Discovery of DS-6930, a potent selective PPARγ modulator. Part I: Lead identification

Accepted Manuscript

Discovery of DS-6930, a Potent Selective PPARγ Modulator. Part I: Lead Iden-

tification

Tsuyoshi Shinozuka, Tomoharu Tsukada, Kunihiko Fujii, Eri Tokumaru,

Kousei Shimada, Yoshiyuki Onishi, Yumi Matsui, Satoko Wakimoto, Masanori

Kuroha, Tsuneaki Ogata, Kazushi Araki, Jun Ohsumi, Ryoko Sawamura,

Nobuaki Watanabe, Hideki Yamamoto, Kazunori Fujimoto, Yoshiro Tani,

Makoto Mori, Jun Tanaka

PII:

S0968-0896(18)31308-7

https://doi.org/10.1016/j.bmc.2018.09.006

BMC 14531

DOI:

Reference:

Bioorganic & Medicinal Chemistry

To appear in:

Received Date:

Revised Date:

Accepted Date:

20 July 2018

3 September 2018

5 September 2018

Please cite this article as: Shinozuka, T., Tsukada, T., Fujii, K., Tokumaru, E., Shimada, K., Onishi, Y., Matsui, Y.,

Wakimoto, S., Kuroha, M., Ogata, T., Araki, K., Ohsumi, J., Sawamura, R., Watanabe, N., Yamamoto, H., Fujimoto,

K., Tani, Y., Mori, M., Tanaka, J., Discovery of DS-6930, a Potent Selective PPARγ Modulator. Part I: Lead

Identification, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.09.006

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers

we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and

review of the resulting proof before it is published in its final form. Please note that during the production process

errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Bioorganic & Medicinal Chemistry

Discovery of DS-6930, a Potent Selective PPARγ Modulator. Part I: Lead

Identification

Tsuyoshi Shinozuka,^*,†Tomoharu Tsukada,^†Kunihiko Fujii,^†Eri Tokumaru,^†Kousei Shimada,^†Yoshiyuki

Onishi,^†Yumi Matsui,^‡Satoko Wakimoto,^†Masanori Kuroha,^†Tsuneaki Ogata,^†Kazushi Araki,^†Jun

Ohsumi,^†Ryoko Sawamura,^†Nobuaki Watanabe,^†Hideki Yamamoto,^†Kazunori Fujimoto,^†Yoshiro Tani,^†

Makoto Mori^†and Jun Tanaka^†

^†R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan

^‡Daiichi Sankyo RD Novare Co., Ltd., 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan

ARTICLE INFO

ABSTRACT

Article history:

Received

Revised

Accepted

Available online

The lead identification of a novel potent selective PPARγ agonist, DS-6930 is reported. To avoid

PPARγ-related adverse effects, a partial agonist was designed to prevent the direct interaction

with helix 12 of PPARγ-LBD. Because the TZD group is known to interact with helix 12, the TZD

in efatutazone (CS-7017) was replaced to discover novel PPARγ intermediate partial agonist 8i.

The optimization of 8i yielded 13ac with high potency in vitro. Compound 13ac exhibited robust

plasma glucose lowering effects comparable to those of rosiglitazone (3 mg/kg) in Zucker diabetic

fatty rats. Upon toxicological evaluation, compound 13ac (300 mg/kg) induced hemodilution to a

lower extent than rosiglitazone; however, 13ac elevated liver enzyme activities. X-ray

crystallography revealed no direct interaction of 13ac with helix 12, and the additional lipophilic

interactions are also suggested to be related to the maximum transcriptional activity of 13ac.

Keywords:

PPARγ

DS-6930

benzimidazole

1. Introduction

phosphorylation inhibitory activity exhibited potent in vivo

efficacy in rodents,^13,14suggesting that PPARγ phosphorylation is

Type 2 diabetes (T2DM) is a global epidemic that is a leading

cause of death even in developing countries.¹Out of the estimated

425 million people with diabetes, 90% suffer from T2DM.¹It is an

economically debilitating disorder that critically affects national

healthcare budgets.¹Commercially marketed drugs for T2DM

include metformin, sulfonylureas, dipeptidyl peptidase-IV

inhibitors, sodium-dependent glucose co-transporter 2 inhibitors,

GLP-1 receptor agonists and peroxisome proliferator-activated

receptor γ (PPARγ) agonists.^2,3However, drugs with less adverse

effects remain to be identified.⁴Thiazolidinedione (TZD)-based

PPARγ full agonists, pioglitazone and rosiglitazone improve

insulin sensitivity by restoring plasma insulin and glucose levels

in T2DM patients.^5,6However, they are associated with adverse

effects, such as weight gain, peripheral edema, hepatotoxicity,

bone fracture, carcinogenicity and cardiovascular risks.^6-10These

adverse effects limit their usage. If such adverse effects are

avoided, PPARγ modulators present an attractive therapeutic

approach.¹¹

the underlying mechanism of antidiabetic efficacy, while PPARγ

agonist activity is related to adverse effects.¹⁵We adopted an

ordinary approach to tackle PPARγ-related adverse effects. We

believe there is still scope for the development of safer PPARγ

partial agonists by the regulation of cofactors. In this paper, we

report the lead identification of a novel potent selective PPARγ

agonist, DS-6930 (I, Figure 1) through conventional PPARγ

reporter assays.

We carried out this study based on TZD-based PPARγ full

agonists, such as rivoglitazone (CS-011, II, Figure 1)¹⁶,

efatutazone (CS-7017, III, Figure 1),¹⁷and non-TZD partial

PPARγ agonist, (-)-cercosporamide derivative (IV, Figure 1)^18-20

.

The stabilization of helix 12 dynamics of PPARγ-ligand binding

domain (LBD) is known to play a critical role in the PPARγ

transcriptional activity of PPARγ full agonists.^21,22TZD moiety of

PPARγ full agonists interacts directly with Tyr473 on helix 12.^21,22

On the other hand, (-)-cercosporamide derivative (IV) binds to

PPARγ-LBD without direct interaction to helix12.^18-20Such an

interaction is proposed to partially activate the PPARγ receptor,

which results in diminished PPARγ-related adverse effects

through the selective recruitment of cofactors.^18-20,23-29Several

PPARγ partial agonists are known to lack the direct interaction

with this helix.^23-29INT131 showed partially activated PPARγ

target genes without direct hydrogen bonding with helix 12.²³The

Although the particular mechanism of PPARγ agonism for

antidiabetic efficacy is not fully elucidated, the inhibition of

PPARγ phosphorylation by cyclin-dependent kinase 5 (Cdk5) is

reported to be involved in the mechanism of antidiabetic efficacy

of PPARγ modulators, rather than PPARγ transcriptional

activity.¹²Furthermore, non-PPARγ agonists with PPARγ

absence of interaction between benzimidazolone-based PPARγ

partial agonist and Tyr473 on helix 12 was also reported.^24,25In

both cases, mutagenesis studies on Tyr473 indicated that this

residue was nonessential for the transcriptional activity.^23-25

Another study concluded that partial agonists activate PPARγ

using a hexlix 12-independent mechanism.²⁹Consequently, the

avoidance of such interaction is expected to identify novel partial

agonists. Efatutazone is known as one of the most potent full

PPARγ agonists (EC₅₀: 0.038 nM). Derivatization of efatutazone

would lead to the discovery of partial agonists with enough in vitro

potency, even though replacement of the TZD group leads to a

significant drop in the activity. Thus, to acquire potent partial

PPARγ agonists with robust efficacy in vivo, our research initiated

with modification of the TZD moiety of III to prevent the direct

interaction with Tyr473 on helix 12.

Scheme 1. Synthesis of Compounds 8. Reagents and

conditions: (a) NaH, DMA; (b) H₂, Pd/C, EtOH; (c) glycolic acid,

HCl, 1,4-dioxane, 53% (3 steps); (d) Boc₂O, i-PrOH, 26%; (e)

ADDP, PBu₃, toluene; (f) HCl, 1,4-dioxane, 67% (2 steps, 7h),

63% (2 steps, 7i), 67% (2 steps, 7j), 53% (2 steps, 8a), 53% (2

steps, 8b), 87% (2 steps, 8c), 92% (2 steps, 8d), 68% (2 steps, 8e),

83% (2 steps, 8f), 92% (2 steps, 8g); (g) NaOH, 1,4-dioxane, H₂O,

60 °C, 66% (8h), 61% (8i), 77% (8j).

3-Benzoic acid derivatives 13 listed in Tables 2 and 3 were

synthesized using the same synthetic scheme as that used for

synthesizing compounds 8 (Scheme 2). Suitably substituted

phenols were reacted with nitrobenzene

1

to provide

corresponding nitro ethers 9. After reducing nitro group,

benzimidazoles 11 were obtained by the treatment of 10 with

glycolic acid under an acidic condition. Because several diamines

10 indicated instability towards chromatographic purification,

diamines 10 were converted to benzimidazoles 11 without

purification. After Mitsunobu reaction of benzimidazoles 11 with

methyl 3-hydroxybenzoate, the final saponification of methyl

esters 12 gave desired carboxylic acids 13a-13ae.

I; DS-6930

II; rivoglitazone (CS-011)

PPAR EC ₅₀: 43 nM

PPAR EC ₅₀: 41 nM

PPAR E _max: 68%

PPAR E _max: 126%

a

b

1

9

10

III; efatutazone (CS-7017)

PPAR EC ₅₀: 0.038 nM

IV; (-)-cercosporamide derivative

PPAR EC ₅₀: 12 nM

c

d

PPAR E _max: 100%

PPAR E _max: 64%

Figure 1. Structures of DS-6930 (I), rivoglitazone (CS-011, II),

efatutazone (CS-7017, III) and (-)-cercosporamide derivative (IV) with

PPARγ-agonist activities in COS-7 cells.

11

12: Y = Me

13: Y = H

e

Scheme 2. Synthesis of Compounds 13a-13ae. Reagents and

conditions: (a) NaH, DMF, 80 °C, 99% (9d), 99% (9g), 99% (9h),

99% (9i), 56% (9n), 92% (9r), 69% (9ab), 99% (9ac), 99% (9ad),

99% (9ae); (b) Fe, NH₄Cl, EtOH, H₂O, reflux; (c) glycolic acid,

HCl, 1,4-dioxane,H₂O, reflux, 73% (11a, 3 steps), 72% (11b, 3

steps), 80% (11c, 3 steps), 29% (11d, 2 steps), 72% (11e, 3 steps),

63% (11f, 3 steps), 88% (11g, 2 steps), 20% (11h, 2 steps), 78%

(11i, 2 steps), 61% (11j, 3 steps), 89% (11k, 3 steps), 87% (11l, 3

steps), 75% (11m, 3 steps), 91% (11n, 2 steps), 57% (11o, 3 steps),

75% (11p, 3 steps), 76% (11q, 3 steps), 83% (11r, 2 steps), 60%

(11s, 3 steps), 88% (11t, 3 steps), 69% (11u, 3 steps), 86% (11v, 3

steps), 85% (11w, 3 steps), 87% (11x, 3 steps), 70% (11y, 3 steps),

83% (11z, 3 steps), 77% (11aa, 3 steps), 58% (11ab, 2 steps), 57%

(11ac, 2 steps), 49% (11ad, 2 steps), 45% (11ae, 2 steps); (d)

ADDP, PBu₃, CH₂Cl₂, 66% (12a), 58% (12b), 86% (12c), 81%

(12d), 81% (12e), 85% (12f), 83% (12g), 97% (12h), 44% (12i),

89% (12j), 65% (12k), 75% (12l), 81% (12m), 91% (12n), 81%

(12o), 78% (12p), 84% (12q), 78% (12r), 60% (12s), 80% (12t),

86% (12u), 77% (12v), 65% (12w), 78% (12x), 49% (12y), 82%

(12z), 80% (12aa), 74% (12ab), 99% (12ac), 32% (12ad), 67%

(12ae); (e) NaOH, 1,4-dioxane, H₂O, reflux, 70% (13a), 68%

(13b), 93% (13c), 37% (13d), 98% (13e), 60% (13f), 68% (13g),

84% (13h), 89% (13i), 89% (13j), 89% (13k), 86% (13l), 86%

(13m), 81% (13n), 89% (13o), 86% (13p), 48% (13q), 91% (13r),

85% (13s), 82% (13t), 76% (13u), 89% (13v), 93% (13w), 48%

(13x), 95% (13y), 94% (13z), 81% (13aa), 73% (13ab), 72%

(13ac), 86% (13ad), 70% (13ae).

2. RESULTS and DISCUSSION

2.1. Chemical synthesis

4-Amino-3,5-dimethylphenoxy derivatives 8 (Table 1) were

synthesized by nucleophilic aromatic substitution as a key step

(Scheme 1). Nitrobenzene 1 was reacted with 4-amino-3,5-

dimethylphenol to afford nitro ether 2, and reduced under a

hydrogenolytic condition to yield diamine 3. Reacting the

unpurified compound 3 with glycolic acid under an acidic

condition afforded benzimidazole 4. In this reaction scheme, the

protection of the amino group of 4-amino-3,5-dimethylphenoxy

moiety was not required due to the low nucleophilicity of the

hindered amino group. However, the protection of this amino

group was essential for the subsequent Mitsunobu reaction

conducted with a variety of phenols to yield compounds 6. Finally,

BOC group was deprotected to afford target molecules 8a–8g.

Carboxylic acids 8h–8j were synthesized by hydrolyzing esters

7h–7j.

a

c

1

2: Y = NO ₂

3: Y = NH ₂

b

e

Bicyclic ring analogues 13af–13ah listed in Table 3 were

synthesized from amides 15 as intermediates (Scheme 3).

4: P = H

5: P = Boc

6a-6g: P = Boc

8a-8g: P = H

d

f

Diamines

10

were

coupled

with

[3-

6h-6j: P = Boc, X = CO ₂Me or CO ₂Et

f

7h-7j: P = H, X = CO ₂Me or CO ₂Et

(methoxycarbonyl)phenoxy]acetic acid (14) under the usual

condition to provide amides 15. Benzimidazoles 12 were obtained

g

8h-8j: P = H, X = CO ₂

H

by the treatment of amides 15 with HCl. The saponification of

methyl esters 12 provided carboxylic acids 13af-13ah.

8g

8h

8i

4-CN

395

6094

299

797

79

52

73

91

2-CO₂H

3-CO₂H

4-CO₂H

a

b

8j

^aLuciferase activity in COS-7 cells after treatment with the test

compound. Values on single experiment run in octuplicate unless

otherwise noted. HCl salts of all the test compounds were used.

^bValue represented as mean ± S.E.M. Value on five independent

experiments run in octuplicate.

1

9

10

c

d

The modifications of left-hand side phenyl ring in compound 8i

are summarized in Table 2. The removal of all substituents from

this phenyl ring only led to a 2.8-fold reduction in potency in vitro

(Compound 13a). This modification enhanced the maximum

transcriptional activity due to unclear reasons (E_max= 90%). In

vitro ADME profiling showed that this modification decreases

solubility, while both compounds 8i and 13a exhibited high

stability against human liver microsomes. SAR studies were

performed to identify compounds with high potency and low

maximum transcriptional activity. The introduction of a fluorine

atom at positions 3 and 4 increased the potencies by several folds

retaining the maximum transcriptional activities (Compounds 13c

and 13d), while 2-fluoro derivative 13b showed diminished

potency. The microsomal stability of 3-fluoro-substituted

compound 13c was robust. Upon evaluation of selectivity over

PPARα, fluoro-substituted compounds 13c and 13d were found to

exhibit high selectivity over PPARα; both compounds caused less

than 50% activation of PPARα at 10 μM. This series of compounds

caused no PPARδ transcriptional activity at 10 μM (Data not

shown). These compounds were then evaluated for preliminary in

vivo efficacy. Plasma glucose (PG) lowering effects in

hyperglycemic KK/Ta mice were assessed after they were

administered 0.03% (ca. 30 mg/kg/day, assuming constant food

intake) of the compounds through their diets. After 3 days, abilities

to induce PG reduction (% change vs vehicle control) and body

weight gain (% change vs vehicle control) as well as plasma

concentrations of the test compounds were determined (n = 3).

Both compounds 13c and 13d significantly reduced PG levels in

KK/Ta mice (52.1 and 56.7%, respectively vs vehicle control).

They also increased body weight by 3–4%, similar to full

agonists.³⁰Despite its modest potency in vitro, 3-fluoro derivative

13c exhibited potent in vivo efficacy due to its high plasma

concentration. Methyl substitution enhanced in vitro potency by

over ten-fold, and all methyl substituted compounds 13e–13g had

EC₅₀values less than 100 nM. Among these methyl derivatives, 3-

and 4-substituted compounds (13f and 13g) significantly reduced

PG levels in KK/Ta mice. 2-Methyl derivative 13e failed to exhibit

potent in vivo efficacy, despite showing in vitro potency and

plasma exposure comparable to those of 3-methyl derivative 13f.³¹

Among methyl substituted derivatives, 2- and 3-substituted

compounds (13e and 13f) exhibited modest microsomal stability,

while 4-derivative 13g displayed high microsomal stability. A

high in vitro potency was also observed in 3- or 4-chloro

substituted compounds (13h and 13i). 4-Chloro derivative 13i

indicated excellent selectivity over PPARα, while 3-chloro

analogue 13h exhibited remarkably enhanced PPARα activity.

Methoxy substitutions were not found to be suitable for good in

vitro potency (Compounds 13j–13l), while 3-ethoxy derivative

13m showed high in vitro potency. Nevertheless, 13m failed to

exhibit potent in vivo efficacy in KK/Ta mice. Because 3-

substitution was one of the optimal substation patterns for in vitro

potency, several 3-substituted derivatives were synthesized to find

out 3-trifluoromethoxy and 3-trifluoromethyl substituted

compounds 13n and 13o, which exhibited high potencies in vitro

as well as in vivo. Similar to 3-chloro analogue 13h, a diminished

12: Y = Me

13: Y = H

e

15

14

Scheme 3. Synthesis of Compounds 13af-13ah. Reagents and

conditions: (a) NaH, DMF, 80 °C, 27% (9af); (b) Fe, NH₄Cl,

EtOH, H₂O, reflux; (c) WSC•HCl, HOBt•H₂O, CH₂Cl₂, 86%

(15af, 2 steps), 94% (15ag, 3 steps), 93% (15ah, 3 steps); (d) HCl,

EtOAc, 60 °C, 84% (12af), 47% (12ag), 79% (12ah); (e) NaOH,

1,4-dioxane, H₂O, reflux, 63% (13af), 74% (13ag), 98% (13ah).

2.2. In vitro activity, in vitro ADME properties and in vivo

efficacy in KK/Ta mice

PPARγ transcriptional activities were evaluated by GAL4-

PPAR-LBD reporter gene assays using COS-7 cells. The

maximum transcriptional activity (E_max) of each test compound

was calculated relative to that of rosiglitazone (100%). As shown

in Table 1, PPARγ full agonist rosiglitazone exhibited EC₅₀= 396

nM. To prevent the direct interaction with Tyr473 on helix 12, the

removal of TZD-methyl group from III was examined, which

resulted in several thousand-fold reduction in potency in vitro

(Compound 8a, EC₅₀= 318 nM). As expected, 8a closed to a

partial agonist (E_max= 73%). Therefore, we decided to introduce

several small functional groups to avoid the direct interaction with

Tyr473. When methyl ketone, nitrile or carboxylic acid was

introduced in the right-hand side benzene ring, 3-position was

found to be optimal for the potency. 3-Subtituted ketone 8c and

nitrile 8f exhibited 4.9- and 7.4-fold higher potencies, respectively,

than that of 8a. Compounds 8c (E_max= 86%) and 8f (E_max= 92%)

assumed a full agonist, while 3-carboxylic acid derivative 8i

closed to a partial agonist (E_max= 73%). Accordingly, 8i was

further modified to enhance its potency in vitro.

Table 1. PPARγ Transcriptional Activities of Compounds 8^a

2

3

4

Compound

X

EC₅₀(nM)

396 ± 133^b

E_max(%)

100

rosiglitazone

4-

III

0.038

100

8a

8b

8c

8d

8e

8f

H

318

356

65

73

96

86

70

80

92

2-COMe

3-COMe

4-COMe

2-CN

340

391

43

3-CN

selectivity over PPARα was observed in 3-trifluoromethyl

derivative 13o.

Table 2. PPAR Transcriptional Activities, In Vitro ADME Profiles and In Vivo Efficacies of Compounds 13

3

4

5

2

6

In vivo efficacies in KK/Ta mice^f

PPARγ

PPARα PPARα

PPARγ

Solubility

Human

Compound

X

Log D^c

1.4

PG

Body

Plasma

MS (%)^e

EC₅₀

E_max

(%)^b

E_max(%)^a

(μg/mL) ^d

reduction

(%)

weight

gain (%) (μg/mL)

concentration

(nM)^a

(nM)^b

3-Me, 4-

NH₂, 5-

Me

8iⁱ

299

73

NT^g

11

88

NT^g

13aⁱ

13b

H

845

90

1.8

1.7

2.0

12

100

NT^g

2-F

1434

125

>10000 21

>10000 22

52.1 ±

9.1**

3.2 ±

0.97

13c

13d

13e

13f

3-F

201

122

56

75

80

76

73

103

62

2.0

2.4

2.3

2.4

2.7

2.6

2.2

19

2.2

3.8

7.9

0

98

1.74 ± 0.87

56.7 ±

6.4**

4-F

>10000 29

>10000 16

>10000 19

>10000 28

NT^g

62

3.4 ± 2.2 0.32 ± 0.092

26.9 ±

12.3

3.7 ±

2-Me

3-Me

4-Me

3-Cl

4-Cl

0.10 ± 0.005

0.69

56.9 ±

0.85**

0.090 ±

3.2 ± 1.2

79

69

0.022

48.4 ±

9.7*

13g

13hⁱ

13i

40

91

4.8 ± 1.1 0.51 ± 0.051

2.8 ± 1.4 0.19 ± 0.020

4.1 ± 1.2 ND^h

59.6 ±

3.1**

86

ND^h

69

90

34.4 ±

10.7*

126

>10000

8

4

2.0

NT^g

13jⁱ

13k

13l

2-OMe

3-OMe

4-OMe

474

235

307

60

92

71

1.3

1.8

12

74

NT^g

>10000 30

>10000

5.4

9.3

68

7

100

22.0 ±

4.9

13m

13n

13oⁱ

3-OEt

3-OCF₃

3-CF₃

40

56

69

81

>10000 37

>10000 17

2.4

3.3

2.8

0

NT^g

98

3.3 ± 3.1 ND^h

41.9 ±

4.1**

113

23

0.9

0

5.3 ± 2.3 0.37 ± 0.018

45.1 ±

16.6*

0.090 ±

3.8 ± 1.7

ND^h

56

85

0.031

b

^aLuciferase activity in COS-7 cells after treatment with the test compound. Values on single experiment run in octuplicate. PPARα

activity (%) in COS-7 cells. Values on single experiment run in octuplicate. The maximum transcriptional activity (E_max) of the test

compound is expressed relative to that of the reference compound as 100%.^{32 c}Distribution coefficients (Log D) were measured based on

d

partition between 1-octanol and PBS (pH = 7.4). Aqueous thermodynamic solubility at pH 6.8. Values on single experiment run in

duplicate. ^eHuman microsomal stability assessed based on test compound (%) remaining after 0.5 h of incubation with human liver

f

microsomes. PG reduction (% change in PG level vs vehicle control), body weight gain (% change in body weight vs vehicle control)

and plasma concentration (μg/mL) of the test compounds in hyperglycemic KK/Ta mice after the oral administration of 0.03% (ca. 30

mg/kg/day, assuming constant food intake) of the test compounds through their diet on day 3 (n = 3). Data are represented as mean ±

S.E.M. Statistical significance compared to vehicle treatment is denoted by *p < 0.05 and **p < 0.01 as determined by the Student T-test.

