Discovery of Gemilukast (ONO-6950), a Dual CysLT1 and CysLT2 Antagonist As a Therapeutic Agent for Asthma

Article

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Discovery of Gemilukast (ONO-6950), a Dual CysLT₁and CysLT₂

Antagonist As a Therapeutic Agent for Asthma

Satoshi Itadani,*^,†Kentaro Yashiro,^†Yoshiyuki Aratani,^†Tetsuya Sekiguchi,^†Atsushi Kinoshita,^†

Hideki Moriguchi,^†Nobukazu Ohta,^†Shinya Takahashi,^†Akiharu Ishida,^†Yohei Tajima,^†

Katsuya Hisaichi,^†Masaki Ima,^†Junya Ueda,^†Hiromu Egashira,^†Tomohiko Sekioka,^‡Michiaki Kadode,^‡

Yasuo Yonetomi,^‡Takafumi Nakao,^‡Atsuto Inoue,^‡Hiroaki Nomura,^‡Tetsuya Kitamine,^‡

Manabu Fujita,^‡Takeshi Nabe,^∥,⊥Yoshiyuki Yamaura,^§Naoya Matsumura,^§Akira Imagawa,^†

Yoshisuke Nakayama,^†Jun Takeuchi,^†and Kazuyuki Ohmoto*^,†

‡

§

^†Medicinal Chemistry Research Laboratories, Department of Biology & Pharmacology and Pharmaceutical Technology

Laboratories, Ono Pharmaceutical Co., Ltd. 3-1-1 Sakurai, Shimamoto, Mishima, Osaka 618-8585, Japan

^∥Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge, Hirakata, Osaka 573-0101, Japan

^⊥Department of Pharmacology, Kyoto Pharmaceutical University, 5 Nakauchi Misasagi, Yamashina, Kyoto 607-8414, Japan

S

* Supporting Information

ABSTRACT: An orally active dual CysLT₁and CysLT₂

antagonist possessing a distinctive structure which consists of

triple bond and dicarboxylic acid moieties is described.

Gemilukast (ONO-6950) was generated via isomerization of

the core indole and the incorporation of a triple bond into a

lead compound. Gemilukast exhibited antagonist activities with

IC₅₀values of 1.7 and 25 nM against human CysLT₁and

human CysLT₂, respectively, and potent eﬃcacy at an oral dose of 0.1 mg/kg given 24 h before LTD₄challenge in a CysLT₁-

dependent guinea pig asthmatic model. In addition, gemilukast dose-dependently reduced LTC₄-induced bronchoconstriction in

both CysLT₁- and CysLT₂-dependent guinea pig asthmatic models, and it reduced antigen-induced constriction of isolated

human bronchi. Gemilukast is currently being evaluated in phase II trials for the treatment of asthma.

severity is linked to the amount of LTD₄production. The

involvement of CysLT₂in severe asthma is anticipated.

Therefore, dual CysLT₁and CysLT₂antagonists may be

more attractive as therapeutics for asthma.

As shown in Figure 2, two potent CysLT₂selective

antagonists have been reported.^21,22However, there are no

reports of eﬀective dual antagonists.²³⁻²⁷

INTRODUCTION

■

The cysteinyl leukotrienes (CysLTs), LTC₄, LTD₄, and LTE₄

are lipid mediators derived from arachidonic acid via the 5-

lipoxygenase pathway,¹⁻⁴and they are involved in various

inﬂammatory diseases, including asthma and allergic rhinitis.

CysLTs are known to cause constriction of smooth muscle and

the migration of inﬂammatory cells via certain receptors.

Pharmacological studies have revealed the existence of two

classes of receptors to which CysLTs can bind. They are the

CysLT₁and CysLT₂receptors, which were identiﬁed and

cloned in 1999 and 2000, respectively.^5,6Figure 1 shows several

CysLT₁selective antagonists. Pranlukast, montelukast, and

zaﬁrlukast, which are in clinical use, have demonstrated clinical

beneﬁt for the treatment of bronchial asthma and allergic

rhinitis. Nonetheless, approximately half of the patients treated

with these agents are nonresponders.⁷⁻⁹Thus, more eﬀective

drugs than CysLT₁-selective antagonists are needed.

CysLT₂receptors were reported to be expressed on airway

smooth muscle cells,⁶inﬂammatory cells,¹⁰⁻¹³and vascular

endothelial cells¹⁴similar to CysLT₁receptors. In addition, the

urine concentration of LTE₄, which is a metabolite of LTD₄, is

increased in aspirin-sensitive asthmatics or severe asthmatic

patients.¹⁵⁻²⁰LTD₄likely binds to both receptors in severe

asthma but more tightly to CysLT₁than CysLT₂because the

In our previous report,²⁸(2S)-4-(3-carboxypropyl)-8-{[4-(4-

phenylbutoxy)benzoyl]amino}-3,4-dihydro-2H-1,4-benzoxa-

zine-2-carboxylic acid (ONO-2050297, the S-isomer of 1) was

identiﬁed as the ﬁrst potent dual antagonist, with IC₅₀values of

17 and 0.87 nM for human CysLT₁(hCysLT₁) and human

CysLT₂(hCysLT₂), respectively. As shown in Table 1, the PK

proﬁle of 1 was very poor, and its bioavailability was only 1.5%

in rats.²⁸However, further modiﬁcation resulted in 4-(3-

(carboxymethyl)-4-{(E)-2-[4-(4-phenoxybutoxy)phenyl]-

vinyl}-1H-indol-1-yl)butanoic acid (2, ONO-4310321), which

had a signiﬁcantly improved PK proﬁle in rats.²⁹Compound 2

demonstrated a reduction in bronchoconstriction in a dose-

dependent manner and complete inhibition at an oral dose of

10 mg/kg in a CysLT₁-dependent guinea pig bronchoconstric-

Received: May 7, 2015

A

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Figure 1. CysLT₁-selective antagonists. IC₅₀values were quoted from refs 5 (CysLT₁antagonist activities) and 6 (CysLT₂antagonist activities).

previously reported.²⁹Table 2 shows that indole-1-acetic acid

derivative 3, an isomer, is a more potent dual antagonist, with

IC₅₀values of 1.5 and 6.0 nM against hCysLT₁and hCysLT₂,

respectively. In addition, both compounds 2 and 3 were orally

administered 1 h before LTD₄challenge at 1 mg/kg, and

compound 3 showed potent in vivo eﬃcacy (97% inhibition of

bronchoconstriction) compared to compound 2 (48%

inhibition). Indole-1-acetic acid derivatives appeared more

Figure 2. CysLT₂-selective antagonists. IC₅₀values were quoted from

amenable to further modiﬁcation, although their bioavailability

refs 21 (HAMI3378) and 22 (BayCysLT₂).

in guinea pigs was moderate.

As shown in Table 2, both compounds 2 and 3 demonstrated

much lower bioavailability in guinea pigs than in rats. Generally,

tion model. However, compound 2 was unﬁt as a clinical

the more basic pH of the small intestine reduces the

permeability of acidic compounds. Indeed, compounds 2 and

3 showed lower permeability at pH 7.4 than at pH 6.2 (Table

2). The pH in the small intestine of guinea pigs is greater than

that for other species (pH 7.6−8.2 for guinea pigs, pH 6.5−7.1

for rats, pH 6.6−7.5 for dogs, pH 5.5−6.0 for monkeys, and pH

5.0−7.0 for humans),^30,31and this basic pH explains why these

compounds are less bioavailable in guinea pigs. This species

diﬀerence should be considered when evaluating compounds in

guinea pigs.

candidate because the strength and duration of its in vivo

eﬃcacy were inadequate.

In this article, further modiﬁcation to create an orally active

dual CysLT₁and CysLT₂antagonist compatible with once daily

administration will be described. After structural optimizations

based on the duration of eﬃcacy and penetration into lung

tissue, 4,4′-[4-ﬂuoro-7-(2-{4-[4-(3-ﬂuoro-2-methylphenyl)-

butoxy]phenyl}ethynyl)-2-methyl-1H-indole-1,3-diyl]-

dibutanoic acid (35, ONO-6950, gemilukast) was identiﬁed as a

clinical candidate.

Table 3 shows the eﬀects of introducing a methyl group at

the 2-position of the indole and other substitutions on the

terminal phenyl group. As indicated by a comparison of

compounds 9−16 and 4−8, the incorporation of the methyl

RESULTS AND DISCUSSION

Lead Optimization. The isomer of the core indole ring was

initially investigated. The indole-3-acetic acid derivative 2 was

■

Table 1. Pharmacokinetic Proﬁles and In Vivo Eﬃcacy of 1 and 2

a

Compound 1: Rat bioavailability was calculated based on an intravenous (iv) dose of 1 mg/kg and an oral administration (po) dose of 30 mg/kg.

b

guinea pig bioavailability was calculated based on an iv dose of 0.1 mg/kg and a po dose of 30 mg/kg. Mean values (n = 3). Compound 2: Rat and

guinea pig bioavailability was calculated based on an iv dose of 1 mg/kg and a po dose of 10 mg/kg. Mean values (n = 3). IC₅₀values and 95%

conﬁdence intervals of each compound were obtained with Prism 5 software (GraphPad) (n ≥ 2). LTD₄-induced bronchoconstriction model

dependent on CysLT₁. Mean values (n = 3).

c

d

B

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Table 2. Indole-3-acetic Acid (2) and Indole-1-acetic Acid (3)

a

Assay protocols are provided in the Experimental Section. IC₅₀values and 95% conﬁdence intervals of each compound were obtained with Prism 5

b

software (GraphPad) (n ≥ 2). Eﬀects on a LTD₄-induced bronchoconstriction model dependent on CysLT₁. The values represent the mean for n =

3. The compounds were orally administered 1 h before LTD₄challenge. pK_a: Predicted by ADMET Predictor version 7 (Simulations Plus, Inc.). F

(%) was calculated using the mean of AUC (po) and the mean of AUC (iv) (n = 3).

c

d

group into the indole improved permeability at pH 7.4. This

improvement appears to result from the diminished acidity of

the 1-acetic acid moiety due to the electron donation of the

methyl group.

In addition, the hCysLT₂antagonist activity of 2H-indole-1-

acetic acid derivatives tended to be much weaker in guinea pigs

(IC₅₀value of >100 nM, data not shown) compared with

humans. Ultimately, 2-methyl-indole was considered a more

suitable core structure.

Upon the addition of substitutions to the terminal phenyl

group, no signiﬁcant diﬀerences in hCysLT₁or hCysLT₂

antagonist activities were achieved. However, the compounds

possessing certain substituents on the terminal phenyl group

had a tendency toward improved antagonist activities and

permeability. Thus, these compounds were evaluated in a

CysLT₁-dependent guinea pig asthmatic model at doses of 1,

0.3, or 0.1 mg/kg. The compounds were orally administered 1

h before LTD₄challenge.

As shown in Table 3, some of the compounds demonstrated

high inhibitory activity at 0.3 mg/kg. In particular, compounds

8 and 16, both possessing a 3-chloro-2-methylphenyl group at

the terminus, exhibited approximately 50% inhibition at 0.1

mg/kg. Both compounds appeared equivalent. However,

compound 16 was selected for further proﬁling because 2-

methyl-indole-1-acetic acid derivatives are more promising in

terms of the species diﬀerences in antagonist activities and

permeability, as discussed above.

Lakshminarayana et al. reported that tuberculosis drugs

whose K_pvalues are at least 2 can be eﬃcient.³²In addition, the

K_pvalue of montelukast was reported to be greater than 5 at 24

h according to its FDA pharmacology review.³³Thus, the 2-

methyl-indole derivative represented by 16 appears to possess a

proﬁle appropriate for the treatment agent of asthma.

Further Optimization of Advanced Lead toward a

Once-Daily Drug. From Figure 3, it appeared that there was

room to improve the pharmacokinetic properties of compound

16 as a clinical candidate, although it was active in the in vivo

model. In particular, the permeability of 16 was still low. Two

options for improving the low permeability were deduced:

reducing (1) ﬂexibility and (2) acidity. In a situation where

target proteins are related to lipid mediators, in our experience

there is a trend toward an elongated and ﬂapping compound as

a ligand. Thus, it is diﬃcult to make dual antagonists rigid. For

example, shortening or ﬁxing the side chain to reduce ﬂexibility

decreases antagonist activity. Therefore, we focused on the

trans-double bond in the core structure and attempted its

replacement with a triple bond. Because a triple bond is more

linear than a double bond, the potential spatial area occupied by

the side chain was expected to decrease.

Compounds possessing a triple bond instead of the double

bond were synthesized and evaluated as shown in Table 5.

Interestingly, compound 19, which corresponds to compound

16, retained dual antagonist activities; if anything, hCysLT₂

antagonist activity increased. Thus, the compatibility of the

triple bond was conﬁrmed. In addition, the permeability of 19

was improved by P_app(pH 7.4) = 3.2 × 10⁻⁶cm/s as expected.

The acidity of carboxylic acids should correlate with distance

from the indole ring. As previously reported for the

benzoxazine analogues,²⁸the length of the carboxylic acid

side chain is an important factor that aﬀects antagonist

activities. Thus, several analogues with various carboxylic acid

side chains lengths were synthesized to better balance

Table 4 shows the pharmacokinetic proﬁle of 16 in rats,

guinea pigs, and dogs. Compound 16 demonstrated a low oral

bioavailability of 11% in guinea pigs, whereas it demonstrated

good oral bioavailability of 56% and a long half-life of 8.9 h in

rats. As shown above, compound 16 showed in vivo eﬃcacy

even at the lowest dose. Thus, the concentration of 16 in the

plasma and lung tissue of guinea pigs was measured (Figure 3).

The concentration of 16 remained high (K_pvalue of 15) for

over 24 h in lung tissue.

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Table 3. Introduction of a Methyl Group into the 2-Position of the Indole and Substitutions on the Terminal Phenyl Group

a

Assay protocols are provided in the Experimental Section. IC₅₀values and 95% conﬁdence intervals of each compound were obtained with Prism 5

b

c

software (GraphPad) (n ≥ 2). Eﬀects on LTD₄-induced bronchoconstriction model dependent on CysLT₁(n = 3). IC₅₀values were quoted from

refs 5 (CysLT₁antagonist activity) and 6 (CysLT₂antagonist activity). IC₅₀values were quoted from ref 21.

d

Table 4. Pharmacokinetic Parameters of 16

dose (mg/kg)

a

b

species

rat

iv

po

iv t_1/2(h)

iv CL (mL/min/kg)

V_dss(L/kg)

po AUC (μg·h/mL)

F

(%)

56

1

3

10

1

8.9 8.5

5.3 0.7

4.7 5.0

1.1 0.7

1.8 0.1

0.30 0.14

0.23 0.01

0.38 0.33

39 12

10 4.1

guinea pig

dog

11

21

5.5 1.1

0.65 0.16

a

b

The value are expressed as the mean standard deviation (n = 3). F (%) was calculated using the mean of AUC (po) and the mean of AUC (iv).

permeability with dual antagonist activities. Compound 25 was

A triple bond is generally considered unstable under a certain

condition. Thus, the stability of 25 was assessed under various

severe conditions. Surprisingly, 25 was stable under acidic

the best among these analogues in Table 5.

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These compounds were evaluated in a LTD₄-induced

bronchoconstriction model at an oral dose of 0.3 or 0.1 mg/

kg 24 h prior to LTD₄challenge. As shown in Table 7, 2,3,4,6-

tetraﬂuorophenyl derivatives 32 and 33 and 3-ﬂuoro-2-

methylphenyl derivatives 34 and 35 demonstrated more than

70% inhibition of bronchoconstriction at 0.3 mg/kg.

Furthermore, compounds 34 and 35 demonstrated potent in

vivo eﬃcacy (approximately 70% inhibition) and a long

duration of action even at 0.1 mg/kg. Compound 35 (ONO-

6950, gemilukast) was ultimately chosen as a clinical candidate

based upon further evaluation, including a safety study.

Further Biological Proﬁling of 35. Table 8 shows that 35

demonstrated acceptable bioavailability (35% in rats, 51% in

dogs, 56% in guinea pigs) and low clearance values (0.68 mL/

min/kg in rats, 1.10 mL/min/kg in guinea pigs, 0.64 mL/min/

kg in dogs). In particular, the clearance of 35 in dogs was much

lower compared to 16, and this resulted in an improved

pharmacokinetic proﬁle in dogs. For example, 35 had a long

half-life (7.6 h) and abundant blood exposure (28.2 μg·h/mL).

An excellent pharmacokinetic proﬁle for 35 in humans was

expected because much lower clearance (0.2 mL/min/kg) in

humans was estimated based upon allometric scaling of the

animal results.^34,35As shown in Figure 4, 2-methyl-indole

derivatives such as 35 tended to penetrate lung tissue well.

Thus, a ratio (K_pvalue) of the concentration of 35 in lung

tissue to the concentration in plasma was calculated from the

observed data in guinea pigs. As expected, the K_pvalue was very

high: approximately 8 at an oral dose of 3 mg/kg or 30 at an

oral dose of 0.3 mg/kg.

Figure 3. Concentration of 16 in plasma or lung tissue of guinea pigs.

The value are expressed as the mean SD (n = 4).

conditions (1 mol/L H₂SO₄in DME/H₂O at 50 °C for 24 h)

or basic conditions (1 mol/L NaOH in DME/EtOH/H₂O at

50 °C for 24 h). Furthermore, degradation of 25 in ambient

light was not observed even under the conditions where the

compounds with a double bond such as 16 easily decomposed.

In the case of 16, the isomerization of the trans-double bond by

ambient light caused oxidation and decomposition.

Next, the eﬀects of substitution at the benzene ring of the

core indole were investigated. Table 6 shows 4-, 5-, and 6-ﬂuoro

derivatives of 25. Only compound 29 exhibited dual antagonist

activities comparable to 25. Table 6 also shows the in vivo

eﬃcacies and PK proﬁles of 25 and 29. Both compounds were

evaluated in a LTD₄-induced bronchoconstriction model. They

demonstrated suﬃcient eﬃcacies at an oral dose even 24 h

before LTD₄challenge. Compound 29 demonstrated similar in

vivo eﬃcacy despite its poor PK proﬁle, likely attributable to

better penetration into lung tissue.

Regarding the risk assessment of 35, the result (11%

inhibition at 10 μM) of an in vitro hERG assay indicated low

risk for QT interval prolongation, and the Ames test was

negative. Further evaluation of 35 as a clinical candidate was

then conducted.

Table 7 shows the optimization of substitutions to the phenyl

group in the 2-methyl-indole-1-butanoic acid derivatives 25 and

29. All compounds exhibited potent antagonist activities against

not only hCysLT₁and hCysLT₂but also guinea pig CysLT₁

(gCysLT₁) and guinea pig CysLT₂(gCysLT₂).

The value are expressed as the mean

SE (n = 8). The

compound was administered once daily.

