(s, 2 H), 2.54 (s, 3 H), 2.26 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃) δ

158.70, 154.46, 149.95, 147.94, 145.58, 137.54, 133.78, 131.52, 130.55,

128.30, 127.95, 127.51, 123.02, 122.38, 122.22, 118.62, 118.01, 116.31,

110.53, 42.70, 24.30, 17.71. IR (KBr) 3416, 3011, 1511, 750 cm⁻¹.

HRMS-ESI m/z [M + H]⁺calcd for C₂₃H₂₂N₇396.1931, found

396.1938. HPLC purity: 100%.

6.87 (td, 1 H, J = 7.6, 1.6 Hz), 6.84 (dd, 1 H, J = 8.0, 1.6 Hz), 6.77 (td,

1 H, J = 7.8, 1.6 Hz), 6.69 (dd, 1 H, J = 7.8, 1.6 Hz), 4.58 (s, 2 H), 3.91

(s, 3 H), 2.53 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃) δ 158.74,

154.42, 149.92, 148.16, 148.13, 147.29, 137.51, 137.33, 133.68, 131.57,

128.41, 127.84, 122.52, 122.04, 121.59, 118.34, 117.98, 116.22, 110.93,

109.79, 55.65, 42.78, 24.22. IR (KBr) 3414, 1514, 1252, 1030, 746

cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₃H₂₂N₇O 412.1880,

found 412.1888. HPLC purity: 100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-2-(triﬂuoromethoxy)aniline (12i). The

residue was puriﬁed by MPLC on silica gel using CHCl₃/MeOH

(24:1) as eluent to give the titled compound as a white solid. Yield

21%; mp 162.9−164.9 °C. ¹H NMR (400 MHz, CDCl₃) δ 10.42 (br s,

1 H), 8.96 (br s, 1 H), 8.37 (s, 1 H), 7.82 (d, 1 H, J = 9.2 Hz), 7.78 (d,

1 H, J = 9.2 Hz), 7.45 (t, 1 H, J = 7.8 Hz), 7.25−7.18 (m, 2 H), 7.15

(td, 1 H, J = 7.8, 1.2 Hz), 7.01 (d, 1 H, J = 7.6 Hz), 6.81 (dd, 1 H, J =

8.0, 1.2 Hz), 6.77 (td, 1 H, J = 7.8, 1.6 Hz), 4.85 (t, 1 H, J = 5.6 Hz),

4.61 (d, 2 H, J = 5.6 Hz), 2.52 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃)

δ 157.11, 154.94, 154.71, 150.27, 149.26, 145.44, 140.75, 139.53,

136.89, 133.17, 131.06, 129.45, 128.83, 128.06, 123.36, 121.12, 121.07

(q, J = 257.0 Hz), 119.19, 118.24, 117.00, 112.85, 41.12, 22.47. IR

(KBr) 3424, 3073, 1612, 1517, 1319, 1252, 1218, 1171, 750 cm⁻¹.

HRMS-ESI m/z [M + H]⁺calcd for C₂₃H₁₉F₃N₇O 466.1598, found

466.1617. HPLC purity: 100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-2-(methylthio)aniline (12j). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (24:1) as

eluent to give the titled compound as a yellow solid. Yield 67%; mp

1

122.3−124.3 °C. H NMR (400 MHz, CDCl₃) δ 11.34 (br s, 1 H),

8.99 (s, 1 H), 8.34 (s, 1 H), 7.83 (br d, 1 H, J = 9.2 Hz), 7.72 (br d, 1

H, J = 9.2 Hz), 7.43 (t, 1 H, J = 7.8 Hz), 7.35 (dd, 1 H, J = 7.6, 1.6

Hz), 7.21 (br d, 1 H, J = 7.6 Hz), 7.12 (td, 1 H, J = 7.8, 1.6 Hz), 6.95

(d, 1 H, J = 8.0 Hz), 6.70 (td, 1 H, J = 7.6, 1.2 Hz), 6.65 (dd, 1 H, J =

8.0, 1.2 Hz), 5.49 (br t, 1 H, J = 4.8 Hz), 4.55 (d, 2 H, J = 4.8 Hz), 2.33

(s, 3 H), 2.32 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃) δ 158.59,

154.46, 149.94, 147.89, 147.84, 147.38, 137.66, 133.74, 133.60, 131.48,

129.50, 128.26, 127.94, 122.27, 122.17, 121.21, 118.81, 118.17, 116.28,

110.93, 42.65, 24.04, 18.51. IR (KBr) 3370, 3071, 2924, 1585, 1502,

1314 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₃H₂₂N₇S 428.1652,

found 428.1643. HPLC purity: 96.15%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-2-ethylaniline (12f). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as

eluent to give the titled compound as a yellow solid. Yield 65%; mp

169.0−171.0 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.97 (br s, 1 H), 8.37

(s, 1 H), 7.83 (dd, 1 H, J = 9.2, 1.6 Hz), 7.78 (dd, 1 H, J = 9.2, 0.8 Hz),

7.46 (t, 1 H, J = 7.8 Hz), 7.23 (br d, 1 H, J = 7.6 Hz), 7.16−7.11 (m, 2

H), 7.01 (d, 1 H, J = 7.6 Hz), 6.80 (td, 1 H, J = 7.6, 1.2 Hz), 6.71 (dd,

1 H, J = 8.4, 1.2 Hz), 4.60 (s, 2 H), 2.60 (q, 2 H, J = 7.6 Hz), 2.52 (s, 3

H), 1.32 (t, 3 H, J = 7.6 Hz). ¹³C NMR (100 MHz, CDCl₃/CD₃OD)

δ 158.56, 153.81, 149.49, 148.34, 148.26, 144.85, 137.41, 132.31,

132.00, 129.11, 128.03, 127.81, 127.05, 122.30, 122.12, 118.59, 118.41,

115.69, 110.60, 41.92, 24.05, 23.62, 12.93 (1 peak was not observed).

IR (KBr) 3421, 3071, 1510, 753 cm⁻¹. HRMS-ESI m/z [M + H]⁺

calcd for C₂₄H₂₄N₇410.2088, found 410.2092. HPLC purity: 100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-2-isopropylaniline (12g). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (24:1) as eluent

to give the titled compound as a yellow solid. Yield 67%; mp 155.5−

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-2-vinylaniline (12k). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as eluent

to give the titled compound as a yellow solid. Yield 62%; mp 171.7−

1

173.7 °C. H NMR (400 MHz, CDCl₃) δ 8.96 (br s, 1 H), 8.35 (s, 1

H), 7.80 (dd, 1 H, J = 9.2, 1.6 Hz), 7.73 (dd, 1 H, J = 9.2, 0.8 Hz), 7.44

(t, 1 H, J = 7.8 Hz), 7.28 (dd, 1 H, J = 7.6, 1.2 Hz), 7.22 (br d, 1 H, J =

8.0 Hz), 7.15 (td, 1 H, J = 7.8, 1.2 Hz), 6.99 (d, 1 H, J = 7.6 Hz), 6.79

(dd, 1H, overlapped, J = 17.2, 11.2 Hz), 6.78 (td, 1 H, overlapped, J =

7.6, 0.8 Hz), 6.68 (dd, 1 H, J = 8.2, 1.0 Hz), 5.62 (dd, 1 H, J = 17.2, 1.4

Hz), 5.34 (dd, 1 H, J = 11.2, 1.4 Hz), 4.53 (s, 2 H), 2.45 (s, 3 H). ¹³C

NMR (100 MHz, CDCl₃) δ 158.57, 154.56, 150.01, 147.95, 147.57,

144.46, 137.86, 133.64, 132.69, 131.43, 129.35, 128.11, 127.69, 127.34,

125.33, 122.41, 121.87, 119.02, 117.14, 116.39, 111.52, 42.61, 24.14. IR

(KBr) 3421, 3079, 1507, 751 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for

C₂₄H₂₂N₇408.1931, found 408.1940. HPLC purity: 95.01%.

1

157.5 °C. H NMR (400 MHz, CDCl₃) δ 8.97 (br s, 1 H), 8.37

(s, 1 H), 7.82 (dd, 1 H, J = 9.2, 1.6 Hz), 7.76 (dd, 1 H, J = 9.2, 0.8 Hz),

7.45 (t, 1 H, J = 7.8 Hz), 7.23 (br d, 1 H, overlapped, J = 8.0 Hz), 7.21

(dd, 1 H, overlapped, J = 7.6, 1.6 Hz), 7.12 (td, 1 H, J = 7.6, 1.6 Hz),

7.00 (d, 1 H, J = 7.6 Hz), 6.83 (td, 1 H, J = 7.6, 1.2 Hz), 6.71 (dd, 1 H,

J = 8.0, 1.2 Hz), 4.58 (s, 2 H), 3.00 (heptet, 1 H, J = 6.8 Hz), 2.50

(s, 3 H), 1.31 (d, 6 H, J = 6.8 Hz). ¹³C NMR (100 MHz, CDCl₃) δ

158.70, 154.46, 149.95, 147.92, 144.26, 137.50, 133.80, 133.38, 131.52,

128.25, 127.92, 127.10, 125.45, 122.47, 122.15, 119.05, 117.87, 116.29,

111.38, 42.95, 27.40, 24.32, 22.67 (2 C) (1 peak was not observed). IR

(KBr) 3402, 3072, 2962, 1579, 1508, 1453, 1313 cm⁻¹. HRMS-ESI

m/z [M + H]⁺calcd for C₂₅H₂₆N₇424.2244, found 424.2254. HPLC

purity: 99.53%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-2-methoxyaniline (12h). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (24:1) as eluent

to give the titled compound as a yellow solid. Yield 51%; mp 77.1−

79.1 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.98 (br s, 1 H), 8.37 (s, 1 H),

7.83 (dd, 1 H, J = 9.2, 1.6 Hz), 7.78 (dd, 1 H, J = 9.2, 0.8 Hz), 7.45

(t, 1 H, J = 7.6 Hz), 7.22 (br d, 1 H, J = 7.6 Hz), 7.00 (d, 1 H, J = 7.6 Hz),

2-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)benzonitrile (12l). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as eluent

to give the titled compound as a yellow solid. Yield 51%; mp 260.9−

1

262.9 °C. H NMR (400 MHz, CDCl₃) δ 8.98 (br s, 1 H), 8.36 (s, 1

H), 7.83 (dd, 1 H, J = 9.2, 1.6 Hz), 7.76 (dd, 1 H, J = 9.2, 0.8 Hz), 7.47

(t, 1 H, J = 7.8 Hz), 7.40 (br d, 1 H, overlapped, J = 7.6 Hz), 7.38

(t, 1 H, J = 7.8 Hz), 7.25 (br d, 1 H, J = 8.0 Hz), 7.00 (d, 1 H, J = 7.6),

6.80 (d, 1 H, J = 8.4 Hz), 6.75 (td, 1 H, J = 8.0, 0.8 Hz), 5.32 (br t, 1

H, J = 5.6 Hz), 4.62 (d, 2 H, J = 5.6 Hz), 2.41 (s, 3 H). ¹³C NMR (100

MHz, CDCl₃/CD₃OD) δ 158.53, 153.82, 149.70, 149.46, 148.43,

146.82, 137.44, 134.64, 132.96, 132.29, 131.93, 129.74, 127.79, 122.33,

122.03, 118.67, 117.89, 117.65, 115.65, 111.34, 96.22, 40.98, 24.00. IR

(KBr) 3376, 3083, 2931, 2215, 1578, 1514, 1315 cm⁻¹. HRMS-ESI

4226

dx.doi.org/10.1021/jm500115w | J. Med. Chem. 2014, 57, 4213−4238

Journal of Medicinal Chemistry

Article

m/z [M + H]⁺calcd for C₂₃H₁₉N₈407.1727, found 407.1751. HPLC

purity: 100%.

to give the titled compound as a white yellow solid. Yield 83%; mp

208.9−210.9 °C. H NMR (400 MHz, CDCl₃/CD₃OD) δ 8.89 (br s,

1

N-(2-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-

yl)-1H-imidazol-2-yl)methylamino)phenyl)methanesulfonamide

(12n). The residue was puriﬁed by MPLC on silica gel using CHCl₃/

MeOH (12:1) as eluent to give the titled compound as a yellow solid.

Yield 21%; mp 195.2−197.2 °C. ¹H NMR (400 MHz, CD₃OD) δ 9.18

(dd, 1 H, J = 1.6, 0.8 Hz), 8.41 (s, 1 H), 7.87 (dd, 1 H, J = 9.2, 1.6 Hz),

7.75 (dd, 1 H, J = 9.2, 0.8 Hz), 7.66 (t, 1 H, J = 7.8 Hz), 7.38 (br d,

1 H, J = 7.6 Hz), 7.20 (dd, 1 H, J = 7.8, 1.4 Hz), 7.16 (d, 1 H,

overlapped, J = 7.6 Hz), 7.15 (td, 1 H, overlapped, J = 8.0, 1.6 Hz),

6.82 (dd, 1 H, J = 8.0, 1.2 Hz), 6.71 (td, 1 H, J = 8.0, 1.4 Hz), 4.59

(s, 2 H), 3.05 (s, 3 H), 2.48 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃) δ

158.46, 153.91, 149.52, 148.60, 147.93, 144.74, 137.46, 131.96, 131.26,

130.12, 129.70, 128.30, 128.00, 122.34, 121.45, 121.42, 118.61, 118.12,

115.56, 112.23, 40.99, 39.01, 24.08. IR (KBr) 3417, 3305, 3094, 1600,

1515, 1457, 1318, 1147 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for

C₂₃H₂₃N₈O₂S 475.1659, found 475.1668. HPLC purity: 100%.

N-(2-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-

yl)-1H-imidazol-2-yl)methylamino)phenyl)acetamide (12o). The

residue was puriﬁed by MPLC on silica gel using CHCl₃/MeOH (12:1)

as eluent to give the titled compound as a yellow solid. Yield 63%; mp

183.2−185.2 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.56 (br s, 1 H),

9.58 (s, 1 H), 9.36 (s, 1 H), 8.50 (s, 1 H), 8.00 (br d, 1 H, J = 9.2 Hz),

7.82 (d, 1 H, J = 9.2 Hz), 7.71 (t, 1 H, J = 7.8 Hz), 7.44 (br s, 1 H),

7.16 (d, 1 H, overlapped, J = 8.0 Hz), 7.15 (d, 1 H, overlapped, J = 7.6

Hz), 7.03 (td, 1 H, J = 7.6, 1.2 Hz), 6.81 (d, 1 H, J = 7.6 Hz), 6.61

(td, 1 H, J = 7.6, 1.2 Hz), 5.65 (t, 1 H, J = 6.0 Hz), 4.45 (d, 2 H, J =

6.0 Hz), 2.47 (br s, 3 H), 2.09 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃/

CD₃OD) δ 171.09, 158.43, 153.94, 149.52, 148.98, 148.10, 141.96,

137.42, 131.96, 128.17, 128.02, 126.83, 126.78, 123.53, 123.42, 122.32,

121.27, 118.83, 118.42, 115.43, 112.34, 41.30, 24.14, 23.31. IR (KBr)

3206, 1663, 1601, 1515, 1453, 1315, 750 cm⁻¹. HRMS-ESI m/z

[M + H]⁺calcd for C₂₄H₂₃N₈O 439.1989, found 439.2003. HPLC

purity: 100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3-ﬂuoroaniline (12p). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as

eluent to give the titled compound as a white pink solid. Yield 33%;

mp 161.6−163.6 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.94 (t, 1 H, J =

1.4 Hz), 8.36 (s, 1 H), 7.79 (dd, 1 H, J = 9.2, 1.6 Hz), 7.75 (dd, 1 H,

J = 9.2, 0.8 Hz), 7.46 (t, 1 H, J = 7.8 Hz), 7.23 (d, 1 H, J = 8.0 Hz),

7.13−7.07 (m, 1 H), 7.01 (d, 1 H, J = 7.6 Hz), 6.47−6.41 (m, 2 H),

6.37 (dt, 1 H, J = 8.8, 2.4 Hz), 4.49 (s, 2 H), 2.49 (s, 3 H). ¹³C NMR

(100 MHz, CDCl₃) δ 164.14 (d, J = 242.2 Hz), 158.67, 154.52,

149.97, 149.27 (d, J = 10.1 Hz), 147.73, 147.27, 137.73, 133.47,

131.40, 130.73 (d, J = 10.1 Hz), 128.47, 128.03, 122.41, 121.92,

118.14, 116.37, 109.20 (d, J = 2.3 Hz), 105.32 (d, J = 21.7 Hz), 100.34

(d, J = 25.5 Hz), 42.31, 24.18. ¹⁹F NMR (376 MHz, CDCl₃) −112.19.

