Synthesis and structure-activity relationships of 2-acylamino-4,6-diphenylpyridine derivatives as novel antagonists of GPR54

Article abstract of DOI:10.1016/j.bmc.2010.04.036

GPR54 is a G protein-coupled receptor (GPCR) which was formerly an orphan receptor. Recent functional study of GPR54 revealed that the receptor has an essential role to modulate sex-hormones including GnRH. Though antagonists of GPR54 are expected to be novel drugs for sex-hormone dependent diseases such as prostate cancer or endometriosis, small molecule GPR54 antagonists have not been reported. We have synthesized a series of 2-acylamino-4,6-diphenylpyridines to identify potent GPR54 antagonists. Detailed structure-activity relationship studies led to compound 9l with an IC₅₀ value of 3.7 nM in a GPR54 binding assay, and apparent antagonistic activity in a cellular functional assay.

Full text of DOI:10.1016/j.bmc.2010.04.036

Bioorganic & Medicinal Chemistry 18 (2010) 3841–3859
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationships of 2-acylamino-4,6-diphenyl-
pyridine derivatives as novel antagonists of GPR54
a
a
b
b
Toshitake Kobayashi a, , Satoshi Sasaki , Naoki Tomita , Seiji Fukui , Noritaka Kuroda ,
*
Masaharu Nakayama b, Atsushi Kiba a, Yoshihiro Takatsu a, Tetsuya Ohtaki a, Fumio Itoh b, Atsuo Baba a
a Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd, 10 Wadai, Tsukuba-shi, Ibaraki 300-4293, Japan
b Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd, 2-17-85, Jusohonmachi, 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 has an essential role to modulate sex-hormones including
GnRH. Though antagonists of GPR54 are expected to be novel drugs for sex-hormone dependent diseases
such as prostate cancer or endometriosis, small molecule GPR54 antagonists have not been reported. We
have synthesized a series of 2-acylamino-4,6-diphenylpyridines to identify potent GPR54 antagonists.
Detailed structure–activity relationship studies led to compound 9l with an IC50 value of 3.7 nM in a
GPR54 binding assay, and apparent antagonistic activity in a cellular functional assay.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 2 April 2010
Revised 14 April 2010
Accepted 15 April 2010
Available online 20 April 2010
Keywords:
GPR54 antagonist
2-Acylamino-4,6-diphenylpyridine
Metastin
Kisspeptin
1. Introduction
In a recent study, GPR54-metastin signaling was revealed to be
essential to initiate gonadotropin secretion.7 Central or peripheral
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.1 GPR54 was identified
as an orphan receptor in rat in 1999,2 and later Ohtaki et al. discov-
ered that a 54-amino-acid product of a gene called Kiss-1 was its
endogenous ligand.3 As 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.5a In 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,6 Individuals with IHH fail to undergo puberty and are
infertile because of failure to secrete the gonadotropic hormones,
such as follicle stimulating hormone (FSH) and luteinizing hor-
mone (LH) from the pituitary.
administration of metastin stimulates a dose-dependent rise in
serum levels of LH and FSH in adult rat,7,8 mice,9 sheep, macaques,
and humans,10 while 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,11 therefore, 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,11,12 Thus, 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 gonadtropic hormones and such com-
pounds would be novel orally available drugs for sex hormone-
dependant diseases, including prostate cancer and endometriosis,
however, small molecule GPR54 antagonists have yet to be
reported.13
High-throughput screening of our proprietary compound
collection identified hit compound 1 (Fig. 1) with an IC50 value of
1.2 lM in a receptor binding assay (RBA). Since this compound also
showed antagonistic activity against stimulation of metastin
(40–54) in a cellular Ca2+ mobilization assay, we anticipated that
this class of compound would be appropriate as leads for a
GPR54 antagonist. In this report we describe the structure–activity
relationship study that was performed to modify the 4-phenyl ring,
* Corresponding author. Tel.: +81 29 864 6465; fax: +81 29 864 6308.
E-mail address: Kobayashi_Toshitake@takeda.co.jp (T. Kobayashi).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.036

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T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
Scheme 1. The hydroxy groups of 2a–k were protected by tert-
butyldimethylsilyl (TBDMS), methoxymethyl (MOM), or ethoxy-
methyl (EOM) groups to give 3a–m. These ketones were treated
with the corresponding benzaldehydes, malononitrile, and ammo-
nium acetate to afford the 2-amino-4,6-diphenylpyridines 4a–p.
Compounds with an amino group on the 4-phenyl ring and their
amide derivatives were synthesized as shown in Scheme 2. Acyla-
tion of the 2-amino group of compounds 4a–l with an excess
amount of acid chloride in pyridine gave the diacylated com-
pounds, which were easily converted to the monoacylated com-
pounds in basic conditions, followed by reduction of the nitro
group to yield the anilines 6a–m. Cleavage of the protective group
of the phenolic hydroxy group gave 7a–d, which possess amino
groups on the 4-phenyl ring and phenol at the 6-position of the
pyridine ring.
Figure 1. Structure of the hit compound 1 and synthetic strategy.
6-phenyl ring, and 2-acylamino group, as shown in Figure 1, and
led to the discovery of compound 9l as a potent GPR54 antagonist.
Condensation of anilines 6 with the corresponding carboxylic
acids afforded amides 8a,b,d–m. For compounds having TBDMS
as the protecting group of the hydroxy group, partial or complete
deprotection was observed after condensation. Compound 8c was
synthesized from 7d, which was obtained by deprotection of a
2. Chemistry
The 2-amino-4,6-diphenylpyridines 4a–p, the key intermedi-
ates of 2-acylamino derivatives, were prepared as shown in
Scheme 1. Synthesis of 2-aminopyridine derivatives. Reagents and conditions: (a) TBDMSCl, imidazole, DMF, rt; (b) MOMCl, NaH, THF, rt; (c) EOMCl, NaH, THF, rt; (d)
benzaldehyde, malononitrile, NH4OAc, toluene, reflux.
Scheme 2. Synthesis of 2-acylamino-4,6-diphenylpyridines with an amino group on the 4-phenyl ring and their amide derivatives. Reagents and conditions: (a) R2COCl,
pyridine, rt, then purification by basic silica gel; (b) R2COCl, pyridine, rt, then ammonia solution, rt; (c) iron powder, NH4Cl, EtOH, H2O, reflux; (d) TBAF, THF, 0 °C; (e) R6CO2H,
WSCD, HOBt, DMF, rt; (f) 4 M HCl EtOAc solution, rt or 10% HCl MeOH solution, rt; (g) TFA, rt.

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
3843
Scheme 3. Synthesis of 2-acylamino-4,6-diphenylpyridines with carboxylic acid groups on the 4-phenyl ring and their amide derivatives. Reagents and conditions: (a) (1)
R2COCl, pyridine, rt., then 2 M Na2CO3, THF, MeOH, rt; (2) 4 M NaOH, EtOH, THF, rt; (b) (1) R2COCl, pyridine, rt, then purification by basic silica gel; (2) 4 M NaOH, EtOH, THF,
rt; (c) R2COCl, pyridine, rt, then 4 M NaOH, rt; (d) concd HCl, THF, rt; (e) H2NR7, WSCD, HOBt, DMF, rt; (f) 4 M HCl EtOAc solution, rt.
Scheme 4. Synthesis of 2-acylamino-4,6-diphenylpyridine 19 with an aminomethyl group on the 4-phenyl ring. Reagents and conditions: (a) LiBH4, THF, 50 °C; (b) CBr4, PPh3,
CH2Cl2, rt; (c) NaN3, DMF, rt; (d) 2-thenoyl chloride, pyridine, rt, then purification by basic silica gel; (e) H2 (3 atm), 10% Pd/C, EtOH, EtOAc, rt; (f) concd HCl, THF, rt.
TBDMS group in advance by TBAF. Removal of the protecting
groups of 8a–m, both on the amide side chain and the phenolic hy-
droxy group, afforded the amines 9a–m.
The synthesis of compounds bearing a carboxy group on the 4-
phenyl ring and their amide derivatives is outlined in Scheme 3.
Acylation of 2-aminopyridines 4m–p by the method described ear-
lier and hydrolysis of the esters gave 10a–d. Cleavage of the MOM
group of 10a in acidic condition yielded 11 having a carboxy group
on the 4-phenyl ring and a phenol at the 6-position of the pyridine
ring.
Condensation of carboxylic acids 10a–d with amines provided
12a–e, which were treated with HCl to remove the MOM group
and afford 13a–e.
Compound 19 having an aminomethyl moiety on the 4-phenyl
ring was prepared as described in Scheme 4. Reduction of the ester
of compound 4m afforded the alcohol 14, followed by bromination
and introduction of an azide to give compound 16. After acylation
of the 2-amino group with 2-thenoyl chloride, the azide was
hydrogenated under three atm hydrogen atmosphere to yield 18,
which was converted to 19 by treatment with HCl.
ketones 20a,b to yield the 2-amino-4,6-diphenylpyridines 21a,b,
followed by acylation of the amino group to provide 22a,b. After
hydrolysis of ester 22a, carboxylic acid 22c was converted to the
tert-butoxycarbonyl(Boc)-protected aniline 22d by Curtius rear-
rangement reaction. Reduction of the nitro group of compounds
22b,d gave anilines 23a,b, and for 23b, subsequent condensation
with Boc-protected b-alanine yielded 24. Treatment of 24 with
HCl to remove the Boc group provided 25.
In order to optimize the amine side chain on the 4-phenyl ring
and the 2-acylamino group, a combinatorial protocol was applied
as summarized in Scheme 6. Acylation of the amino group of 4p
and hydrolysis of the ester gave carboxylic acid 26, followed by
condensation with amines, cleavage of MOM group, and purifica-
tion with HPLC to afford compound 27. We planned to synthesize
168 compounds constructed by combining 14 acyl groups with
each of 12 amines and actually obtained 148 compounds discard-
ing samples that had insufficient quality or quantity.
3. Results and discussion
The synthesis of compounds without a phenolic hydroxy group
on the 6-phenyl ring is summarized in Scheme 5. The four compo-
nents condensation reaction described above was applied to
All compounds synthesized were initially evaluated by their
binding affinities for human GPR54 against 125I-labeled human

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T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
Scheme 5. Synthesis of 2-acylamino-4,6-diphenylpyridines without
a phenolic hydroxy group on the 6-phenyl ring. Reagents and conditions: (a) benzaldehyde,
malononitrile, NH4OAc, toluene, reflux; (b) benzoyl chloride, pyridine, rt, then NaOMe, MeOH, rt; (c) 2-thenoyl chloride, pyridine, rt, then purification by basic silica gel; (d)
4 M NaOH, MeOH, THF, rt; (e) DPPA, Et3N, toluene, reflux, then t-BuOH, reflux; (f) H2 (3 atm), 10% Pd/C, THF, 2-propanol, rt; (g) iron powder, NH4Cl, EtOH, H2O, reflux; (h) N-
Boc-b-alanine, WSCD, HOBt, DMF, rt; (i) 4 M HCl EtOAc solution, CH2Cl2, rt.
We concluded that the b-alanine amide is the optimum linker be-
tween the amino group and the phenyl ring. Compound 13b, which
is an inverse amide derivative of 9b, also showed potent activity,
while 9b was twofold more active. Shifting the amine side chain
to the 20- or 40-position of the phenyl ring (13a,c) resulted in de-
creased potency.
Regarding modification of the acyl group at the 2-position of the
pyridine ring, replacement of the 2-thienyl group with a phenyl
group retained activity, though the 2-thienyl group was preferable
(9b vs 9c).
Next, we conducted modification of the 6-phenyl ring (Table 2).
Scheme 6. Synthesis of 2-acylamino-4,6-diphenylpyridines by
a combinatorial
protocol. Reagents and conditions: (a) R2COCl, pyridine, rt; (b) 4 M NaOH, THF,
MeOH, rt; (c) HNR7R8, WSCD, HOBt, triethylamine, DMAP, DMF, CH2Cl2, 50 °C then
purification by HPLC; (d) TFA, rt, then purification by HPLC.
Initially, investigation of the effect of a hydroxy group at the 20-po-
sition of the phenyl ring was conducted. Methylation of the hydro-
xy group attenuated activity (7a vs 23a), suggesting that it may be
important as a proton donor. Then we replaced the hydroxy group
to an amino group as an alternative proton donor, however, it re-
sulted in moderate activity (9c vs 25). Thus a proton donor at the
20-position of the 6-phenyl ring was considered essential for the
affinity to GPR54 and the phenolic hydroxy group was appropriate
as the proton donor.
metastin (40–54) in a receptor binding assay. For compounds hav-
ing potent affinities for GPR54, antagonistic activities were evalu-
ated in a cellular Ca2+ mobilization assay against stimulation of
metastin (40–54). The results are shown in Tables 1–4.
We initially modified the hydroxymethyl group on the 4-phenyl
ring of hit compound 1 (Table 1). Replacement of the hydroxy-
methyl group by a carboxylic acid (11) or amine (7a) retained activ-
ity, while introduction of an aminomethyl group (19) resulted in
threefold enhancement of activity compared to 1. As we speculated
that introduction of an amino group at this position is preferable,
detailed modification of the aminomethyl group was conducted.
Introduction of a glycine amide at the 30-position of the phenyl ring
significantly improved activity with an IC50 value of 51 nM (9a)
with apparent antagonistic activity. Next, the length of the methy-
lene linker was examined. Introduction of a b-alanine amide (9b)
improved potency further compared with the glycine amide 9a,
With the information above, we introduced a second substitu-
ent on the 6-phenyl ring to optimize the proton donor ability of
the phenol. Introduction of a chloro (9e) or methoxy group (9f)
at the 50-position of the phenyl ring reduced activity compared
to the unsubstituted compound 9b, suggesting substitution at this
position is sterically limited. Compounds having fluoro (9g) or
chloro (9h) groups at 40-position of the phenyl ring improved
potency compared to 9b, though the methoxy group (9k) showed
decreased activity. These results indicate that an electron-with-
drawing group, especially a fluoro group, is preferable at 40-posi-
tion of the phenyl ring.
though the c-amino butyryl amide 9d slightly decreased activity.