Table 5. Length of Two Carboxylic Acid Side Chains of 2-Methyl-indole Derivatives

a

IC₅₀(nM)

compd

m

n

hCysLT₁

hCysLT₂

permeability PAMPA × 10⁻⁶cm/s pH 7.4

17

18

19

20

21

22

23

24

25

26

27

28

1

2

3

4

1

2

3

1

2

3

1

2

3

1

2

3

0.47 (0.29−0.76)

0.32 (0.20−0.50)

0.22 (0.17−0.27)

12 (8.3−16)

10 (6.2−17)

17 (13−23)

5.5 (3.6−8.5)

0.79 (0.54−1.2)

1.5 (1.2−2.0)

65 (56−75)

25 (17−36)

56 (46−69)

0.89 (0.81−0.97)

5.7 (4.9−6.8)

0.88 (0.71−1.1)

96 (84−110)

78 (48−127)

22 (14−35)

55 (46−65)

15 (11−20)

5.5 (4.3−7.1)

248 (73−847)

60 (26−138)

30 (23−41)

<0.05

0.7

3.2

7.2

3.6

3.0

4.4

7.2

7.9

1.0

4.6

5.0

a

Assay protocols are provided in the Experimental Section. IC₅₀values and 95% conﬁdence intervals of each compound were obtained with Prism 5

software (GraphPad) (n ≥ 2).

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Table 6. Incorporation of a Fluorine Atom into the Core Indole Ring

a

b

c

IC₅₀(nM)

In vivo eﬃcacy , po % Inhibition

pharmacokinetics guinea pig

c

d

compd

R1

R2

R3

hCysLT₁

hCysLT₂

0.3 mg/kg, 1 h

1 mg/kg, 24 h

AUC (μg·h/mL)

F

(%)

66

52

25

29

30

31

H

F

H

F

H

F

1.5 (1.2−2.0)

3.4 (2.1−5.3)

16 (6.6−38)

11 (8.2−15)

5.5 (4.3−7.1)

12 (9.9−15)

31 (15−63)

48 (33−70)

96

99

97

79

10.5 2.7

3.4 0.9

H

a

Assay protocols are provided in the Experimental Section. IC₅₀values and 95% conﬁdence intervals of each compound were obtained with Prism 5

b

software (GraphPad) (n ≥ 2). Eﬀects on LTD₄-induced bronchoconstriction model dependent on CysLT₁. The values represent the means for n =

6. Guinea pig cassette dosing at 0.2 mg/kg, iv and 1 mg/kg, po (nonfasted). The value are expressed as the mean SD (n = 3). F (%) was

c

d

calculated using the mean of AUC (po) and the mean of AUC (iv).

Table 7. Substitution on the Terminal Phenyl Group of 2-Methyl-indole-1-butanoic Acid Derivatives

a

Assay protocols are provided in the Experimental Section. IC₅₀values and 95% conﬁdence intervals of each compound were obtained with Prism 5

b

software (GraphPad) (n ≥ 6). Eﬀects on LTD₄-induced bronchoconstriction model dependent on CysLT₁. The values represent the means for n =

6.

Asthmatic Model Mediated via CysLT₂in Guinea Pigs.

LTC₄is reported to be a guinea pig CysLT₂selective ligand,

unlike the case in humans.^36,37It is diﬃcult to evaluate

pharmacological eﬀects involving CysLT₂in guinea pigs

because LTC₄is immediately metabolized by γ-glutamyltrans-

peptidase. Yonetomi et al. reported a novel asthmatic model

mediated via the CysLT₂receptor in guinea pigs.³⁸In the

reported CysLT₂dependent model, the metabolism of LTC₄

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Table 8. Pharmacokinetic Parameters of 35 in Rats and Dogs

dose (mg/kg)

c

d

species

iv

po

iv t_1/2(h)

CL (mL/min/kg)

V_dss(L/kg)

po C_max(μg/mL)

po AUC (μg·h/mL)

F

(%)

35

a

rat

1

9.6 3.1

7.1 3.8

0.68 0.26

1.10 0.10

0.24 0.06

0.31 0.19

0.14 0.05

1.19 0.59

1.01 0.29

3.69 0.74

9.33 1.45

8.51 1.49

28.2 8.70

b

guinea pig

0.2

1

56

51

a

dog

7.6 1.0

0.64 0.24

a

b

c

d

Single intravenous and oral administration. Cassette dosing. The value are expressed as the mean standard deviation (n = 3). F (%) was

calculated using the mean of AUC (po) and the mean of AUC (iv).

Figure 4. Plasma and lung tissue concentrations of 35 in guinea pigs.

was inhibited, and the eﬀect of montelukast (CysLT₁selective

antagonist) on LTC₄-induced bronchoconstriction was only

partial. In contrast, as shown in Figure 5, 35 (dual CysLT₁and

Figure 6. Eﬀect of compound 35 on antigen-induced constriction of

isolated human bronchi. (a) Concentration of both montelukast and

BayCysLT₂RA was 10 nmol/L (n = 4). (b) Concentration of 35 was

100 nmol/L (n = 4).

Figure 5. Eﬃcacy of 35 in an asthmatic model mediated via CysLT₂in

guinea pigs. (a) ML (montelukast) was orally administered at 0.1 mg/

kg. Each column represents the means SE of 5−10 animals.

As a result, the inhibitory activity of 35 (blue line) was

comparable to that of a combination treatment of montelukast

and BayCysLT₂RA (green line). Recently, the expression of

CysLT₂in airways of asthma subjects was reported to be higher

than for nonasthma subjects.⁴⁰This result suggests that dual

CysLT₁and CysLT₂antagonists could be more eﬀective and

useful.

CysLT₂antagonist) signiﬁcantly reduced the bronchoconstric-

tion in a dose-dependent manner. Thus, 35 is expected to be a

more useful treatment agent for asthma because of the potential

involvement of CysLT₂in severe symptoms.

Antigen-Induced Constriction in Isolated Human

Bronchi. A study using human tissue increases the probability

of success in clinical trials. An evaluation of 35 using human

bronchi was performed. Isolated human bronchi were passively

sensitized by incubation with human atopic serum, and the

resulting bronchial strip was suspended in a Magnus bath. Test

compound or vehicle was applied 30 min before mite antigen

challenge. In addition, a cyclooxygenase inhibitor (indometha-

cin) and an antihistamine drug (pyrilamine) were applied 10

and 5 min, respectively, before antigen challenge such that the

antigen-induced contractile response was dependent on

CysLTs. The detailed procedure is described in the

Experimental Section.

CHEMISTRY

■

Syntheses of the compounds herein are outlined in Schemes

1−6. Syntheses of 38a−f are described in Scheme 1.

Compounds 36a−f were subjected to Sonogashira coupling

with 3-butyn-1-ol. The resulting acetylene derivatives 37a−f

were converted to alcohol derivatives 38a−f with Pd/C under a

hydrogen atmosphere. Tosyl derivatives 38aa and 38ba were

aﬀorded by tosylation of 38a and 38b.

Compounds 4−16 were prepared as shown in Scheme 2.

Commercially available indole derivatives 39a or 39b were

converted to ketoesters 40a or 40b by Friedel−Crafts acylation

followed by reduction with sodium borohydride to prepare

monoester 41a or 41b. N-Alkylation of 41a and 41b with ethyl

bromoacetate using K₂CO₃as the base aﬀorded diesters 42a

Figure 6 shows that 100 nmol/L of 35 reduced the

constriction of isolated human bronchi. The in vitro CysLT₁

or CysLT₂antagonist activity of 35 was several times weaker

than that of montelukast (CysLT₁) or BayCysLT₂RA³⁹

(CysLT₂), so compound 35 was used at a higher concentration.

G

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a

Scheme 1. Syntheses of Side Chains 38a−f

a

Reagents and conditions: (a) 3-butyn-1-ol, PdCl₂(PPh₃)₂, Ph₃P, CuI, Et₃N, rt−80 °C, 33−99%; (b) H₂gas, 10% Pd−C(wet), EtOH, rt, 52−95%;

(c) p-toluenesulfonyl chloride, Et₃N, Me₃N·HCl, toluene, rt, 80−95%.

a

Scheme 2. Syntheses of Compounds 4−16

a

Reagents and conditions: (a) AlCl₃, ethyl succinyl chloride, CH₂Cl₂, 0 °C, 83%−34%; (b) NaBH₄, BF₃·OEt₂, THF, 0 °C, 54%−76%; (c) ethyl

bromoacetate, Cs₂CO₃, DMF, 50 °C, 99%−86%; (d) 4-acetoxystyrene, palladium(II)acetate, trio-tolylphoshine, Et₃N, dioxane, 100 °C; (e) K₂CO₃,

THF, EtOH, 50 °C, 89−96% (2 steps); (f) 4-phenyl-1-butanol, 4-(2-methoxyphenyl)-1-butanol, 4-(3-chlorophenyl)-1-butanol, or 38a−g, ADDP,

PPh₃, CH₂Cl₂, rt; (g) NaOHaq, EtOH−DME, rt-50 °C, 51−97% (2 steps).

and 42b. Heck reaction of 42a and 42b and 4-acetoxystyrene,

deprotection of the acetoxy group, and Mitsunobu reaction

with the corresponding alcohols gave diesters 44a−m.

Carboxylic acid derivatives 4−16 were aﬀorded by alkaline

hydrolysis of 44a−m.

Compound 3 was prepared as shown in Scheme 3. Heck

reaction of the indole derivative 42a with 1-(4-phenoxybutoxy)-

4-vinylbenzene followed by alkaline hydrolysis gave carboxylic

acid derivative 3.

Syntheses of 17−19 and 23−28 are described in Scheme 4.

N-alkylation of indole derivatives 46a, 46b, and 41b prepared

via Fischer indole synthesis was performed with the

corresponding bromide to aﬀord diesters 47a and 47b, 42b,

and 47d−i. These compounds were subjected to Sonogashira

coupling with 4-ethynylphenyl acetate, and then the acetoxy

group was deprotected. The resulting phenols 48a−i were

converted to dicarboxylic acid derivatives 17−19 and 23−28 by

O-alkylation with tosylate 38ba, which was followed by alkaline

hydrolysis.

Syntheses of 20−22 are described in Scheme 5. N-Alkylation

and selective deprotection of indole derivatives 46a, 46b, and

41b resulted in monocarboxylic acids 51a−c. For one-carbon

extension of 51a−c, diazoketone intermediates 52a−c were

prepared with the use of trimethylsilyldiazomethane and treated

under Wolﬀ rearrangement conditions. The resulting C1

extended diesters 53a−c were converted to dicarboxylic acid

compounds 20−22 as in Scheme 4.

H

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a

Scheme 3. Synthesis of Compound 3

a

Reagents and conditions: (a) 1-(4-phenoxybutoxy)-4-vinylbenzene, Pd₂(dba)₃, tert-Bu₃P, Cy₂NMe, 1,4-dioxane, 100 °C; (b) NaOHaq, EtOH−

DME, rt−50 °C, 92% (2 steps).

a

Scheme 4. Syntheses of Compounds 17−19 and 23−28

a

Reagents and conditions: (a) H₂SO₄, EtOH, 4-oxopentanoic acid (n = 1), 5-oxohexanoic acid (n = 2), or 6-oxoheptanoic acid (n = 3), 100 °C, 58−

60%; (b) Br-(CH₂)_m-CO₂Et (m = 1−4) Cs₂CO₃, rt−50 °C, 58−99%; (c) 4-ethynylphenyl acetate, Pd(tert-Bu₃P)₂, ⁱPr₂NH, CH₃CN, rt; (d) K₂CO₃,

DME−EtOH, rt, 62−78% (2 steps); (e) 38ba, Cs₂CO₃, DMF, rt; (f) NaOHaq, EtOH−DME, rt−50 °C, 67−97% (2 steps).

Scheme 6 shows the preparation of the ﬂuorine-substituted

derivatives 29−35. These compounds were also synthesized

with the use of the corresponding synthetic materials according

to the method described in Scheme 4.

As shown in Figure 7, the structure of 35 (gemilukast) was

conﬁrmed with an X-ray crystallographic analysis of a single

crystal (see Supporting Information). Interestingly, the terminal

alkyloxyphenyl ring is orthogonal to the indole ring. In general,

two aromatic rings at either end of a triple bond tend to be in

the same plane. The ﬂuorine atom at the 4-position of the

indole is thought to be causally related to this twisted formation

based on the tendencies of its analogues. This conformation is

considered reasonable for gemilukast and its analogues.

CONCLUSION

■

In summary, the isomerization of the core indole and the

incorporation of a triple bond signiﬁcantly improved in vivo

eﬃcacy, and 35 (ONO-6950, gemilukast) was determined to be

a novel, highly potent, and orally available dual CysLT₁and

CysLT₂antagonist. Gemilukast showed good PK properties in

rats, guinea pigs, and dogs without species diﬀerences,

promising excellent PK in humans. Gemilukast also showed

excellent eﬃcacy in two guinea pig asthmatic models: 74%

inhibition of bronchoconstriction in a CysLT₁-dependent

model at an oral dose of 0.1 mg/kg 24 h before LTD₄

challenge, and 87% inhibition of bronchoconstriction in both

CysLT₁- and CysLT₂-dependent models at an oral dose of 3

I

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a

Scheme 5. Syntheses of Compounds 20−22

a

Reagents and conditions: (a) 2-methyl-2-propanyl bromoacetate, Cs₂CO₃, 45 °C, 100%; (b) HCO₂H, CH₂Cl₂, rt, 91−100%; (c) isobutyl

chloroformate, triethylamine, THF then CH₂N₂/diethyl ether, −10 °C to 0 °C; (d) silver(I) benzoate, triethylamine, EtOH, rt−50 °C, 44−52% (2

steps); (e) 4-ethynylphenyl acetate, Pd(Pt-Bu₃)₂, ⁱPr₂NH, CH₃CN, rt; (f) K₂CO₃, DME−EtOH, rt−50 °C, 35−84% for 2 steps; (g) 38ba, Cs₂CO₃,

DMF, rt; (h) NaOHaq, DME−EtOH, rt−50 °C, 86−95%.

a

Scheme 6. Syntheses of Compounds 29−35

a

Reagents and conditions: (a) H₂SO₄, EtOH, 6-oxoheptanoic acid 100 °C, 35−75%; (b) ethyl 4-bromobutanoate, Cs₂CO₃, 55 °C, 95−97%; (c) 4-

ethynylphenyl acetate, Pd(tert-Bu₃P)₂, ⁱPr₂NH, CH₃CN, rt; (d) K₂CO₃, DME−EtOH, rt, 77−95% (2 steps); (e) 38aa or 38ba, Cs₂CO₃, DMF, rt, or

38a or 38e, ADDP, PPh₃, CH₂Cl₂, rt; (f) NaOHaq, EtOH−DME, rt−50 °C, 72−98% (2 steps).

J

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Figure 7. ORTEP drawing of compound 35 (ONO-6950, gemilukast). Data and reﬁnement statistics are summarized in Supporting Information.

36a (1.0 g 5.29 mmol) in Et₃N (5.0 mL) at room temperature. The

reaction mixture was stirred for 1 h at 80 °C. Pr₂O was added to the

mg/kg 24 h before LTC₄challenge. Furthermore, gemilukast

reduced the antigen-induced constriction of isolated human

bronchi, and its eﬀect was comparable to that of combination

treatment with antagonists against CysLT₁and CysLT₂. On the

basis of the strength of the data presented herein, gemilukast

was selected as a clinical candidate. Gemilukast is currently

being evaluated in phase II clinical trials. The results will be

reported in due course.

i

reaction mixture; this mixture was ﬁltered and the ﬁltrate was

concentrated. The residue was puriﬁed by column chromatography on

silica gel (n-hexane/EtOAc) to give 37a (357 mg, 40%). 37a (313 mg,

1.76 mmol) and platinum dioxide (30 mg) in EtOH (2.0 mL) were

stirred for 3 h under a hydrogen atmosphere. The catalyst was

removed by ﬁltration, and the ﬁltrate was concentrated to give 38a

1

(304 mg, 95%). H NMR (300 MHz, CDCl₃) δ 1.14−1.44 (m, 1H),

1.57−1.73 (m, 4H), 2.16−2.26 (m, 3H), 2.62 (t, J = 7.2 Hz, 2H),

3.61−3.76 (m, 2H), 6.82−6.90 (m, 1H), 6.90−6.95 (m, 1H), 7.02−

7.12 (m, 1H).

EXPERIMENTAL SECTION

■

General Remarks. Analytical samples were homogeneous as

conﬁrmed by TLC, and spectroscopic results were consistent with the

assigned structures. NMR spectra were recorded as designated on

either a Varian Mercury 300 spectrometer or INOVA-500

spectrometer using deuterated chloroform (CDCl₃), deuterated

methanol (CD₃OD), or deuterated dimethyl sulfoxide (DMSO-d₆)

as the solvent. Fast atom bombardment (FABMS, HRMS) and

electron ionization (EI) were performed on a JEOL JMS-DX303HF

spectrometer. Atmospheric pressure chemical ionization (APCI) mass

spectra were determined with a Hitachi M-1200H spectrometer. IR

spectra were measured on a JASCO FTIR-430 spectrometer.

Elemental analyses were performed with a Perkin−Elmer PE2400

series II CHNS/O analyzer and were only indicated as the elements

within 0.4% of the theoretical values unless otherwise noted. Purity

analyses were performed on the following HPLC systems: HPLC,

Agilent Technologies 1200 series with Unison UK-C18 (4.6 mm ×

150 mm). Method A: Unison UK-C18 (4.6 mm × 150 mm), eluting

over 60 min with 30−80% acetonitrile in 20 mM KH₂PO₄aq (pH =

2.0), ﬂow rate of 1 mL/min, column temperature of 35 °C, detection

with UV (210 nm). Method B: Unison UK-C18 (4.6 mm × 150 mm),

eluting over 60 min with 30−80% acetonitrile in 20 mM K₂HPO₄aq

(pH = 7.0), ﬂow rate of 1 mL/min, column temperature of 35 °C,

detection with UV (210 nm). Column chromatography was performed

with silica gel [Merck Silica Gel 60 (0.063−0.200 μm), Wako gel C-

200, or Fuji Silysia FL60D]. Thin layer chromatography was

performed with silica gel (Merck TLC or HPTLC plates, Silica Gel

60 F254) or with a medium-pressure preparative liquid chromatograph

W-prep 2XY (manufactured by Yamazen Corporation; column: main

column size S-5L, inject column size SS-2L). The following

abbreviations for solvents and reagents are used: DMF, N,N-

dimethylformamide; DMSO, dimethyl sulfoxide; EtOH, ethanol;

EtOAc, ethyl acetate; MeOH methanol; THF, tetrahydrofuran;

CH₂Cl₂, dichloromethane; tert-BuOMe, tert-butyl methyl ether;

ⁱPr₂O, diisopropyl ether; CH₃CN, acetonitrile; Et₃N, triethylamine;

TFA, triﬂuoroacetic acid; ADDP, 1,1′-(azodicarbonyl)-dipiperidine.

All animal experiments were approved by the Animal Ethical

Committee of Ono Pharmaceutical Co., Ltd. and were performed in

accordance with institutional animal care guidelines.

4-(3-Chloro-2-methylphenyl)-1-butanol (38b). Compound 38b

was prepared from compound 36b in a manner similar to that

described for compound 38a. ¹H NMR (300 MHz, CDCl₃) δ 1.27 (t, J

= 5.1 Hz, 1H), 1.61−1.68 (m, 4H), 2.35 (s, 3H), 2.62−2.72 (m, 2H),

3.63−3.75 (m, 2H), 7.00−7.09 (m, 2H), 7.17−7.26 (m, 1H).