IR (KBr) 3277, 3076, 1619, 1592, 1502, 1460 cm⁻¹. HRMS-ESI m/z

[M + H]⁺calcd for C₂₂H₁₉FN₇400.1680, found 400.1689. HPLC

purity: 100%.

1 H), 8.30 (s, 1 H), 7.75 (dd, 1 H, J = 9.2, 1.6 Hz), 7.69 (d, 1 H, J =

9.2 Hz), 7.44 (br t, 1 H, J = 7.6 Hz), 7.12 (br d, 1 H, J = 7.6 Hz), 7.00

(d, 1 H, J = 8.0 Hz), 6.97 (t, 1 H, overlapped, J = 8.0 Hz), 6.82−6.77

(m, 2 H), 6.58 (ddd, 1 H, J = 8.2, 2.4, 0.8 Hz), 4.41 (s, 2 H), 2.49

(s, 3 H). ¹³C NMR (100 MHz, CDCl₃/CD₃OD) δ 158.55, 153.88,

149.53, 148.91, 148.27, 147.63, 137.54, 132.13, 131.92, 130.66, 129.30,

127.85, 123.24, 122.43, 121.84, 121.04, 118.70, 115.87, 115.78, 111.80,

41.38, 24.02. IR (KBr) 3268, 3074, 1595, 1501, 1317, 1252 cm⁻¹.

HRMS-ESI m/z [M + H]⁺calcd for C₂₂H₁₉BrN₇460.0880, found

460.0890. HPLC purity: 99.54%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3-methylaniline (12s). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as eluent

to give the titled compound as a white solid. Yield 74%; mp 111.7−

1

113.7 °C. H NMR (400 MHz, CDCl₃) δ 8.96 (dd, 1 H, J = 1.6,

1.2 Hz), 8.37 (s, 1 H), 7.82 (dd, 1 H, J = 9.2, 1.6 Hz), 7.78 (dd, 1 H,

J = 9.2, 1.2 Hz), 7.46 (t, 1 H, J = 7.8 Hz), 7.22 (br d, 1 H, J = 8.0 Hz),

7.11 (t, 1 H, J = 7.8 Hz), 7.01 (d, 1 H, J = 7.6 Hz), 6.63 (dt, 1 H, J =

7.6, 0.8 Hz), 6.57−6.53 (m, 2 H), 4.55 (s, 2 H), 2.54 (s, 3 H), 2.29 (s,

3 H). ¹³C NMR (100 MHz, CDCl₃) δ 158.74, 154.44, 149.91, 148.07,

147.89, 147.59, 139.46, 137.39, 133.65, 131.49, 129.48, 128.44, 127.87,

122.44, 122.13, 119.91, 117.93, 116.25, 114.39, 110.50, 42.78, 24.32,

21.75. IR (KBr) 3487, 3278, 1601, 754 cm⁻¹. HRMS-ESI m/z [M + H]⁺

calcd for C₂₃H₂₂N₇396.1931, found 396.1941. HPLC purity: 97.84%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3-ethylaniline (12t). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as

eluent to give the titled compound as a white solid. Yield 84%; mp

1

89.7−91.7 °C. H NMR (400 MHz, CDCl₃) δ 8.96 (d, 1 H, J = 1.2

Hz), 8.37 (s, 1 H), 7.82 (dd, 1 H, J = 9.2, 1.6 Hz), 7.77 (dd, 1 H, J =

9.2, 1.2 Hz), 7.45 (t, 1 H, J = 7.8 Hz), 7.22 (br d, 1 H, J = 8.0 Hz), 7.14

(t, 1 H, J = 7.8 Hz), 7.01 (d, 1 H, J = 7.6 Hz), 6.66 (dd, 1 H, J = 7.6,

0.8 Hz), 6.58 (d, 1 H, J = 2.0 Hz), 6.55 (dd, 1 H, J = 7.6, 2.0 Hz), 4.55

(s, 2 H), 2.58 (q, 2 H, J = 7.6 Hz), 2.53 (s, 3 H), 1.21 (t, 3 H, J = 7.6

Hz). ¹³C NMR (100 MHz, CDCl₃) δ 158.73, 154.43, 149.91, 148.01,

147.92, 147.63, 145.88, 137.41, 133.57, 131.51, 129.57, 128.44, 127.90,

122.39, 122.14, 118.71, 117.92, 116.25, 113.35, 110.70, 42.79, 29.09,

24.32, 15.64. IR (KBr) 3248, 3045, 1602, 1499, 1317, 754 cm⁻¹.

HRMS-ESI m/z [M + H]⁺calcd for C₂₄H₂₄N₇410.2088, found

410.2096. HPLC purity: 99.42%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3-isopropylaniline (12u). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (24:1) as eluent

to give the titled compound as a yellow solid. Yield 78%; mp 158.8−

1

160.8 °C. H NMR (400 MHz, CDCl₃) δ 8.96 (dd, 1 H, J = 1.4, 0.8

Hz), 8.35 (s, 1 H), 7.79 (dd, 1 H, J = 9.2, 1.4 Hz), 7.73 (dd, 1 H, J =

9.2, 0.8 Hz), 7.44 (t, 1 H, J = 7.8 Hz), 7.21 (d, 1 H, J = 7.6 Hz), 7.11

(t, 1 H, J = 7.8 Hz), 6.99 (dd, 1 H, J = 7.8, 0.4 Hz), 6.67 (d, 1 H, J =

7.6 Hz), 6.57 (t, 1 H, J = 2.4 Hz), 6.50 (ddd, 1 H, J = 8.0, 2.4, 0.8 Hz),

4.51 (s, 2 H), 2.80 (heptet, 1 H, J = 6.8 Hz), 2.47 (s, 3 H), 1.20 (d, 6

H, J = 6.8 Hz). ¹³C NMR (100 MHz, CDCl₃) δ 158.74, 154.42,

150.55, 149.91, 148.00, 147.93, 147.61, 137.40, 133.56, 131.53, 129.59,

128.44, 127.91, 122.42, 122.14, 117.89, 117.26, 116.25, 112.14, 110.77,

42.83, 34.32, 24.35, 24.08 (2 C). IR (KBr) 3418, 3314, 2962, 1599,

1499, 1317 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₅H₂₆N₇

424.2244, found 424.2257. HPLC purity: 100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3-chloroaniline (12q). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as eluent

to give the titled compound as a yellow solid. Yield 52%; mp 180.8−

1

182.8 °C. H NMR (400 MHz, CDCl₃) δ 8.95 (t, 1 H, J = 1.6 Hz),

8.37 (s, 1 H), 7.81 (dd, 1 H, J = 9.2, 1.6 Hz), 7.77 (dd, 1 H, J = 9.2, 1.2

Hz), 7.47 (t, 1 H, J = 7.8 Hz), 7.24 (d, 1 H, J = 8.0 Hz), 7.10 (t, 1 H,

J = 8.0 Hz), 7.02 (d, 1 H, J = 7.6 Hz), 6.75 (ddd, 1 H, J = 8.0, 2.0, 0.8

Hz), 6.69 (t, 1 H, J = 2.0 Hz), 6.57 (ddd, 1 H, J = 8.0, 2.4, 0.8 Hz),

4.51 (s, 2 H), 2.52 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃/CD₃OD) δ

158.57, 153.99, 149.60, 148.73, 148.15, 147.66, 137.61, 135.08, 132.21,

131.87, 130.42, 129.16, 127.93, 122.48, 121.73, 118.69, 118.23, 115.90,

112.99, 111.48, 41.45, 24.07. IR (KBr) 3261, 2927, 1599, 1577 1503,

1468 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₂H₁₉ClN₇

416.1385, found 416.1398. HPLC purity: 100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3-methoxyaniline (12v). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (40:1) as eluent

to give the titled compound as a yellow solid. Yield 62%; mp 196.2−

1

198.2 °C. H NMR (400 MHz, CDCl₃) δ 8.95 (br s, 1 H), 8.37 (s,

1 H), 7.81 (dd, 1 H, J = 9.2, 1.6 Hz), 7.76 (dd, 1 H, J = 9.2, 0.8 Hz),

7.45 (t, 1 H, J = 7.6 Hz), 7.22 (d, 1 H, J = 7.6 Hz), 7.11 (t, 1 H, J = 8.0

Hz), 7.01 (d, 1 H, J = 7.6 Hz), 6.36−6.30 (m, 2 H), 6.27 (t, 1 H, J =

2.4 Hz), 4.52 (s, 2 H), 3.75 (s, 3 H), 2.51 (s, 3 H). ¹³C NMR

(100 MHz, CDCl₃) δ 161.02, 158.74, 154.47, 149.94, 148.96, 148.01,

147.65, 137.45, 133.67, 131.49, 130.41, 128.45, 127.90, 122.41, 122.17,

117.94, 116.29, 106.39, 103.95, 99.78, 55.30, 42.68, 24.32. IR (KBr)

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3-bromoaniline (12r). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as eluent

4227

dx.doi.org/10.1021/jm500115w | J. Med. Chem. 2014, 57, 4213−4238

Journal of Medicinal Chemistry

Article

3286, 1600, 1502, 1210, 1165 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd

for C₂₃H₂₂N₇O 412.1880, found 412.1891. HPLC purity: 100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3-(triﬂuoromethoxy)aniline (12w). The

residue was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH

(20:1) as eluent to give the titled compound as a white solid. Yield

35%; mp 170.7−172.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.94 (br s,

1 H), 8.37 (s, 1 H), 7.80 (dd, 1 H, J = 9.2, 1.6 Hz), 7.77 (dd, 1 H, J =

9.2, 0.8 Hz), 7.46 (t, 1 H, J = 7.8 Hz), 7.23 (br d, 1 H, J = 8.0 Hz), 7.19

(t, 1 H, J = 8.2 Hz), 7.02 (d, 1 H, J = 7.6 Hz), 6.64−6.60 (m, 2 H),

6.55 (br s, 1 H), 4.62 (br s, 1 H), 4.52 (s, 2 H), 2.52 (s, 3 H). ¹³C

NMR (100 MHz, CDCl₃) δ 158.74, 154.48, 150.55, 149.94, 148.92,

147.88, 146.89, 137.62, 133.76, 131.38, 130.60, 128.57, 127.90, 122.34,

122.28, 120.60 (q, J = 255.4 Hz), 118.06, 116.34, 111.54, 110.53,

105.76, 42.34, 24.19. IR (KBr) 3396, 3074, 1616, 1500, 1318, 1257,

1220, 1160 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₃H₁₉F₃N₇O

466.1598, found 466.1624. HPLC purity: 100%.

(m, 2 H), 7.22−7.17 (m, 2 H), 7.15 (dt, 1 H, J = 7.6, 2.0 Hz), 7.10 (dd,

1 H, J = 8.4, 0.8 Hz), 6.86 (ddd, 1 H, J = 7.6, 2.0, 0.8 Hz), 4.51 (s, 2 H),

2.50 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃/CD₃OD) δ 171.10, 158.53,

153.81, 149.44, 148.53, 147.85, 147.71, 137.41, 134.05, 131.93, 131.65,

129.78, 129.49, 127.82, 122.36, 121.76, 118.74, 117.12, 117.04, 115.60,

111.58, 41.11, 24.01. IR (KBr) 3337, 3183, 1664, 1581 cm⁻¹. HRMS-ESI

m/z [M + H]⁺calcd for C₂₃H₂₁N₈O 425.1833, found 425.1837. HPLC

purity: 99.37%.

2-(3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)phenyl)acetonitrile (12ab). The resi-

due was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1)

as eluent to give the titled compound as a yellow solid. Yield 30%; mp

1

260.1−262.1 °C. H NMR (400 MHz, CDCl₃) δ 10.76 (br s, 1 H),

8.94 (br s, 1 H), 8.37 (s, 1 H), 7.81 (br d, 1 H, J = 9.2 Hz), 7.76 (br d,

1 H, J = 9.2 Hz), 7.46 (t, 1 H, J = 7.6 Hz), 7.21 (br d, 1 H, J = 7.6 Hz),

7.20−7.15 (m, 1 H), 7.01 (d, 1 H, J = 7.6 Hz), 6.69 (dd, 1 H, J = 7.6,

0.8 Hz), 6.64−6.62 (m, 2 H), 4.53 (br s, 1 H, overlapped), 4.52 (br s,

2 H, overlapped), 3.65 (s, 2 H), 2.50 (s, 3 H). ¹³C NMR (100 MHz,

CDCl₃/CD₃OD) δ 158.51, 153.81, 149.46, 148.33, 148.21, 147.83,

137.50, 131.96, 131.04, 130.11, 129.38, 127.84, 122.38, 121.85, 118.68,

118.30, 117.56, 115.67, 112.65, 112.39, 41.38, 23.98, 23.48 (1 peak was

not observed). IR (KBr) 3077, 2933, 2247, 1601, 1500, 1316 cm⁻¹.

HRMS-ESI m/z [M + H]⁺calcd for C₂₄H₂₁N₈421.1884, found

421.1876. HPLC purity: 97.46%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3-(methylthio)aniline (12x). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (24:1) as

eluent to give the titled compound as a white solid. Yield 82%; mp

1

145.8−147.8 °C. H NMR (400 MHz, CDCl₃) δ 11.06 (br s, 1 H),

8.94 (s, 1 H), 8.35 (s, 1 H), 7.78 (dd, 1 H, J = 9.2, 1.2 Hz), 7.74 (d, 1

H, J = 9.2 Hz), 7.45 (t, 1 H, J = 7.8 Hz), 7.21 (br d, 1 H, J = 8.0 Hz),

7.07 (t, 1 H, J = 7.8 Hz), 6.99 (d, 1 H, J = 7.6 Hz), 6.64 (dd, 1 H, J =

7.6, 2.0 Hz), 6.54 (t, 1 H, J = 1.6 Hz), 6.42 (dd, 1 H, J = 8.0, 1.6 Hz),

4.47 (s, 2 H) 4.42 (br s, 1 H), 2.45 (s, 3 H), 2.40 (s, 3 H). ¹³C NMR

(100 MHz, CDCl₃) δ 158.67, 154.41, 149.87, 147.97, 147.88, 147.58,

139.81, 137.51, 133.35, 131.43, 129.89, 128.62, 127.89, 122.27, 122.12,

118.13, 116.68, 116.24, 111.25, 110.21, 42.38, 24.21, 15.71. IR (KBr)

3402, 3074, 2927, 1589, 1503, 1317 cm⁻¹. HRMS-ESI m/z [M + H]⁺

calcd for C₂₃H₂₂N₇S 428.1652, found 428.1664. HPLC purity: 99.10%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3-vinylaniline (12y). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as

eluent to give the titled compound as a yellow solid. Yield 75%; mp

165.3 °C. ¹H NMR (400 MHz, CDCl₃) δ 10.59 (br s, 1 H), 8.94 (s, 1

H), 8.37 (s, 1 H), 7.81 (d, 1 H, J = 9.2 Hz), 7.77 (d, 1 H, J = 9.2 Hz),

7.45 (t, 1 H, J = 7.8 Hz), 7.20 (br d, 1 H, overlapped, J = 7.6 Hz), 7.15

(t, 1 H, overlapped, J = 7.8 Hz), 7.00 (d, 1 H, J = 8.0 Hz), 6.86 (d, 1 H,

J = 7.6 Hz), 6.75 (t, 1 H, J = 2.0 Hz), 6.63 (dd, 1 H, overlapped, J =

17.6, 10.8 Hz), 6.61 (dd, 1 H, overlapped, J = 8.0, 2.0 Hz), 5.69 (dd, 1

H, J = 17.6, 0.8 Hz), 5.21 (dd, 1 H, J = 10.8, 0.8 Hz), 4.55 (s, 2 H),

4.39 (br s, 1 H), 2.51 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃) δ 158.58,

154.56, 150.01, 147.95, 147.59, 144.47, 137.85, 132.70, 131.45, 132.70,

131.45, 129.35, 128.09, 127.70, 125.33, 122.40, 121.93, 119.03, 118.14,

117.17, 116.40, 111.52, 42.63, 24.15. IR (KBr) 3272, 3078, 1584, 1501,

754 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₄H₂₂N₇408.1931,

found 408.1944. HPLC purity: 98.39%.