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
3845
Table 1
Human GPR54 binding affinities and antagonistic activities (1)
Compound
R1
R2
RBA hGPR54 IC50
(lM)
Ca2+ mobilization assay hGPR54 IC50
(lM)
1
11
7a
19
9a
9b
9c
9d
13a
13b
13c
30-CH2OH
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
Phenyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
1.5
9.9
2.1
0.57
0.051
0.021
0.051
0.034
33
58% inhibition at 10 lM
30-CO2H
NT
NT
NT
2.8
1.1
24
1.4
NT
5.1
NT
30-NH2
30-CH2NH2
30-NHCOCH2NH2
30-NHCO(CH2)2NH2
30-NHCO(CH2)2NH2
30-NHCO(CH2)3NH2
20-CONH(CH2)2NH2
30-CONH(CH2)2NH2
40-CONH(CH2)2NH2
0.045
0.12
RBA = receptor binding assay. NT = not tested.
Table 2
Human GPR54 binding affinities and antagonistic activities (2)
Compound
R1
R2
R3
RBA hGPR54 IC50
(lM)
Ca2+ mobilization hGPR54 IC50
(l
M)
7a
23a
9b
9c
25
9e
9f
99
9h
9i
NH2
NH2
2-Thienyl
2-Thienyl
2-Thienyl
Phenyl
20-OH
2.1
>30
0.021
0.051
0.89
NT
NT
1.1
24
NT
NT
NT
3.6
4.1
6.3
0.91
NT
20-OMe
NHCO(CH2)2NH2
NHCO(CH2)2NH2
NHCO(CH2)2NH2
NHCO(CH2)2NH2
NHCO(CH2)2NH2
NHCO(CH2)2NH2
NHCO(CH2)2NH2
NHCO(CH2)2NH2
NHCO(CH2)2NH2
NHCO(CH2)2NH2
20-OH
20-OH
Phenyl
20-NH2
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
Phenyl
20-OH-50-CI
20-OH-50-OMe
20-OH-40-F
20-OH-40-CI
20-OH-40-Br
20-OH-40-Me
20-OH-40-OMe
0.49
0.48
0.0074
0.0098
0.019
0.012
0.12
9j
9k
RBA = receptor binding assay. NT = not tested.
In order to optimize the amine moiety on the 4-phenyl ring and
2-acylamino group, we applied a combinatorial protocol to synthe-
size 148 compounds that satisfied both quality and quantity crite-
ria. After rough RBA screening, compounds with high activity were
evaluated for their IC50 value, as shown in Table 3. The results
show that primary amines connected with a linear linker were
suitable as the 30-substituent of the 4-phenyl ring. For the acyl
group, a 2-furyl group was most appropriate and a phenyl group
bearing a methoxy group at the 20- or 40-position was comparable
to a 2-thienyl group.
On the basis of the earlier results, we synthesized compounds
with combinations of substituents that showed high potency (Ta-
ble 4). As expected, these compounds exhibited markedly high po-
tency, especially, compound 9l showed the most potent affinity
with an IC50 value of 3.7 nM, almost 1000-fold more active com-
pared to hit compound 1, with appropriate antagonistic activity
of GPR54 in a cellular functional assay.
We succeeded to obtain an X-ray crystal structure of compound
13b from methanol as shown in Figure 2. In the crystal structure,
the 4-phenyl ring is twisted perpendicular to the pyridine ring,
which may be caused by the steric repulsion of the cyano group.
In contrast, the pyridine ring and 6-phenyl ring exist on same plane
because of an intramolecular hydrogen bond between the pyridine
nitrogen atom and the phenolic hydroxy group.
The intramolecular hydrogen bond is also indicated by the 1H
NMR spectrum in DMSO-d6 using tetramethylsilane as internal
standard (0 ppm).14 As shown in Table 5, the chemical shift of
the phenolic hydroxy group at the 20-position, which can form an
intramolecular hydrogen bond, was around 12 ppm (7a). On the
other hand, the chemical shifts of the hydroxy groups at 30- or
40-position, which cannot make hydrogen bonds with the pyridine
nitrogen atom, were approximately 10 ppm (7b,c).
In order to confirm the importance of the stereo disposition
mentioned above towards the affinity for GPR54, we prepared

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T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
Table 3
Table 5
Human GPR54 binding affinities for compounds synthesized by
protocol
a
combinatorial
The position of the phenolic hydroxy group on the 6-phenyl ring and the 1H NMR
chemical shifts in DMSO-d6
R7
R2
RBA hGPR54 IC50 (lM)
Compound
7a
7b
7c
Compound
Position of OH
20
30
40
27a
27b
27c
27d
2-Thienyl
2-Furyl
20-MeOPh
40-MeOPh
0.030
0.014
0.045
0.031
Chemical shift of OH in 1H NMR
11.87 ppm
9.69 ppm
10.07 ppm
DMSO-d6 was used as solvent.
27e
27f
27g
2-Furyl
0.015
0.032
0.032
20-MeOPh
40-MeOPh
compound 9m bearing a methyl group at the 5-position of the pyr-
idine ring, which sterically hinders the intramolecular hydrogen
bond (Fig. 3). As we expected, the chemical shift of the phenolic hy-
droxy group of 9m was 9.89 ppm, which indicates cleavage of the
intramolecular hydrogen bond. Moreover, the receptor binding
affinity of 9m was 100-fold less than that of 9b, which may form
the hydrogen bond as estimated by the chemical shift of the 20-
OH at 11.79 ppm.
RBA = receptor binding assay.
Table 4
Human GPR54 binding affinities and antagonistic activities (3)
These experimental results suggest the active conformation of
the 4,6-diphenyl pyridines is almost the same as the crystal struc-
ture of compound 13b and, particularly, the intramolecular hydro-
gen bond between the phenol and pyridine nitrogen atom appears
to be essential for binding affinity.
4. Conclusion
Compound
R1
RBA hGPR54
IC50 M)
Ca2+ mobilization
In an attempt to find novel GPR54 antagonists, we synthesized
2-acylamino-4,6-diphenylpyridine derivatives and evaluated their
activities. Since compound 19, with a methylamino group at the
30-position of the 4-phenyl ring showed moderate activity, detailed
investigation of the amine side chain was conducted and led to
identification of the b-alanine amide derivative 9b having potent
GPR54 binding affinity and antagonistic activity. Further investiga-
tion to optimize the 6-phenyl ring revealed that the hydroxy group
at the 20-position is essential for activity and introduction of an
electron-withdrawing group at the 40-position improved potency.
With the use of combinatorial chemistry technology, a 2-furoyl
(l
hGPR54 IC50
(lM)
9l
0.0037
0.0080
0.011
0.46
13d
13e
0.66
2.5
RBA = receptor binding assay.
Figure 2. X-ray crystal structure of compound 13b.

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
3847
1
Figure 3. Human GPR54 binding affinities and H NMR chemical shifts of the 20-OH in DMSO-d6.
group was found to be the most suitable 2-acyl group, and finally
these structure–activity relationship studies led to compound 9l
with an IC50 value of 3.7 nM in a GPR54 binding assay. Moreover,
these compounds showed apparent antagonistic activity in a cellu-
lar functional assay. In vivo examination, using these compounds
for a proof-of-concept study as a drug for sex-hormone dependent
disease, is now ongoing.
7.00 (1H, dd, J = 9.0, 3.3 Hz), 7.14 (1H, d, J = 9.0 Hz), 7.25 (1H, d,
J = 3.3 Hz).
5.3. 1-[2-(Ethoxymethoxy)-4-fluorophenyl]ethanone (3f)
Compound 3f was prepared from 1-(4-fluoro-2-hydroxy-
phenyl)ethanone (2f) and chloromethyl ethyl ether in a manner
similar to that described for 3d as a colorless oil in 90% yield: 1H
NMR (CDCl3) d 1.25 (3H, t, J = 7.2 Hz), 2.61 (3H, s), 3.76 (2H, q,
J = 7.2 Hz), 5.32 (2H, s), 6.74 (1H, tt, J = 8.1, 2.4 Hz), 6.96 (1H, dd,
J = 10.8, 2.4 Hz), 7.78 (1H, t, J = 8.1 Hz).
5. Experimental
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). Elemental
analyses were carried out by Takeda Analytical Laboratories. Reac-
tion progress was determined by thin layer chromatography (TLC)
analysis on Silica Gel 60 F254 plate (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), basic silica gel (ChromatorexNH, 100–200 mesh, Fuji
5.4. 1-[4-Chloro-2-(methoxymethoxy)phenyl]ethanone (3g)
Compound 3g was prepared from 1-(4-chloro-2-hydroxy-
phenyl)ethanone (2g) and chloromethyl methyl ether in a manner
similar to that described for 3d as a colorless oil in 68% yield: 1H
NMR (CDCl3) d 2.62 (3H, s), 3.53 (3H, s), 5.28 (2H, s), 7.03 (1H,
dd, J = 8.4, 2.1 Hz), 7.22 (1H, d, J = 2.1 Hz), 7.68 (1H, d, J = 8.7 Hz).
5.5. 1-[4-Bromo-2-(methoxymethoxy)phenyl]ethanone (3h)
Compound 3h was prepared from 1-(4-bromo-2-hydroxy-
phenyl)ethanone (2h) and chloromethyl methyl ether in a manner
similar to that described for 3d as a colorless oil in 79% yield: 1H
NMR (CDCl3) d 2.61 (3H, s), 3.52 (3H, s), 5.28 (2H, s), 7.19 (1H,
dd, J = 8.4, 1.8 Hz), 7.37 (1H, d, J = 1.8 Hz), 7.60 (1H, d, J = 8.4 Hz).
silysia chemical Ltd) or Purif-Pack (SI 60 lM or NH 60 lM, Fuji
Silysia, Ltd). Commercial reagents and solvents were used without
additional purification.
5.6. 1-[2-(Ethoxymethoxy)-4-methylphenyl]ethanone (3i)
5.1. 1-[5-Chloro-2-(methoxymethoxy)phenyl]ethanone (3d)
Compound 3i was prepared from 1-(2-hydroxy-4-methyl-
phenyl)ethanone (2i) and chloromethyl ethyl ether in a manner
similar to that described for 3d as a colorless oil in 75% yield: 1H
NMR (CDCl3) d 1.26 (3H, t, J = 7.2 Hz), 2.37 (3H, s), 2.61 (3H, s),
3.76 (2H, q, J = 7.2 Hz), 5.32 (2H, s), 6.84 (1H, d, J = 7.8 Hz), 7.01
(1H, s), 7.65 (1H, d, J = 7.8 Hz).
To an ice-cooled solution of 1-(5-chloro-2-hydroxyphenyl)etha-
none (2d, 5.0 g, 29.3 mmol) in THF (50 ml) was added a 60% sus-
pension of NaH in mineral oil (1.2 g, 29.3 mmol). After stirring at
0 °C for 30 min, to the mixture was added chloromethyl methyl
ether (4.7 g, 58.6 mmol) and the whole mixture was stirred at
room temperature for 16 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 chromatogra-
phy (EtOAc/hexane = 1/4) to give 3d (5.6 g, 89%) as a colorless oil:
1H NMR (CDCl3) d 2.63 (3H, s), 3.51 (3H, s), 5.26 (2H, s), 7.15 (1H, d,
J = 9.0 Hz), 7.37 (1H, dd, J = 9.0, 2.7 Hz), 7.67 (1H, d, J = 2.7 Hz).
5.7. 1-[4-Methoxy-2-(methoxymethoxy)phenyl]ethanone (3j)
Compound 3j was prepared from 1-(2-hydroxy-4-methoxy-
phenyl)ethanone (2j) and chloromethyl methyl ether in a manner
similar to that described for 3d as a colorless oil in 70% yield: 1H
NMR (CDCl3) d 2.60 (3H, s), 3.52 (3H, s), 3.84 (3H, s), 5.28 (2H, s),
6.58 (1H, dd, J = 8.7, 1.8 Hz), 6.70 (1H, d, J = 1.8 Hz), 7.81 (1H, d,
J = 8.7 Hz).
5.2. 1-[5-Methoxy-2-(methoxymethoxy)phenyl]ethanone (3e)
Compound 3e was prepared from 1-(2-hydroxy-5-methoxy-
phenyl)ethanone (2e) and chloromethyl methyl ether in a manner
similar to that described for 3d as a colorless oil in 79% yield: 1H
NMR (CDCl3) d 2.64 (3H, s), 3.51 (3H, s), 3.80 (3H, s), 5.21 (2H, s),
5.8. 1-[4-Fluoro-2-(methoxymethoxy)phenyl]ethanone (3k)
Compound 3k was prepared from 1-(4-fluoro-2-hydroxy-
phenyl)ethanone (2f) and chloromethyl methyl ether in a manner