4-(3-Chloro-2-methylphenyl)butyl 4-methylbenzenesulfonate

(38ba). Triethylamine (7.9 mL, 56.9 mmol), trimethylamine hydro-

chloride (272 mg, 2.84 mmol), and p-toluenesulfonyl chloride (7.05 g,

37.0 mmol) were added to a solution of compound 38b (5.65 g, 28.4

mmol) in toluene (57 mL) at 0 °C, and the mixture was stirred for 2.5

h at 0 °C. The reaction mixture was poured into H₂O, and the

resulting mixture was extracted with EtOAc. The organic layer was

washed with water and saturated brine, dried over anhydrous sodium

sulfate, and concentrated. The residue was puriﬁed by column

chromatography on silica gel (n-hexane/EtOAc) to give 38ba (9.49 g,

95%). ¹H NMR (300 MHz, CDCl₃) δ 1.51−1.64 (m, 2H), 1.64−1.78

(m, 2H), 2.29 (s, 3H), 2.44 (s, 3H), 2.52−2.65 (m, 2H), 4.05 (t, J =

6.2 Hz, 2H), 6.93−7.05 (m, 2H), 7.21 (dd, J = 7.7 Hz, 1.5 Hz, 1H),

7.33 (d, J = 8.2 Hz, 2H), 7.78 (d, J = 8.2 Hz, 2H).

4-(3-Fluoro-2-methylphenyl)butyl 4-Methylbenzenesulfonate

(38aa). Compound 38aa was prepared from compound 38a in a

1

manner similar to that described for compound 38ba. H NMR (300

MHz, CDCl₃) δ 1.51−1.64 (m, 2H), 1.65−1.78 (m, 2H), 2.15 (d, J =

2.2 Hz, 3H), 2.44 (s, 3H), 2.57 (t, J = 7.7 Hz, 2H), 4.05 (t, J = 6.2 Hz,

2H), 6.77−6.91 (m, 2H), 6.97−7.13 (m, 1H), 7.33 (d, J = 8.4 Hz,

2H), 7.78 (d, J = 8.4 Hz, 2H).

4-(4,5-Diﬂuoro-2-methylphenyl)-1-butanol (38c). Compound 38c

was prepared from compound 36c in a manner similar to that

1

described for compound 38a. H NMR (300 MHz, CDCl₃) δ 1.59−

1.69 (m, 5H), 2.24 (s, 3H), 2.53−2.58 (m, 2H), 3.67−3.71 (m, 2H),

6.89−6.95 (m, 2H).

4-(2,3,6-Triﬂuorophenyl)-1-butanol (38d). Compound 38d was

prepared from compound 36d in a manner similar to that described

1

for compound 38a. H NMR (300 MHz, CDCl₃) δ 1.14−1.15 (m,

1H), 1.52−1.82 (m, 4H), 2.63−2.86 (m, 2H), 3.58−3.77 (m, 2H),

6.71−6.81 (m, 1H), 6.88−7.05 (m, 1H).

4-(2,3,5,6-Tetraﬂuorophenyl)-1-butanol (38e). Compound 38e

was prepared from compound 36e in a manner similar to that

4-(3-Fluoro-2-methylphenyl)-1-butanol (38a). 3-Butyn-1-ol (0.38

mL, 5.0 mmol), PdCl₂(PPh₃)₂(175 mg, 0.26 mmol), Ph₃P (131 mg,

0.53 mmol), and CuI (95 mg, 0.53 mmol) were added to a solution of

1

described for compound 38a. H NMR (300 MHz, CDCl₃) δ 1.20−

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1.35 (m, 1H), 1.52−1.80 (m 4H), 2.69−2.87 (m, 2H), 3.56−3.77 (m,

2H), 6.82−7.01 (m, 1H).

97%). ¹H NMR (300 MHz, CDCl₃) δ 1.25 (t, J = 7.2 H, 3H), 1.26 (t, J

= 7.2 Hz, 3H), 1.93−2.09 (m, 2H), 2.36 (t, J = 7.4 Hz, 2H), 2.69−2.82

(m, 2H), 4.12 (q, J = 7.2 Hz, 2H), 4.23 (q, J = 7.2 Hz, 2H), 5.22 (s,

2H), 6.80 (s, 1H), 6.94 (dd, J = 7.9, 7.6 Hz, 1H), 7.33 (dd, J = 7.6, 1.0

Hz, 1H), 7.52 (dd, J = 7.9, 1.1 Hz, 1H).

Ethyl 4-[7-Bromo-1-(2-ethoxy-2-oxoethyl)-2-methyl-1H-indol-3-

yl]butanoate (42b). Compound 42b was prepared from compound

41b in a manner similar to that described for compound 42a. ¹H NMR

(300 MHz, CDCl₃) δ 1.23 (t, J = 7.2 Hz, 3H), 1.26 (t, J = 7.2 Hz, 3H),

1.86−1.97 (m, 2H), 2.27 (s, 3H), 2.30 (t, J = 7.5 Hz, 2H), 2.74 (t, J =

7.2 Hz, 2H), 4.11 (q, J = 7.2 Hz, 2H), 4.23 (q, J = 7.2 Hz, 2H), 5.27 (s,

2H), 6.90 (t, J = 7.8 Hz, 1H), 7.25 (dd, J = 7.5, 0.9 Hz, 1H), 7.44 (dd,

J = 7.5, 0.9 Hz, 1H).

4-(2,3,4,6-Tetraﬂuorophenyl)-1-butanol (38f). Compound 38f was

prepared from compound 36f in a manner similar to that described for

1

compound 38a. H NMR (300 MHz, CDCl₃) δ 1.15−1.39 (m, 1H),

1.51−1.79 (m, 4H), 2.68 (t, J = 6.0 Hz, 2H), 3.67 (t, J = 5.9 Hz, 2H),

6.71 (m, 1H).

4-(2,3,4,5-Tetraﬂuorophenyl)-1-butanol (38g). Compound 38g

was prepared from compound 36g in a manner similar to that

1

described for compound 38a. H NMR (300 MHz, CDCl₃) δ 1.39

(brs, 1H), 1.60−1.74 (m, 4H), 2.63−2.68 (m, 2H), 3.65−3.69 (m,

2H), 6.75−6.84 (m, 1H).

Ethyl 4-(7-Bromo-1H-indol-3-yl)-4-oxobutanoate (40a). AlCl₃

(1.36 g, 10.2 mmol) and 39a (1.0 g, 5.10 mmol) were added to

ethyl 4-chloro-4-oxobutanoate (1.68 g, 10.2 mmol) in CH₂Cl₂at 0 °C,

which was followed by stirring for 5 h at room temperature. H₂O was

added to the reaction mixture, and the resulting mixture was extracted

with EtOAc. The organic layer was washed sequentially with water and

saturated brine, dried over anhydrous sodium sulfate, and concen-

trated. The residue was stirred for 30 min in EtOAc and ﬁltered to

obtain a solid. The resulting solid was dried to give 40a (1.37 g, 83%).

¹H NMR (300 MHz, DMSO-d₆) δ 1.27 (t, J = 7.2 Hz, 3H), 2.79 (t, J =

Ethyl 4-{1-(2-Ethoxy-2-oxoethyl)-7-[(E)-2-(4-hydroxyphenyl)-

vinyl]-1H-indol-3-yl}butanoate (43a). Dicyclohexylmethylamine

t

(3.24 mL, 15.1 mmol) and a solution of Bu₃P (7.08 mL, 2.52

mmol, 10 w/w % hexane solution) were added to a solution of 42a

(5.00 g, 12.6 mmol), 4-acetoxystyrene (2.02 mL, 13.2 mmol), and

tris(dibenzylideneacetone)dipalladium(0) (1.15 g, 1.26 mmol) in

dioxane (60 mL) at room temperature under an argon atmosphere.

The reaction mixture was stirred for 3 h at 100 °C. The reaction

mixture was ﬁltered, the ﬁltrate was poured into water, and the

resulting mixture was extracted with EtOAc. The organic layer was

washed sequentially with aqueous saturated ammonium chloride

solution, water, and saturated brine, dried over anhydrous sodium

sulfate, and concentrated. The residue was puriﬁed by column

chromatography on silica gel (n-hexane/EtOAc) to give acetoxy

derivative (5.90 g, 98%). Potassium carbonate (857 mg, 6.2 mmol)

was added to a solution of the acetoxy derivative (5.90 g, 12.3 mmol)

in THF (40 mL) and EtOH (40 mL), and the mixture was stirred for

17 h at room temperature. The reaction mixture was poured into

water, and the resulting mixture was extracted with EtOAc. The

organic layer was washed sequentially with water, saturated aqueous

ammonium chloride solution, and saturated brine, dried over

anhydrous sodium sulfate, and concentrated. The residue was puriﬁed

by column chromatography on silica gel (n-hexane/EtOAc) to give

43a (4.61 g, 87%). ¹H NMR (300 MHz, CDCl₃) δ 1.19 (t, J = 7.2 Hz,

3H), 1.25 (t, J = 7.2 Hz, 3H), 2.78 (t, J = 7.5 Hz, 2H), 2.04 (t, J = 7.5

Hz, 2H), 2.39 (t, J = 7.5 Hz, 2H), 4.13 (q, J = 7.2 Hz, 2H), 4.16 (q, J =

7.2 Hz, 2H), 4.98 (s, 2H), 5.19 (s, 1H), 6.80 (s, 1H), 6.82 (d, J = 15.0

Hz, 1H), 6.83 (d, J = 8.7 Hz, 2H), 7.10 (t, J = 7.5 Hz, 1H), 7.24 (d, J =

7.5 Hz, 1H), 7.38 (d, J = 8.7 Hz, 2H), 7.42 (d, J = 15.0 Hz, 1H),

7.51(d, J = 7.5 Hz, 1H).

6.9 Hz, 2H), 3.23 (t, J = 6.9 Hz, 2H), 4.17 (q, J = 7.2 Hz, 2H), 7.16 (t,

J = 7.8 Hz, 1H), 7.43 (dd, J = 7.8, 0.9 Hz, 1H), 7.96 (t, J = 0.9 Hz,

1H), 8.30 (dd, J = 7.8, 0.9 Hz, 1H), 8.76 (brs, 1H).

Ethyl 4-(7-Bromo-2-methyl-1H-indol-3-yl)-4-oxobutanoate

(40b). Compound 40b was prepared from compound 39b in a

1

manner similar to that described for compound 40a. H NMR (300

MHz, DMSO-d₆) δ 1.28 (t, J = 7.2 Hz, 3H), 2.79 (s, 3H), 2.79 (t, J =

6.6 Hz, 2H), 3.28 (t, J = 6.6 Hz, 2H), 4.19 (q, J = 7.2 Hz, 2H), 7.09 (t,

J = 8.1 Hz, 1H), 7.33 (d, J = 8.1, Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H),

8.75 (brs, 1H).

Ethyl 4-(7-Bromo-1H-indol-3-yl)butanoate (41a). NaBH₄(118

mg, 3.11 mmol) and boron triﬂuoride−ethyl ether complex (0.66 mL,

5.19 mmol) were added to compound 40a (560 mg, 1.73 mmol) in

THF at −20 °C, which was followed by stirring for 2.5 h at −10 °C.

Saturated ammonium chloride solution was added to the reaction

mixture, and the resulting mixture was extracted with EtOAc. The

organic layer was washed sequentially with water and saturated brine,

dried over anhydrous sodium sulfate, and concentrated. The residue

was puriﬁed by column chromatography on silica gel (n-hexane/

1

EtOAc) to give 41a (292 mg, 54%). H NMR (300 MHz, CDCl₃) δ

1.24 (t, J = 7.2 Hz, 3H), 1.98−2.08 (m, 2H), 2.36 (t, J = 7.5 Hz, 2H),

2.79 (t, J = 7.2 Hz, 2H), 4.12 (q, J = 7.2 Hz, 2H), 6.99 (t, J = 7.5 Hz,

1H), 7.10−7.08 (m, 1H), 7.34 (dd, J = 7.5, 0.6 Hz, 1H), 7.55 (dt, J =

7.5, 0.6 Hz, 1H), 8.12 (brs, 1H).

Ethyl 4-(7-Bromo-2-methyl-1H-indol-3-yl)butanoate (41b). Pro-

cedure 1. Starting with 40b, following the procedures above resulted

in compound 41a.

Ethyl 4-{1-(2-Ethoxy-2-oxoethyl)-7-[(E)-2-(4-hydroxyphenyl)-

vinyl]-2-methyl-1H-indol-3-yl}butanoate (43b). Compound 43b

was prepared from compound 42b in a manner similar to that

1

described for compound 43a. H NMR (300 MHz, CDCl₃) δ 1.17−

1.29 (m, 6H), 1.90−1.98 (m, 2H), 2.29(s, 3H), 2.32 (t, J = 7.3 Hz,

2H), 2.76 (t, J = 7.3 Hz, 2H), 4.11 (q, J = 7.0 Hz, 2H), 4.20 (q, J = 7.2

Hz, 2H), 4.89 (s, 1H), 4.97 (s, 2H), 6.77−6.86 (m, 3H), 7.06 (t, J =

7.3 Hz, 1H), 7.15 (d, J = 7.3 Hz, 1H), 7.35−7.46 (m, 4H).

Procedure 2. Fisher indole synthesis: Concentrated H₂SO₄(12

mL) was added to a solution of 45 (25 g, 112 mmol) and 6-

oxoheptanoic acid (16.1 g, 112 mmol) in EtOH (120 mL) at room

temperature. The reaction mixture was stirred for 1.5 h at reﬂux. The

resulting mixture was extracted with EtOAc. The organic layer was

washed sequentially with water and saturated brine, dried over

anhydrous sodium sulfate, and concentrated. The residue was puriﬁed

by column chromatography on silica gel (n-hexane/EtOAc) to give

41b (27.5 g, 76%). ¹H NMR (300 MHz, CDCl₃) δ 1.23 (t, J = 7.2 Hz,

3H), 1.89−2.00 (m, 2H), 2.30 (t, J = 7.2 Hz, 2H), 2.39 (s, 3H), 2.71

(t, J = 7.2 Hz, 2H), 4.10 (q, J = 7.2 Hz, 2H), 6.94 (t, J = 8.1 Hz, 1H),

7.24 (d, J = 8.1 Hz, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.91 (brs, 1H).

Ethyl 4-[7-Bromo-1-(2-ethoxy-2-oxoethyl)-1H-indol-3-yl]-

butanoate (42a). Cs₂CO₃(8.4 g, 25.8 mmol) and ethyl bromoacetate

(1.51 mL, 13.5 mmol) were added to compound 41a (4.0 g, 12.9

mmol) in DMF (40 mL) at 0 °C, which was followed by stirring for 1

h at 0 °C. H₂O was added to the reaction mixture, and the resulting

mixture was extracted with EtOAc. The organic layer was washed

sequentially with water and saturated brine, dried over anhydrous

sodium sulfate, and concentrated. The residue was puriﬁed by column

chromatography on silica gel (n-hexane/EtOAc) to give 42a (4.98 g,

4-(1-(Carboxymethyl)-7-{(E)-2-[4-(4-phenoxybutoxy)phenyl]-

vinyl}-1H-indol-3-yl)butanoic Acid (3). 1-(4-Phenoxybutoxy)-4-vinyl-

benzene²⁸(0.78 g, 2.91 mmol), tris(dibenzylideneacetone)-

dipalladium(0) (243 mg, 0.27 mmol), dicyclohexylmethylamine

(0.62 g, 3.2 mmol), and a solution of tert-Bu₃P (1.08 g, 0.53 mmol,

10 w/w % hexane solution) were added to a solution of 42a (1.05 g,

2.65 mmol) in dioxane (14 mL) at room temperature under an argon

atmosphere. The reaction mixture was stirred for 3 h at 105 °C. The

reaction mixture was then ﬁltered, the ﬁltrate was poured into water,

and the resulting mixture was extracted with EtOAc. The organic layer

was washed sequentially with aqueous saturated ammonium chloride

solution, water, and saturated brine, dried over anhydrous sodium

sulfate, and concentrated. The residue was puriﬁed by column

chromatography on silica gel (n-hexane/EtOAc) to give 44n (1.29 g,

83%). A 2 mol/L aqueous solution of sodium hydroxide (2.8 mL) was

added to a solution of 44n (1.1 g, 1.9 mmol) in DME (18 mL), and

the mixture was stirred for 5 h at room temperature. Hydrochloric acid

(1 mol/L) and water were added to the reaction mixture, and the

resulting mixture was extracted with EtOAc. The organic layer was

L

DOI: 10.1021/acs.jmedchem.5b00741

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Journal of Medicinal Chemistry

Article

washed with water and brine sequentially, dried over anhydrous

sodium sulfate, and concentrated. The residue was washed with ⁱPr₂O

to give 3 (890 mg, 90%). H NMR (300 MHz, DMSO-d₆) δ 1.78−

Hz, 1 H), 7.43−7.55 (m, 3H), 12.20 (brs, 1H), 13.10 (brs, 1H). MS

(FAB, neg) m/z 524 (M − H)⁻. HRMS (FAB, neg) C₃₃H₃₅NO₅(M −

H)⁻calcd mass 524.2437, found 524.2446. HPLC purity 96%

(method B).

1

1.93 (m, 6H), 2.28 (t, J = 7.2 Hz, 2H), 2.67 (t, J = 7.4 Hz, 2H), 3.97−

4.12 (m, 4H), 5.13 (s, 2H), 6.86−6.97 (m, 6H), 7.02 (dd, J = 7.6 Hz,

1H), 7.08 (s, 1H), 7.22−7.31 (m, 3H), 7.40−7.58 (m, 4H), 12.29 (s,

2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 23.67, 25.07, 25.39, 33.30,

50.40, 66.92, 67.11, 113.62, 114.39, 114.64, 118.02, 119.02, 120.14,

120.36, 122.41, 123.07, 127.65, 128.84, 129.24, 129.40, 129.89, 130.14,

133.62, 158.23, 158.56, 171.15, 174.37. MS (FAB, neg) m/z 526 (M −

H)⁻. HRMS (FAB, neg) C₃₂H₃₃NO₆(M − H)⁻calcd mass 526.2230,

found 526.2233. LCMS (ELSD) RT = 1.17 min (>98%). HPLC purity

96% (method A).

4-(1-(Carboxymethyl)-7-{(E)-2-[4-(4-phenylbutoxy)phenyl]vinyl}-

1H-indol-3-yl)butanoic Acid (4). Compound 4 was prepared from

compound 43a in a manner similar to that described for compound

16. ¹H NMR (300 MHz, DMSO-d₆) δ 1.66−1.78 (m, 4H), 1.78−1.93

(m, 2H), 2.28 (t, J = 7.5 Hz, 2H), 2.58−2.76 (m, 4H), 3.91−4.10 (m,

2H), 5.11 (s, 2H), 6.87−6.95 (m, 4H), 7.01 (t, J = 7.8 Hz, 1H), 7.07

(s, 1H), 7.14−7.32 (m, 6H), 7.41−7.57 (m, 3H), 12.05 (brs, 2H). MS

(FAB, neg) m/z 510 (M − H)⁻. HRMS (FAB, neg) C₃₂H₃₃NO₅(M −

H)⁻calcd mass 510.2280, found 510.2269. HPLC purity 95%

(method B).