The plasma concentration of the test compound was measured on day 3 (n = 3). Each value represents the mean ± S.D. ^gNot tested. ^hNot

determined. ⁱHCl salt.

To further enhance in vitro potency and in vivo efficacy,

another substituent was introduced on the left-hand side phenyl

ring as shown in Table 3. Initially, compounds were tested for

synergic effects with difluoro derivatives. When another fluorine

atom was introduced at positions 3, 4, 5, or 6 on benzene ring of

2-fluoro derivative 13b, 2,3-, 2,4- and 2,5-substitution

(Compounds 13p–13r) indicated severalfold improved potencies,

whereas 2,6-substituted compound 13s did not exhibit an

improved potency. Further enhancement of the potency was

achieved in 3,4- and 3,5-substituted compounds (Compounds 13t

and 13u). Among difluoro-substituted compounds, 2,5- and 3,4-

difluoro derivatives (13r and 13t) exhibited excellent in vivo

efficacies in KK/Ta mice. In terms of PPARα selectivity, only 2,3-

difluoro derivative 13p indicated diminished selectivity. Because

2-methyl substitution significantly enhanced in vitro potency

(Compound 13e in Table 1), 2-methyl substituted 4- or 5-fluoro

derivatives (13v and 13w) were synthesized to find excellent

potencies in vitro (EC₅₀= 22 and 39 nM, respectively). Compound

13v exhibited potent PG reduction in KK/Ta mice, while 13w did

not. Although 13v indicated robust in vivo efficacy, modest

microsomal stability was still an issue. 2-Fluoro-substituted 4-

methyl and 5-methyl derivatives (Compounds 13x and 13y)

showed less potencies in vitro with better microsomal stabilities

than compounds 13v and 13w. Both 13x and 13y showed potent

in vivo efficacies. To further enhance in vitro potencies of 3,4- and

3,5-substituted derivatives, a methyl group was incorporated. As

expected, 3-fluoro-4-methyl derivative 13z exhibited excellent

potency in vitro (EC₅₀= 4.4 nM) with modest partial agonist

affinity (E_max= 80%). Compound 13z showed over 50% PG

reduction in vivo. Moreover, the 3-fluoro-4-methyl substitution

enhanced PPARγ potency without PPARα activation. 3-Methyl-4-

fluoro derivative 13aa and 3-fluoro-5-methyl derivative 13ab also

exhibited excellent potencies in vitro. Both compounds yielded the

same plasma exposures in KK/Ta mice and reduced PG to the

same extent. It should be noticed that 13aa showed moderate

microsomal stability (69%), while 13ab possessed robust stability

(100%). Because the incorporation of chloro group was expected

to increase in vitro potency similar to methyl group (Compounds

13f and 13h, Table 2), a chloride atom was incorporated to yield

compounds 13ac–13ae; they exhibited excellent potencies in vitro.

3-Fluoro-4-chloro derivative 13ac and 3-fluoro-5-chloro analogue

13ad not only exhibited strong potencies in vitro, but also robust

efficacies in KK/Ta mice. 3-Chloro-4-fluoro derivative 13ae

exhibited attenuated in vitro potency and in vivo efficacy. The

enhancement of PPARα activity in 13ae may relate to the 3-chloro

substituent, because enhanced PPARα activity was observed in 3-

chloro analogue 13h. Because 3,4-disubstituted compounds

possessed optimal characteristics, fused ring derivatives were

synthesized. Dihydrobenzofurans 13af and 13ag retained high

potencies in vitro. Compound 13ag exhibited E_max= 95% due to

unclear reasons. Although 13ag exhibited potent in vitro potency

as well as in vivo efficacy, a moderate microsomal stability was

observed. The aromatization of 13ag provided benzofuran 13ah,

which showed attenuated potency in vitro with high microsomal

stability.

Table 3. PPAR Transcriptional Activities, In Vitro ADME Profiles and In Vivo Efficacies of Compounds 13

3

4

5

2

6

In vivo efficacies in KK/Ta mice^f

PPARγ

PPARα

PPARγ

Solubility

Body

weight

gain

Human

PG

reduction

(%)

Plasma

Compound

X

Log D^c

MS (%)^e

EC₅₀

E_max

(%)^b

E_max(%)^a

(μg/mL) ^d

concentration

(μg/mL)

(nM)^a

(nM)^b

(%)

35.8 ±

12.9

4.3 ±

1.4

13p

13q

2-F, 3-F 230

2-F, 4-F 143

86

77

ND^h

52

17

2.1

1.9

0

88

ND^h

37.6 ±

9.6*

3.7 ±

0.92

>10000

11

100

0.37 ± 0.074

51.2 ±

4.7**

3.3 ±

1.7

13r

13s

13tⁱ

2-F, 5-F 134

2-F, 6-F 1326

3-F, 4-F 118

79

76

71

>10000

36

19

39

1.9

1.8

2.0

5.7

NT^g

0

98

0.70 ± 0.67

NT^g

85

NT^g

56.0 ±

3.6**

3.4 ±

1.4

100

0.16 ± 0.017

39.5 ±

11.7*

4.4 ±

2.3

13u

3-F, 5-F 95

54

>10000

33

19

17

16

37

40

28

29

2.3

2.5

2.3

2.4

2.7

2.3

2.7

10

1.7

0.6

0.5

11

4.4

0

NT^g

59

0.36 ± 0.11

0.19 ± 0.048

ND^h

2-Me,

22

54.7 ±

5.0**

2.8 ±

2.9

13v

66

4-F

2-Me,

39

26.4 ±

7.4

5.1 ±

2.7

13w

13x

84

64

5-F

2-F, 4-

92

53.4 ±

10.1**

2.5 ±

3.1

90

92

0.55 ± 0.140

0.12 ± 0.039

0.47 ± 0.11

0.19 ± 0.010

0.19 ± 0.041

0.57 ± 0.23

0.37 ± 0.064

0.17 ± 0.021

Me

2-F, 5-

139

49.4 ±

18.4*

1.6 ±

2.4

13y

111

80

83

Me

3-F, 4-

4.4

56.8 ±

1.2**

4.2 ±

1.8

13z

91

Me

3-Me,

62

49.4 ±

6.7**

3.5 ±

1.5

13aaⁱ

13ab

13ac

13ad

13aeⁱ

61

69

4-F

3-F, 5-

21

58.3 ±

3.1**

1.7 ±

2.4

84

0.5

0

100

78

Me

3-F, 4-

Cl

43.2 ±

6.7**

4.4 ±

3.0

68 ± 28 ^j

82 ± 4.0^j

78

>10000^j32 ± 2.7^j2.7

3-F, 5-

Cl

57.5 ±

4.2**

3.1 ±

2.9

23

64

>10000

ND^h

43

52

2.8

0.9

0

3-Cl, 4-

F

30.0 ±

14.4

3.7 ±

3.4

79

NT^g

13afⁱ

87

60

>10000

8

1.7

7.6

87

NT^g

41.6 ±

7.3**

4.8 ±

3.6

13ag

13ah

21

95

79

>10000

12

37

2.0

2.4

16

62

0.36 ±0.058

0.38 ± 0.060

48.3 ±

10.5**

2.5 ±

1.7

188

0.8

100

^aLuciferase activity in COS-7 cells after treatment with the test compound. Values on single experiment run in octuplicate unless

b

otherwise noted. PPARα activity (%) in COS-7 cells. Values on single experiment run in octuplicate unless otherwise noted. The

maximum transcriptional activity (E_max) of the test compound is expressed relative to that of the reference compound as 100%.³²

d

^cDistribution coefficients (Log D) were measured after partition between 1-octanol and PBS (pH = 7.4). Aqueous thermodynamic

e

solubility at pH 6.8. Values on single experiment run in duplicate. Human microsomal stability. Test compound (%) remaining after a

0.5 h of incubation with human liver microsomes. ^fPG reduction (% change in PG level vs vehicle control), body weight gain (% change

in body weight vs vehicle control) and plasma concentration (μg/mL) of the test compounds in hyperglycemic KK/Ta mice after the oral

administration of 0.03% (ca. 30 mg/kg/day assuming constant food intake) of compounds through their diet on day 3 (n = 3). Data are

represented as mean ± S.E.M. Statistical significance compared to vehicle treatment is denoted by *p < 0.05 and **p < 0.01 as determined

by the Student T-test. The plasma concentration of the test compound was measured on day 3 (n = 3). Each value represents the mean ±

g

h

i

j

S.D. Not tested. Not determined. HCl salt. Values represented as mean ± S.E.M. Values on 13 independent experiments run in

octuplicate.

2.3. In Vivo Efficacy in ZDF Rats

body weight gain (vs vehicle control) similar to the full agonist,

rosiglitazone. Compounds 13g and 13r increased the body weight

of ZDF rats by 13.0 and 9.5%, respectively, whereas rosiglitazone

increased the body weight by 12.6%. Although compound 13v

exhibited potent PG reduction in KK/Ta mice (Table 3), 13v

suffered from poor efficacy in ZDF rats due to low plasma

exposure. Compounds 13x, 13z and 13ag also significantly

reduced PG levels. Despite their similar effects on PG levels,

compounds 13z and 13ag induced relatively high body weight

gains (13.8 and 15.6%, respectively, vs vehicle control), while 13x

moderately increased the body weight (8.5% vs vehicle control).

Note that compound 13ag indicated a full agonist affinity. When

compound 13ac was administered at 0.3, 3 and 30 mg/kg, a clear

PK/PD correlation was observed. In this assessment, body weight

was also increased in a dose-dependent manner.

Several compounds were selected for further pharmacological

profiling. These compounds were assessed for their abilities to

reduce PG in Zucker diabetic fatty (ZDF) rats after administration

at 3 mg/kg/day p.o. for 14 days as shown in Table 4 (n = 5). After

this treatment duration, an additional administration was

performed to evaluate PK parameters (Table 4). The

administration of rosiglitazone reduced PG levels by 40.3% vs

vehicle control. Compounds 13g and 13r indicated robust stability

against rat liver microsomes, and were expected to exhibit potent

efficacy in ZDF rats because both compounds already significantly

reduced PG levels in KK/Ta mice. As expected, both compounds

exhibited excellent efficacies in ZDF rats (58.6 and 61.2% PG

reduction, respectively, vs vehicle control) with statistical

significance. Administration of this series of compounds caused

Table 4. Plasma Glucose (PG) Reduction (%) in Zucker Diabetic Fatty (ZDF) Rats After the Oral administration of the Test

Compounds on Day 14 with PK parameters (n = 5)

In vivo efficacies in ZDF rats^b

PK parameters in ZDF rats^c

Rat

Compound

microsomal

Body weight

gain (%)

Dose (mg/kg)

PG reduction (%)

C_max(g/mL)

T_max(h)

AUC_{0-24 h}(h·g/mL)

stability (%)^a

rosiglitazone

13g

NT^d

100

95

3

40.3 ± 16.2

12.6 ± 3.3*

13.0 ± 2.4**

9.5 ± 1.5**

11.9 ± 2.8*

8.5 ± 2.9*

ND^e

3

58.6 ± 8.8**

61.2 ± 10.1*

22.0 ± 17.9

1.11 ± 0.11

0.52 ± 0.05

0.042 ± 0.01

0.30 ± 0.03

0.79 ± 0.11

0.18 ± 0.13

0.92 ± 0.33

8.08 ± 1.78

0.080 ± 0.01

2.00 ± 0.00

4.00 ± 0.00

2.40 ± 0.89

2.00 ± 0.00

6.80 ± 9.65

2.40 ± 0.89

2.00 ± 0.00

3.20 ± 1.10

7.10 ± 0.75

3.87 ± 0.29

0.42 ± 0.04

3.16 ± 0.39

5.53 ± 0.83

1.74 ± 0.43

6.75 ± 0.85

55.77 ± 16.70

0.85 ± 0.06

b

13r

3

13v

47

3

13x

98

3

61.7 ± 10.2*

59.3 ± 10.9**

16.6 ± 12.9

13z

100

94

3

13.8 ± 2.1**

2.3 ± 2.9

13ac

13ag

0.3

3

94

43.8 ± 13.8**

74.9 ± 0.8**

69.7 ± 0.9**

12.4 ± 3.1**

16.7 ± 1.5**

15.6 ± 1.6**

94

30

3

79

^aRat microsomal stability assessed based on test compound (%) remaining after 0.5 h of incubation with rat liver microsomes. PG

reduction (% change in PG level vs vehicle control) and body weight (% change in body weight vs vehicle control) in ZDF rats after the

oral administration of 3 mg/kg of the test compounds in 0.5% methylcellulose on day 14. Data are represented as mean ± S.E.M. Statistical

significance compared to vehicle treatment is denoted by *p < 0.05 and **p < 0.01 as determined by the Student T-test. ^cPK parameters

were acquired after the administration of the test compounds to ZDF rats on day 15. Each value represents the mean ± S.D. ^dNot tested.

^eNot dertermined.

2.4. Monkey PK Profile

compounds as shown in Table 5. The selected compounds were

orally administered to male cynomolgus monkeys at 3 mg/kg (n =

2-3). Total body clearance (CL), distribution volume at steady state

(V_ss) and F value (%) were calculated after intravenous

administration (1 mg/kg) of the compounds to the same monkeys.

Based on pharmacological results in rodents, Compounds 13g,

13r, 13x, 13z and 13ac were selected as candidates for further

evaluation.³³Monkey PK study was performed with these

All compounds exhibited ideal T_1/2values for once daily

administration in clinical studies. One of the reasons for this

excellent half-life is the robust stability against monkey liver

microsomes (Table 5). 4-Methyl-substituted derivatives 13g, 13x

and 13z exhibited higher CL values than other compounds.

Moreover, 13g and 13x exhibited higher V_ssand lower AUC

values; nevertheless, these PK parameters were acceptable for

clinical candidate selection. Halogenated compounds 13r and

13ac exhibited excellent PK parameters with the lowest CL and V_ss

as well as the highest AUC values, despite the dosage being only

3 mg/kg. Consequently, compound 13ac with higher in vitro

potency was selected for further toxicological evaluation.

Table 5. PK Parameters of the test Compounds in Cynomolgus Monkeys^a

Compd

Monkey

microsomal

stability

(%)^b

C_max(μg/mL) T_max(h)

T_1/2(h)

AUC_last

(h·μg/mL)

F (%)

CL

V_ss(L/kg)

(mL/min/kg)

13g^c

13r^c

100

1.02 ± 0.12

5.63 ± 1.9

1.00 ± 0.0

10.4 ± 3.1

16.1 ± 4.7

4.87 ± 0.54

40.9 ± 7.0

34 ± 3.9

65 ± 11

3.58 ± 0.52

0.80 ± 0.014

4.16 ± 0.62

0.56 ± 0.14

0.39 ± 0.014

1.23 ± 0.46

1.00 ± 0.0

0.33 ±

0.0071

1.50 ± 0.71

16.1 ±

0.21

2.87 ± 0.071

24 ± 0.64

13x^c

13z^c

91

2.06 ± 0.11

4.44 ± 1.5

1.50 ± 0.71

1.67 ± 0.6

13.8 ± 9.6

18.3 ± 6.6

8.44 ± 2.9

40.2 ±4.3

28 ± 9.8

25 ± 2.7

1.80 ± 0.66

0.32 ± 0.028

0.20 ± 0.090

13ac^d

100

0.32 ± 0.035

^aThe test compounds in 0.5% methylcellulose were administered to male cynomolgus monkeys at 3 mg/kg (p.o.). Total body clearance

(CL), distribution volume at steady state (V_ss) and F value were calculated after intravenous (1 mg/kg) administration of the test

compounds. Each value represents the mean ± S.D. ^bMonkey microsomal stability assessed based on test compound (%) remaining after

0.5 h of incubation with monkey liver microsomes. ^cn = 2. ^dn = 3.

with hemodilution.^18,24,36The administration of rosiglitazone

significantly increased the weight of the heart (% of body weight)

at 30 mg/kg (p < 0.05) and higher doses (p < 0.01). Compound

13ac significantly increased the weight of the heart (p < 0.05) at

300 mg/kg, which was the maximum dose studied (Figure 2c). The

heart weight gain after the administration of 13ac at 300 mg/kg

(9.9%) was lower than that after rosiglitazone administration at

100 mg/kg (24.7%) with statistical significance (p < 0.05). These

results clearly demonstrate that 13ac causes PPARγ-related

adverse effects; however, these effects are milder than those of

rosiglitazone. Nevertheless, compound 13ac elevated the activities

of liver enzymes. As shown in Figure 2d, it elevated aspartate

transaminase (AST) activity but without clear dose-dependency.

Alanine transaminase (ALT) activity was also elevated (data not

shown). These elevations in liver enzyme activities were not

accompanied with any histopathological changes. Although these

changes in liver enzyme activities by 13ac were modest, 13ac was

considered hepatotoxic because rosiglitazone did not affect liver

enzyme activities in this study. Therefore, further derivatization is

deemed essential to avoid such adverse effects.

2.5. In Vivo Toxicological Evaluation of Compound 13ac

In vivo toxicological results of 13ac are summarized in Figure

2. Adverse effects were assessed after repeated oral administration

of 13ac (30, 100 and 300 mg/kg/day) to female Wistar–Imamichi

rats for 28 days. Rosiglitazone administration served as control.

TK parameters were obtained on day 28 from a satellite group of

female rats as shown in Table 6 (n = 2).³⁴At the pharmacologically

effective dose of 0.3 mg/kg (C_max, 0.18 μg/mL, AUC_{0–24 h}, 1.74

h·μg/mL), a high plasma exposure of 13ac was observed. No death

or clinical abnormalities were observed during the administration

period in any test group. As shown in Figure 2a, rosiglitazone

significantly (p < 0.01) reduced the number of red blood cells

(RBCs) to cause hemodilution in a dose-dependent manner (5.9,

8.9 and 14.2% reduction at 30, 100 and 300 mg/kg, respectively).³⁵

On the other hand, no remarkable change in RBC counts was

observed in 13ac-administered groups. At 300 mg/kg, compound

13ac affected RBC counts; however, they were not statistically

significant. Similar results were obtained after hematocrit analysis

(Figure 2b). The weight of the heart is reported to be associated

a)

b)

c)

d)

Figure 2. In vivo toxicological evaluation of compound 13ac. (a) RBC numbers, (b) hematocrit (%), (c) heart weight (% of body

weight), and (d) aspartate aminotransferase (AST) level in Wistar–Imamichi rats after repeated administration of 13ac or

rosiglitazone (30, 100 and 300 mg/kg) for 4 weeks. Data are represented as mean ± S.D. (n = 5). Statistical significance

compared to vehicle treatment is denoted by *p < 0.05, **p < 0.01 as determined by the Student T-test.

Table 6. TK Parameters of 13ac (n = 2)^a

Dose

C_max

T_max(h)

AUC_{0-24 h}

(mg/kg/day)

(μg/mL)

(h·μg/mL)

30

9.1 ± 2.0

14.9 ± 3.2

22.6 ± 13

4.0 ± 0.0

5.0 ± 4.2

3.0 ± 1.4

93.7 ± 7.6

192 ± 26

190 ± 57

100

300

a

TK parameters were acquired from a satellite group of female Wistar–Imamichi rats on day 28. Each value represents the mean ±

S.D.

2.6. X-ray crystal structure of 13ac bound to PPARγ-LBD

observed in rosiglitazone, they were considered responsible for the

more potent pharmacological activities of 13ac than rosiglitazone.

The binding mode of 13ac to PPARγ-LBD was determined at

1.8 Å resolution with Rcryst and Rfree values of 0.204 and 0.229,

respectively (Figure 3). The overall structure adopted a canonical

three-layered α-helical sandwich structure, which is typical of a

nuclear receptor LBD (Figure 3a). Compound 13ac utilizes the

same ligand-binding pocket as rosiglitazone. As shown in Figure

3b, 13ac forms hydrophobic and hydrophilic interactions with

PPARγ-LBD, including two direct hydrogen bonds, two water-

mediated hydrogen bonds and van der Waals contacts. The

benzoic acid group of 13ac forms direct hydrogen bonds with side

chains of Tyr327 and Lys367 as well as a water-mediated

hydrogen bond with Ser289. 1-Methylbenzimidazole moiety of

13ac forms a water-mediated hydrogen bond with the main-chain

nitrogen of Ser342. Additional lipophilic interactions of 4-chloro-

3-fluorophenoxy group in 13ac were observed. 4-Chloro-3-

fluorophenoxy group occupies an additional binding site

surrounded by a β-sheet, helix 2’ and helix 3, and makes lipophilic

contacts with Ile249, Leu255, Gly258, Glu259, Ile262, Arg280,

Ile341 and Met348. Because such lipophilic interactions were not

Typical PPARγ full agonists stabilize the PPARγ LBD in active

conformation by interacting with Tyr473 on helix 12,^21,22while

several PPARγ partial agonists that selectively regulate the

recruitment of cofactors lack the direct interaction with helix

12.^23,24,27Full agonist rosiglitazone forms a tight direct hydrogen

bond with Tyr473 (Figure 3c).^21,37On the other hand, the crystal

structure of PPARγ-LBD with 13ac shows that this series of

compounds also do not directly interact with helix12 (Figure 3d).

