2-Acylamino-4,6-diphenylpyridine derivatives as novel GPR54 antagonists with good brain exposure and in vivo efficacy for plasma LH level in male rats

Bioorganic & Medicinal Chemistry 18 (2010) 5157–5171

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Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

2-Acylamino-4,6-diphenylpyridine derivatives as novel GPR54 antagonists

with good brain exposure and in vivo efﬁcacy for plasma LH level in male rats

a

b

a

Toshitake Kobayashi ^a,, Satoshi Sasaki , Naoki Tomita , Seiji Fukui , Masaharu Nakayama , Atsushi Kiba ,

*

Masami Kusaka ^a, Shin-ichi Matsumoto ^b, Masashi Yamaguchi ^b, Fumio Itoh ^b, Atsuo Baba ^a

^aPharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd, 10 Wadai, Tsukuba-shi, Ibaraki 300-4293, Japan

^bPharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd, 17-85, Jusohonmachi 2-Chome, Yodogawa-ku, Osaka 532-8686, Japan

a r t i c l e i n f o

a b s t r a c t

Article history:

GPR54 is a G protein-coupled receptor (GPCR) which was formerly an orphan receptor. Recent functional

study of GPR54 revealed that the receptor plays an essential role to modulate sex-hormones including

GnRH. Thus, antagonists of GPR54 are expected to be novel drugs for sex-hormone dependent diseases

such as prostate cancer or endometriosis. We recently reported 2-acylamino-4,6-diphenylpyridines as

the ﬁrst small molecule GPR54 antagonists with high potency. However, the representative compound

1 showed low brain exposure, where GPR54 acts as a modulator of gonadotropins by binding with its

endogenous ligand, metastin. In order to discover compounds that have not only potent GPR54 antago-

nistic activity but also good brain permeability, we focused on converting the primary amine on the side

chain to a secondary or tertiary amine, and ﬁnally we identiﬁed 15a containing a piperazine group. This

compound exhibited high afﬁnity to human and rat GPR54, apparent antagonistic activity, and high brain

exposure. In addition, intravenous administration of 15a to castrated male rat suppressed plasma LH

level, which indicates the possibility of a small molecule GPR54 antagonist as a novel drug for sex-hor-

mone dependent diseases.

Received 26 April 2010

Revised 21 May 2010

Accepted 22 May 2010

Available online 1 June 2010

Keywords:

GPR54 antagonist

2-Acylamino-4,6-diphenylpyridine

Metastin

Kisspeptin

1. Introduction

such as follicle stimulating hormone (FSH) and luteinizing hor-

mone (LH) from the pituitary.

GPR54 (OT7T175, AXOR12) is a G protein-coupled receptor

(GPCR) that is highly expressed in brain, including hypothalamus

and pituitary as well as peripheral regions.¹GPR54 was identiﬁed

as an orphan receptor in rat in 1999,²and later Ohtaki et al. discov-

ered that a 54-amino-acid product of a gene called Kiss-1 was its

endogenous ligand.³As the Kiss-1 gene was originally isolated as

a tumor metastasis gene, the peptide product was named ‘meta-

stin’. Later others also isolated the same peptide and named it

‘kisspeptin’.⁴

After much study to understand the function of GPR54, several

ﬁndings were reported that suggest GPR54 plays an important role

in reproduction and pubertal development. The phenotype of

GPR54-null mice was consistent with lack of steroid sex-hormone

production.^5aIn the mutant male mice, the serum testosterone le-

vel was similar to that found in normal females. In the case of the

mutant females, they did not show the rise in estradiol normally

found during estrus. Moreover, mutations of GPR54 were found

in patients with idiopathic hypogonadoropic hypogonadism

(IHH).^5a,6Individuals with IHH fail to undergo puberty and are

infertile because of failure to secrete the gonadotropic hormones,

In a recent study, GPR54-metastin signaling was revealed to be

essential to initiate gonadotropin secretion.⁷Central or peripheral

administration of metastin stimulates a dose-dependent rise in

serum levels of LH and FSH in adult rat,^7,8mice,⁹sheep, macaques,

and humans,¹⁰while metastin stimulation causes no effect on gon-

adotropin secretion in mice lacking a functional GPR54 gene.⁵

Moreover, the effect is blocked by pretreatment with a GnRH

antagonist,^7,9,10b,11therefore, GPR54-metastin signaling is sup-

posed to be up stream of GnRH–GnRH receptor signaling. Further-

more, GPR54 mRNA expression was observed in GnRH neurons of

rats and mice, suggesting GPR54 may act directly on GnRH neu-

rons.^10a,11,12Thus, GPR54 is expected to play a key role on hypo-

thalamus-pituitary-gonadal (HPG) axis. These facts suggest that

small molecule GPR54 antagonists may suppress the release of

gonadotropic hormones and such compounds would be novel

drugs for sex-hormone dependent diseases, including prostate can-

cer and endometriosis.

We have previously reported 2-acylamino-4,6-diphenylpyri-

dine derivatives as the ﬁrst small molecule GPR54 antagonists.¹³

By a structure–activity relationship (SAR) study, we revealed that

the 2-acylamino-4,6-diphenylpyridine derivatives bearing a pri-

mary amine on their side chain showed high antagonistic activities

for GPR54. We then conducted in vivo evaluation using compounds

* Corresponding author. Tel.: +81 29 864 6465; fax: +81 29 864 6308.

E-mail address: Kobayashi_Toshitake@takeda.co.jp (T. Kobayashi).

doi:10.1016/j.bmc.2010.05.061

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T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 5157–5171

including 1, which has potent binding activity (Table 1, hGPR54

IC₅₀= 7.4 nM), however, those compounds did not show in vivo

efﬁcacy. After detailed investigation, exposure of these compounds

in brain was revealed to be unsatisfactory, and compounds with a

primary amine moiety appeared to be the substrate of P-glycopro-

tein (P-gp) transporters by Caco-2 permeability test. For example,

the ratio of basal-to-apical (B-to-A) versus apical-to-basal (A-to-

B) ﬂux of compound 1 was 49, and these compounds were sup-

posed to have low blood–brain-barrier (BBB) penetration. As men-

tioned above, metastin acts on GPR54 in the brain to modulate

gonadotropins, therefore these compounds were thought to be

unsuitable for in vivo evaluation. We also found that modiﬁcation

of the primary amine at the side chain to secondary or tertiary

amines improves Caco-2 permeability as well as enhances lipophil-

icity, though in vitro GPR54 antagonistic potency declined. For

example, compound 2, which possesses a dimethylamino moiety,

showed good Caco-2 permeability and high concentration in rat

brain in a rat cassette dosing test, while the afﬁnity for GPR54

was deteriorated (Table 1).

Based on this information, we focused on modifying the pri-

mary amine moiety to secondary or tertiary amines to identify

compounds with good brain permeability as well as potent

GPR54 antagonistic activity (Fig. 1). Other parts of the compounds

were ﬁxed to the substituents of compound 3,¹³which has the

most potent afﬁnity for GPR54 (hGPR54 IC₅₀= 3.7 nM) in our pre-

vious study; the 2-position of the pyridine ring is a 2-furoylamine

and the 6-position is a 4-ﬂuoro-2-hydroxybenzene.

2. Chemistry

The synthesis of pyridine derivatives 6a–i and 7a–j, which pos-

sess a secondary or tertiary amine on their side chain is shown in

Scheme 1. Compounds 5a–j were synthesized by condensation of

the known aniline 4¹³and tert-butoxycarbonyl(Boc)-protected

amino acids. Deprotection of Boc and methoxymethyl (MOM)

groups of 5a–j with HCl gave 3 and 6a–i. Amines 3 and 6d–g were

converted to 7a–j under the conditions of reductive alkylation

using NaBH₃(CN). In the case of 6f, a large amount of acetaldehyde

(20 equiv) was needed to afford the diethylated compound 7i.

Alkylation of amine 6e with a large excess of acetone gave only

the monoalkylated compound 7e.

Compounds 10a–c having N,N-dialkylated glycine moiety on

their side chain were prepared as Scheme 2. Aniline 4 was treated

with chloroacetyl chloride to give compound 8. Amination of com-

pound 8 afforded 9a–c, followed by deprotection of MOM group

with HCl to produce 10a–c.

The synthesis of pyridine derivatives 15a–d bearing a pipera-

zine or homopiperazine moiety on the 4-phenyl group is shown

in Scheme 3. The four component condensation reaction of ketone

11,¹³3-bromobenzaldehyde, malononitrile, and ammonium ace-

tate constructed the pyridine ring to give compound 12. Acylation

of the amine at the 2-position of the pyridine ring with furoyl chlo-

ride gave the diacylated compound, which was converted to the

monoacylated compound 13 by treatment with basic silica gel.

The coupling reaction of 13 with piperazine or homopiperazine

derivatives, catalyzed by Pd complex, afforded compounds

14a–d. Treatment with HCl gave deprotected compounds 15a–d.

Compounds 21a and 21b, with a methylene linker inserted be-

tween a piperazine or homopiperazine ring and the phenyl ring,

were synthesized as described in Scheme 4. The reduction of the

methyl ester 16¹³afforded alcohol 17. After mesylation of the hy-

droxy group of 17, displacement with a Boc-protected piperazine

or homopiperazine was conducted to give compounds 19a and

19b. Treatment with TFA or HCl to remove the protecting groups

afforded compounds 20a and 20b. For compound 20a, reductive

alkylation yielded N-methyl (21a) and N-ethyl (21b) derivatives.

The synthesis of the inverse-amide type compound 24 is

shown in Scheme 5. Carboxylic acid 22¹³was condensed with

N,N-dimethylethylenediamine to give amide 23, followed by

deprotection of MOM group with HCl to afford amine 24.

Conversion of the amide linker between the phenyl ring and

amino group to ethylene is shown in Scheme 6. Reductive alkyl-

ation of 4 with N-Boc-2-aminoacetaldehyde gave 25, followed by

treatment with HCl to afford amine 26. Subsequent methylation

of the amino group provided 27.

Herein, we report the SAR studies of 2-acylamino-4,6-diphenyl-

pyridine derivatives with secondary or tertiary amines on the side

chains, their brain exposure, and the efﬁcacy of in vivo examination.

Table 1

Receptor binding assay for human GPR54, Caco-2 permeability, log D, and plasma and

brain exposure data of compounds 1 and 2

Compound

1

2

hGPR54 RBA

Caco-2 permeability

Log D at pH 7.4

IC₅₀(nM)

B-to-A/A-to-B

2.14 3.07

2.2 39.2

Plasma (ng/ml) 13.4 29.6

Kp 0.16 1.32

7.4

49

180

1

iv rat cassette dosing test (0.2 mpk, 30 min) Brain (ng/g)

3. Results and discussion

RBA = receptor binding assay.

Kp = the brain-to-plasma concentration ratio.

All compounds synthesized were initially evaluated by their

binding afﬁnities for human GPR54 against ¹²⁵I-labeled human

metastin (40–54) in a receptor binding assay.

Figure 1. The strategy of identiﬁcation of compounds with good brain permeability as well as potent GPR54 binding activity.

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 5157–5171

5159

Scheme 1. Synthesis of compounds with a secondary or tertiary amine on their side chain. Reagents and conditions: (a) carboxylic acid, WSCD, HOBt, DMF, rt; (b) 4 M HCl

EtOAc solution, EtOAc, rt; (c) (i) aldehyde or ketone, NaBH₃(CN), AcOH, NEt₃, MeOH, rt; (ii) 4 M HCl EtOAc solution, EtOAc, rt.

Scheme 2. Synthesis of compounds with a glycine amide as their side chain. Reagents and conditions: (a) chloroacetyl chloride, NEtⁱPr₂, THF, 0 °C; (b) amine, K₂CO₃, DMF, rt;

(c) 4 M HCl EtOAc solution, MeOH, rt.

The result for compounds possessing an alkylated b-alanine

amide moiety is shown in Table 2. Monomethylamine derivative

6d showed comparable activity to primary amine 3, however, the

afﬁnities of dimethylamine derivative 7a and other tertiary amine

derivatives (7b, 7c) were weakened. These results reconﬁrmed that

the amine group at the end of the side chain is a critical component

for activity. Next, we introduced a methyl or ethyl group onto the

7c, 7g, and 7i, introduction of small alkyl groups as R is allowed.

Furthermore, the afﬁnity relationship between secondary amines

and tertiary amines was retained. When R was methyl, secondary

amines 7d and 7e were more potent than the tertiary amines 7f

and 7g, and when R was ethyl, the tendency appeared more clearly

(7h vs 7i).

As introduction of an alkyl group at the a-position of the amino

a-position of the amino group to investigate the sterical tolerance

group was allowed, cyclic amine derivatives 6g–i and 7j were

around the amine moiety. Comparing the potency of compounds

synthesized to evaluate the effect of ﬁxing the amine moiety.

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T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 5157–5171

Scheme 3. Synthesis of compounds bearing a piperazine or homopiperazine ring. Reagents and conditions: (a) 3-bromobenzaldehyde, NH₄OAc, malononitrile, toluene,

reﬂux; (b) 2-furoyl chloride, pyridine, 0 °C then puriﬁcation by basic silica gel; (c) amine, Pd₂(dba)₃, XPhos, NaO^tBu, toluene, reﬂux; (d) 4 M HCl EtOAc solution, MeOH, rt.

Scheme 4. Synthesis of compounds possessing a methylene linker between a piperazine or piperazine ring and the phenyl ring. Reagents and conditions: (a) LiBH₄, THF,

45 °C; (b) MsCl, NEt₃, CH₂Cl₂, rt; (c) amine, NEt₃, THF, rt; (d) TFA, CH₂Cl₂, rt for 19a; (e) 4 M HCl EtOAc solution, MeOH, rt for 19b; (f) (i) aldehyde, NaBH₃(CN), AcOH, Et₃N,

MeOH, rt; (ii) 4 M HCl EtOAc solution, EtOAc, rt.