1-(3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)phenyl)ethanone (12ac). The residue

was puriﬁed by MPLC on silica gel using CHCl₃/MeOH (10:1) as

eluent to give the titled compound as a yellow solid. Yield 33%; mp

1

154.3−156.3 °C. H NMR (400 MHz, CDCl₃) δ 11.09 (br s, 1 H),

8.92 (s, 1 H), 8.35 (s, 1 H), 7.79 (d, 1 H, J = 9.2 Hz), 7.74 (d, 1 H, J =

9.2 Hz), 7.44 (t, 1 H, J = 7.8 Hz), 7.30 (d, 1 H, J = 7.6 Hz), 7.26−7.24

(m, 1 H), 7.23 (t, 1 H, overlapped, J = 7.8 Hz), 7.19 (br d, 1 H, J =

8.0 Hz), 7.00 (d, 1 H, J = 8.0 Hz), 6.86 (dd, 1 H, J = 8.0, 1.8 Hz), 4.68

(br s, 1 H), 4.52 (d, 2 H, J = 5.2 Hz), 2.53 (s, 3 H), 2.47 (s, 3 H). ¹³C

NMR (100 MHz, CDCl₃/CD₃OD) δ 199.91, 158.63, 154.07, 149.66,

148.09, 147.81, 147.67, 137.99, 137.61, 131.83, 129.70, 129.30, 128.08,

122.55, 121.35, 119.09, 118.78, 118.68, 115.93, 111.41, 41.18,

26.78, 24.12 (1 peak was not observed). IR (KBr) 3362, 1676,

1600, 1318, cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₄H₂₂N₇O

424.1880, found 424.1876. HPLC purity: 95.06%.

Methyl 3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-

2-yl)-1H-imidazol-2-yl)methylamino)benzoate (12ad). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (10:1) as

eluent to give the titled compound as a yellow solid. Yield 34%; mp

271.2−273.2 °C. ¹H NMR(400 MHz, CDCl₃) δ 8.94 (br s, 1 H), 8.34

(s, 1 H), 7.77 (dd, 1 H, J = 9.2, 1.6 Hz), 7.72 (dd, 1 H, J = 9.2, 0.8 Hz),

7.44 (t, 1 H, J = 7.8 Hz), 7.39 (dt, 1 H, J = 7.6, 1.2 Hz), 7.32 (dd, 1 H,

J = 2.4, 1.6 Hz), 7.22 (d, 1 H, overlapped, J = 8.0 Hz), 7.19 (t, 1 H,

overlapped, J = 8.0 Hz), 6.98 (d, 1 H, J = 7.6 Hz), 6.81 (ddd, 1 H, J =

8.0, 2.4, 0.8 Hz), 4.49 (s, 2 H), 3.84 (s, 3 H), 2.43 (s, 3 H). ¹³C NMR

(100 MHz, CDCl₃/CD₃OD) δ 167.98, 158.58, 153.95, 149.57, 148.30,

147.65, 147.64, 137.50, 132.07, 131.91, 130.94, 129.48, 129.34, 127.94,

122.40, 121.78, 119.46, 118.62, 118.03, 115.79, 113.62, 52.24, 41.44,

24.10. IR (KBr) 3291, 1715, 1600, 1287 cm⁻¹. HRMS-ESI m/z [M +

H]⁺calcd for C₂₄H₂₂N₇O₂440.1829, found 440.1812. HPLC purity:

97.89%.

3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)benzonitrile (12z). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as eluent

to give the titled compound as a yellow solid. Yield 91%; mp 238.3−

1

240.3 °C. H NMR (400 MHz, CDCl₃) δ 8.94 (t, 1 H, J = 1.2 Hz),

8.38 (s, 1 H), 7.80 (dd, 1 H, J = 9.2, 0.8 Hz), 7.77 (dd, 1 H, J = 9.2, 1.6

Hz), 7.55 (t, 1 H, J = 8.0 Hz), 7.31 (d, 1 H, J = 8.0 Hz), 7.25 (t, 1 H,

J = 8.0 Hz), 7.08 (d, 1 H, J = 8.0 Hz), 7.02 (dt, 1 H, J = 7.6, 1.2 Hz),

6.96−6.92 (m, 2 H), 4.56 (s, 2 H), 2.62 (s, 3 H). ¹³C NMR

(100 MHz, CDCl₃/CD₃OD) δ 158.55, 153.89, 149.52, 148.32, 148.02,

147.16, 137.58, 132.24, 131.89, 130.10, 129.46, 127.82, 122.46, 121.85,

121.46, 119.43, 118.74, 117.70, 115.79, 115.79, 115.36, 112.69, 41.07,

24.01. IR (KBr) 3280, 2925, 2229, 1598, 1507, 1445, 1321, 1258,

1178 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₃H₁₉N₈407.1727,

found 407.1732. HPLC purity: 100%.

N-(3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)phenyl)methanesulfonamide (12ae).

The residue was puriﬁed by MPLC on silica gel using CHCl₃/MeOH

(93:7) as eluent to give the titled compound as a light-brown solid. Yield

1

14%; mp 11.7−113.7 °C. H NMR (400 MHz, CDCl₃) δ 8.92 (br s,

1 H), 8.35 (s, 1 H), 7.78 (dd, 1 H, J = 9.2, 1.6 Hz), 7.73 (dd, 1 H, J =

9.2, 0.8 Hz), 7.46 (t, 1 H, J = 7.6 Hz), 7.21 (br d, 1 H, J = 7.6 Hz), 7.11

(t, 1 H, overlapped, J = 8.2 Hz), 7.10 (br s, 1 H, overlapped), 7.01 (d,

1 H, J = 7.6 Hz), 6.58 (t, 1 H, J = 2.0 Hz), 6.54 (ddd, 1 H, J = 8.2, 2.0,

0.8 Hz), 6.49 (ddd, 1 H, J = 8.2, 2.0, 0.8 Hz), 4.50 (s, 2 H), 2.94 (s, 3

H), 2.51 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃/CD₃OD) δ 158.56,

154.02, 149.60, 148.56, 148.15, 147.95, 138.63, 137.68, 131.83, 131.76,

130.43, 129.35, 127.99, 122.57, 121.42, 118.78, 115.90, 110.14, 109.87,

104.73, 41.27, 38.91, 24.04. IR (KBr) 3411, 3271, 3089, 1607, 1509,

3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)benzamide (12aa). The residue was

puriﬁed by MPLC on silica gel using CHCl₃/MeOH (16:1) as eluent

to give the titled compound as a yellow solid. Yield 54%; mp 184.4−

1

186.4 °C. H NMR (400 MHz, CD₃OD) δ 9.06 (br s, 1 H), 8.34

(s, 1 H), 7.83 (br d, 1 H, J = 9.2 Hz), 7.71 (d, 1 H, J = 9.2 Hz), 7.59−7.55

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1462, 1321, 1148 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for

C₂₃H₂₃N₈O₂S 475.1659, found 475.1673. HPLC purity: 95.10%.

N-(3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)phenyl)acetamide (12af). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as

eluent to give the titled compound as a yellow solid. Yield 31%; mp

(2 C), 128.43, 128.30, 127.89, 122.53, 122.10, 117.88, 116.26, 113.69

(2 C), 43.15, 24.37, 20.56. IR (KBr) 3314, 3010, 1521, 754 cm⁻¹.

HRMS-ESI m/z [M + H]⁺calcd for C₂₃H₂₂N₇396.1931, found

396.1931. HPLC purity: 99.58%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-4-isopropylaniline (12ak). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (24:1) as

eluent to give the titled compound as an oﬀ-white solid. Yield 68%; mp

151.1−153.1 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.96 (t, 1 H, J = 1.2

Hz), 8.36 (s, 1 H), 7.81 (dd, 1 H, J = 9.2, 1.6 Hz), 7.75 (d, 1 H, J = 9.2

Hz), 7.44 (t, 1 H, J = 7.8 Hz), 7.21 (d, 1 H, J = 8.0 Hz), 7.06 (m, 2 H),

6.99 (d, 1 H, J = 7.6 Hz), 6.64 (m, 2 H), 4.50 (s, 2 H), 2.80 (heptet, 1

H, J = 6.8 Hz), 2.48 (s, 3 H), 1.19 (d, 6 H, J = 6.8 Hz). ¹³C NMR (100

MHz, CDCl₃/CD₃OD) δ 158.58, 153.85, 149.50, 148.38, 148.34,

145.40, 139.29, 137.41, 132.19, 132.00, 129.13, 127.84, 127.27 (2 C),

122.29, 122.06, 118.53, 115.71, 113.51 (2 C), 42.27, 33.20, 24.14

(2 C), 24.08. IR (KBr) 3364, 2959, 1614, 1519, 1462, 1315 cm⁻¹.

HRMS-ESI m/z [M + H]⁺calcd for C₂₅H₂₆N₇424.2244, found

424.2256. HPLC purity: 98.98%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-4-methoxyaniline (12al). The residue was

puriﬁed by MPLC on silica gel using CHCl₃/MeOH (39:1) as eluent

to give the titled compound as a pale-yellow solid. Yield 84%; mp

197.8−199.8 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.96 (br s, 1 H), 8.37

(s, 1 H), 7.82 (dd, 1 H, J = 9.2, 1.6 Hz), 7.77 (dd, 1 H, J = 9.2, 0.8 Hz),

7.45 (t, 1 H, J = 7.8 Hz), 7.22 (br d, 1 H, J = 7.6 Hz), 7.01 (br d, 1 H,

J = 8.0 Hz), 6.80 (m, 2 H), 6.70 (m, 2 H), 4.50 (s, 2 H), 3.74 (s, 3 H),

2.53 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃/CD₃OD) δ 158.60,

154.05, 153.91, 153.00, 149.56, 148.41, 148.18, 141.45, 137.40, 131.97,

127.82, 122.64, 122.25, 118.46, 115.79, 115.04 (2 C), 114.99 (2 C),

114.84, 55.81, 43.04, 24.15. IR (KBr) 3376, 1514, 1242, 1036 cm⁻¹.

HRMS-ESI m/z [M + H]⁺calcd for C₂₃H₂₂N₇O 412.1880, found

412.1894. HPLC purity: 100%.

1

118.8−120.8 °C. H NMR (400 MHz, DMSO-d₆) δ 8.97 (br s, 1 H),

8.35 (s, 1 H), 7.79 (dd, 1 H, J = 9.2, 1.6 Hz), 7.73 (d, 1 H, J = 9.2 Hz),

7.46 (t, 1 H, J = 7.8 Hz), 7.38 (br s, 1 H), 7.24 (d, 1 H, J = 8.0 Hz),

7.15 (br s, 1 H), 7.09 (t, 1 H, J = 8.0 Hz), 7.01 (d, 1 H, J = 7.6 Hz),

6.69 (br d, 1 H, J = 8.0 Hz), 6.41 (dd, 1 H, J = 8.0, 1.6 Hz), 4.50

(s, 2 H), 2.51 (s, 3 H), 2.13 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃/

CD₃OD) δ 169.82, 158.55, 153.91, 149.52, 148.50, 148.15, 147.96,

139.25, 137.47, 131.95, 129.75, 127.92, 122.39, 121.65, 118.75, 115.67,

110.15, 110.05, 109.56, 104.74, 104.64, 41.44, 24.07 (2 C). IR (KBr)

3272, 3085, 1669, 1611, 1551, 1317, 1258 cm⁻¹. HRMS-ESI m/z

[M + H]⁺calcd for C₂₄H₂₃N₈O 439.1989, found 439.2004. HPLC

purity: 98.90%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-4-ﬂuoroaniline (12ag). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as eluent

to give the titled compound as a yellow solid. Yield 85%; mp 156.4−

1

158.4 °C. H NMR (400 MHz, CDCl₃) δ 8.95 (dd, 1 H, J = 1.6, 1.2

Hz), 8.37 (s, 1 H), 7.80 (dd, 1 H, J = 9.2, 1.6 Hz), 7.76 (dd, 1 H, J =

9.2, 1.2 Hz), 7.46 (t, 1 H, J = 7.6 Hz), 7.23 (d, 1 H, J = 7.6 Hz), 7.01

(d, 1 H, J = 7.6 Hz), 6.92−6.87 (m, 2 H), 6.65−6.62 (m, 2 H), 4.49 (s,

2 H), 2.51 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃) δ 158.75, 156.61

(d, J = 235.3 Hz), 154.44, 149.89, 148.04, 147.55, 143.82 (d, J = 2.3

Hz), 137.47, 133.63, 131.42, 128.57, 127.85, 122.36, 122.22, 118.02,

116.20 (d, 2 C, J = 13.2 Hz), 115.90, 114.38 (d, 2 C, J = 7.0 Hz),

43.22, 24.27. ¹⁹F NMR (376 MHz, CDCl₃) −126.29. IR (KBr) 3275,

3070, 1575, 1512, 1465, 1315, 1253 cm⁻¹. HRMS-ESI m/z [M + H]⁺

calcd for C₂₂H₁₉FN₇400.1680, found 400.1686. HPLC purity: 100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-4-chloroaniline (12ah). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as eluent

to give the titled compound as a yellow solid. Yield 57%; mp 154.3−

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-4-(triﬂuoromethoxy)aniline (12am). The

residue was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH

(20:1) as eluent to give the titled compound as a white solid. Yield

1

156.3 °C. H NMR (400 MHz, CDCl₃) δ 8.95 (t, 1 H, J = 1.4 Hz),

34%; mp 125.3−127.3 °C. H NMR (400 MHz, CDCl₃) δ 8.95 (s, 1

8.38 (s, 1 H), 7.81 (dd, 1 H, overlapped, J = 9.2, 1.4 Hz), 7.78 (dd, 1

H, overlapped, J = 9.2, 1.2 Hz), 7.47 (t, 1 H, J = 7.6 Hz), 7.23 (br d, 1

H, J = 7.6 Hz), 7.16 (m, 2 H), 7.02 (br d, 1 H, J = 7.6 Hz), 6.65 (m, 2

H), 4.51 (s, 2 H), 2.54 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃) δ

158.60, 154.58, 150.01, 147.58, 147.49, 146.02, 137.90, 133.68, 131.37,

129.44 (2 C), 128.22, 128.07, 123.61, 122.46, 121.89, 118.10, 116.46,

114.61 (2 C), 42.55, 24.16. IR (KBr) 3286, 2928, 1597, 1502, 1315,

1253 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₂H₁₉ClN₇

416.1385, found 416.1397. HPLC purity: 100%.

H), 8.38 (s, 1 H), 7.81 (dd, 1 H, J = 9.2, 1.6 Hz), 7.78 (dd, 1 H, J =

9.2, 0.8 Hz), 7.46 (t, 1 H, J = 7.8 Hz), 7.23 (d, 1 H, J = 8.0 Hz), 7.08

(m, 2 H), 7.02 (d, 1 H, J = 7.6 Hz), 6.70 (m, 2 H), 4.53 (s, 2 H), 2.54

(s, 3 H). ¹³C NMR (100 MHz, CDCl₃/CD₃OD) δ 158.57, 153.85,

149.49, 148.38, 147.78, 146.40, 141.09 (d, 2 C, J = 1.6 Hz), 137.48,

132.31, 131.93, 129.31, 127.79, 122.44 (2 C), 122.37, 122.05, 120.69

(q, J = 253.8 Hz), 118.66, 115.74, 113.54, 41.83, 24.02. IR (KBr) 3294,

3075, 1515, 1317, 1255, 1206, 1161 cm⁻¹. HRMS-ESI m/z [M + H]⁺

calcd for C₂₃H₁₉F₃N₇O 466.1598, found 466.1625. HPLC purity:

100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-4-bromoaniline (12ai). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as eluent

to give the titled compound as a white solid. Yield 59%; mp 118.1−

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-4-(methylthio)aniline (12an). The residue

was puriﬁed by MPLC on silica gel using CHCl₃/MeOH (24:1) as

eluent to give the titled compound as a yellow solid. Yield 74%; mp

1

120.1 °C. H NMR (400 MHz, CDCl₃) δ 8.94 (br s, 1 H), 8.38 (s, 1

1

H), 7.82−7.77 (m, 2 H), 7.46 (t, 1 H, J = 7.6 Hz), 7.29 (m, 2 H), 7.21

(d, 1 H, J = 7.6 Hz), 7.02 (d, 1 H, J = 7.6 Hz), 6.61 (m, 2 H), 4.51 (s, 2

H), 4.44 (br s, 1 H), 2.54 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃) δ

158.67, 154.47, 149.90, 147.87, 147.28, 146.48, 137.64, 133.51, 132.26

(2 C), 131.29, 128.57, 127.92, 122.36, 122.09, 118.13, 116.32, 114.98

(2 C), 110.57, 42.46, 24.22. IR (KBr) 3276, 3084, 3013, 1594, 1500,

1465, 1315, 1254 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for

C₂₂H₁₉BrN₇460.0880, found 460.0886. HPLC purity: 100%.