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T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
similar to that described for 3d as a colorless oil in 92% yield: 1H
NMR (CDCl3) d 2.62 (3H, s), 3.53 (3H, s), 5.28 (2H, s), 6.70–6.80
(1H, m), 6.93 (1H, dd, J = 10.8, 2.4 Hz), 7.79 (1H, dd, J = 8.7, 6.9 Hz).
39% yield: 1H NMR (DMSO-d6) d 3.31 (3H, s), 3.77 (3H, s), 5.14
(2H, s), 7.02 (1H, d, J = 9.0, 3.3 Hz), 7.11 (2H, br s), 7.17 (1H, d,
J = 9.0 Hz), 7.29 (1H, s), 7.33 (1H, d, J = 3.3 Hz), 7.87 (1H, t,
J = 8.1 Hz), 8.12 (1H, d, J = 7.8 Hz), 8.39 (1H, dd, J = 7.8, 1.8 Hz),
8.44 (1H, d, J = 1.8 Hz).
5.9. 1-[2-(Methoxymethoxy)phenyl]propan-1-one (3l)
Compound 3l was prepared from 1-(2-hydroxyphenyl)propan-
1-one (2k) and chloromethyl methyl ether in a manner similar to
that described for 3d as a colorless oil in 82% yield: 1H NMR (CDCl3)
d 1.18 (3H, t, J = 7.2 Hz), 3.01 (2H, q, J = 7.2 Hz), 3.51 (3H, s), 5.27
(2H, s), 7.05 (1H, td, J = 7.5, 0.6 Hz), 7.18 (1H, dd, J = 8.4, 0.6 Hz),
7.42 (1H, td, J = 7.5, 1.8 Hz), 7.65 (1H, dd, J = 7.5, 1.8 Hz).
5.15. 2-Amino-6-[2-(ethoxymethoxy)-4-fluorophenyl]-4-(3-
nitrophenyl)pyridine-3-carbonitrile (4f)
Compound 4f was prepared from 3f and 3-nitrobenzaldehyde in
a manner similar to that described for 4b as a yellow solid in 19%
yield: 1H NMR (CDCl3) d 1.21 (3H, t, J = 7.2 Hz), 3.70 (2H, q,
J = 7.2 Hz), 5.28 (2H, s), 5.39 (2H, br s), 6.85 (1H, t, J = 8.4 Hz),
7.01 (1H, dd, J = 10.8, 2.4 Hz), 7.38 (1H, s), 7.73 (1H, t, J = 8.1 Hz),
7.84 (1H, t, J = 7.8 Hz), 8.00 (1H, d, J = 7.8 Hz), 8.37 (1H, dq,
J = 8.1, 1.2 Hz), 8.46 (1H, t, J = 2.4 Hz).
5.10. 1-[2-(Methoxymethoxy)phenyl]ethanone (3m)
Compound 3m was prepared from 1-(2-hydroxyphenyl)etha-
none (2a) and chloromethyl methyl ether in a manner similar to
that described for 3d as a colorless oil in 100% yield: 1H NMR
(CDCl3) d 2.64 (3H, s), 3.50 (3H, s), 5.29 (2H, s), 7.05 (1H, t,
J = 7.5 Hz), 7.13 (1H, d, J = 8.4 Hz), 7.44 (1H, t, J = 7.8 Hz), 7.71
(1H, d, J = 7.8 Hz).
5.16. 2-Amino-6-[4-chloro-2-(methoxymethoxy)phenyl]-4-(3-
nitrophenyl)pyridine-3-carbonitrile (4g)
Compound 4g was prepared from 3g and 3-nitrobenzaldehyde
in a manner similar to that described for 4b as a yellow solid in
25% yield: mp 196–198 °C; 1H NMR (CDCl3) d 3.48 (3H, s), 5.24
(2H, s), 5.40 (2H, br s), 7.13 (1H, dd, J = 8.4, 2.1 Hz), 7.26 (1H, d,
J = 2.1 Hz), 7.39 (1H, s), 7.71–7.81 (2H, m), 8.01 (1H, dt, J = 8.1,
0.9 Hz), 8.37 (1H, dq, J = 8.1, 0.9 Hz), 8.46 (1H, t, J = 1.8 Hz).
5.11. 2-Amino-6-(3-{[tert-butyl(dimethyl)silyl]oxy}phenyl)-4-
(3-nitrophenyl)pyridine-3-carbonitrile (4b)
A mixture of 1-(3-{[tert-butyl(dimethyl)silyl]oxy}phenyl)etha-
none15 (3b, 9.86 g, 39.7 mmol), 3-nitrobenzaldehyde (6.00 g,
39.7 mmol), malononitrile (2.62 g, 39.7 mmol), and NH4OAc
(4.60 g, 59.6 mmol) in toluene (30 ml) was refluxed for 16 h. After
cooling to room temperature, the reaction mixture was concen-
trated in vacuo and the residue was partitioned between saturated
aqueous NaHCO3 and 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 = 1/4) and triturated with Et2O/hexane to give 4b (1.88 g,
11%) as a colorless solid: 1H NMR (CDCl3) d 0.25 (6H, s), 1.01 (9H,
s), 5.42 (2H, s), 6.96 (1H, d, J = 8.1 Hz), 7.17 (1H, s), 7.34 (1H, t,
J = 8.1 Hz), 7.49 (1H, t, J = 2.1 Hz), 7.57 (1H, d, J = 8.1 Hz), 7.74
(1H, t, J = 8.1 Hz), 7.99 (1H, dt, J = 7.5, 1.2 Hz), 8.38 (1H, dq,
J = 8.4, 1.2 Hz), 8.47 (1H, t, J = 1.2 Hz).
5.17. 2-Amino-6-[4-bromo-2-(methoxymethoxy)phenyl]-4-(3-
nitrophenyl)pyridine-3-carbonitrile (4h)
Compound 4h was prepared from 3h and 3-nitrobenzaldehyde
in a manner similar to that described for 4b as a yellow solid in 27%
yield: mp 201–202 °C; 1H NMR (CDCl3) d 3.48 (3H, s), 5.23 (2H, s),
5.40 (2H, br s), 7.29 (1H, dd, J = 8.4, 1.8 Hz), 7.40 (2H, d, J = 7.5 Hz),
7.73 (2H, t, J = 8.1 Hz), 8.00 (1H, dt, J = 7.5, 1.2 Hz), 8.38 (1H, dt,
J = 8.1, 1.2 Hz), 8.46 (1H, t, J = 1.8 Hz).
5.18. 2-Amino-6-[2-(ethoxymethoxy)-4-methylphenyl]-4-(3-
nitrophenyl)pyridine-3-carbonitrile (4i)
Compound 4i was prepared from 3i and 3-nitrobenzaldehyde in
a manner similar to that described for 4b as a yellow solid in 31%
yield: mp 148–149 °C; 1H NMR (CDCl3) d 1.20 (3H, t, J = 7.2 Hz),
2.40 (3H, s), 3.70 (2H, q, J = 7.2 Hz), 5.27 (2H, s), 5.38 (2H, br s),
6.95 (1H, dd, J = 8.4, 0.9 Hz), 7.05 (1H, s), 7.42 (1H, s), 7.69–7.75
(2H, m), 8.02 (1H, d, J = 7.8 Hz), 8.36 (1H, dq, J = 8.4, 0.9 Hz), 8.46
(1H, t, J = 1.8 Hz).
5.12. 2-Amino-6-(4-{[tert-butyl(dimethyl)silyl]oxy}phenyl)-4-
(3-nitrophenyl)pyridine-3-carbonitrile (4c)
Compound 4c was prepared from 1-(4-{[tert-butyl(dimethyl)-
silyl]oxy}phenyl)ethanone16 (3c) and 3-nitrobenzaldehyde in a
manner similar to that described for 4b as a colorless amorphous so-
lid in 11% yield: 1H NMR (CDCl3) d 0.24 (6H, s), 1.00 (9H, s), 5.38 (2H,
br s), 6.94 (2H, d, J = 8.7 Hz), 7.16 (1H, s), 7.73 (1H, t, J = 8.0 Hz), 7.91–
8.01 (3H, m), 8.36 (1H, d, J = 8.4 Hz), 8.47 (1H, s).
5.19. 2-Amino-6-[4-methoxy-2-(methoxymethoxy)phenyl]-4-
(3-nitrophenyl)pyridine-3-carbonitrile (4j)
5.13. 2-Amino-6-[5-chloro-2-(methoxymethoxy)phenyl]-4-(3-
nitrophenyl)pyridine-3-carbonitrile (4d)
Compound 4j was prepared from 3j and 3-nitrobenzaldehyde in
a manner similar to that described for 4b as a yellow solid in 22%
yield: mp 171–172 °C; 1H NMR (CDCl3) d 3.47 (3H, s), 3.86 (3H, s),
5.24 (2H, s), 5.37 (2H, br s), 6.69 (1H, dd, J = 8.7, 2.4 Hz), 6.78 (1H, d,
J = 2.4 Hz), 7.44 (1H, s), 7.72 (1H, t, J = 8.0 Hz), 7.86 (1H, d,
J = 8.7 Hz), 8.01 (1H, dd, J = 6.6, 1.2 Hz), 8.36 (1H, d, J = 8.4 Hz),
8.47 (1H, d, J = 2.1 Hz).
Compound 4d was prepared from 3d and 3-nitrobenzaldehyde
in a manner similar to that described for 4b as a light brown solid
in 11% yield: mp 192–193 °C: 1H NMR (CDCl3) d 3.46 (3H, s), 5.22
(2H, s), 5.42 (2H, br s), 7.18 (1H, d, J = 9.0 Hz), 7.35 (1H, dd, J = 9.0,
2.4 Hz), 7.41 (1H, s), 7.74 (1H, t, J = 7.1 Hz), 7.83 (1H, d, J = 2.4 Hz),
8.02 (1H, d, J = 8.7 Hz), 8.36 (1H, d, J = 8.4 Hz), 8.46 (1H, s).
5.20. 2-Amino-6-[4-fluoro-2-(methoxymethoxy)phenyl]-4-(3-
nitrophenyl)pyridine-3-carbonitrile (4k)
5.14. 2-Amino-6-[5-methoxy-2-(methoxymethoxy)phenyl]-4-
(3-nitrophenyl)pyridine-3-carbonitrile (4e)
Compound 4k was prepared from 3k and 3-nitrobenzaldehyde
in a manner similar to that described for 4b as a pale yellow solid
in 34% yield: mp 202–203 °C; 1H NMR (CDCl3) d 3.48 (3H, s), 5.23
(2H, s), 5.39 (2H, s), 6.80–6.90 (1H, m), 6.99 (1H, dd, J = 10.8,
Compound 4e was prepared from 3e and 3-nitrobenzaldehyde
in a manner similar to that described for 4b as a yellow solid in