Since modifications of the left-hand side phenoxy group provide

compounds with a variety of agonist efficacy (Emax = 54-125%),

the lipophilic interactions of substituted phenoxy group with β-

sheet, helix 2’ and helix 3 may relate to the magnitude of agonist

efficacy. Therefore, avoiding the interaction with Tyr473 and the

additional lipophilic interactions of substituted phenoxy group

may relate to the maximum transcriptional activity of 13ac.³⁸The

superior safety profile of 13ac was caused due to this distinct

binding mode through the selective recruitment of cofactors. It was

later confirmed with the same molecular template DS-6930 (I,

Figure 1), which is disclosed in a subsequent paper.

a)

b)

c)

d)

Figure 3. X-ray crystal structure of compound 13ac bound to PPARγ-LBD (PDB 5Z5S). (a) Schematic representation of

PPARγ-LBD with 13ac. Helix 3 (residues 277–303) and helix 12 (residues 467–477) of PPARγ-LBD and PGC-1 peptide,

respectively, are colored in yellow, magenta and green. Other parts of the PPARγ-LBD are colored in gray. The bound form of

13ac is depicted as a CPK model with carbon atoms in cyan, oxygen atoms in red and nitrogen atoms in blue. (b) Details of the

binding model of 13ac to PPARγ-LBD. Residues of PPARγ-LBD involved in the binding of 13ac, residue 473 on helix 12 and

13ac are shown as stick models. Hydrogen bonds are marked as red dotted lines. (c) The binding mode of rosiglitazone to

PPARγ-LBD. A tight hydrogen bond with Tyr473 was observed (PDB 2PRG). (d) The binding mode of 13ac to PPARγ-LBD.

13ac lacks the direct interaction with Tyr473.

robust pharmacological activities of 13ac with fewer PPARγ-

related adverse effects. Upon toxicological evaluation, compound

13ac was found to cause modest hepatotoxicity. Therefore, we

further sought to synthesize compounds with minimal

hepatotoxicity, which will be reported in a proceeding paper.

3. CONCLUSIONS

By modifying TZD moiety of full agonist efatutazone (CS-

7017), a novel PPARγ agonist 8i was designed such that it lacks

direct interaction with Tyr473 on helix 12 of PPARγ-LBD. The

left-hand side benzene ring in compound 8i was optimized to yield

13ac, which exhibited high in vitro potency. The positions of

halogen atoms dictated pharmacological activity; 3-chloro-4-

fluoro analog 13ae exhibited attenuated activity in vitro and in

vivo. Compound 13ac demonstrated robust PG lowering effects

similar to rosiglitazone in ZDF rats, whereas 13ac caused fewer

PPARγ-related adverse effects, including hemodilution in Wistar–

Imamichi rats. The analysis of the binding mode of compound

13ac with PPARγ-LBD revealed no direct interaction between

13ac and Tyr473 on helix 12. In addition, the lipophilic

interactions of 4-chloro-3-fluorophenoxy group of 13ac were

observed. This binding mode is proposed to be the reason for

4. EXPERIMENTAL SECTION

4.1. General Procedures

Starting reagents were purchased from commercial suppliers

and were used without further purification unless otherwise

specified. Chromatographic elution was carried under continuous

monitoring by TLC using silica gel 60F254 (Merck & Co., Inc.) as

the stationary phase; the mobile phase was the elution solvent used

in column chromatography. A UV detector was used for detection.

Silica gel SK-85 (230–400 mesh) or silica gel SK-34 (70–230

mesh), manufactured by Merck & Co., Inc., or Chromatorex NH

(200–350 mesh), manufactured by Fuji Silysia Chemical Ltd., was

used as the column packing silica gel. H NMR spectra were

A solution of 7 or 12 (0.400 mmol) in 1 M NaOH (10 mL, 10

mmol) and 1,4-dioxane (10 mL) was stirred at 60 °C for 2 h.

Concentrated HCl (1.5 mL) was added to the cooled mixture, and

the mixture was concentrated to obtain a residue. The resulting

solid was washed with water, followed by EtOAc to obtain 8 or 13

as an HCl salt. Alternatively, the cooled reaction mixture was

neutralized by adding an equimolar volume of 1 M HCl, and the

precipitated solid was collected by filtration to provide the free

form of 13.

1

obtained on Varian Unity 400- and 500-MHz spectrometers.

Spectra were recorded in the indicated solvent at ambient

temperature; chemical shifts are reported in ppm (δ) relative to the

solvent peak. Resonance patterns are represented with the

following notations: br (broad signal), s (singlet), d (doublet), t

(triplet), q (quartet) and m (multiplet). MS analysis was carried out

by FAB, EI, or ESI. HRMS was carried out using an LC-MS

system composed of a Waters Xevo Q-ToF MS and an Acquity

UHPLC system. Elemental analyses were carried out on a

Microcorder JM10 and a Dionex ICS-1500. The purity was

assessed by reversed-phase HPLC analysis (column, Inertsil ODS-

3, 4.6 × 250 mm; eluent, MeCN/0.1% Et3N•HCl aqueous solution;

flow rate, 1 mL/min; wavelength, 254 nm). All assay compounds

were ≥ 95% pure.

4.7. General Procedure for Amidation to Prepare 15 (General

Procedure F)

A solution of crude 10 prepared from 9 (30.0 mmol), 14 (6.30

g, 30.0 mmol), HOBt•H₂O (4.05 g, 30.0 mmol) and WSC•HCl

(5.73 g, 30.0 mmol) in CH₂Cl₂(100 mL) was stirred at room

temperature for 18 h under N₂. Water was added to the reaction

mixture, and the mixture was extracted with CH₂Cl₂several times.

The combined organic layers were dried over anhydrous Na₂SO₄

and concentrated under reduced pressure. The residue was purified

by silica gel chromatography to obtain 15.

4.2. General Procedure for S_NAr Reaction to Prepare 9 (General

Procedure A)

NaH (30.0 mmol) was added to a solution of 1 (8.60 g, 30.0

mmol) and substituted phenol (30.0 mmol) in DMF (150 mL) at

room temperature under N₂, and the mixture was stirred at 80 °C

for 10 h. Water was added to the cooled mixture, and the mixture

was extracted with EtOAc several times. The combined organic

layers were washed with water, dried over anhydrous Na₂SO₄, and

concentrated under reduced pressure. The crude residue was

purified by silica gel column chromatography to obtain purified

compound 9, or crude 9 was used directly for the next reaction

without further purification.

4.8. General Procedure for Benzimidazole Ring Annulation of 15

to Prepare 12 (General Procedure G)

A solution of 15 (32.8 mmol) in 4 M HCl in EtOAc (165 mL)

was stirred at 60 °C for 4 h. After cooling the reaction mixture, the

precipitated solid was collected by filtration to obtain 12 as an HCl

salt.

4.8.1. [6-(4-Amino-3,5-dimethylphenoxy)-1-methyl-

1H-benzimidazol-2-yl] methanol (4).

4.3. General Procedure for Reduction of Nitro Group, Followed

by Benzimidazole Ring Annulation to Prepare 11 (General

Procedure B)

NaH (55%, 4.02 g, 92.1 mmol) was added to a solution of 1

(26.4 g, 92.1 mmol) and 4-amino-3,5-dimethylphenol (12.6 g,

mmol) in DMA (200 mL) at 0 °C under N₂, and the mixture was

stirred at room temperature for 24 h. Water was added to the

reaction mixture at 0 °C, and the mixture was extracted with

EtOAc twice. The combined organic layers were washed with

water, dried over anhydrous Na₂SO₄, and concentrated under

reduced pressure to obtain crude 2. A solution of crude 2 and Pd/C

(10%, 7.60 g) in EtOH (500 mL) was stirred under H₂at room

temperature for 24 h. The catalyst was filtered off, and the filtrate

was concentrated under reduced pressure to obtain crude 3. A

solution of crude 3, glycolic acid (10.5 g, 138 mmol) in 4 M HCl

in 1,4-dioxane (200 mL) was stirred under reflux for 2 h. The

cooled reaction mixture was poured slowly into 10% NaHCO₃

aqueous solution, followed by extraction with EtOAc twice. The

combined organic layers were washed with brine, dried over

anhydrous MgSO₄, and concentrated under reduced pressure. The

crude residue was purified by recrystallization from EtOAc/i-Pr₂O

to obtain 4 (14.7 g, 53%, 3 steps). ¹H-NMR (400 MHz, DMSO-d₆)

δ 2.07 (6H, s), 3.72 (3H, s), 4.38 (2H, s), 4.67 (2H, d, J = 5.9 Hz),

5.53 (1H, t, J = 5.9 Hz), 6.57 (2H, s), 6.78 (1H, dd, J = 2.4, 8.6

Hz), 7.02 (1H, d, J = 2.0 Hz), 7.49 (1H, d, J = 8.6 Hz); HRMS

(ESI) m/z: [M + H]⁺calcd for C₁₇H₂₀N₃O₂, 298.1556; found

298.1558.

A solution of 9 (30.0 mmol), iron powder (8.37 g, 150 mmol)

and NH₄Cl (0.803 g, 15.0 mmol) in water (80 mL) and EtOH (160

mL) was stirred under reflux for 5 h. The cooled reaction mixture

was filtered through a pad of celite, and the filtrate was extracted

with EtOAc several times. The combined organic layers were

dried over anhydrous Na₂SO₄, and concentrated under reduced

pressure to obtain crude 10. A solution of crude 10, glycolic acid

(3.42 g, 45.0 mmol) in 4 M HCl in 1,4-dioxane (200 mL) was

stirred under reflux for 2 h. The cooled reaction mixture was

poured slowly into 10% NaHCO₃aqueous solution to precipitate

a solid, which was collected by filtration, washed with

EtOAc/hexane (1:1), and finally washed with water to obtain 11.

4.4. General Procedure for Mitsunobu Reaction, Followed by

Deprotection to Prepare 7 or 8 (General Procedure C)

A solution of 5 (1.00 mmol), PBu₃(2.00 mmol), ADDP (2.00

mmol) and substituted phenol (1.50 mmol) in toluene (5 mL) was

stirred for 10 h under N₂. After concentration of the reaction

mixture under reduced pressure, the residue was purified by silica

gel chromatography to obtain 6. A solution of 6 in 4 M HCl in 1,4-

dioxane (10 mL) was stirred for 2 h. The precipitated solid was

collected by filtration and washed with EtOAc to obtain 7 or 8.

4.8.2. tert-Butyl (4-{[2-(hydroxymethyl)-1-methyl-

1H-benzimidazol-6-yl]oxy}-2,6-

dimethylphenyl)carbamate (5).

4.5. General Procedure for Mitsunobu Reaction to Prepare 12

(General Procedure D)

A solution of 4 (13.0 g, 43.7 mmol) and BOC₂O (19.0 g, 87.0

mmol) in i-PrOH (150 mL) was stirred for 16 h. Water was added

to the cooled mixture, and the mixture was extracted with EtOAc

several times. Combined organic layers were successively washed

with water and brine. The organic layer was dried over anhydrous

Na₂SO₄and concentrated under reduced pressure. The residue was

purified by silica gel chromatography (EtOAc/MeOH, 90:10). The

resulting form was crystallized from EtOAc and hexane to obtain

A solution of 11 (1.00 mmol), PBu₃(2.00 mmol), ADDP (2.00

mmol) and methyl 3-hydroxybenzoate (0.23 g, 1.5 mmol) in

toluene (5 mL) was stirred for 10 h under N₂. After concentrating

the reaction mixture under reduced pressure, the residue was

purified by silica gel chromatography to obtain 12.

4.6. General Procedure for Hydrolysis to Prepare 8 or 13

(General Procedure E)

5 (4.50 g, 26%). ¹H-NMR (400 MHz, CDCl₃) δ 1.26 (9H, s), 2.21

(6H, s), 3.75 (3H, s), 4.89 (2H, s), 6.67 (2H, s), 6.93 (1H, d, J =

2.0 Hz), 6.96 (1H, dd, J = 2.0, 9.0 Hz), 7.63 (1H, d, J = 9.0 Hz).

(1H, d, J = 8.8 Hz); HRMS (ESI) m/z: [M + H]⁺calcd for

C₂₅H₂₆N₃O₃, 416.1974; found 416.1951.

4.8.9. 1-(4-{[6-(4-Amino-3,5-dimethylphenoxy)-1-

methyl-1H-benzimidazol-2-

yl]methoxy}phenyl)ethanone•2 HCl (8d).

4.8.3. Ethyl 2-{[6-(4-Amino-3,5-dimethylphenoxy)-

1-methyl-1H-benzimidazol-2-

yl]methoxy}benzoate•2HCl (7h).

Compound 8d was prepared according to general procedure C

(yield, 92%, 2 steps). ¹H-NMR (500 MHz, DMSO-d₆) δ 2.33 (6H,

s), 2.54 (3H, s), 3.93 (3H, s), 5.71 (2H, s), 6.80 (2H, s), 7.15 (1H,

dd, J = 2.0, 8.8 Hz), 7.30 (2H, dd, J = 7.3, 7.8 Hz), 7.57 (1H, d, J

= 1.5 Hz), 7.79 (1H, d, J = 8.8 Hz), 8.00 (2H, d, J = 8.8 Hz) ;

HRMS (ESI) m/z: [M + H]⁺calcd for C₂₅H₂₆N₃O₃, 416.1974;

found 416.1982; Anal. calcd for C₂₄H₂₂N₄O₂•2HCl•0.40H₂O: C,

60.23; H, 5.22; N, 11.71; Cl, 14.82; found C, 60.31; H, 5.04; N,

11.65; Cl, 14.64.

Compound 7h was prepared according to general procedure C

(yield, 67%, 2 steps). ¹H-NMR (400 MHz, DMSO-d₆) δ 1.20 (3H,

t, J = 7.2 Hz), 2.33 (6H, s), 3.94 (3H, s), 4.23 (2H, q, J = 7.2 Hz),

5.64 (2H, s), 6.79 (2H, s), 7.11 (1H, d, J = 8.8 Hz), 7.13 (1H, dd,

J = 7.3, 7.8 Hz), 7.45 (1H, d, J = 8.4 Hz), 7.55 (1H, s), 7.60 (1H,

ddd, J = 1.4, 7.3, 8.4 Hz), 7.72 (1H, dd, J = 1.4, 7.8 Hz), 7.77 (1H,

d, J = 8.8 Hz); HRMS (ESI) m/z: [M + H]⁺calcd for C₂₆H₂₈N₃O₄,

446.2080; found 446.2100.

4.8.4. Methyl 3-{[6-(4-amino-3,5-dimethylphenoxy)-

1-methyl-1H-benzimidazol-2-

yl]methoxy}benzoate•2HCl (7i).

4.8.10. 2-{[6-(4-Amino-3,5-dimethylphenoxy)-1-

methyl-1H-benzimidazol-2-

yl]methoxy}benzonitrile•2HCl (8e).

Compound 7i was prepared according to general procedure C

(yield, 63%, 2 steps). ¹H-NMR (400 MHz, DMSO-d₆) δ 2.25 (6H,

s), 3.87 (3H, s), 3.87 (3H, s), 5.60 (2H, s), 6.74 (2H, s), 7.03 (1H,

d, J = 8.8 Hz), 7.41 (1H, s), 7.45 (1H, d, J = 8.3 Hz), 7.52 (1H, dd,

J = 7.8, 8.3 Hz), 7.63 (1H, d, J = 7.8 Hz), 7.69 (1H, s), 7.72 (1H,

d, J = 8. 8 Hz); HRMS (ESI) m/z: [M + H]⁺calcd for C₂₅H₂₆N₃O₄,

432.1923; found 432.1904.

Compound 8e was prepared according to general procedure C

(yield, 68%, 2 steps). ¹H-NMR (400 MHz, DMSO-d₆) δ 2.33 (6H,

s), 3.93 (3H, s), 5.73 (2H, s), 6.80 (2H, s), 7.06 (1H, dd, J = 2.0,

9.0 Hz), 7.19 (1H, t, J = 7.4 Hz), 7.50 (1H, d, J = 2.0 Hz), 7.55

(1H, d, J = 8.6 Hz), 7.71–7.76 (2H, m), 7.80 (1H, dd, J = 1.6, 7.4

Hz) ; HRMS (ESI) m/z: [M + H]⁺calcd for C₂₄H₂₃N₄O₂, 399.1821;

found 399.1850.

4.8.5. Ethyl 4-{[6-(4-amino-3,5-dimethylphenoxy)-

1-methyl-1H-benzimidazol-2-

4.8.11. 3-{[6-(4-Amino-3,5-dimethylphenoxy)-1-

methyl-1H-benzimidazol-2-

yl]methoxy}benzoate•2HCl (7j).

yl]methoxy}benzonitrile•2HCl (8f).

Compound 7j was prepared according to general procedure C

(yield, 67%, 2 steps). ¹H-NMR (400 MHz, DMSO-d₆) δ 1.31 (3H,

t, J = 7.0 Hz), 2.12 (6H, s), 3.90 (3H, s), 4.29 (2H, q, J = 7.0 Hz),

5.66 (2H, s), 6.71 (2H, s), 7.10 (1H, d, J = 8.8 Hz), 7.29 (2H, d, J

= 8.8 Hz), 7.51 (1H, s), 7.75 (1H, d, J = 8.8 Hz), 7.98 (2H, d, J =

8.8 Hz); HRMS (ESI) m/z: [M + H]⁺calcd for C₂₆H₂₈N₃O₄,

446.2080; found 446.2080.

Compound 8f was prepared according to general procedure C

(yield, 83%, 2 steps). ¹H-NMR (400 MHz, DMSO-d₆) δ 2.31 (6H,

s), 3.89 (3H, s), 5.65 (2H, s), 6.78 (2H, s), 7.10 (1H, dd, J = 1.6,

8.6 Hz), 7.51–7.53 (3H, m), 7.56–7.60 (1H, m), 7.71 (1H, s), 7.76

(1H, d, J = 9.0 Hz) ; HRMS (ESI) m/z: [M + H]⁺calcd for

C₂₄H₂₃N₄O₂, 399.1821; found 399.1847.

4.8.12. 4-{[6-(4-Amino-3,5-dimethylphenoxy)-1-

methyl-1H-benzimidazol-2-

yl]methoxy}benzonitrile•2HCl (8g).

Compound 8g was prepared according to general procedure C

(yield, 92%, 2 steps). ¹H-NMR (400 MHz, DMSO-d₆) δ 2.32 (6H,

s), 3.89 (3H, s), 5.68 (2H, s), 6.78 (2H, s), 7.09 (1H, d, J = 9.0 Hz),

7.36 (2H, d, J = 8.6 Hz), 7.51 (1H, s), 7.76 (1H, d, J = 8.6 Hz),

7.87 (2H, d, J = 9.0 Hz); HRMS (ESI) m/z: [M + H]⁺calcd for

C₂₄H₂₃N₄O₂, 399.1821; found 399.1841.

4.8.6. 2,6-Dimethyl-4-{[1-methyl-2-

(phenoxymethyl)-1H-benzimidazol-6-

yl]oxy}aniline•2 HCl (8a).

Compound 8a was prepared according to general procedure C

(yield, 53%, 2 steps). ¹H-NMR (400 MHz, DMSO-d₆) δ: 2.28 (6H,

s), 3.88 (3H, s), 5.56 (2H, s), 6.74 (2H, s), 6.99 (1H, t, J = 7.0 Hz),

7.08-7.14 (3H, m), 7.32 (2H, t, J = 8.0 Hz), 7.52 (1H, d, J = 2.0

Hz), 7.73 (1H, d, J = 8.6 Hz); HRMS (ESI) m/z: [M + H]⁺calcd

for C₂₃H₂₄N₃O₂, 374.1869; found 374.1860.

4.8.13. 2-{[6-(4-Amino-3,5-dimethylphenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoic

acid•2HCl (8h).

4.8.7. 1-(2-{[6-(4-Amino-3,5-dimethylphenoxy)-1-

methyl-1H-benzimidazol-2-

yl]methoxy}phenyl)ethanone•2 HCl (8b).

Compound 8h was prepared according to general procedure E

(yield, 66%). ¹H-NMR (400 MHz, DMSO-d₆) δ 2.30 (6H, s), 3.92

(3H, s), 5.62 (2H, s), 6.87 (2H, s), 7.10 (1H, m), 7.11 (1H, m), 7.43

(1H, d, J = 8.3 Hz), 7.55 (1H, m), 7.58 (1H, s), 7.69 (1H, dd, J =

1.4, 7.6 Hz), 7.75 (1H, d, J = 8. 8 Hz); HRMS (ESI) m/z: [M + H]⁺

calcd for C₂₄H₂₃N₃O₄, 418.1767; found 418.1742.

Compound 8b was prepared according to general procedure C

(yield, 53%, 2 steps). ¹H-NMR (500 MHz, DMSO-d₆) δ 2.23 (6H,

s), 2.57 (3H, s), 3.84 (3H, s), 5.47 (2H, s), 6.71 (2H, s), 6.98 (1H,

d, J = 2.4 Hz), 7.02–7.07 (2H, m), 7.33 (1H, d, J = 8.2 Hz), 7.46–

7.50 (1H, m), 7.70 (1H, dd, J = 1.6, 7.4 Hz), 7.72 (1H, d, J = 8.6

Hz); HRMS (ESI) m/z: [M + H]⁺calcd for C₂₅H₂₆N₃O₃, 416.1974;

found 416.1945.

4.8.14. 3-{[6-(4-Amino-3,5-dimethylphenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoic

acid•2HCl (8i).

4.8.8. 1-(3-{[6-(4-Amino-3,5-dimethylphenoxy)-1-

methyl-1H-benzimidazol-2-

yl]methoxy}phenyl)ethanone•2 HCl (8c).