Scheme 5. Synthesis of inverse-amide type compound 24. Reagents and conditions: (a) N,N-dimethylethylenediamine, WSCD, HOBt, DMF, rt; (b) 4 M HCl EtOAc solution,

MeOH, rt.

Compound 7j showed similar afﬁnity to the linear analogue 7f. Sec-

ondary amines 6g and 6h increased activity compared to tertiary

amine 7j, and the (S)-isomer 6g was more potent than the (R)-iso-

mer 6h. Compound 6i, which has a piperidine instead of the pyrrol-

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 5157–5171

5161

Scheme 6. Synthesis of compound 27 having an ethylene linker between the phenyl ring and amino group. Reagents and conditions: (a) N-Boc-2-aminoacetaldehyde,

NaBH(OAc)₃, AcOH, THF, rt; (b) 4 M HCl EtOAc solution, EtOAc, rt; (c) formaldehyde, NaBH₃(CN), AcOH, MeOH, rt.

Table 2

The afﬁnities of compounds possessing an alkylated glycinyl

group, for human type GPR54, are described in Table 3. The ten-

dency of the afﬁnity was similar to that of compounds with an

alkylated b-alaninyl moiety. The primary amine 6a was the most

potent, secondary amine 6b slightly reduced the afﬁnity, and ter-

tiary amine 10a was the weakest. The diethylamine 10b had re-

duced afﬁnity compared with the dimethyl amine 10a, while the

cyclic amine derivative 10c retained afﬁnity. As the proline deriv-

ative 6c resulted in a twofold decrease of afﬁnity compared to 6b,

ﬁxing the linker was not preferable, unlike for the b-alaninyl

analogue.

The binding afﬁnities of b-alanine amide derivatives for human type GPR54

Compound

R

R¹

R²

hGPR54 RBA IC₅₀(nM)

Reducing hydrogen bonds, especially proton donors, and

enhancing lipophilicity appropriately are common strategies to

improve brain penetration.¹⁴We prepared compound 27, bearing

an amine linker instead of the polar amide linker in expectation

of increasing brain permeation, however, the potency declined

compared to that of the corresponding amide analogue 10a (Table

4). We speculated that converting the linker amide to methylene

increased the ﬂexibility of the terminal amino group, which is

essential for the afﬁnity, and therefore it could not be located at

an appropriate position. With the hypothesis above, we planned

to ﬁx the conformation of the linker and designed compound

15c, having a piperazine ring instead of the amide linker. In the

overlay of stable conformations of 10a and 15c, calculated by

MOE,¹⁵the nitrogen atoms of 10a and 15c were located at similar

positions (Fig. 2). Encouraged by this result, we prepared 15c and

its analogues and evaluated their afﬁnities for human type

GPR54 in a binding assay (Table 4). As expected, 15c showed al-

most the same afﬁnity as 10a. Removal of the methyl group of

3

H

Me

Et

3.7

4.6

10

16

6d

7a

7b

7c

Me

Et

7d

7e

7f

Me

H

Me

Et

Racemate

10

9.7

18

17

i-Pr

Me

Et

7g

7h

7i

Et

H

Et

Racemate

8.0

22

6g

6h

6i

–(CH₂)₃–

–(CH₂)₄–

–(CH₂)₃–

H

Me

(S)-Isomer

(R)-Isomer

Racemate

(S)-Isomer

7.9

10

14

18

7j

24

20

RBA = receptor binding assay.

Table 3

The binding afﬁnities of glycine amide derivatives for human type GPR54

Table 4

The binding afﬁnities of compounds without amide linker for human GPR54

Compound

R¹

R²

R

hGPR54 RBA IC₅₀(nM)

6a

6b

6c

10a

10b

10c

H

Me

–(CH₂)₃–

Me

Et

H

4.1

8.4

16

12

30

15

Compound

m

n

R³

hGPR54 RBA IC₅₀(nM)

(R)-Isomer

10a

27

12

30

Me

Et

H

–(CH₂)⁴–

15a

15b

15c

15d

20a

20b

21a

21b

0

1

2

1

2

1

H

Me

Et

H

Me

Et

3.6

7.3

15

11

9.7

25

18

20

RBA = receptor binding assay.

idine of b-proline, also showed potent activity, though it was

racemic.

Compound24, whichis aninverse-amide analogueof 7a, reduced

the activity, indicating that the anilide derivatives are preferable.

RBA = receptor binding assay.

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T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 5157–5171

ability to penetrate the brain. The brain and plasma levels of com-

pounds in rats after 30 min of 0.2 mg/kg intravenous cassette

administration are shown in Table 5. Generally, tertiary amine

derivatives were superior to secondary amines regarding brain

exposure (6b vs 10a, 6d vs 7a, 15a vs 15d). Especially, tertiary

amines 10a and 15d showed high concentration in brain and good

brain-to-plasma concentration ratio (Kp). In the case of secondary

amines, although almost all compounds showed low concentration

in brain, compound 15a, with amide linker converted to

a

piperazine ring, showed relatively high brain exposure, as was

expected.

Caco-2 permeability test for 7a, 10a, 15a, and 15d, as represen-

tative compounds, was conducted to evaluate their P-gp suscepti-

bility (Table 5). A value of B-to-A/A-to-B above 3.0 indicates that a

compound is a P-gp substrate. Compound 7a, which was revealed

to be a substrate of P-gp, exhibited low concentration in brain and

low Kp value compared to compound 15a, without efﬂux effect by

P-gp. From the brain exposure data of compounds 10a, 15a, and

15d, lipophilicity can be seen to be essential for permeability to

brain, when the effect of P-gp mediated efﬂux is negligible. Espe-

cially, a log D value above three looks preferable for the brain pen-

etration considering the high brain concentration of 10a

(log D = 3.31) and 15d (log D = 4.10).

Next we investigated the afﬁnity of compounds for rat type

GPR54 (Table 5). The result of a binding assay using rat type

GPR54 was approximately parallel to that of human type GPR54,

although the afﬁnities were attenuated. Particularly, the rat recep-

tor binding afﬁnity of compound 10a, having high brain permeabil-

Figure 2. Overlay of the 4-phenyl moiety of 10a (green) and 15c (purple) in stable

conformations calculated by MOE.¹⁵

15c increased potency (15a) and converting the methyl group to an

ethyl group retained the afﬁnity (15c vs 15d). A compound bearing

a homopiperazine ring instead of the piperazine ring also showed

potent activity (15a vs 15b). Insertion of a methylene linker be-

tween the phenyl group and piperazine ring resulted in decreased

activity, regardless of it being a secondary amine (15a vs 20a) or

tertiary amine (15c vs 21a, 15d vs 21b).

Selected compounds with potent afﬁnities for human type

GPR54 in a receptor binding assay were then evaluated by their

Table 5

Brain and plasma exposure data in rats, Caco-2 permeability, and rat GPR54 binding afﬁnities

Compound

R⁴

iv rat cassette dosing test (0.2 mpk, 30 min)

Concentration (plasma; ng/ml, brain; ng/g)

Caco-2 permeability

B-to-A/A-to-B

Log D at pH 7.4

RBA IC₅₀(nM)

Rat GPR54

Plasma

Brain

11.7

<5

6b

6d

6g

6h

NT

2.66

2.13

2.29

2.13

26

20

26

38

Kp

—

Plasma

Brain

Kp

5.2

<5

—

Plasma

Brain

Kp

11.6

3.7

0.32

Plasma

Brain

Kp

5.5

2.0

0.36

Plasma

Brain

Kp

15.9

8.1

0.51

7a

4.3

2.95

47

Plasma

Brain

Kp

19.3

80.3

4.16

10a

15a

15d

1.4

1.2

1.0

3.31

2.72

4.10

130

15

Plasma

Brain

Kp

14.0

13.2

0.94

Plasma

Brain

23.8

93.1

47

Kp

3.91

Kp = the brain-to-plasma concentration ratio. NT = not tested. RBA = receptor binding assay.

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 5157–5171

5163

ity, was reduced remarkably. On the other hand, compound 15a,

which showed moderate brain penetration in spite of a secondary

amine, exhibited high afﬁnity for rat GPR54. Considering both

brain permeability and afﬁnity for rat GPR54, 15a and 15d were se-

lected for further examinations.

by 32%. This result is the ﬁrst example of a small molecule GPR54

antagonist suppressing the plasma LH level and suggests the pos-

sibility of GPR54 antagonists as drugs for sex-hormone dependent

diseases such as prostate cancer and endometriosis.

In order to evaluate the antagonistic activities of 15a and 15d, a

cellular Ca²⁺mobilization assay was carried out using metastin

(40–54) as a stimulator. The result is shown in Figure 3. Compound

4. Conclusion

To identify compounds having potent GPR54 antagonistic

activity and brain permeability, chemical modiﬁcation focused

on the side chain on the 4-phenyl ring was performed. As a re-

sult, secondary or tertiary amine derivatives 10a, 15a, and 15d

were discovered, which had high afﬁnity for human GPR54 and

good brain permeability. Excluding 10a, because of low potency

in a rat receptor binding assay, the cellular Ca²⁺mobilization as-

say was conducted for 15a and 15d. Unfortunately 15d did not

suppress the Ca²⁺inﬂux completely at adequately high concen-

tration, however, 15a showed fully potent antagonistic activity.

Finally, compound 15a was selected for an in vivo examination,

and the intravenous injection of 15a for castrated male rats

apparently suppressed the plasma LH level. The result indicates

the possibility of small molecule GPR54 antagonists for treat-

ment of sex-hormone dependent diseases such as prostate cancer

and endometriosis.

15a exhibited fully potent antagonist activity (IC₅₀= 0.93 lM),

however, 15d did not suppress the Ca²⁺inﬂux completely at an

adequately high concentration. We concluded that 15d was not

suitable for further investigation, and 15a was selected for

in vivo study.

Finally we tested the effect of 15a on the plasma LH level in cas-

trated male rats. Administration of 15a intravenously at 0.22 mg/

kg was repeated ﬁve times at 10 min intervals, ending up with a to-

tal dosage at 1.1 mg/kg. The result of plasma LH level measurement

is shown in Figure 4. The ﬁrst administration was performed at

0 min, which is indicated by an arrow, and the following four

administrations are indicated with arrowheads. The vehicle

administration did not show any effects on plasma LH level during

180 min of blood collection. On the contrary, 60 min after the

administration of compound 15a the plasma LH level was reduced

5. Experimental section

Melting points were determined on a Büchi melting point appa-

ratus and were not corrected. Proton nuclear magnetic resonance

(¹H NMR) spectra were recorded on Bruker DPX300 (300 MHz)

instruments. Chemical shifts are reported as d values (ppm) down-

ﬁeld from internal tetramethylsilane of the indicated solution.

Peak multiplicities are expressed as follows: s, singlet; d, doublet;

t, triplet; q, quartet; dd, doublet of doublet; ddd, doublet of doublet

of doublets; dt, doublet of triplet; br s, broad singlet; m, multiplet.

Coupling constants (J values) are given in hertz (Hz). Element anal-

yses were carried out by Takeda Analytical Laboratories. Reaction

progress was determined by thin layer chromatography (TLC) anal-

ysis on Silica Gel 60 F₂₅₄plate (Merck) or NH TLC plates (Fuji Silysia

chemical Ltd). Chromatographic puriﬁcation was carried on silica

gel columns 60 (0.063–0.200 mm or 0.040–0.063 mm, Merck), ba-

sic silica gel (ChromatorexNH, 100–200 mesh, Fuji Silysia Chemical

Figure 3. The antagonistic activities of 15a (solid line) and 15d (broken line)

against metastin (40–54) in CHO cell.

Ltd) or Purif-Pack (SI 60 lM or NH 60 lM, Fuji Silysia, Ltd). Com-

mercial reagents and solvents were used without additional

puriﬁcation.

5.1. tert-Butyl {2-[(3-{3-cyano-6-[4-ﬂuoro-2-(methoxyme-

thoxy)phenyl]-2-[(furan-2-ylcarbonyl)amino]pyridin-4-yl}

phenyl)amino]-2-oxoethyl}carbamate (5a)

To an ice-cooled mixture of N-{4-(3-aminophenyl)-3-cyano-6-

[4-ﬂuoro-2-(methoxymethoxy)phenyl]pyridin-2-yl}furan-2-car-

boxamide¹³(4, 0.20 g, 0.44 mmol), N-(tert-butoxycarbonyl)glycine

(0.15 g, 0.88 mmol), and HOBt (0.12 g, 0.88 mmol) in DMF (5 ml)

was added WSCD (0.17 g, 0.88 mmol). After stirring at room tem-

perature for 40 h, the reaction mixture was diluted with saturated

aqueous NaHCO₃and extracted with EtOAc. The extract was

washed with brine, dried over MgSO₄, and concentrated in vacuo.

The residue was puriﬁed by silica gel column chromatography

(EtOAc/hexane = 3/2). Recrystallization from EtOAc/hexane gave

5a (0.21 g, 78%) as colorless crystals: mp 119–121 °C; ¹H NMR

(DMSO-d₆) d 1.49 (9H, s), 3.47 (3H, s), 3.95 (2H, d, J = 6.0 Hz),

5.10–5.25 (1H, m), 5.30 (2H, s), 6.61 (1H, q, J = 1.8 Hz), 6.84–6.91

(1H, m), 7.02 (1H, dd, J = 10.5, 2.4 Hz), 7.39 (1H, dd, J = 3.3,

1.5 Hz), 7.48–7.61 (4H, m), 7.97 (1H, s), 8.00–8.03 (1H, m), 8.20–

8.35 (1H, br s), 8.81 (1H, s).

Figure 4. Effect of compound 15a on plasma LH level in castrated male rats. Values

*

**

are means SE, determined from eight experiments.

compared with the vehicle control.