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
3849
2.4 Hz), 7.38 (1H, s), 7.73 (1H, t, J = 7.8 Hz), 7.84 (1H, dd, J = 8.7,
6.9 Hz), 7.96–8.05 (1H, m), 8.35–8.40 (1H, m), 8.45–8.50 (1H, m).
ane = 5/95 to 50/50) and recrystallization from EtOAc/hexane to
give 5a (0.59 g, 47%) as a colorless solid: mp 106–108 °C; 1H
NMR (CDCl3) d 0.12 (6H, s), 0.83 (9H, s), 6.95 (1H, dd, J = 0.9,
9.3 Hz), 7.13 (1H, t, J = 7.5 Hz), 7.18–7.21 (1H, m), 7.37 (1H, t,
J = 7.8 Hz), 7.67 (1H, dd, J = 4.8, 0.9 Hz), 7.75 (1H, t, J = 8.1 Hz),
7.78–7.81 (1H, m), 7.85 (1H, dd, J = 7.5, 1.5 Hz), 7.97 (1H, s), 8.05
(1H, d, J = 7.5 Hz), 8.41 (1H, d, J = 7.5 Hz), 8.46 (1H, s), 8.49 (1H, s).
5.21. 2-Amino-6-[2-(methoxymethoxy)phenyl]-5-methyl-4-(3-
nitrophenyl)pyridine-3-carbonitrile (4l)
Compound 4l was prepared from 3l and 3-nitrobenzaldehyde in
a manner similar to that described for 4b as a pale yellow amor-
phous solid in 37% yield: 1H NMR (CDCl3) d 1.82 (3H, s), 3.42
(3H, s), 5.17 (2H, s), 5.25 (2H, s), 7.13 (1H, t, J = 6.6 Hz), 7.23–
7.28 (2H, m), 7.37–7.42 (1H, m), 7.72–7.74 (2H, m), 8.25 (1H, br
s), 8.34–8.37 (1H, m).
5.27. N-[6-(3-{[tert-Butyl(dimethyl)silyl]oxy}phenyl)-3-cyano-
4-(3-nitrophenyl)pyridin-2-yl]thiophene-2-carboxamide (5b)
Compound 5b was prepared from 4b in a manner similar to that
described for 5a as a pale yellow amorphous solid in 98% yield: 1H
NMR (CDCl3) d 0.23 (6H, s), 0.99 (9H, s), 6.87 (1H, d, J = 9.0 Hz), 7.15
(1H, dd, J = 4.8, 3.9 Hz), 7.56 (1H, s), 7.62–7.69 (2H, m), 7.86 (1H,
dd, J = 3.6, 0.6 Hz), 7.93–7.99 (3H, m), 8.31 (1H, dd, J = 6.9,
1.8 Hz), 8.41 (1H, t, J = 1.8 Hz), 8.83 (1H, s).
5.22. Methyl 3-{2-amino-3-cyano-6-[2-(methoxymethoxy)-
phenyl]pyridin-4-yl}benzoate (4m)
Compound 4m was prepared from 3m and methyl 3-form-
ylbenzoate in a manner similar to that described for 4b as a color-
less solid in 28% yield: mp 129–130 °C; 1H NMR (CDCl3) d 3.45 (3H,
s), 3.95 (3H, s), 5.22 (2H, s), 5.35 (2H, br s), 7.14 (1H, t, J = 7.5 Hz),
7.22 (1H, d, J = 7.5 Hz), 7.37–7.42 (2H, m), 7.61 (1H, t, J = 7.8 Hz),
7.77 (1H, dd, J = 7.8, 1.8 Hz), 7.85–7.89 (1H, m), 8.15–8.19 (1H,
m), 8.28 (1H, t, J = 1.7 Hz).
5.28. N-[6-(4-{[tert-Butyl(dimethyl)silyl]oxy}phenyl)-3-cyano-
4-(3-nitrophenyl)pyridin-2-yl]thiophene-2-carboxamide (5c)
Compound 5c was prepared from 4c in a manner similar to that
described for 5a as a pale yellow amorphous solid in 78% yield: 1H
NMR (CDCl3) d 0.23 (6H, s), 1.00 (9H, s), 6.97 (1H, d, J = 8.1 Hz), 7.17
(1H, dd, J = 4.8, 3.9 Hz), 7.35 (1H, t, J = 8.1 Hz), 7.52 (1H, t,
J = 1.8 Hz), 7.62–7.66 (3H, m), 7.74 (1H, t, J = 8.1 Hz), 7.83 (1H,
dd, J = 3.9, 0.9 Hz), 8.06 (1H, d, J = 7.8 Hz), 8.38 (1H, d, J = 8.1 Hz),
8.66 (1H, s).
5.23. Methyl 2-{2-amino-3-cyano-6-[2-(methoxymethoxy)-
phenyl]pyridin-4-yl}benzoate (4n)
Compound 4n was prepared from 3m and methyl 2-form-
ylbenzoate in a manner similar to that described for 4b as an or-
ange solid in 19% yield: mp 109–110 °C; 1H NMR (DMSO-d6) d
3.31 (3H, s), 3.68 (3H, s), 5.14 (2H, s), 6.93 (2H, s), 7.02 (1H, s),
7.13 (1H, t, J = 8.0 Hz), 7.20 (1H, d, J = 7.8 Hz), 7.37–7.49 (2H, m),
7.63 (1H, t, J = 8.2 Hz), 7.71–7.80 (2H, m), 7.95 (1H, dd, J = 7.8,
1.8 Hz).
5.29. N-[6-(2-{[tert-Butyl(dimethyl)silyl]oxy}phenyl)-3-cyano-
4-(3-nitrophenyl)pyridin-2-yl]benzamide (5d)
Compound 5d was prepared from 4a and benzoyl chloride in a
manner similar to that described for 5a as a colorless solid in 77%
yield: 1H NMR (CDCl3) d 0.12 (6H, s), 0.80 (9H, s), 6.94–6.97 (1H,
m), 7.09–7.15 (1H, m), 7.33–7.39 (1H, m), 7.52–7.57 (2H, m),
7.61–7.66 (1H, m), 7.76 (1H, t, J = 7.8 Hz), 7.83 (1H, dd, J = 7.8,
1.8 Hz), 7.98 (1H, s), 8.00–8.09 (3H, m), 8.38–8.42 (1H, m), 8.50
(1H, t, J = 1.8 Hz), 8.57 (1H, br s).
5.24. Methyl 4-{2-amino-3-cyano-6-[2-(methoxymethoxy)-
phenyl]pyridin-4-yl}benzoate (4o)
Compound 4o was prepared from 3m and methyl 4-form-
ylbenzoate in a manner similar to that described for 4b as a yellow
solid in 35% yield: mp 170–171 °C; 1H NMR (CDCl3) d 3.43 (3H, s),
3.96 (3H, s), 5.21 (2H, s), 5.36 (2H, br s), 7.14 (1H, td, J = 7.5, 0.9 Hz),
7.21 (1H, dd, J = 8.4, 0.9 Hz), 7.35–7.42 (2H, m), 7.69 (2H, d,
J = 8.4 Hz), 7.77 (1H, dd, J = 7.2, 1.8 Hz), 8.18 (2H, d, J = 8.4 Hz).
5.30. N-{6-[5-Chloro-2-(methoxymethoxy)phenyl]-3-cyano-4-
(3-nitrophenyl)pyridin-2-yl}thiophene-2-carboxamide (5e)
To a solution of 4d (0.50 g, 1.2 mmol) in pyridine (30 ml) was
added 2-theonyl chloride (0.45 g, 3.0 mmol) and the whole was
stirred at room temperature for 16 h. To the solution was added
2-theonyl chloride (0.98 g, 6.7 mmol) and the mixture was stirred
at room temperature for 72 h. To the solution was added 28% aque-
ous ammonia solution (3 ml) and the mixture was stirred at room
temperature for 1 h. After the solvent was evaporated in vacuo, the
residue was diluted with water and extracted with EtOAc twice.
The combined organic layer was washed with brine, dried over
MgSO4, and concentrated in vacuo. The residue was purified by sil-
ica gel column chromatography (EtOAc/hexane = 1/2 to 1/1) and
recrystallization from EtOAc/Et2O to give 5e (0.36 g, 57%) as a col-
orless solid: mp 161–162 °C; 1H NMR (DMSO-d6) d 3.48 (3H, s),
5.27 (2H, s), 7.19–7.26 (3H, m), 7.39–7.42 (1H, m), 7.68 (1H, d,
J = 4.8 Hz), 7.75–7.83 (2H, m), 7.95 (1H, s), 7.99 (1H, s), 8.13–8.52
(3H, m).
5.25. Methyl 3-{2-amino-3-cyano-6-[4-fluoro-2-(methoxy-
methoxy)phenyl]pyridin-4-yl}benzoate (4p)
Compound 4p was prepared from 3k and methyl 3-form-
ylbenzoate in a manner similar to that described for 4b as a yellow
solid in 35% yield: mp 155–158 °C; 1H NMR (CDCl3) d 3.46 (3H, s),
3.95 (3H, s), 5.22 (2H, s), 5.36 (2H, br s), 6.85 (1H, td, J = 8.4, 2.4 Hz),
6.98 (1H, dd, J = 10.8, 2.4 Hz), 7.34 (1H, s), 7.61 (1H, t, J = 7.8 Hz),
7.75–7.90 (2H, m), 8.10–8.20 (1H, m), 8.27 (1H, s).
5.26. N-[6-(2-{[tert-Butyl(dimethyl)silyl]oxy}phenyl)-3-cyano-
4-(3-nitrophenyl)pyridin-2-yl]thiophene-2-carboxamide (5a)
To a solution of 2-amino-6-(2-{[tert-butyl(dimethyl)silyl]oxy}-
phenyl)-4-(3-nitrophenyl)pyridine-3-carbonitrile17 (4a, 1.0 g, 2.2
mmol) in pyridine (10 ml) was added 2-thenoyl chloride (0.6 ml,
5.6 mmol) at room temperature and the whole was stirred at room
temperature for 20 h. After the solvent was evaporated in vacuo,
the residue 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/hex-
5.31. N-{3-Cyano-6-[5-methoxy-2-(methoxymethoxy)phenyl]-
4-(3-nitrophenyl)pyridin-2-yl}thiophene-2-carboxamide (5f)
Compound 5f was prepared from 4e in a manner similar to that
described for 5e as a pale yellow amorphous solid in 24% yield: 1H
NMR (CDCl3) d 3.47 (3H, s), 3.86 (3H, s), 5.20 (2H, s), 7.00 (1H, dd,

3850
T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
J = 9.0, 3.3 Hz), 7.19–7.26 (3H, m), 7.47 (1H, d, J = 3.3 Hz), 7.67 (1H,
d, J = 4.5 Hz), 7.45–7.83 (2H, m), 8.00 (1H, s), 8.14 (1H, d, J = 7.8 Hz),
8.40 (1H, d, J = 7.8 Hz), 8.53 (1H, t, J = 2.1 Hz).
C25H17FN4O6: C, 61.48; H, 3.51; N, 11.47. Found: C, 61.58; H,
3.69; N, 11.43.
5.38. N-{3-Cyano-6-[2-(methoxymethoxy)phenyl]-5-methyl-4-
(3-nitrophenyl)pyridin-2-yl}furan-2-carboxamide (5m)
5.32. N-{3-Cyano-6-[2-(ethoxymethoxy)-4-fluorophenyl]-4-(3-
nitrophenyl)pyridin-2-yl}thiophene-2-carboxamide (5g)
Compound 5m was prepared from 4l and 2-furoyl chloride in a
manner similar to that described for 5a as a colorless amorphous
solid in 100% yield: 1H NMR (DMSO-d6) d 2.00 (3H, s), 3.42 (3H,
s), 5.17 (2H, s), 6.59 (1H, q, J = 1.8 Hz), 7.16 (1H, td, J = 7.5,
0.9 Hz), 7.26–7.37 (3H, m), 7.41–7.47 (1H, m), 7.55 (1H, q,
J = 0.9 Hz), 7.74–7.82 (2H, m), 8.32 (1H, s), 8.37–8.41 (1H, m),
8.71 (1H, s).
Compound 5g was prepared from 4f in a manner similar to that
described for 5e as a yellow powder in 70% yield: mp 166–167 °C;
1H NMR (CDCl3) d 1.22 (3H, t, J = 6.9 Hz), 3.73 (2H, q, J = 6.9 Hz),
5.33 (2H, s), 6.88 (1H, t, J = 7.2 Hz), 7.05 (1H, d, J = 10.8 Hz), 7.20
(1H, t, J = 4.4 Hz), 7.63–7.84 (3H, m), 7.97–8.02 (2H, m), 8.13 (1H,
d, J = 7.8 Hz), 8.39–8.51 (3H, m).
5.39. N-[4-(3-Aminophenyl)-6-(2-{[tert-butyl(dimethyl)silyl]-
oxy}phenyl)-3-cyanopyridin-2-yl]thiophene-2-carboxamide (6a)
5.33. N-{6-[4-Chloro-2-(methoxymethoxy)phenyl]-3-cyano-4-
(3-nitrophenyl)pyridin-2-yl}thiophene-2-carboxamide (5h)
A mixture of 5a (0.20 g, 0.36 mmol), iron powder (0.12 g,
2.16 mmol), and NH4Cl (0.13 g, 2.3 mmol) in 50% aqueous EtOH
(20 ml) was stirred under reflux for 20 min. After cooling to room
temperature, the solution was diluted with saturated aqueous
NaHCO3 and extracted with EtOAc. The extract was washed with
brine, dried over MgSO4, and concentrated in vacuo to give 6a
(0.18 g, 95%) as an amorphous solid: 1H NMR (DMSO-d6) d 0.18
(6H, s), 0.76 (9H, s), 5.39–5.43 (2H, m), 6.71–6.81 (2H, m), 6.88
(1H,s), 7.03 (1H, d, J = 8.1 Hz), 7.12–7.31(3H, m), 7.44 (1H, t,
J = 7.7 Hz), 7.79–7.92 (2H, m), 7.96 (1H, d, J = 4.8 Hz), 8.14 (1H, d,
J = 3.0 Hz), 11.46 (1H, s).
Compound 5h was prepared from 4g in a manner similar to that
described for 5e as a pale yellow amorphous solid in 74% yield: 1H
NMR (CDCl3) d 3.51 (3H, s), 5.29 (2H, s), 7.14–7.21 (2H, m), 7.30
(1H, d, J = 1.8 Hz), 7.67 (1H, d, J = 5.1 Hz), 7.75–7.82 (2H, m),
7.92–7.98 (2H, m), 8.13 (1H, d, J = 7.8 Hz), 8.41 (1H, d, J = 7.5 Hz),
8.43–8.52 (2H, m).
5.34. N-{6-[4-Bromo-2-(methoxymethoxy)phenyl]-3-cyano-4-
(3-nitrophenyl)pyridin-2-yl}thiophene-2-carboxamide (5i)
Compound 5i was prepared from 4h in a manner similar to that
described for 5e as a yellow solid in 87% yield: mp 166–167 °C; 1H
NMR (CDCl3) d 3.51 (3H, s), 5.29 (2H, s), 7.20 (1H, t, J = 4.4 Hz), 7.32
(1H, dd, J = 8.1, 1.5 Hz), 7.45 (1H, s), 7.67 (1H, d, J = 4.4 Hz), 7.75–
7.87 (3H, m), 7.98 (1H, s), 8.13 (1H, d, J = 7.5 Hz), 8.41 (1H, dd,
J = 8.1, 1.5 Hz), 8.47 (1H, s), 8.51 (1H, t, J = 1.5 Hz).
5.40. N-[4-(3-Aminophenyl)-6-(3-{[tert-butyl(dimethyl)-
silyl]oxy}phenyl)-3-cyanopyridin-2-yl]thiophene-2-carboxamide
(6b)
Compound 6b was prepared from 5b in a manner similar to that
described for 6a as a yellow amorphous solid in 89% yield: 1H NMR
(CDCl3) d 0.21 (6H, s), 0.99 (9H, s), 3.84–3.99 (2H, br s), 6.74 (1H,
dd, J = 8.1, 1.8 Hz), 6.90–6.97 (3H, m), 7.06 (1H, t, J = 4.4 Hz),
7.18–7.31 (2H, m), 7.49–7.61 (4H, m), 7.76 (1H, d, J = 3.6 Hz),
8.91(1H, s).
5.35. N-{3-Cyano-6-[2-(ethoxymethoxy)-4-methylphenyl]-4-(3-
nitrophenyl)pyridin-2-yl}thiophene-2-carboxamide (5j)
Compound 5j was prepared from 4i in a manner similar to that
described for 5e as a yellow solid in 50% yield: mp 121–123 °C; 1H
NMR (CDCl3) d 1.21 (3H, t, J = 6.8 Hz), 2.42 (3H, s), 3.73 (2H, q,
J = 6.8 Hz), 5.33 (2H, s), 6.98 (1H, d, J = 7.8 Hz), 7.10 (1H, s), 7.19
(1H, t, J = 4.5 Hz), 7.65–7.87 (4H, m), 8.01 (1H, br s), 8.14 (1H, d,
J = 7.8 Hz), 8.40 (1H, dt, J = 7.5, 0.9 Hz), 8.49–8.52 (2H, m).
5.41. N-[4-(3-Aminophenyl)-6-(4-{[tert-butyl(dimethyl)silyl]-
oxy}phenyl)-3-cyanopyridin-2-yl]thiophene-2-carboxamide (6c)
Compound 6c was prepared from 5c in a manner similar to that
described for 6a as a yellow amorphous solid in 91% yield: 1H NMR
(CDCl3) d 0.23 (6H, s), 1.00 (9H, s), 3.80–4.00 (2H, br s), 6.74 (1H, dd,
J = 7.8, 1.8 Hz), 6.84–6.96 (4H, m), 7.11 (1H, dd, J = 4.8, 3.9 Hz), 7.21
(1H, t, J = 7.8 Hz), 7.52 (1H, s), 7.58 (1H, d, J = 3.9 Hz), 7.81 (1H, d,
J = 2.7 Hz), 7.93 (2H, d, J = 8.7 Hz), 8.82(1H, s).
5.36. N-{3-Cyano-6-[4-methoxy-2-(methoxymethoxy)phenyl]-
4-(3-nitrophenyl)pyridin-2-yl}benzamide (5k)
Compound 5k was prepared from 4j and benzoyl chloride in a
manner similar to that described for 5e as a yellow amorphous so-
lid in 95% yield: 1H NMR (CDCl3) d 3.49 (3H, s), 3.83 (3H, s), 5.27
(2H, s), 6.64 (1H, dd, J = 8.7, 2.1 Hz), 6.77 (1H, d, J = 2.1 Hz), 7.48–
7.62 (3H, m), 7.73 (1H, t, J = 8.1 Hz), 7.94 (1H, d, J = 8.7 Hz), 8.00–
8.02 (3H, m), 8.11 (1H, d, J = 7.2 Hz), 8.36 (1H, d, J = 7.2 Hz), 8.49
(1H, d, J = 1.8 Hz), 8.86 (1H, s).
5.42. N-{4-(3-Aminophenyl)-6-[5-chloro-2-(methoxymethoxy)-
phenyl]-3-cyanopyridin-2-yl}thiophene-2-carboxamide (6e)
Compound 6e was prepared from 5e in a manner similar to that
described for 6a as a light brown solid in 74% yield: mp 180–
182 °C; 1H NMR (CDCl3) d 3.45 (3H, s), 5.23 (2H, s), 6.69–6.91
(1H, m), 7.04–7.08 (2H, m), 7.19–7.38 (4H, m), 7.64 (1H, d,
J = 7.5 Hz), 7.81–7.88 (3H, m), 8.30–8.60 (1H, m).
5.37. N-{3-Cyano-6-[4-fluoro-2-(methoxymethoxy)phenyl]-4-
(3-nitrophenyl)pyridin-2-yl}furan-2-carboxamide (5l)
Compound 5l was prepared from 4k and 2-furoyl chloride in a
manner similar to that described for 5a as a colorless solid in
90% yield: mp 177–179 °C; 1H NMR (DMSO-d6) d 3.37 (3H, s),
5.36 (2H, s), 6.77 (1H, dd, J = 3.4, 1.9 Hz), 7.01–7.10 (1H, m), 7.18
(1H, dd, J = 11.3, 2.3 Hz), 7.55 (1H, d, J = 3.8 Hz), 7.90–8.06 (3H,
m), 8.11 (1H, s), 8.17–8.23 (1H, m, J = 8.3 Hz), 8.43–8.48 (1H, m),
8.55 (1H, t, J = 1.9 Hz), 11.37 (1H, br s). Anal. Calcd for
5.43. N-{4-(3-Aminophenyl)-3-cyano-6-[5-methoxy-2-(methoxy-
methoxy)phenyl]pyridin-2-yl}thiophene-2-carboxamide (6f)
Compound 6f was prepared from 5f in a manner similar to that
described for 6a as a yellow amorphous solid in 99% yield: 1H NMR
(CDCl3) d 3.42 (3H, s), 3.85 (3H, s), 5.16 (2H, s), 6.84 (1H, d,
J = 7.8 Hz), 6.97 (1H, dd, J = 9.0, 3.3 Hz), 7.03–7.06 (2H, m),