Compound 8c was prepared according to general procedure C

(yield, 87%, 2 steps). ¹H-NMR (500 MHz, DMSO-d₆) δ 2.32 (6H,

s), 2.61 (3H, s), 3.93 (3H, s), 5.68 (2H, s), 6.79 (2H, s), 7.14 (1H,

dd, J = 2.4, 8.8 Hz), 7.46 (1H, dd, J = 2.9, 8.3 Hz), 7.53 (1H, d, J

= 7.8 Hz), 7.55 (1H, s), 7.67 (1H, d, J = 7.8 Hz), 7.70 (1H, s), 7.78

Compound 8i was prepared according to general procedure E

(yield, 61%). Mp, 235–239 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ

2.30 (6H, s), 3.91 (3H, s), 5.65 (2H, s), 6.78 (2H, s), 7.11 (1H, dd,

J = 2. 0, 8.8 Hz), 7.43 (1H, d, J = 7.8 Hz), 7.49 (1H, dd, J = 7.8,

7.8 Hz), 7.51 (1H, d, J = 2.0 Hz), 7.63 (1H, d, J = 7.8 Hz), 7.76

(1H, d, J = 8.8 Hz); HRMS (ESI) m/z: [M + H]⁺calcd for

C₂₄H₂₃N₃O₄, 418.1767; found 418.1802.

4.8.15. 4-{[6-(4-Amino-3,5-dimethylphenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoic

acid•2HCl (8j).

Compound 8j was prepared according to general procedure E

(yield, 77%). ¹H-NMR (400 MHz, DMSO-d₆) δ 2.30 (6H, s), 3.89

(3H, s), 5.64 (2H, s), 6.77 (2H, s), 7.08 (1H, d, J = 8. 8 Hz), 7.25

(2H, d, J = 8.8 Hz), 7.49 (1H, s), 7.75 (1H, d, J = 8.8 Hz), 7.95

(2H, d, J = 8. 8 Hz); HRMS (ESI) m/z: [M + H]⁺calcd for

C₂₄H₂₃N₃O₄, 418.1767; found 418.1752.

(3H, s), 6.85–6.92 (4H, m), 7.43 (1H, t, J = 8. 6 Hz), 7.93 (1H, t, J

= 8. 6 Hz).

4.8.24. tert-Butyl [5-(3-chloro-5-fluorophenoxy)-2-

nitrophenyl]methylcarbamate (9ad).

Compound 9ad was prepared according to general procedure A

1

(yield, 99%). H-NMR (400 MHz, CDCl₃) δ 1.33 (6H, s), 1.51

(3H, s), 3.28 (3H, s), 6.74 (1H, dt, J = 2.4, 9.0 Hz), 6.90–6.93 (3H,

m), 7.00 (1H, d, J = 7.8 Hz), 7.98 (1H, d, J = 8.6 Hz).

4.8.25. tert-Butyl [5-(3-chloro-4-fluorophenoxy)-2-

nitrophenyl]methylcarbamate (9ae).

4.8.16. tert-Butyl [5-(4-fluorophenoxy)-2-

nitrophenyl]methylcarbamate (9d).

Compound 9ae was prepared according to general procedure A

Compound 9d was prepared according to general procedure A

1

(yield, 99%). H-NMR (400 MHz, CDCl₃) δ 1.32 (9H, s), 3.26

1

(yield, 99%). H-NMR (400 MHz, CDCl₃) δ 1.33 (6H, s), 1.50

(3H, s), 6.79–6.85 (2H, m), 6.95–6.97 (1H, m), 7.15–7.18 (2H, m),

7.91–7.93 (1H, m).

(3H, s), 3.26 (3H, s), 6.81 (1H, dd, J = 2.7, 9.0 Hz), 6.85 (1H, br),

7.07–7.17 (4H, m), 7.93–7.97 (1H, m).

4.8.26. tert-Butyl [5-(2,3-dihydro-1-benzofuran-5-

yloxy)-2-nitrophenyl] methylcarbamate (9af).

4.8.17. tert-Butyl methyl[5-(4-methylphenoxy)-2-

nitrophenyl]carbamate (9g).

Compound 9af was prepared according to general procedure A

(yield, 27%) ¹H-NMR (500 MHz, CDCl₃) δ1.32 (6H, s), 1.50 (3H,

s), 3.24–3.27 (2H, m), 3.25 (3H, s), 4.64 (2H, t, J = 8.8 Hz), 6.77–

63.83 (4H, m), 6.96 (1H, s), 7.91 (1H, d, J = 9.3 Hz).

Compound 9g was prepared according to general procedure A

(yield, 99%). H-NMR (400 MHz, CDCl₃) δ 1.32 (6H, s), 1.49

(3H, br), 2.39 (3H, s), 3.24 (3H, s), 6.80–6.82 (2H, m), 6.99 (2H,

d, J = 8.2 Hz), 7.23 (2H, d, J = 7.8 Hz), 7.92 (1H, dd, J = 1.6, 8.6

Hz).

1

4.8.27. (1-Methyl-6-phenoxy-1H-benzimidazol-2-

yl)methanol (11a).

4.8.18. tert-Butyl [5-(3-chlorophenoxy)-2-

nitrophenyl]methylcarbamate (9h).

Compound 11a was prepared according to general procedure A,

followed by procedure B (yield, 73%; 3 steps). H-NMR (400

MHz, DMSO-d₆) δ 3.77 (3H, s), 4.70 (2H, s), 6.90 (1H, dt, J = 2.0,

8.6 Hz), 6.96 (2H, d, J = 7.8 Hz), 7.08 (1H, t, J = 7.4 Hz), 7.29

(1H, s), 7.36 (2H, t, J = 8.6 Hz), 7.60 (1H, dd, J = 1.6 Hz).

1

Compound 9h was prepared according to general procedure A,

1

(yield, 99%). H-NMR (400 MHz, CDCl₃) δ 1.33 (6H, s), 1.50

(3H, s), 3.27 (3H, s), 6.87 (1H, dd, J = 2.7, 8.6 Hz), 6.89 (1H, br),

7.01 (1H, d, J = 8.1 Hz), 7.12 (1H, t, J = 2.0 Hz), 7.24–7.26 (1H,

m), 7.38 (1H, t, J = 8.2 Hz), 7.96 (1H, d, J = 9.0 Hz).

4.8.28. [6-(2-Fluorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methanol (11b).

4.8.19. tert-Butyl [5-(4-chlorophenoxy)-2-

nitrophenyl]methylcarbamate (9i).

Compound 11b was prepared according to general procedure A,

followed by procedure B (yield, 72%; 3 steps). H-NMR (400

MHz, CDCl₃) δ 3.74 (3H, s), 4.88 (2H, s), 6.91 (1H, d, J = 2.4 Hz),

6.98 (1H, dd, J = 2.4, 9.0 Hz), 7.00–7.03 (1H, m), 7.09 (1H, d, J =

2.7 Hz), 7.11 (1H, d, J = 3.1 Hz), 7.17–7.22 (1H, m), 7.63 (1H, d,

J = 9.0 Hz).

1

Compound 9i was prepared according to general procedure A

1

(yield, 99%). H-NMR (400 MHz, CDCl₃) δ 1.32 (6H, s), 1.49

(3H, br), 2.39 (3H, s), 3.24 (3H, s), 6.80–6.82 (2H, m), 6.99 (2H,

d, J = 8.2 Hz), 7.23 (2H, d, J = 7.8 Hz), 7.92 (1H, dd, J = 1.6, 8.6

Hz).

4.8.29. [6-(3-Fluorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methanol (11c).

4.8.20. tert-Butyl methyl{2-nitro-5-[3-

(trifluoromethoxy)phenoxy]phenyl}carbamate (9n).

Compound 11c was prepared according to general procedure A,

Compound 9n was prepared according to general procedure A

1

followed by procedure B (yield, 80%; 3 steps). H-NMR (400

1

(yield, 56%). H-NMR (400 MHz, CDCl₃) δ 1.33 (6H, s), 1.50

MHz, DMSO-d₆) δ 3.80 (3H, s), 4.71 (2H, d, J = 5.5 Hz), 5.59 (1H,

t, J = 5.5 Hz), 6.75–6.82 (2H, m), 6.87–6.97 (2H, m), 7.33–7.42

(2H, m), 7.62 (1H, d, J = 8. 6 Hz).

(3H, s), 3.27 (3H, s), 6.89–6.91 (2H, m), 6.99 (1H, s.), 7.05 (1H,

d, J = 8.6 Hz), 7.13 (1H, d, J = 7.8 Hz), 7.47 (1H, t, J = 8.2 Hz),

7.97 (1H, d, J = 8.6 Hz).

4.8.30. [6-(4-Fluorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methanol (11d).

4.8.21. tert-Butyl [2-amino-5-(2,5-

difluorophenoxy)phenyl]methylcarbamate (9r).

Compound 11d was prepared according to general procedure B

(yield, 29%; 2 steps). ¹H-NMR (400 MHz, CDCl₃) δ 3.75 (3H, s),

4.89 (2H, s), 6.90 (1H, d, J = 2.0 Hz), 6.94–7.05 (5H, m), 7.64 (1H,

d, J = 8.6 Hz).

Compound 9r was prepared according to general procedure A

(yield, 92%). H-NMR (400 MHz, CDCl₃) δ 1.32 (6H, s), 1.50

(3H, br), 3.28 (3H, s), 6.81–7.07 (4H, m), 7.17–7.26 (1H, m), 7.96

(1 H, d, J = 9.0 Hz).

1

4.8.31. [1-Methyl-6-(2-methylphenoxy)-1H-

benzimidazol-2-yl] methanol (11e).

4.8.22. tert-Butyl [5-(3-fluoro-5-methylphenoxy)-2-

nitrophenyl]methylcarbamate (9ab).

Compound 11e was prepared according to general procedure A,

Compound 9ab was prepared according to general procedure A

1

followed by procedure B (yield, 72%; 3 steps). H-NMR (400

1

(yield, 69%). H-NMR (400 MHz, CDCl₃) δ 1.33 (6H, s), 1.51

MHz, CDCl₃) δ 2.28 (3H, s), 3.73 (3H, s), 4.86 (2H, s), 6.80 (1H,

d, J = 2.0 Hz), 6.83–6.87 (1H, m), 6.91 (1H, dd, J = 2.0, 8.6 Hz),

7.03–7.08 (1H, m), 7.12–7.18 (1H, m), 7.24–7.29 (1H, m), 7.59

(1H, d, J = 8.6 Hz).

(3H, s), 2.38 (3H, s), 3.27 (3H, s), 6.63 (1H, dt, J = 2.4, 9.4 Hz),

6.71 (1H, s), 6.80 (1H, d, J = 8.6 Hz), 6.86–6.88 (2H, m), 7.95 (1H,

d, J = 8.2 Hz).

4.8.23. tert-Butyl [5-(4-chloro-3-fluorophenoxy)-2-

nitrophenyl]methylcarbamate (9ac).

4.8.32. [1-Methyl-6-(3-methylphenoxy)-1H-

benzimidazol-2-yl] methanol (11f).

Compound 9ac was prepared according to general procedure A

Compound 11f was prepared according to general procedure A,

1

(yield, 99%). H-NMR (400 MHz, CDCl₃) δ 1.32 (9H, s), 3.26

1

followed by procedure B (yield, 63%; 3 steps). H-NMR (400

4.8.41. {1-Methyl-6-[3-(trifluoromethyl)phenoxy] -

1H-benzimidazol-2-yl}methanol (11o).

MHz, CDCl₃) δ 2.32 (3H, s), 3.75 (3H, s), 4.90 (2H, s), 6.80 (2H,

s), 6.90 (1H, d, J = 7.4 Hz), 6.94–7.02 (2H, m), 7.21 (1H, t, J = 7.8

Hz), 7.65 (1H, d, J = 8.6 Hz).

Compound 11o was prepared according to general procedure A,

1

followed by procedure B (yield, 57%; 3 steps). H-NMR (400

4.8.33. [1-Methyl-6-(4-methylphenoxy)-1H-

benzimidazol-2-yl] methanol (11g).

Compound 11g was prepared according to general procedure B

(yield, 89%; 2 steps). ¹H-NMR (400 MHz, CDCl₃) δ 2.34 (3H, s),

3.74 (3H, s), 4.86 (2H, s), 6.87–6.92 (3H, m), 6.95 (1H, dd, J = 2.3,

8.6 Hz), 7.14 (2H, d, J = 7.8 Hz), 7.59 (1H, d, J = 8.6 Hz).

MHz, CDCl₃) δ 3.78 (3H, s), 4.93 (2H, s), 6.97–7.04 (2H, m),

7.09–7.18 (1H, m), 7.21 (1H, s), 7.33 (1H, d, J = 8.6 Hz), 7.43 (1H,

t, J = 7.8 Hz), 7.71 (1H, d, J = 8.2 Hz).

4.8.42. [6-(2,3-Difluorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methanol (11p).

Compound 11p was prepared according to general procedure A,

followed by procedure B (yield, 75%; 3 steps). H-NMR (400

MHz, CDCl₃) δ 3.77 (3H, s), 4.90 (2H, s), 6.73 (1H, t, J = 8.2 Hz),

6.90–7.02 (4H, m), 7.66 (1H, d, J = 8.6 Hz).

4.8.34. [6-(3-Chlorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methanol (11h).

1

Compound 11h was prepared according to general procedure B

(yield, 20%; 2 steps). ¹H-NMR (400 MHz, CDCl₃) δ 3.79 (3H, s),

4.91 (2H, s), 6.88 (1H, d, J = 8.6 Hz), 6.95 (1H, s), 6.98–7.01 (2H,

m), 7.05 (1H, d, J = 8.2 Hz), 7.24 (1H, d, J = 8.2 Hz), 7.69 (1H, d,

J = 9.4 Hz).

4.8.43. [6-(2,4-Difluorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methanol (11q).

Compound 11q was prepared according to general procedure A,

1

followed by procedure B (yield, 76%; 3 steps). H-NMR (400

4.8.35. [6-(4-Chlorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methanol (11i).

MHz, CDCl₃) δ 3.74 (3H, s), 4.58 (1H, br), 4.87 (2H, s), 6.79–6.90

(2H, m), 6.90–7.07 (3H, m), 7.61 (1H, d, J = 8.6 Hz).

Compound 11i was prepared according to general procedure B

(yield, 78%; 2 steps). ¹H-NMR (400 MHz, CDCl₃) δ 3.76 (3H, s),

4.90 (2H, s), 6.86-7.02 (4H, m), 7.26-7.30 (2H, m), 7.65 (1H, d, J

= 9.2 Hz).

4.8.44. [6-(2,5-Difluorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methanol (11r).

Compound 11r was prepared according to general procedure B

(yield, 83%; 2 steps). ¹H-NMR (400 MHz, CDCl₃) δ 3.77 (3H, s),

4.91 (2H, s), 6.60–6.71 (1H, m), 6.71–6.82 (1H, m), 6.97–7.06 (2

H, m), 7.09–7.21 (1H, m), 7.68 (1H, d, J = 9.0 Hz).

4.8.36. [6-(2-Methoxyphenoxy)-1-methyl-1H-

benzimidazol-2-yl] methanol (11j).

Compound 11j was prepared according to general procedure A,

1

4.8.45. [6-(2,6-Difluorophenoxy)-1-methyl-1H-

followed by procedure B (yield, 61%; 3 steps). H-NMR (400

benzimidazol-2-yl] methanol (11s).

Compound 11s was prepared according to general procedure A,

followed by procedure B (yield, 60%; 3 steps). H-NMR (500

MHz, CDCl₃) δ 3.73 (3H, s), 3.87 (3H, s), 4.88 (2H, s), 6.91–6.95

(3H, m), 6.97 (1H, dd, J = 2.0, 8.6 Hz), 7.03 (1H, dd, J = 1.6, 8.6

Hz), 7.10–7.15 (1H, m), 7.63 (1H, d, J = 8.2 Hz).

1

MHz, CDCl₃) δ 3.73 (3H, s), 4.88 (2H, s), 6.88 (1H, d, J = 2.4 Hz),

6.95 (1H, dd, J = 2.0, 8.8 Hz), 7.03 (2H, t, J = 7.8 Hz), 7.14–7.20

(1H, m), 7.62 (1H, d, J = 8.8 Hz).

4.8.37. [6-(3-Methoxyphenoxy)-1-methyl-1H-

benzimidazol-2-yl] methanol (11k).

Compound 11k was prepared according to general procedure A,

1

4.8.46. [6-(3,4-Difluorophenoxy)-1-methyl-1H-

followed by procedure B (yield, 89%; 3 steps). H-NMR (400

benzimidazol-2-yl] methanol (11t).

Compound 11t was prepared according to general procedure A,

followed by procedure B (yield, 88%; 3 steps). H-NMR (400

MHz, CDCl₃) δ 3.78 (3H, s), 4.90 (2H, s), 6.68–6.73 (1H, m),

6.78–6.83 (1H, m), 6.95 (1H, s), 6.95–6.98 (1H, m), 7.11 (1H, q,

J = 9.4 Hz), 7.67 (1H, dd, J = 1.2, 8.2 Hz).

MHz, CDCl₃) δ 3.76 (3H, s), 3.77 (3H, s), 4.08 (2H, s), 6.54–6.57

(2H, m), 6.64 (1H, ddd, J = 1.2, 2.4, 8.2 Hz), 6.96 (1H, d, J = 2.0

Hz), 6.99 (1H, dd, J = 2.0, 8.6 Hz), 7.21 (1H, t, J = 8.6 Hz), 7.63

(1H, dd, J = 0.8, 8.6 Hz).

1

4.8.38. [6-(4-Methoxyphenoxy)-1-methyl-1H-

benzimidazol-2-yl] methanol (11l).

4.8.47. [6-(3,5-Difluorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methanol (11u).

Compound 11l was prepared according to general procedure A,

followed by procedure B (yield, 87%; 3 steps). H-NMR (400

1

MHz, CDCl₃) δ 3.73 (3H, s), 3.81 (3H, s), 4.85 (2H, s), 5.23 (1H,

br), 6.81 (1H, d, J = 2.4 Hz), 6.89 (2H, d, J = 9.0 Hz), 6.88–6.98

(1H, m), 6.98 (2H, d, J = 9.0 Hz), 7.57 (1H, d, J = 9.0 Hz).

Compound 11u was prepared according to general procedure A,

followed by procedure B (yield, 69%; 3 steps). H-NMR (400

MHz, CDCl₃) δ 3.80 (3H, s), 4.92 (2H, s), 6.42–6.55 (3H, m), 7.00

(1H, dd, J = 2.4, 8.6 Hz), 7.04 (1H, d, J = 2.4 Hz), 7.71 (1H, d, J

= 8.6 Hz).

1

4.8.39. [6-(3-Ethoxyphenoxy)-1-methyl-1H-

benzimidazol-2-yl] methanol (11m).

4.8.48. [6-(4-Fluoro-2-methylphenoxy)-1-methyl-

1H-benzimidazol-2-yl] methanol (11v).

Compound 11m was prepared according to general procedure

A, followed by procedure B (yield, 75%; 3 steps). ¹H-NMR (400

MHz, CDCl₃) δ 1.38 (3H, t, J = 7.0 Hz), 3.75 (3H, s), 3.99 (2H, q,

J = 7.0 Hz), 4.90 (2H, s), 6.53–6.58 (2H, m), 6.83 (1H, ddd, J =

0.8, 2.4, 8.2 Hz), 6.99–7.02 (2H, m), 7.20 (1H, t, J = 8.2 Hz), 7.66

(1H, d, J = 8.2 Hz).

Compound 11v was prepared according to general procedure A,

1

followed by procedure B (yield, 86%; 3 steps). H-NMR (400

MHz, CDCl₃) δ2.25 (3H, s), 3.73 (3H, s), 4.86 (2H, s), 6.73 (1H,

d, J = 2.3 Hz), 6.84–6.90 (3H, m), 6.96–7.00 (1H, m), 7.59 (1H, d,

J = 8.6 Hz).

4.8.40. {1-Methyl-6-[3-(trifluoromethoxy)phenoxy] -

1H-benzimidazol-2-yl}methanol (11n).

4.8.49. [6-(5-Fluoro-2-methylphenoxy)-1-methyl-

1H-benzimidazol-2-yl] methanol (11w).

Compound 11n was prepared according to general procedure B

(yield, 91%; 2 steps). ¹H-NMR (400 MHz, CDCl₃) δ 3.79 (3H, s),

4.91 (2H, s), 6.85 (1H, s), 6.89 (1H, dd, J = 2.4, 8.6 Hz), 6.92–6.95

(1H, m), 6.98–7.01 (2H, m), 7.32 (1H, t, J = 8.2 Hz), 7.67 (1H, d,

J = 9.0 Hz).

Compound 11w was prepared according to general procedure

A, followed by procedure B (yield, 85%; 3 steps). ¹H-NMR (500

MHz, CDCl₃) δ 2.26 (3H, s), 3.77 (3H, s), 4.88 (2H, s), 6.50 (1H,

dd, J = 2.4, 10.3 Hz), 6.73 (1H, dt, J = 2.4, 8.3 Hz), 6.87 (1H, d, J

= 2.4 Hz), 6.93 (1H, dd, J = 2.4, 8.8 Hz), 7.16–7.20 (1H, m), 7.63

(1H, d, J = 8.8 Hz).

4.8.50. [6-(2-Fluoro-4-methylphenoxy)-1-methyl-

1H-benzimidazol-2-yl] methanol (11x).

(3H, s), 5.69 (2H, s), 7.02 (2H, d, J = 8.7 Hz), 7.12 (1H, dd, J =

2.2, 8.8 Hz), 7.15 (1H, t, J = 7. 5 Hz), 7.41 (2H, dd, J = 7.5, 8.7

Hz), 7.48 (1H, ddd, J = 1.3, 2.6, 8.2 Hz), 7.54 (1H, dd, J = 7.5, 8.2

Hz), 7.59 (1H, d, J = 2.2 Hz), 7.66 (1H, ddd, J = 1.3, 1.5, 7.5 Hz),

7.72 (1H, dd, J = 1.5, 2.6 Hz), 7.80 (1H, d, J = 8.8 Hz); HRMS

(ESI) m/z: [M + H]⁺calcd for C₂₃H₂₁N₂O₄, 389.1501; found

389.1518.

Compound 11x was prepared according to general procedure A,

1

followed by procedure B (yield, 87%; 3 steps). H-NMR (400

MHz, CDCl₃) δ 2.35 (3H, s), 3.72 (3H, s), 4.60 (1H, br), 4.86 (2H,

s), 6.85 (1H, d, J = 2.0 Hz), 6.86–6.96 (3H, m), 7.01 (1H, dd, J =

1.6, 11.4 Hz), 7.60 (1H, d, J = 8.26 Hz).