P <0.05 and

P <0.01

5164

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 5157–5171

5.2. tert-Butyl {2-[(3-{3-cyano-6-[4-ﬂuoro-2-(methoxy-

methoxy)phenyl]-2-[(furan-2-ylcarbonyl)amino]pyridin-4-yl}

phenyl)amino]-2-oxoethyl}methylcarbamate (5b)

from MeOH/EtOAc gave 6a (0.16 g, 100%) as a pale yellow solid:

mp 220–236 °C; ¹H NMR (DMSO-d₆) d 3.85 (2H, d, J = 5.7 Hz),

6.79–6.87 (3H, m), 7.47 (1H, d, J = 7.8 Hz), 7.54 (1H, d, J = 3.3 Hz),

7.62 (1H, t, J = 7.8 Hz), 7.82 (1H, d, J = 8.1 Hz), 8.00 (1H, s), 8.02

(1H, t, J = 0.9 Hz), 8.16–8.26 (5H, m), 10.87 (1H, s), 11.34 (1H, s),

12.33 (1H, s); Anal. Calcd for C₂₅H₁₈FN₅O₄ꢀHClꢀH₂O: C, 57.09; H,

4.02; N, 13.32. Found: C, 56.84; H, 3.83; N, 13.09.

Compound 5b was prepared from 4 (500 mg, 1.1 mmol) and N-

(tert-butoxycarbonyl)-N-methylglycine (410 mg, 2.2 mmol) in a

manner similar to that described for 5a. Compound 5b (540 mg,

78%) was obtained as a colorless solid: mp 162–164 °C (EtOAc/hex-

ane); ¹H NMR (CDCl₃) d 1.51 (9H, s), 3.03 (3H, s), 3.48 (3H, s), 4.00

(2H, s), 5.31 (2H, s), 6.61 (1H, dd, J = 3.6, 1.8 Hz), 6.84–6.90 (1H, m),

7.03 (1H, dd, J = 10.8, 2.4 Hz), 7.39 (1H, dd, J = 3.3, 0.6 Hz), 7.45 (3H,

br s), 7.59–7.61 (1H, m), 7.97–8.05 (3H, m), 8.60 (1H, br s), 8.82

(1H, br s).

5.7. N-[3-Cyano-6-(4-ﬂuoro-2-hydroxyphenyl)-4-{3-[(N-methyl-

glycyl)amino]phenyl}pyridin-2-yl]furan-2-carboxamide hydro-

chloride (6b)

Compound 6b was prepared from 5b (500 mg, 0.79 mmol) in a

manner similar to that described for 6a. Compound 6b (380 mg,

92%) was obtained as a pale yellow solid: mp 294–296 °C (MeOH);

¹H NMR (DMSO-d₆) d 2.64 (3H, s), 4.00 (2H, s), 6.79–6.89 (3H, m),

7.47 (1H, d, J = 8.1 Hz), 7.55 (1H, dd, J = 3.6, 0.6 Hz), 7.62 (1H, t,

J = 7.8 Hz), 7.83 (1H, d, J = 8.1 Hz), 8.02 (1H, s), 8.08 (1H, dd,

J = 1.8, 0.6 Hz), 8.16 (1H, s), 8.24 (1H, dd, J = 8.7, 6.9 Hz), 9.03 (1H,

br s), 11.04 (1H, s), 11.33 (1H, s), 12.34 (1H, s); Anal. Calcd for

5.3. tert-Butyl (2R)-2-[(3-{3-cyano-6-[4-ﬂuoro-2-(methoxy-

methoxy)phenyl]-2-[(furan-2-ylcarbonyl)amino]pyridin-4-yl}

phenyl)carbamoyl]pyrrolidine-1-carboxylate (5c)

Compound 5c was prepared from 4 (40 mg, 0.087 mmol) and N-

(tert-butoxycarbonyl)-D-proline (40 mg, 0.19 mmol) in a manner

similar to that described for 5a. Compound 5c (39 mg, 68%) was

obtained as a colorless amorphous solid: ¹H NMR (CDCl₃) d 1.51

(10H, s), 1.85–2.00 (2H, m), 2.45–2.65 (1H, br), 3.30–3.50 (5H,

m), 4.40–4.55 (1H, m), 5.32 (2H, s), 6.61 (1H, q, J = 1.8 Hz), 6.84–

6.91 (1H, m), 7.03 (1H, dd, J = 10.8, 2.4 Hz), 7.39 (1H, dd, J = 6.6,

0.6 Hz), 7.48 (3H, br s), 7.59–7.61 (1H, m), 7.98 (1H, s), 8.03 (1H,

dd, J = 8.7, 6.9 Hz), 8.12 (1H, s), 8.81 (1H, s), 9.65–9.85 (1H, m).

C

₂₆H₂₀FN₅O₄ꢀHCl: C, 59.72; H, 3.86; N, 13.34. Found: C, 59.83; H,

4.06; N, 13.42.

5.8. N-(3-{3-Cyano-6-(4-ﬂuoro-2-hydroxyphenyl)-2-[(furan-2-

ylcarbonyl)amino]pyridin-4-yl}phenyl)-

chloride (6c)

D-prolinamide hydro-

Compound 6c was prepared from 5c (190 mg, 0.29 mmol) in a

manner similar to that described for 6a. Compound 6c (122 mg,

5.4. tert-Butyl {3-[(3-{3-cyano-6-[4-ﬂuoro-2-(methoxy-

methoxy)phenyl]-2-[(furan-2-ylcarbonyl)amino]pyridin-4-yl}

phenyl)amino]-3-oxopropyl}methylcarbamate (5e)

77%) was obtained as

a pale yellow solid: mp 216–220 °C

(MeOH/Et₂O); ¹H NMR (DMSO-d₆) d 1.91–2.07 (3H, m), 2.39–2.51

(1H, m), 3.22–3.29 (2H, m), 4.41 (1H, t, J = 7.2 Hz), 6.79–6.87 (3H,

m), 7.47–7.55 (2H, m), 7.63 (1H, t, J = 7.8 Hz), 7.84 (1H, d,

J = 8.4 Hz), 8.02 (1H, s), 8.08 (1H, d, J = 0.9 Hz), 8.16 (1H, s), 8.23

(1H, t, J = 7.8 Hz), 9.00–9.60 (2H, m), 11.02 (1H, s), 11.30–11.40

Compound 5e was prepared from 4 (500 mg, 1.1 mmol) and N-(

tert-butoxycarbonyl)-N-methyl-b-alanine (440 mg, 2.2 mmol) in a

manner similar to that described for 5a. Compound 5e (530 mg,

75%) was obtained as a colorless solid: mp 172–174 °C (EtOAc/hex-

ane); ¹H NMR (CDCl₃) d 1.46 (9H, s), 2.69 (2H, br s), 2.91 (3H, s),

3.47 (3H, s), 3.64 (2H, t, J = 6.3 Hz), 5.31 (2H, s), 6.61 (1H, dd,

J = 3.3, 1.5 Hz), 6.84–6.91 (1H, m), 7.03 (1H, dd, J = 10.8, 2.4 Hz),

7.38 (1H, dd, J = 3.6, 0.6 Hz), 7.44–7.51 (2H, m), 7.59–7.60 (2H,

m), 7.96 (1H, s), 8.02 (1H, dd, J = 8.4, 6.9 Hz), 8.14 (1H, br s), 8.82

(1H, br s), 9.07 (1H, br s).

(1H, m), 12.33 (1H, s); ½a _D²⁵

ꢁ

= +19.1 (c 1.0, DMSO); Anal. Calcd for

C

₂₈H₂₂FN₅O₄ꢀHClꢀH₂O: C, 59.42; H, 4.45; N, 12.37. Found: C,

59.17; H, 4.52; N, 12.29.

5.9. N-[3-Cyano-6-(4-ﬂuoro-2-hydroxyphenyl)-4-{3-[(N-methyl-

b-alanyl)amino]phenyl}pyridin-2-yl]furan-2-carboxamide

hydrochloride (6d)

5.5. tert-Butyl 2-{2-[(3-{3-cyano-6-[4-ﬂuoro-2-(methoxy-

methoxy)phenyl]-2-[(furan-2-ylcarbonyl)amino]pyridin-4-yl}

phenyl)amino]-2-oxoethyl}piperidine-1-carboxylate (5j)

Compound 6d was prepared from 5e (500 mg, 0.78 mmol) in a

manner similar to that described for 6a. Compound 6d (380 mg,

91%) was obtained as a pale yellow solid: mp 253–255 °C (MeOH);

¹H NMR (DMSO-d₆) d 2.58 (3H, t, J = 5.4 Hz), 2.86 (2H, t, J = 6.9 Hz),

3.16–3.22 (2H, m), 6.79–6.88 (3H, m), 7.41 (1H, d, J = 7.8 Hz), 7.53–

7.60 (2H, m), 7.83 (1H, d, J = 8.1 Hz), 8.01 (1H, s), 8.08 (1H, dd,

J = 1.5, 0.6 Hz), 8.14 (1H, s), 8.23 (1H, dd, J = 8.1, 6.6 Hz), 8.74

(2H, br s), 10.61 (1H, s), 11.31 (1H, s), 12.33 (1H, s); Anal. Calcd

for C₂₇H₂₂FN₅O₄ꢀHClꢀH₂O: C, 58.54; H, 4.55; N, 12.64. Found: C,

58.80; H, 4.71; N, 12.70.

Compound 5j was prepared from 4 (190 mg, 0.41 mmol) and [1-

(tert-butoxycarbonyl)piperidine-2-yl]acetic

acid

(130 mg,

0.53 mmol) in a manner similar to that described for 5a. Com-

pound 5j (230 mg, 82%) was obtained as a pale yellow solid: mp

154–156 °C (EtOAc/hexane); ¹H NMR (CDCl₃) d 1.43 (9H, s), 1.52–

1.76 (6H, m), 2.57 (1H, dd, J = 5.4 Hz, 16.2 Hz), 2.83–2.96 (2H, m),

3.47 (3H, s), 3.98 (1H, d, J = 14.7 Hz), 4.89 (1H, br s), 5.31 (2H, s),

6.61 (1H, dd, J = 3.3, 1.8 Hz), 6.84–6.91 (1H, m), 7.03 (1H, dd,

J = 10.8, 2.4 Hz), 7.38 (1H, dd, J = 3. 6, 0.9 Hz), 7.43–7.50 (2H, m),

7.53–7.61 (2H, m), 7.97 (1H, s), 8.02 (1H, dd, J = 9.0, 6.9 Hz), 8.16

(1H, br s), 8.81 (1H, br s), 9.01 (1H, br s).

5.10. N-[3-Cyano-6-(4-ﬂuoro-2-hydroxyphenyl)-4-{3-[(piperi-

din-2-ylacetyl)amino]phenyl}pyridin-2-yl]furan-2-carbox-

amide hydrochloride (6i)

Compound 6i was prepared from 5j (150 mg, 0.22 mmol) in a

manner similar to that described for 6a. Compound 6i (97 mg,

77%) was obtained as a yellow solid: mp 277–278 °C (MeOH/

Et₂O); ¹H NMR (DMSO-d₆) d 1.51–1.88 (6H, m), 2.73–2.98 (3H,

m), 3.26 (1H, d, J = 12.6 Hz), 6.79–6.88 (3H, m), 7.42 (1H, d,

J = 7.8 Hz), 7.54–7.61 (2H, m), 7.82 (1H, d, J = 9.0 Hz), 8.02 (1H, br

s), 8.08 (1H, d, J = 0.9 Hz), 8.14 (1H, s), 8.23–8.26 (1H, m), 8.78–

8.91 (2H, m), 10.65 (1H, s), 11.32 (1H, s), 12.34 (1H, s); Anal. Calcd

5.6. N-{3-Cyano-6-(4-ﬂuoro-2-hydroxyphenyl)-4-[3-(glycyla-

mino)phenyl]pyridin-2-yl}furan-2-carboxamide hydrochloride

(6a)

A mixture of 5a (0.20 g, 0.32 mmol) and 4 M HCl EtOAc solution

(4 ml) in EtOAc (16 ml) was stirred at room temperature for 40 h.

The precipitate was collected by ﬁltration and recrystallization

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 5157–5171

5165

for C₃₀H₂₆FN₅O₄ꢀHClꢀ0.5H₂O: C, 61.59; H, 4.82; N, 11.97. Found: C,

5.14. N-[3-Cyano-6-(4-ﬂuoro-2-hydroxyphenyl)-4-(3-{[(2R)-

pyrrolidin-2-ylacetyl]amino}phenyl)pyridin-2-yl]furan-2-

carboxamide hydrochloride (6h)

61.28; H, 4.90; N, 12.05.