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
3851
7.16–7.34 (4H, m), 7.40 (1H, br s), 7.63 (1H, d, J = 4.8 Hz), 7.80 (1H,
d, J = 3.6 Hz), 7.85–8.00 (1H, m).
1H NMR (DMSO-d6) d 5.42 (2H, s), 6.75 (1H, d, J = 7.8 Hz), 6.84 (1H,
d, J = 7.5 Hz), 6.89–6.95 (2H, m), 7.21 (1H, d, J = 7.8 Hz), 7.26–7.37
(2H, m), 7.64 (1H, s), 7.66 (1H, d, J = 8.1 Hz), 7.90 (1H, s), 7.97 (1H,
d, J = 0.6 Hz), 8.16 (1H, d, J = 3.0 Hz), 9.69 (1H, s), 11.32 (1H, s).
Anal. Calcd for C23H16N4O2S: C, 66.97; H, 3.91; N, 13.58. Found:
C, 66.93; H, 3.91; N, 13.69.
5.44. N-{4-(3-Aminophenyl)-6-[4-chloro-2-(methoxymethoxy)-
phenyl]-3-cyanopyridin-2-yl}thiophene-2-carboxamide (6h)
Compound 6h was prepared from 5h in a manner similar to that
described for 6a as a green amorphous solid in 100% yield: 1H NMR
(CDCl3) d 3.39 (3H, s), 3.70–4.00 (2H, m), 5.15 (2H, s), 6.72 (1H, d,
J = 7.2 Hz), 6.90–7.20 (5H, m), 7.54 (1H, br s), 7.65–7.80 (4H, m),
8.54 (1H, br s).
5.50. N-[4-(3-Aminophenyl)-3-cyano-6-(4-hydroxyphenyl)-
pyridin-2-yl]thiophene-2-carboxamide (7c)
Compound 7c was prepared from 6c in a manner similar to that
described for 7a as a light brown solid in 51% yield: mp 264–
265 °C; 1H NMR (DMSO-d6) d 5.40 (2H, s), 6.75 (2H, d, J = 7.8 Hz),
6.82 (1H, d, J = 7.8 Hz), 6.88–6.92 (3H, m), 7.20–7.30 (2H, m),
7.86 (1H, s), 7.96 (1H, d, J = 4.8 Hz), 8.11–8.16 (3H, m), 10.07 (1H,
br s), 11.35 (1H, br s). Anal. Calcd for C23H16N4O2S: C, 66.97; H,
3.91; N, 13.58. Found: C, 66.82; H, 3.90; N, 13.63.
5.45. N-{4-(3-Aminophenyl)-3-cyano-6-[4-methoxy-2-(methoxy-
methoxy)phenyl]pyridin-2-yl}benzamide (6k)
Compound 6k was prepared from 5k in a manner similar to that
described for 6a as a yellow amorphous solid in 100% yield: 1H
NMR (CDCl3) d 3.42 (3H, s), 3.80 (3H, s), 5.20 (2H, s), 6.62 (1H,
dd, J = 9.0, 2.1 Hz), 6.75–6.77 (2H, m), 6.96–7.00 (2H, m), 7.25
(1H, t, J = 7.8 Hz), 7.46 (2H, t, J = 7.4 Hz), 7.53 (1H, d, J = 7.2 Hz),
7.85–7.98 (4H, m), 8.85 (1H, s).
5.51. N-[4-(3-Aminophenyl)-3-cyano-6-(2-hydroxyphenyl)-
pyridin-2-yl]benzamide (7d)
A mixture of 5d (0.80 g, 1.5 mmol), iron powder (0.41 g,
7.3 mmol), and NH4Cl (0.40 g, 7.5 mmol) in EtOH (10 ml), THF
(5 ml) and water (5 ml) was stirred under reflux for 2 h. After cool-
ing to room temperature, the solution was diluted with water 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 = 1/2) to give
N-[4-(3-aminophenyl)-6-(2-{[tert-butyl(dimethyl)silyl]oxy}phe-
nyl)-3-cyanopyridin-2-yl]benzamide (6d, 0.55 g, 72%) as a yellow
amorphous solid.
5.46. N-{4-(3-Aminophenyl)-3-cyano-6-[4-fluoro-2-(methoxyme-
thoxy)phenyl]pyridin-2-yl}furan-2-carboxamide (6l)
Compound 6l was prepared from 5l in a manner similar to
that described for 6a as a colorless solid in 90% yield: mp
204–206 °C; 1H NMR (DMSO-d6) d 3.36 (3H, s), 5.38 (2H, s), 5.45
(2H, s), 6.71–6.81 (3H, m), 6.85 (1H, d, J = 1.9 Hz), 6.99–7.09
(1H, m), 7.13–7.27 (2H, m), 7.52 (1H, d, J = 3.4 Hz), 7.92–7.99
(2H, m), 8.02 (1H, s), 11.09 (1H, br s). Anal. Calcd for
C25H19FN4O4ꢀH2O: C, 63.02; H, 4.44; N, 11.75. Found: C, 63.28;
H, 4.07; N, 11.59.
Compound 7d was prepared from 6d in a manner similar to
that described for 7a as
a yellow solid in 65% yield: mp
259–261 °C; 1H NMR (DMSO-d6) d 5.45 (2H, br s), 6.76–6.79
(1H, m), 6.82–6.85 (1H, m), 6.90 (1H, t, J = 2.1 Hz), 6.94–7.01
(2H, m), 7.25 (1H, t, J = 7.8 Hz), 7.36–7.42 (1H, m), 7.59–7.64
(2H, m), 7.67–7.72 (1H, m), 8.05–8.08 (2H, m), 8.12 (1H, s),
8.15 (1H, dd, J = 8.1, 1.5 Hz), 11.41(1H, s), 11.99 (1H, s). Anal.
Calcd for C25H18N4O2: C, 73.88; H, 4.46; N, 13.78. Found: C,
73.60; H, 4.42; N, 13.73.
5.47. N-{4-(3-Aminophenyl)-3-cyano-6-[2-(methoxymethoxy)-
phenyl]-5-methylpyridin-2-yl}furan-2-carboxamide (6m)
Compound 6m was prepared from 5m in a manner similar to
that described for 6a as a colorless amorphous solid in 92% yield:
1H NMR (CDCl3) d 2.00 (3H, s), 3.38 (3H, s), 3.89 (2H, br s), 5.13
(2H, s), 6.53 (1H, q, J = 1.8 Hz), 6.69–6.77 (3H, m), 7.12 (1H, td,
J = 7.8, 0.9 Hz), 7.22–7.42 (5H, m), 7.50 (1H, s), 8.82 (1H, s).
5.52. tert-Butyl {2-[(3-{3-cyano-6-(2-hydroxyphenyl)-2-
[(thiophen-2-ylcarbonyl)amino]pyridin-4-yl}phenyl)amino]-
2-oxoethyl}carbamate (8a)
5.48. N-[4-(3-Aminophenyl)-3-cyano-6-(2-hydroxyphenyl)-
pyridin-2-yl]thiophene-2-carboxamide (7a)
To an ice-cooled solution of 6a (0.13 g, 0.25 mmol), [(tert-butox-
ycarbonyl)amino]acetic acid (86 mg, 0.50 mmol), and HOBt (66 mg,
0.50 mmol) in DMF (4 ml) was added WSCD (94 mg, 0.50 mmol).
After stirring at room temperature for 17 h, the reaction mixture
was diluted with saturated aqueous NaHCO3 and extracted with
EtOAc twice. The combined extracts were washed with brine, dried
over MgSO4, and concentrated in vacuo. The residue was purified by
silica gel column chromatography (EtOAc/hexane = 1/1 to 3/2) to
give a mixture of tert-butyl {2-[(3-{6-(2-{[tert-butyl(dimethyl)silyl]-
oxy}phenyl)-3-cyano-2-[(thiophen-2-ylcarbonyl)amino]pyridin-4-
yl}phenyl)amino]-2-oxoethyl}carbamate and 8a (0.10 g) as a pale
yellow solid.
The mixture was treated with TBAF in a manner similar to that
described for 7a to give 8a as a yellow solid in 40% yield from 6a:
mp 206–215 °C; 1H NMR (CDCl3) d 1.56 (9H, s), 3.98 (2H, d,
J = 6.0 Hz), 5.20–5.30 (1H, m), 6.90 (1H, t, J = 7.5 Hz), 7.10 (1H, d,
J = 7.5 Hz), 7.19 (1H, dd, J = 4.8, 3.9 Hz), 7.35–7.40 (2H, m), 7.50
(1H, t, J = 7.8 Hz), 7.60 (1H, d, J = 8.1 Hz), 7.67–7.69 (2H, m),
7.77–7.81 (2H, m), 7.95 (1H, s), 8.45–8.50 (1H, m), 8.80 (1H, s),
13.15–13.25 (1H, m).
To an ice-cooled solution of 6a (0.33 g, 0.63 mmol) in THF (6 ml)
was added TBAF (1 M in THF, 2 ml). After stirring at 0 °C for 40 min,
the reaction mixture was diluted with water and extracted with
EtOAc. The extract was washed with water and brine, dried over
MgSO4, and concentrated in vacuo. The residue was treated with
CHCl3 and the precipitated solid was collected by filtration. The
mother liquid was purified by silica gel column chromatography
(EtOAc/hexane = 2/98 to 70/30) to give a yellow solid. The solids
were combined and recrystallized from DMF/Et2O to give 7a
(45 mg, 17%) as a yellow solid: mp 283–284 °C; 1H NMR (DMSO-
d6) d 5.44 (2H, s), 6.72–6.85 (2H, m), 6.88 (1H, s), 7.18–7.46 (3H,
m), 8.00 (1H, d, J = 4.8 Hz), 8.08–8.19 (3H, m), 11.39 (1H, s),
11.87 (1H, s). Anal. Calcd for C23H16N4O2S: C, 66.97; H, 3.91; N,
13.58. Found: C, 66.77; H, 4.02; N, 13.60.
5.49. N-[4-(3-Aminophenyl)-3-cyano-6-(3-hydroxyphenyl)-
pyridin-2-yl]thiophene-2-carboxamide (7b)
Compound 7b was prepared from 6b in a manner similar to that
described for 7a as light brown solid in 57% yield: mp 246–247 °C;