4-{1-(Carboxymethyl)-7-[(E)-2-{4-[4-(3-chlorophenyl)butoxy]-

phenyl}vinyl]-1H-indol-3-yl}butanoic Acid (5). Compound 5 was

prepared from compound 43a in a manner similar to that described for

compound 16. ¹H NMR (300 MHz, DMSO-d₆) δ 1.67−1.77 (m, 4H),

1.78−1.91 (m, 2H), 2.28 (t, J = 7.5 Hz, 2H), 2.61−2.72 (m, 4H),

3.96−4.04 (m, 2H), 5.11 (s, 2H), 6.91 (d, J = 15.9 Hz, 1H), 6.92 (d, J

= 9.0 Hz, 2H), 7.01 (t, J = 7.5 Hz, 1H), 7.07 (s, 1H), 7.16−7.57 (m,

9H), 12.15 (brs, 2H). MS (FAB, neg) m/z 544 (M − H)⁻. HRMS

(FAB, neg) C₃₂H₃₂ClNO₅(M − H)⁻calcd mass 544.1891, found

544.1895. HPLC purity 93% (method B).

4-{1-(Carboxymethyl)-7-[(E)-2-{4-[4-(2,3,6-triﬂuorophenyl)-

butoxy]phenyl}vinyl]-1H-indol-3-yl}butanoic Acid (6). Compound 6

was prepared from compound 43a in a manner similar to that

described for compound 16. ¹H NMR (300 MHz, DMSO-d₆) δ 1.62−

1.92 (m, 6H), 2.28 (t, J = 7.2 Hz, 2H), 2.66 (t, J = 7.2 Hz, 2H), 2.70−

2.79 (m, 2H), 4.00 (t, J = 6.0 Hz, 2H), 5.13 (s, 2H), 6.87−6.96 (m,

3H), 7.01 (t, J = 7.5 Hz, 1H), 7.08 (s, 1H), 7.10−7.17 (m, 1H), 7.25

(d, J = 7.5 Hz, 1H), 7.30−7.57 (m, 5H),12.31 (brs, 1H), 12.85 (brs,

1H). MS (FAB, neg) m/z 564 (M − H)⁻. HRMS (FAB, neg)

C₃₃H₃₀F₃NO₅(M − H)⁻calcd mass 564.1998, found 564.1996.

HPLC purity 99% (method B).

4-{1-(Carboxymethyl)-7-[(E)-2-{4-[4-(3-ﬂuoro-2-methylphenyl)-

butoxy]phenyl}vinyl]-1H-indol-3-yl}butanoic Acid (7). Compound 7

was prepared from compound 43a in a manner similar to that

described for compound 16. ¹H NMR (300 MHz, DMSO-d₆) δ 1.59−

1.92 (m, 6H), 2.17 (d, J = 2.0 Hz, 3H), 2.22−2.34 (m, 2H), 2.61−2.73

(m, 4H), 4.02 (t, J = 6.2 Hz, 2H), 5.13 (s, 2H), 6.86−7.05 (m, 6H),

7.06−7.18 (m, 2H), 7.26 (d, J = 7.3 Hz, 1H), 7.40−7.58 (m, 4H),

12.15 (brs, 1H), 12.95 (brs, 1H). MS (FAB, neg) m/z 542 (M − H)⁻.

HRMS (FAB, neg) C₃₃H₃₄FNO₅(M − H)⁻calcd mass 542.5343,

found 542.2343. HPLC purity 95% (method B).

4-{1-(Carboxymethyl)-7-[(E)-2-{4-[4-(3-chloro-2-methylphenyl)-

butoxy]phenyl}vinyl]-1H-indol-3-yl}butanoic Acid (8). Compound 8

was prepared from compound 43a in a manner similar to that

described for compound 16. ¹H NMR (300 MHz, DMSO-d₆) δ 1.53−

1.94 (m, 6H), 2.23−2.35 (m, 5H), 2.60−2.77 (m, 4H), 4.02 (t, J = 5.9

Hz, 2H), 5.13 (s, 2H), 6.86−6.97 (m, 3H), 7.02 (t, J = 7.7 Hz, 1H),

7.06−7.18 (m, 3H), 7.22−7.29 (m, 2H), 7.40−7.57 (m, 4H), 12.21

(brs, 1H), 12.81 (brs, 1H). MS (FAB, neg) m/z 558 (M − H)⁻.

HRMS (FAB, neg) C₃₃H₃₄ClNO₅(M − H)⁻calcd mass 558.2047,

found 558.2058. HPLC purity 96% (method B).

4-{1-(Carboxymethyl)-7-[(E)-2-{4-[4-(2-methoxyphenyl)butoxy]-

phenyl}vinyl]-2-methyl-1H-indol-3-yl}butanoic Acid (10). Com-

pound 10 was prepared from compound 43b in a manner similar to

1

that described for compound 16. H NMR (300 MHz, DMSO-d₆) δ

1.62−1.80 (m, 6H), 2.21 (t, J = 7.3 Hz, 2H), 2.25 (s, 3H), 2.61 (t, J =

7.3 Hz, 2H), 2.66 (t, J = 7.1 Hz, 2H), 3.77 (s, 3H), 3.95−4.04 (m,

2H), 5.01 (s, 2H), 6.82−7.02 (m, 6H), 7.11−7.21 (m, 3H), 7.39 (d, J

= 7.7 Hz, 1H), 7.48 (d, J = 8.6 Hz, 2H), 7.50 (d, J = 15.7 Hz, 1H),

12.05 (brs, 1H), 13.40 (brs, 1H). MS (FAB, neg) m/z 554 (M − H)⁻.

HRMS (FAB, neg) C₃₄H₃₇NO₆(M − H)⁻calcd mass 554.2543, found

554.2532. HPLC purity 97% (method B).

4-{1-(Carboxymethyl)-2-methyl-7-[(E)-2-{4-[4-(2,3,6-

triﬂuorophenyl)butoxy]phenyl}vinyl]-1H-indol-3-yl}butanoic Acid

(11). Compound 11 was prepared from compound 43b in a manner

similar to that described for compound 16. ¹H NMR (300 MHz,

DMSO-d₆) δ 1.64−1.84 (m, 6H), 2.22 (t, J = 7.2 Hz, 2H), 2.24−2.30

(s, 3H), 2.67 (t, J = 7.2 Hz, 2H), 2.70−2.80 (m, 2H), 3.97−4.08 (m,

2H), 5.02 (s, 2H), 6.81−7.06 (m, 4H), 7.05−7.21 (m, 2H), 7.28−7.44

(m, 2H), 7.44−7.60 (m, 3H), 12.06 (brs, 1H), 13.10 (brs, 1H). MS

(FAB, neg) m/z 578 (M − H)⁻. HRMS (FAB, neg) C₃₃H₃₂F₃NO₅(M

− H)⁻calcd mass 578.2154, found 578.2151. HPLC purity 95%

(method B).

4-{1-(Carboxymethyl)-2-methyl-7-[(E)-2-{4-[4-(2,3,5,6-

tetraﬂuorophenyl)butoxy]phenyl}vinyl]-1H-indol-3-yl}butanoic Acid

(12). Compound 12 was prepared from compound 43b in a manner

similar to that described for compound 16. ¹H NMR (300 MHz,

DMSO-d₆) δ 1.65−1.84 (m, 6H), 2.21 (t, J = 7.2 Hz, 2H), 2.25 (s,

3H), 2.62−2.71 (m, 2H), 2.75−2.85 (m, 2H), 3.97−4.08 (m, 2H),

4.97−5.09 (m, 2H), 6.83−7.04 (m, 4H), 7.14 (d, J = 7.1 Hz, 1H),

7.36−7.41 (m, 1H), 7.44−7.56 (m, 3H), 7.64−7.83 (m, 1H), 12.11

(brs, 1H), 13.07 (brs, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ 9.85,

22.12, 22.97, 25.16, 25.72, 28.08, 33.00, 47.13, 66.98, 104.33 (m),

110.58, 114.65, 117.13, 118.95, 120.13, 120.64 (m), 122.31, 123.03,

127.59, 128.75, 129.89, 130.24, 133.50, 134.11, 144.18 (m), 145.22

(m), 158.17, 171.19, 174.42. MS (FAB, neg) m/z 596 (M − H)⁻.

HRMS (FAB, neg) C₃₃H₃₁F₄NO₅(M − H)⁻calcd mass 596.2060,

found 596.2060. HPLC purity 95% (method B).

4-{1-(Carboxymethyl)-2-methyl-7-[(E)-2-{4-[4-(2,3,4,5-

tetraﬂuorophenyl)butoxy]phenyl}vinyl]-1H-indol-3-yl}butanoic Acid

(13). Compound 13 was prepared from compound 43b in a manner

similar to that described for compound 16. ¹H NMR (300 MHz,

DMSO-d₆) δ 1.65−1.85 (m, 6H), 2.21 (t, J = 7.3 Hz, 2H), 2.25 (s,

3H), 2.62−2.76 (m, 4H), 3.90−4.09 (m, 2H), 5.02 (s, 2H), 6.87 (d, J

= 15.9 Hz, 1H), 6.89−7.04 (m, 3H), 7.14 (d, J = 7.3 Hz, 1H), 7.33−

7.42 (m, 2H), 7.43−7.58 (m, 3H), 12.02 (brs, 1H), 13.01 (brs, 1H).

¹³C NMR (125 MHz, DMSO-d₆) δ 27.23, 27.95, 33.00, 47.12, 67.09,

110.59, 112.09 (m), 114.65, 117.13, 118.95, 120.13, 122.31, 123.02,

125.87 (m), 127.58, 128.76, 129.87, 130.25, 133.49, 134.11, 137.66

(m), 139.74 (m), 145.04 (m), 146.05 (m), 158.19, 171.19, 174.42. MS

(FAB, neg) m/z 596 (M − H)⁻. HRMS (FAB, neg) C₃₃H₃₁F₄NO₅(M

− H)⁻calcd mass 596.2060, found 596.2060. HPLC purity 95%

(method B).

4-{1-(Carboxymethyl)-2-methyl-7-[(E)-2-{4-[4-(2,3,4,6-

tetraﬂuorophenyl)butoxy]phenyl}vinyl]-1H-indol-3-yl}butanoic Acid

(14). Compound 14 was prepared from compound 43b in a manner

similar to that described for compound 16. ¹H NMR (300 MHz,

DMSO-d₆) δ 1.60−1.84 (m, 6H), 2.21 (t, J = 7.1 Hz, 2H), 2.25 (s,

3H), 2.61−2.77 (m, 4H), 3.99 (t, J = 5.9 Hz, 2H), 4.99 (s, 2H), 6.82−

6.94 (m, 3H), 6.98 (t, J = 7.6 Hz, 1H), 7.14 (d, J = 7.3 Hz, 1H), 7.38

(d, J = 7.3 Hz, 1H), 7.41−7.58 (m, 4H), 12.20 (brs, 1H), 13.10 (brs,

1H). ¹³C NMR (125 MHz, DMSO-d₆) δ 9.85, 21.45, 22.98, 25.28,

25.73, 28.06, 33.01, 47.24, 66.99, 101.31 (m), 110.52, 114.63, 114.95

(m), 117.11, 118.90, 120.07, 122.31, 123.08, 127.58, 128.73, 129.89,

130.17, 133.50, 134.11, 136.38 (m), 148.17(m), 148.93 (m), 154.94

(m), 158.15, 171.22, 174.42. MS (FAB, neg) m/z 596 (M − H)⁻.

HRMS (FAB, neg) C₃₃H₃₁F₄NO₅(M − H)⁻calcd mass 596.2060,

found 596.2060. HPLC purity 98% (method B).

4-[1-(Carboxymethyl)-2-methyl-7-{(E)-2-[4-(4-phenylbutoxy)-

phenyl]vinyl}-1H-indol-3-yl]butanoic Acid (9). Compound 9 was

prepared from compound 43b in a manner similar to that described

1

for compound 16. H NMR (300 MHz, DMSO-d₆) δ 1.61−1.84 (m,

6H), 2.13−2.30 (m, 5H), 2.58−2.76 (m, 4H), 3.93−4.07 (m, 2H),

5.01 (s, 2H), 6.81−7.03 (m, 4H), 7.09−7.32 (m, 6H), 7.38 (d, J = 7.3

M

DOI: 10.1021/acs.jmedchem.5b00741

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Article

4-{1-(Carboxymethyl)-7-[(E)-2-{4-[4-(3-ﬂuoro-2-methylphenyl)-

butoxy]phenyl}vinyl]-2-methyl-1H-indol-3-yl}butanoic Acid (15).

Compound 15 was prepared from compound 43b in a manner

similar to that described for compound 16. ¹H NMR (300 MHz,

DMSO-d₆) δ 1.59−1.85 (m, 6H), 2.17 (d, J = 2.2 Hz, 3H), 2.21 (t, J =

7.4 Hz, 2H), 2.25 (s, 3H), 2.60−2.74 (m, 4H), 4.02 (t, J = 6.1 Hz,

2H), 5.01 (s, 2H), 6.81−7.05 (m, 6H), 7.08−7.18 (m, 2H), 7.38 (d, J

= 7.5 Hz, 1H), 7.47 (d, J = 8.6 Hz, 2H), 7.49 (d, J = 16.5 Hz, 1H),

12.10 (brs, 1H), 13.20 (brs, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ

9.85, 10.01, 22.98, 25.72, 26.16, 28.36, 31.97, 33.00, 47.13, 67.25,

110.590, 112.31 (J_C−F= 23.0 Hz), 114.660, 117.130, 118.950, 120.130,

122.200, 122.320, 122.990, 124.61 (J_C−F= 2.8 Hz), 126.67(J_C−F= 9.1

Hz), 127.590, 128.760, 129.830, 130.260, 133.490, 134.100, 143.08

(J_C−F= 4.1 Hz), 158.260, 160.70 (J_C−F= 239.9 Hz), 171.190, 174.420.

MS (FAB, neg) m/z 556 (M − H)⁻. HRMS (FAB, neg) C₃₄H₃₆FNO₅

(M − H)⁻calcd mass 556.2499, found 556.2502. HPLC purity 98%

(method A).

7.4 Hz, 2H), 5.27 (s, 2H), 6.92 (dd, J = 7.5, 7.8 Hz, 1H), 7.27 (dd, J =

7.5, 1.0 Hz, 1H), 7.44 (dd, J = 7.8, 1.0, Hz, 1H).

Ethyl 4-[7-Bromo-3-(2-ethoxy-2-oxoethyl)-2-methyl-1H-indol-1-

yl]butanoate (47d). Compound 47d was prepared from compound

46a in a manner similar to that described for compound 42a. ¹H NMR

(300 MHz, CDCl₃) δ 1.22 (t, J = 7.2 Hz, 3H), 1.26 (t, J = 7.2 Hz, 3H),

1.99−2.14 (m, 2H), 2.40 (t, J = 7.2 Hz, 2H), 2.42 (s, 3H), 3.67 (s,

2H), 4.11 (q, J = 7.2 Hz, 2H), 4.14 (q, J = 7.2 Hz, 2H), 4.47−4.58 (m,

2H), 6.91 (t, J = 7.8, 1.0 Hz, 1H), 7.29 (dd, J = 7.8, 1.0 Hz, 1H), 7.47

(dd, J = 7.8, 1.0 Hz, 1H).

Ethyl 4-[7-Bromo-3-(3-ethoxy-3-oxopropyl)-2-methyl-1H-indol-1-

yl]butanoate (47e). Compound 47e was prepared from compound

46b in a manner similar to that described for compound 42a. ¹H NMR

(300 MHz, CDCl₃) δ 1.22 (t, J = 6.9 Hz, 3H), 1.26 (t, J = 6.8 Hz, 3H),

1.99−2.09 (m, 2H), 2.36−2.41 (m, 2H), 2.39 (s, 3H), 2.55 (t, J = 7.9

Hz, 2H), 3.02 (t, J = 7.9 Hz, 2H), 4.07−4.17 (m, 4H), 4.48−4.53 (m,

2H), 6.90 (dd, J = 7.5, 7.8 Hz, 1H), 7.28 (d, J = 7.5 Hz, 1H), 7.44 (d, J

= 7.8 Hz, 1H).

4-{1-(Carboxymethyl)-7-[(E)-2-{4-[4-(3-chloro-2-methylphenyl)-

butoxy]phenyl}vinyl]-2-methyl-1H-indol-3-yl}butanoic Acid (16).

38b (37 mg, 0.19 mmol), ADDP (98 mg, 0.37 mmol), and PPh₃

(94 mg, 0.37 mmol) were added to a solution of 43b (70 mg, 0.16

mmol) in CH₂Cl₂(1.0 mL), and the mixture was stirred for 15 h at

room temperature. Triphenylphosphine oxide was removed by

ﬁltration, and the ﬁltrate was concentrated. The residue was puriﬁed

by column chromatography on silica gel (n-hexane/EtOAc) to give

44m (109 mg, quant). A 2 mol/L aqueous solution of sodium

hydroxide (1.0 mL) was added to a mixture of 44m (98 mg, 0.16

mmol), DME (2.0 mL), and EtOH (2.0 mL), and the mixture was

stirred for 2.5 h at 50 °C. The reaction mixture was acidiﬁed with 2

mol/L hydrochloric acid and extracted with EtOAc. The organic layer

was washed sequentially with water and saturated brine, dried over

anhydrous sodium sulfate, and concentrated. The residue was stirred

Diethyl 4,4′-(7-Bromo-2-methyl-1H-indole-1,3-diyl)dibutanoate

(47f). Compound 47f was prepared from compound 41b in a manner

1

similar to that described for compound 42a. H NMR (300 MHz,

CDCl₃) δ 1.23 (t, J = 7.2 Hz, 3H), 1.25 (t, J = 7.2 Hz, 3H), 1.82−2.12

(m, 4H), 2.29 (t, J = 7.8 Hz, 2H), 2.36 (s, 3H), 2.39 (t, J = 7.5 Hz,

2H), 2.73 (t, J = 7.5 Hz, 2H), 4.10 (q, J = 7.2 Hz, 2H), 4.13 (q, J = 7.2

Hz, 2H), 4.46−4.55 (m, 2H), 6.88 (t, J = 7.5 Hz, 1H), 7.27 (dd, J =

7.5, 0.9 Hz, 1H), 7.43 (dd, J = 7.5, 0.9 Hz, 1H).

Ethyl 5-[7-Bromo-3-(2-ethoxy-2-oxoethyl)-2-methyl-1H-indol-1-

yl]pentanoate (47g). Compound 47g was prepared from compound

46a in a manner similar to that described for compound 42a. ¹H NMR

(300 MHz, CDCl₃) δ 1.17−1.29 (m, 6H), 1.65−1.87 (m, 4H), 2.35 (t,

J = 7.0 Hz, 2H), 2.39 (s, 3H), 3.66 (s, 2H), 4.10 (q, J = 7.2 Hz, 2H),

4.12 (q, J = 7.2 Hz, 2H), 4.39−4.52 (m, 2H), 6.89 (t, J = 7.8, 7.5 Hz,

1H), 7.27 (dd, J = 7.5, 0.9 Hz, 1H), 7.45 (dd, J = 7.8, 0.9 Hz, 1H).

Ethyl 5-[7-Bromo-3-(3-ethoxy-3-oxopropyl)-2-methyl-1H-indol-1-

yl]pentanoate (47h). Compound 47h was prepared from compound

46b in a manner similar to that described for compound 42a. ¹H NMR

(300 MHz, CDCl₃) δ 1.19−1.28 (m, 6H), 1.69−1.95 (m, 4H), 2.33−

2.36 (m, 2H), 2.36 (s, 3H), 2.54 (t, J = 7.8 Hz, 2H), 3.01 (t, J = 7.8

Hz, 2H), 4.05−4.15 (m, 4H), 4.41−4.46 (m, 2H), 6.87 (dd, J = 7.8,

7.5 Hz, 1H), 7.27 (dd, J = 7.5, 0.9 Hz, 1H), 7.41 (dd, J = 7.8, 0.9 Hz,

1H).