5.11. N-[3-Cyano-6-(4-ﬂuoro-2-hydroxyphenyl)-4-(3-{[(2S)-pyrr

olidin-2-ylacetyl]amino}phenyl)pyridin-2-yl]furan-2-carbox-

amide hydrochloride (6g)

Compound 6h was prepared from 4 (400 mg, 0.87 mmol) and

[(2R)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]acetic

acid¹⁶

(300 mg, 1.31 mmol) in a manner similar to that described for

6g. Compound 6h (340 mg, 70%) was obtained as a yellow solid:

mp 269–270 °C (MeOH); ¹H NMR (DMSO-d₆) d 1.55–1.68 (1H,

m), 1.81–1.97 (2H, m), 2.10–2.20 (1H, m), 2.93 (2H, d, J = 6.9 Hz),

3.13–3.21 (2H, m), 3.76–3.85 (1H, m), 6.79–6.89 (3H, m), 7.41

(1H, d, J = 7.8 Hz), 7.54–7.61 (2H, m), 7.81–7.84 (1H, m), 8.04

(1H, br s), 8.08–8.09 (1H, m), 8.15 (1H, s), 8.23 (1H, dd, J = 8.4,

6.6 Hz), 8.94 (1H, br s), 9.18 (1H, br s), 10.64 (1H, s), 11.33 (1H,

To a solution of compound 4 (500 mg, 1.1 mmol), [(2S)-1-(tert-

butoxtcabonyl)pyrrolidin-2-yl]acetic acid¹⁶(300 mg, 1.3 mmol),

and HOBt (180 mg, 1.3 mmol) in DMF (5 ml) was added WSCD

(250 mg, 1.3 mmol) and the mixture was stirred at room temper-

ature for 5 days. The solution was diluted with EtOAc, washed

with water three times and brine, dried over MgSO₄, and concen-

trated in vacuo. The residue was puriﬁed by silica gel column

chromatography (hexane to hexane/EtOAc = 1/1) and basic silica

gel column chromatography (hexane to EtOAc) to give tert-butyl

(2S)-2-{2-[(3-{3-cyano-6-[4-ﬂuoro-2-(methoxymethoxy)phenyl]-

2-[(furan-2-ylcarbonyl)amino]pyridin-4-yl}phenyl)amino]-2-oxo-

ethyl}pyrrolidine-1-carboxylate (5h) as an amorphous solid. After

the solid was dissolved in EtOAc (5 ml) and MeOH (2 ml), 4 M HCl

EtOAc solution (5 ml) was added and the mixture was stirred at

room temperature overnight. The solvent was evaporated and

the residue was recrystallized from MeOH/Et₂O to give 6g

(390 mg, 63%) as a pale yellow solid: mp 268–269 °C; ¹H NMR

(DMSO-d₆) d 1.55–1.68 (1H, m), 1.81–1.97 (2H, m), 2.10–2.20

(1H, m), 2.93 (2H, d, J = 6.9 Hz), 3.13–3.21 (2H, m), 3.76–3.85

(1H, m), 6.79–6.89 (3H, m), 7.41 (1H, d, J = 7.8 Hz), 7.54–7.61

(2H, m), 7.81–7.84 (1H, m), 8.04 (1H, br s), 8.08–8.09 (1H, m),

8.15 (1H, s), 8.23 (1H, dd, J = 8.4, 6.6 Hz), 8.94 (1H, br s), 9.18

(1H, br s), 10.64 (1H, s), 11.33 (1H, s), 12.33 (1H, s);

s), 12.33 (1H, s); ½a _D²⁵

= ꢂ14.7 (c 1.0, DMSO); Anal. Calcd for

ꢁ

C

₂₉H₂₄FN₅O₄ꢀHCl: C, 61.98; H, 4.48; N, 12.46. Found: C, 61.77; H,

4.53; N, 12.34.

5.15. N-[3-Cyano-4-(3-{[3-(ethylamino)pentanoyl]amino}

phenyl)-6-(4-ﬂuoro-2-hydroxyphenyl)pyridin-2-yl]furan-2-

carboxamide hydrochloride (7h)

To a solution of 6f (150 mg, 0.27 mmol), acetaldehyde (97%,

26 mg, 0.60 mmol), and AcOH (0.1 ml) in MeOH (10 ml) was added

NaBH₃(CN) (38 mg, 0.60 mmol) and the mixture was stirred at

room temperature for 4 h. The mixture was diluted with saturated

aqueous NaHCO₃and extracted with EtOAc. The organic layer was

washed with brine, dried over MgSO₄, and concentrated in vacuo. A

solution of the residue in EtOAc (5 ml) was treated with 4 M HCl

EtOAc solution (0.5 ml) at room temperature and then concen-

trated. The residue was recrystallized from MeOH/Et₂O to give

7h (137 mg, 88%) as a pale yellow solid: mp 233–238 °C; ¹H

NMR (DMSO-d₆) d 0.96 (3H, t, J = 7.5 Hz), 1.23 (3H, t, J = 7.2 Hz),

1.61–1.84 (2H, m), 2.80–2.95 (2H, m), 3.02 (2H, q, J = 7.2 Hz),

3.48 (1H, br s), 6.79–6.88 (3H, m), 7.42 (1H, d, J = 7.8 Hz), 7.53–

7.61 (2H, m), 7.83 (1H, d, J = 9.0 Hz), 8.01 (1H, d, J = 1.5 Hz), 8.07

(1H, t, J = 0.9 Hz), 8.21 (1H, s), 8.26 (1H, t, J = 7.8 Hz), 8.50–8.85

(2H, m), 10.70 (1H, s), 11.25–11.40 (1H, m), 12.34 (1H, br s); Anal.

Calcd for C₃₀H₂₈FN₅O₄ꢀHClꢀ0.5H₂O: C, 61.38; H, 5.15; N, 11.93.

Found: C, 61.21; H, 5.18; N, 11.92.

½

a ²_D⁵

ꢁ

= +16.6 (c 1.0, DMSO); Anal. Calcd for C₂₉H₂₄FN₅O₄ꢀ

HClꢀ0.25H₂O: C, 61.49; H, 4.54; N, 12.36. Found: C, 61.33; H,

4.52; N, 12.41.

5.12. N-[4-{3-[(3-Aminobutanoyl)amino]phenyl}-3-cyano-6-(4-

ﬂuoro-2-hydroxyphenyl)pyridin-2-yl]furan-2-carboxamide

hydrochloride (6e)

Compound 6e was prepared from 4 (500 mg, 1.09 mmol) and

N-(tert-butoxycarbonyl)-3-aminobutanoic acid (443 mg, 2.18

mmol) in a manner similar to that described for 6g. Compound

6e (411 mg, 70%) was obtained as a yellow solid: mp 213–215 °C

(MeOH/Et₂O); ¹H NMR (DMSO-d₆) d 1.26 (3H, d, J = 6.6 Hz), 2.60–

2.85 (2H, m), 3.50–3.70 (1H, m), 6.75–6.90 (3H, m), 7.41 (1H, d,

J = 8.1 Hz), 7.54 (1H, d, J = 3.9 Hz), 7.59 (1H, d, J = 7.8 Hz), 7.83

(1H, d, J = 8.1 Hz), 7.90–8.10 (5H, m), 8.14 (1H, s), 8.23 (1H, t,

J = 7.5 Hz), 10.60 (1H, s), 11.31 (1H, s), 12.34 (1H, s); Anal. Calcd

for C₂₇H₂₂FN₅O₄ꢀHClꢀ1.5H₂O: C, 57.60; H, 4.65; N. 12.44. Found:

C, 57.49; H, 4.66; N, 12.38.

5.16. N-[3-Cyano-4-{3-[(N,N-dimethyl-b-alanyl)amino]phenyl}-

6-(4-ﬂuoro-2-hydroxyphenyl)pyridin-2-yl]furan-2-carbox-

amide hydrochloride (7a)

Compound 7a was prepared from 3 (120 mg, 0.23 mmol) and

formaldehyde (37%, 37 lL, 0.46 mmol) in a manner similar to that

described for 7h. Compound 7a (65 mg, 51%) was obtained as a

pale yellow solid: mp 240–241 °C (MeOH/Et₂O); ¹H NMR (DMSO-

d₆) d 2.79 (6H, s), 2.93 (2H, t, J = 7.2 Hz), 3.30–3.45 (2H, m), 6.75–

6.90 (3H, m), 7.41 (1H, d, J = 7.8 Hz), 7.50–7.65 (2H, m), 7.82 (1H,

d, J = 8.4 Hz), 8.01 (1H, s), 8.08 (1H, s), 8.14 (1H, s), 8.20–8.30

(1H, m), 9.99 (1H, br s), 10.65 (1H, s), 11.32 (1H, s), 12.33 (1H,

s); Anal. Calcd for C₂₈H₂₄FN₅O₄ꢀHClꢀ0.5H₂O: C, 60.16; H, 4.69; N.

12.53. Found: C, 60.44; H, 4.66; N, 12.68.

5.13. N-[4-{3-[(3-Aminopentanoyl)amino]phenyl}-3-cyano-6-

(4-ﬂuoro-2-hydroxyphenyl)pyridin-2-yl]furan-2-carboxamide

hydrochloride (6f)

Compound 6f was prepared from 4 (120 mg, 0.26 mmol) and

N-(tert-butoxycarbonyl)-3-aminopentanoic acid (114 mg, 0.52

mmol) in a manner similar to that described for 6g. Compound

6f (105 mg, 73%) was obtained as a yellow solid: mp 212–214 °C

(MeOH/Et₂O); ¹H NMR (DMSO-d₆) d 0.95 (3H, t, J = 7.6 Hz), 1.55–

1.75 (2H, m), 2.65–2.90 (2H, m), 3.46 (1H, br s), 6.75–6.90 (3H,

m), 7.42 (1H, d, J = 7.6 Hz), 7.54 (1H, d, J = 3.6 Hz), 7.58 (1H, t,

J = 8.0 Hz), 7.83 (1H, d, J = 8.8 Hz), 8.01 (4H, br s), 8.08 (1H, s),

8.14 (1H, s), 8.23 (1H, t, J = 7.6 Hz), 10.63 (1H, s), 11.31 (1H, s),

12.33 (1H, s); Anal. Calcd for C₂₈H₂₄FN₅O₄ꢀHClꢀ1.5H₂O: C, 58.28;

H, 4.89; N. 12.14. Found: C, 58.36; H, 4.77; N, 12.13.

5.17. N-[3-Cyano-4-{3-[(N-ethyl-N-methyl-b-alanyl)amino]

phenyl}-6-(4-ﬂuoro-2-hydroxyphenyl)pyridin-2-yl]furan-2-

carboxamide hydrochloride (7b)

Compound 7b was prepared from 6d (150 mg, 0.28 mmol) and

acetaldehyde (90%, 41 mg, 0.84 mmol) in a manner similar to that

described for 7h. Compound 7b (92 mg, 58%) was obtained as a

pale yellow solid: mp 223–224 °C (MeOH); ¹H NMR (DMSO-d₆) d

1.25 (3H, t, J = 7.2 Hz), 2.75 (3H, d, J = 3.9 Hz), 2.95 (2H, t,

J = 7.2 Hz), 3.07–3.44 (4H, m), 6.79–6.88 (3H, m), 7.41 (1H, d,

5166

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 5157–5171

J = 8.1 Hz), 7.53–7.61 (2H, m), 7.81–7.84 (1H, m), 8.01 (1H, s), 8.08

(1H, dd, J = 1.8, 0.9 Hz), 8.14 (1H, s), 8.21–8.26 (1H, m), 9.97 (1H, br

s), 10.65 (1H, s), 11.32 (1H, s), 12.33 (1H, d, J = 1.2 Hz); Anal. Calcd

for C₂₉H₂₆FN₅O₄ꢀHClꢀ0.5H₂O: C, 60.79; H, 4.93; N, 12.22. Found: C,

60.46; H, 5.06; N, 12.00.

Calcd for C₂₉H₂₆FN₅O₄ꢀHClꢀ0.5H₂O: C, 60.79; H, 4.93; N. 12.22.

Found: C, 60.84; H, 4.85; N, 12.27.

5.22. N-[3-Cyano-4-(3-{[3-(diethylamino)butanoyl]amino}

phenyl)-6-(4-ﬂuoro-2-hydroxyphenyl)pyridin-2-yl]furan-2-

carboxamide hydrochloride (7g)

5.18. N-[3-Cyano-4-{3-[(N,N-diethyl-b-alanyl)amino]phenyl}-6-

(4-ﬂuoro-2-hydroxyphenyl)pyridin-2-yl]furan-2-carboxamide

hydrochloride (7c)

Compound 7g was prepared from 6e (100 mg, 0.19 mmol) and

acetaldehyde (97%, 17 mg, 0.38 mmol) in a manner similar to that

described for 7h. Compound 7g (60 mg, 54%) was obtained as a

pale yellow solid: mp 193–195 °C (MeOH/Et₂O); ¹H NMR (DMSO-

d₆) d 1.25–1.45 (9H, m), 2.70–2.85 (1H, m), 3.05–3.30 (5H, m),

3.92 (1H, br s), 6.75–6.90 (3H, m), 7.43 (1H, d, J = 7.8 Hz), 7.54

(1H, d, J = 3.0 Hz), 7.58 (1H, t, J = 7.8 Hz), 7.82 (1H, d, J = 9.0 Hz),

8.01 (1H, s), 8.08 (1H, s), 8.15 (1H, s), 8.24 (1H, t, J = 7.5 Hz), 9.79

(1H, s), 10.68 (1H, s), 11.31 (1H, s), 12.35 (1H, s); Anal. Calcd for

Compound 7c was prepared from 3 (200 mg, 0.38 mmol) and

acetaldehyde (97%, 0.6 ml) in a manner similar to that described

for 7h. Compound 7c (194 mg, 88%) was obtained as a pale yellow

solid: mp 209–213 °C (MeOH/Et₂O); ¹H NMR (DMSO-d₆) d 1.24 (6H,

t, J = 7.2 Hz), 2.94 (2H, t, J = 7.2 Hz), 3.08–3.24 (4H, m), 3.34–3.39

(2H, m), 6.79–6.88 (3H, m), 7.41 (1H, d, J = 7.8 Hz), 7.54–7.60 (2H,

m), 7.83 (1H, d, J = 8.4 Hz), 8.01 (1H, s), 8.08 (1H, s), 8.14 (1H, s),

8.23 (1H, t, J = 7.8 Hz), 10.07 (1H, br s), 10.67 (1H, s), 11.32 (1H,

s), 12.34 (1H, s); Anal. Calcd for C₃₀H₂₈FN₅O₄ꢀHClꢀ0.5H₂O: C,

61.38; H, 5.15; N, 11.93. Found: C, 61.23; H, 4.95; N, 11.80.

C

₃₁H₃₀FN₅O₄ꢀHClꢀ1.5H₂O: C, 60.14; H, 5.54; N. 11.31. Found: C,

60.51; H, 5.89; N, 11.25.