3852
T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
5.53. tert-Butyl {3-[(3-{3-cyano-6-(2-hydroxyphenyl)-2-
[(thiophen-2-ylcarbonyl)amino]pyridin-4-yl}phenyl)amino]-
3-oxopropyl}carbamate (8b)
7.15–7.22 (2H, m), 7.43–7.50 (3H, m), 7.62–7.68 (2H, m), 7.81 (1H,
d, J = 3.3 Hz), 7.95 (2H, br s), 7.98–8.10 (1H, m), 8.45–8.64 (1H, m).
5.58. tert-Butyl {3-[(3-{3-cyano-6-[2-(ethoxymethoxy)-
4-fluorophenyl]-2-[(thiophen-2-ylcarbonyl)amino]pyridin-
4-yl}phenyl)amino]-3-oxopropyl}carbamate (8g)
To an ice-cooled solution of 6a (0.30 g, 0.57 mmol), 3-[(tert-
butoxycarbonyl)amino]propanoic acid (0.11 g, 0.57 mmol), and
HOBt (77 mg, 0.57 mmol) in DMF (8 ml) was added WSCD
(0.11 g, 0.57 mmol). After stirring at room temperature for 12 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 = 1/
1 to 2/1) and recrystallization from CHCl3/Et2O to give 8b (0.13 g,
40%) as a pale yellow solid: mp 230–231 °C; 1H NMR (DMSO-d6)
d 1.38 (9H, s), 2.40–2.54 (2H, m), 3.22–3.27 (2H, m), 6.89–7.01
(3H, m), 7.28–7.42 (3H, m), 7.54 (1H, t, J = 8.0 Hz), 7.80 (1H, d,
J = 8.1 Hz), 7.99 (2H, s), 8.12–8.14 (3H, m), 10.24 (1H, s), 11.44
(1H, s), 11.75–11.85 (1H, m).
A mixture of 5g (0.20 g, 0.39 mmol), iron powder (0.13 g,
2.31 mmol), and NH4Cl (0.13 g, 2.50 mmol) in 50% aqueous EtOH
(20 ml) was stirred under reflux for 2 h. After cooling to room tem-
perature, the solution was diluted with saturated aqueous NaHCO3
and extracted with EtOAc. The extract was washed with brine, dried
over MgSO4, and concentrated in vacuo to give N-{4-(3-aminophe-
nyl)-3-cyano-6-[2-(ethoxymethoxy)-4-fluorophenyl]pyridin-2-yl}-
thiophene-2-carboxamide (6g, 0.15 g, 79%) as an amorphous solid.
Compound 8g was prepared from 6g and 3-[(tert-butoxycar-
bonyl)amino]propanoic acid in a manner similar to that described
for 8b as a yellow solid in 63% yield: 1H NMR (CDCl3) d 1.21 (3H, t,
J = 7.2 Hz), 1.44 (9H, s), 2.63 (2H, t, J = 6.0 Hz), 3.49 (2H, q,
J = 6.0 Hz), 3.71 (2H, q, J = 7.2 Hz), 5.12–5.20 (1H, m), 5.34 (2H, s),
6.85 (1H, td, J = 8.4, 2.4 Hz), 7.05 (1H, d, J = 10.5 Hz), 7.17 (1H, t,
J = 4.5 Hz), 7.42–7.50 (2H, m), 7.60–7.65 (2H, m), 7.75–7.80 (1H,
m), 7.93–7.98 (3H, m), 8.08–8.20 (1H, m), 8.52 (1H, br s).
5.54. tert-Butyl {3-[(3-{3-cyano-6-(2-hydroxyphenyl)-2-[(phenyl-
carbonyl)amino]pyridin-4-yl}phenyl)amino]-3-oxopropyl}-
carbamate (8c)
Compound 8c was prepared from 7d and 3-[(tert-butoxycar-
bonyl)amino]propanoic acid in a manner similar to that described
for 8b as a pale yellow solid in 74% yield: mp 220–221 °C; 1H NMR
(DMSO-d6) d 1.38 (9 H, s), 2.53–2.55 (2H, m), 3.21–3.27 (2H, m),
6.88 (1H, br s), 6.95–7.01 (2H, m), 7.37–7.43 (2H, m), 7.52–7.73
(4H, m), 7.82 (1H, brd, J = 8.4 Hz), 8.00 (1H, br s), 8.06–8.08 (2H,
m), 8.13–8.17 (2H, m), 10.24 (1H, br s), 11.45 (1H, br s), 11.92
(1H, br s). Anal. Calcd for C33H31N5O5: C, 68.62; H, 5.41; N, 12.12.
Found: C, 68.37; H, 5.36; N, 12.11.
5.59. tert-Butyl {3-[(3-{6-[4-chloro-2-(methoxymethoxy)-
phenyl]-3-cyano-2-[(thiophen-2-ylcarbonyl)amino]pyridin-
4-yl}phenyl)amino]-3-oxopropyl}carbamate (8h)
Compound 8h was prepared from 6h and 3-[(tert-butoxycar-
bonyl)amino]propanoic acid in a manner similar to that described
for 8b as a colorless solid in 54% yield: mp 194–195 °C; 1H NMR
(DMSO-d6) d 1.37 (9H, s), 2.50–2.55 (2H, m), 3.22–3.30 (2H, m),
3.36 (3H, s), 5.41 (2H, s), 6.80–6.95 (1H, m), 7.25–7.29 (2H, m),
7.37–7.39 (2H, m), 7.54 (1H, td, J = 7.8, 3.0 Hz), 7.72 (1H, d,
J = 7.5 Hz), 7.94–7.98 (2H, m), 8.02 (1H, s), 8.15 (1H, s), 8.16 (1H,
s), 10.24 (1H, s), 11.46 (1H, s).
5.55. tert-Butyl {4-[(3-{3-cyano-6-(2-hydroxyphenyl)-2-[(thio-
phen-2-ylcarbonyl)amino]pyridin-4-yl}phenyl)amino]-4-
oxobutyl}carbamate (8d)
Compound 8d was prepared from 6a and 4-[(tert-butoxycar-
bonyl)amino]butanoic acid in a manner similar to that described
for 8b as a colorless amorphous solid in 91% yield: 1H NMR
5.60. tert-Butyl {3-[(3-{6-[4-bromo-2-(methoxymethoxy)-
phenyl]-3-cyano-2-[(thiophen-2-ylcarbonyl)amino]pyridin-
4-yl}phenyl)amino]-3-oxopropyl}carbamate (8i)
(DMSO-d6)
d 1.56 (9H, s), 1.66–1.75 (2H, m), 2.35 (2H, t,
J = 7.4 Hz), 2.89–3.01 (2H, m), 6.80–6.90 (1H, m), 6.95–7.01 (2H,
m), 7.31–7.59 (4H, m), 7.70–7.80 (1H, m), 7.90–8.05 (3H, m), 8.14
(2H, d, J = 7.8 Hz), 10.21 (1H, s), 11.45 (1H, s), 11.80–11.90 (1H, m).
A mixture of 5i (0.15 g, 0.27 mmol), iron powder (88 mg,
1.59 mmol), and NH4Cl (92 mg, 1.72 mmol) in 50% aqueous EtOH
(10 ml) was stirred under reflux for 2 h. After cooling to room tem-
perature, the solution was diluted with saturated aqueous NaHCO3
and extracted with EtOAc. The extract was washed with brine, dried
over MgSO4, and concentrated in vacuo to give N-{4-(3-aminophe-
nyl)-6-[4-bromo-2-(methoxymethoxy)phenyl]-3-cyanopyridin-2-
yl}thiophene-2-carboxamide (6i, 0.14 g, 100%) as an amorphous
solid.
Compound 8i was prepared from 6i and 3-[(tert-butoxycar-
bonyl)amino]propanoic acid in a manner similar to that described
for 8b as a yellow solid in 63% yield: mp 144–145 °C; 1H NMR
(CDCl3) d 1.44 (9H, t, J = 7.2 Hz), 2.64 (2H, t, J = 6.0 Hz), 3.45–3.54
(5H, m), 5.10–5.18 (1H, m), 5.30 (2H, s), 7.18 (1H, t, J = 4.4 Hz),
7.26–7.32 (2H, m), 7.45–7.58 (3H, m), 7.61–7.66 (2H, m), 7.79–
7.81 (2H, m), 7.91–8.02 (2H, m), 8.40–8.50 (1H, m).
5.56. tert-Butyl {3-[(3-{6-[5-chloro-2-(methoxymethoxy)-
phenyl]-3-cyano-2-[(thiophen-2-ylcarbonyl)amino]pyridin-
4-yl}phenyl)amino]-3-oxopropyl}carbamate (8e)
Compound 8e was prepared from 6e and 3-[(tert-butoxycar-
bonyl)amino]propanoic acid in a manner similar to that described
for 8b as a colorless solid in 69% yield: mp 230–231 °C; 1H NMR
(DMSO-d6) d 1.38 (9H, s), 3.21–3.28 (2H, m), 3.78 (3H, s), 5.37
(2H, s), 6.80–6.90 (1H, m), 7.27–7.40 (3H, m), 7.52–7.58 (2H, m),
7.73 (1H, d, J = 7.8 Hz), 7.98 (2H, d, J = 3.0 Hz), 8.06–8.16 (3H, m),
10.24 (1H, s), 11.41–11.50 (1H, m).
5.57. tert-Butyl {3-[(3-{3-cyano-6-[5-methoxy-2-(methoxy-
methoxy)phenyl]-2-[(thiophen-2-ylcarbonyl)amino]pyridin-
4-yl}phenyl)amino]-3-oxopropyl}carbamate (8f)
5.61. tert-Butyl {3-[(3-{3-cyano-6-[4-methoxy-2-(methoxyme-
thoxy)phenyl]-2-[(phenylcarbonyl)amino]pyridin-4-yl}phenyl)-
amino]-3-oxopropyl}carbamate (8k)
Compound 8f was prepared from 6f and 3-[(tert-butoxycar-
bonyl)amino]propanoic acid in a manner similar to that described
for 8b as a yellow solid in 63% yield: mp 167–168 °C; 1H NMR
(CDCl3) d 1.43 (9H, s), 2.62 (2H, t, J = 5.7 Hz), 3.43 (3H, s), 3.49 (2H,
q, J = 5.9 Hz), 3.84 (3H, s), 5.12 (2H, s), 6.97 (1H, dd, J = 9.0, 3.3 Hz),
Compound 8k was prepared from 6k and 3-[(tert-butoxycar-
bonyl)amino]propanoic acid in a manner similar to that described
for 8b as a colorless amorphous solid in 32% yield: 1H NMR (CDCl3)
d 1.44 (9H, s), 2.64 (2H, t, J = 6.0 Hz), 3.48–3.55 (5H, m), 3.86 (3H,