Ethyl 5-[7-Bromo-3-(4-ethoxy-4-oxobutyl)-2-methyl-1H-indol-1-

yl]pentanoate (47i). Compound 47i was prepared from compound

41b in a manner similar to that described for compound 42a. ¹H NMR

(300 MHz, CDCl₃) δ 1.24 (t, J = 7.1 Hz, 3H), 1.25 (t, J = 7.2 Hz, 3H),

1.70−1.81 (m, 4H), 1.85−1.94 (m, 2H), 2.27−2.40 (m, 4H), 2.34 (s,

3H), 2.73 (t, J = 7.4 Hz, 2H), 4.07−4.16 (m, 4H), 4.42−4.47 (m, 2H),

6.88 (dd, J = 7.8, 7.5 Hz, 1H), 7.27 (dd, J = 7.5, 1.1 Hz, 1H), 7.43 (dd,

J = 7.8, 1.1 Hz, 1H).

Diethyl 2,2′-{7-[(4-Hydroxyphenyl)ethynyl]-2-methyl-1H-indole-

1,3-diyl}diacetate (48a). Compound 48a was prepared from

compound 47a in a manner similar to that described for compound

43a. ¹H NMR (300 MHz, CDCl₃) δ 1.11 (t, J = 7.2 Hz, 3H), 1.27 (t, J

= 7.2 Hz, 3H), 2.32 (s, 3H), 3.73 (s, 2H), 4.12 (q, J = 7.2 Hz, 2H),

4.16 (q, J = 7.2 Hz, 2H), 5.35 (s, 2H), 5.72 (s, 1H), 6.62 (d, J = 8.7

Hz, 2H), 7.06 (t, J = 7.6 Hz, 1H), 7.31 (dd, J = 7.4, 1.2 Hz, 1H), 7.35

(d, J = 8.7 Hz, 2H), 7.50 (dd, J = 7.6, 1.2 Hz, 1H).

Ethyl 3-{1-(2-Ethoxy-2-oxoethyl)-7-[(4-hydroxyphenyl)ethynyl]-2-

methyl-1H-indol-3-yl}propanoate (48b). Compound 48b was

prepared from compound 47b in a manner similar to that described

for compound 43a. ¹H NMR (300 MHz, CDCl₃) δ 1.12 (t, J = 7.1 Hz,

3H), 1.22 (t, J = 7.2 Hz, 3H), 2.32 (s, 3H), 2.59 (t, J = 7.5 Hz, 2H),

3.05 (t, J = 7.5 Hz, 2H), 4.11(q, J = 7.1 Hz, 2H), 4.13 (q, J = 7.2 Hz,

2H), 5.12 (s, 1H), 5.43 (s, 2H), 6.81−6.84 (m, 2H), 7.04 (dd, J = 7.8,

7.5 Hz, 1H), 7.31 (dd, J = 7.5, 1.0 Hz, 1H), 7.43−7.46 (m, 2H), 7.49

(dd, J = 7.8, 1.0 Hz, 1H).

i

for 30 min in Pr₂O and ﬁltered to obtain a solid. The resulting solid

was dried to give 16 (82 mg, 92%). ¹H NMR (300 MHz, DMSO-d₆) δ

1.52−1.86 (m, 6H), 2.21 (t, J = 7.3 Hz, 2H), 2.25 (s, 3H), 2.31 (s,

3H), 2.61−2.79 (m, 4H), 4.02 (t, J = 6.2 Hz, 2H), 5.01 (s, 2H), 6.81−

7.05 (m, 4H), 7.07−7.19 (m, 3H), 7.22−7.31 (m, 1H), 7.34−7.43 (m,

1H), 7.43−7.59 (m, 3H), 12.20 (brs, 1H), 13.31 (brs, 1H). ¹³C NMR

(125 MHz, DMSO-d₆) δ 9.85, 15.50, 22.98, 25.72, 26.21, 28.35, 33.00,

33.06, 47.13, 67.24, 110.58, 114.66, 117.13, 118.95, 120.13, 122.32,

123.00, 126.63, 126.86, 127.59, 127.86, 128.76, 129.84, 130.26, 133.27,

133.50, 133.83, 134.11, 142.74, 158.25, 171.20, 174.42. MS (FAB, neg)

m/z 572 (M − H)⁻. HRMS (FAB, neg) C₃₄H₃₆ClNO₅(M − H)⁻

calcd mass 572.2204, found 572.2201. HPLC purity 95% (method A).

Ethyl (7-Bromo-2-methyl-1H-indol-3-yl)acetate (46a). Com-

pound 46a was prepared from compound 45 in a manner similar to

that described for compound 41b (Procedure 2). ¹H NMR (300 MHz,

CDCl₃) δ 1.23 (t, J = 7.2 Hz, 3H), 2.44 (s, 3H), 3.65 (s, 2H), 4.12 (q,

J = 7.2 Hz, 2H), 6.96 (t, J = 7.8 Hz, 1H), 7.25 (dd, J = 7.8, 0.9 Hz,

1H), 7.41−7.50 (m, 1H), 8.01 (s, 1H).

Ethyl 3-(7-Bromo-2-methyl-1H-indol-3-yl)propanoate (46b).

Compound 46b was prepared from compound 45 in a manner

1

similar to that described for compound 41b (Procedure 2). H NMR

(300 MHz, CDCl₃) δ 1.21 (t, J = 7.2 Hz, 3H), 2.42 (s, 3H), 2.59 (t, J =

6.6 Hz, 2H), 3.01 (t, J = 7.2 Hz, 2H), 4.10 (q, J = 7.2 Hz, 2H), 6.95 (t,

J = 7.8 Hz, 1H), 7.25 (dd, J = 7.8, 0.9 Hz, 1H), 7.43 (d, J = 7.8 Hz,

1H), 7.92 (brs, 1H).

Diethyl 2,2′-(7-Bromo-2-methyl-1H-indole-1,3-diyl)diacetate

(47a). Compound 47a was prepared from compound 46a in a

1

manner similar to that described for compound 42a. H NMR (300

MHz, CDCl₃) δ 1.22 (t, J = 7.2 Hz, 3H), 1.27 (t, J = 7.2 Hz, 3H), 2.33

(s, 3H), 3.68 (s, 2H), 4.11 (q, J = 7.2 Hz, 2H), 4.24 (q, J = 7.2 Hz,

2H), 5.29 (s, 2H), 6.92 (t, J = 7.8 Hz, 1H), 7.23−7.31 (m, 1H), 7.47

(dd, J = 7.8, 1.1 Hz, 1H).

Ethyl 3-[7-Bromo-1-(2-ethoxy-2-oxoethyl)-2-methyl-1H-indol-3-

yl]propanoate (47b). Compound 47b was prepared from compound

41b in a manner similar to that described for compound 42a. ¹H NMR

(300 MHz, CDCl₃) δ 1.20−1.32 (m, 6H), 2.31 (s, 3H), 2.57 (t, J = 7.2

Hz, 2H), 3.03 (t, J = 7.5 Hz, 2H), 4.10 (q, J = 7.2 Hz, 2H), 4.26 (q, J =

Ethyl 4-{1-(2-Ethoxy-2-oxoethyl)-7-[(4-hydroxyphenyl)ethynyl]-2-

methyl-1H-indol-3-yl}butanoate (48c). Compound 48c was prepared

from compound 42b in a manner similar to that described for

compound 43a. H NMR (300 MHz, CDCl₃) δ 1.12 (t, J = 7.1 Hz,

3H), 1.22 (t, J = 7.2 Hz, 3H), 1.86−1.98 (m, 2H), 2.29 (s, 3H), 2.28−

1

N

DOI: 10.1021/acs.jmedchem.5b00741

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

2.36 (m, 2H), 2.76 (t, J = 7.4 Hz, 2H), 4.11 (q, J = 7.2 Hz, 2H), 4.13

(q, J = 7.2 Hz, 2H), 5.09 (s, 1H), 5.44 (s, 2H), 6.82 (d, J = 8.8 Hz,

2H), 7.03 (dd, J = 7.8, 7.5 Hz, 2H), 7.30 (dd, J = 7.5, 1.1 Hz, 1H), 7.44

(d, J = 8.8 Hz, 2H), 7.49 (dd, J = 7.8, 1.1 Hz, 1H).

was washed sequentially with water and saturated brine, dried over

anhydrous sodium sulfate, and concentrated. The residue was stirred

for 30 min in Pr₂O and ﬁltered to obtain a solid. The resulting solid

i

was dried to give 17 (88 mg, 89%). ¹H NMR (300 MHz, DMSO-d₆) δ

1.57−1.85 (m, 4H), 2.28 (s, 3H), 2.31 (s, 3H), 2.65−2.76 (m, 2H),

3.62 (s, 2H), 4.00−4.10 (m, 2H), 5.40 (s, 2H), 6.94−7.04 (m, 3H),

7.08−7.17 (m, 2H), 7.18−7.28 (m, 2H), 7.42−7.54 (m, 3H), 12.15

(brs, 1H), 13.02 (brs, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ 9.83,

15.50, 26.16, 28.26, 29.87, 33.04, 45.45, 67.36, 85.83, 92.69, 104.52,

105.18, 114.39, 114.78, 118.85, 126.23, 126.64, 126.86, 127.86, 128.64,

132.52, 133.27, 133.83, 134.09, 135.82, 142.71, 158.81, 170.56, 172.73.

MS (FAB, neg) m/z 542 (M − H)⁻. HRMS (FAB, neg)

C₃₂H₃₀ClNO₅(M − H)⁻calcd mass 542.1734, found 542.1725.

HPLC purity 97% (method A).

Ethyl 4-{3-(2-Ethoxy-2-oxoethyl)-7-[(4-hydroxyphenyl)ethynyl]-2-

methyl-1H-indol-1-yl}butanoate (48d). Compound 48d was pre-

pared from compound 47d in a manner similar to that described for

1

compound 43a. H NMR (300 MHz, CDCl₃) δ 1.12 (t, J = 7.2 Hz,

3H), 1.25 (t, J = 7.2 Hz, 3H), 2.07−2.20 (m, 2H), 2.30 (t, J = 7.4 Hz,

2H), 2.40 (s, 3H), 3.70 (s, 2H), 4.08 (q, J = 7.2 Hz, 2H), 4.14 (q, J =

7.2 Hz, 2H), 4.53−4.64 (m, 2H), 5.50 (s, 1H), 6.71 (d, J = 8.8 Hz,

2H), 7.05 (t, J = 7.8 Hz, 1H), 7.33 (dd, J = 7.8, 1.1 Hz, 1H), 7.38 (d, J

= 8.8 Hz, 2H), 7.51 (dd, J = 7.8, 1.1 Hz, 1H).

Ethyl 4-{3-(3-Ethoxy-3-oxopropyl)-7-[(4-hydroxyphenyl)ethynyl]-

2-methyl-1H-indol-1-yl}butanoate (48e). Compound 48e was

prepared from compound 47e in a manner similar to that described

3-[1-(Carboxymethyl)-7-({4-[4-(3-chloro-2-methylphenyl)-

butoxy]phenyl}ethynyl)-2-methyl-1H-indol-3-yl]propanoic Acid

(18). Compound 18 was prepared from compound 48b and 38ba in

1

for compound 43a. H NMR (300 MHz, CDCl₃) δ 1.19−1.25 (m,

1

a manner similar to that described for compound 17. H NMR (300

6H), 2.12−2.19 (m, 2H), 2.28−2.33 (m, 2H), 2.39 (s, 3H), 2.58 (t, J =

7.7 Hz, 2H), 3.04 (t, J = 7.7 Hz, 2H), 4.05−4.14 (m, 4H), 4.62 (t, J =

7.5 Hz, 2H), 5.35 (s, 1H), 6.81−6.84 (m, 2H), 7.03 (dd, J = 7.8, 7.2

Hz, 1H), 7.32 (d, J = 7.2 Hz, 1H), 7.41−7.44 (m, 2H), 7.49 (d, J = 7.8

Hz, 1H).

MHz, DMSO-d₆) δ 1.59−1.72 (m, 2H), 1.72−1.85 (m, 2H), 2.27 (s,

3H), 2.31 (s, 3H), 2.40−2.47 (m, 2H), 2.67−2.73 (m, 2H), 2.91 (t, J =

7.5 Hz, 2H), 4.05 (t, J = 6.3 Hz, 2H), 5.38 (s, 2H), 7.00 (t, J = 8.0 Hz,

3H), 7.08−7.17 (m, 2H), 7.18−7.23 (m, 1H), 7.23−7.29 (m, 1H),

7.45−7.54 (m, 3H), 12.06 (brs, 1H), 12.91 (brs, 1H). ¹³C NMR (125

MHz, DMSO-d₆) δ 9.68, 15.50, 19.33, 26.16, 28.26, 33.04, 34.90,

45.36, 67.36, 85.92, 92.65, 104.51, 110.11, 114.42, 114.79, 118.59,

118.73, 126.12, 126.64, 126.87, 127.86, 128.25, 132.50, 133.27, 133.83,

134.23, 134.63, 142.71, 158.80, 170.63, 173.89. MS (FAB, neg) m/z

556 (M − H)⁻. HRMS (FAB, neg) C₃₃H₃₂ClNO₅(M − H)⁻calcd

mass 556.1891, found 556.1890. HPLC purity 95% (method B).

4-[1-(Carboxymethyl)-7-({4-[4-(3-chloro-2-methylphenyl)-

butoxy]phenyl}ethynyl)-2-methyl-1H-indol-3-yl]butanoic Acid (19).

Compound 19 was prepared from compound 48c and 38ba in a

Diethyl 4,4′-{7-[(4-Hydroxyphenyl)ethynyl]-2-methyl-1H-indole-

1,3-diyl}dibutanoate (48f). Compound 48f was prepared from

compound 47f in a manner similar to that described for compound

43a. ¹H NMR (300 MHz, CDCl₃) δ 1.22 (t, J = 7.2 Hz, 3H), 1.26 (t, J

= 7.2 Hz, 3H), 1.82−1.99 (m, 2H), 2.05−2.21 (m, 2H), 2.29−2.35 (m,

7H), 2.75 (t, J = 7.5 Hz, 2H), 4.04−4.14 (m, 4H), 4.62 (t, J = 7.2 Hz,

2H), 5.39 (s, 1H), 6.83 (d, J = 8.7 Hz, 2H), 7.01 (t, J = 7.8 Hz, 1H),

7.30 (dd, J = 7.8, 1.2 Hz, 1H), 7.42 (d, J = 8.7 Hz, 2H), 7.48 (dd, J =

7.2, 1.2 Hz, 1H).

Ethyl 5-{3-(2-Ethoxy-2-oxoethyl)-7-[(4-hydroxyphenyl)ethynyl]-2-

methyl-1H-indol-1-yl}pentanoate (48g). Compound 48g was pre-

pared from compound 47g in a manner similar to that described for

1

manner similar to that described for compound 17. H NMR (300

MHz, DMSO-d₆) δ 1.58−1.86 (m, 6H), 2.21 (t, J = 7.2 Hz, 2H), 2.25

(s, 3H), 2.31 (s, 3H), 2.62−2.75 (m, 4H), 3.99−4.09 (m, 2H), 5.31−

5.46 (m, 2H), 6.94−7.04 (m, 3H), 7.08−7.16 (m, 2H), 7.20 (d, J = 7.1

Hz, 1H), 7.22−7.29 (m, 1H), 7.44−7.54 (m, 3H), 12.09 (brs, 1H),

12.88 (brs, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ 26.16, 28.25,

32.96, 33.03, 45.34, 67.36, 85.94, 92.62, 104.47, 110.73, 114.43,

114.79, 118.59, 118.65, 126.06, 126.64, 126.86, 127.85, 128.57, 132.48,

133.27, 133.83, 134.25, 134.48, 142.71, 158.79, 170.67, 174.38. MS

(FAB, neg) m/z 570 (M − H)⁻. HRMS (FAB, neg) C₃₄H₃₄ClNO₅(M

− H)⁻calcd mass 570.2047, found 570.2048. HPLC purity 95%

(method B).

1

compound 43a. H NMR (300 MHz, CDCl₃) δ 1.19 (t, J = 7.2 Hz,

3H), 1.26 (t, J = 7.2 Hz, 3H), 1.56−1.70 (m, 2H), 1.74−1.90 (m, 2H),

2.24 (t, J = 7.4 Hz, 2H), 2.37 (s, 3H), 3.71 (s, 2H), 4.06 (q, J = 7.2 Hz,

2H), 4.15 (q, J = 7.2 Hz, 2H), 4.45−4.59 (m, 2H), 5.69 (s, 1H), 6.65

(d, J = 8.8 Hz, 2H), 7.04 (t, J = 7.8 Hz, 1H), 7.29−7.40 (m, 3H), 7.50

(dd, J = 8.7, 1.1 Hz, 1H).

Ethyl 5-{3-(3-Ethoxy-3-oxopropyl)-7-[(4-hydroxyphenyl)ethynyl]-

2-methyl-1H-indol-1-yl}pentanoate (48h). Compound 48h was

prepared from compound 47h in a manner similar to that described

1

for compound 43a. H NMR (300 MHz, CDCl₃) δ 1.17−1.24 (m,

6H), 1.64−1.86 (m, 4H), 2.25 (t, J = 7.4 Hz, 2H), 2.37 (s, 3H), 2.57

(t, J = 7.8 Hz, 2H), 3.04 (t, J = 7.8 Hz, 2H), 4.03−4.14 (m, 4H), 4.54

(t, J = 7.7 Hz, 2H), 5.36 (s, 1H), 6.81−6.84 (m, 2H), 7.02 (dd, J = 7.8,

7.7 Hz, 1H), 7.31 (dd, J = 7.7, 0.9 Hz, 1H), 7.41−7.44 (m, 2H), 7.48

(dd, J = 7.8, 0.9 Hz, 1H).

4-[3-(Carboxymethyl)-7-({4-[4-(3-chloro-2-methylphenyl)-

butoxy]phenyl}ethynyl)-2-methyl-1H-indol-1-yl]butanoic Acid (23).

Compound 23 was prepared from compound 48d and 38ba in a

1

manner similar to that described for compound 17. H NMR (300

MHz, DMSO-d₆) δ 1.57−1.86 (m, 4H), 1.87−2.05 (m, 2H), 2.20 (t, J

= 7.4 Hz, 2H), 2.31 (s, 3H), 2.33−2.38 (m, 3H), 2.64−2.79 (m, 2H),

3.61 (s, 2H), 4.05 (t, J = 6.2 Hz, 2H), 4.49−4.62 (m, 2H), 6.93−7.04

(m, 3H), 7.06−7.18 (m, 2H), 7.19−7.28 (m, 2H), 7.41−7.54 (m, 3H),

12.25 (brs, 2H). MS (FAB, neg) m/z 570 (M − H)⁻. HRMS (FAB,

neg) C₃₄H₃₄ClNO₅(M − H)⁻calcd mass 570.2047, found 570.2026.

HPLC purity 95% (method B).