106.0−108.0 °C. H NMR (400 MHz, CDCl₃) δ 11.08 (br s, 1 H),

8.94 (s, 1 H), 8.35 (s, 1 H), 7.78 (dd, 1 H, J = 9.2, 1.6 Hz), 7.73 (d, 1

H, J = 9.2 Hz), 7.45 (t, 1 H, J = 7.8 Hz), 7.21 (br d, 1 H, J = 8.0 Hz),

7.17 (m, 2 H), 6.99 (d, 1 H, J = 7.6 Hz), 6.60 (m, 2 H), 4.46 (s, 2 H),

2.45 (s, 3 H), 2.37 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃) δ 158.69,

154.46, 149.90, 147.92, 147.61, 146.10, 137.57, 133.27, 131.46, 131.01

(2 C), 128.64, 127.99, 126.33, 122.32, 122.00, 118.12, 116.27, 114.12

(2 C), 42.51, 24.27, 18.66. IR (KBr) 3392, 3083, 2924, 1597, 1504,

1314 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₃H₂₂N₇S 428.1652,

found 428.1656. HPLC purity: 100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-4-methylaniline (12aj). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as eluent

to give the titled compound as a yellow solid. Yield 65%; mp 122.4−

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-4-vinylaniline (12ao). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as eluent

to give the titled compound as a white solid. Yield 67%; mp 121.2−

1

124.4 °C. H NMR (400 MHz, CDCl₃) δ 8.96 (t, 1 H, J = 1.2 Hz),

8.37 (s, 1 H), 7.82 (dd, 1 H, J = 9.2, 1.6 Hz), 7.77 (dd, 1 H, J = 9.2, 0.8

Hz), 7.45 (t, 1 H, J = 7.8 Hz), 7.22 (br d, 1 H, J = 8.0 Hz), 7.03 (m, 2

H), 7.02 (d, 1 H, overlapped, J = 7.6 Hz), 6.65 (m, 2 H), 4.53 (s, 2 H),

2.53 (s, 3 H), 2.25 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃) δ 158.76,

154.44, 149.93, 148.09, 147.96, 145.25, 137.37, 133.71, 131.54, 130.08

1

123.2 °C. H NMR (400 MHz, CDCl₃) δ 10.39 (br s, 1 H), 8.96 (s, 1

H), 8.37 (s, 1 H), 7.82 (dd, 1 H, J = 9.2, 1.6 Hz), 7.78 (dd, 1 H, J = 9.2,

0.8 Hz), 7.45 (t, 1 H, J = 7.6 Hz), 7.28 (m, 2 H), 7.22 (br d, 1 H, J = 7.6

Hz), 7.01 (d, 1 H, J = 7.6 Hz), 6.69 (m, 2 H), 6.61 (dd, 1 H, J = 17.6,

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10.8 Hz), 5.55 (dd, 1 H, J = 17.6, 0.8 Hz), 5.05 (dd, 1 H, J = 10.8, 0.8

Hz), 4.56, (s, 2 H), 2.53 (s, 3 H). ¹³C NMR (125 MHz, CDCl₃) δ

158.78, 154.39, 149.89, 148.21, 147.48, 147.25, 137.31, 136.49, 131.53,

128.91, 128.64, 127.83, 127.59 (2 C), 122.66, 122.11, 117.95, 116.22,

113.65, 113.35 (2 C), 110.49, 42.65, 24.36. IR (KBr) 3275, 3079, 1610,

1520, 754 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₄H₂₂N₇408.1931,

found 408.1943. HPLC purity: 95.14%.

69%; mp 121.4−123.4 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.93 (br s,

1 H), 8.38 (s, 1 H), 7.80 (dd, 1 H, J = 9.2, 1.6 Hz), 7.76 (d, 1 H, J =

9.2 Hz), 7.46 (t, 1 H, J = 7.8 Hz), 7.22 (br d, 1 H, J = 8.0 Hz), 7.09 (d,

2 H, J = 8.8 Hz), 7.02 (d, 1 H, J = 7.6 Hz), 6.67 (d, 2 H, J = 8.8 Hz),

6.34 (br s, 1 H), 4.52 (br s, 3 H), 2.93 (s, 3 H), 2.54 (s, 3 H). ¹³C

NMR (100 MHz, CDCl₃/CD₃OD) δ 158.57, 154.08, 149.64, 148.14,

147.89, 146.11, 137.69, 132.18, 131.77, 129.22, 127.99, 127.20, 125.55,

122.53, 121.57, 118.70, 115.97 (2 C), 113.82 (2 C), 41.84, 38.73,

24.11. IR (KBr) 3406, 3260, 3023, 1608, 1574, 1517, 1320, 1149,

974 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₃H₂₃N₈O₂S

475.1659, found 475.1673. HPLC purity: 100%.

N-(4-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-

yl)-1H-imidazol-2-yl)methylamino)phenyl)acetamide (12av). The

residue was puriﬁed by MPLC on silica gel using CHCl₃/MeOH

(12:1) as eluent to give the titled compound as a yellow solid. Yield

55%; mp 236.0−238.0 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.96 (br s,

1 H), 8.37 (s, 1 H), 7.81 (dd, 1 H, J = 9.2, 1.6 Hz), 7.76 (dd, 1 H, J =

9.2, 0.8 Hz), 7.46 (t, 1 H, J = 7.8 Hz), 7.27 (m, 2 H), 7.23 (d, 1 H, J =

8.0 Hz), 7.10 (br s, 1 H), 7.01 (d, 1 H, J = 7.6 Hz), 6.66 (m, 2 H), 4.50

(s, 2 H), 2.53 (s, 3 H), 2.13 (s, 3 H). ¹³C NMR (100 MHz, DMSO-d₆)

δ 167.37, 157.18, 154.27, 148.99, 147.62, 144.44, 137.28, 131.80,

129.41, 127.82, 121.77, 121.53, 120.78 (2 C), 119.76, 118.39, 114.82,

112.58 (2 C), 41.20, 23.84, 23.71 (2 peaks were not observed). IR

(KBr) 3283, 1677, 1548, 1517, 1464, 1307, 1239, 747 cm⁻¹. HRMS-

ESI m/z [M + H]⁺calcd for C₂₄H₂₃N₈O 439.1989, found 439.2004.

HPLC purity: 100%.

4-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)benzonitrile (12ap). The residue was

puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as eluent

to give the titled compound as a yellow solid. Yield 75%; mp 179.1−

1

181.1 °C. H NMR (400 MHz, CDCl₃) δ 8.93 (t, 1 H, J = 1.2 Hz),

8.37 (s, 1 H), 7.79 (dd, 1 H, overlapped, J = 9.2, 1.6 Hz), 7.76 (dd,

1 H, overlapped, J = 9.2, 0.8 Hz), 7.48 (t, 1 H, J = 7.8 Hz), 7.43 (m,

2 H), 7.24 (d, 1H, J = 8.0 Hz), 7.03 (d, 1 H, J = 7.6 Hz), 6.68 (m,

2 H), 5.12 (br s, 1 H), 4.54 (d, 2 H, J = 4.0 Hz), 2.51 (s, 3 H). ¹³C

NMR (100 MHz, CDCl₃/CD₃OD) δ 158.53, 153.89, 151.10, 149.50,

148.28, 146.86, 137.63, 133.77 (2 C), 132.23, 131.83, 129.49, 127.80,

122.50, 121.80, 120.35, 118.78, 115.80, 112.67 (2 C), 99.09, 40.63,

23.98. IR (KBr) 3363, 3075, 2926, 2215, 1607, 1526, 1319 cm⁻¹.

HRMS-ESI m/z [M + H]⁺calcd for C₂₃H₁₉N₈407.1727, found

407.1726. HPLC purity: 100%.

2-(4-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)phenyl)acetonitrile (12ar). The resi-

due was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1)

as eluent to give the titled compound as a yellow solid. Yield 37%; mp

1

192.7−194.7 °C. H NMR (400 MHz, CDCl₃) δ 10.78 (br s, 1 H),

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-2,3-diﬂuoroaniline (12aw). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as

eluent to give the titled compound as a white solid. Yield 46%; mp

176.0−178.0 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.96 (br s, 1 H), 8.38

(s, 1 H), 7.82 (dd, 1 H, J = 9.2, 1.6 Hz), 7.78 (dd, 1 H, J = 9.2, 0.8 Hz),

7.47 (t, 1 H, J = 7.6 Hz), 7.24 (d, 1 H, J = 7.6 Hz), 7.02 (dd, 1 H, J =

7.6, 0.4 Hz), 6.94−6.88 (m, 1 H), 6.60−6.51 (m, 2 H), 4.71 (br s, 1

H), 4.58 (d, 2 H, J = 3.6 Hz), 2.51 (s, 3 H). ¹³C NMR (100 MHz,

CDCl₃) δ 158.69, 154.48, 150.83 (dd, J = 244.2, 14.4 Hz), 149.94,

147.91, 147.06, 140.06 (dd, J = 238.9, 14.4 Hz), 137.73 (d, J = 3.0 Hz),

137.65, 133.82, 131.31, 128.57, 127.85, 124.43 (dd, J = 9.1, 4.5 Hz),

122.34, 122.21, 118.25, 116.31, 107.83, 106.41 (d, J = 17.4 Hz), 42.30,

23.97. ¹⁹F NMR (376 MHz, CDCl₃) −139.18, −160.99. IR (KBr) 3079,

3005, 1635, 1576, 1534, 1489, 1465 cm⁻¹. HRMS-ESI m/z [M + H]⁺

calcd for C₂₂H₁₈F₂N₇418.1586, found 418.1598. HPLC purity: 100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-2,3-dichloroaniline (12ax). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as

eluent to give the titled compound as a white solid. Yield 46%; mp

89.1−91.1 °C. ¹H NMR (400 MHz, CDCl₃) δ 11.02 (br s, 1 H), 8.97

(s, 1 H), 8.36 (s, 1 H), 7.81 (dd, 1 H, J = 9.2, 1.6 Hz), 7.75 (dd, 1 H,

J = 9.2, 0.8 Hz), 7.46 (t, 1 H, J = 8.0 Hz), 7.24 (d, 1 H, J = 8.0 Hz),

7.03 (t, 1 H, overlapped, J = 8.0 Hz), 7.00 (d, 1 H, overlapped, J = 8.0

Hz), 6.85 (dd, 1 H, J = 8.0, 1.6 Hz), 6.63 (dd, 1 H, J = 8.0, 1.6 Hz),

5.15 (t, 1 H, J = 5.6 Hz), 4.57 (d, 2 H, J = 5.6 Hz), 2.43 (s, 3 H). ¹³C

NMR (100 MHz, CDCl₃) δ 158.57, 154.53, 149.97, 147.71, 146.95,

144.95, 137.86, 133.86, 133.28, 131.35, 128.47, 128.17, 127.96, 122.45,

122.03, 119.57, 118.29, 118.03, 116.39, 109.80, 42.46, 24.04. IR (KBr)

3411, 3077, 1586, 1501, 1315 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd

for C₂₂H₁₈Cl₂N₇450.0995, found 450.1006. HPLC purity: 100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-2,3-dimethylaniline (12ay). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as

eluent to give the titled compound as an oﬀ-white solid. Yield 36%; mp

114.9−116.9 °C. ¹H NMR(400 MHz, CDCl₃) δ 8.97 (s, 1 H), 8.37 (s,

1 H), 7.83 (dd, 1 H, J = 9.2, 1.6 Hz), 7.78 (dd, 1 H, J = 9.2, 0.8 Hz),

7.45 (t, 1 H, J = 7.8 Hz), 7.22 (br d, 1 H, J = 7.6 Hz), 7.03 (t, 1 H,

overlapped, J = 7.8 Hz), 7.02 (d, 1 H, overlapped, J = 8.0 Hz), 6.69 (d,

1 H, J = 7.6 Hz), 6.58 (d, 1 H, J = 8.0 Hz), 4.59 (s, 2 H), 2.54 (s, 3 H),

2.31 (s, 3 H), 2.17 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃) δ 158.73,

154.43, 149.94, 148.06, 148.03, 145.56, 137.44, 137.13, 133.76, 131.53,

128.33, 127.89, 126.57, 122.48, 122.14, 121.42, 120.94, 117.98, 116.26,

108.82, 43.04, 24.32, 20.83, 12.82. IR (KBr): 3421, 2927, 1589, 754 cm⁻¹.

8.94 (br s, 1 H), 8.36 (s, 1 H), 7.80 (dd, 1 H, J = 9.2, 1.2 Hz), 7.76 (d,

1 H, J = 9.2 Hz), 7.46 (t, 1 H, J = 7.8 Hz), 7.22 (br d, 1 H, J = 7.6 Hz),

7.11 (d, 2 H, J = 8.4 Hz), 7.01 (d, 1 H, J = 8.0 Hz), 6.67 (d, 2 H, J =

8.4 Hz), 4.50 (br s, 3 H), 3.61 (s, 2 H), 2.49 (s, 3 H). ¹³C NMR (100

MHz, CDCl₃/CD₃OD) δ 158.56, 153.92, 149.55, 148.19, 147.91,

147.29, 137.57, 132.06, 131.89, 129.21, 129.03 (2 C), 127.90, 122.45,

121.73, 119.09, 118.68, 115.81, 113.65 (2 C), 41.54, 24.04, 22.69

(1 peak was not observed). IR (KBr) 3089, 2926, 2257, 1624, 1530,

1318 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₄H₂₁N₈421.1884,

found 421.1886. HPLC purity: 96.86%.

1-(4-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)phenyl)ethanone (12as). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as

eluent to give the titled compound as a yellow solid. Yield 39%; mp

1

165.3−167.3 °C. H NMR(400 MHz, CDCl₃) δ 8.96 (t, 1 H, J =

1.4 Hz), 8.37 (s, 1 H), 7.81 (d, 2 H, J = 8.8 Hz), 7.79−7.77 (m, 2 H),

7.54 (t, 1 H, J = 7.8 Hz), 7.30 (d, 1 H, J = 8.0 Hz), 7.07 (d, 1 H,

J = 7.6 Hz), 6.70 (d, 2 H, J = 8.8 Hz), 4.63 (s, 2 H), 2.59 (s, 3 H), 2.48

(s, 3 H). ¹³C NMR (100 MHz, CDCl₃/CD₃OD) δ 197.53, 158.54,

154.16, 151.79, 149.72, 147.79, 147.31, 137.95, 131.85, 131.72, 131.00

(2 C), 129.13, 128.14, 127.31, 122.78, 121.05, 118.87, 116.10, 112.01

(2 C), 40.65, 26.04, 23.97. IR (KBr) 3274, 1652, 1596, 1279 cm⁻¹.

HRMS-ESI m/z [M + H]⁺calcd for C₂₄H₂₂N₇O 424.1880, found

424.1890. HPLC purity: 96.91%.

Methyl 4-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-

2-yl)-1H-imidazol-2-yl)methylamino)benzoate (12at). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as

eluent to give the titled compound as a yellow solid. Yield 41%; mp

1

150.4−152.4 °C. H NMR(400 MHz, CDCl₃) δ 10.52 (br s, 1 H),

8.94 (s, 1 H), 8.37 (s, 1 H), 7.89 (m, 2 H), 7.81 (br d, 1 H, J = 9.6

Hz), 7.78 (br d, 1 H, J = 9.6 Hz), 7.46 (t, 1 H, J = 8.0 Hz), 7.21 (d, 1

H, J = 8.0 Hz), 7.02 (d, 1 H, J = 8.0 Hz), 6.68 (m, 2 H), 4.90 (t, 1 H,

J = 5.6 Hz), 4.58 (d, 2 H, J = 5.6 Hz), 3.85 (s, 3 H), 2.51 (s, 3 H). ¹³C

NMR (100 MHz, CDCl₃/CD₃OD) δ 167.72, 158.56, 154.00, 151.54,

149.60, 148.18, 147.30, 137.64, 132.25, 131.84, 131.63 (2 C), 129.28,

127.92, 122.51, 121.71, 119.07, 118.74, 115.91, 112.01 (2 C), 51.73,

40.94, 24.04. IR (KBr) 3273, 1698, 1605, 1281 cm⁻¹. HRMS-ESI m/z

[M + H]⁺calcd for C₂₄H₂₂N₇O₂440.1829, found 440.1825. HPLC

purity: 100%.