5.23. N-[3-Cyano-4-(3-{[3-(diethylamino)pentanoyl]amino}

phenyl)-6-(4-ﬂuoro-2-hydroxyphenyl)pyridin-2-yl]furan-2-

carboxamide hydrochloride (7i)

5.19. N-[3-Cyano-4-(3-{[3-(ethylamino)butanoyl]amino}

phenyl)-6-(4-ﬂuoro-2-hydroxyphenyl)pyridin-2-yl]furan-2-

carboxamide hydrochloride (7d)

Compound 7i was prepared from 6f (150 mg, 0.22 mmol) and

acetaldehyde (97%, 268 mg, 5.46 mmol) in a manner similar to that

described for 7h. Compound 7i (130 mg, 79%) was obtained as a

pale yellow solid: mp 171–175 °C (MeOH/Et₂O); ¹H NMR (DMSO-

d₆) d 0.98 (3H, t, J = 7.2 Hz), 1.28–1.33 (6H, m), 1.60–1.78 (1H,

m), 1.86–2.06 (1H, m), 2.76 (1H, dd, J = 16.2, 6.6 Hz), 3.04–3.39

(5H, m), 3.80 (1H, br s), 6.79–6.88 (3H, m), 7.42 (1H, d,

J = 8.1 Hz), 7.54–7.61 (2H, m), 7.83 (1H, d, J = 8.7 Hz), 8.02 (1H, s),

8.08 (1H, d, J = 0.6 Hz), 8.15 (1H, s), 8.23 (1H, t, J = 7.8 Hz), 9.61

(1H, br s), 10.78 (1H, s), 11.31 (1H, s), 12.34 (1H, s); Anal. Calcd

for C₃₂H₃₂FN₅O₄ꢀHClꢀH₂O: C, 61.58; H, 5.65; N, 11.22. Found: C,

61.43; H, 5.69; N, 11.20.

Compound 7d was prepared from 6e (100 mg, 0.19 mmol) and

acetaldehyde (97%, 14 mg, 0.28 mmol) in a manner similar to that

described for 7h. Compound 7d (83 mg, 77%) was obtained as a

pale yellow solid: mp 249–255 °C (MeOH/Et₂O); ¹H NMR (DMSO-

d₆) d 1.23 (3H, t, J = 7.2 Hz), 1.30 (3H, d, J = 6.3 Hz), 2.69–2.77

(1H, m), 2.91–3.02 (3H, m), 3.60 (1H, q, J = 5.7 Hz), 6.78–6.86

(3H, m), 7.41 (1H, d, J = 7.5 Hz), 7.53–7.60 (2H, m), 7.83 (1H, d,

J = 8.1 Hz), 8.01 (1H, d, J = 1.2 Hz), 8.14 (1H, s), 8.21 (1H, s), 8.23

(1H, t, J = 7.8 Hz), 8.60–8.90 (2H, m), 10.65 (1H, s), 11.30–11.50

(1H, m), 12.30–12.50 (1H, m); Anal. Calcd for C₂₉H₂₆FN₅O₄ꢀHCl:

C, 61.76; H, 4.83; N, 12.42. Found: C, 61.39; H, 4.81; N, 12.38.

5.24. N-{3-Cyano-6-(4-ﬂuoro-2-hydroxyphenyl)-4-[3-({[(2S)-1-

methylpyrrolidin-2-yl]acetyl}amino)phenyl]pyridin-2-yl}

furan-2-carboxamide hydrochloride (7j)

5.20. N-{3-Cyano-6-(4-ﬂuoro-2-hydroxyphenyl)-4-[3-({3-[(1-

methylethyl)amino]butanoyl}amino)phenyl]pyridin-2-

yl}furan-2-carboxamide hydrochloride (7e)

Compound 7j was prepared from 6g (200 mg, 0.36 mmol) and

formaldehyde (37%, 60 mg, 0.74 mmol) in a manner similar to that

described for 7h. Compound 7j (180 mg, 87%) was obtained as a

pale yellow solid: mp 257–258 °C (MeOH); ¹H NMR (DMSO-d₆) d

1.67–1.79 (1H, m), 1.87–2.06 (2H, m), 2.24–2.35 (1H, m), 2.79–

2.93 (4H, m), 3.00–3.24 (2H, m), 3.52–3.73 (2H, m), 6.79–6.88

(3H, m), 7.42 (1H, d, J = 8.1 Hz), 7.53–7.60 (2H, m), 7.81–7.84

(1H, m), 8.01 (1H, t, J = 1.8 Hz), 8.08 (1H, dd, J = 1.5 Hz, 0.6 Hz),

8.15 (1H, s), 8.21–8.26 (1H, m), 10.48 (1H, br s), 10.70 (1H, s),

Compound 7e was prepared from 6e (200 mg, 0.37 mmol) and

acetone (0.2 ml) in a manner similar to that described for 7h. Com-

pound 7e (170 mg, 79%) was obtained as a pale yellow solid: mp

197–199 °C (MeOH/Et₂O); ¹H NMR (DMSO-d₆) d 1.26–1.33 (9H,

m), 2.75 (1H, dd, J = 15.9, 7.8 Hz), 2.94 (1H, dd, J = 15.9, 4.8 Hz),

3.44 (1H, br s), 3.68 (1H, br s), 6.79–6.88 (3H, m), 7.42 (1H, d,

J = 7.8 Hz), 7.53–7.61 (2H, m), 7.84 (1H, d, J = 8.1 Hz), 8.01 (1H, s),

8.08 (1H, d, J = 1.2 Hz), 8.14 (1H, s), 8.21–8.26 (1H, m), 8.62 (1H,

br s), 8.76 (1H, br s), 10.65 (1H, s), 11.31 (1H, s), 12.34 (1H, s); Anal.

Calcd for C₃₀H₂₈FN₅O₄ꢀHClꢀ1.5H₂O: C, 59.55; H, 5.33; N, 11.57.

Found: C, 59.51; H, 5.32; N, 11.61.

11.31 (1H, s), 12.35 (1H, s); ½a _D²⁵

= ꢂ6.6 (c 1.0, DMSO); Anal. Calcd

ꢁ

for C₃₀H₂₆FN₅O₄ꢀHClꢀ0.25H₂O: C, 62.07; H, 4.77; N, 12.06. Found: C,

61.84; H, 4.65; N, 12.15.

5.21. N-[3-Cyano-4-(3-{[3-(dimethylamino)butanoyl]amino}

phenyl)-6-(4-ﬂuoro-2-hydroxyphenyl)pyridin-2-yl]furan-2-

carboxamide hydrochloride (7f)

5.25. N-(4-{3-[(Chloroacetyl)amino]phenyl}-3-cyano-6-[4-

ﬂuoro-2-(methoxymethoxy)phenyl]pyridin-2-yl)furan-2-

carboxamide (8)

Compound 7f was prepared from 6e (100 mg, 0.19 mmol) and

formaldehyde (37%, 30 lL, 0.37 mmol) in a manner similar to that

To an ice-cooling solution of 4 (0.50 g, 1.1 mmol) and N,N-diiso-

propylethylamine (0.17 g, 1.3 mmol) in THF (5 ml) was added chlo-

roacetyl chloride (0.10 ml, 1.3 mmol) and the mixture was stirred

at the same temperature for 3 h. The solution was diluted with

EtOAc, washed with water and brine, dried over MgSO₄, and con-

centrated in vacuo. The residue was puriﬁed by silica gel column

chromatography (EtOAc). Recrystallization from EtOAc/hexane

gave 8 (0.55 g, 93%) as a pale yellow solid: mp 174–176 °C; ¹H

NMR (CDCl₃) d 3.47 (3H, s), 4.23 (2H, d, J = 2.1 Hz), 5.30 (2H, s),

described for 7h. Compound 7f (68 mg, 64%) was obtained as a pale

yellow solid: mp 233–234 °C (MeOH/Et₂O); ¹H NMR (DMSO-d₆) d

1.30 (3H, d, J = 6.6 Hz), 2.65–2.85 (7H, m), 2.90–3.10 (1H, m),

3.70–3.90 (1H, m), 6.75–6.90 (3H, m), 7.43 (1H, d, J = 7.5 Hz),

7.54 (1H, d, J = 3.3 Hz), 7.59 (1H, t, J = 7.8 Hz), 7.82 (1H, d,

J = 9.0 Hz), 8.00 (1H, s), 8.08 (1H, s), 8.15 (1H, s), 8.20–8.30 (1H,

m), 10.03 (1H, br s), 10.65 (1H, s), 11.32 (1H, s), 12.34 (1H, s); Anal.

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 5157–5171

5167

6.62 (1H, dd, J = 3.3, 1.5 Hz), 6.84–6.91 (1H, m), 7.03 (1H, dd,

J = 10.8, 2.4 Hz), 7.40 (1H, dd, J = 3.6, 0.9 Hz), 7.53–7.66 (4H, m),

7.97 (1H, s), 8.00–8.05 (2H, m), 8.38 (1H, br s), 8.82 (1H, br s).

5.30. N-[3-Cyano-4-{3-[(N,N-diethylglycyl)amino]phenyl}-6-(4-

ﬂuoro-2-hydroxyphenyl)pyridin-2-yl]furan-2-carboxamide

hydrochloride (10b)

Compound 10b was prepared from 9b (150 mg, 0.26 mmol) in a

manner similar to that described for 6a. Compound 10b (130 mg,

5.26. N-(3-Cyano-4-{3-[(N,N-dimethylglycyl)amino]phenyl}-6-

[4-ﬂuoro-2-(methoxymethoxy)phenyl]pyridin-2-yl)furan-2-

carboxamide (9a)

89%) was obtained as

a pale yellow solid: mp 262–263 °C

(MeOH/Et₂O); ¹H NMR (DMSO-d₆) d 1.25 (6H, t, J = 7.2 Hz), 3.26

(4H, br s), 4.20 (2H, d, J = 3.9 Hz), 6.79–6.89 (3H, m), 7.49 (1H, d,

J = 8.1 Hz), 7.54 (1H, dd, J = 3.6, 0.6 Hz), 7.63 (1H, t, J = 8.1 Hz),

7.82–7.86 (1H, m), 8.03–8,04 (1H, m), 8.08 (1H, dd, J = 1.5,

0.6 Hz), 8.16 (1H, s), 8.23 (1H, dd, J = 8.4, 6.9 Hz), 9.75 (1H, br s),

11.18 (1H, s), 11.34 (1H, s), 12.31 (1H, s); Anal. Calcd for

A mixture of compound 8 (200 mg, 0.37 mmol), dimethylamine

hydrochloride (60 mg, 0.74 mmol), and K₂CO₃(150 mg, 0.11 mmol)

in DMF (2 ml) was stirred at room temperature for 14 h. The solu-

tion was diluted with EtOAc, washed with water twice and brine,

dried over MgSO₄, and concentrated in vacuo. The residue was puri-

ﬁed by basic silica gel column chromatography (hexane to EtOAc).

Recrystallization from EtOAc/hexane gave 9a (110 mg, 55%) as a

colorless solid: mp 174–176 °C; ¹H NMR (CDCl₃) d 2.41 (6H, s),

3.11 (2H, s), 3.47 (3H, s), 5.30 (2H, s), 6.62 (1H, q, J = 1.8 Hz),

6.84–6.91 (1H, m), 7.02 (1H, dd, J = 10.8 Hz, 2.4 Hz), 7.39 (1H, dd,

J = 3.6 Hz, 0.9 Hz), 7.45–7.59 (2H, m), 7.60 (1H, s), 7.73–7.77 (1H,

m), 7.98–8.04 (3H, m), 8.81 (1H, s), 9.30 (1H, s).

C

₂₉H₂₆FN₅O₄ꢀHClꢀ0.25H₂O: C, 61.27; H, 4.88; N, 12.32. Found: C,

61.22; H, 4.85; N, 12.30.

5.31. N-[3-Cyano-6-(4-ﬂuoro-2-hydroxyphenyl)-4-{3-[(pyrr-

olidin-1-ylacetyl)amino]phenyl}pyridin-2-yl]furan-2-carbox-

amide hydrochloride (10c)

Compound 10c was prepared from 9c (150 mg, 0.26 mmol) in a

manner similar to that described for 6a. Compound 10c (140 mg,

5.27. N-(3-Cyano-4-{3-[(N,N-diethylglycyl)amino]phenyl}-6-[4-

ﬂuoro-2-(methoxymethoxy)phenyl]pyridin-2-yl)furan-2-

carboxamide (9b)

96%) was obtained as

a pale yellow solid: mp 203–205 °C

(MeOH/Et₂O); ¹H NMR (DMSO-d₆) d 1.94–2.00 (4H, m), 3.17 (2H,

br s), 3.63 (2H, br s), 4.32 (2H, s), 6.79–6.90 (3H, m), 7.47 (1H, d,

J = 8.1 Hz), 7.55 (1H, dd, J = 0.9 Hz, 3.6 Hz), 7.63 (1H, t, J = 8.1 Hz),

7.82–7.85 (1H, m), 8.03 (1H, br s), 8.08 (1H, dd, J = 0.6 Hz,

1.5 Hz), 8.16 (1H, s), 8.24 (1H, dd, J = 6.9 Hz, 8.7 Hz), 10.33 (1H,

br s), 11.10 (1H, s), 11.35 (1H, s), 12.33 (1H, s); Anal. Calcd for

Compound 9b was prepared from 8 (300 mg, 0.56 mmol) and

diethylamine (0.12 ml, 1.2 mmol) in a manner similar to that de-

scribed for 9a. Compound 9b (230 mg, 72%) was obtained as a col-

orless solid: mp 126–128 °C (EtOAc/hexane); ¹H NMR (CDCl₃) d

1.11 (6H, t, J = 7.2 Hz), 2.68 (4H, q, J = 7.2 Hz), 3.17 (2H, s), 3.47

(3H, s), 5.30 (2H, s), 6.62 (1H, dd, J = 3.6, 1.8 Hz), 6.84–6.91 (1H,

m), 7.03 (1H, dd, J = 10.8, 2.4 Hz), 7.39 (1H, dd, J = 3.6, 0.9 Hz),

7.45–7.54 (2H, m), 7.59–7.60 (1H, m), 7.67–7.70 (1H, m), 7.98–

8.05 (3H, m), 8.82 (1H, s), 9.58 (1H, br s).