Ethyl 5-{3-(4-Ethoxy-4-oxobutyl)-7-[(4-hydroxyphenyl)ethynyl]-2-

methyl-1H-indol-1-yl}pentanoate (48i). Compound 48i was pre-

pared from compound 47i in a manner similar to that described for

1

compound 43a. H NMR (300 MHz, CDCl₃) δ 1.20 (t, J = 7.1 Hz,

3H), 1.24 (t, J = 7.2 Hz, 3H), 1.65−1.97 (m, 6H), 2.26 (t, J = 7.5 Hz,

2H), 2.31 (t, J = 8.0 Hz, 2H), 2.34 (s, 3H), 2.75 (t, J = 7.5 Hz, 2H),

4.07 (q, J = 7.1 Hz, 2H), 4.10 (q, J = 7.2 Hz, 2H), 4.58 (t, J = 7.7 Hz,

2H), 5.36 (s, 1H), 6.82−6.85 (m, 2H), 7.01 (dd, J = 8.0, 7.4 Hz, 1H),

7.31 (dd, J = 7.4, 1.1 Hz, 1H), 7.41−7.44 (m, 2H), 7.48 (dd, J = 8.0,

1.1 Hz, 1H).

4-[3-(2-Carboxyethyl)-7-({4-[4-(3-chloro-2-methylphenyl)-

butoxy]phenyl}ethynyl)-2-methyl-1H-indol-1-yl]butanoic Acid (24).

Compound 24 was prepared from compound 48e and 38ba in a

1

manner similar to that described for compound 17. H NMR (300

2,2′-[7-({4-[4-(3-Chloro-2-methylphenyl)butoxy]phenyl}ethynyl)-

2-methyl-1H-indole-1,3-diyl]diacetic Acid (17). 38ba (65 mg, 0.18

mmol) and Cs₂CO₃(109 mg, 0.33 mmol) were added to a solution of

48a (70 mg) in DMF (0.5 mL), and the reaction mixture was stirred

for 14 h at room temperature. The reaction mixture was ﬁltered and

concentrated. The residue was puriﬁed by column chromatography on

silica gel (n-hexane/EtOAc) to give the ester derivative 49a. A 2 mol/

L aqueous solution of sodium hydroxide (0.6 mL) was added to a

mixture of 49a, DME (1.5 mL), and EtOH (1.5 mL), and the mixture

was stirred for 7 h at 50 °C. The reaction mixture was acidiﬁed with 2

mol/L hydrochloric acid and extracted with EtOAc. The organic layer

MHz, DMSO-d₆) δ 1.59−1.72 (m, 2H), 1.72−1.83 (m, 2H), 1.86−

2.02 (m, 2H), 2.13−2.22 (m, 2H), 2.31 (s, 3H), 2.35 (s, 3H), 2.39−

2.46 (m, 2H), 2.65−2.77 (m, 2H), 2.84−2.99 (m, 2H), 4.05 (t, J = 6.3

Hz, 2H), 4.47−4.60 (m, 2H), 6.94−7.04 (m, 3H), 7.09−7.18 (m, 2H),

7.19−7.30 (m, 2H), 7.45−7.55 (m, 3H), 12.10 (s, 2H). MS (FAB,

neg) m/z 584 (M − H)⁻. HRMS (FAB, neg) C₃₅H₃₆ClNO₅(M −

H)⁻calcd mass 584.2204, found 584.2206. HPLC purity 99%

(method A).

4,4′-[7-({4-[4-(3-Chloro-2-methylphenyl)butoxy]phenyl}ethynyl)-

2-methyl-1H-indole-1,3-diyl]dibutanoic Acid (25). Compound 25

was prepared from compound 48f and 38ba in a manner similar to

O

DOI: 10.1021/acs.jmedchem.5b00741

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1

that described for compound 17. H NMR (300 MHz, DMSO-d₆) δ

ⁱPr₂O and ﬁltered to obtain a solid. The resulting solid was dried to

give 51a (768 mg, 93%). H NMR (300 MHz, CDCl₃) δ 1.22 (t, J =

7.2 Hz, 3H), 2.34 (s, 3H), 3.68 (s, 2H), 4.11 (q, J = 7.2 Hz, 2H), 5.37

(s, 2H), 6.94 (t, J = 7.8 Hz, 1H), 7.29 (dd, J = 7.8, 0.9 Hz, 1H), 7.48

(dd, J = 7.8, 0.9 Hz, 1H).

1

1.59−1.87 (m, 6H), 1.88−2.03 (m, 2H), 2.12−2.23 (m, 4H), 2.31 (s,

3 H), 2.33 (s, 3H), 2.61−2.77 (m, 4H), 4.05 (t, J = 6.2 Hz, 2H), 4.54

(t, J = 7.5 Hz, 2H), 6.93−7.03 (m, 3H), 7.07−7.18 (m, 2H), 7.19−

7.29 (m, 2H), 7.42−7.58 (m, 3H), 12.05 (s, 2H). ¹³C NMR (125

MHz, DMSO-d₆) δ 9.74, 15.49, 22.82, 25.74, 26.14, 26.61, 28.25,

30.48, 33.01, 33.03, 42.57, 67.36, 86.39, 91.64, 104.35, 110.76, 114.44,

114.94, 118.40, 118.70, 126.21, 126.63, 126.85, 127.85, 128.66, 132.42,

133.27, 133.47, 133.83, 134.09, 142.71, 158.82, 173.70, 174.38. MS

(FAB, neg) m/z 598 (M − H)⁻. HRMS (FAB, neg) C₃₆H₃₈ClNO₅(M

− H)⁻calcd mass 598.2360, found 598.2371. HPLC purity 98%

(method B)

[7-Bromo-3-(3-ethoxy-3-oxopropyl)-2-methyl-1H-indol-1-yl]-

acetic Acid (51b). Compound 51b was prepared from compound 50b

1

in a manner similar to that described for compound 51a. H NMR

(300 MHz, DMSO-d₆) δ 1.12 (t, J = 7.2 Hz, 3H), 2.25 (s, 3H), 2.47−

2.55 (m, 2H), 2.93 (t, J = 7.2 Hz, 2H), 3.99 (q, J = 7.2 Hz, 2H), 5.23

(s, 2H), 6.90 (t, J = 7.7 Hz, 1H), 7.22 (dd, J = 7.7, 0.9 Hz, 1H), 7.47

(dd, J = 7.7, 0.9 Hz, 1H), 13.01 (s, 1H).

5-[3-(Carboxymethyl)-7-({4-[4-(3-chloro-2-methylphenyl)-

butoxy]phenyl}ethynyl)-2-methyl-1H-indol-1-yl]pentanoic Acid

(26). Compound 26 was prepared from compound 48g and 38ba in

[7-Bromo-3-(4-ethoxy-4-oxobutyl)-2-methyl-1H-indol-1-yl]acetic

Acid (51c). Compound 51c was prepared from compound 50c in a

1

manner similar to that described for compound 51a. H NMR (300

1

a manner similar to that described for compound 17. H NMR (300

MHz, DMSO-d₆) δ 1.15 (t, J = 7.2 Hz, 3H), 1.65−1.83 (m, 2H),

2.20−2.32 (m, 5H), 2.67 (t, J = 7.5 Hz, 2H), 4.03 (q, J = 7.2 Hz, 2H),

5.24 (s, 2H), 6.90 (t, J = 7.8 Hz, 1H), 7.21 (dd, J = 7.8, 0.9 Hz, 1H),

7.47 (dd, J = 7.8, 0.9 Hz, 1H), 13.01 (s, 1H).

MHz, DMSO-d₆) δ 1.43−1.58 (m, 2H), 1.58−1.85 (m, 6H), 2.19 (t, J

= 7.3 Hz, 2H), 2.31 (s, 3H), 2.35 (s, 3H), 2.64−2.76 (m, 2H), 3.61 (s,

2H), 4.04 (t, J = 6.3 Hz, 2H), 4.45−4.64 (m, 2H), 6.91−7.03 (m, 3H),

7.07−7.18 (m, 2H), 7.19−7.29 (m, 2H), 7.40−7.55 (m, 3H), 12.15

(brs, 2H). MS (FAB, neg) m/z 584 (M − H)⁻. HRMS (FAB, neg)

C₃₅H₃₆ClNO₅(M − H)⁻calcd mass 584.2204, found 584.2200.

HPLC purity 96% (method B).

Ethyl 3-[7-Bromo-3-(2-ethoxy-2-oxoethyl)-2-methyl-1H-indol-1-

yl]propanoate (53a). Triethylamine (0.42 mL, 3.0 mmol) and

isobutyl chloroformate (0.25 mL, 2.6 mmol) were added to a solution

of 51a (708 mg, 2.0 mmol) in THF (10.0 mL) at −10 °C. The

reaction mixture was stirred for 3 h at −10 °C. Then CH₂N₂/diethyl

ether (10 mL) was added to the reaction mixture at −10 °C. The

reaction mixture was stirred for 3 h at 0 °C. H₂O was added to the

reaction mixture, and the resulting mixture was extracted with EtOAc.

The organic layer was washed sequentially with water and saturated

brine, dried over anhydrous sodium sulfate, and concentrated. The

residue was puriﬁed by column chromatography on silica gel (n-

hexane/EtOAc) to give 52a. Triethylamine (0.16 mL, 1.15 mmol) and

silver(I) benzoate (30 mg, 0.13 mmol) were added to a solution of 52a

in EtOH (4.0 mL), and the reaction mixture was stirred for 3 h at 50

°C. The reaction mixture was ﬁltered, the ﬁltrate was poured into

water, and the resulting mixture was extracted with EtOAc. The

organic layer was washed sequentially with aqueous saturated

ammonium chloride solution, water, and saturated brine, dried over

anhydrous sodium sulfate, and concentrated. The residue was puriﬁed

by column chromatography on silica gel (n-hexane/EtOAc) to give

53a (408 mg, 51%, 2 steps). ¹H NMR (300 MHz, CDCl₃) δ 1.23 (t, J

= 7.2 Hz, 3H), 1.25 (t, J = 7.2, 3H), 1.82−1.98 (m, 2H), 2.29 (t, J =

7.2 Hz, 2H), 2.36 (s, 3H), 2.68−2.81 (m, 4H), 4.10 (q, J = 7.2 Hz,

2H), 4.15 (q, J = 7.2 Hz, 2H), 4.72−4.84 (m, 2H), 6.90 (t, J = 7.8 Hz,

1H), 7.28 (dd, J = 7.5, 0.9 Hz, 1H), 7.43 (dd, J = 7.8, 0.9 Hz, 1H).

Diethyl 3,3′-(7-Bromo-2-methyl-1H-indole-1,3-diyl)dipropanoate

(53b). Compound 53b was prepared from compound 51b in a manner

5-[3-(2-Carboxyethyl)-7-({4-[4-(3-chloro-2-methylphenyl)-

butoxy]phenyl}ethynyl)-2-methyl-1H-indol-1-yl]pentanoic Acid

(27). Compound 27 was prepared from compound 48h and 38ba in

1

a manner similar to that described for compound 17. H NMR (300

MHz, DMSO-d₆) δ 1.41−1.57 (m, 2H), 1.59−1.84 (m, 6H), 2.18 (t, J

= 7.2 Hz, 2H), 2.31 (s, 3H), 2.34 (s, 3H), 2.38−2.46 (m, 2H), 2.65−

2.76 (m, 2H), 2.90 (t, J = 7.4 Hz, 2H), 4.04 (t, J = 6.2 Hz, 2H), 4.47−

4.61 (m, 2H), 6.92−7.02 (m, 3H), 7.07−7.17 (m, 2H), 7.20 (dd, J =

7.3, 0.9 Hz, 1H), 7.23−7.28 (m, 1H), 7.43−7.53 (m, 3H), 11.99 (brs,

2H). MS (FAB, neg) m/z 598 (M − H)⁻. HRMS (FAB, neg)

C₃₆H₃₈ClNO₅(M − H)⁻calcd mass 598.2360, found 598.2352.

HPLC purity 99% (method B).

5-[3-(3-Carboxypropyl)-7-({4-[4-(3-chloro-2-methylphenyl)-

butoxy]phenyl}ethynyl)-2-methyl-1H-indol-1-yl]pentanoic Acid

(28). Compound 28 was prepared from compound 48i and 38ba in

1

a manner similar to that described for compound 17. H NMR (300

MHz, DMSO-d₆) δ 1.43−1.57 (m, 2H), 1.59−1.85 (m, 8H), 2.13−

2.24 (m, 4H), 2.31 (s, 3H), 2.33 (s, 3H), 2.62−2.75 (m, 4H), 3.99−

4.09 (m, 2H), 4.50−4.60 (m, 2H), 6.93−7.01 (m, 3H), 7.07−7.17 (m,

2H), 7.20 (dd, J = 7.4, 0.8 Hz, 1H), 7.23−7.28 (m, 1H), 7.43−7.52

(m, 3H), 11.96 (brs, 2H). MS (FAB, neg) m/z 612 (M − H)⁻. HRMS

(FAB, neg) C₃₇H₄₀ClNO₅(M − H)⁻calcd mass 612.2517, found

612.2516. HPLC purity 99% (method B).

Ethyl 2-Methyl-2-propanyl 2,2′-(7-bromo-2-methyl-1H-indole-

1,3-diyl)diacetate (50a). Compound 50a was prepared from

compound 46a in a manner similar to that described for compound

42a. ¹H NMR (300 MHz, CDCl₃) δ 1.22 (t, J = 7.2 Hz, 3H), 1.46 (s,

9H), 2.33 (s, 3H), 3.68 (s, 2H), 4.11 (q, J = 7.2 Hz, 2H), 5.20 (s, 2H),

6.92 (t, J = 7.8 Hz, 1H), 7.24−7.31 (m, 1H), 7.47 (dd, J = 7.8, 1.0 Hz,

1H).

1

similar to that described for compound 53a. H NMR (300 MHz,

CDCl₃) δ 1.22 (t, J = 7.2 Hz, 3H), 1.25 (t, J = 7.2 Hz, 3H), 2.38 (s,

3H), 2.49−2.60 (m, 2H), 2.67−2.81 (m, 2H), 2.95−3.06 (m, 2H),

4.09 (q, J = 7.2 Hz, 2H), 4.15 (q, J = 7.2 Hz, 2H), 4.71−4.83 (m, 2H),

6.90 (t, J = 7.8 Hz, 1H), 7.28 (dd, J = 7.8, 1.0 Hz, 1H), 7.42 (dd, J =

7.8, 1.0 Hz, 1H).

Ethyl 4-[7-Bromo-1-(3-ethoxy-3-oxopropyl)-2-methyl-1H-indol-3-

yl]butanoate (53c). Compound 53c was prepared from compound

51c in a manner similar to that described for compound 53a. ¹H NMR

(300 MHz, CDCl₃) δ 1.23 (t, J = 7.2 Hz, 3H), 1.25 (t, J = 7.2 Hz, 3H),

1.82−1.98 (m, 2H), 2.29 (t, J = 7.2 Hz, 2H), 2.36 (s, 3H), 2.68−2.81

(m, 4H), 4.10 (q, J = 7.2 Hz, 2H), 4.15 (q, J = 7.2 Hz, 2H), 4.72−4.84

(m, 2H), 6.90 (t, J = 7.8 Hz, 1H), 7.28 (dd, J = 7.8, 0.9 Hz, 1H), 7.43

(dd, J = 7.8, 0.9 Hz, 1H).

Ethyl 3-(7-Bromo-2-methyl-1-{2-[(2-methyl-2-propanyl)oxy]-2-

oxoethyl}-1H-indol-3-yl)propanoate (50b). Compound 50b was

prepared from compound 46b in a manner similar to that described

for compound 42a. ¹H NMR (300 MHz, CDCl₃) δ 1.22 (t, J = 7.2 Hz,

3H), 1.45 (s, 9H), 2.30 (s, 3H), 2.51−2.62 (m, 2H), 2.98−3.08 (m,

2H), 4.10 (q, J = 7.2 Hz, 2H), 5.17 (s, 2H), 6.90 (dd, J = 7.8, 7.6 Hz,

1H), 7.26 (dd, J = 7.6, 1.0 Hz, 1H), 7.43 (dd, J = 7.8, 1.1 Hz, 1H).

Ethyl 4-(7-Bromo-2-methyl-1-{2-[(2-methyl-2-propanyl)oxy]-2-

oxoethyl}-1H-indol-3-yl)butanoate (50c). Compound 50c was

prepared from compound 41b in a manner similar to that described

for compound 42a. ¹H NMR (300 MHz, CDCl₃) δ 1.24 (t, J = 7.2 Hz,

3H), 1.44 (s, 9H), 1.84−1.98 (m, 2H), 2.24−2.34 (m, 5H), 2.74 (t, J =

7.4 Hz, 2H), 4.10 (q, J = 7.2 Hz, 2H), 5.17 (s, 2H), 6.88 (t, J = 7.6 Hz,

1H), 7.24 (dd, J = 7.6, 1.0 Hz, 1H), 7.42 (dd, J = 7.6, 1.0 Hz, 1H).

[7-Bromo-3-(2-ethoxy-2-oxoethyl)-2-methyl-1H-indol-1-yl]acetic

Acid (51a). HCO₂H (15 mL) was added to a solution of 50a (890 mg,

2.17 mmol) in CH₂Cl₂(10 mL), and the reaction mixture was stirred

for 14 h at room temperature. The residue was stirred for 30 min in

Ethyl 3-{3-(2-Ethoxy-2-oxoethyl)-7-[(4-hydroxyphenyl)ethynyl]-2-

methyl-1H-indol-1-yl}propanoate (54a). Compound 54a was pre-

pared from compound 53a in a manner similar to that described for

1

compound 43a. H NMR (300 MHz, CDCl₃) δ 1.11 (t, J = 7.2 Hz,

3H), 1.26 (t, J = 7.2 Hz, 3H), 2.39 (s, 3H), 2.77−2.89 (m, 2H), 3.70

(s, 2H), 3.99 (q, J = 7.2 Hz, 2H), 4.15 (q, J = 7.2 Hz, 2H), 4.74−4.89

(m, 2H), 5.65−5.77 (m, 1H), 6.60 (d, J = 8.6 Hz, 2H), 7.04 (dd, J =

7.8, 1.0 Hz, 1H), 7.28−7.39 (m, 3H), 7.48 (dd, J = 7.8, 1.0 Hz, 1H).

Diethyl 3,3′-{7-[(4-Hydroxyphenyl)ethynyl]-2-methyl-1H-indole-

1,3-diyl}dipropanoate (54b). Compound 54b was prepared from

P

DOI: 10.1021/acs.jmedchem.5b00741

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

compound 53b in a manner similar to that described for compound

43a. ¹H NMR (300 MHz, CDCl₃) δ 1.12 (t, J = 7.2 Hz, 3H), 1.22 (t, J

= 7.2 Hz, 3H), 2.39 (s, 3H), 2.52−2.62 (m, 2H), 2.80−2.89 (m, 2H),

2.98−3.08 (m, 2H), 4.01 (q, J = 7.2 Hz, 2H), 4.11 (q, J = 7.2 Hz, 2H),

4.83−4.94 (m, 2H), 5.16 (s, 1H), 6.81 (d, J = 8.7 Hz, 2H), 7.04 (dd, J

= 7.8, 7.4 Hz, 1H), 7.32 (dd, J = 7.4, 1.1 Hz, 1H), 7.42 (d, J = 8.7 Hz,

2H), 7.48 (dd, J = 7.8, 1.1 Hz, 1H).