N-(4-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)phenyl)methanesulfonamide (12au).

The residue was puriﬁed by MPLC on silica gel using CHCl₃/MeOH

(12:1) as eluent to give the titled compound as a yellow solid. Yield

4230

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HRMS-ESI m/z [M + H]⁺calcd for C₂₄H₂₄N₇410.2088, found

410.2092. HPLC purity: 99.28%.

7.45 (t, 1 H, J = 7.6 Hz), 7.22 (br d, 1 H, J = 8.0 Hz), 7.00 (d, 1 H, J = 7.6

Hz), 6.98 (d, 1 H, J = 8.0 Hz), 6.57 (d, 1 H, J = 2.4 Hz), 6.50 (dd, 1 H,

J = 8.0, 2.4 Hz), 4.53 (s, 2 H), 2.54 (s, 3 H), 2.20 (s, 3 H), 2.16 (s, 3 H).

¹³C NMR (100 MHz, CDCl₃) δ 158.74, 154.42, 149.91, 148.14, 148.07,

145.65, 137.76, 137.33, 133.67, 131.53, 130.59, 128.43, 127.86, 127.13,

122.55, 122.06, 117.89, 116.22, 115.50, 110.86, 43.16, 24.35, 20.17, 18.87.

IR (KBr) 3280, 3074, 1508 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for

C₂₄H₂₄N₇410.2088, found 410.2093. HPLC purity: 98.94%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-2,3-dimethoxyaniline (12az). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (30:1) as

eluent to give the titled compound as a yellow solid. Yield 40%; mp

186.7−188.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.96 (br s, 1 H), 8.34

(s, 1 H), 7.80 (dd, 1 H, J = 9.2, 1.6 Hz), 7.72 (d, 1 H, J = 9.2 Hz), 7.42

(t, 1 H, J = 7.6 Hz), 7.20 (br d, 1 H, J = 7.6 Hz), 6.96 (d, 1 H, J =

7.6 Hz), 6.88 (td, 1 H, J = 8.2, 0.4 Hz), 6.36 (d, 1 H, overlapped, J =

8.0 Hz), 6.34 (d, 1 H, overlapped, J = 8.2 Hz), 4.52 (s, 2 H), 3.84

(s, 3 H), 3.81 (s, 3 H), 2.43 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃) δ

158.64, 154.48, 152.68, 149.97, 148.10, 147.88, 141.74, 137.48, 135.90,

133.67, 131.52, 128.24, 127.98, 124.75, 122.30, 122.13, 117.89, 116.27,

104.92, 102.87, 60.37, 55.91, 42.67, 24.25. IR (KBr) 3400, 1601,

1480, 1134 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₄H₂₄N₇O₂

442.1986, found 442.2000. HPLC purity: 98.39%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3,4-dimethoxyaniline (12be). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (30:1) as

eluent to give the titled compound as a yellow solid. Yield 33%; mp

1

135.5−137.5 °C. H NMR (400 MHz, CDCl₃) δ 8.94 (dd, 1 H, J =

1.6, 0.8 Hz), 8.34 (s, 1 H), 7.78 (dd, 1 H, J = 9.2, 1.6 Hz), 7.72 (dd, 1

H, J = 9.2, 0.8 Hz), 7.44 (t, 1 H, J = 8.0 Hz), 7.21 (br d, 1 H, J = 8.0

Hz), 6.99 (d, 1 H, J = 8.0 Hz), 6.71 (d, 1 H, J = 8.4 Hz), 6.30 (d, 1 H,

J = 2.4 Hz), 6.19 (dd, 1 H, J = 8.4, 2.4 Hz), 4.46 (s, 2 H), 3.78 (s, 3 H),

3.76 (s, 3 H), 2.47 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃) δ 158.71,

154.46, 150.21, 149.91, 147.95, 147.88, 142.68, 142.14, 137.44, 133.39,

131.45, 128.53, 127.94, 122.24, 122.19, 117.90, 116.27, 113.20,

104.16, 99.85, 56.73, 55.98, 43.39, 24.35. IR (KBr) 3367, 1515,

1233, 1027 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₄H₂₄N₇O₂

442.1986, found 442.1999. HPLC purity: 100%.

3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)phthalonitrile (12ba). The residue

was puriﬁed by MPLC on silica gel using CHCl₃/MeOH (19:1) as

eluent to give the titled compound as a yellow solid. Yield 26%; mp

241.1−243.1 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.99 (br s, 1 H), 8.35

(s, 1 H), 7.84 (dd, 1 H, J = 9.2, 1.6 Hz), 7.74 (dd, 1 H, J = 9.2, 0.8 Hz),

7.50 (t, 1 H, J = 7.8 Hz), 7.37 (t, 1 H, J = 8.2 Hz), 7.27 (br d, 1 H, J =

7.6 Hz), 7.05 (d, 1 H, J = 8.8 Hz), 7.01 (d, 1 H, overlapped, J = 8.0

Hz), 6.98 (d, 1 H, overlapped, J = 7.6 Hz), 5.94 (br t, 1 H, J = 5.6 Hz),

4.66 (d, 2 H, J = 5.6 Hz), 2.30 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃/

CD₃OD) δ 158.53, 153.91, 150.66, 149.53, 148.23, 145.93, 137.75,

134.49, 132.14, 131.83, 129.93, 127.85, 122.59, 122.06, 121.70, 118.83,

116.15, 115.98, 115.80 (2 C), 114.79, 97.65, 40.56, 23.95. IR (KBr)

3367, 3082, 2928, 2224, 1592, 1315 cm⁻¹. HRMS-ESI m/z [M + H]⁺

calcd for C₂₄H₁₈N₉432.1680, found 432.1683. HPLC purity: 100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3,4-diﬂuoroaniline (12bb). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as

eluent to give the titled compound as a white solid. Yield 31%; mp

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3,5-diﬂuoroaniline (12bf). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as

eluent to give the titled compound as a pale-yellow solid. Yield 47%;

mp 199.5−201.5 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.94 (t, 1 H, J =

1.2 Hz), 8.38 (s, 1 H), 7.81 (dd, 1 H, overlapped, J = 9.2, 1.6 Hz), 7.79

(dd, 1 H, overlapped, J = 9.2, 1.2 Hz), 7.47 (t, 1 H, J = 7.8 Hz), 7.24

(d, 1 H, J = 8.0 Hz), 7.03 (d, 1 H, J = 7.6 Hz), 6.24−6.19 (m, 3 H),

4.71 (br s, 1 H), 4.50 (s, 2 H), 2.55 (s, 3 H). ¹³C NMR (100 MHz,

CDCl₃/CD₃OD) δ 164.19 (dd, 2 C, J = 243.0, 15.5 Hz), 158.57,

153.98, 149.99 (t, J = 13.2 Hz), 149.60, 148.19, 147.28, 137.67, 132.13,

131.88, 129.33, 127.93, 122.55, 121.66, 118.78, 115.89, 95.90 (dd, 2 C,

J = 20.1, 8.5 Hz), 93.1 (t, J = 25.9 Hz), 41.22, 24.02. ¹⁹F NMR (376

MHz, CDCl₃/CD₃OD) −110.32. IR (KBr) 3268, 3086, 1634, 1596,

1524, 1494, 1318 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for

C₂₂H₁₈F₂N₇418.1586, found 418.1599. HPLC purity: 99.11%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3,5-dichloroaniline (12bg). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as

eluent to give the titled compound as a pale-yellow solid. Yield 39%;

mp 240.5−242.5 °C. ¹H NMR (400 MHz, CDCl₃/CD₃OD) δ 8.90 (s,

1 H), 8.24 (s, 1 H), 7.71 (dd, 1 H, J = 9.2, 1.6 Hz), 7.64 (dd, 1 H, J =

9.2, 0.8 Hz), 7.45 (t, 1 H, J = 7.6 Hz), 7.16 (d, 1 H, J = 7.6 Hz), 6.99

(d, 1 H, J = 7.6 Hz), 6.57 (t, 1 H, J = 1.6 Hz), 6.51 (d, 2 H, J = 1.6

Hz), 4.36 (s, 2 H), 2.45 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃/

CD₃OD) δ 158.95, 154.12, 150.07, 149.87, 148.98, 147.90, 138.23,

135.91 (2 C), 132.58, 132.12, 130.31, 128.36, 123.07, 122.03, 119.50,

117.82, 115.92, 111.62 (2 C), 41.30, 24.11. IR (KBr) 3422, 3115,

1600, 1500 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₂H₁₈Cl₂N₇

450.0995, found 450.1002. HPLC purity: 100%.

1

188.9−190.9 °C. H NMR (400 MHz, CDCl₃) δ 8.94 (t, 1 H, J = 1.4

Hz), 8.38 (s, 1 H), 7.81 (dd, 1 H, overlapped, J = 9.2, 1.6 Hz), 7.78 (dd, 1

H, overlapped, J = 9.2, 1.2 Hz), 7.47 (t, 1 H, J = 7.8 Hz), 7.24 (d, 1 H, J =

8.0 Hz), 7.03 (d, 1 H, J = 7.6 Hz), 7.04−6.95 (m, 1 H), 6.52 (ddd, 1 H,

J = 12.4, 6.8, 2.8 Hz), 6.42−6.37 (m, 1 H), 4.48 (s, 2 H), 2.55 (s, 3 H).

¹³C NMR (100 MHz, CDCl₃/CD₃OD) δ 158.56, 153.82, 150.84 (dd, J =

243.8, 13.1 Hz), 149.47, 148.37, 147.60, 144.59, 143.47 (dd, J = 238.3,

13.1 Hz), 137.50, 132.16, 131.94, 129.40, 127.79, 122.39, 121.97, 118.68,

117.53 (dd, J = 17.8, 1.5 Hz), 115.70, 108.36 (dd, J = 5.4, 3.1 Hz), 101.95

(d, J = 20.9 Hz), 41.89, 24.01. ¹⁹F NMR (376 MHz, CDCl₃/CD₃OD)

−137.48, −152.61. IR (KBr) 3271, 3074, 1635, 1601, 1574, 1523,

1464 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₂H₁₈F₂N₇418.1586,

found 418.1599. HPLC purity: 100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3,4-dichloroaniline (12bc). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as

eluent to give the titled compound as a yellow solid. Yield 53%; mp

1

194.3−196.3 °C. H NMR (400 MHz, CDCl₃) δ 8.94 (t, 1 H, J =

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3,5-dimethylaniline (12bh). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as

eluent to give the titled compound as a yellow solid. Yield 69%; mp

1.2 Hz), 8.37 (s, 1 H), 7.80 (dd, 1 H, overlapped, J = 9.2, 1.6 Hz), 7.77

(dd, 1 H, overlapped, J = 9.2, 1.2 Hz), 7.47 (t, 1 H, J = 8.0 Hz), 7.24

(br d, 1 H, J = 8.0 Hz), 7.20 (d, 1 H, J = 8.8 Hz), 7.03 (d, 1 H, J = 8.0

Hz), 6.77 (d, 1 H, J = 2.8 Hz), 6.53 (dd, 1 H, J = 8.8, 2.8 Hz), 4.47

(s, 2 H), 2.51 (s, 3 H). ¹³C NMR (100 MHz, CDCl₃/CD₃OD) δ

158.69, 153.94, 149.62, 148.45, 147.64, 147.37, 137.79, 132.92, 132.13,

131.81, 130.83, 129.80, 128.05, 122.70, 121.76, 120.66, 119.00, 115.82,

114.43, 112.98, 41.34, 24.04. IR (KBr) 3400, 3288, 2927, 1597, 1500,

1317 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₂H₁₈Cl₂N₇

450.0995, found 450.1008. HPLC purity: 100%.

1

185.3−187.3 °C. H NMR (400 MHz, CDCl₃) δ 8.95 (d, 1 H, J =

1.2 Hz), 8.36 (s, 1 H), 7.79 (dd, 1 H, J = 9.2, 1.6 Hz), 7.74 (dd, 1 H,

J = 9.2, 0.8 Hz), 7.45 (t, 1 H, J = 7.8 Hz), 7.22 (d, 1 H, J = 8.0 Hz),

6.99 (d, 1 H, J = 7.6 Hz), 6.44 (br s, 1 H), 6.31 (s, 2 H), 4.49 (s, 2 H),

2.48 (s, 3 H), 2.21 (s, 6 H). ¹³C NMR (100 MHz, CDCl₃) δ 158.73,

154.42, 149.90, 148.11, 147.98, 147.66, 139.34 (2 C), 137.34, 133.64,

131.50, 128.44, 127.85, 122.51, 122.08, 120.97, 117.90, 116.21 111.44

(2 C), 42.83, 24.33, 21.62 (2 C). IR (KBr) 3279, 3009, 1601, 1504,

1467, 1319 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₄H₂₄N₇

410.2088, found 410.2097. HPLC purity: 100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3,4-dimethylaniline (12bd). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (20:1) as

eluent to give the titled compound as an oﬀ-white solid. Yield 68%; mp

167.7−169.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.96 (d, 1 H, J = 1.2 Hz),

8.37 (s, 1 H), 7.82 (dd, 1 H, J = 9.2, 1.6 Hz), 7.77 (dd, 1 H, J = 9.2, 0.8 Hz),

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3,5-dimethoxyaniline (12bi). The residue

was puriﬁed by MPLC on silica gel using CH₂Cl₂/MeOH (19:1) as

4231

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eluent to give the titled compound as a yellow solid. Yield 74%; mp

188.6−190.6 °C. H NMR (400 MHz, CDCl₃) δ 10.43 (br s, 1 H),

8.94 (s, 1 H), 8.37 (s, 1 H), 7.81 (d, 1 H, J = 9.2 Hz), 7.77 (d, 1 H, J =

9.2 Hz), 7.45 (t, 1 H, J = 7.6 Hz), 7.20 (d, 1 H, J = 7.6 Hz), 7.01 (d,

1 H, J = 7.6 Hz), 5.96 (t, 1 H, J = 2.0 Hz), 5.92 (d, 2 H, J = 2.0 Hz),

4.52 (d, 2 H, J = 2.8 Hz), 4.41 (br s, 1 H), 3.75 (s, 6 H), 2.54 (s, 3 H).

¹³C NMR (100 MHz, CDCl₃) δ 161.92 (2 C), 158.67, 154.42, 149.89,

(d, 2 H, J = 5.6 Hz), 3.66 (s, 2 H), 2.59 (br s, 4 H), 2.42 (s, 3 H),

1.81−1.75 (m, 4 H). ¹³C NMR (125 MHz, CDCl₃/CD₃OD) δ 158.53,

153.84, 150.62 (d, J = 238.9 Hz), 149.49, 148.05, 147.72, 137.27,

136.01, 135.92, 135.91 (d, J = 12.1 Hz), 132.01, 127.73, 124.31 (d, J =

4.1 Hz), 123.61 (d, J = 12.8 Hz), 122.12, 120.10, 120.07, 118.55,

115.61, 112.29, 53.88 (2 C), 52.77 (d, J = 2.1 Hz), 41.68, 24.13, 23.20

(2 C). IR (KBr) 3430, 3067, 2964, 2802, 1626, 1511, 1319 cm⁻¹.

HRMS-ESI m/z [M + H]⁺calcd for C₂₇H₂₈FN₈483.2415, found

483.2449. HPLC purity: 98.71%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-2-ﬂuoro-3-(morpholinomethyl)aniline

(14e). The residue was puriﬁed by MPLC on silica gel using CH₂Cl₂/

MeOH (22:3) as eluent to give the titled compound as a brown solid.

Yield 64%; mp 61.8−63.8 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.97 (s,

1 H), 8.36 (s, 1 H), 7.82 (dd, 1 H, J = 9.2, 2.0 Hz), 7.76 (dd, 1 H, J =

9.2, 0.8 Hz), 7.46 (t, 1 H, J = 7.6 Hz), 7.23 (br d, 1 H, J = 7.6 Hz), 7.00

(d, 1 H, J = 7.6 Hz), 6.95 (t, 1 H, J = 8.0 Hz), 6.76 (t, 1 H, J = 7.6 Hz),

6.70 (td, 1 H, J = 8.0, 1.6 Hz), 4.67 (br s, 1 H), 4.54 (d, 2 H, J = 4.8

Hz), 3.73 (m, 4 H), 3.59 (s, 2 H), 2.53 (br s, 4 H), 2.47 (s, 3 H). ¹³C

NMR (125 MHz, CDCl₃) δ 158.78, 154.39, 150.56 (d, J = 238.8 Hz),

149.89, 148.22, 147.29, 137.31, 136.03 (d, J = 12.3 Hz), 133.95,

131.55, 128.58, 127.81, 124.37 (d, J = 4.3 Hz), 123.60 (d, J = 12.3 Hz),

122.67, 122.11, 120.43 (d, J = 3.1 Hz), 118.01, 116.21, 111.97 (d,

J = 2.8 Hz), 66.96 (2 C), 55.92 (d, J = 2.1 Hz), 53.49 (2 C), 42.52,

24.32. IR (KBr) 3425, 3071, 2960, 2858, 1627, 1511, 1452, 1318,

1116, 755 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₇H₂₈FN₈O

499.2365, found 499.2402. HPLC purity: 99.37%.