C

₂₉H₂₄FN₅O₄ꢀHClꢀH₂O: C, 60.05; H, 4.69; N, 12.07. Found: C,

59.90; H, 4.69; N, 12.02.

5.32. 2-Amino-4-(3-bromophenyl)-6-[4-ﬂuoro-2-(methoxyme-

thoxy)phenyl]pyridine-3-carbonitrile (12)

A

mixture of 1-[4-ﬂuoro-2-(methoxymethoxy)phenyl]etha-

5.28. N-(3-Cyano-6-[4-ﬂuoro-2-(methoxymethoxy)phenyl]-4-

{3-[(pyrrolidin-1-ylacetyl)amino]phenyl}pyridin-2-yl)furan-2-

carboxamide (9c)

none¹³(11, 5.0 g, 25.3 mmol), 3-bromobenzaldehyde (4.68 g,

25.23 mmol), malononitrile (1.67 g, 25.3 mmol), and NH₄OAc

(2.92 g, 37.9 mmol) in toluene (60 ml) was stirred under reﬂux

for 18 h. The reaction mixture was diluted with saturated aque-

ous NaHCO₃and extracted with EtOAc. The extract was washed

with brine, dried over MgSO₄, and concentrated in vacuo. The

residue was recrystallized from EtOAc/hexane to give 12

(3.28 g, 30%) as a yellow solid: mp 165–166 °C; ¹H NMR (CDCl₃)

d 3.46 (3H, s), 5.22 (2H, s), 5.34 (2H, br s), 6.81–6.88 (1H, m),

6.98 (1H, dd, J = 10.8, 2.4 Hz), 7.29 (1H, s), 7.39 (1H, t,

J = 7.8 Hz), 7.57–7.65 (2H, m), 7.73 (1H, t, J = 1.8 Hz), 7.79 (1H,

dd, J = 8.7, 6.9 Hz).

Compound 9c was prepared from 8 (250 mg, 0.47 mmol) and

pyrrolidine (37 mg, 0.52 mmol) in a manner similar to that de-

scribed for 9a. Compound 9c (180 mg, 67%) was obtained as a col-

orless solid: mp 152–154 °C (EtOAc/hexane); ¹H NMR (CDCl₃) d

1.87–1.91 (4H, m), 2.73 (4H, br s), 3.32 (2H, s), 3.47 (3H, s), 5.30

(2H, s), 6.62 (1H, dd, J = 1.8 Hz, 3.6 Hz), 6.84–6.91 (1H, m), 7.02

(1H, dd, J = 2.4 Hz, 10.8 Hz), 7.39–7.40 (1H, m), 7.45–7.54 (2H,

m), 7.59–7.60 (1H, m), 7.69–7.73 (1H, m), 7.98–8.04 (3H, m),

8.82 (1H, br s), 9.28 (1H, br s).

5.33. N-{4-(3-Bromophenyl)-3-cyano-6-[4-ﬂuoro-2-(methoxy-

methoxy)phenyl]pyridin-2-yl}furan-2-carboxamide (13)

5.29. N-[3-Cyano-4-{3-[(N,N-dimethylglycyl)amino]phenyl}-6-

(4-ﬂuoro-2-hydroxyphenyl)pyridin-2-yl]furan-2-carboxamide

hydrochloride (10a)

To a solution of 12 (2.2 g, 5.1 mmol) in pyridine (30 ml) was

added 2-furoyl chloride (1.7 g, 12.8 mmol) at room temperature

and the whole was stirred at room temperature for 18 h. The reac-

tion mixture was diluted with EtOAc, washed with water and

brine, dried over MgSO₄, and concentrated in vacuo. The residue

was puriﬁed by basic silica gel column chromatography (EtOAc/

hexane = 1/1 to 2/1) and triturated with diisopropyl ether to give

13 (2.4 g, 90%) as a colorless solid: ¹H NMR (CDCl₃) d 3.49 (3H,

s), 5.27 (2H, s), 6.62 (1H, q, J = 1.8 Hz), 6.85–6.91 (1H, m), 7.01

(1H, dd, J = 10.8, 1.8 Hz), 7.39–7.46 (2H, m), 7.60 (1H, d,

J = 0.9 Hz), 7.66–7.69 (2H, m), 7.80 (1H, t, J = 1.8 Hz), 7.90 (1H, s),

8.01 (1H, t, J = 7.8 Hz), 8.81 (1H, s).

Compound 10a was prepared from 9a (110 mg, 0.20 mmol) in a

manner similar to that described for 6a. Compound 10a (102 mg,

95%) was obtained as

a pale yellow solid: mp 217–222 °C

(MeOH/Et₂O); ¹H NMR (DMSO-d₆) d 2.88 (6H, s), 4.19 (2H, s),

6.79–6.88 (3H, m), 7.48 (1H, d, J = 7.2 Hz), 7.54 (1H, d, J = 3.6 Hz),

7.63 (1H, t, J = 7.8 Hz), 7.83 (1H, d, J = 7.8 Hz), 8.02 (1H, s), 8.08

(1H, s), 8.16 (1H, s), 8.23 (1H, t, J = 7.8 Hz), 9.97 (1H, s), 11.06

(1H, s), 11.34 (1H, s), 12.32 (1H, s); Anal. Calcd for

C

₂₇H₂₂FN₅O₄ꢀHClꢀ1.5H₂O: C, 59.29; H, 4.79; N, 12.80. Found: C,

59.28; H, 4.57; N, 12.56.

5168

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 5157–5171

5.34. tert-Butyl 4-(3-{3-cyano-6-[4-ﬂuoro-2-(methoxymethoxy)

phenyl]-2-[(furan-2-ylcarbonyl)amino]pyridin-4-yl}phenyl)

piperazine-1-carboxylate (14a)

73%) was obtained as a pale yellow solid: mp 266–273 °C (MeOH/

Et₂O); ¹H NMR (DMSO-d₆) d 3.26 (4H, s), 3.50 (4H, s), 6.79–6.87

(3H, m), 7.22 (2H, t, J = 9.0 Hz), 7.30 (1H, s), 7.48–7.54 (2H, m),

8.08 (1H, s), 8.16 (1H, s), 8.25 (1H, t, J = 8.1 Hz), 8.80–9.20 (2H,

m), 11.10–11.50 (1H, m), 12.43 (1H, br s); Anal. Calcd for

A mixture of 13 (300 mg, 0.57 mmol), tert-butyl piperazine-1-

carboxylate (128 mg, 0.69 mmol), Xphos (55 mg, 0.11 mmol),

NaO^tBu (83 mg, 0.86 mmol), and Pd₂(dba)₃(53 mg, 0.057 mmol)

in toluene (15 ml) was stirred under reﬂux for 2 h. The reaction

mixture was diluted with saturated aqueous NaHCO₃and ex-

tracted with EtOAc. The extract was washed with brine, dried over

MgSO₄, and concentrated in vacuo. The residue was puriﬁed by sil-

ica gel column chromatography (EtOAc/hexane = 1/1) to give 14a

(300 mg, 84%) as a pale brown amorphous solid: ¹H NMR (CDCl₃)

d 1.49 (9H, s), 3.24 (4H, t, J = 5.1 Hz), 3.45 (3H, s), 3.61 (4H, t,

J = 5.1 Hz), 5.20 (2H, s), 6.61 (1H, q, J = 1.8 Hz), 6.84–6.90 (1H, m),

6.98–7.14 (3H, m), 7.27 (1H, s), 7.39–7.46 (2H, m), 7.58 (1H, t,

J = 0.9 Hz), 7.93 (1H, s), 8.00 (1H, dd, J = 8.7, 6.9 Hz), 8.84(1H, s).

C

₂₇H₂₂FN₅O₃ꢀHClꢀH₂O: C, 59.29; H, 4.79; N, 12.80. Found: C,

59.61; H, 4.97; N, 12.76.

5.39. N-{3-Cyano-4-[3-(1,4-diazepan-1-yl)phenyl]-6-(4-ﬂuoro-

2-hydroxyphenyl)pyridin-2-yl}furan-2-carboxamide hydro-

chloride (15b)

Compound 15b was prepared from 14b (140 mg, 0.22 mmol) in

a manner similar to that described for 6a. Compound 15b (108 mg,

86%) was obtained as a pale yellow solid: mp 254–259 °C (MeOH/

Et₂O); ¹H NMR (DMSO-d₆) d 2.09–2.20 (2H, m), 3.10–3.20 (2H, m),

3.30–3.40 (2H, m), 3.61 (2H, t, J = 5.7 Hz), 3.75–3.95 (2H, m), 6.79–

6.86 (3H, m), 6.99–7.07 (3H, m), 7.43 (1H, t, J = 7.8 Hz), 7.53 (1H, d,

J = 3.6 Hz), 8.08 (1H, t, J = 1.5 Hz), 8.13 (1H, s), 8.22 (1H, dd, J = 9.9,

6.9 Hz), 8.96 (2H, br s), 11.23 (1H, s), 12.41 (1H, s); Anal. Calcd for

5.35. tert-Butyl 4-(3-{3-cyano-6-[4-ﬂuoro-2-(methoxymethoxy)

phenyl]-2-[(furan-2-ylcarbonyl)amino]pyridin-4-yl}phenyl)-

1,4-diazepane-1-carboxylate (14b)

C

₂₈H₂₄FN₅O₃ꢀHClꢀ0.5H₂O: C, 61.94; H, 4.83; N, 12.90. Found: C,

61.86; H, 4.82; N, 12.64.

Compound 14b was prepared from 13 (300 mg, 0.57 mmol) and

tert-butyl homopiperazine-1-carboxylate (138 mg, 0.69 mmol) in a

manner similar to that described for 14a. Compound 14b (155 mg,

42%) was obtained as a pale yellow amorphous solid: ¹H NMR

(CDCl₃) d 1.39 (4H, s), 1.44 (5H, s), 2.00–2.15 (2H, m), 3.20–3.40

(2H, m), 3.46 (3H, s), 3.58–3.70 (6H, br s), 5.25 (2H, s), 6.61 (1H,

q, J = 1.8 Hz), 6.83–6.95 (3H, m), 6.99–7.03 (2H, m), 7.34–7.39

(2H, m), 7.59 (1H, t, J = 0.9 Hz), 7.94 (1H, s), 8.03 (1H, t,

J = 7.8 Hz), 8.81 (1H, s).

5.40. N-{3-Cyano-6-(4-ﬂuoro-2-hydroxyphenyl)-4-[3-(4-

methylpiperazin-1-yl)phenyl]pyridin-2-yl}furan-2-carbox-

amide hydrochloride (15c)

Compound 15c was prepared from 14c (95 mg, 0.18 mmol) in a

manner similar to that described for 6a. Compound 15c (82 mg,

80%) was obtained as

a pale yellow solid: mp 254–259 °C

(MeOH/Et₂O); ¹H NMR (DMSO-d₆) d 2.83 (3H, s), 3.10–3.30 (4H,

m), 3.40–3.65 (2H, m), 3.80–4.15 (2H, m), 6.79–6.87 (3H, m),

7.20–7.27 (2H, m), 7.32 (1H, s), 7.48–7.54 (2H, m), 8.08 (1H, s),

8.16 (1H, s), 8.25 (1H, dd, J = 9.9, 6.9 Hz), 10.62 (1H, br s), 11.25

(1H, s), 11.44 (1H, s); Anal. Calcd for C₂₈H₂₄FN₅O₃ꢀHClꢀH₂O: C,

60.93; H, 4.93; N, 12.68. Found: C, 61.32; H, 4.85; N, 12.69.

5.36. N-{3-Cyano-6-[4-ﬂuoro-2-(methoxymethoxy)phenyl]-4-

[3-(4-methylpiperazin-1-yl)phenyl]pyridin-2-yl}furan-2-

carboxamide (14c)

Compound 14c was prepared from 13 (300 mg, 0.57 mmol) and

1-methylpiperazine (69 mg, 0.69 mmol) in a manner similar to

that described for 14a. Compound 14c (103 mg, 53%) was obtained

as a pale yellow amorphous solid: ¹H NMR (CDCl₃) d 2.37 (3H, s),

2.60 (4H, t, J = 5.1 Hz), 3.32 (4H, t, J = 5.1 Hz), 3.46 (3H, s), 5.24

(2H, s), 6.61 (1H, q, J = 1.8 Hz), 6.85–6.91 (1H, m), 7.01 (1H, dd,

J = 10.8, 2.4 Hz), 7.06–7.12 (2H, m), 7.24–7.26 (1H, m), 7.39–7.45

(2H, m), 7.59 (1H, s), 7.93 (1H, s), 8.00 (1H, dd, J = 8.7, 7.2 Hz),

8.81 (1H, s).

5.41. N-{3-Cyano-4-[3-(4-ethylpiperazin-1-yl)phenyl]-6-(4-

ﬂuoro-2-hydroxyphenyl)pyridin-2-yl}furan-2-carboxamide

hydrochloride (15d)

Compound 15d was prepared from 14d (90 mg, 0.16 mmol) in a

manner similar to that described for 6a. Compound 15d (77 mg,

82%) was obtained as

a pale yellow solid: mp 218–222 °C

(MeOH/Et₂O); ¹H NMR (DMSO-d₆) d 1.29 (3H, t, J = 7.2 Hz), 3.10–

3.25 (6H, m), 3.59 (2H, d, J = 11.4 Hz), 3.98 (2H, d, J = 11.4 Hz),

6.79–6.87 (3H, m), 7.20–7.27 (2H, m), 7.33 (1H, s), 7.48–7.54

(2H, m), 8.08 (1H, t, J = 0.9 Hz), 8.16 (1H, s), 8.25 (1H, dd, J = 9.6,

6.9 Hz), 10.53 (1H, br s), 11.25 (1H, s), 12.44 (1H, s); Anal. Calcd

for C₂₉H₂₆FN₅O₃ꢀHClꢀH₂O: C, 61.54; H, 5.16; N, 12.37. Found: C,

61.72; H, 5.30; N, 12.34.