4.8.59. Methyl 3-{[6-(2-fluorophenoxy)-1-methyl-

1H-benzimidazol-2-yl] methoxy}benzoate (12b).

4.8.51. [6-(2-Fluoro-5-methylphenoxy)-1-methyl-

1H-benzimidazol-2-yl] methanol (11y).

Compound 12b was prepared according to general procedure D

(yield, 58%). H-NMR (400 MHz, CDCl₃) δ 3.81 (3H, s), 3.92

Compound 11y was prepared according to general procedure A,

followed by procedure B (yield, 70%; 3 steps). H-NMR (400

1

(3H, s), 5.39 (2H, s), 6.96 (1H, s), 7.00–7.05 (2H, m), 7.07–7.13

(2H, m), 7.20 (1H, t, J = 9.5 Hz), 7.29 (1H, d, J = 7.3 Hz), 7.37

(1H, t, J = 7.3 Hz), 7.69 (1H, d, J = 7.8 Hz), 7.70–7.74 (2H, m).

MHz, CDCl₃) δ 2.27 (3H, s), 3.75 (3H, s), 4.89 (2H, s), 6.78–6.84

(1H, m), 6.85–6.91 (1H, m), 6.92 (1H, d, J = 2.4 Hz), 6.98 (1H, dd,

J = 2.2, 8.8 Hz), 7.07 (1H, dd, J = 8.6, 10. 6 Hz), 7.64 (1H, d, J =

9.0 Hz).

4.8.60. Methyl 3-{[6-(3-fluorophenoxy)-1-methyl-

1H-benzimidazol-2-yl] methoxy}benzoate (12c).

4.8.52. [6-(3-Fluoro-4-methylphenoxy)-1-methyl-

1H-benzimidazol-2-yl] methanol (11z).

Compound 11z was prepared according to general procedure A,

followed by procedure B (yield, 83%; 3 steps). H-NMR (400

MHz, CDCl₃) δ 2.24 (3H, d, J = 2.0 Hz), 3.76 (3H, s), 4.90 (2H,

s), 6.63–6.72 (2H, m), 6.92–7.01 (2H, m), 7.11 (1H, t, J = 9.0 Hz),

7.65 (1H, d, J = 8.6 Hz).

Compound 12c was prepared according to general procedure D

(yield, 86%). H-NMR (400 MHz, CDCl₃) δ 3.84 (3H, s), 3.93

1

(3H, s), 5.41 (2H, s), 6.89 (1H, dt, J = 2.4, 10.2 Hz), 6.76–6.80

(2H, m), 7.02–7.04 (2H, m), 7.23–7.31 (2H, m), 7.38 (1H, t, J =

7.4 Hz), 7.69 (1H, dt, J = 1.2, 7.4 Hz), 7.73 (1H, dd, J = 1.2, 2.4

Hz), 7.74–7.77 (1H, m); HRMS (ESI) m/z: [M + H]⁺calcd for

C₂₃H₂₀FN₂O₄, 407.1407; found 407.1396.

4.8.53. [6-(4-Fluoro-3-methylphenoxy)-1-methyl-

4.8.61. Methyl 3-{[6-(4-fluorophenoxy)-1-methyl-

1H-benzimidazol-2-yl] methoxy}benzoate (12d).

1H-benzimidazol-2-yl] methanol (11aa).

Compound 11aa was prepared according to general procedure

A, followed by procedure B (yield, 77%; 3 steps). ¹H-NMR (400

MHz, CDCl₃) δ 2.25 (3H, d, J = 2.0 Hz), 3.78 (3H, s), 4.05 (1H,

br), 4.91 (2H, s), 6.75–6.81 (1H, m), 6.83 (2H, dd, J = 2.9, 6.1 Hz),

6.87 (1H, d, J = 2.0 Hz), 6.96 (2H, d, J = 8.6 Hz), 7.60 (1H, d, J =

8.6 Hz).

Compound 12d was prepared according to general procedure D

1

(yield, 81%). H-NMR (400 MHz, CDCl₃) δ 3.82 (3H, s), 3.92

(3H, s), 5.39 (2H, s), 6.94–7.05 (5H, m), 7.29 (1H, br), 7.38 (1H,

t, J = 7.8 Hz), 7.69 (1H, d, J = 7.8 Hz), 7.71–7.74 (2H, m).

4.8.62. Methyl 3-{[1-methyl-6-(2-methylphenoxy)-

1H-benzimidazol-2-yl] methoxy}benzoate (12e).

4.8.54. [6-(3-Fluoro-5-methylphenoxy)-1-methyl-

1H-benzimidazol-2-yl] methanol (11ab).

Compound 12e was prepared according to general procedure D

(yield, 81%). H-NMR (400 MHz, CDCl₃) δ 2.29 (3H, s), 3.79

(3H, s), 3.92 (3H, s), 5.38 (2H, s), 6.84 (1H, d, J = 2.3 Hz), 6.88

(1H, d, J = 8.2 Hz), 6.97 (1 H, dd, J = 2.3, 9.0 Hz), 7.04–7.10 (1H,

m), 7.13–7.19 (1H, m), 7.25–7.32 (2H, m), 7.37 (1H, t, J = 7.8 Hz),

7.66–7.73 (3H, m).

1

Compound 11ab was prepared according to general procedure

1

B (yield, 58% 2 steps). H-NMR (400 MHz, CDCl₃) δ 2.31 (3H,

s), 3.78 (3H, s), 4.92 (2H, s), 6.49 (1H, dt, J = 2.0, 10.2 Hz), 6.58

(1H, s), 6.61 (1H, d, J = 11.0 Hz), 6.99–7.01 (2H, m), 7.69 (1H, d,

J = 9.4 Hz).

4.8.63. Methyl 3-{[1-methyl-6-(3-methylphenoxy)-

1H-benzimidazol-2-yl] methoxy}benzoate (12f).

4.8.55. [6-(4-Chloro-3-fluorophenoxy)-1-methyl-

1H-benzimidazol-2-yl] methanol (11ac).

Compound 12f was prepared according to general procedure D

(yield, 85%). H-NMR (400 MHz, CDCl₃) δ 3.81 (3H, br), 3.92

(3H, s), 5.39 (2H, s), 6.78–6.83 (2H, m), 6.91 (1H, d, J = 7.8 Hz),

6.97–7.04 (2H, m), 7.21 (1H, t, J = 8.0 Hz), 7.27–7.32 (1H, m),

7.38 (1H, t, J = 7.8 Hz), 7.69 (1H, td, J = 1.2, 1.4, 7.6 Hz), 7.71–

7.75 (2H, m).

Compound 11ac was prepared according to general procedure

B (yield, 57%; 2 steps). ¹H-NMR (400 MHz, CDCl₃) δ 3.78 (3H,

s), 4.88 (2H, s), 6.69 (1H, dd, J = 3.9, 10.1 Hz), 6.73 (1H, dd, J =

3.2, 10.1 Hz), 6.93–6.95 (2H, m), 7.28 (1H, t, J = 8.7 Hz), 7.05

(1H, d, J = 8.7 Hz).

1

4.8.56. [6-(3-Chloro-5-fluorophenoxy)-1-methyl-

1H-benzimidazol-2-yl] methanol (11ad).

4.8.64. Methyl 3-{[1-methyl-6-(4-methylphenoxy)-

1H-benzimidazol-2-yl] methoxy}benzoate (12g).

Compound 11ad was prepared according to general procedure

B (yield, 49%; 2 steps). ¹H-NMR (400 MHz, CDCl₃) δ 3.80 (3H,

s), 4.91 (2H, s), 6.58 (1H, dt, J = 2.4, 10.2 Hz), 6.98–7.02 (2H, m),

7.23 (1H, dd, J = 2.0, 9.4 Hz), 7.32 (1H, d, J = 2.0 Hz), 7.70 (1H,

d, J = 8.6 Hz).

Compound 12g was prepared according to general procedure D

1

(yield, 83%). H-NMR (500 MHz, CDCl₃) δ 2.34 (3H, s), 3.80

(3H, s), 3.92 (3H, s), 5.39 (2H, s), 6.89–6.97 (3H, m), 7.01 (1H,

dd, J = 2.4, 8.8 Hz), 7.14 (2H, d, J = 8.3 Hz), 7.27–7.31 (1H, m),

7.37 (1H, t, J = 8.1 Hz), 7.66–7.74 (3H, m).

4.8.57. [6-(3-Chloro-4-fluorophenoxy)-1-methyl-

1H-benzimidazol-2-yl] methanol (11ae).

4.8.65. Methyl 3-{[6-(3-Chlorophenoxy)-1-methyl-

1H-benzimidazol-2-yl] methoxy}benzoate (12h).

Compound 11ae was prepared according to general procedure

B (yield, 45%; 2 steps). H-NMR (400 MHz, CDCl₃) δ 3.78 (3H,

s), 4.89 (2H, s), 6.85–7.00 (4H, m), 7.09 (1H, t, J = 8. 6 Hz), 7.05

(1H, d, J = 8.8 Hz).

1

Compound 12h was prepared according to general procedure D

1

(yield, 97%). H-NMR (400 MHz, CDCl₃) δ 3.85 (3H, s), 3.93

(3H, s), 5.41 (2H, s), 6.89 (1H, ddd, J = 0.8, 3.1, 8.2 Hz), 6.97 (1H,

t, J = 2.0 Hz), 7.01–7.07 (3H, m), 7.24 (1H, d, J = 8.2 Hz), 7.29–

7.32 (1H, m), 7.39 (1H, t, J = 7.8 Hz), 7.70 (1H, dt, J = 1.1, 7.4

Hz), 7.73–7.74 (1H, m), 7.77 (1H, d, J = 9.4 Hz).

4.8.58. Methyl 3-[(1-methyl-6-phenoxy-1H-

benzimidazol-2-yl)methoxy]benzoate•HCl (12a).^{3 9}

Compound 12a was prepared according to general procedure D

(yield, 66%). ¹H-NMR (400 MHz, DMSO-d₆) δ 3.88 (3H, s), 3.93

4.8.66. Methyl 3-{[6-(4-Chlorophenoxy)-1-methyl-

1H-benzimidazol-2-yl] methoxy}benzoate (12i).

7.69 (1H, d, J = 7.8 Hz), 7.73 (1H, dd, J = 1.5, 2.9 Hz), 7.74 (1H,

d, J = 8.3 Hz).

Compound 12i was prepared according to general procedure D

(yield, 44%). ¹H-NMR (400 MHz, DMSO-d₆) δ 3.82 (3H, s), 3.93

(3H, s), 5.40 (2H, s), 6.91-7.02 (4H, m), 7.24-7.31 (3H, m), 7.38

(1H, t, J = 7.9 Hz), 7.68-7.76 (3H, m).

4.8.74. Methyl 3-{[6-(2,4-difluorophenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoate

(12q).

Compound 12q was prepared according to general procedure D

1

4.8.67. Methyl 3-{[6-(2-methoxyphenoxy)-1-methyl-

(yield, 84%). H-NMR (400 MHz, CDCl₃) δ 3.81 (3H, s), 3.92

1H-benzimidazol-2-yl] methoxy}benzoate (12j).

(3H, s), 5.39 (2H, s), 6.81–6.88 (1H, m), 6.90 (1H, d, J = 2.4 Hz),

6.94–7.07 (3H, m), 7.29 (1H, d, J = 1.6 Hz), 7.37 (1H, t, J = 8.2

Hz), 7.66–7.76 (3H, m).

Compound 12j was prepared according to general procedure D

1

(yield, 89%). H-NMR (400 MHz, CDCl₃) δ 3.79 (3H, s), 3.87

(3H, s), 3.92 (3H, s), 5.38 (2H, s), 6.89–6.96 (3H, m), 7.00 (1H,

dd, J = 2.4, 8.6 Hz), 7.03 (1H, dd, J = 1.2, 7.8 Hz), 7.13 (1 H, ddd,

J = 1.4, 7.0, 8.2 Hz), 7.29 (1 H, ddd, J = 0.8, 2.4, 8.2 Hz), 7.37 (1

H, t, J = 7.8 Hz), 7.68 (1 H, dt, J = 1.5, 7.8 Hz), 7.69 (1 H, d, J =

8.6 Hz), 7.72 (1 H, dd, J = 1.6, 2.7 Hz).

4.8.75. Methyl 3-{[6-(2,5-difluorophenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoate

(12r).

Compound 12r was prepared according to general procedure D

(yield, 78%). ¹H-NMR (400 MHz, CDCl₃) δ 3.84 (3 H, s), 3.92 (3

H, s), 5.40 (2 H, s), 6.65–6.71 (1 H, m), 6.73–6.80 (1 H, m), 7.01–

7.05 (2 H, m), 7.11–7.18 (1 H, m), 7.29 (1 H, dd, J = 2.7, 8.2 Hz),

7.38 (1 H, t, J = 8.0 Hz), 7.67–7.77 (3 H, m).

4.8.68. Methyl 3-{[6-(3-methoxyphenoxy)-1-methyl-

1H-benzimidazol-2-yl] methoxy}benzoate (12k).

Compound 12k was prepared according to general procedure D

1

(yield, 65%). H-NMR (400 MHz, CDCl₃) δ 3.77 (3H, s), 3.89

4.8.76. Methyl 3-{[6-(2,6-difluorophenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoate

(12s).

(3H, s), 5.57 (2H, s), 6.55–6.58 (2H, m), 6.61–6.69 (1H, m), 7.00–

7.07 (2H, m), 7.19–7.25 (2H, m), 7.38 (1H, t, J = 7.8 Hz), 7.84

(1H, d, J = 8.6 Hz), 7.78 (1H, d, J = 7.4 Hz), 8.03 (1H, s).

Compound 12s was prepared according to general procedure D

1

(yield, 60%). H-NMR (400 MHz, CDCl₃) δ 3.80 (3H, s), 3.92

4.8.69. Methyl 3-{[6-(4-methoxyphenoxy)-1-methyl-

(3H, s), 5.37 (2H, s), 6.89 (1H, d, J = 2.4 Hz), 6.98 (1H, dd, J =

2.4, 8.6 Hz), 7.01–7.07 (2H, m), 7.14–7.21 (1H, m), 7.28 (1H, ddd,

J = 1.2, 2.7, 8.2 Hz), 7.37 (1H, t, J = 7.8 Hz), 7.66–7.71 (3H, m);

MS (FAB) m/z: 425 [M + H]⁺.

1H-benzimidazol-2-yl] methoxy}benzoate (12l).

Compound 12l was prepared according to general procedure D

1

(yield, 75%). H-NMR (400 MHz, CDCl₃) δ 3.79 (3H, s), 3.81

(3H, s), 3.92 (3H, s), 5.38 (2H, s), 6.88–6.91 (3H, m), 6.97–7.01

(3H, m), 7.27–7.31 (1H, m), 7.37 (1H, t, J = 7.8 Hz), 7.66–7.73

(3H, m).

4.8.77. Methyl 3-{[6-(3,4-difluorophenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoate

(12t).

4.8.70. Methyl 3-{[6-(3-ethoxyphenoxy)-1-methyl-

1H-benzimidazol-2-yl] methoxy}benzoate (12m).

Compound 12t was prepared according to general procedure D

1

(yield, 80%). H-NMR (400 MHz, CDCl₃) δ 3.81 (3H, s), 3.92

Compound 12m was prepared according to general procedure

D (yield, 81%). ¹H-NMR (400 MHz, CDCl₃) δ 1.39 (3H, t, J = 7.0

Hz), 3.82 (3H, s), 3.93 (3H, s), 3.99 (2H, q, J = 7.0 Hz), 5.40 (2H,

s), 6.54–6.58 (2H, m), 6.63 (1H, dd, J = 2.4, 8.2 Hz), 7.01–7.05

(2H, m), 7.21 (1H, t, J = 8.2 Hz), 7.30 (1H, ddd, J = 0.8, 2.4, 5.9

Hz), 7.38 (1H, t, J = 7.8 Hz), 7.69 (1H, d, J = 7.8 Hz), 7.72–7.74

(2H, m).

(3H, s), 5.39 (2H, s), 6.77–6.85 (2H, m), 6.92–7.00 (3H, m), 7.29

(1H, ddd, J = 0.8, 2.4, 8.2 Hz), 7.38 (1H, t, J = 7.8 Hz), 7.67–7.73

(3H, m).

4.8.78. Methyl 3-{[6-(3,5-difluorophenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoate

(12u).

Compound 12u was prepared according to general procedure D

(yield, 86%). H-NMR (400 MHz, CDCl₃) δ 3.86 (3H, s), 3.92

(3H, s), 3.93 (9H, s), 5.41 (2H, s), 6.44–6.55 (2H, m), 7.03 (1H,

dd, J = 2.2, 8.8 Hz), 7.07 (1H, d, J = 2.4 Hz), 7.27–7.33 (2H, m),

7.39 (1H, t, J = 7.8 Hz), 7.67–7.71 (1H, m), 7.73 (1H, dd, J = 1.4,

2.5 Hz), 7.78 (1H, d, J = 8.6 Hz).

4.8.71. Methyl 3-({1-methyl-6-[3-

(trifluoromethoxy)phenoxy] -1H-benzimidazol-2-

yl}methoxy)benzoate (12n).

1

Compound 12n was prepared according to general procedure D

1

(yield, 91%). H-NMR (400 MHz, CDCl₃) δ 3.85 (3H, s), 3.93

(3H, s), 5.42 (2H, s), 6.86 (1H, s), 6.91 (1H, dd, J = 2.4, 7.4 Hz),

6.93–6.96 (1H, m), 7.02–7.06 (2H, m), 7.29–7.34 (2H, m), 7.39

(1H, t, J = 7.4 Hz), 7.69–7.74 (2H, m), 7.77 (1H, d, J = 8.2 Hz).

4.8.79. Methyl 3-{[6-(4-fluoro-2-methylphenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoate

(12v).

4.8.72. Methyl 3-({1-methyl-6-[3-

(trifluoromethyl)phenoxy] -1H-benzimidazol-2-

yl}methoxy)benzoate (12o).

Compound 12v was prepared according to general procedure D

1

(yield, 77%). H-NMR (400 MHz, CDCl₃) δ 2.25 (3H, s), 3.79

(3H, s), 3.92 (3H, s), 5.38 (2H, s), 6.77 (1H, d, J = 2.3 Hz), 6.84–

7.02 (4H, m), 7.25–7.33 (1H, m), 7.37 (1H, t, J = 8.0 Hz), 7.66–

7.74 (3H, m).

Compound 12o was prepared according to general procedure D

(yield, 81%). H-NMR (400 MHz, CDCl₃) δ 3.85 (3H, s), 3.93

(3H, s), 5.41 (2H, s), 7.01–7.06 (2H, m), 7.15 (1H, dd, J = 2.4, 8.2

Hz), 7.23 (1H, s), 7.28–7.45 (4H, m), 7.69 (1H, dt, J = 1.2, 7.8 Hz),

7.73 (1H, dd, J = 1.6, 2.7 Hz), 7.77 (1H, dd, J = 0.8, 9.0 Hz).

1

4.8.80. Methyl 3-{[6-(5-fluoro-2-methylphenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoate

(12w).

Compound 12w was prepared according to general procedure

D (yield, 65%). ¹H-NMR (400 MHz, CDCl₃) δ 2.27 (3H, s), 3.82

(3H, s), 3.92 (3H, s), 5.40 (2H, s), 6.53 (1H, dd, J = 2.7, 9.8 Hz),

6.74 (1H, dt, J = 2.7, 8.2 Hz), 6.91 (1H, d, J = 2.0 Hz), 6.98 (1H,

dd, J = 2.0, 8.6 Hz), 7.17–7.22 (1H, m), 7.27–7.32 (1H, m), 7.38

(1H, t, J = 8.2 Hz), 7.67–7.76 (3H, m).

4.8.73. Methyl 3-{[6-(2,3-difluorophenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoate

(12p).

Compound 12p was prepared according to general procedure D

1

(yield, 78%). H-NMR (500 MHz, CDCl₃) δ 3.84 (3H, s), 3.93

(3H, s), 5.40 (2H, s), 6.75 (1H, dt, J = 2.0, 6.85 Hz), 6.91–7.04

(4H, m), 7.30 (1H, dd, J = 2.4, 8.3 Hz), 7.38 (1H, t, J = 7.8 Hz),

4.8.81. Methyl 3-{[6-(2-fluoro-4-methylphenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoate

(12x).

4.8.88. Methyl 3-{[6-(3-chloro-4-fluorophenoxy)-1-

methyl-1H-benzimidazol-2-

yl]methoxy}benzoate•HCl (12ae).^{3 9}

Compound 12x was prepared according to general procedure D

(yield, 78%). H-NMR (400 MHz, CDCl₃) δ 2.36 (3H, s), 3.80

Compound 12ae was prepared according to general procedure

D (yield, 67%). H-NMR (400 MHz, DMSO-d₆) δ 3.87 (3H, s),

1

(3H, s), 3.92 (3H, s), 5.38 (2H, s), 6.88–7.04 (5H, m), 7.29 (1H, s),

7.37 (1H, t, J = 8.0 Hz), 7.65–7.75 (3H, m).

3.92 (3H, s), 5.66 (2H, s), 7.06 (1H, ddd, J = 3.0, 3.9, 9.1 Hz), 7.19

(1H, dd, J = 2.2, 8.9 Hz), 7.28 (1H, dd, J = 3.0, 6.3 Hz), 7.46 (1H,

dd, J = 8.9, 9.1 Hz), 7.47 (1H, ddd, J = 1.1, 2.2, 8.2 Hz), 7.53 (1H,

dd, J = 7.6, 8.2 Hz), 7.59 (1H, d, J = 2.2 Hz), 7.65 (1H, ddd, J =

1.1, 1.5, 7.6 Hz), 7.71 (1H, dd, J = 1.5, 2.2 Hz), 7.79 (1H, d, J =

8.9 Hz); HRMS (ESI) m/z: [M + H]⁺calcd for C₂₃H₁₉ClFN₂O₄,

441.1017; found 441.1018.

4.8.82. Methyl 3-{[6-(2-fluoro-5-methylphenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoate

(12y).