1

149.49, 147.99, 147.61, 137.46, 133.28, 131.45, 128.60, 127.91, 122.21,

122.13, 118.06, 116.20, 92.40 (2 C), 90.84, 55.34, 42.43, 24.25. IR

(KBr) 3310, 1605, 1204, 1154, 754 cm⁻¹. HRMS-ESI m/z [M + H]⁺

calcd for C₂₄H₂₄N₇O₂442.1986, found 442.2005. HPLC purity: 100%.

N³-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-4-ﬂuoro-N¹,N¹-dimethylbenzene-1,3-diamine

(14a). The residue was puriﬁed by MPLC on silica gel using CH₂Cl₂/

MeOH (10:1) as eluent to give the titled compound as a yellow solid.

1

Yield 42%; mp 198.8−200.8 °C. H NMR (400 MHz, CDCl₃) δ 8.95

(t, 1 H, J = 1.2 Hz), 8.35 (s, 1 H), 7.78 (dd, 1 H, J = 9.2, 1.6 Hz), 7.70

(dd, 1 H, J = 9.2, 1.2 Hz), 7.43 (t, 1 H, J = 7.8 Hz), 7.21 (d, 1 H, J =

8.0 Hz), 6.94 (d, 1 H, J = 7.6 Hz), 6.80−6.73 (m, 1 H), 6.14 (br d,

1 H, J = 7.6 Hz), 6.00−5.95 (m, 1 H), 4.49 (s, 2 H), 2.79 (s, 6 H), 2.31

(s, 3 H). ¹³C NMR (100 MHz, CDCl₃/CD₃OD) δ 158.59, 153.92,

149.56, 148.50, 147.99, 147.87, 145.59 (d, J = 229.1 Hz), 137.33,

136.00 (d, J = 12.3 Hz), 132.84, 131.96, 127.72, 122.54, 122.17,

118.40, 115.80, 114.76 (d, J = 19.3 Hz), 114.66 (d, J = 18.6 Hz),

102.41 (d, J = 7.0 Hz), 99.07 (d, J = 1.6 Hz), 41.78, 41.71 (2 C), 24.15.

IR (KBr) 3425, 3090, 2955, 1615, 1528, 1257, 1200 cm⁻¹. HRMS-ESI

m/z [M + H]⁺calcd for C₂₄H₂₄FN₈443.2102, found 443.2130. HPLC

purity: 100%.

3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)-2-((dimethylamino)methyl)-

benzonitrile (14f). The residue was puriﬁed by MPLC on silica gel

using CH₂Cl₂/MeOH (20:1) as eluent to give the titled compound as

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-5-((dimethylamino)methyl)-2-ﬂuoroani-

line (14b). The residue was puriﬁed by MPLC on silica gel using

CH₂Cl₂/MeOH (17:3) as eluent to give the titled compound as a

1

a yellow solid. Yield 72%; mp 172.4−174.3 °C. H NMR (400 MHz,

1

DMSO-d₆) δ 12.70 (br s, 1 H), 9.54 (s, 1 H), 8.50 (s, 1 H), 7.99 (dd, 1

H, J = 9.2, 2.0 Hz), 7.83 (dd, 1 H, J = 9.2, 0.8 Hz), 7.72 (t, 1 H, J = 7.8

Hz), 7.52 (br s, 1 H), 7.32 (t, 1 H, J = 7.6 Hz), 7.17−7.13 (m, 2 H),

7.07 (d, 1 H, J = 8.0 Hz), 7.02 (dd, 1 H, J = 7.6, 0.8 Hz), 4.48 (d, 2 H,

J = 5.6 Hz), 3.68 (s, 2 H), 2.47 (s, 3 H), 2.23 (s, 6 H). ¹³C NMR (125

MHz, CDCl₃/CD₃OD) δ 158.58, 153.92, 153.76, 149.41, 147.87,

147.49, 137.20, 134.03, 131.76, 129.25, 127.77, 127.55, 125.33, 122.88,

122.09, 121.39, 118.30, 115.73, 114.77, 113.22, 59.66, 44.73 (2 C),

41.40, 24.09 (1 peak was not observed). IR (KBr) 3202, 3089, 2954,

2826, 2225, 1586, 1506, 1465, 1316 cm⁻¹. HRMS-ESI m/z [M + H]⁺

calcd for C₂₆H₂₆N₉464.2306, found 464.2343. HPLC purity: 98.88%.

3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)-2-(pyrrolidin-1-ylmethyl)-

benzonitrile (14g). The residue was puriﬁed by MPLC on silica gel

using CH₂Cl₂/MeOH (20:1) as eluent to give the titled compound as

brown solid. Yield 72%; mp 134.1−136.1 °C. H NMR (400 MHz,

CDCl₃) δ 9.06 (s, 1 H), 8.34 (s, 1 H), 7.84 (dd, 1 H, J = 9.2, 1.6 Hz),

7.72 (d, 1 H, J = 9.2 Hz), 7.48 (t, 1 H, J = 7.6 Hz), 7.32 (br s, 1 H),

7.18 (br s, 1 H), 7.00 (d, 1 H, J = 7.6 Hz), 6.90 (dd, 1 H, J = 11.2, 8.0

Hz), 6.55−6.51 (m, 1 H), 4.78 (br s, 1 H), 4.59 (d, 2 H, J = 6.4 Hz),

3.54 (s, 2 H), 2.50 (s, 3 H), 2.34 (s, 6 H). ¹³C NMR (125 MHz,

CDCl₃) δ 158.47, 154.31, 151.66 (d, J = 239.4 Hz), 149.83, 149.56,

149.02, 147.15, 146.73, 137.22, 136.34 (d, J = 12.3 Hz), 131.78,

127.96, 122.29, 121.93, 118.82 (d, J = 6.9 Hz), 118.32, 115.82, 115.27,

114.66 (d, J = 18.6 Hz), 113.41 (d, J = 3.3 Hz), 63.39, 44.30 (2 C),

41.19, 24.44. IR (KBr) 3413, 3082, 2950, 1619, 1527, 1454 cm⁻¹.

HRMS-ESI m/z [M + H]⁺calcd for C₂₅H₂₆FN₈457.2259, found

457.2292. HPLC purity: 100%.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-3-((dimethylamino)methyl)-2-ﬂuoroani-

line (14c). The residue was puriﬁed by MPLC on silica gel using

CH₂Cl₂/MeOH (17:3) as eluent to give the titled compound as a

brown solid. Yield 56%; mp 51.8−53.8 °C. ¹H NMR (400 MHz,

CDCl₃) δ 8.99 (br s, 1 H), 8.36 (s, 1 H), 7.83 (dd, 1 H, J = 9.2, 1.6

Hz), 7.76 (d, 1 H, J = 9.2 Hz), 7.46 (t, 1 H, J = 7.8 Hz), 7.24 (br s, 1

H), 6.99 (d, 1 H, overlapped, J = 8.0 Hz), 6.96 (t, 1 H, overlapped, J =

8.0 Hz), 6.76−6.70 (m, 2 H), 4.73 (br s, 1 H), 4.56 (d, 2 H, J = 5.6

Hz), 3.60 (s, 2 H), 2.50 (s, 3 H), 2.36 (s, 6 H). ¹³C NMR (100 MHz,

CDCl₃) δ 158.80, 154.43, 150.60 (d, J = 239.2 Hz), 149.94, 148.28,

147.20, 137.27, 136.12 (d, J = 11.6 Hz), 131.62, 127.88, 124.57 (d, J =

4.6 Hz), 123.40 (d, J = 11.6 Hz), 122.65, 122.07, 120.59 (d, J = 3.1

Hz), 117.92, 116.21, 112.52, 44.96 (2 C), 42.48, 24.45. IR (KBr) 3425,

3075, 2951, 2827, 1628, 1512, 1454, 1318 cm⁻¹. HRMS-ESI m/z

[M + H]⁺calcd for C₂₅H₂₆FN₈457.2259, found 457.2288. HPLC

purity: 100%.

1

a brown solid. Yield 62%; mp 206.7−208.5 °C. H NMR (400 MHz,

DMSO-d₆) δ 12.71 (br s, 1 H), 9.52 (s, 1 H), 8.50 (s, 1 H), 7.98 (dd, 1

H, J = 9.2, 1.6 Hz), 7.83 (d, 1 H, J = 9.2 Hz), 7.71 (t, 1 H, J = 7.8 Hz),

7.51 (br s, 1 H), 7.37 (t, 1 H, J = 5.2 Hz), 7.31 (t, 1 H, J = 8.0 Hz),

7.16 (d, 1 H, J = 7.6 Hz), 7.05 (d, 1 H, J = 8.0 Hz), 7.00 (dd, 1 H, J =

8.0, 1.2 Hz), 4.47 (d, 2 H, J = 5.2 Hz), 3.87 (s, 2 H), 2.51 (br s, 4 H),

2.47 (s, 3 H), 1.74 (br s, 4 H). ¹³C NMR (100 MHz, CDCl₃/CD₃OD)

δ 158.62, 153.82, 149.48, 147.97, 147.58, 137.34, 134.12, 131.98,

129.10, 127.89, 127.68, 126.35, 123.03, 122.21, 121.41, 118.57, 118.43,

115.73, 114.60, 112.60, 56.11, 53.62 (2 C), 41.42, 24.15, 23.64 (2 C)

(1 peak was not observed). IR (KBr) 3432, 3180, 3089, 2964, 2225,

1585, 1507, 1465, 1317 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for

C₂₈H₂₈N₉490.2462, found 490.2441. HPLC purity: 99.24%.

3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)-2-(morpholinomethyl)benzonitrile

(14h). The residue was puriﬁed by MPLC on silica gel using CH₂Cl₂/

MeOH (20:1) as eluent to give the titled compound as a yellow solid.

N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methyl)-2-ﬂuoro-3-(pyrrolidin-1-ylmethyl)aniline

(14d). The residue was puriﬁed by MPLC on silica gel using CH₂Cl₂/

MeOH (22:3) as eluent to give the titled compound as a brown solid.

1

Yield 48%; mp 241.5−243.6 °C. H NMR (400 MHz, DMSO-d₆) δ

12.70 (br s, 1 H), 9.48 (br s, 1 H), 8.50 (s, 1 H), 7.95 (dd, 1 H, J = 9.2,

1.6 Hz), 7.84 (d, 1 H, J = 9.2 Hz), 7.71 (t, 1 H, J = 7.6 Hz), 7.44 (br s,

1 H), 7.34 (t, 1 H, J = 8.0 Hz), 7.28 (t, 1 H, J = 5.6 Hz), 7.16 (d, 1 H,

J = 7.6 Hz), 7.07 (d, 1 H, J = 8.4 Hz), 7.03 (dd, 1 H, J = 7.6, 1.2 Hz),

4.49 (d, 2 H, J = 5.6 Hz), 3.76 (s, 2 H), 3.58 (m, 4 H), 2.48 (s, 3 H),

2.41 (br s, 4 H). ¹³C NMR (100 MHz, CDCl₃/CD₃OD) δ 158.61,

1

Yield 70%; mp 54.6−56.6 °C. H NMR (400 MHz, CDCl₃) δ 11.11

(br s, 1 H), 8.98 (s, 1 H), 8.35 (s, 1 H), 7.81 (dd, 1 H, J = 9.2, 1.6 Hz),

7.73 (d, 1 H, J = 9.2 Hz), 7.44 (t, 1 H, J = 7.8 Hz), 7.23 (br s, 1 H),

6.97 (d, 1 H, J = 8.0 Hz), 6.91 (t, 1 H, J = 8.0 Hz), 6.75 (td, 1 H, J =

8.0, 1.2 Hz), 6.65 (td, 1 H, J = 8.0, 1.2 Hz), 4.66 (br s, 1 H), 4.51

4232

dx.doi.org/10.1021/jm500115w | J. Med. Chem. 2014, 57, 4213−4238

Journal of Medicinal Chemistry

Article

153.87, 149.52, 148.19, 147.16, 137.48, 132.00, 129.93, 127.74, 122.34,

121.63, 118.58, 118.35, 115.77, 115.19, 113.98, 66.50 (2 C), 60.68,

52.80 (2 C), 41.16, 24.14 (5 peaks were not observed). IR (KBr) 3221,

3090, 2962, 2225, 1585, 1507, 1464, 1317 cm⁻¹. HRMS-ESI m/z [M +

H]⁺calcd for C₂₈H₂₈N₉O 506.2411, found 506.2412. HPLC purity:

98.89%.

3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)-4-(dimethylamino)benzonitrile

(14m). The residue was puriﬁed by MPLC on silica gel using CH₂Cl₂/

MeOH (20:1) as eluent to give the titled compound as a white solid.

Yield 76%; mp 200.5−202.6 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.96

(t, 1 H, J = 1.6 Hz), 8.38 (s, 1 H), 7.82 (dd, 1 H, J = 9.2, 1.6 Hz), 7.79

(dd, 1 H, J = 9.2, 0.8 Hz), 7.53 (t, 1 H, J = 7.8 Hz), 7.31 (d, 1 H, J =

8.0 Hz), 7.08−7.03 (m, 3 H), 6.87 (d, 1 H, J = 1.6 Hz), 5.44 (br s,

1 H), 4.55 (s, 2 H), 2.74 (s, 6 H), 2.58 (s, 3 H). ¹³C NMR (100 MHz,

CDCl₃/CD₃OD) δ 158.53, 153.93, 149.58, 148.35, 147.01, 145.20,

142.47, 137.53, 133.00, 131.93, 129.11, 127.82, 122.73, 122.33, 122.27,

120.03, 119.38, 118.64, 115.84, 112.83, 106.94, 43.17 (2 C), 41.71,

24.13. IR (KBr) 3356, 3081, 2949, 2837, 2224, 1572, 1515 cm⁻¹.

HRMS-ESI m/z [M + H]⁺calcd for C₂₅H₂₄N₉450.2149, found

450.2170. HPLC purity: 100%.

3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)-5-(dimethylamino)benzonitrile

(14i). The residue was puriﬁed by MPLC on silica gel using CH₂Cl₂/

MeOH (20:1) as eluent to give the titled compound as a white solid.

1

Yield 79%; mp 203.2−205.2 °C. H NMR (400 MHz, CDCl₃) δ 8.94

(t, 1 H, J = 1.2 Hz), 8.38 (s, 1 H), 7.80 (dd, 1 H, overlapped, J = 9.2,

1.6 Hz), 7.78 (dd, 1 H, overlapped, J = 9.2, 1.2 Hz), 7.49 (t, 1 H, J =

7.6 Hz), 7.25 (d, 1 H, J = 7.6 Hz), 7.04 (d, 1 H, J = 7.6 Hz), 6.38 (dd,

1 H, J = 2.4, 1.2 Hz), 6.30 (t, 1 H, J = 1.6 Hz), 6.18 (t, 1 H, J = 2.2

Hz), 4.53 (s, 2 H), 2.92 (s, 6 H), 2.56 (s, 3 H). ¹³C NMR (125 MHz,

CDCl₃/CD₃OD) δ 158.56, 153.91, 151.50, 149.53, 148.89, 148.41,

147.47, 137.43, 132.44, 131.86, 129.34, 127.76, 122.30, 122.15, 120.24,

118.54, 115.78, 112.97, 106.30, 104.49, 100.86, 41.48, 40.20 (2 C),

24.11. IR (KBr) 3391, 3085, 2928, 2228, 1593 cm⁻¹. HRMS-ESI m/z

[M + H]⁺calcd for C₂₅H₂₄N₉450.2149, found 450.2169. HPLC

purity: 100%.