5.37. N-{3-Cyano-4-[3-(4-ethylpiperazin-1-yl)phenyl]-6-[4-

ﬂuoro-2-(methoxymethoxy)phenyl]pyridin-2-yl}furan-2-

carboxamide (14d)

Compound 14d was prepared from 13 (300 mg, 0.57 mmol) and

1-ethylpiperazine (79 mg, 0.69 mmol) in a manner similar to that

described for 14a. Compound 14d (94 mg, 30%) was obtained as

a pale yellow amorphous solid: ¹H NMR (CDCl₃) d 1.14 (3H, t,

J = 7.2 Hz), 2.49 (2H, q, J = 7.2 Hz), 2.63 (4H, t, J = 5.1 Hz), 3.30

(4H, t, J = 5.1 Hz), 3.46 (3H, s), 5.24 (2H, s), 6.61 (1H, q,

J = 1.8 Hz), 6.71–6.91 (1H, m), 7.00 (1H, dd, J = 10.8, 2.4 Hz), 7.06–

7.12 (2H, m), 7.23–7.26 (1H, m), 7.38–7.45 (2H, m), 7.59 (1H, q,

J = 0.9 Hz), 7.93 (1H, s), 8.00 (1H, dd, J = 8.7, 6.6 Hz), 8.81 (1H, s).

5.42. N-{3-Cyano-6-[4-ﬂuoro-2-(methoxymethoxy)phenyl]-4-

[3-(hydroxymethyl)phenyl]pyridin-2-yl}furan-2-carboxamide

(17)

To a solution of methyl 3-{3-cyano-6-[4-ﬂuoro-2-(methoxyme-

thoxy)phenyl]-2-[(furan-2-ylcarbonyl)amino]pyridin-4-yl}benzo-

ate¹³(16, 640 mg, 1.28 mmol) in THF (30 ml) was added LiBH₄

(56 mg, 2.55 mmol) and the mixture was stirred at 45 °C for 96 h.

The reaction mixture was diluted with saturated aqueous NaHCO₃

and extracted with EtOAc. The extract was washed with brine,

dried over MgSO₄, and concentrated in vacuo. The residue was

puriﬁed by silica gel column chromatography (EtOAc/hexane =

2/1) to give 17 (350 mg, 58%) as a yellow amorphous solid: ¹H

NMR (CDCl₃) d 3.45 (3H, s), 4.80 (2H, s), 5.24 (2H, s), 6.60 (1H, q,

5.38. N-[3-Cyano-6-(4-ﬂuoro-2-hydroxyphenyl)-4-(3-piperazin-

1-ylphenyl)pyridin-2-yl]furan-2-carboxamide hydrochloride

(15a)

Compound 15a was prepared from 14a (300 mg, 0.48 mmol) in

a manner similar to that described for 6a. Compound 15a (194 mg,

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 5157–5171

5169

J = 1.8 Hz), 6.83–6.90 (1H, m), 7.00 (1H, dd, J = 10.5, 2.4 Hz), 7.38

(1H, dd, J = 3.3, 0.6 Hz), 7.51–7.62 (4H, m), 7.70 (1H, s), 7.92 (1H,

s), 7.96 (1H, dd, J = 8.7, 6.9 Hz), 8.84 (1H, br s).

Calcd for C₂₈H₂₄FN₅O₃ꢀC₂HF₃O₂ꢀ1.5H₂O: C, 56.43; H, 4.40; N,

10.96. Found: C, 56.33; H, 4.09; N, 10.65.

5.47. N-{3-Cyano-4-[3-(1,4-diazepan-1-ylmethyl)phenyl]-6-(4-

ﬂuoro-2-hydroxyphenyl)pyridin-2-yl}furan-2-carboxamide

dihydrochloride (20b)

5.43. 3-{3-Cyano-6-[4-ﬂuoro-2-(methoxymethoxy)phenyl]-2-

[(furan-2-ylcarbonyl)amino]pyridin-4-yl}benzyl methane-

sulfonate (18)

Compound 20b was prepared from 19b (75 mg, 0.11 mmol) in a

manner similar to that described for 6a. Compound 20b (63 mg,

98%) was obtained as a pale yellow solid: mp 199–206 °C (MeOH/

Et₂O); ¹H NMR (DMSO-d₆) d 2.10–2.30 (2H, m), 3.10–3.80 (8H, m),

4.53 (2H, br s), 6.80–6.87 (3H, m), 7.55 (1H, d, J = 3.6 Hz), 7.73

(1H, t, J = 7.5 Hz), 7.85 (2H, br s), 8.09 (2H, br s), 8.37–8.43 (2H,

m), 9.32 (1H, br s), 9.60 (1H, br s), 11.34 (1H, s), 11.82 (1H, s),

12.54 (1H, s); Anal. Calcd for C₂₉H₂₆FN₅O₃ꢀ2HClꢀ1.5H₂O: C, 56.96;

H, 5.11; N, 11.45. Found: C, 57.05; H, 5.15; N, 11.43.

To a solution of 17 (90 mg, 0.19 mmol) and triethylamine

(38 mg, 0.38 mmol) in CH₂Cl₂(5 ml) was added methanesulfonyl

chloride (44 mg, 0.38 mmol). After stirring at room temperature

for 1 h, the reaction mixture was diluted with saturated aqueous

NaHCO₃and extracted with EtOAc. The extract was washed with

brine, dried over MgSO₄, and concentrated in vacuo. The residue

was puriﬁed by silica gel column chromatography (EtOAc/hex-

ane = 1/1) to give 18 (60 mg, 57%) as a colorless amorphous solid:

¹H NMR (CDCl₃) d 3.02 (3H, s), 3.47 (3H, s), 5.27 (2H, s), 5.34 (2H, s),

6.62 (1H, q, J = 1.8 Hz), 6.85–6.92 (1H, m), 7.02 (1H, dd, J = 10.5,

2.4 Hz), 7.40 (1H, dd, J = 3.6, 0.6 Hz), 7.60–7.61 (3H, m), 7.72–

7.76 (2H, m), 7.94 (1H, s), 8.02 (1H, dd, J = 8.7, 6.9 Hz), 8.81 (1H, s).

5.48. N-[3-Cyano-6-(4-ﬂuoro-2-hydroxyphenyl)-4-{3-[(4-meth

ylpiperazin-1-yl)methyl]phenyl}pyridin-2-yl]furan-2-carbox-

amide dihydrochloride (21a)

Compound 21a was prepared from 20a (60 mg, 0.083 mmol)

and formaldehyde (37%, 0.5 ml, 3.20 mmol) in a manner similar

to that described for 7h. Compound 21a (40 mg, 82%) was obtained

as a pale yellow solid: mp 205–210 °C (MeOH/Et₂O); ¹H NMR

(DMSO-d₆) d 2.73 (3H, s), 3.30–4.50 (10H, m), 6.80–6.87 (3H, m),

7.54 (1H, d, J = 3.6 Hz), 7.62–7.81 (3H, m), 7.90–8.05 (1H, m),

8.08 (1H, s), 8.32–8.39 (2H, m), 11.32 (1H, s), 12.51 (1H, br s); Anal.

Calcd for C₂₉H₂₆FN₅O₃ꢀ2HClꢀ1.5H₂O: C, 56.96; H, 5.11; N, 11.45.

Found: C, 56.93; H, 5.16; N, 11.45.

5.44. tert-Butyl 4-(3-{3-cyano-6-[4-ﬂuoro-2-(methoxymethoxy)

phenyl]-2-[(furan-2-ylcarbonyl)amino]pyridin-4-yl}benzyl)

piperazine-1-carboxylate (19a)

A solution of 18 (60 mg, 0.11 mmol), tert-butyl piperazine-1-

carboxylate (24 mg, 0.13 mmol), and triethylamine (14 mg,

0.13 mmol) in THF (5 ml) was stirred at room temperature for

18 h. The mixture was diluted with saturated aqueous NaHCO₃

and extracted with EtOAc. The organic layer was washed with

brine, dried over MgSO₄, and concentrated in vacuo. The residue

was puriﬁed by silica gel column chromatography (EtOAc/hex-

ane = 1/1 to 2/1) to give 19a (40 mg, 57%) as a pale yellow amor-

phous solid: ¹H NMR (CDCl₃) d 1.45 (9H, s), 2.44 (4H, s), 3.46

(7H, s), 3.61 (2H, s), 5.25 (2H, s), 6.62 (1H, q, J = 1.8 Hz), 6.89–

6.91 (1H, m), 7.01 (1H, dd, J = 10.8, 2.4 Hz), 7.40 (1H, q,

J = 2.4 Hz), 7.49–7.60 (4H, m), 7.72 (1H, s), 7.92 (1H, s), 8.00 (1H,

dd, J = 8.7, 6.6 Hz), 8.82 (1H, s).

5.49. N-[3-Cyano-4-{3-[(4-ethylpiperazin-1-yl)methyl]phenyl}-

6-(4-ﬂuoro-2-hydroxyphenyl)pyridin-2-yl]furan-2-carbox-

amide dihydrochloride (21b)

Compound 21b was prepared from 20a (60 mg, 0.083 mmol)

and acetaldehyde (97%, 0.5 ml, 9.53 mmol) in a manner similar to

that described for 7h. Compound 21b (41 mg, 83%) was obtained

as a pale yellow solid: mp 215–222 °C (MeOH/Et₂O); ¹H NMR

(DMSO-d₆) d 1.25 (3H, t, J = 7.2 Hz), 3.10–4.50 (12H, m), 6.80–

6.87 (3H, m), 7.54 (1H, d, J = 3.6 Hz), 7.68–7.82 (3H, m), 7.95–

8.05 (1H, m), 8.09 (1H, s), 8.33–8.39 (2H, m), 11.33 (1H, s), 12.52

(1H, br s); Anal. Calcd for C₃₀H₂₈FN₅O₃ꢀ2HClꢀ1.5H₂O: C, 57.60; H,

5.32; N, 11.20. Found: C, 57.88; H, 5.23; N, 11.14.

5.45. tert-Butyl 4-(3-{3-cyano-6-[4-ﬂuoro-2-(methoxymethoxy)

phenyl]-2-[(furan-2-ylcarbonyl)amino]pyridin-4-yl}benzyl)-

1,4-diazepane-1-carboxylate (19b)

Compound 19b was prepared from 18 (100 mg, 0.18 mmol) and

tert-butyl homopiperazine-1-carboxylate (54 mg, 0.27 mmol) in a

manner similar to that described for 19a. Compound 19b (80 mg,

68%) was obtained as a pale yellow amorphous solid: ¹H NMR

(DMSO-d₆) d 1.45 (9H, s), 1.75–1.94 (2H, m), 2.62–2.78 (4H, m),

3.43–3.55 (7H, m), 3.72 (2H, s), 5.25 (2H, s), 6.61 (1H, q,

J = 1.8 Hz), 6.84–6.90 (1H, m), 7.00 (1H, dd, J = 10.8, 2.4 Hz), 7.39

(1H, d, J = 3.6 Hz), 7.49–7.59 (4H, m), 7.69 (1H, s), 7.92 (1H, s),

8.00 (1H, dd, J = 8.7, 6.9 Hz), 8.85 (1H, s).

5.50. N-{3-Cyano-4-(3-{[2-(dimethylamino)ethyl]carbamoyl}

phenyl)-6-[4-ﬂuoro-2-(methoxymethoxy)phenyl]pyridin-2-yl}

furan-2-carboxamide (23)

Compound 23 was prepared from 3-{3-cyano-6-[4-ﬂuoro-2-

(methoxymethoxy)phenyl]-2-[(furan-2-ylcarbonyl)amino]pyridin-

4-yl}benzoic acid¹³(22, 200 mg, 0.41 mmol) and N,N-dimethyleth-

ylenediamine (43 mg, 0.49 mmol) in a manner similar to that

described for 5a. Compound 23 (90 mg, 39%) was obtained as a

pale brown amorphous solid: ¹H NMR (CDCl₃) d 2.29 (6H, s), 2.56

(2H, t, J = 5.7 Hz), 3.47 (3H, s), 3.56 (2H, q, J = 5.5 Hz), 5.27 (2H,

s), 6.61 (1H, q, J = 1.8 Hz), 6.84–6.90 (1H, m), 7.01 (1H, dd,

J = 10.8, 2.4 Hz), 7.13 (1H, br s), 7.39 (1H, dd, J = 3.6, 0.6 Hz),

7.59–7.64 (2H, m), 7.83–7.86 (1H, m), 7.95–8.01 (3H, m), 8.15

(1H, d, J = 1.5 Hz), 8.70–9.10 (1H, m).

5.46. N-{3-Cyano-6-(4-ﬂuoro-2-hydroxyphenyl)-4-[3-(pipera-

zin-1-ylmethyl)phenyl]pyridin-2-yl}furan-2-carbox- amide

triﬂuoroacetate (20a)

To a solution of 19a (50 mg, 0.078 mmol) in CH₂Cl₂(2 ml) was

added TFA (1 ml) and the mixture was stirred at room temperature

for 1 h. The solution was diluted with Et₂O and the precipitate was

collected by ﬁltration. Recrystallization from MeOH/Et₂O gave 20a

(29 mg, 35%) as a colorless solid: mp 144–150 °C; ¹H NMR (DMSO-

d₆) d 2.66 (4H, br s), 3.13 (4H, br s), 3.73 (2H, s), 6.80–6.87 (3H, m),

7.52–7.74 (5H, m), 8.08 (1H, d, J = 0.9 Hz), 8.18 (1H, s), 8.26 (1H, dd,

J = 9.6, 6.9 Hz), 8.59 (2H, br s), 11.29 (1H, s), 12.40 (1H, s); Anal.