Compound 12y was prepared according to general procedure D

1

(yield, 49%). H-NMR (400 MHz, CDCl₃) δ 2.27 (3H, s), 3.82

4.8.89. Methyl 3-{[6-(2,3-dihydro-1-benzofuran-5-

yloxy)-1-methyl-1H-benzimidazol-2-

yl]methoxy}benzoate•HCl (12af).

Compound 12af was prepared according to general procedure

G (yield, 84%). ¹H-NMR (400 MHz, CDCl₃) δ 3.25 (2H, t, J = 8.6

Hz), 3.93 (3H, s), 4.00 (3H, s), 4.64 (2H, t, J = 8.6 Hz), 5.88 (2H,

s), 6.80–6.85 (2H, m), 6.93 (2H, d, J = 2.0 Hz), 7.24–7.27 (1H, m),

7.42–7.50 (2H, m), 7.68 (1H, d, J = 2.4 Hz), 7.73 (1H, dt, J = 1.6,

7.0 Hz), 7.91 (1H, d, J = 9.0 Hz).

(3H, s), 3.92 (3H, s), 5.39 (2H, s), 6.82 (1H, s), 6.86–6.92 (1H, m),

6.95 (1H, d, J = 2.4 Hz), 7.01 (1H, dd, J = 2.4, 9.0 Hz), 7.07 (1H,

dd, J = 8.2, 10.6 Hz), 7.28–7.31 (1H, m), 7.38 (1H, t, J = 7.8 Hz),

7.67–7.73 (3H, m).

4.8.83. Methyl 3-{[6-(3-fluoro-4-methylphenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoate

(12z).

Compound 12z was prepared according to the general

1

procedure D (yield, 82%). H-NMR (400 MHz, CDCl₃) δ 2.24

4.8.90. Methyl 3-{[6-(2,3-dihydro-1-benzofuran-6-

yloxy)-1-methyl-1H-benzimidazol-2-

yl]methoxy}benzoate•HCl (12ag).

Compound 12ag was prepared according to general procedure

G (yield, 47%). ¹H-NMR (500 MHz, DMSO-d₆) δ 3.15 (2H, t, J =

8.8 Hz), 3.88 (3H, s), 3.93 (3H, s), 4.57 (2H, t, J = 8.8 Hz), 5.69

(2H, s), 6.47 (2H, s), 7.16 (1H, d, J = 8.8 Hz), 7.21 (1H, d, J = 7.8

Hz), 7.47–7.56 (3H, m), 7.66 (1H, d, J = 8.3 Hz), 7.72 (1H, s), 7.78

(1H, d, J = 9.3 Hz).

(3H, d, J = 1.6 Hz), 3.82 (3H, s), 3.92 (3H, s), 5.40 (2H, s), 6.66–

6.71 (2H, m), 6.98–7.04 (2H, m), 7.11 (1H, t, J = 9.0 Hz), 7.27–

7.32 (1H, m), 7.38 (1H, t, J = 8.0 Hz), 7.67–7.70 (1H, m), 7.71–

7.75 (2H, m).

4.8.84. Methyl 3-{[6-(4-fluoro-3-methylphenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoate

(12aa).

Compound 12aa was prepared according to general procedure

D (yield, 80%). ¹H-NMR (400 MHz, CDCl₃) δ 2.25 (3H, d, J = 2.0

Hz), 3.81 (3H, s), 3.92 (3H, s), 5.39 (2H, s), 6.76–6.86 (2H, m),

6.92–7.00 (3H, m), 7.27–7.31 (1H, m), 7.38 (1H, t, J = 8.0 Hz),

7.71 (3H, d, J = 8.6 Hz).

4.8.91. Methyl 3-{[6-(1-benzofuran-6-yloxy)-1-

methyl-1H-benzimidazol-2-

yl]methoxy}benzoate•HCl (12ah).

Compound 12ah was prepared according to the general

procedure G (yield, 79%). H-NMR (400 MHz, CDCl₃) δ 3.93

4.8.85. Methyl 3-{[6-(3-fluoro-5-methylphenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoate

(12ab).

Compound 12ab was prepared according to general procedure

D (yield, 74%). ¹H-NMR (400 MHz, CDCl₃) δ 2.30 (3H, s), 3.84

(3H, s), 3.93 (3H, s), 5.40 (2H, s), 6.49 (1H, dt, J = 2.4, 10.2 Hz),

6.58 (1H, s), 6.60 (1H, d, J = 10.6 Hz), 7.00–7.03 (2H, m), 7.28–

7.31 (1H, m), 7.38 (1H, t, J = 7.8 Hz), 7.68 (1H, dt, J = 1.6, 8.6

Hz), 7.72–7.76 (2H, m).

1

(3H, s), 4.00 (3H, s), 5.89 (2H, s), 6.82 (1H, dd, J = 0.8, 2.0 Hz),

7.02 (1H, dd, J = 2.0, 8.6 Hz), 7.03 (1H, d, J = 2.4 Hz), 7.24 (1H,

d, J = 1.2 Hz), 7.32 (1H, dd, J = 2.4, 9.0 Hz), 7.42–7.49 (2H, m),

7.63 (1H, d, J = 8.6 Hz), 7.68 (2H, d, J = 2.0 Hz), 7.74 (1H, dt, J

= 1.6, 7.0 Hz), 7.94 (1H, d, J = 9.4 Hz).

4.8.92. 3-[(1-Methyl-6-phenoxy-1H-benzimidazol-2-

yl)methoxy]benzoic acid•HCl (13a).

Compound 13a was prepared according to general procedure E

(yield, 70%). ¹H-NMR (400 MHz, DMSO-d₆) δ 3.95 (3H, s), 5.72

(2H, s), 7.04 (2H, d, J = 8. 8 Hz), 7.16 (1H, t, J = 7.5 Hz), 7.21

(1H, dd, J = 2.4, 8.8 Hz), 7.41 (2H, dd, J = 7.5, 8.8 Hz), 7.46 (1H,

ddd, J = 1.1, 2.6, 8.2 Hz), 7.50 (1H, dd, J = 7.5, 8.2 Hz), 7.62 (1H,

d, J = 2.2 Hz), 7.64 (1H, ddd, J = 1.1, 1.5, 7.5 Hz), 7.71 (1H, dd, J

= 1.5, 2.6 Hz), 7.82 (1H, d, J = 8. 8 Hz); HRMS (ESI) m/z: [M +

H]⁺calcd for C₂₂H₁₉N₂O₄, 375.1345; found 375.1344.

4.8.86. Methyl 3-{[6-(4-chloro-3-fluorophenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoate

(12ac).

Compound 12ac was prepared according to general procedure

D (yield, 99%). ¹H-NMR (400 MHz, CDCl₃) δ 3.85 (3H, s), 3.93

(3H, s), 5.41 (2H, s), 6.74 (1H, ddd, J = 1.2, 2.8, 9.0 Hz), 6.78 (1H,

dd, J = 2.7, 10.2 Hz), 7.00–7.03 (2H, m), 7.28–7.33 (2H, m), 7.38

(1H, t, J = 7.4 Hz), 7.69 (1H, dt, J = 1.6, 7.4 Hz), 7.73 (1H, dd, J

= 1.6, 2.7 Hz), 7.77 (1H, d, J = 8.2 Hz); HRMS (ESI) m/z: [M +

H]⁺calcd for C₂₃H₁₉ClFN₂O₄, 441.1017; found 441.1071.

4.8.93. 3-{[6-(2-Fluorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methoxy}benzoic acid (13b).

Compound 13b was prepared according to general procedure E

(yield, 68%). Mp, 205–212 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ

3.81 (3H, s), 5.47 (2H, s), 6.94 (1H, dd, J = 2.5, 8.8 Hz), 7.04–7.09

(1H, m), 7.18 (2H, ddd, J = 3.1, 3.3, 6.1 Hz), 7.30 (1H, d, J = 2.4

Hz), 7.35–7.42 (2H, m), 7.45 (1H, t, J = 7.8 Hz), 7.58 (1H, d, J =

7.4 Hz), 7.66 (1H, d, J = 8.6 Hz), 7.63 (1H, s), 13.03 (1H, br); MS

(FAB) m/z: 393 [M + H]⁺; Anal. calcd for C₂₄H₂₂N₂O₅•0.14H₂O:

C, 66.91; H, 4.41; N, 7.09; F, 4.81; found C, 66.86; H, 4.48; N,

7.08; F, 4.80.

4.8.87. Methyl 3-{[6-(3-chloro-5-fluorophenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoate

(12ad).

Compound 12ad was prepared according to general procedure

D (yield, 32%). ¹H-NMR (400 MHz, CDCl₃) δ 3.87 (3H, s), 3.93

(3H, s), 5.42 (2H, s), 6.59 (1H, dt, J = 2.4, 9.8 Hz), 6.75 (1H, s)

6.80 (1H, dt, J = 2.4, 7.8 Hz), 7.03 (1H, dd, J = 2.0, 8.6 Hz), 7.07

(1H, d, J = 2.4 Hz), 7.31 (1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.39 (1H,

t, J = 7.8 Hz), 7.70 (1H, dd, J = 1.2, 7.8 Hz), 7.74 (1H, s), 7.79

(1H, d, J = 8.6 Hz).

4.8.94. 3-{[6-(3-Fluorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methoxy}benzoic acid (13c).

4.8.100. 3-{[6-(4-Chlorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methoxy}benzoic acid (13i).

Compound 13c was prepared according to general procedure E

(yield, 93%). Mp, 236–237 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ

3.83 (3H, s), 5.48 (2H, s), 6.77–6.84 (2H, m), 6.92 (1H, ddt, J =

0.8, 2.4, 8.2 Hz), 6.99 (1H, dd, J = 2.4, 8.6 Hz), 7.35–7.41 (2H,

m), 7.42 (1H, d, J = 2.0 Hz), 7.46 (1H, t, J = 7.8 Hz), 7.58 (1H, dt,

J = 1.2, 7.4 Hz), 7.64 (1H, dd, J = 1.6, 2.7 Hz), 7.70 (1H, d, J =

8.6 Hz), 13.05 (1H, br); HRMS (ESI) m/z: [M + H]⁺calcd for

C₂₂H₁₈FN₂O₄, 393.1251; found 393.1223; Anal. calcd for

C₂₂H₁₇FN₂O₄: C, 67.34; H, 4.37; N, 7.14; F, 4.84; found C, 67.41;

H, 4.33; N, 7.13; F, 5.03.

Compound 13i was prepared according to general procedure E

(yield, 89%). ¹H-NMR (400 MHz, DMSO-d₆) δ 3.78 (3H, s), 5.44

(2H, s), 6.91-6.98 (3H, m), 7.33-7.37 (4H, m), 7.42 (1H, t, J = 7.8

Hz), 7.54 (1H, dt, J = 1.3, 7.7 Hz), 7.60-7.61 (1H, m), 7.65 (1H, d,

J = 8.2 Hz), 13.04 (1H, br); Anal. calcd for C₂₂H₁₇ClN₂O₄: C,

64.63; H, 4.19; N, 6.85; Cl, 8.67; found C, 64.54; H, 4.20; N, 6.84;

Cl, 8.90.

4.8.101. 3-{[6-(2-Methoxyphenoxy)-1-methyl-1H-

benzimidazol-2-yl] methoxy}benzoic acid•0.13HCl

(13j).

4.8.95. 3-{[6-(4-Fluorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methoxy}benzoic acid (13d).

Compound 13j was prepared according to general procedure E

(yield, 89%). Mp, 193–196 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ

3.76 (3H, s), 3.77 (3H, s), 5.44 (2H, s), 6.81 (1H, dd, J = 2.4, 8.6

Hz), 6.91–6.97 (2H, m), 7.09 (1H, d, J = 2.4 Hz), 7.15–7.17 (2H,

m), 7.38 (1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.44 (1H, t, J = 7.4 Hz),

7.57 (1H, dt, J = 1.2, 7.4 Hz), 7.59 (1H, d, J = 8.6 Hz), 7.63 (1H,

dd, J = 1.6, 2.7 Hz), 13.04 (1H, br); MS (FAB) m/z: 405 [M + H]⁺;

Anal. calcd for C₂₃H₂₀N₂O₅•0.13HCl: C, 67.55; H, 4.96; N, 6.85;

Cl, 1.08; found C, 67.76; H, 4.90; N, 6.92; Cl, 1.08.

Compound 13d was prepared according to general procedure E

(yield, 37%). Mp, 250–254 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ

3.81 (3H, s), 5.46 (2H, s), 6.93 (1H, dd, J = 2.4, 8.8 Hz), 7.01–7.04

(2H, m), 7.19 (2H, t, J = 8.8 Hz), 7.30 (1H, d, J = 2.4 Hz), 7.37–

7.39 (1H, m), 7.45 (1H, t, J = 7.8 Hz), 7.57 (2H, d, J = 7.8 Hz),

7.66 (1H, d, J = 8.8 Hz), 7.63 (1H, s), 13.03 (1H, br); MS (FAB)

m/z: 393 [M + H]⁺; Anal. calcd for C₂₂H₁₇FN₂O₄•0.20H₂O: C,

66.73; H, 4.43; N, 7.07; F, 4.80; found C, 66.75; H, 4.55; N, 7.06;

F, 4.91.

4.8.102. 3-{[6-(3-Methoxyphenoxy)-1-methyl-1H-

benzimidazol-2-yl] methoxy}benzoic acid (13k).

4.8.96. 3-{[1-Methyl-6-(2-methylphenoxy)-1H-

benzimidazol-2-yl] methoxy}benzoic acid (13e).

Compound 13k was prepared according to general procedure E

(yield, 89%). Mp, 210–214 °C; ¹H-NMR (400 MHz, CDCl₃) δ 3.77

(3H, s), 3.89 (3H, s), 5.57 (2H, s), 6.55–6.58 (2H, m), 6.61–6.69

(1H, m), 7.00–7.07 (2H, m), 7.19–7.25 (2H, m), 7.38 (1H, t, J =

7.8 Hz), 7.84 (1H, d, J = 8.6 Hz), 7.78 (1H, d, J = 7.4 Hz), 8.03

(1H, s); MS (FAB) m/z: 405 [M + H]⁺; Anal. calcd for C₂₃H₂₀N₂O₅:

C, 68.31; H, 4.98; N, 6.93; found C, 68.04; H, 4.97; N, 6.91.

Compound 13e was prepared according to general procedure E

(yield, 98%). ¹H-NMR (500 MHz, DMSO-d₆) δ 2.25 (3H, s), 3.79

(3H, s), 5.45 (2H, s), 6.81 (1H, d, J = 7.8 Hz), 6.86 (1H, dd, J =

2.4, 8.8 Hz), 7.06 (1H, t, J = 7.3 Hz), 7.14–7.21 (2H, m), 7.32 (1H,

d, J = 7.3 Hz), 7.36–7.41 (1H, m), 7.45 (1H, t, J = 8.1 Hz), 7.57

(1H, d, J = 7.3 Hz), 7.63 (2H, d, J = 8.3 Hz), 13.03 (1H, s); MS

(FAB) m/z: 389 [M + H]⁺; Anal. calcd for C₂₃H₂₀N₂O₄•0.20H₂O:

C, 70.47; H, 5.25; N, 7.15; found C, 70.44; H, 5.12; N, 7.16.

4.8.103. 3-{[6-(4-Methoxyphenoxy)-1-methyl-1H-

benzimidazol-2-yl] methoxy}benzoic acid (13l).

Compound 13l was prepared according to general procedure E

(yield, 86%). Mp, 208–211 °C; ¹H-NMR (500 MHz, CDCl₃) δ 3.79

(3H, s), 3.81 (3H, s), 5.36 (2H, br), 6.87–6.93 (3H, m) 6.98 (3H,

d, J = 8.3 Hz), 7.20–7.29 (1H, m), 7.36 (1H, s), 7.65–7.76 (3H, m);

4.8.97. 3-{[1-Methyl-6-(3-methylphenoxy)-1H-

benzimidazol-2-yl] methoxy}benzoic acid (13f).

Compound 13f was prepared according to general procedure E

(yield, 60%). ¹H-NMR (400 MHz, DMSO-d₆) δ 2.27 (3H, s), 3.81

(3H, s), 5.47 (2H, s), 6.74–6.83 (2H, m), 6.87–6.97 (2H, m), 7.32

(1H, d, J = 2.4 Hz), 7.36–7.41 (1H, m), 7.45 (1H, t, J = 8.0 Hz),

7.58 (1H, dt, J = 1.2, 1.4, 7.6 Hz), 7.62–7.68 (2H, m); MS (FAB)

m/z: 389 [M + H]⁺; Anal. calcd for C₂₃H₂₀N₂O₄•0.33H₂O: C,

70.04; H, 5.28; N, 7.10; found C, 69.99; H, 5.16; N, 7.15.

MS (FAB) m/z: 405 [M

+

H]⁺; Anal. calcd for

C₂₃H₂₀N₂O₅•1.5H₂O: C, 64.03; H, 5.37; N, 6.49; found C, 63.96;

H, 5.30; N, 6.52.

4.8.104. 3-{[6-(3-Ethoxyphenoxy)-1-methyl-1H-

benzimidazol-2-yl] methoxy}benzoic acid (13m).

Compound 13m was prepared according to general procedure

E (yield, 86%). ¹H-NMR (400 MHz, DMSO-d₆) δ 1.29 (3H, t, J =

6.7 Hz), 3.82 (3H, s), 3.98 (2H, q, J = 7.0 Hz), 5.47 (2H, s), 6.48–

6.52 (2H, m), 6.66 (1H, ddd, J = 1.2, 2.4, 8.2 Hz), 6.95 (1H, dd, J

= 2.4, 8.6 Hz), 7.23 (1H, dt, J = 0.8, 7.4 Hz), 7.34 (1H, d, J = 2.4

Hz), 7.38 (1H, ddd, J = 1.2, 2.4, 8.2 Hz), 7.45 (1H, t, J = 7.8 Hz),

7.58 (1H, dt, J = 1.2, 7.4 Hz), 7.64 (1H, dd, J = 1.6, 2.4 Hz), 7.67

(1H, d, J = 8.6 Hz), 13.03 (1H, br); MS (FAB) m/z: 419 [M + H]⁺.

4.8.98. 3-{[1-Methyl-6-(4-methylphenoxy)-1H-

benzimidazol-2-yl] methoxy}benzoic acid (13g).

Compound 13g was prepared according to general procedure E

(yield, 68%). Mp > 300 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ 2.28

(3H, s), 3.80 (3H, s), 5.46 (2H, s), 6.87–6.94 (3H, m), 7.17 (2H, d,

J = 9.0 Hz), 7.26 (1H, d, J = 2.3 Hz), 7.36–7.41 (1H, m), 7.45 (1H,

t, J = 8.0 Hz), 7.58 (1H, d, J = 7.8 Hz), 7.62–7.67 (2H, m), 13.05

(1H, s); MS (FAB) m/z: 389 [M + H]⁺; Anal. calcd for C₂₃H₂₀N₂O₄:

C, 71.12; H, 5.19; N, 7.21; found C, 71.17; H, 5.09; N, 7.29.

4.8.105. 3-({1-Methyl-6-[3-

(trifluoromethoxy)phenoxy] -1H-benzimidazol-2-

yl}methoxy)benzoic acid (13n).

4.8.99. 3-{[6-(3-Chlorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methoxy}benzoic acid•0.83HCl

(13h).

Compound 13n was prepared according to general procedure E

(yield, 81%). Mp, 221–227 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ

3.83 (3H, s), 5.48 (2H, s), 6.95–6.97 (2H, m), 7.00 (1H, dd, J =

2.4, 8.6 Hz), 7.07–7.09 (1H, m), 7.36–7.39 (1H, m), 7.43–7.49

(3H, m), 7.58 (1H, dt, J = 1.2, 7.8 Hz), 7.64 (1H, dd, J = 1.2, 2.4

Hz), 7.71 (1H, d, J = 8.2 Hz), 13.08 (1H, br); MS (FAB) m/z: 459

[M + H]⁺; Anal. calcd for C₂₃H₁₇F₃N₂O₅: C, 60.26; H, 3.74; N,

6.11; found C, 60.00; H, 3.70; N, 6.23.

Compound 13h was prepared according to general procedure E

(yield, 84%). Mp, 218–222 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ

3.91 (3H, s), 5.62 (2H, s), 6.98 (1H, dd, J = 3.1, 9.0 Hz), 7.04 (1H,

t, J = 2.0 Hz), 7.17 (1H, dd, J = 2.4, 9.0 Hz), 7.18–7.20 (1H, m),

7.40 (1H, d, J = 8.2 Hz), 7.42–7.44 (1H, m), 7.49 (1H, t, J = 7.8

Hz), 7.61 (1H, s), 7.62 (1H, d, J = 7.4 Hz), 7.69 (1H, s), 7.79 (1H,

d, J = 9.0 Hz); MS (FAB) m/z: 409 [M + H]⁺; Anal. calcd for

C₂₂H₁₇ClN₂O₄•0.83HCl: C, 60.16; H, 4.09; N, 6.38; Cl, 14.80;

found C, 60.13; H, 3.98; N, 6.49; Cl, 14.96.

4.8.106. 3-({1-Methyl-6-[3-

(trifluoromethyl)phenoxy] -1H-benzimidazol-2-

yl}methoxy)benzoic acid•0.1HCl (13o).

Compound 13o was prepared according to general procedure E

(yield, 89%). ¹H-NMR (400 MHz, DMSO-d₆) δ 3.84 (3H, s), 5.48

(2H, s), 7.02 (1H, dd, J = 2.4, 9.0 Hz), 7.23–7.27 (2H, m), 7.33–

7.48 (4H, m), 7.57–7.61 (2H, m), 7.64 (1H, s), 7.72 (1H, d, J = 8.6

Hz); MS (FAB) m/z: 443 [M

C₂₃H₁₇F₃N₂O₄•0.10HCl: C, 61.93; H, 3.86; F, 12.78; N, 6.28; Cl,

0.79; found C, 61.78; H, 3.85; F, 12.55; N, 6.37; Cl, 0.83.

Hz), 7.02 (1H, dd, J = 2.4, 8.6 Hz), 7.30–7.36 (1H, m), 7.42 (1H,

t, J = 7.8 Hz), 7.48 (1H, d, J = 2.4 Hz), 7.57 (1H, d, J = 7.4 Hz),

7.63 (1H, dd, J = 1.6, 2.4 Hz), 7.72 (1H, d, J = 8.6 Hz); MS (FAB)

m/z: 411 [M + H]⁺; Anal. calcd for C₂₂H₁₆F₂N₂O₄•H₂O: C, 61.68;

H, 4.24; F, 8.87; N, 6.54; found C, 61.58; H, 3.95; F, 9.06; N, 6.51.