3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)-4-((dimethylamino)methyl)-

benzonitrile (14n). The residue was puriﬁed by MPLC on silica gel

using CH₂Cl₂/MeOH (10:1) as eluent to give the titled compound as

1

a white solid. Yield 79%; mp 188.5−190.7 °C. H NMR (400 MHz,

CDCl₃) δ 8.99 (s, 1 H), 8.36 (s, 1 H), 7.86 (dd, 1 H, J = 9.2, 1.6 Hz),

7.78 (dd, 1 H, J = 9.2, 0.8 Hz), 7.47 (t, 1 H, J = 7.6 Hz), 7.27 (br d, 1

H, J = 7.6 Hz), 7.09 (d, 1 H, J = 7.6 Hz), 7.01 (d, 1 H, J = 7.6 Hz),

6.98 (dd, 1 H, J = 7.6, 1.6 Hz), 6.93 (br s, 1 H), 4.59 (s, 2 H), 3.63 (s,

2 H), 2.53 (s, 3 H), 2.33 (s, 6 H). ¹³C NMR (100 MHz, CDCl₃) δ

158.71, 154.39, 149.93, 148.16, 146.86, 137.30, 131.58, 128.13, 122.75,

122.06, 121.40, 119.59, 117.80, 116.26, 113.06, 112.54, 63.45, 45.06

(2 C), 41.80, 24.54. IR (KBr) 3212, 3080, 2951, 2229, 1576, 1525 cm⁻¹.

HRMS-ESI m/z [M + H]⁺calcd for C₂₆H₂₆N₉464.2306, found

464.2341. HPLC purity: 99.47%.

3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)-5-((dimethylamino)methyl)-

benzonitrile (14j). The residue was puriﬁed by MPLC on silica gel

using CH₂Cl₂/MeOH (10:1) as eluent to give the titled compound as

1

a light-brown solid. Yield 65%; mp 124.1−127.1 °C. H NMR (400

MHz, CDCl₃) δ 9.01 (s, 1 H), 8.36 (s, 1 H), 7.83 (dd, 1 H, J = 9.2, 1.6

Hz), 7.76 (dd, 1 H, J = 9.2, 0.8 Hz), 7.49 (t, 1 H, J = 7.6 Hz), 7.28 (br

d, 1 H, J = 7.6 Hz), 7.15 (br s, 1 H), 7.03 (d, 1 H, J = 7.6 Hz), 6.94 (s,

1 H), 6.82 (dd, 1 H, J = 2.0, 1.2 Hz), 4.86 (br s, 1 H), 4.55 (d, 2 H, J =

5.6 Hz), 3.48 (s, 2 H), 2.54 (s, 3 H), 2.32 (s, 6 H). ¹³C NMR (125

MHz, CDCl₃/CD₃OD) δ 158.55, 153.87, 149.50, 148.38, 148.34,

146.99, 140.28, 137.27, 131.93, 127.72, 122.17, 122.00, 119.26, 118.54,

117.56, 115.63, 115.29, 112.76, 63.64, 45.11 (2 C), 40.92, 24.20 (3

peaks were not observed). IR (KBr) 3267, 3073, 2949, 2229, 1601,

1457 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₆H₂₆N₉464.2306,

found 464.2341. HPLC purity: 100%.

3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)-4-(pyrrolidin-1-ylmethyl)-

benzonitrile (14o). The residue was puriﬁed by MPLC on silica gel

using CH₂Cl₂/MeOH (20:1) as eluent to give the titled compound as

1

a yellow solid. Yield 60%; mp 143.9−145.8 °C. H NMR (400 MHz,

CDCl₃) δ 10.40 (br s, 1 H), 8.96 (s, 1 H), 8.36 (s, 1 H), 7.84 (dd, 1 H,

J = 9.2, 1.6 Hz), 7.77 (d, 1 H, J = 9.2 Hz), 7.46 (t, 1 H, J = 7.6 Hz),

7.24 (br d, 1 H, J = 7.6 Hz), 7.09 (d, 1 H, J = 7.6 Hz), 7.00 (d, 1 H, J =

7.6 Hz), 6.96 (dd, 1 H, J = 7.6, 1.6 Hz), 6.86 (br s, 1 H), 4.55 (s, 2 H),

3.74 (s, 2 H), 2.54 (br s, 4 H), 2.51 (s, 3 H), 1.81 (br s, 4 H). ¹³C

NMR (125 MHz, CDCl₃) δ 158.83, 154.43, 149.97, 148.15, 147.80,

146.75, 137.28, 134.59, 131.57, 129.96, 129.08, 127.92, 122.78, 123.00,

122.05, 121.36, 119.71, 117.59, 116.32, 112.68, 112.20, 59.74, 53.82

(2 C), 41.75, 24.54, 23.87 (2 C). IR (KBr) 3218, 3078, 2963, 2228,

1576, 1445 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₈H₂₈N₉

490.2462, found 490.2443. HPLC purity: 99.53%.

3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)-5-(pyrrolidin-1-ylmethyl)-

benzonitrile (14k). The residue was puriﬁed by MPLC on silica gel

using CH₂Cl₂/MeOH (10:1) as eluent to give the titled compound as

1

a yellow solid. Yield 46%; mp 165.0−167.0 °C. H NMR (400 MHz,

CDCl₃) δ 8.99 (s, 1 H), 8.36 (s, 1 H), 7.82 (dd, 1 H, J = 9.2, 1.6 Hz),

7.77 (d, 1 H, J = 9.2 Hz), 7.48 (t, 1 H, J = 7.6 Hz), 7.27 (br d, 1 H, J =

7.6 Hz), 7.13 (br s, 1 H), 7.03 (d, 1 H, J = 7.6 Hz), 6.97 (br s, 1 H),

6.81 (dd, 1 H, J = 2.4, 1.2 Hz), 4.81 (br s, 1 H), 4.54 (d, 2 H, J = 5.6

Hz), 3.62 (s, 2 H), 2.57 (br s, 4 H), 2.54 (s, 3 H), 1.79 (br s, 4 H). ¹³C

NMR (125 MHz, CDCl₃) δ 158.69, 154.26, 149.74, 147.74, 147.60,

146.06, 142.18, 137.19, 134.30, 131.26, 128.00, 127.62, 122.63, 122.22,

122.03, 119.27, 117.81, 117.68, 116.15, 114.14, 112.79, 59.94, 54.12

(2 C), 41.86, 24.25, 23.44 (2 C). IR (KBr) 3396, 3287, 3079, 2963,

2229, 1600, 1458 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₈H₂₈N₉

490.2462, found 490.2456. HPLC purity: 100%.

3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)-4-(morpholinomethyl)benzonitrile

(14p). The residue was puriﬁed by MPLC on silica gel using CH₂Cl₂/

MeOH (20:1) as eluent to give the titled compound as a yellow solid.

Yield 68%; mp 191.0−192.8 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.97

(s, 1 H), 8.37 (s, 1 H), 7.84 (dd, 1 H, J = 9.2, 1.6 Hz), 7.79 (dd, 1 H,

J = 9.2, 1.2 Hz), 7.49 (t, 1 H, J = 7.8 Hz), 7.29 (br d, 1 H, J = 8.0 Hz),

7.12 (d, 1 H, J = 7.6 Hz), 7.03 (d, 1 H, J = 7.6 Hz), 6.99 (dd, 1 H, J =

7.6, 1.6 Hz), 6.93 (br s, 1 H), 4.57 (s, 2 H), 3.75 (br s, 4 H,

overlapped), 3.72 (s, 2 H, overlapped), 2.55 (br s, 7 H). ¹³C NMR

(125 MHz, CDCl₃) δ 158.87, 154.46, 149.97, 148.14, 147.69, 146.19,

137.37, 134.57, 131.48, 130.97, 128.07, 127.79, 126.92, 122.94, 122.15,

121.29, 119.58, 117.56, 116.38, 112.82, 112.59, 67.03 (2 C), 62.56,

53.21 (2 C), 41.62, 24.58. IR (KBr) 3220, 3079, 2962, 2228, 1574,

1453 cm⁻¹. HRMS-ESI m/z [M + H]⁺calcd for C₂₈H₂₈N₉O 506.2411,

found 506.2401. HPLC purity: 100%.

General Procedure for the Preparation of the 2- or 4-((4-([1,2,4]-

Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-

2-yl)methylamino)benzamide (12m and 12aq). To a stirred

solution of 12l (0.52 mmol) or 12ap (0.52 mmol) in EtOH (2 mL)

were added 34.5% H₂O₂(1.82 mmol) and 1 N NaOH (1.55 mmol) at

room temperature. The mixture was stirred at room temperature for

2−3 h, cooled to 0 °C, and then neutralized with 1 N HCl to pH 8.

After removal of the solvent, the residue was extracted with 20%

MeOH in CHCl₃(3 × 100 mL). The combined organic layers was

3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)-5-(morpholinomethyl)benzonitrile

(14l). The residue was puriﬁed by MPLC on silica gel using CH₂Cl₂/

MeOH (20:1) as eluent to give the titled compound as a light-yellow

solid. Yield 69%; mp 122.2−124.7 °C. ¹H NMR (400 MHz, CDCl₃) δ

8.97 (s, 1 H), 8.38 (s, 1 H), 7.82 (dd, 1 H, J = 9.2, 1.6 Hz), 7.78 (dd, 1

H, J = 9.2, 0.8 Hz), 7.48 (t, 1 H, J = 7.6 Hz), 7.28 (br d, 1 H, J = 7.6

Hz), 7.06 (br s, 1 H, overlapped), 7.04 (d, 1 H, J = 7.6 Hz), 7.01 (s, 1

H), 6.84 (dd, 1 H, J = 2.0, 1.2 Hz), 4.79 (br s, 1 H), 4.54 (d, 2 H, J =

7.6 Hz), 3.70 (m, 4 H), 3.48 (s, 2 H), 2.55 (s, 3 H), 2.47 (br s, 4 H).

¹³C NMR (125 MHz, CDCl₃/CD₃OD) δ 158.63, 153.90, 149.52,

148.25, 148.21, 146.90, 139.97, 137.37, 134.16, 131.85, 127.68, 122.27,

122.13, 119.37, 118.53, 118.11, 115.79, 114.63, 112.75, 66.72 (2 C),

62.76, 53.51 (2 C), 41.16, 24.18 (2 peaks were not observed). IR

(KBr) 3269, 3079, 2958, 2229, 1600, 1457 cm⁻¹. HRMS-ESI m/z

[M + H]⁺calcd for C₂₈H₂₈N₉O 506.2411, found 506.2407. HPLC

purity: 100%.

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dried over anhydrous Na₂SO₄, ﬁltered, and then evaporated to dryness

under reduced pressure. The residue was puriﬁed by MPLC on silica

gel to aﬀord the titled compounds as a solid.

give a clear solution under nitrogen atmosphere. The CH₂Cl₂solution

was cooled to 0 °C, and to it was added 1.0 M HCl in Et₂O (0.32 mL,

0.32 mmol). The mixture was stirred at room temperature for 15 min,

and solvent was evaporated by nitrogen gas bubbling into the solution.

The solid was dried using a rotary evaporator in vacuo at 40 °C for 16

2-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)benzamide (12m). The residue was

puriﬁed by MPLC on silica gel using CHCl₃/MeOH (23:2) as eluent

to give the titled compound as a yellow solid. Yield 84%; mp 121.8−

1

h to aﬀord the titled compound as an oﬀ-white solid. Yield 90%. H

NMR (400 MHz, DMSO-d₆) δ 9.48 (t, 1 H, J = 1.2 Hz), 8.60 (s, 1 H),

7.92 (dd, 1 H, J = 9.2, 1.2 Hz), 7.86 (dd, 1 H, overlapped, J = 9.2, 1.6

Hz), 7.85 (t, 1 H, overlapped, J = 8.0 Hz), 7.64 (d, 1 H, J = 8.0 Hz),

7.49 (t, 1 H, J = 7.8 Hz), 7.37 (d, 1 H, J = 8.0 Hz), 7.24 (dd, 1 H, J =

7.6, 0.8 Hz), 7.10 (dd, 1 H, J = 8.0, 0.8 Hz), 4.81 (s, 2 H), 4.61

(s, 2 H), 2.89 (s, 6 H), 2.52 (s, 3 H).

1

123.8 °C. H NMR (400 MHz, CDCl₃) δ 9.04 (dd, 1 H, J = 1.6, 0.8

Hz), 8.48 (br s, 1 H), 8.33 (s, 1 H), 7.84 (dd, 1 H, J = 9.2, 1.6 Hz),

7.72 (d, 1 H, J = 9.2 Hz), 7.41 (t, 1 H, J = 7.8 Hz), 7.37 (dd, 1 H, J =

7.6, 1.2 Hz), 7.29 (td, 1 H, J = 8.4, 1.2 Hz), 7.21 (br d, 1 H, J = 8.0

Hz), 6.92 (d, 1 H, J = 7.6 Hz), 6.83 (d, 1 H, J = 8.4 Hz), 6.62 (td, 1 H,

J = 8.0, 1.0 Hz), 6.25 (br s, 2 H), 4.59 (d, 2 H, J = 5.2 Hz), 2.32 (s, 3 H).

¹³C NMR (100 MHz, CDCl₃) δ 172.65, 158.52, 154.21, 149.72, 149.68,

Synthesis of 3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-

methylpyridin-2-yl)-1H-imidazol-2-yl)methylamino)-4-

(dimethylamino)benzonitrile Hydrochloride (14m·HCl). A

stirred suspension of 14m (0.05 g, 0.11 mmol) in a mixture of

CH₂Cl₂(3 mL) and CHCl₃(0.5 mL) was heated at 50 °C to give a

clear solution under nitrogen atmosphere. The solution was cooled to

0 °C, and to it was added 1.0 M HCl in Et₂O (0.33 mL, 0.33 mmol).

The mixture was stirred at room temperature for 15 min, and solvent

was evaporated by nitrogen gas bubbling into the solution. The solid

was dried using a rotary evaporator in vacuo at 40 °C for 16 h to aﬀord

the titled compound as an oﬀ-white solid. Yield 100%. ¹H NMR

(400 MHz, DMSO-d₆) δ 9.44 (dd, 1 H, J = 1.6, 0.8 Hz), 8.64 (s, 1 H),

7.99 (dd, 1 H, J = 9.2, 0.8 Hz), 7.84 (t, 1 H, overlapped, J = 7.8 Hz),

7.82 (dd, 1 H, J = 9.2, 0.8 Hz), 7.51 (d, 1 H, J = 8.0 Hz), 7.39 (d, 1 H,

J = 7.6 Hz), 7.15 (dd, 2 H, overlapped, J = 7.6, 0.8 Hz), 7.14 (d, 1 H,

overlapped, J = 1.2 Hz), 4.79 (s, 2 H), 2.71 (s, 6 H), 2.56 (s, 3 H).

Luciferase Assay. To establish HaCaT (3TP-luc) and 4T1 (3TP-

luc) stable cells, cells were seeded on six-well plates. Cells were

allowed to adhere overnight and then transfected with the p3TP-luc

(neo) expression plasmid using PEI reagent (Sigma-Aldrich).

Transfected cells were cultured for 4 weeks in the presence of G418

(500 μg/mL). Several single clones were isolated and measured luciferase

activity. The clone showing response to TGF-β1 treatment was used for

reporter assay. HaCaT (3TP-luc) stable cells or 4T1 (3TP-luc) stable cells

were seeded at 2.5 × 10⁴cells/well or 5 × 10⁵cells/well in 96-well plate,

respectively, and were allowed to adhere overnight. Cells were

concomitantly treated with TGF-β1 (2 ng/mL) and indicated

concentrations of ALK5 inhibitors in 0.2% FBS medium and incubated

for 24 h at 37 °C in 5% CO₂. Cell lysates were prepared using Luciferase

Assay System (Promega) according to the manufacturer’s instruction, and

luminescence was measured by a luminometer, Micro Lumat Plus

(Berthold, Germany).

148.64, 148.26, 137.27, 133.87, 132.78, 131.61, 129.37, 128.61, 127.91,

122.10, 122.03, 118.64, 116.13, 115.88, 114.20, 112.52, 41.58, 23.94. IR

(KBr) 3336, 3185, 1654, 1514, 1315 cm⁻¹. HRMS-ESI m/z [M + Na]⁺

calcd for C₂₃H₂₀N₈NaO 447.1652, found 447.1680. HPLC purity: 99.52%.