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
3853
s), 5.10–5.20 (1H, m), 5.31 (2H, s), 6.69 (1H, dd, J = 8.7, 2.1 Hz), 6.82
(1H, s), 7.48–7.63 (6H, m), 7.91–8.04 (6H, m).
(2H, m), 7.37–7.45 (2H, m), 7.56–7.64 (3H, m), 7.68–7.85 (5H,
m), 8.00 (1H, br s), 8.05–8.08 (2H, m), 8.14 (1H, dd, J = 8.1,
1.5 Hz), 8.17 (1H, s), 10.46 (1H, br s), 11.49 (1H, br s), 11.87 (1H,
br s). Anal. Calcd for C28H23N5O3ꢀC2HF3O2: C, 60.91; H, 4.09; N,
11.84. Found: C, 60.75; H, 4.17; N, 11.90.
5.62. tert-Butyl {3-[(3-{3-cyano-6-[4-fluoro-2-(methoxymethoxy)-
phenyl]-2-[(furan-2-ylcarbonyl)amino]pyridin-4-yl}phenyl)-
amino]-3-oxopropyl}carbamate (8l)
5.67. N-[4-{3-[(4-Aminobutanoyl)amino]phenyl}-3-cyano-6-(2-
hydroxyphenyl)pyridin-2-yl]thiophene-2-carboxamide
hydrochloride (9d)
Compound 8l was prepared from 6l and 3-[(tert-butoxycar-
bonyl)amino]propanoic acid in a manner similar to that described
for 8b as a colorless solid in 19% yield: mp 198–200 °C; 1H NMR
(DMSO-d6) d 1.38 (9H, s), 2.48–2.55 (2H, m), 3.20–3.28 (2H, m),
3.37 (3H, s), 5.40 (2H, s), 6.77 (1H, dd, J = 3.4, 1.9 Hz), 6.88 (1H, t,
J = 5.7 Hz), 7.01–7.10 (1H, m), 7.18 (1H, dd, J = 11.3, 2.6 Hz), 7.38
(1H, d, J = 7.5 Hz), 7.50–7.59 (2H, m), 7.71 (1H, d, J = 8.3 Hz),
7.95–8.06 (3H, m), 8.11 (1H, br s), 10.24 (1H, br s), 11.28 (1H, br
s). Anal. Calcd for C33H32FN5O7: C, 62.95; H, 5.12; N, 11.12. Found:
C, 62.76; H, 5.10; N, 11.10.
Compound 9d was prepared from 8d in a manner similar to that
described for 9a as a pale yellow solid in 41% yield: mp 236–
238 °C; 1H NMR (DMSO-d6) d 1.83–1.93 (2H, m), 2.47–2.51 (2H,
m), 2.87 (2H, t, J = 7.8 Hz), 6.95–7.03 (2H, m), 7.30 (1H, t,
J = 4.4 Hz), 7.38–7.41 (2H, m), 7.56 (1H, t, J = 8.0 Hz), 7.60–7.70
(3H, m), 7.99–8.02 (2H, m), 8.13–8.15 (3H, m), 10.35 (1H, s),
11.40–11.90 (2H, m). Anal. Calcd for C27H23N5O3SꢀHClꢀ0.5H2O: C,
59.71; H, 4.64; N, 12.90. Found: C, 60.02; H, 4.50; N, 13.02.
5.63. tert-Butyl {3-[(3-{3-cyano-2-[(furan-2-ylcarbonyl)amino]-
6-[2-(methoxymethoxy)phenyl]-5-methylpyridin-4-yl}phenyl)-
amino]-3-oxopropyl}carbamate (8m)
5.68. N-{4-[3-(b-Alanylamino)phenyl]-6-(5-chloro-2-hydroxy-
phenyl)-3-cyanopyridin-2-yl}thiophene-2-carboxamide
trifluoroacetate (9e)
Compound 8m was prepared from 6m and 3-[(tert-butoxycar-
bonyl)amino]propanoic acid in a manner similar to that described
for 8b as a colorless amorphous solid in 73% yield: 1H NMR (CDCl3)
d 1.44 (9H, s), 2.01 (3H, s), 2.61 (2H, t, J = 5.7 Hz), 3.41 (3H, s), 3.49
(2H, q, J = 5.9 Hz), 5.19 (2H, s), 5.20–5.30 (1H, m), 6.57 (1H, q,
J = 1.8 Hz), 7.12–7.16 (2H, m), 7.24–7.35 (3H, m), 7.38–7.53 (4H,
m), 7.65–7.85 (1H, m), 7.97 (1H, br s), 8.70 (1H, s).
Compound 9e was prepared from 8e in a manner similar to that
described for 9c as a yellow solid in 95% yield: mp 197–204 °C; 1H
NMR (DMSO-d6) d 2.76 (2H, t, J = 6.6 Hz), 3.10–3.13 (2H, m), 7.05
(1H, d, J = 8.7 Hz), 7.31 (1H, t, J = 4.4 Hz), 7.41–7.45 (2H, m), 7.58
(1H, t, J = 8.0 Hz), 7.60–7.85 (4H, m), 8.01 (2H, d, J = 5.1 Hz), 8.14
(1H, d, J = 3.6 Hz), 8.21 (1H, d, J = 2.7 Hz), 8.26 (1H, s), 10.46 (1H,
s), 11.44 (1H, s), 11.26(1H, s). Anal. Calcd for C26H20ClN5O3SꢀC2H-
F3O2ꢀH2O: C, 51.74; H, 3.56; N, 10.77. Found: C, 51.74; H, 3.57; N,
10.77.
5.64. N-{3-Cyano-4-[3-(glycylamino)phenyl]-6-(2-hydroxy-
phenyl)pyridin-2-yl}thiophene-2-carboxamide dihydrochloride
(9a)
5.69. N-{4-[3-(b-Alanylamino)phenyl]-3-cyano-6-(2-hydroxy-
5-methoxyphenyl)pyridin-2-yl}thiophene-2-carboxamide
hydrochloride (9f)
To a solution of 8a (60 mg, 0.11 mmol) in EtOAc (5 ml) was
added 4 M HCl EtOAc solution (2 ml) and the whole was stirred
at room temperature for 2 h. The precipitated solid was collected
by filtration and washed with EtOAc to give 9a (53 mg, 95%) as a
yellow solid: mp 210–217 °C; 1H NMR (DMSO-d6) d 3.85 (2H, t,
J = 5.7 Hz), 6.96–7.05 (2H, m), 7.31 (1H, t, J = 2.6 Hz), 7.37–7.49
(2H, m), 7.62 (1H, t, J = 8.0 Hz), 7.83 (1H, d, J = 9.0 Hz), 8.00–8.04
(2H, m), 8.13–8.25 (6H, m), 10.97 (1H, s), 11.09 (1H, s). Anal. Calcd
for C25H19N5O3Sꢀ2HClꢀ0.5H2O: C, 54.45; H, 4.02; N, 12.70. Found: C,
54.65; H, 4.01; N, 12.61.
Compound 9f was prepared from 8f in a manner similar to that
described for 9a as an orange solid in 100% yield: mp 197–201 °C;
1H NMR (DMSO-d6) d 2.79 (2H, t, J = 6.6 Hz), 3.10 (2H, q, J = 6.0 Hz),
3.78 (3H, s), 6.95–7.05 (2H, m), 7.31 (1H, t, J = 4.4 Hz), 7.40 (1H, d,
J = 7.5 Hz), 7.55–7.63 (2H, m), 7.82–8.00 (4H, m), 8.01 (2H, d,
J = 4.0 Hz), 8.15 (1H, d, J = 4.0 Hz), 8.22 (1H, s), 10.55 (1H, s),
11.20–11.45 (1H, m), 11.49 (1H, s). Anal. Calcd for
C27H23N5O4SꢀHClꢀ2.5H2O: C, 54.50; H, 4.91; N, 11.77. Found: C,
54.46; H, 4.78; N, 11.75.
5.65. N-{4-[3-(b-Alanylamino)phenyl]-3-cyano-6-(2-hydroxy-
phenyl)pyridin-2-yl}thiophene-2-carboxamide hydrochloride
(9b)
5.70. N-{4-[3-(b-Alanylamino)phenyl]-3-cyano-6-(4-fluoro-2-
hydroxyphenyl)pyridin-2-yl}thiophene-2-carboxamide
hydrochloride (9g)
Compound 9b was prepared from 8b in a manner similar to that
described for 9a as a yellow solid in 88% yield: mp 223–228 °C; 1H
NMR (DMSO-d6) d 2.78 (2H, t, J = 6.8 Hz), 3.10–3.17 (2H, m), 6.95–
7.04 (2H, m), 7.30–7.33 (1H, m), 7.40 (1H, t, J = 7.8 Hz), 7.58 (1H, t,
J = 7.8 Hz), 7.80–8.00 (4H, m), 8.00–8.02 (1H, m), 8.12–8.18 (3H,
m), 10.54 (1H, s), 11.50 (1H, s), 11.79 (1H, s). Anal. Calcd for
C26H21N5O3SꢀHClꢀ1.5H2O: C, 57.19; H, 4.43; N, 12.83. Found: C,
57.36; H, 4.58; N, 12.85.
Compound 9g was prepared from 8g in a manner similar to that
described for 9a as a yellow solid in 64% yield: mp 217–220 °C; 1H
NMR (DMSO-d6) d 2.80 (2H, t, J = 6.8 Hz), 3.05–3.15 (2H, m), 6.81–
6.90 (2H, m), 7.31 (1H, t, J = 4.4 Hz), 7.41 (1H, t, J = 7.8 Hz), 7.57
(1H, t, J = 7.8 Hz), 7.83 (1H, d, J = 8.4 Hz), 7.85–8.02 (5H, m),
8.15–8.25 (3H, m), 10.58 (1H, s), 11.47 (1H, s), 12.25 (1H, s). Anal.
Calcd for C26H20FN5O3SꢀHClꢀ1.5H2O: C, 55.27; H, 4.28; N, 12.40.
Found: C, 55.20; H, 4.31; N, 12.39.
5.66. N-{4-[3-(b-Alanylamino)phenyl]-3-cyano-6-(2-hydroxy-
phenyl)pyridin-2-yl}benzamide trifluoroacetate (9c)
5.71. N-{4-[3-(b-Alanylamino)phenyl]-6-(4-chloro-2-hydroxy-
phenyl)-3-cyanopyridin-2-yl}thiophene-2-carboxamide
trifluoroacetate (9h)
A solution of 8c (0.15 g, 0.26 mmol) in TFA (5 ml) was stirred at
room temperature for 90 min. The solvent was evaporated in vacuo
and the residual solid was recrystallized from EtOH/Et2O to give 9c
(0.12 g, 78%) as a pale yellow solid: mp 225–226 °C; 1H NMR
(DMSO-d6) d 2.76 (2H, t, J = 6.9 Hz), 3.12 (2H, br s), 6.95–7.02
Compound 9h was prepared from 8h in a manner similar to that
described for 9c as a pale green solid in 58% yield: mp 206–209 °C;