4.8.113. 3-{[6-(4-Fluoro-2-methylphenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoic acid

(13v).

+

H]⁺; Anal. calcd for

Compound 13v was prepared according to general procedure E

(yield, 89%). Mp, 217–218 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ

2.22 (3H, s), 3.78 (3H, s), 5.45 (2H, s), 6.82–6.91 (2H, m), 6.98–

7.05 (1H, m), 7.12 (1H, d, J = 2.3 Hz), 7.20 (1H, dd, J = 3.1, 9.4

Hz), 7.35–7.40 (1H, m), 7.45 (1H, t, J = 7.8 Hz), 7.55–7.59 (1H,

m), 7.60–7.65 (2H, m), 13.03 (1H, s); MS (FAB) m/z: 407 [M +

H]⁺; Anal. calcd for C₂₃H₁₉FN₂O₄•0.20H₂O: C, 67.38; H, 4.77; N,

6.83; found C, 67.44; H, 4.47; F, 4.75; N, 6.90.

4.8.107. 3-{[6-(2,3-Difluorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methoxy}benzoic acid (13p).

Compound 13p was prepared according to general procedure E

(yield, 86%). ¹H-NMR (400 MHz, DMSO-d₆) δ 3.82 (3H, s), 5.47

(2H, s), 6.84 (1H, tt, J = 2.0, 7.0 Hz), 7.01 (1H, dd, J = 2.0, 8.6

Hz), 7.13–7.24 (2H, m), 7.36–7.41 (2H, m), 7.45 (1H, t, J = 7.4

Hz), 7.57 (1H, dt, J = 1.6, 7.8 Hz), 7.63 (1H, dd, J = 1.6, 2.7 Hz),

7.68 (1H, d, J = 8.6 Hz), 13.00 (1H, br); MS (FAB) m/z: 411 [M +

H]⁺; Anal. calcd for C₂₂H₁₆F₂N₂O₄•0.17H₂O: C, 63.92; H, 3.98; F,

9.19; N, 6.78; found C, 63.88; H, 3.80; F, 9.11; N, 6.90.

4.8.114. 3-{[6-(5-Fluoro-2-methylphenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoic acid

(13w).

Compound 13w was prepared according to general procedure

E (yield, 93%). Mp, 208–213 °C; ¹H-NMR (400 MHz, DMSO-d₆)

δ 2.24 (3H, s), 3.82 (3H, s), 5.47 (2H, s), 6.57 (1H, dd, J = 2.5,

10.4 Hz), 6.85–6.95 (2H, m), 7.29 (1H, d, J = 2.3 Hz), 7.31–7.41

(2H, m), 7.46 (1H, t, J = 7.8 Hz), 7.58 (1H, d, J = 7.8 Hz), 7.63–

7.66 (1H, m), 7.67 (1H, d, J = 8.6 Hz), 13.04 (1H, s); MS (FAB)

m/z: 407 [M + H]⁺; Anal. calcd for C₂₃H₁₉FN₂O₄•0.50H₂O: C,

66.50; H, 4.85; N, 6.74; found C, 66.87; H, 5.16; N, 6.59.

4.8.108. 3-{[6-(2,4-Difluorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methoxy}benzoic acid (13q).

Compound 13q was prepared according to general procedure E

(yield, 48%). ¹H-NMR (400 MHz, DMSO-d₆) δ 3.80 (3H, s), 5.45

(2H, s), 6.93 (1H, dd, J = 2.5, 8.8 Hz), 7.05–7.12 (1H, m), 7.19

(1H, dt, J = 5.9, 9.2 Hz), 7.25 (1H, d, J = 2.4 Hz), 7.32–7.37 (1H,

m), 7.43 (1H, t, J = 7.82 Hz), 7.44–7.51 (1H, m), 7.54–7.59 (1H,

m), 7.62 (1H, dd, J = 1.4, 2.5 Hz), 7.64 (1H, d, J = 8.6 Hz); MS

(FAB) m/z: 411 [M + H]⁺; Anal. calcd for C₂₂H₁₆F₂N₂O₄•0.50H₂O:

C, 63.01; H, 4.09; F, 9.06; N, 6.68; found C, 63.13; H, 3.86; F,

9.20; N, 6.70.

4.8.115. 3-{[6-(2-Fluoro-4-methylphenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoic acid

(13x).

Compound 13x was prepared according to general procedure E

(yield, 48%). Mp, 230–235 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ

2.31 (3H, s), 3.79 (3H, s), 5.45 (2H, s), 6.90 (1H, dd, J = 2.4, 9.0

Hz), 6.99–7.02 (2H, m), 7.21 (1H, t, J = 6.7 Hz), 7.21 (1H, s),

7.35–7.40 (1H, m), 7.45 (1H, t, J = 7.8 Hz), 7.55–7.59 (1 H, m),

7.63 (1H, d, J = 8.6 Hz), 7.63 (1H, dd, J = 1.6, 2.4 Hz), 13.03 (1H,

4.8.109. 3-{[6-(2,5-Difluorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methoxy}benzoic acid (13r).

Compound 13r was prepared according to general procedure E

(yield, 91%). Mp, 224–227 °C; ¹H-NMR (500 MHz, DMSO-d₆) δ

3.83 (3H, s), 5.47 (2H, s), 6.88–6.95 (1H, m), 6.98–7.05 (2H, m),

7.38 (2H, s), 7.41–7.48 (2H, m), 7.58 (1H, d, J = 7.3 Hz), 7.63 (1H,

s), 7.68 (1H, d, J = 8.8 Hz), 13.03 (1H, br); MS (FAB) m/z: 411

[M + H]⁺; Anal. calcd for C₂₂H₁₆F₂N₂O₄•0.25H₂O: C, 63.69; H,

4.01; F, 9.16; N, 6.75; found C, 63.84; H, 4.05; F, 9.22; N, 6.83.

br), MS (FAB) m/z: 407 [M

+

H]⁺; Anal. calcd for

C₂₃H₁₉FN₂O₄•0.20H₂O: C, 67.38; H, 4.77; F, 4.63; N, 6.83; found

C, 67.46; H, 4.72; F, 4.75; N, 6.89.

4.8.116. 3-{[6-(2-Fluoro-5-methylphenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoic acid

(13y).

4.8.110. 3-{[6-(2,6-difluorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methoxy}benzoic acid (13s).

Compound 13y was prepared according to general procedure E

(yield, 95%). Mp, 227–233 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ

2.23 (3H, s), 3.81 (3H, s), 5.46 (2H, s), 6.87 (1H, s), 6.93 (1H, dd,

J = 2.4, 9.0 Hz), 6.94–6.99 (1H, m), 7.22–7.31 (2H, m), 7.39 (1H,

s), 7.45 (1H, t, J = 7.8 Hz), 7.58 (1H, d, J = 7.4 Hz), 7.62–7.68

(2H, m); MS (FAB) m/z: 407 [M + H]⁺; Anal. calcd for

C₂₃H₁₉FN₂O₄•0.20H₂O: C, 67.38; H, 4.77; F, 4.63; N, 6.83; found

C, 67.43; H, 4.71; F, 4.80; N, 6.87.

Compound 13s was prepared according to general procedure E

(yield, 85%). ¹H-NMR (400 MHz, DMSO-d₆) δ 3.79 (3H, s), 5.43

(2H, s), 6.89 (1H, dd, J = 2.4, 8.6 Hz), 7.17 (1H, d, J = 2.4 Hz),

7.29–7.42 (5H, m), 7.55 (1H, dt, J = 1.6, 7.8 Hz), 7.60–7.62 (2H,

m); MS (FAB) m/z: 411 [M + H]⁺.

4.8.111. 3-{[6-(3,4-Difluorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methoxy}benzoic acid•0.1HCl

(13t).

4.8.117. 3-{[6-(3-Fluoro-4-methylphenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoic acid

(13z).

Compound 13t was prepared according to general procedure E

(yield, 82%). Mp, 256–260 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ

3.82 (3H, s), 5.47 (2H, s), 6.77–6.84 (1H, m), 6.97 (1H, dd, J =

2.4, 8.6 Hz), 7.10–7.18 (1H, m), 7.35–7.48 (4H, m), 7.55–7.70

(3H, m); MS (FAB) m/z: 411 [M + H]⁺; Anal. calcd for

C₂₂H₁₆F₂N₂O₄•0.10HCl: C, 67.37; H, 4.69; F, 4.63; N, 6.83; Cl,

0.86; found C, 67.24; H, 4.70; F, 4.56; N, 7.00; Cl, 0.64.

Compound 13z was prepared according to general procedure E

(yield, 94%). Mp, 220–223 °C; ¹H-NMR (400 MHz, DMSO-d₆) δ

2.18 (3H, s), 3.82 (3H, s), 5.47 (2H, s), 6.72 (6H, dd, J = 2.4, 8.6

Hz), 6.80 (6H, dd, J = 2.5, 11.1 Hz), 6.95 (6H, dd, J = 2.4, 8.6 Hz),

7.25 (6H, t, J = 8.6 Hz), 7.35–7.40 (2H, m), 7.36 (1H, d, J = 2.4

Hz), 7.45 (1H, t, J = 7.8 Hz), 7.58 (1H, d, J = 7.8 Hz), 7.67 (1H,

d, J = 8.6 Hz), 7.63–7.69 (1H, m), 13.08 (1H, br); MS (FAB) m/z:

407 [M + H]⁺; Anal. calcd for C₂₃H₁₉FN₂O₄: C, 67.97; H, 4.71; F,

4.67; N, 6.89; found C, 67.58; H, 4.63; F, 4.67; N, 6.91.

4.8.112. 3-{[6-(3,5-Difluorophenoxy)-1-methyl-1H-

benzimidazol-2-yl] methoxy}benzoic acid (13u).

Compound 13u was prepared according to general procedure E

(yield, 76%). ¹H-NMR (400 MHz, DMSO-d₆) δ 3.85 (3H, s), 5.47

(2H, s), 6.69 (2H, dd, J = 2.2, 8.8 Hz), 6.95 (1H, tt, J = 2.2, 9.3

4.8.118. 3-{[6-(4-Fluoro-3-methylphenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoic

acid•0.1HCl (13aa).

4.8.123. 3-{[6-(2,3-Dihydro-1-benzofuran-5-yloxy)-

1-methyl-1H-benzimidazol-2-yl] methoxy}benzoic

acid 0.17 HCl (13af).

Compound 13aa was prepared according to general procedure

E (yield, 81%). H-NMR (500 MHz, DMSO-d₆) δ 2.20 (3H, s),

Compound 13af was prepared according to general procedure

E (yield, 63%). ¹H-NMR (400 MHz, DMSO-d₆) δ 3.16 (2H, t, J =

8.6 Hz), 3.79 (3H, s), 4.52 (2H, t, J = 8.6 Hz), 5.45 (2H, s), 6.73–

6.78 (2H, m), 6.88 (1H, dd, J = 2.7, 9.0 Hz), 6.93 (1H, dd, J = 0.8,

2.4 Hz), 7.18 (1H, d, J = 2.4 Hz), 7.38 (1H, ddd, J = 1.2, 2.7, 8.2

Hz), 7.45 (1H, t, J = 7.4 Hz), 7.57 (1H, dt, J = 1.2, 9.0 Hz), 7.61

(1H, d, J = 8.6 Hz), 7.63 (1H, dd, J = 1.6, 2.7 Hz), 13.06 (1H, br);

1

3.81 (3H, s), 5.46 (2H, s), 6.80–6.87 (1 H, m), 6.89–6.95 (2H, m),

7.12 (1H, t, J = 9.0 Hz), 7.28 (1H, d, J = 2.0 Hz), 7.36–7.41 (1H,

m), 7.45 (1H, t, J = 7.8 Hz), 7.57 (1H, d, J = 7.8 Hz), 7.62–7.67

(2H, m), 13.03 (1H, br); MS (FAB) m/z: 407 [M + H]⁺; Anal. calcd

for C₂₃H₁₉FN₂O₄•0.10HCl: C, 67.37; H, 4.69; F, 4.63; N, 6.83; Cl,

0.86; found C, 67.24; H, 4.70; F, 4.56; N, 7.00; Cl, 0.64.

MS (FAB) m/z: 417 [M

+

H]⁺; Anal. calcd for

C₂₄H₂₀N₂O₅•0.17HCl•1.25H₂O: C, 64.77; H, 5.13; N, 6.29; Cl,

1.33; found C, 64.91; H, 4.71; N, 6.23; Cl, 1.26.

4.8.119. 3-{[6-(3-Fluoro-5-methylphenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoic acid

(13ab).

4.8.124. 3-{[6-(2,3-Dihydro-1-benzofuran-6-yloxy)-

1-methyl-1H-benzimidazol-2-yl] methoxy}benzoic

acid (13ag).

Compound 13ab was prepared according to general procedure

E (yield, 73%). Mp, 224–226 °C; ¹H-NMR (400 MHz, DMSO-d₆)

δ 2.25 (3H, s), 3.83 (3H, s), 5.47 (2H, s), 6.58–6.60 (2H, m), 6.75

(1H, d, J = 10.6 Hz), 6.96 (1H, dd, J = 2.4, 8.6 Hz), 7.37–7.39 (2H,

m), 7.45 (1H, t, J = 7.4 Hz), 7.57 (1H, dt, J = 1.2, 7.8 Hz), 7.64

(1H, dd, J = 1.2, 2.4 Hz), 7.68 (1H, d, J = 8.6 Hz), 13.03 (1H, s);

MS (FAB) m/z: 407 [M + H]⁺; Anal. calcd for C₂₃H₁₉FN₂O₄: C,

67.97; H, 4.71; N, 6.89; found C, 68.13; H, 4.57; N, 6.95.

Compound 13ag was prepared according to general procedure

E (yield, 74%). Mp, 213–217 °C; ¹H-NMR (400 MHz, DMSO-d₆)

δ 3.13 (2H, t, J = 8.4 Hz), 3.81 (3H, s), 4.55 (2H, t, J = 8.6 Hz),

5.46 (2H, s), 6.39–6.44 (2H, m), 6.91 (1H, dd, J = 2.4, 8.6 Hz),

7.16 (1H, d, J = 7.8 Hz), 7.28 (1H, d, J = 2.4 Hz), 7.36–7.40 (1H,

m), 7.45 (1H, t, J = 7.8 Hz), 7.58 (1H, dt, J = 1.2, 1.4, 7.6 Hz),

7.62–7.66 (2H, m), 13.04 (1H, br); MS (FAB) m/z: 417 [M + H]⁺;

Anal. calcd for C₂₄H₂₀N₂O₅•0.33H₂O: C, 68.24; H, 4.93; N, 6.63;

found C, 68.34; H, 4.84; N, 6.79.

4.8.120. 3-{[6-(4-Chloro-3-fluorophenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoic acid

(13ac).

4.8.125. 3-{[6-(1-Benzofuran-6-yloxy)-1-methyl-1H-

benzimidazol-2-yl] methoxy}benzoic acid (13ah).

Compound 13ac was prepared according to general procedure

E (yield, 72%). Mp, 234–236 °C; ¹H-NMR (400 MHz, DMSO-d₆)

δ 3.84 (3H, s), 5.48 (2H, s), 6.83 (1H, ddd, J = 1.2, 2.7, 9.0 Hz),

7.01 (1H, dd, J = 2.4, 8.6 Hz), 7.10 (1H, dd, J = 2.7, 10.6 Hz), 7.39

(1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.44–7.48 (2H, m), 7.54 (1H, t, J =

9.0 Hz), 7.58 (1H, dt, J = 1.6, 7.8 Hz), 7.64 (1H, dd, J = 1.6, 2.4

Hz), 7.71 (1H, d, J = 9.0 Hz), 13.05 (1H, br); HRMS (ESI) m/z:

[M + H]⁺calcd for C₂₂H₁₇ClFN₂O₄, 427.0861; found 427.0853;

Anal. calcd for C₂₂H₁₆ClFN₂O₄: C, 61.91; H, 3.78; N, 6.56; Cl,

8.31; found C, 61.72; H, 3.70; N, 6.63; Cl, 8.54.

Compound 13ah was prepared according to general procedure

1

E (yield, 98%). H-NMR (400 MHz, DMSO-d₆) δ 3.81 (3H, s),

5.47 (2H, s), 6.95 (1H, dd, J = 0.8, 2.4 Hz), 6.97 (1H, dd, J = 2.4,

4.3 Hz), 6.99 (1H, dd, J = 20, 3.9 Hz), 7.22 (1H, d, J = 2.7 Hz),

7.33 (1H, d, J = 2.4 Hz), 7.37–7.40 (1H, m), 7.45 (1H, t, J = 7.4

Hz), 7.58 (1H, dt, J = 1.2, 7.4 Hz), 7.62–7.68 (3H, m), 7.95 (1H,

d, J = 2.4 Hz), 13.05 (1H, br); MS (FAB) m/z: 415 [M + H]⁺; Anal.

calcd for C₂₄H₁₈N₂O₅•0.20H₂O: C, 68.96; H, 4.44; N, 6.70; found

C, 69.18; H, 4.32; N, 6.61.

4.8.121. 3-{[6-(3-Chloro-5-fluorophenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoic acid

(13ad).

4.8.126. [3-(Methoxycarbonyl)phenoxy]acetic acid

(14).

Compound 13ad was prepared according to general procedure

E (yield, 86%). Mp, 209–213 °C; ¹H-NMR (400 MHz, DMSO-d₆)

δ 3.84 (3H, s), 5.48 (2H, s), 6.83–6.86 (2H, m), 7.02 (1H, dd, J =

2.4, 9.0 Hz), 7.15 (1H, dt, J = 2.0, 8.6 Hz), 7.39 (1H, ddd, J = 1.2,

2.4, 8.2 Hz), 7.45 (1H, t, J = 7.4 Hz), 7.49 (1H, d, J = 2.4 Hz), 7.58

(1H, dt, J = 1.2, 7.4 Hz), 7.65 (1H, dd, J = 1.2, 2.4 Hz), 7.72 (1H,

d, J = 8.6 Hz), 13.04 (1H, s); MS (FAB) m/z: 427 [M + H]⁺; Anal.

calcd for C₂₂H₁₆ClFN₂O₄•0.2H₂O: C, 61.39; H, 3.84; N, 6.51; Cl,

8.24; found C, 61.56; H, 3.76; N, 6.53; Cl, 8.53.

A solution of tert-butyl bromoacetate (506 g, 2.59 mol), methyl

3-hydroxybenzoate (395 g, 2.60 mol) and K₂CO₃(789 g, 5.71 mol)

in DMF (2.0 L) was stirred at room temperature for 2 h under N₂.

The reaction mixture was concentrated and water (1.0 L) was

added, followed by extraction with EtOAc (2.0 L). The organic

layer was washed with water (1.0 L) twice, dried over anhydrous

Na₂SO₄, and concentrated under reduced pressure to obtain crude

tert-butyl [3-(methoxycarbonyl)phenoxy]acetate as a colorless oil.

A

solution

of

crude

tert-butyl

[3-

(methoxycarbonyl)phenoxy]acetate in TFA (1.0 kg), anisole (100

mL) and CH₂Cl₂(1.0 L) was stirred at room temperature for 48 h

under N₂. The reaction mixture was concentrated, and the residue

was crystallized from i-Pr₂O to obtain 14 (467 g, 86%) as a white

solid. H-NMR (400 MHz, CDCl₃) δ 3.93 (3H, s), 4.76 (2H, s),

7.17 (1H, dd, J = 2.4, 8.2 Hz), 7.39 (1H, t, J = 7.8 Hz), 7.57–7.58

(1H, m), 7.72 (1H, d, J = 7.4 Hz).

4.8.122. 3-{[6-(3-Chloro-4-fluorophenoxy)-1-

methyl-1H-benzimidazol-2-yl] methoxy}benzoic

acid•0.13HCl (13ae).

Compound 13ae was prepared according to general procedure

1

E (yield, 70%). H-NMR (400 MHz, DMSO-d₆) δ 3.94 (3H, s),

5.69 (2H, s), 7.07 (1H, ddd, J = 3.0, 3.9, 9.1 Hz), 7.22 (1H, dd, J

= 2.2, 8.9 Hz), 7.29 (1H, dd, J = 3.0, 6.2 Hz), 7.44 (1H, ddd, J =

1.1, 2.7, 8.2 Hz), 7.46 (1H, dd, J = 9.1, 9.1 Hz), 7.50 (1H, dd, J =

7.6, 8.2 Hz), 7.63 (1H, d, J = 2.2 Hz), 7.64 (1H, ddd, J = 1.1, 1.3,

7.6 Hz), 7.70 (1H, dd, J = 1.3, 2.7 Hz), 7.82 (1H, d, J = 8. 9 Hz);

HRMS (ESI) m/z: [M + H]⁺calcd for C₂₂H₁₇ClFN₂O₄, 427.0861;

found 427.0854; Anal. calcd for C₂₂H₁₆ClFN₂O₄•0.13HCl: C,

61.25; H, 3.77; N, 6.49; Cl, 9.25; F, 4.40; found C, 61.40; H, 3.76;

N, 6.52; Cl, 8.95; F, 4.86.

4.8.127. Methyl 3-[2-({2-[(tert-

butoxycarbonyl)(methyl)amino] -4-(2,3-dihydro-1-

benzofuran-5-yloxy)phenyl}amino)-2-

oxoethoxy]benzoate (15af).

Compound 15af was prepared according to general procedure

1

F (yield, 86%; 2 steps). H-NMR (400 MHz, CDCl₃) δ 1.41 (9H,

br), 3.10 (3H, br), 3.21 (2H, t, J = 8.6 Hz), 3.93 (3H, s), 4.60 (2H,

t, J = 8.6 Hz), 4.67 (2H, s), 6.73–6.87 (4H, m), 6.90 (1H, d, J = 2.0

Hz), 7.20 (1H, dd, J = 2.4, 7.8 Hz), 7.26–7.27 (1H, m), 7.41 (1H,

t, J = 7.8 Hz), 7.63 (1H, s), 7.74 (1H, d, J = 7.4 Hz).