Ethyl 4-{1-(3-Ethoxy-3-oxopropyl)-7-[(4-hydroxyphenyl)ethynyl]-

2-methyl-1H-indol-3-yl}butanoate (54c). Compound 54c was

prepared from compound 53c in a manner similar to that described

for compound 43a. ¹H NMR (300 MHz, CDCl₃) δ 1.12 (t, J = 7.2 Hz,

3H), 1.24 (t, J = 7.2 Hz, 3H), 1.85−1.99 (m, 2H), 2.30 (t, J = 7.4 Hz,

2H), 2.36 (s, 3H), 2.74 (t, J = 7.4 Hz, 2H), 2.79−2.91 (m, 2H), 4.01

(q, J = 7.2 Hz, 2H), 4.10 (q, J = 7.2 Hz, 2H), 4.82−4.94 (m, 2H), 5.08

(s, 1H), 6.80 (d, J = 8.8 Hz, 2H), 7.02 (t, J = 7.8 Hz, 1H), 7.31 (dd, J =

7.4, 1.1 Hz, 1H), 7.41 (d, J = 8.8 Hz, 2H), 7.47 (dd, J = 7.8, 1.1 Hz,

1H).

3-[3-(Carboxymethyl)-7-({4-[4-(3-chloro-2-methylphenyl)-

butoxy]phenyl}ethynyl)-2-methyl-1H-indol-1-yl]propanoic Acid

(20). Compound 20 was prepared from compound 54a in a manner

similar to that described for compound 17. ¹H NMR (300 MHz,

DMSO-d₆) δ 1.56−1.85 (m, 4H), 2.31 (s, 3H), 2.37 (s, 3H), 2.63−

2.80 (m, 4H), 3.60 (s, 2H), 4.04 (t, J = 6.1 Hz, 2H), 4.69−4.86 (m,

2H), 6.95 (d, J = 8.6 Hz, 2H), 7.00 (t, J = 7.7 Hz, 1H), 7.08−7.18 (m,

2H), 7.21−7.30 (m, 2H), 7.41−7.56 (m, 3H), 12.30 (brs, 2H). MS

(FAB, neg) m/z 556 (M − H)⁻. HRMS (FAB, neg) C₃₃H₃₂ClNO₅(M

− H)⁻calcd mass 556.1891, found 556.1903. HPLC purity 95%

(method B).

J = 7.2 Hz, 2H), 6.89 (dd, J = 9.4, 8.5 Hz, 1H), 7.33 (dd, J = 8.5, 5.5

Hz, 1H), 7.90 (s, 1H).

Diethyl 4,4′-(7-Bromo-4-ﬂuoro-2-methyl-1H-indole-1,3-diyl)-

dibutanoate (58a). Compound 58a was prepared from compound

57a in a manner similar to that described for compound 42a. ¹H NMR

(300 MHz, CDCl₃) δ 1.21−1.29 (m, 6H), 1.81−2.11 (m, 4H), 2.31 (t,

J = 7.2 Hz, 2H), 2.34 (s, 3H), 2.39 (t, J = 7.2 Hz, 2H), 2.81 (t, J = 7.2

Hz, 2H), 4.05−4.17 (m, 4H), 4.49 (t, J = 7.8 Hz, 2H), 6.56 (dd, J =

10.2 Hz, 8.4 Hz, 1H), 7.14 (dd, J = 8.4, 4.8 Hz, 1H).

Diethyl 4,4′-(7-Bromo-5-ﬂuoro-2-methyl-1H-indole-1,3-diyl)-

dibutanoate (58b). Compound 58b was prepared from compound

57b in a manner similar to that described for compound 42a. ¹H NMR

(300 MHz, CDCl₃) δ 1.25 (t, J = 7.2 Hz, 3H), 1.26 (t, J = 7.2 Hz, 3H),

1.79−1.95 (m, 2H), 1.96−2.10 (m, 2H), 2.29 (t, J = 7.4 Hz, 2H), 2.35

(s, 3H), 2.38 (t, J = 7.4 Hz, 2H), 2.68 (t, J = 7.4 Hz, 2H), 4.05−4.20

(m, 4H), 4.40−4.53 (m, 2H), 7.01−7.14 (m, 2H).

Diethyl 4,4′-(7-Bromo-6-ﬂuoro-2-methyl-1H-indole-1,3-diyl)-

dibutanoate (58c). Compound 58c was prepared from compound

57c in a manner similar to that described for compound 42a. ¹H NMR

(300 MHz, CDCl₃) δ 1.18−1.33 (m, 6H), 1.81−1.95 (m, 2H), 1.97−

2.11 (m, 2H), 2.29 (t, J = 7.3 Hz, 2H), 2.34 (s, 3H), 2.40 (t, J = 7.2

Hz, 2H), 2.70 (t, J = 7.5 Hz, 2H), 4.10 (q, J = 7.2 Hz, 2H), 4.13 (q, J =

7.2 Hz, 2H), 4.42−4.54 (m, 2H), 6.88 (dd, J = 9.2, 8.5 Hz, 1H), 7.32

(dd, J = 8.5, 5.1 Hz, 1H).

Diethyl 4,4′-{4-Fluoro-7-[(4-hydroxyphenyl)ethynyl]-2-methyl-

1H-indole-1,3-diyl}dibutanoate (59a). Compound 59a was prepared

from compound 58a in a manner similar to that described for

1

compound 43a. H NMR (300 MHz, CDCl₃) δ 1.21 (t, J = 7.2 Hz,

3H), 1.23 (t, J = 7.2 Hz, 3H), 1.82−1.99 (m, 2H), 2.05−2.21 (m, 2H),

2.21−2.38 (m, 7H), 2.81 (t, J = 7.2 Hz, 2H), 4.04−4.13 (m, 4H), 4.61

(t, J = 7.5 Hz, 2H), 5.43 (s, 1H), 6.66 (dd, J = 10.8, 8.4 Hz, 1H), 6.82

(d, J = 8.4 Hz, 2H), 7.21 (dd, J = 8.1, 5.1 Hz, 1H), 7.40 (d, J = 8.4 Hz,

2H).

3,3′-[7-({4-[4-(3-Chloro-2-methylphenyl)butoxy]phenyl}ethynyl)-

2-methyl-1H-indole-1,3-diyl]dipropanoic Acid (21). Compound 21

was prepared from compound 54b in a manner similar to that

described for compound 17. ¹H NMR (300 MHz, DMSO-d₆) δ 1.56−

1.86 (m, 4H), 2.30 (s, 3H), 2.36 (s, 3H), 2.41 (t, J = 7.5 Hz, 2H),

2.62−2.75 (m, 4H), 2.89 (t, J = 7.5 Hz, 2H), 4.03 (t, J = 6.1 Hz, 2H),

4.66−4.85 (m, 2H), 6.89−7.04 (m, 3H), 7.07−7.17 (m, 2H), 7.20−

7.29 (m, 2H), 7.44−7.54 (m, 3H), 12.41 (brs, 2H). MS (FAB, neg)

m/z 570 (M − H)⁻. HRMS (FAB, neg) C₃₄H₃₄ClNO₅(M − H)⁻

calcd mass 570.2047, found 570.2039. HPLC purity 98% (method B).

4-[1-(2-Carboxyethyl)-7-({4-[4-(3-chloro-2-methylphenyl)-

butoxy]phenyl}ethynyl)-2-methyl-1H-indol-3-yl]butanoic Acid (22).

Compound 22 was prepared from compound 54c in a manner similar

to that described for compound 17. ¹H NMR (300 MHz, DMSO-d₆) δ

1.57−1.86 (m, 6H), 2.19 (t, J = 7.3 Hz, 2H), 2.30 (s, 3H), 2.35 (s,

3H), 2.59−2.77 (m, 6H), 4.04 (t, J = 6.4 Hz, 2H), 4.69−4.83 (m, 2H),

6.90−7.04 (m, 3H), 7.07−7.18 (m, 2H), 7.19−7.29 (m, 2H), 7.44−

7.54 (m, 3H), 12.30 (brs, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ

9.63, 15.49, 22.83, 25.78, 26.15, 28.26, 33.02, 33.10, 35.95, 67.33,

86.12, 91.73, 104.33, 110.85, 114.44, 114.73, 118.50, 118.61, 126.26,

126.63, 126.85, 127.85, 128.76, 132.55, 133.21, 133.26, 133.82, 134.04,

142.70, 158.73, 172.43, 174.49. MS (FAB, neg) m/z 584 (M − H)⁻.

HRMS (FAB, neg) C₃₅H₃₆ClNO₅(M − H)⁻calcd mass 584.2204,

found 584.2200. HPLC purity 99% (method A).

Ethyl 4-(7-Bromo-4-ﬂuoro-2-methyl-1H-indol-3-yl)butanoate

(57a). Compound 57a was prepared from compound 56a in a

manner similar to that described for compound 41b (procedure 2). ¹H

NMR (300 MHz, CDCl₃) δ 1.22 (t, J = 7.2 Hz, 3H), 1.86−2.03 (m,

2H), 2.32 (t, J = 7.4 Hz, 2H), 2.38 (s, 3H), 2.78 (t, J = 7.4 Hz, 2H),

4.08 (q, J = 7.2 Hz, 2H), 6.63 (dd, J = 10.7, 8.4 Hz, 1H), 7.11 (dd, J =

8.4, 4.2 Hz, 1H), 7.96 (s, 1H).

Ethyl 4-(7-Bromo-5-ﬂuoro-2-methyl-1H-indol-3-yl)butanoate

(57b). Compound 57b was prepared from compound 56b in a

manner similar to that described for compound 41b (procedure 2). ¹H

NMR (300 MHz, CDCl₃) δ 1.24 (t, J = 7.2 Hz, 3H), 1.84−1.99 (m,

2H), 2.30 (t, J = 7.3 Hz, 2H), 2.39 (s, 3H), 2.67 (t, J = 7.4 Hz, 2H),

4.11 (q, J = 7.2 Hz, 2H), 7.05 (dd, J = 8.9, 2.2 Hz, 1H), 7.11 (dd, J =

9.3, 2.2 Hz, 1H), 7.87 (s, 1H).

Diethyl 4,4′-{5-Fluoro-7-[(4-hydroxyphenyl)ethynyl]-2-methyl-

1H-indole-1,3-diyl}dibutanoate (59b). Compound 59b was prepared

from compound 58b in a manner similar to that described for

1

compound 43a. H NMR (300 MHz, CDCl₃) δ 1.23 (t, J = 7.2 Hz,

3H), 1.25 (t, J = 7.2 Hz, 3H), 1.82−1.97 (m, 2H), 2.07−2.20 (m, 2H),

2.26−2.34 (m, 4H), 2.35 (s, 3H), 2.70 (t, J = 7.4 Hz, 2H), 4.08 (q, J =

7.2 Hz, 2H), 4.12 (q, J = 7.2 Hz, 2H), 4.52−4.64 (m, 2H), 5.22−5.32

(m, 1H), 6.84 (d, J = 8.8 Hz, 2H), 7.04 (dd, J = 9.7, 2.6 Hz, 1H), 7.13

(dd, J = 9.2, 2.6 Hz, 1H), 7.43 (d, J = 8.8 Hz, 2H).

Diethyl 4,4′-{6-Fluoro-7-[(4-hydroxyphenyl)ethynyl]-2-methyl-

1H-indole-1,3-diyl}dibutanoate (59c). Compound 59c was prepared

from compound 58c in a manner similar to that described for

1

compound 43a. H NMR (300 MHz, CDCl₃) δ 1.15−1.33 (m, 6H),

1.82−1.99 (m, 2H), 2.06−2.22 (m, 2H), 2.23−2.40 (m, 7H), 2.72 (t, J

= 7.4 Hz, 2H), 4.09 (q, J = 7.2 Hz, 2H), 4.11 (q, J = 7.2 Hz, 2H),

4.50−4.66 (m, 2H), 5.40 (s, 1H), 6.77−6.93 (m, 3H), 7.37 (dd, J =

8.6, 5.3 Hz, 1H), 7.45 (d, J = 8.8 Hz, 2H).

4,4′-[7-({4-[4-(3-Chloro-2-methylphenyl)butoxy]phenyl}ethynyl)-

4-ﬂuoro-2-methyl-1H-indole-1,3-diyl]dibutanoic Acid (29). Com-

pound 29 was prepared from compound 59a in a manner similar to

1

that described for compound 17. H NMR (300 MHz, DMSO-d₆) δ

1.57−1.84 (m, 6H), 1.86−2.03 (m, 2H), 2.12−2.23 (m, 4H), 2.30 (s,

3H), 2.32 (s, 3H), 2.65−2.79 (m, 4H), 4.05 (t, J = 6.3 Hz, 2H), 4.55

(t, J = 7.7 Hz, 2H), 6.76 (dd, J = 10.8, 8.2 Hz, 1H), 6.99 (d, J = 9.0 Hz,

2H), 7.07−7.30 (m, 4H), 7.48 (d, J = 9.0 Hz, 2H), 12.08 (brs, 2H).

¹³C NMR (125 MHz, DMSO-d₆) δ 9.54, 15.49, 23.95, 26.14, 26.49,

28.25, 30.40, 32.83, 33.03, 42.70, 67.36, 85.49, 91.32, 101.31 (J_C−F

=

3.3 Hz), 104.52 (J_C−F= 20.6 Hz), 109.2 (J_C−F= 3.3 Hz), 114.31,

114.93, 116.46 (J_C−F= 19.1 Hz), 126.63, 126.85, 127.05 (J_C−F= 8.3

Hz), 127.85, 132.42, 133.27, 133.83, 134.87, 136.13 (J_C−F= 12.9 Hz),

142.70, 155.98 (J_C−F= 246.8 Hz), 158.85, 173.62, 174.30. MS (FAB,

neg) m/z 616 (M − H)⁻. HRMS (FAB, neg) C₃₆H₃₇ClFNO₅(M −

H)⁻calcd mass 616.2266, found 616.2272. HPLC purity 99%

(method A).

Ethyl 4-(7-Bromo-6-ﬂuoro-2-methyl-1H-indol-3-yl)butanoate

(57c). Compound 57c was prepared from compound 56c in a manner

4,4′-[7-({4-[4-(3-Chloro-2-methylphenyl)butoxy]phenyl}ethynyl)-

5-ﬂuoro-2-methyl-1H-indole-1,3-diyl]dibutanoic Acid (30). Com-

pound 30 was prepared from compound 59b in a manner similar to

1

similar to that described for compound 41b (procedure 2). H NMR

(300 MHz, CDCl₃) δ 1.23 (t, J = 7.2 Hz, 3H), 1.85−2.01 (m, 2H),

2.30 (t, J = 7.4 Hz, 2H), 2.38 (s, 3H), 2.70 (t, J = 7.4 Hz, 2H), 4.10 (q,

1

that described for compound 17. H NMR (300 MHz, DMSO-d₆) δ

Q

DOI: 10.1021/acs.jmedchem.5b00741

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1.58−1.85 (m, 6H), 1.86−2.01 (m, 2H), 2.11−2.24 (m, 4H), 2.30 (s,

3H), 2.33 (s, 3H), 2.59−2.76 (m, 4H), 4.05 (t, J = 6.2 Hz, 2H), 4.51

(t, J = 7.6 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H), 7.04 (dd, J = 9.9, 2.6 Hz,

1H), 7.08−7.18 (m, 2H), 7.25 (dd, J = 7.0, 2.4 Hz, 1H), 7.29 (dd, J =

9.3, 2.6 Hz, 1H), 7.50 (d, J = 8.8 Hz, 2H), 12.22 (s, 2 H). MS (FAB,

neg) m/z 616 (M − H)⁻. HRMS (FAB, neg) C₃₆H₃₇ClFNO₅(M −

H)⁻calcd mass 616.2266, found 616.2272. HPLC purity 98%

(method A).

Gemilukast, ONO-6950). Compound 35 was prepared from

compound 59a in a manner similar to that described for compound

17. ¹H NMR (300 MHz, DMSO-d₆) δ 1.64−1.71 (m, 2H), 1.74−1.83

(m, 4H), 1.93−2.00 (m 2H), 2.17−2.23 (m, 7H), 2.35 (s, 3H), 2.69 (t,

J = 7.5 Hz, 2H), 2.75 (t, J = 7.5 Hz, 2H), 4.07 (t, J = 6.0 Hz, 2H), 4.57

(t, J = 7.5 Hz, 2H), 6.78 (dd, J = 10.5, 8.5 Hz, 1H), 6.96−7.04 (m,

4H), 7.12−7.18 (m, 1H), 7.22 (dd, J = 8.5, 5.0 Hz, 1H), 7.50 (d, J =

9.0 Hz, 2H), 12.09 (brs, 2H). ¹³C NMR (DMSO-d₆) δ 9.58, 10.02 (J

= 5.5 Hz), 24.98, 26.13, 26.52, 28.30, 30.42, 31.98 (J

= 2.8

4,4′-[7-({4-[4-(3-Chloro-2-methylphenyl)butoxy]phenyl}ethynyl)-

6-ﬂuoro-2-methyl-1H-indole-1,3-diyl]dibutanoic Acid (31). Com-

pound 31 was prepared from compound 59c in a manner similar to

C−F

Hz), 32.84, 42.74, 67.39, 85.52, 91.34, 101.32 (J _C−F= 3.8 Hz), 104.56

(J = 21 Hz), 109.02, 112.37 (J = 23 Hz), 114.31, 114.95

C−F

1

that described for compound 17. H NMR (300 MHz, DMSO-d₆) δ

116.56 (J

= 20 Hz), 122.30 (J

= 16 Hz), 124.65 (J

= 2.8

C−F

1.57−1.85 (m, 6H), 1.88−2.01 (m, 2H), 2.14−2.25 (m, 4H), 2.30 (s,

3H), 2.31 (s, 3H), 2.59−2.76 (m, 4H), 4.05 (t, J = 6.2 Hz, 2H), 4.50

(t, J = 7.1 Hz, 2H), 6.94 (dd, J = 10.0, 8.7 Hz, 1H), 7.00 (d, J = 8.8 Hz,

2H), 7.07−7.19 (m, 2H), 7.26 (dd, J = 6.8, 2.2 Hz, 1H), 7.42−7.55

(m, 3H), 12.09 (s, 2H). MS (FAB, neg) m/z 616 (M − H)⁻. HRMS

(FAB, neg) C₃₆H₃₇ClFNO₅(M − H)⁻calcd mass 616.2266, found

616.2272. HPLC purity 98% (method A).

Hz), 126.72 (J _C−F= 9.1 Hz), 127.08 (J _C−F= 7.8 Hz), 132.47, 134.96

(J

= 0.9 Hz), 136.14 (J

= 13 Hz), 143.08 (J

= 4.1 Hz),

C−F

156.00 (J

= 246 Hz), 158.88, 160.72 (J

= 240 Hz), 173.69,

C−F

174.37. MS (FAB, neg) m/z 600 (M − H)⁻. HRMS (FAB, neg)

C₃₆H₃₇F₂NO₅(M − H)⁻calcd mass 600.2562, found 600.2562.

HPLC purity >99% (method A). Elemental analysis calculated (%) for

C₃₆H₃₇F₂NO₅: C 71.86, H 6.20, N 2.33. Found: C 71.88, H 6.19, N

2.25.