5.51. N-[3-Cyano-4-(3-{[2-(dimethylamino)ethyl]carbamoyl}

phenyl)-6-(4-ﬂuoro-2-hydroxyphenyl)pyridin-2-yl]furan-2-

carboxamide hydrochloride (24)

Compound 24 was prepared from 23 (80 mg, 0.14 mmol) in a

manner similar to that described for 6a. Compound 24 (60 mg,

5170

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 5157–5171

76%) was obtained as a colorless solid: mp 200–204 °C (MeOH/

Et₂O); ¹H NMR (DMSO-d₆) d 2.84 (6H, s), 3.26–3.33 (2H, m),

3.65–3.69 (2H, m), 6.80–6.87 (3H, m), 7.54 (1H, d, J = 3.6 Hz),

7.76 (1H, t, J = 7.8 Hz), 7.93 (1H, d, J = 7.8 Hz), 8.08–8.14 (2H, m),

8.26–8.35 (3H, m), 9.03 (1H, t, J = 5.4 Hz), 9.89 (1H, br s), 11.32

(1H, s), 12.43 (1H, s); Anal. Calcd for C₂₈H₂₄FN₅O₄ꢀHClꢀ1.5H₂O: C,

58.28; H, 4.89; N, 12.14. Found: C, 58.49; H, 4.69; N, 12.12.

gen) in 5% CO₂/95% air atmosphere. Cells were harvested within

50% conﬂuency in Ca²⁺–Mg²⁺-free phosphate buffered saline con-

taining 1% EDTA and centrifuged (2500 rpm, 4 °C, 10 min, HIMAC

rotor #RP24A-132). After twice washed with the same buffer, cells

were homogenized in ice-cold homogenization buffer (10 mM

NaHCO₃, 2 mM EGTA, 0.2 mM Magnesium acetate, 10

peptin, 10 g/ml E-64, 100 g/ml o-phenanthroline, 90

phenylmethylsulfonyl ﬂuoride (PMSF), 10 g/ml Pepstatin A, pH

lg/ml Leu-

l

g/ml

l

5.52. tert-Butyl {2-[(3-{3-cyano-6-[4-ﬂuoro-2-(methoxy-

methoxy)phenyl]-2-[(furan-2-ylcarbonyl)amino]pyridin-4-yl}

phenyl)amino]ethyl}carbamate (25)

7.3 at 25 °C) and collected by centrifugation (30,000 rpm, 1 h,

4 °C, HIMAC rotor #RP42-767). Pellets were resuspended in ice-

cold stock buffer (20 mM Tris, 250 mM sucrose, 2 mM EGTA,

10

90

at ꢂ80 °C.

l

g/ml Leupeptin, 10

lg/ml E-64, 100 lg/ml o-phenanthroline,

To a solution of 4 (500 mg, 1.09 mmol), N-Boc-2-aminoacetal-

dehyde (347 mg, 2.18 mmol), and AcOH (1.0 ml) in THF (10 ml)

was added NaBH(OAc)₃(462 mg, 2.18 mmol) and the whole was

stirred at room temperature for 18 h. The mixture was diluted with

saturated aqueous NaHCO₃and extracted with EtOAc. The organic

layer was washed with brine, dried over MgSO₄, and concentrated

in vacuo. The residue was puriﬁed by silica gel column chromatog-

raphy (EtOAc/hexane = 1/1) and recrystallization from Et₂O to give

25 (395 mg, 60%) as a yellow solid: mp 145–146 °C; ¹H NMR

(CDCl₃) d 1.45 (9H, s), 3.33–3.52 (7H, m), 4.11 (1H, br s), 4.34

(1H, br s), 5.25 (2H, s), 6.61 (1H, q, J = 1.8 Hz), 6.75 (1H, dd,

J = 8.1, 1.8 Hz), 6.85–7.03 (4H, m), 7.26 (1H, s), 7.32 (1H, t,

J = 7.8 Hz), 7.38 (1H, dd, J = 3.0, 0.6 Hz), 7.92 (1H, s), 8.01 (1H, dd,

J = 8.4, 6.6 Hz), 8.80 (1H, s).

g/ml PMSF, 10 g/ml Pepstatin A, pH 7.4 at 25 °C) and stored

l

5.56. Receptor binding assay

The procedure of this binding assay was based on the method of

Ohtaki et al.¹⁷The frozen membranes were suspended in binding

buffer (20 mM Tris, 2.5 mM magnesium acetate, 2 mM EGTA,

100

10

l

g/ml o-phenanthroline, 90

g/ml Pepstatin A, 10 g/ml E-64, and 1 mg/ml BSA, pH 7.4).

lg/ml PMSF, 10 lg/ml Leupeptin,

l

Membranes were incubated with 135 pM ¹²⁵I-metatsin (40–54)

(the Peptide Institute products labeled with ¹²⁵I by lactoperoxidase

method) and test compounds at 25 °C for 60 min. The reaction

mixtures were ﬁltrated using GF/C ﬁlter plates pretreated with

0.3% polyethyleneimine by a cell harvester (PerkinElmer). After ﬁl-

5.53. N-[4-{3-[(2-Aminoethyl)amino]phenyl}-3-cyano-6-(4-

ﬂuoro-2-hydroxyphenyl)pyridin-2-yl]furan-2-carboxamide

dihydrochloride (26)

tration, the ﬁlter plates were washed with three times 300 lL buf-

fer (50 mM Tris, 5 mM MgCl₂, 1 mM EDTA, 0.05% CHAPS, 0.05%

NaN₃, 0.1% BSA, pH 7.4). The ﬁlter plates were dried and the radio-

activity was determined after addition of 30

TopCount (PerkinElmer). Nonspeciﬁc binding was deﬁned in the

presence of 1.8 M metastin (45–54).

lL Microscint-0 using

Compound 26 was prepared from 25 (200 mg, 0.33 mmol) in a

manner similar to that described for 6a. Compound 26 (120 mg,

69%) was obtained as a yellow solid: mp 203–205 °C (MeOH); ¹H

l

NMR (DMSO-d₆)

d

2.91–3.11 (2H, m), 3.31–3.46 (2H, m),

5.57. Preparation of CHO cell lines

6.74–6.98 (6H, m), 7.29–7.41 (1H, m), 7.53 (1H, d, J = 3.6 Hz),

7.90–8.15 (5H, m), 8.18–8.29 (1H, m), 11.24 (1H, s), 12.39 (1H, br

s); Anal. Calcd for C₂₅H₂₀FN₅O₃ꢀ2HClꢀH₂O: C, 54.75; H, 4.41; N,

12.77. Found: C, 54.81; H, 4.32; N, 12.77.

CHO cell lines stably expressing human GPR54 (cell line: h175-

KB33, h175-16) were used for Ca²⁺mobilization assays. Cells were

cultured in Eagle’s Minimum Essential Medium (MEM) supple-

mented with 10% dFBS, 100 U/ml penicillin, and 100

mycin in 5% CO₂/95% air atmosphere.

lg/ml strepto-

5.54. N-[3-Cyano-4-(3-{[2-(dimethylamino)ethyl](methyl)

amino}phenyl)-6-(4-ﬂuoro-2-hydroxyphenyl)pyridin-2-yl]

furan-2-carboxamide (27)

5.58. Ca²⁺Mobilization assay

To a solution of 26 (50 mg, 0.094 mmol), formaldehyde (37%,

46 mg, 0.57 mmol), and AcOH (0.10 ml) in MeOH (5 ml) was added

NaBH₃(CN) (35 mg, 0.56 mmol) and the whole was stirred at room

temperature for 3 days. The mixture was diluted with saturated

aqueous NaHCO₃and extracted with EtOAc. The organic layer

was washed with brine, dried over MgSO₄, and concentrated in va-

cuo. The residue was puriﬁed by recrystallization from EtOAc/hex-

ane to give 27 (36 mg, 77%) as a yellow solid: mp 195–197 °C; ¹H

NMR (CDCl₃) d 2.33 (6H, s), 2.57 (2H, t, J = 7.5 Hz), 3.06 (3H, s),

3.55 (2H, t, J = 7.5 Hz), 6.59–6.68 (2H, m), 6.79 (1H, dd, J = 10.6,

2.6 Hz), 6.84–6.93 (3H, m), 7.36–7.47 (2H, m), 7.59–7.67 (2H, m),

7.80 (1H, dd, J = 9.1, 6.4 Hz), 9.21 (1H, s), 13.60 (1H, d, J = 1.3 Hz);

Anal. Calcd for C₂₈H₂₆FN₅O₃ꢀ0.25H₂O: C, 66.72; H, 5.30; N, 13.89.

Found: C, 66.45; H, 5.12; N, 13.78.

Cells were plated at 30,000 cells/well in 96-well plate (type

3904, Corning) and cultured overnight. The cells were incubated

in Ca²⁺assay kit solution (115 mM NaCl, 5.4 mM KCl, 0.8 mM

MgCl₂, 1.8 mM CaCl₂, 20 mM Hepes, 13.8 mM

D-glucose; pH 7.4,

Dojin) containing 0.1% BSA, 2.5 g/ml Fluo-3AM, 1.25 mM proben-

l

ecid and 0.04% Pluronic F-127 for 60 min at 37 °C. They were

washed with Ca²⁺assay kit solution containing 0.1% BSA and

1.25 mM probenecid at 37 °C. After washing cells, the Ca²⁺mobili-

zation was detected in a ﬂuorometric imaging plate reader (FLIPR,

Molecular Devices) as described in the FLIPR system manual.

Antagonist activities of test compounds in h175-KB33 and h175-

16 were determined as inhibitory response to 30 nM and 100 pM

metastin (40–54), respectively. The 50% inhibitory concentration

(IC₅₀) was used to calculate by non-linear regression analysis in

GraphPad Prism software (GraphPad Software Inc.).

5.55. Preparation of CHO membranes for GPR54 binding assay

5.59. Brain and plasma concentration in rats

CHO cell lines stably expressing human GPR54 (h175-KB34) or

rat GPR54 (r175-KB29) were used for receptor binding assays. Cells

were cultured in Dulbecco’s Modiﬁed Eagle Medium (D-MEM) sup-

plemented with 10% dialyzed fetal bovine serum (dFBS), 1ꢃ Non-

Test compounds were administrated intravenously as a cassette

dosing to male Crj/CD(SD)(IGS) rats. After dosing, blood samples

were collected into heparinized tubes, centrifuged to obtain

plasma. The brain was immediately harvested, weighted, homoge-

Essential Amino Acid (Invitrogen), 50 lg/ml gentamycin (Invitro-

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 5157–5171

5171

nized with saline to 20% (w/v). The plasma and brain homogenate

samples were deproteinized with acetonitrile, followed by centri-

fugation to obtain supernatants. The supernatants were diluted

with 0.01 M ammonium formate (0.2% formic acid) in water/aceto-

nitrile (9:1, v/v) containing internal standard. The supernatants

were injected into a LC/MS/MS system to measure the compound

concentrations.

tion at 15,000 rpm for 10 min. The supernatant was stored at

ꢂ20 °C until used for Radioimmunoassay (RIA) to measure the

plasma LH levels.

References and notes

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Caco-2 monolayers were grown to conﬂuence on collagen-

coated, microporous, polycarbonate membranes in 12-well Costar

Transwell^Òplates. The permeability assay buffer was Hanks’ Bal-

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lL were taken from

l

tion was performed in duplicate. The permeability through a cell-

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P_app¼ ðdC_r=dtÞ ꢃ V_r=A ꢃ C₀

ð1Þ

Percent Recovery ¼ 100 ꢃ ððV_rꢃ C^finalÞ þ ðV_dꢃ C^finalÞÞ=ðV_dꢃ C₀Þ

r

d

ð2Þ

where, dC_r/dt is the slope of the cumulative concentration in the re-

ceiver compartment versus time in l

mol/L s^ꢂ1. V_ris the volume of

the receiver compartment in cm³. V_dis the volume of the donor

compartment in cm³. A is the area of the cell monolayer (1.1 cm²

for 12-well Transwell^Ò). C₀is the nominal concentration of the dos-

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2571 (S)-isomer was prepared in the same manner.

ing solution in

l

mol/L. C^f_r^inalis the cumulative receiver concentra-

tion in

l

mol/L at the end of the incubation period. C^f_d^inalis the

concentration of the donor in

lmol/L at the end of the incubation

period.

5.61. In vivo evaluation in castrated rat model

Male Wister rats were purchased from SLC Japan. These animals

were 11 weeks old when subjected for the test, and castration was

performed 7 days prior to the test. The compound 15a was sus-

pended in 20% DMSO-40% PEG400 to the ﬁnal concentration at

1.5 mmol/L. Administration of the compound 15a at 0.22 mg/kg

(0.8 ml/kg, n = 8) was repeated ﬁve times with 10 min intervals,

through the jugular catheter, ending up with a total dosage at

1.1 mg/kg. The vehicle without compound was used as a negative

control (n = 8). The blood sample was collected through the jugular

catheter immediately before the administration and 25, 45, 60, 90,

120 and 180 min after the administration. The plasma was pre-

pared by the treatment with aprotinin–EDTA and the centrifuga-

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Article Doi

2-Acylamino-4,6-diphenylpyridine derivatives as novel GPR54 antagonists with good brain exposure and in vivo efficacy for plasma LH level in male rats

DOI: 10.1016/j.bmc.2010.05.061

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Article abstract of DOI:10.1016/j.bmc.2010.05.061

Full text of DOI:10.1016/j.bmc.2010.05.061

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