4.8.128. Methyl 3-[2-({2-[(tert-

octuplicate, except for 13ac, which are from thirteen independent

butoxycarbonyl)(methyl)amino] -4-(2,3-dihydro-1-

benzofuran-6-yloxy)phenyl}amino)-2-

oxoethoxy]benzoate (15ag).

experiments run in octuplicate.

4.10. Measurement of Log D.

Compound 15ag was prepared according to general procedure

F Yield: 94% (3 steps). H-NMR (400 MHz, CDCl₃) δ 1.41 (9H,

Equal amounts of PBS and 1-octanol were shaken and left

overnight. The upper layer (1-octanol) and lower layer (PBS) were

collected individually. Each test compound was dissolved in 1-

octanol or PBS (200 mM). The same amount of either PBS or 1-

octanol was added and the mixture was shaken vigorously for 30

min at room temperature followed by centrifugation at 2100g for

5 min at room temperature. Then, both phases were separated and

assayed by HPLC and LC-MS. Log D7.4 was calculated by the

following equation:

1

br), 3.11 (3H, br), 3.19 (2H, t, J = 8.6 Hz), 3.93 (3H, s), 4.62 (2H,

t, J = 8.6 Hz), 4.68 (2H, s), 6.48–6.50 (3H, m), 6.86 (1H, br), 6.94

(1H, d, J = 3.9 Hz), 7.12 (1H, d, J = 7.4 Hz), 7.21 (1H, dd, J = 2.0,

7.8 Hz), 7.42 (1H, t, J = 8.2 Hz), 7.64 (1H, s), 7.75 (1H, d, J = 7.8

Hz).

4.8.129. Methyl 3-[2-({4-(1-benzofuran-6-yloxy)-2-

[(tert-

butoxycarbonyl)(methyl)amino]phenyl}amino) -2-

oxoethoxy]benzoate (15ah).

Log D7.4 = log (peak area of compound in 1-octanol/peak area

of compound in PBS).

Compound 15ah was prepared according to general procedure

1

4.11. Solubility Assay.

F (yield, 93%; 3 steps). H-NMR (400 MHz, CDCl₃) δ 1.42 (9H,

br), 3.11 (3H, br), 3.93 (3H, s), 4.68 (2H, s), 6.76 (1H, d, J = 0.8,

2.0 Hz), 6.88 (1H, br), 6.95 (1H, br), 6.99 (1H, dd, J = 2.0, 8.6 Hz),

7.17 (1H, s), 7.21 (1H, dd, J = 2.0, 8.6 Hz), 7.42 (1H, t, J = 7.8

Hz), 7.54 (1H, d, J = 8.6 Hz) , 3.19 (2H, t, J = 8.6 Hz), 7.62 (1H,

d, J = 2.0 Hz), 7.64 (1H, br), 7.75 (1H, dt, J = 1.2, 7.8 Hz).

After lyophilization of 10 mM DMSO solution of the test

compounds, aqueous neutral solution (pH 6.8) was added, stirred,

and allowed to stand at room temperature for at least 4 hours. After

allowing to filter by suction through Uni Filter (Uni Filter), the

concentration of the filtrate was measured by HPLC-UV

methodologies. Data represent single experiment run in duplicate.

4.9. Measurement of PPARγ Transcriptional Activity.

4.12. Hypoglycemic effect in KK mice.

GAL4-human PPARγ chimera receptor expression vector, pM-

h PPARγ, expressed the LBD of PPARγ as a fusion protein with

the DNA-DB of yeast transcription factor GAL4. The pG5luc

vector (included in CheckMate Mammalian Two-Hybrid System,

Promega Corporation), which contains five tandem repeats of a

GAL4-binding DNA sequence upstream of minimal TATA to

facilitate the detection of firefly luciferase activity induced by a

GAL4-h PPARγ fusion protein, was used as a GAL4-dependent

reporter vector. Dulbecco’s Modified Eagle Medium (DMEM,

Invitrogen Corporation) containing 10% (v/v) heat-inactivated

fetal bovine serum (FBS, Invitrogen Corporation) was prepared as

a culture medium. The COS-7 cells were cultured in the culture

medium at 37 °C with 5% CO₂(5% CO₂, 95% air). The COS-7

cells were cultured to the confluent in 75-cm²culture flasks. Cells

were transfected with 4.8 μg of the pM-h PPARγ and 19.2 μg of

pG5luc using Lipofectamine 2000 (Invitrogen Corporation) in

Opti-MEM I reduced serum medium (Opti-MEM I, Invitrogen

Corporation) according to the manufacturer’s instruction. After the

transfection, the COS-7 cells were harvested and re-seeded in 96-

well white plates and cultured for about 24 h in a CO₂incubator.

The pM-h PPARα expression vector was used for the GAL4-h

PPARα-LBD reporter gene assay.

All experimental procedures were performed in accordance

with the in-house guidelines of the Institutional Animal Care and

Use Committee of Daiichi Sankyo Co., Ltd. Six-week-old male

KK mice were purchased from CLEA Japan, Inc. and then were

fed until 14 to 15 weeks old to develop diabetes. Before the study,

the each PG level was measured by Glucoloader GXT (A&T

Corp.), and individuals having a PG level of about 350 mg/dL or

more were selected. The test compound was administered to mice

with a diet admixture containing 0.03% of the test compound for

3 days (n = 3). A separate group in which the mice were fed only

with diet was a control group. The body weight was measured and

blood was collected from the tail vein to measure the PG level. The

glucose lowering rate was determined by the following formula.

PG reduction (%) = [(PG level (Control group) - PG level

(Compound-administered group))/ PG level (Control group)] x

100

The plasma concentration of the test compound was measured

from the same blood sample on day 3 (n = 3).

4.13. Hypoglycemic effect in ZDF rats.

The serial dilutions of the test compounds (1, 3, 10, 30, 100,

300, 1000, 3000, 10000 nM) and the control solution (0.1%

DMSO) were added into the individual wells in the 96-well plates,

and the cells were further incubated for about 24 h in a CO₂

incubator. A luciferase assay was performed using a Picagene LT

2.0 Luminescence Reagent (TOYO INK, Co., Ltd.) according to

the manufacturer’s instruction. The light intensity in each well was

measured using a Multimode Microplate Reader (Analyst GT,

Molecular Devices, Inc.). Rosiglitazone and 2-(4-tert-

Butylphenoxy)-3-[4-[2-[(4-pyridin-2-

All experimental procedures were performed in accordance

with the in-house guideline of the Institutional Animal Care and

Use Committee of Daiichi Sankyo Co., Ltd. Six-week-old male

Zucker diabetic fatty rats were purchased from Charles River

Japan, Inc. and then were fed until 8 weeks old. Before the study,

the each PG level was measured by Glucoloader GXT (A&T

Corp.), and individuals having a PG level of about 300 mg/dL or

more were selected. The test compound (0.5%methylcellulose)

was orally administered to ZDF rats once daily for 14 days (n = 5).

The body weight was measured and blood was collected from the

tail vein to measure the PG level. The glucose lowering rate was

determined by the following formula.

ylbenzoyl)amino]ethoxy]phenyl]propionic acid³²were used as

positive references for the PPARγ and PPARα, respectively. The

maximum transcriptional activity of the test compound alone was

defined as the maximum efficacy (E_max, %). The concentration of

the test compound indicating a half value of E_maxwas defined as

the EC₅₀value. Values of each parameter were determined by

nonlinear curve fitting using GraphPad Prism 4.0 (GraphPad

Software Inc.). Data represent single experiment run in

PG reduction (%) = [(PG level (Control group) - PG level

(Compound-administered group))/ PG level (Control group)] x

100

4.14. Monkey Pharmacokinetic Study.

8. REFERENCES

All experimental procedures were performed in accordance

with the in-house guideline of the Institutional Animal Care and

Use Committee of Daiichi Sankyo Co., Ltd. For the determination

of test compound exposures in male cynomolgus monkeys, blood

samples were taken at several time points postdose. The plasma

was separated from blood by centrifugation, and stored at −70 °C

until use for measurement of plasma concentration. The

determination of the plasma concentration of the compound was

performed by LC-MS/MS method using API 4000QTRAP

(Applied Biosystems/MDS SCIEX). PK parameters were

calculated using a non-compartmental analysis techniques by the

computer software WinNonlin Professional version 4.0.1.

(Pharsight Corporation).

1. International Diabetes Federation, IDF Diabetes Atlas Eighth

Edition. http://www.diabetesatlas.org/

2. Mona, A.; Smith, R. E. Y. Nature Rev. Drug Discov. 2007, 6, 777-

778.

3. Chatterjee, S.; Khunti, K.; Davies, M. J. Lancet 2017, 389, 2239-

2251.

4. Philippe, J.; Raccah, D. Int. J. Clin. Pract. 2009, 63, 321-332.

5. Yki-Järvinen, H. N. Engl. J. Med. 2004, 351, 1106-1118.

6. Eldor, R; DeFronzo, R. A.; Abdul-Ghani, M. Diabetes Care 2013,

36, S162-S174.

7. Nesto, R. W.; Bell, D.; Bonow, R. O.; Fonseca, V.; Grundy, S. M.;

Horton, E. S.; Winter, M. L.; Porte, D.; Semenkovich, C. F.;

Smith, S.; Young, L. H.; Kahn, R. Diabetes Care 2004, 27, 256-

263.

8. Home, P. D.; Pocock, S. J.; Beck-Nielsen, H.; Curtis, P. S.;

Gomis, R.; Hanefeld, M.; Jones, N. P.; Komajda, M.; McMurray,

J. J.; RECORD Study Team. Lancet 2009, 373, 2125-2135.

9. Mudaliar, S.; Chang, A. R.; Henry, R. R. Endocr. Pract. 2003, 9,

406-416.

10. Loke, Y. K.; Singh, S.; Furberg, C. D. CMAJ 2009, 180, 32-39.

11. Pirat, C.; Farce, A.; Lebꢀgue, N.; Renault, N.; Furman, C.; Millet,

R.; Yous, S.; Speca, S.; Berthelot, P.; Desreumaux, P.; Chavatte,

P. J. Med. Chem. 2012, 55, 4027-4061.

12. Choi, J. H.; Banks, A. S.; Estall, J. L.; Kajimura, S.; Bostrom, P.;

Laznik, D.; Ruas, J. L.; Chalmers, M. J.; Kamenecka, T. M.;

Bluher, M.; Griffin, P. R.; Spiegelman, B. M. Nature 2010, 466,

451-456.

13. Rieusset, J.; Touri, F.; Michalik, L.; Escher, P.; Desvergne, B.;

Niesor, E.; Wahli, W. Mol. Endocrinol. 2002, 16, 2628−2644.

14. Choi, J. H.; Banks, A. S.; Kamenecka, T. M.; Busby, S. A.;

Chalmers, M. J.; Kumar, N.; Kuruvilla, D. S.; Shin, Y.; He, Y.;

Bruning, J. B.; Marciano, D. P.; Cameron, M. D.; Laznik, D.;

Jurczak, M. J.; Schurer, S. C.; Vidovic, D.; Shulman, G. I.;

Spiegelman, B. M.; Griffin, P. R. Nature 2011, 477, 477-481.

15. Garcia-Vallvꢁ, S.; Guasch, L.; Tomas-Hernꢂndez, S.; del Bas, J.

M.; Ollendorff, V.; Arola, L.; Pujadas, G.; Mulero, M. J. Med.

Chem. 2015, 58, 5381−5394.

16. Kanda, S.; Nakashima, R.; Takahashi, K.; Tanaka, J.; Ogawa, J.;

Ogata, T.; Yachi, M.; Araki, K.; Ohsumi, J. J. Pharmacol. Sci.

2009, 111, 155-166.

17. Shimazaki, N.; Togashi, N.; Hanai, M.; Isoyama, T.; Wada, K.;

Fujita, T.; Fujiwara, K.; Kurakata, S. Eur. J. Cancer 2008, 44,

1734-1743.

4.15. In Vivo Toxicological Evaluation.

All experimental procedures were performed in accordance

with the in-house guideline of the Institutional Animal Care and

Use Committee of Daiichi Sankyo Co., Ltd. Seven-week-old

female Wistar–Imamichi rats were purchased from Charles River

Japan, Inc. and then were housed for one week. Rats (n = 5) were

orally dosed once daily by gavage with vehicle or test compounds

at indicated doses in 0.5% methylcellulose for 28 days. Animals

were euthanized on the next day of the last dose, and the indicated

tissues and organs were collected for organ weight assessment and

histopathological examination. Blood samples were exsanguinated

via the abdominal aorta and assayed for serum chemistry.

4.16. Protein Crystallography Method.

Histidine-tagged human PPARγ-LBD was expressed and

purified as described previously.²⁰A synthetic peptide with a

sequence derived from PGC-1 (QEAEEPSLLKKLLLAPANT)

was purchased from Sigma-Aldrich. Before crystallization,

PPARγ-LBD was concentrated to 22 mg/mL and mixed with 13ac

and PGC-1 in a molar ratio of 1:4:4. Crystals were obtained by the

hanging drop vapor diffusion technique with a reservoir solution

of 24% (w/v) PEG4000, 200 mM NaSCN and 100 mM Tris-HCl

(pH 8.5). Prior to data collection, crystals were transiently soaked

in the reservoir solution containing additional 8% (v/v) PEG400

as a cryoprotectant. X-ray diffraction data were collected using an

X-ray generator FR-E with detector R-AXIS IV (RIGAKU). The

diffraction data were integrated, scaled using HKL2000,⁴⁰and

converted to structure factors using the CCP4 software suite.⁴¹The

structure of PPARγ-LBD derived from the structure 3V9T.pdb¹⁹

was used as an initial model. Several rounds of manual rebuilding

with O⁴²followed by refinement with CNX⁴³(Accerlys) were

carried out. A Ramachandran plot for the final model was

calculated with RAMPAGE.⁴⁴Figures were created with PyMOL

(v.1.7; Schrödinger).Authors will release the atomic coordinates

and experimental data upon article publication. Coordinates are

available from PDB using accession code 5Z5S.

18. Furukawa, A.; Arita, T.; Fukuzaki, T.; Mori, M.; Honda, T.;

Satoh, S.; Matsui, Y.; Wakabayashi, K.; Hayashi, S.; Nakamura,

K.; Araki, K.; Kuroha, M.; Tanaka, J.; Wakimoto, S.; Suzuki, O.;

Ohsumi, J. Eur. J. Med. Chem. 2012, 54, 522-533.

19. Furukawa, A., Arita, T., Fukuzaki, T., Satoh, S., Mori, M., Honda,

T., Matsui, Y., Wakabayashi, K., Hayashi, S., Araki, K., and

Ohsumi, J. Bioorg. Med. Chem. Lett. 2012, 22, 1348-1351.

20. Wakabayashi, K.; Hayashi, S.; Matsui, Y.; Matsumoto, T.;

Furukawa, A.; Kuroha, M.; Tanaka, N.; Inaba, T.; Kanda, S.;

Tanaka, J.; Okuyama, R.; Wakimoto, S.; Ogata, T.; Araki, K.;

Ohsumi, J. Biol. Pharm. Bull. 2011, 34, 1094−1104.

21. Nolte, R. T.; Wisely, G. B.; Wes-tin, S.; Cobb, J. E.; Lambert, M.

H.; Kurokawa, R.; Rosenfeldk, M. G.; Willson, T. M.; Glass, C.

K.; Mil-burn, M. V. Nature 1998, 395, 137-143.

22. Willson, T. M.; Brown, P. J.; Sternbach, D. D.; Henke, B. R. J.

Med. Chem. 2000, 43, 527-550.

23. Motani, A.; Wang, Z.; Weiszmann, J.; McGee, L. R.; Lee, G.; Liu,

Q.; Staunton, J.; Fang, Z.; Fuentes, H.; Lindstrom, M.; Liu, J.;

Biermann, D. H.; Jaen, J.; Walker, N. P.; Learned, R. M.; Chen, J.

L.; Li, Y. J. Mol. Biol. 2009, 386, 1301-1311.

5. AUTHOR INFORMATION

*Tel: +81-3-3492-3131. Fax: +81-3-5436-8563.

24. Liu, W.; Lau, F.; Liu, K.; Wood, H. B.; Zhou, G.; Chen, Y.; Li,

Y.; Akiyama, T. E.; Castriota, G.; Einstein, M.; Wang, C.;

McCann, M. E.; Doebber, T. W.; Wu, M.; Chang, C. H.;

McNamara, L.; McKeever, B.; Mosley, R. T.; Berger, J. P.;

Meinke, P. T. J. Med. Chem. 2011, 54, 8541-8554.

25. Einstein, M.; Akiyama, T. E.; Castriota, G. A.; Wang, C. F.;

McKeever, B.; Mosley, R. T.; Becker, J. W.; Moller, D. E.;

Meinke, P. T.; Wood, H. B.; Berger, J. P. Mol. Pharmacol. 2008,

73, 62−74.

E-mail:

sinozu.xf6@gmail.com;

shinozuka.tsuyoshi.s5@daiichisankyo.co.jp.

6. ORCID ID

Tsuyoshi Shinozuka: 0000-0002-7785-6080

7. Notes

26. van Marrewijk, L. M.; Polyak, S. W.; Hijnen, M.; Kuruvilla, D.;

Chang, M. R.; Shin, Y.; Kamenecka, T. M.; Griffin, P. R.;

Bruning, J. B. ACS Chem. Biol. 2016, 11, 273−283.

27. Oberfield, J. L.; Collins, J. L.; Holmes, C. P.; Goreham, D. M.;

Cooper, J. P.; Cobb, J. E.; Lenhard, J. M.; Hull-Ryde, E. A.;

The authors declare no competing financial interest.

Mohr, C. P.; Blanchard, S. G.; Parks, D. J.; Moore, L. B.;

Lehmann, J. M.; Plunket, K.; Miller, A. B.; Milburn, M. V.;

Kliewer, S. A.; Willson, T. M. Proc. Natl. Acad. Sci. U.S.A. 1999,

96, 6102-6106.

37. Rivoglitazone showed a similar binding mode to that of

rosiglitazone.

38. PPARγ maximum transcriptional activity is not fully attribute to

the binding mode, and the stabilization of helix 12 and other

regions of the binding pocket is measured by amide H/D exchange

kinetics.²⁹Such measurement is necessary for further discussions.

39. HCl salts of compounds 12a and 12ae were synthesized to

evaluate their in vitro activities; they exhibited EC₅₀(E_max) values

as 1.66 μM (87%) and 0.34 μM (67%), respectively.

40. Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307-

326.

28. Li, Y.; Wang, Z.; Furukawa, N.; Escaron, P.; Weiszmann, J.; Lee,

G.; Lindstrom, M.; Liu, J.; Liu, X.; Xu, H.; Plotnikova, O.; Prasad,

V.; Walker, N.; Learned, R. M.; Chen, J. L. J. Biol. Chem. 2008,

283, 9168-9176.

29. Bruning, J. B.; Chalmers, M. J.; Prasad, S.; Busby, S. A.;

Kamenecka, T. M.; He, Y.; Nettles, K. W.; Griffin, P. R. Structure

2007, 15, 1258−1271.

30. The administration of 0.003% of rosiglitazone for 3 days caused

60% reduction in PG levels and 4.8% body weight gain in

hyperglycemic KK/Ta mice in this assay.

31. The inhibition of PPARγ phosphorylation could have explained

several in vitro/in vivo discrepancies. The inhibitory activities of

this series of compounds against PPARγ phosphorylation were not

evaluated.

32. Takamura, M.; Sakurai, M.; Yamada, E.; Fujita, S.; Yachi, M.;

Takagi, T.; Isobe, A.; Hagisawa, Y.; Fujiwara, T.; Yanagisawa, H.

Bioorg. Med. Chem. 2004, 12, 2419-2439.

33. Compound 13ag was excluded because of its full agonist activity,

despite its robust in vivo efficacy in ZDF rats.

41. Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.;

Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie,

A. G. W.; McCoy, A.; McNicholas, S. J.; Murshudov, G. N.;

Pannu, N. S.; Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin,

A.; Wilson, K. S. Acta Crystallogr.,Sect. D: Biol. Crystallogr.

2011, 67, 235-242.

42. Jones, T. A.; Zou, J. Y.; Cowan, S. W.; Kjeldgaard, M. Acta

Crystallogr. 1991, A47, 110-119.

43. Brunger, A. T. Nat. Protoc. 2007, 2, 2728-2733.

44. Lovell S. C.; Davis I. W.; Arendall W. B.; de Bakker P. I. W.;

Word J. M.; Prisant M. G.; Richardson J. S.; Richardson D. C.

Proteins 2003, 50, 437–450.

34. TK parameters of rosiglitazone were not measured.

35. Horita S, Nakamura M, Satoh N, Suzuki M, Seki G. PPAR Res.

2015, 2015, 646423.

36. Acton III, J. J.; Akiyama, T. E.; Chang, C. H.; Colwell, L.;

Debenham, S.; Doebber, T.; Einstein, M.; Liu, K.; McCann, M.

E.; Moller, D. E.; Muise, E. S.; Tan, Y.; Thompson, J. R.; Wong,

K. K.; Wu, M.; Xu, L.; Meinke, P. T.; Berger, J. P.; Wood, H. B.

J. Med. Chem. 2009, 52, 3846-3854.

Graphical Abstract

To create your abstract, type over the instructions in the

template box below.

Fonts or abstract dimensions should not be changed or altered.

Discovery of DS-6930, a Potent Selective

PPARγ Modulator. Part I: Lead

Identification

Leave this area blank for abstract info.

Tsuyoshi Shinozuka,^*,†Tomoharu Tsukada,^†Kunihiko Fujii,^†Eri Tokumaru,^†Kousei Shimada,^†Yoshiyuki

Onishi,^†Yumi Matsui,^‡Satoko Wakimoto,^†Masanori Kuroha,^†Tsuneaki Ogata,^†Kazushi Araki,^†Jun Ohsumi,^†

Ryoko Sawamura,^†Nobuaki Watanabe,^†Hideki Yamamoto,^†Kazunori Fujimoto,^†Yoshiro Tani,^†Makoto Mori^†

and Jun Tanaka^†

Article Doi

Discovery of DS-6930, a potent selective PPARγ modulator. Part I: Lead identification

DOI: 10.1016/j.bmc.2018.09.006

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2018.09.006

Full text of DOI:10.1016/j.bmc.2018.09.006

Products guided by the article

R&D Labs maybe for 1072002-99-3

Relevant to this article

Hot Product