4,4′-[2-Methyl-7-({4-[4-(2,3,4,6-tetraﬂuorophenyl)butoxy]-

phenyl}ethynyl)-1H-indole-1,3-diyl]dibutanoic Acid (32). Com-

pound 32 was prepared from compound 48f in a manner similar to

In Vitro Assay. Method 1. Calcium mobilization assay with

compounds 1−32: The intracellular calcium mobilization response

was measured using the ﬂuorescent Ca²⁺indicator dye Fura 2-AM

(Dojindo). CHO-K1 cells stably expressing human CysLT₁, guinea pig

CysLT₁, or guinea pig CysLT₂receptors, and the HEK293 cells stably

expressing human CysLT₂receptor were used. Before the assay was

performed, these cells were incubated at 37 °C in 5% CO₂for at least

24 h with the following culture conditions: cells expressing human

CysLT₁receptor (4 × 10⁴cells/well) were cultured with Ham’s F-12

supplemented with 10% vol fetal bovine serum, 100 U/mL penicillin,

100 μg/mL streptomycin, and 600 μg/mL Geneticin. cells expressing

human CysLT₂receptor (10 × 10⁴cells/well) were cultured with

Dulbecco’s Modiﬁed Eagle’s Medium supplemented with 10% vol fetal

bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and

400 μg/mL geneticin. Cells expressing guinea pig CysLT₁or CysLT₂

receptors (3 × 10⁴cells/well) were cultured with Ham’s F-12K

supplemented with 10% vol fetal bovine serum, 100 U/mL penicillin,

100 μg/mL streptomycin, and 500 μg/mL geneticin. After removing

the culture medium, loading medium containing 7.5 μM Fura 2-AM,

20 mmol/L HEPES, and 2.5 mmol/L probenecid was added to each

well. After a 1 h incubation, loading medium was removed and the

cells were incubated at room temperature for 1 h in the assay buﬀer

(Hank’s Balanced Salt Solution containing 20 mM HEPES). The cells

were alternately irradiated at two excitation wavelengths (340 and 380

nm) using FDSS-2000 (Hamamatsu Photonics Co., Ltd., Shizuoka,

Japan) to measure the ratio of ﬂuorescence intensities (f340/f380) at

500 nm. To evaluate the antagonism of the compounds, they were

added 30 min before the addition of the ligand: 100, 100, 10, or 100

nM LTD₄for human CysLT₁, human CysLT₂, guinea pig CysLT₁or

guinea pig CysLT₂receptors, respectively.

Method 2. Calcium mobilization assay of compound 35

(gemilukast): The intracellular calcium response was measured using

Fura 2-AM (Dojindo). Brieﬂy, CHO-K1 cells stably expressing human

or guinea pig CysLT₁or CysLT₂receptors were cultured for at least 24

h (3 × 10⁴cells/well). After removing the culture medium (Ham’s F-

12 supplemented with 10% fetal bovine serum and 500 μg/mL

geneticin), loading medium containing 5 μM Fura 2-AM was added to

each well. After a 1 h incubation, the loading medium was removed

and the cells were incubated at room temperature for 1 h in the assay

buﬀer (Hanks’ Balanced Salt Solution containing 20 mM HEPES).

The cells were alternately irradiated at two excitation wavelengths (340

and 380 nm) using FDSS-3000 (Hamamatsu Photonics Co., Ltd.,

Shizuoka, Japan) to measure the ratio of ﬂuorescence intensities

(f340/f380) at 500 nm. To evaluate the antagonism of the

compounds, they were added 30 min before the addition of the

ligand: 100 nM LTD₄for human CysLT₁receptor, 0.3 nM LTD₄for

human CysLT₂receptor, 10 nM LTD₄for guinea pig CysLT₁receptor,

or 3 nM LTC₄for guinea pig CysLT₂receptor.

1

that described for compound 16. H NMR (300 MHz, DMSO-d₆) δ

1.60−1.82 (m, 6 H) 1.86−2.03 (m, 2 H) 2.13−2.25 (m, 4 H) 2.34 (s,

3 H) 2.60−2.78 (m, 4 H) 4.02 (t, J = 5.8 Hz, 2 H) 4.54 (t, J = 7.1 Hz,

2 H) 6.91−7.04 (m, 3 H) 7.21 (d, J = 7.3 Hz, 1 H) 7.38−7.54 (m, 4

H) 12.07 (brs, 2 H). ¹³C NMR (125 MHz, DMSO-d₆) δ 9.74, 21.43,

22.82, 25.23, 25.74, 26.60, 27.98, 30.47, 33.00, 42.56, 67.12, 86.39,

91.61, 101.33 (m), 104.34, 110.76, 114.49, 114.77, 114.91 (m), 118.40,

118.69, 126.21, 128.66, 132.41, 133.47, 134.08, 136.38 (m), 146.67

(m), 148.93 (m), 154.98, 158.74, 173.69, 174.37. MS (FAB, neg) m/z

622 (M − H)⁻. HRMS (FAB, neg) C₃₅H₃₃F₄NO₅(M − H)⁻calcd

mass 622.2217, found 622.2215. HPLC purity 98% (method A).

4,4′-[4-Fluoro-2-methyl-7-({4-[4-(2,3,4,6-tetraﬂuorophenyl)-

butoxy]phenyl}ethynyl)-1H-indole-1,3-diyl]dibutanoic Acid (33).

Compound 33 was prepared from compound 59a in a manner similar

to that described for compound 16. ¹H NMR (300 MHz, DMSO-d₆) δ

1.57−1.83 (m, 6H), 1.86−2.03 (m, 2H), 2.12−2.24 (m, 4H), 2.32 (s,

3H), 2.60−2.78 (m, 4H), 3.99 (t, J = 5.8 Hz, 2H), 4.53 (t, J = 7.3 Hz,

2H), 6.73 (dd, J = 10.7, 8.3 Hz, 1H), 6.94 (d, J = 8.8 Hz, 2H), 7.18

(dd, J = 8.3, 5.1 Hz, 1H), 7.34−7.49 (m, 1H), 7.45 (d, J = 8.8 Hz, 2H),

12.07 (s, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 9.68, 21.54, 24.08,

25.35, 26.59, 28.08, 30.48, 32.98, 42.74, 67.04, 85.38, 91.11, 101.09

(m), 101.13, 104.42 (d, J

= 20.3 Hz), 108.80 (d, J _C−F= 3.0 Hz),

C−F

114.13, 114.61, 114.50 (m), 116.21 (d, J _C−F= 19.5 Hz), 126.75 (d, J

_C−F= 8.3 Hz), 132.09, 134.53, 135.88 (d, J _C−F= 8.3 Hz), 136.12 (m),

147.81 (m), 148.59 (m), 154.59 (m), 155.60 (m), 158.35, 173.20,

173.88. MS (FAB, neg) m/z 640 (M − H)⁻. HRMS (FAB, neg)

C₃₅H₃₂F₅NO₅(M − H)⁻calcd mass 640.2122, found 640.2101.

HPLC purity 99% (method A). Elemental analysis calculated (%) for

C₃₅H₃₂F₅NO₅: C 65.52, H 5.03, N 2.18. Found: C 65.42, H 5.19, N

2.03.

4,4′-[7-({4-[4-(3-Fluoro-2-methylphenyl)butoxy]phenyl}ethynyl)-

2-methyl-1H-indole-1,3-diyl]dibutanoic Acid (34). Compound 34

was prepared from compound 48f in a manner similar to that

described for compound 17. ¹H NMR (300 MHz, DMSO-d₆) δ 1.59−

1.85 (m, 6H), 1.88−2.02 (m, 2H), 2.13−2.24 (m, 7H), 2.33 (s, 3H),

2.67 (t, J = 7.4 Hz, 4H), 4.05 (t, J = 6.3 Hz, 2H), 4.54 (t, J = 7.4 Hz,

2H), 6.91−7.03 (m, 5H), 7.08−7.17 (m, 1 H), 7.21 (d, J = 6.6 Hz,

1H), 7.43−7.52 (m, 3H), 12.07 (brs, 2H). ¹³C NMR (75 MHz,

DMSO-d₆) δ 10.82, 11.08, 23.85, 26.78, 27.11, 27.64, 29.29, 31.47,

32.96, 33.98, 43.52, 68.19, 87.19, 92.39, 105.07, 111.44, 113.00 (d, J

_C−F= 22.5 Hz), 115.12, 115.56, 119.23 (d, J _C−F= 21.8 Hz), 122.92 (d,

J _C−F= 14.4 Hz), 125.22 (d, J _C−F= 3.0 Hz), 126.85, 127.30 (d, J _C−F

=

9.0 Hz), 129.29, 133.03, 134.06, 134.68, 143.60 (d, J = 3.8 Hz),

C−F

159.33, 159.61, 162.79, 174.21, 174.89. MS (FAB, neg) m/z 582 (M −

H)⁻. HRMS (FAB, neg) C₃₆H₃₈FNO₅(M − H)⁻calcd mass

582.2656, found 582.2661. HPLC purity >99% (method A).

Parallel Artiﬁcial Membrane Permeability Assay (PAMPA).

The PAMPA System (Pion) was used. The System Solution

4,4′-[4-Fluoro-7-(2-{4-[4-(3-ﬂuoro-2-methylphenyl)butoxy]-

phenyl}ethynyl)-2-methyl-1H-indole-1,3-diyl]dibutanoic Acid (35:

R

DOI: 10.1021/acs.jmedchem.5b00741

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Concentrate (Pion) was diluted with water, and DMSO was added to

prepare a system solution (in 5% DMSO). NaOH at 0.5 mol/L was

added using an automated buﬀer preparation system to make three

diﬀerent buﬀers with pH values of 5.0, 6.2, and 7.4. A volume of 150

μL of each buﬀer was added per well in a UV plate to serve as blanks

for UV absorption (190−498 nm). Next, 5 μL of DMSO solution

containing the test compounds at 10 mM was added to 1000 μL of the

system solution, and this was ﬁltered by suction through a 96-well ﬁlter

plate (0.2 μm PVDF hydrophilic membrane, Corning) (ﬁnal

compound concentration of 50 μM). A volume of 150 μL of the

ﬁltrate was added per well in the UV plate to serve as the reference for

UV absorption (190−498 nm). Then, 200 μL of the remaining ﬁltrate

was added per well in the donor plate and used as the donor solution.

A membrane was prepared by dropping 4 μL of oil (GIT-0 Liquid,

Pion) onto the ﬁlter of the acceptor plate. A 200 μL aliquot of the

acceptor solution (Acceptor Sink Buﬀer, pH 7.4, Pion) was added to

each well in the acceptor plate. The acceptor plate was placed on the

donor plate. After a 4 h incubation, 150 μL aliquots were transferred

from the donor and acceptor solutions to the UV plate to measure UV

absorption (190−498 nm). The membrane permeability coeﬃcient

was calculated from the observed values obtained using the PAMPA

Evolution Command software.

Pharmacokinetic Analysis in Rat, Guinea Pig, and Dog. The

pharmacokinetics of compounds were studied following single

intravenous and oral administration. The iv and po solution

formulations contained wellsolve/H₂O = 1/9 (Celeste) and sodium

hydroxide (2 equiv). All iv and po formulations were given as

solutions. Animals were fasted prior to oral dosing in single-dose

studies. Oral bioavailability was estimated using noncrossover study

designs for the rat, guinea pig, and dog (n = 3). Plasma samples were

assayed for compounds using protein precipitation by acetonitrile/

ethanol (70/30, v/v), followed by HPLC/MS/MS analysis employing

negative-ion Turbo IonSpray ionization. Plasma concentration−time

data were analyzed by noncompartmental methods.

Pharmacokinetic Analysis in Guinea Pig Cassette Dosing.

The pharmacokinetics of compounds were studied following single

intravenous and oral administration. The iv and po solution

formulations contained wellsolve/H₂O = 5/95 (Celeste) and sodium

hydroxide (1.2 equiv). All iv and po formulations were given as

cassette dosing solutions containing ﬁve compounds. Animals were

fasted prior to oral dosing in single-dose studies. Oral bioavailability

was estimated using noncrossover study designs for the guinea pig (n =

3). Plasma samples were assayed for compounds using protein

precipitation by acetonitrile/ethanol (70/30, v/v), followed by HPLC/

MS/MS analysis employing negative-ion Turbo IonSpray ionization.

Plasma concentration−time data were analyzed by noncompartmental

methods.

Measurement of the Concentration in Lung. The isolated

lungs were homogenized in saline. The resulting lung homogenate (0.5

mL) was incubated in 2 mol/L NaOH (0.5 mL) at 60 °C for 3 h, and

then diluted 50-fold with PBS. The amount of protein in the sample

was spectrophotometrically determined using the BCA Protein Assay

Kit (Thermo Scientiﬁc). Meanwhile, the amount of compound was

measured by HPLC/MS/MS analysis employing negative-ion Turbo

InoSpray ionization after protein precipitation with ethanol. The

concentrations of compounds in lung tissues were expressed in ng/g

protein.

Eﬀects of Compounds on LTD₄and LTC₄-Induced Broncho-

constriction in Guinea Pigs. Ventilation pressure was measured

using the method of Konzett and Rossler. Brieﬂy, guinea pigs were

̈

anesthetized with intraperitoneal pentobarbital sodium (75 mg/kg). A

polyethylene cannula was inserted into the trachea. The other end of

the cannula was connected to a volume-limited ventilator (model SN-

480-7, Shinano Manufacturing Co., Ltd., Tokyo, Japan), and artiﬁcial

ventilation was provided at a supply rate of 5 mL/stroke and 70

strokes/min. Another catheter was inserted into the jugular vein,

securing the route for administration. The ventilation pressure was

measured via a pneumotachometer (M·I·P·S Co., Ltd., Osaka, Japan)

connected to a lateral port of the tracheal cannula, using a Win-

PULMOS-III system (version 3.6, M·I·P·S Co., Ltd., Osaka, Japan).

After the basal ventilation pressure was conﬁrmed to be stabilized,

LTD₄(0.25 μg/kg) or LTC₄(15 μg/kg) was intravenously

administered via the catheter secured in the jugular vein. In the

experiment in which LTC₄-induced bronchoconstriction was exam-

ined, S-hexyl GSH (30 mg/kg) was intravenously administered 10 s

prior to LTC₄(15 μg/kg) injection. The ventilation pressure was

measured for at least 10 min after LTD₄or 30 min after LTC₄

injection, and then the trachea was completely blocked to obtain the

maximum ventilation pressure. After measurement of the maximum

ventilation pressure, artiﬁcial ventilation was stopped and the animal

was euthanized. The area under the percentage bronchoconstriction

curve (AUC) from 0 to 10 min after LTD₄injection or from 0 to 30

min after LTC₄injection was calculated.

Antigen-Induced Contraction in Isolated Human Bronchial

Strips. As described in the previous report,⁴¹bronchi (outer diameter

2−6 mm) were isolated from macroscopic normal portions of the lung

tissue and cut into 1.5 mm wide spirals. In the experiments to evaluate

of the eﬀects of CysLT antagonists on antigen-induced contraction,

the bronchi were passively sensitized by incubation in human atopic

serum for 2 h at 37 °C. Bronchial strips (2 cm long, outer diameter 2−

4 mm) were then cut and suspended under an isotonic resting tension

of 300 mg at 37 °C in a Magnus bath containing Tyrode’s solution

gassed with 95% O₂−5% CO₂. Before starting the experiments,

acetylcholine (5 μM) and then histamine (10 μM) were repeatedly

applied to the preparations until almost equal contractions were

obtained. When the resting tonus of the histamine-treated smooth

muscle stabilized, guinea pig serum albumin at a ﬁnal concentration of

0.1 mg/mL was added to prevent adsorption of ONO-6950 and

montelukast to the inner wall of the Magnus bath. Immediately after

the addition of guinea pig serum albumin, ONO-6950 (100 nM),

BayCysLT₂RA (10 nM), montelukast (10 nM), or DMSO (ﬁnal

concentration: 0.1%) were applied, and 30−35 min after the addition,

antigen challenge (mite extract from Dermatophagoides farinae, 3 μg/

mL) was performed. As previously reported,^42,43a cyclooxygenase

inhibitor, indomethacin (3 μM), and an antihistamine drug, pyrilamine

(1 μM), were applied 10 and 5 min, respectively, before antigen

challenge. Data were expressed as the % of 10 μM histamine-induced

contraction. Each compound was added at 30 min before the antigen

challenge.

ASSOCIATED CONTENT

* Supporting Information

■

S

This information includes the experimental details for data

collection, data reduction, and reﬁnement statistics of the X-ray

crystallography of 35 (PDF). Molecular formula strings

(XLSX). The Supporting Information is available free of charge

on the ACS Publications website at DOI: 10.1021/

acs.jmedchem.5b00741.

In Vitro Hepatocyte Metabolic Stability. The metabolic

stability of test compounds (1 μM test concentration) was determined

in human cryopreserved hepatocytes (Celsis In Vitro Technologies,

Baltimore, USA, pooled donors). Incubations (ﬁnal incubation volume

= 800 μL) were performed at 37 °C under an atmosphere of 95%/5%

CO₂/O₂using 1 million cells mL⁻¹in hepatocyte incubation media (In

Vitro Technologies, Baltimore, MD, USA). Then, 150 μL aliquots

(cells and media) were taken at zero, 0.5, 1, and 2 h after the start of

the incubation and quenched using 300 μL of acetonitrile/ethanol (7/

3). The resulting samples were centrifuged at 3000g for 10 min. An

aliquot (10 μL) of the supernatant was directly injected onto the LC−

MS/MS for analysis. All incubations were performed in duplicate.

AUTHOR INFORMATION

Corresponding Authors

■

*For S.I.; phone, +81-75-961-1151; fax, +81-75-962-9314; E-

mail, itadani@ono.co.jp.

*For K.O.; phone, +81-75-961-1151; fax, +81-75-962-9314; E-

mail, k.ohmoto@ono.co.jp.

S

DOI: 10.1021/acs.jmedchem.5b00741

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(12) Mellor, E. A.; Frank, N.; Soler, D.; Hodge, M. R.; Lora, J. M.;

Austen, K. F.; Boyce, J. A. Expression of the type 2 receptor for

cysteinyl leukotrienes (CysLT2R) by human mast cells: Functional

distinction from CysLT1R. Proc. Natl. Acad. Sci. U. S. A. 2003, 100,

11589−11593.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank Ms. Tomoko Hirota for the LC/MS and HPLC

measurement and Dr. Rie Omi for X-ray crystallographic

analysis.

(13) Gauvreau, G. M.; Plitt, J. R.; Baatjes, A.; MacGlashan, D. W.

Expression of functional cysteinyl leukotriene receptors by human

basophils. J. Allergy Clin. Immunol. 2005, 116, 80−87.

(14) Sjostrom, M.; Johansson, A.-S.; Schroder, O.; Qiu, H.; Palmblad,

̈

ABBREVIATIONS USED

J.; Haeggstrom, J. Z. Dominant expression of the CysLT receptor

̈

2

■

accounts for calcium signaling by cysteinyl leukotrienes in human

umbilical vein endothelial cells. Arterioscler., Thromb., Vasc. Biol. 2003,

23, 37e−41e.

CysLTs, cysteinyl leukotrienes; hCysLT₁, human CysLT₁;

hCysLT₂, human CysLT2; gCysLT1, guinea pig CysLT1;

gCysLT2, guinea pig CysLT2; LTC₄, leukotriene C4; LTD4,

leukotriene D₄; LTE₄, leukotriene E₄

(15) Montuschi, P. Role of leukotrienes and leukotriene modifiers in

asthma. Pharmaceuticals 2010, 3, 1792−1811.

(16) Dahlen, S. E. Treatment of asthma with antileukotrienes: First

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line or last resort therapy? Eur. J. Pharmacol. 2006, 533, 40−56.

(17) Kumlin, M. Measurement of leukotrienes in humans. Am. J.

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Discovery of Gemilukast (ONO-6950), a Dual CysLT1 and CysLT2 Antagonist As a Therapeutic Agent for Asthma

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