4-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-

1H-imidazol-2-yl)methylamino)benzamide (12aq). The residue was

puriﬁed by MPLC on silica gel using CHCl₃/MeOH (23:2) as eluent

to give the titled compound as a yellow solid. Yield 63%; mp 255.7−

1

257.7 °C. H NMR (400 MHz, CDCl₃/CD₃OD) δ 9.03 (br s, 1 H),

8.32 (s, 1 H), 7.81 (dd, 1 H, J = 9.2, 1.6 Hz), 7.70 (dd, 1 H, J = 9.2, 0.8

Hz), 7.67 (m, 2 H), 7.55 (t, 1 H, J = 7.6 Hz), 7.26 (br d, 1 H, J = 7.6

Hz), 7.09 (d, 1 H, J = 7.6 Hz), 6.71 (m, 2 H), 4.52 (s, 2 H), 2.51

(s, 3 H). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.93, 157.18, 154.28,

150.80, 148.99, 146.98, 137.34, 131.73, 129.01 (2 C), 127.84, 121.80,

121.60, 118.41, 114.86, 111.28 (2 C), 48.60, 23.79 (2 peaks were not

observed). IR (KBr) 3325, 2925, 1650, 1604, 1528, 1389 cm⁻¹.

HRMS-ESI m/z [M + H]⁺calcd for C₂₃H₂₁N₈O 425.1833, found

425.1835. HPLC purity: 100%.

Synthesis of N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-

methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-ﬂuoroaniline

Hydrochloride (12b·HCl). To a stirred solution of 12b in CH₂Cl₂

(1 mL) cooled 0 °C, was added 1.0 M HCl in Et₂O (1.9 mL, 1.88 mmol)

under nitrogen atmosphere. The mixture was stirred at room temperature

for 5 min and evaporated to dryness under reduced pressure. The solid

was dried using a rotary evaporator in vacuo at 40 °C for 16 h to aﬀord

1

the titled compound as a yellow solid. Yield 97%. H NMR (400 MHz,

DMSO-d₆) δ 9.44 (d, 1 H, J = 0.8 Hz), 8.62 (s, 1 H), 7.96 (dd, 1 H, J =

9.2, 0.8 Hz), 7.83 (t, 1 H, J = 8.0 Hz), 7.80 (dd, 1 H, J = 9.2, 1.6 Hz), 7.53

(d, 1 H, J = 8.0 Hz), 7.38 (d, 1 H, J = 7.6 Hz), 7.10 (ddd, 1 H, J = 12.0,

8.0, 1.2 Hz), 7.01 (td, 1 H, J = 7.6, 1.2 Hz), 6.88 (br t, 1 H, J = 8.6 Hz),

6.70−6.64 (m, 1 H), 4.75 (s, 2 H), 2.54 (s, 3 H).

Aqueous Solubility Determination. A standard solution was

prepared by dissolving 1 mg of sample in 10 mL of pH 1.2 or pH 6.8

buﬀer solution at 25 °C. The standard solution was diluted with pH

1.2 or 6.8 buﬀer solution as necessary, and the diluted standard

solutions were scanned by UV (U-3010, Hitachi, Japan) at 225.00−

326.00 nm to obtain a standard curve. A saturated solution was pre-

pared by vortex mixing in pH 1.2 or pH 6.8 buﬀer solution for 10 min,

then standing for 24 h, and then vortex mixing for an additional

10 min in the presence of excess compound. The saturated solution

was ﬁltered to remove excess compound using 0.45 μm Millex ﬁlters,

diluted to at least 4 mL, and then scanned by UV at 225.00−326.00 nm.

Total solubility was then determined by the comparison of the

absorbance of the saturated solution with that of the standard curve.

Pharmacokinetic Study in Rats. Four groups of healthy male

Sprague−Dawley rats (7 weeks and 190−210 g) were received 12b·

HCl, 12z·HCl, 14f·HCl, and 14m·HCl intravenously at a dose of

2 mg/kg, respectively. Another four groups of rats were orally

administered with these four compounds at a dose of 10 mg/kg,

respectively. About 0.2 mL of blood samples were collected from the

common carotid artery at 0, 0.08, 0.16, 0.25, 0.5, 0.75, 1, 2, and 4 h

after intravenous or oral administration. Blood samples were

centrifuged at 12000 rpm for 15 min immediately after sampling,

and about 100 μL of plasma samples were stored at −70 °C until

LC/MS/MS analysis. All animal procedures were approved by the

Institutional Animal Ethics Committee of Ewha Womans University,

Korea. Protein precipitation was conducted on 100 μL of plasma

Synthesis of 3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methyl-

pyridin-2-yl)-1H-imidazol-2-yl)methylamino)benzonitrile Hydro-

chloride (12z·HCl). To a stirred suspension of 12z (4.60 g, 11.32 mmol)

in CH₂Cl₂(500 mL) under nitrogen atmosphere, i-PrOH (50 mL) was

added to give a clear solution. To it, 5.0 M HCl in i-PrOH (4.53 mL,

22.64 mmol) was added dropwise, and the mixture was stirred at room

temperature for 1 h. The yellow suspension was diluted with tert-butyl

methyl ether (1000 mL) and stirred for an additional 3 h. The mixture

was ﬁltered under nitrogen atmosphere and washed with tert-butyl

methyl ether (2 × 100 mL). The ﬁltered solid was again suspended in

tert-butyl methyl ether (400 mL) for 1 h, ﬁltered under nitrogen

atmosphere, and washed with tert-butyl methyl ether (2 × 100 mL). The

solid was thoroughly dried in a vacuum oven over P₂O₅at 50 °C for

12 h. To remove a trace amount of residual solvent, the dried solid was

again suspended in n-heptane (400 mL) for 1.5 h, ﬁltered, and dried in a

vacuum oven over P₂O₅at 40 °C for 4 h to aﬀord the titled compound

1

as a yellow solid. Yield 98%. H NMR (400 MHz, DMSO-d₆) δ 9.47

(d, 1 H, J = 0.8 Hz), 8.66 (s, 1 H), 7.97 (dd, 1 H, J = 9.2, 0.8 Hz), 7.87

(t, 1 H, overlapped, J = 7.8 Hz), 7.85 (dd, 1 H, overlapped, J = 9.2, 1.6

Hz), 7.63 (d, 1 H, J = 7.6 Hz), 7.40 (d, 1 H, J = 8.0 Hz), 7.33 (t, 1 H, J =

7.8 Hz), 7.16 (t, 1 H, J = 1.6 Hz), 7.11 (dd, 1 H, J = 8.4, 1.6 Hz), 7.06

(br d, 1 H, J = 7.6 Hz), 4.79 (s, 2 H), 2.53 (s, 3 H).

Synthesis of 3-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methyl-

pyridin-2-yl)-1H-imidazol-2-yl)methylamino)-2-((dimethylamino)-

methyl)benzonitrile Hydrochloride (14f·HCl). A stirred suspension of

14f (0.05 g, 0.11 mmol) in CH₂Cl₂(4 mL) was heated at 50 °C to

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samples with 3 volumes of CH₃CN containing 12a as analytical

internal standard. After centrifugation, supernatant was analyzed by

API2000 LC-ESI/MS/MS system in a positive MRM mode. Analytical

data were processed using Analyst TM1.4.2. (Applied Biosystems,

Concord, Canada.) Pharmacokinetic parameters were obtained by

noncompartmental analysis of the plasma concentration−time proﬁles

using Kinetica 4.4.1 (Thermo Fisher Scientiﬁc, Inc., Woburn, MA,

USA).

Docking Study. All computational works were performed on the

Tripos Sybyl-X 2.0 (Tripos Inc., St Louis, MO) molecular modeling

package with CentOS Linux 5.4. operating system.⁶²Except the

compound 12b, all ligands were prepared with using sketch modules

and saved as MOL2 format. The X-ray coordinates 12b was used for

P-97 micropipet puller. Internal pipet solution contained (in mM):

K-methanesulfonate 115, KCl 10, MgCl₂1, EGTA 5, HEPES 10,

ATP-Mg 3, GTP-Na 0.3, phosphocreatine 4 (pH was adjusted to 7.21

with 0.1 M KOH). The pipet solutions were stored in aliquots at −10

to −30 °C. The resistance of the patch pipettes, when ﬁlled with

internal solution, ranged between 1.5 and 2.0 MΩ. Once stably patch

clamped in whole-cell mode, HEK293 cells expressing hERG were

held at a potential of −80 mV. hERG mediated currents were evoked

by application of a depolarizing voltage command step to +40 mV for

2 s, followed by a repolarizing command step to −50 mV for 1.5 s.

This voltage command protocol was repeated once every 10 s. Analysis

of current amplitudes was performed with pClamp software. Peak tail

currents from the last 30 s of the baseline period were averaged and

compared to 30 s of data recorded in the presence of the test solutions.

Current inhibition is reported in percent according to Inhibition

(%) = 100 × (1 − (I_test/I_baseline)) where I_testwas the peak tail current

measured in the presence of the test solution, and I_baselinewas the peak

tail current measured prior to exposure to the test solution; each cell

served as its own control. Peak tail current readings were adjusted for

cell rundown by a linear model derived from available baseline data for

each cell. Aberrant waveforms with a holding current more negative

than −250 pA were excluded from analysis.

docking calculation. After sketching all ligands, Gasteiger−Huckel

̈

charges were assigned to all atoms in ligand. Energy minimization was

carried out with the standard tripos force ﬁeld with convergence to

maximum derivatives of 0.001 kcal mol⁻¹Å⁻¹. The X-ray structure of

ALK5 complexed with 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole inhib-

itor was used as a receptor for docking performance (PDB: 1RW8).

Native ligand was extracted, and all water molecules except key water

molecules for water-mediated hydrogen bond network were deleted

from the complex structure. To understand binding poses of candidate

compounds which showed good inhibitory activity against ALK5,

docking study was conducted using Surﬂex-Dock⁶³embedded in

Tripos Sybyl X 2.0 software package. The active site was generated by

constructing protomol based on the native ligand. Docking was

performed using default parameters, and maximum number of poses

per ligand was 20 conformers. Binding aﬃnity of each docking pose of

ligand was calculated by Surﬂex-Dock score and consensus scoring

function (CScore¹).

ASSOCIATED CONTENT

* Supporting Information

■

S

Synthetic procedures for compounds 13b−p and detailed

HPLC conditions for conﬁrming purity of target compounds.

This material is available free of charge via the Internet at

http://pubs.acs.org.

CYP Inhibition Assay. The incubation mixtures (ﬁnal volume,

200 μL) contained 1 mg/mL microsomal protein, 0.1 M potassium

phosphate buﬀer (pH 7.4), 1 mM NADPH, and various P450 enzyme-

speciﬁc substrate cocktail (CYP1A2:50 μM phenacetin, CYP2C9:100

μM tolbutamide, CYP2C19:100 μM S-mephenytoin, CYP2D6:5 μM

dextromethorphan, CYP3A4:5 μM midazolam). The substrate cocktail

was used at concentrations approximately equal to their respective K_m

values. The 12b was used at concentrations from 0.5 nM to 10 μM.

After a 5 min preincubation at 37 °C, the reactions were initiated by

the addition of 1 mM NADPH. The incubations were performed at

37 °C in water bath for 10 min. The reactions were terminated by placing

the tubes on ice, adding 200 μL of cold CH₃CN and agitating with a

vortex mixer before centrifugation. The samples for each enzyme assay

were centrifuged at 3000g for 20 min at 4 °C, and the supernatants

of the individual reaction samples were analyzed by 3200 QTRAP

LC/MS/MS system in a positive MRM mode. Sample analysis was

performed by 3200 QTRAP LC/MS/MS system (Applied Biosystems,

Concord, Canada) in a positive MRM mode. The LC/MS/MS system

consisted of an Agilent 1100 series HPLC system (Agilent Technologies)

equipped with autosampler, binary pump, degasser, and thermostated

column compartment and an 3200 QTRP triple quadrupole mass

spectrometer with a Turbo V Ion spray source (Applied Biosystems).

Analytical data were processed using Analyst 1.4.2. (Applied Biosystems).

IC₅₀values were calculated using GraphPad Prism 4.0.

hERG Assay. The hERG assay was performed at MPI Research,

Inc. in Mattawan, MI. HEK293 cells stably expressing the hERG

potassium channel were plated onto sterile glass coverslips up to

2 days prior to electrophysiological evaluation. Following whole-cell

patch clamp and demonstration of stable hERG current for at least

2 min, baseline testing was conducted, and the test and control articles

were applied in physiological salt solution (PSS) via superfusion at

35.2 to 35.7 °C for 3.0 to 5.7 min. The superfusion ﬂow rate was 3.3 to

3.8 mL per min. The concentration levels for 12b were 1, 10, 30, and

100 μM. PSS containing 0.1% DMSO and cisapride (0.1 μM) were

used as the vehicle and positive control, respectively. Currents were

recorded from HEK293 cells stably expressing hERG using whole-cell

patch clamp technique with a Multiclamp 700A ampliﬁer and pClamp

software. Signals were acquired at 2000 μs/point and ﬁltered at

100 Hz. Patch pipettes were pulled from borosilicate capillary glass

tubing (Sutter Instuments) using a Sutter-Flaming/Brown type, model

AUTHOR INFORMATION

Corresponding Author

*Phone: (82) 2-3277-3025. Fax: (82) 2 3277-2467. E-mail:

dkkim@ewha.ac.kr.

■

Present Addresses

^#For D.S.: Sai Life Sciences Ltd., Unit-II, ICICI Knowledge Park,

Turkapally, Hyderabad, 500078, India. E-mail: dpsreenu@

gmail.com.

^∥For V.B.S.: VVR Organics Pvt. Ltd., 115/2RT, Sanjeeva Reddy

Nagar, Hyderabad, 500038, India. E-mail: baluiict@gmail.com.

^⊥For K.S.R.: College of Pharmacy, School of Health Sciences,

University of Kwazulu-Natal, Durban, 4041, South Africa.

E-mail: kotasudhakar79@gmail.com.

^∇For K.L.: College of Pharmacy, Korea University, 2511 Sejong-ro,

Sejong 339−700, Korea. E-mail: kiholee@korea.ac.kr.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by a grant from Ministry of

Education, Science and Technology, Korea (20082005585)

and a RP-Grant 2012 of Ewha Womans University.

ABBREVIATIONS USED

■

(AcO)₃BHNa, sodium triacetoxyborohydride; AcOH, acetic

acid; ALK5, activin receptor-like kinase 5; AST, aspartate

aminotransferase; AUC, area under curve; BID, twice daily;

t-BuOMe, tert-buty methyl ether; CCl₄, carbon tetrachloride;

CHO, Chinese hamster ovary; C_max, maximum concentration;

CTL, cytotoxic T-lymphocyte; CuCN, cuprous cyanide; DMF,

N,N-dimethylformamide; ECG, electrocardiogram; EMT, epi-

thelial−mesenchymal transition; Et₂O, diethyl ether; EtOAc,

ethyl acetate; EtOH, ethanol; FasL, fas ligand; FDA, food and

drug administration; FOB, functional observational battery;

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obstruction in adults but worsens injury in neonatal mice. J. Physiol.

Renal Physiol. 2013, 304, F481−F490.

(13) Moon, J.-A.; Kim, H.-T.; Cho, I.-S.; Sheen, Y. Y.; Kim, D.-K. IN-

1130, a novel transforming growth factor-β type I receptor kinase

(ALK5) inhibitor, suppresses renal fibrosis in obstructive nephropathy.

Kidney Int. 2006, 70, 1234−1243.

GFP, green ﬂuorescent protein; HEK, human embryonic

kidney; hERG, human Ether-a-go-go-related gene; HPLC, high

̀

performance liquid chromatography; IND, investigational new

drug; MeI, iodomethane; Me₂NH·HCl, dimethylamine hydro-

chloride; MeOH, methanol; MOLCAD, molecular computer-

aided design; mp, melting point; MPLC, medium pressure

liquid chromatography; NaBH₄, sodium borohydride; NaH,

sodium hydride; NaOH, sodium hydroxide; NBS, N-bromosucci-

nimide; NH₄OAc, ammonium acetate; NOAEL, no-observed-

adverse-eﬀect level; NOEL, no-observed-eﬀect level; PBr₃,

phosphorus tribromide; Pd/C, palladium/charcoal; PSS, physio-

logical salt solution; QD, once daily; rt, room temperature; RIPK2,

receptor-interacting protein kinase 2; SnCl₂, stannous chloride;

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Article Doi

Discovery of N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin- 2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline (EW-7197): A highly potent, selective, and orally bioavailable inhibitor of TGF-β type I receptor kinase as cancer immunotherapeutic/antifibrotic agent

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