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T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
1H NMR (DMSO-d6) d 2.76 (2H, q, J = 6.6 Hz), 3.09–3.15 (2H, m),
7.03–7.10 (2H, m), 7.31 (1H, t, J = 4.4 Hz), 7.41 (1H, t, J = 7.5 Hz),
7.58 (1H, t, J = 8.0 Hz), 7.65–7.80 (4H, m), 8.01 (2H, d, J = 4.4 Hz),
8.13–8.19 (3H, m), 10.47 (1H, s), 11.48 (1H, s), 12.09 (1H, s). Anal.
Calcd for C26H20ClN5O3SꢀC2HF3O2ꢀ2H2O: C, 50.34; H, 3.77; N, 10.48.
Found: C, 50.19; H, 3.76; N, 10.51.
5.75. N-{4-[3-(b-Alanylamino)phenyl]-3-cyano-6-(4-fluoro-2-
hydroxyphenyl)pyridin-2-yl}furan-2-carboxamide
hydrochloride (9l)
A solution of 8l (97 mg, 0.15 mmol) in 10% HCl MeOH solution
(6 ml) was stirred at room temperature for 3.5 h. The solvent was
evaporated in vacuo and the residual solid was washed with hot
EtOAc. Recrystallization from EtOH/Et2O gave 9l (26 mg, 32%) as
a yellow solid: mp 269–270 °C; 1H NMR (DMSO-d6) d 2.70–2.80
(2H, m), 3.11 (2H, t, J = 6.8 Hz), 6.76–6.89 (3H, m), 7.41 (1H, d,
J = 7.5 Hz), 7.50 (1H, br s), 7.58 (1H, t, J = 8.1 Hz), 7.60–7.95 (4H,
m), 7.99 (1H, s), 8.03–8.14 (2H, m), 8.22 (1H, dd, J = 9.8, 6.8 Hz),
10.47 (1H, br s), 12.40 (2H, br s). Anal. Calcd for C26H20FN5-
O4ꢀHClꢀ2H2O: C, 55.97; H, 4.52; N, 12.55. Found: C, 55.85; H,
4.42; N, 12.31.
5.72. N-{4-[3-(b-Alanylamino)phenyl]-6-(4-bromo-2-hydroxy-
phenyl)-3-cyanopyridin-2-yl}thiophene-2-carboxamide
hydrochloride (9i)
Compound 9i was prepared from 8i in a manner similar to that
described for 9a as a yellow solid in 100% yield: mp 203–211 °C; 1H
NMR (DMSO-d6) d 2.78 (2H, t, J = 6.6 Hz), 3.05–3.16 (2H, m), 7.17
(1H, t, J = 8.7 Hz), 7.26–7.33 (2H, m), 7.40 (1H, d, J = 7.2 Hz), 7.57
(1H, t, J = 8.1 Hz), 7.80–7.98 (4H, m), 8.00–8.18 (5H, m), 10.53
(1H, s), 11.44 (1H, s), 11.90–12.05 (1H, m). Anal. Calcd for
C26H20BrN5O3SꢀHClꢀH2O: C, 48.50; H, 4.07; N, 10.88. Found: C,
48.40; H, 4.35; N, 11.06.
5.76. N-{4-[3-(b-Alanylamino)phenyl]-3-cyano-6-(2-
hydroxyphenyl)-5-methylpyridin-2-yl}furan-2-carboxamide
hydrochloride (9m)
5.73. N-{4-[3-(b-Alanylamino)phenyl]-3-cyano-6-(2-hydroxy-
4-methylphenyl)pyridin-2-yl}thiophene-2-carboxamide
hydrochloride (9j)
Compound 9m was prepared from 8m in a manner similar to
that described for 9a as a yellow amorphous solid in 77% yield:
1H NMR (DMSO-d6)
d 1.95 (3H, s), 2.78 (2H, t, J = 3.3 Hz),
3.09 (2H, q, J = 3.3 Hz), 6.73 (1H, q, J = 1.8 Hz), 6.90–7.02 (2H, m),
7.10 (1H, d, J = 7.5 Hz), 7.26–7.32 (2H, m), 7.51–7.56 (2H, m),
7.71 (1H, d, J = 8.4 Hz), 7.78 (1H, s), 7.80–8.00 (4H, m), 9.89 (1H,
A mixture of 5j (0.20 g, 0.39 mmol), iron powder (0.13 g,
2.33 mmol), and NH4Cl (0.14 g, 2.53 mmol) in 50% aqueous EtOH
(20 ml) was stirred under reflux for 20 min. After cooling to room
temperature, the solution was diluted with saturated aqueous
NaHCO3 and extracted with EtOAc. The extract was washed with
brine, dried over MgSO4, and concentrated in vacuo to give N-{4-
(3-aminophenyl)-3-cyano-6-[2-(ethoxymethoxy)-4-methylphenyl]-
pyridin-2-yl}thiophene-2-carboxamide (6j, 0.14 g, 74%) as an
amorphous solid.
br s), 10.56 (1H, s), 11.22 (1H, s). Anal. Calcd for C27H23
-
N5O4ꢀHClꢀ1.5H2O: C, 59.50; H, 4.99; N, 12.85. Found: C, 59.86; H,
5.10; N, 12.55.
5.77. 3-{3-Cyano-6-[2-(methoxymethoxy)phenyl]-2-[(thiophen-
2-ylcarbonyl)amino]pyridin-4-yl}benzoic acid (10a)
To an ice-cooled solution of 6j (0.14 g, 0.29 mmol), 3-[(tert-
butoxycarbonyl)amino]propanoic acid (0.11 g, 0.58 mmol), and
HOBt (78 mg, 0.58 mmol) in DMF (8 ml) was added WSCD
(0.11 g, 0.57 mmol). After stirring at room temperature for 18 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 = 1/
1 to 3/2) and recrystallization from EtOAc/Et2O to give tert-butyl
{3-[(3-{3-cyano-6-[2-(ethoxymethoxy)-4-methylphenyl]-2-[(thio-
phen-2-ylcarbonyl)amino]pyridin-4-yl}phenyl)amino]-3-oxopropyl}-
carbamate (8j, 55 mg, 29%) as a yellow solid.
Compound 9j was prepared from 8j in a manner similar to that
described for 9a as a yellow solid in 100% yield: mp 204–210 °C; 1H
NMR (DMSO-d6) d 2.31 (3H, s), 2.79 (2H, t, J = 6.8 Hz), 3.10 (2H, t,
J = 6.0 Hz), 6.79–6.84 (2H, m), 7.31 (1H, t, J = 4.4 Hz), 7.41 (1H, t,
J = 7.8 Hz), 7.57 (1H, t, J = 7.8 Hz), 7.82–7.96 (4H, m), 8.00–8.07
(3H, m), 8.14 (2H, d, J = 3.9 Hz), 10.54 (1H, s), 11.43 (1H, s), 11.91
(1H, s). Anal. Calcd for C27H23N5O3SꢀHClꢀH2O: C, 58.74; H, 4.75;
N, 12.69. Found: C, 58.42; H, 4.90; N, 12.30.
To a solution of 4m (1.5 g, 3.9 mmol) in pyridine (15 ml) was
added 2-thenoyl chloride (1.0 ml, 9.6 mmol) at room temperature
and the whole was stirred at room temperature for 17 h. After the
solvent was evaporated in vacuo, the residue was diluted with
EtOAc, washed with water and brine, dried over MgSO4, and con-
centrated in vacuo. The residue was dissolved into MeOH (15 ml)
and to the solution was added 2 M Na2CO3 (20 ml). After the
whole was stirred at room temperature for 20 h, the mixture
was diluted with water 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 chromatogra-
phy (EtOAc/hexane = 1/1) and recrystallization from EtOAc/hex-
ane to give methyl 3-{3-cyano-6-[2-(methoxymethoxy)phenyl]-
2-[(thiophen-2-ylcarbonyl)amino]pyridin-4-yl}benzoate (0.94 g,
49%) as a colorless solid: mp 159–160 °C; 1H NMR (CDCl3) d
3.32 (3H, s), 3.92 (3H, s), 5.32 (2H, s), 7.19 (1H, t, J = 7.5 Hz),
7.27–7.31 (2H, m), 7.50 (1H, t, J = 7.8 Hz), 7.80 (1H, t, J = 7.8 Hz),
7.91 (1H, d, J = 7.8 Hz), 7.97–8.08 (3H, m), 8.16–8.19 (2H, m),
8.26 (1H, s), 11.49 (1H, s).
To a solution of methyl 3-{3-cyano-6-[2-(methoxymethoxy)-
phenyl]-2-[(thiophen-2-ylcarbonyl)amino]pyridine-4-yl}benzoate
(1.0 g, 2.1 mmol) in THF (15 ml) and EtOH (8 ml) was added 4 M
NaOH (1.5 ml) and the whole was stirred at room temperature
for 16 h. The reaction mixture was neutralized with 1 M HCl and
extracted with EtOAc. The extract was washed with brine, dried
over MgSO4, and concentrated in vacuo to give 10a (1.01 g, 100%)
as a colorless solid: mp 179–180 °C; 1H NMR (DMSO-d6) d 3.36
(3H, s), 5.32 (2H, s), 7.17 (1H, t, J = 7.5 Hz), 7.28–7.31 (2H, m),
7.47–7.53 (1H, m), 7.78 (1H, t, J = 7.8 Hz), 7.92 (1H, dd, J = 7.5,
1.5 Hz), 7.98–8.01 (2H, m), 8.09 (1H, s), 8.14–8.20 (2H, m), 8.29
(1H, s), 11.47 (1H, s), 13.15–13.35 (1H, m).
5.74. N-{4-[3-(b-Alanylamino)phenyl]-3-cyano-6-(2-hydroxy-4-
methoxyphenyl)pyridin-2-yl}benzamide trifluoroacetate (9k)
Compound 9k was prepared from 8k in a manner similar to
that described for 9c as a yellow solid in 86% yield: mp 164–
170 °C; 1H NMR (DMSO-d6) d 2.76 (2H, t, J = 7.1 Hz), 3.12 (2H, q,
J = 5.9 Hz), 3.81 (3H, s), 6.53–6.58 (2H, m), 7.42 (1H, d,
J = 7.5 Hz), 7.55–7.85 (8H, m), 7.97 (1H, s), 8.05–8.14 (4H, m),
10.45 (1H, s), 11.45 (1H, s), 12.40(1H, s). Anal. Calcd for
C29H25N5O4ꢀC2HF3O2ꢀH2O: C, 58.22; H, 4.41; N, 10.95. Found: C,
58.45; H, 4.48; N, 10.92.

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
3855
5.78. 2-{3-Cyano-6-[2-(methoxymethoxy)phenyl]-2-[(thiophen-
2-ylcarbonyl)amino]pyridin-4-yl}benzoic acid (10b)
ate (3.0 g, 5.9 mmol) in THF (40 ml) and EtOH (20 ml) was added
4 M NaOH (4.5 ml) and the whole was stirred at room temperature
for 6 h. The reaction mixture was neutralized with 1 M HCl and ex-
tracted with EtOAc. The extract was washed with brine, dried over
MgSO4, and concentrated in vacuo to give 10d (2.6 g, 92%) as a pale
yellow solid: mp 254–255 °C; 1H NMR (DMSO-d6) d 3.38 (3H, s),
5.36 (2H, s), 6.77 (1H, dd, J = 3.3, 1.8 Hz), 7.05 (1H, td, J = 8.4,
2.4 Hz), 7.17 (1H, dd, J = 11.1, 2.4 Hz), 7.55 (1H, d, J = 3.3 Hz), 7.78
(1H, t, J = 7.8 Hz), 7.95–8.05 (3H, m), 8.08 (1H, s), 8.12–8.20 (1H,
m), 8.27 (1H, s), 11.35 (1H, s).
To a solution of 4n (0.60 g, 1.5 mmol) in pyridine (10 ml) was
added 2-thenoyl chloride (0.57 g, 3.9 mmol) at room temperature
and the whole was stirred at room temperature for 48 h. After the
solvent was evaporated in vacuo, the residue was diluted with
EtOAc, washed with water and brine, dried over MgSO4, and concen-
trated in vacuo. The residue was purified by basic silica gel column
chromatography (EtOAc/hexane = 1/1 to 2/1) and recrystallization
from Et2O to give methyl 2-{3-cyano-6-[2-(methoxymethoxy)-
phenyl]-2-[(thiophen-2-ylcarbonyl)amino]pyridin-4-yl}benzoate
(0.69 g, 90%) as a colorless solid: mp 190–191 °C; 1H NMR (CDCl3) d
3.41 (3H, s), 3.77 (3H, s), 5.22 (2H, s), 7.14–7.26 (4H, m), 7.38–7.46
(2H, m), 7.57–7.70 (3H, m), 7.76 (1H, s), 7.95 (1H, dd, J = 8.1,
1.8 Hz), 8.12 (1H, d, J = 8.0 Hz), 8.38 (1H, s).
Compound 10b was prepared from methyl 2-{3-cyano-6-
[2-(methoxymethoxy)phenyl]-2-[(thiophen-2-ylcarbonyl)amino]-
pyridin-4-yl}benzoate in a manner similar to that described for
10a as a colorless solid in 89% yield: mp 182–183 °C; 1H NMR
(DMSO-d6) d 3.33 (3H, s), 5.26 (2H, s), 7.19 (1H, t, J = 7.7 Hz),
7.26–7.29 (2H, m), 7.45–7.51 (2H, m), 7.67 (1H, t, J = 7.7 Hz), 7.77
(1H, t, J = 7.5 Hz), 7.87 (1H, s), 7.91–7.97 (2H, m), 8.08 (1H, dd,
J = 7.8, 1.2 Hz), 8.14–8.15 (1H, m), 11.35 (1H, s), 13.10 (1H, s).
5.81. 3-{3-Cyano-6-(2-hydroxyphenyl)-2-[(thiophen-2-
ylcarbonyl)amino]pyridin-4-yl}benzoic acid (11)
To a solution of 10a (0.15 g, 0.31 mmol) in THF (10 ml) was
added concd HCl (0.5 ml) with ice-cooling and the whole was stir-
red at room temperature for 1 h. The reaction mixture was diluted
with EtOAc, washed with water and brine, dried over MgSO4, and
concentrated in vacuo. The residual solid was washed with EtOAc
to give 11 (0.12 g, 84%) as a colorless solid: mp 308–309 °C; 1H
NMR (DMSO-d6)
d 6.97–7.02 (2H, m), 7.31 (1H, dd, J = 4.8,
3.9 Hz), 7.41 (1H, t, J = 7.8 Hz), 7.78 (1H, t, J = 7.8 Hz), 8.00–8.03
(2H, m), 8.14–8.18 (3H, m), 8.27–8.30 (2H, m), 11.49 (1H, br s),
11.95 (1H, s), 13.33 (1H, br s). Anal. Calcd for C24H15N3O4S: C,
65.30; H, 3.42; N, 9.52. Found: C, 65.05; H, 3.41; N, 9.41.
5.79. 4-{3-Cyano-6-[2-(methoxymethoxy)phenyl]-2-[(thiophen-
2-ylcarbonyl)amino]pyridin-4-yl}benzoic acid (10c)
5.82. tert-Butyl (2-{[(3-{3-cyano-6-[2-(methoxymethoxy)-
phenyl]-2-[(thiophen-2-ylcarbonyl)amino]pyridin-4-yl}-
phenyl)carbonyl]amino}ethyl)carbamate (12b)
To a solution of 4o (0.30 g, 0.77 mmol) in pyridine (10 ml) was
added 2-thenoyl chloride (0.28 g, 1.9 mmol) at room temperature
and the whole was stirred at room temperature for 18 h. To the
reaction mixture was added 4 M NaOH (2.5 ml) and stirred at room
temperature for 1 h. The solution was diluted with water, neutral-
ized with 5 M HCl, and extracted with EtOAc. The extract was
washed with brine, dried over MgSO4, and concentrated in vacuo.
The residue was purified by recrystallization from EtOAc to give
10c (0.30 g, 81%) as a yellow solid: mp 205–206 °C; 1H NMR
(DMSO-d6) d 3.34 (3H, s), 5.32 (2H, s), 7.19 (1H, t, J = 8.4 Hz),
7.27–7.31 (2H, m), 7.50 (1H, t, J = 7.8 Hz), 7.84–8.00 (4H, m), 8.06
(1H, d, J = 2.4 Hz), 8.14–8.18 (3H, m), 11.47 (1H, s), 13.10–13.40
(1H, m).
Compound 12b was prepared from 10a and tert-butyl (2-ami-
noethyl)carbamate in a manner similar to that described for 8b
as a colorless solid in 89% yield: mp 200–201 °C; 1H NMR (CDCl3)
d 1.35 (9H, s), 3.09–3.16 (2H, m), 3.30–3.33 (5H, m), 5.31 (2H, s),
6.93 (1H, t, J = 5.7 Hz), 7.20 (1H, t, J = 7.5 Hz), 7.27–7.31 (2H, m),
7.47–7.53 (1H, m), 7.72 (1H, t, J = 7.8 Hz), 7.88 (2H, dd, J = 7.8,
1.8 Hz), 7.97–8.06 (3H, m), 8.15–8.17 (2H, m), 8.65 (1H, t,
J = 5.7 Hz), 11.48 (1H, s).
5.83. tert-Butyl (2-{[(4-{3-cyano-6-[2-(methoxymethoxy)-
phenyl]-2-[(thiophen-2-ylcarbonyl)amino]pyridin-4-yl}-
phenyl)carbonyl]amino}ethyl)carbamate (12c)
5.80. 3-{3-Cyano-6-[4-fluoro-2-(methoxymethoxy)phenyl]-2-
[(furan-2-ylcarbonyl)amino]pyridin-4-yl}benzoic acid (10d)
Compound 12c was prepared from 10c and tert-butyl (2-ami-
noethyl)carbamate in a manner similar to that described for 8b
as a colorless solid in 64% yield: 1H NMR (DMSO-d6) d 1.38
(9H, s), 2.49–2.51 (2H, m), 3.10–3.17 (2H, m), 3.34 (3H, s), 5.32
(2H, s), 6.93 (1H, t, J = 5.4 Hz), 7.19 (1H, t, J = 7.5 Hz), 7.27–7.31
(2H, m), 7.49 (1H, td, J = 7.8, 1.8 Hz), 7.81 (2H, d, J = 8.1 Hz),
7.91 (1H, dd, J = 7.5, 1.8 Hz), 7.97 (1H, d, J = 5.1 Hz), 8.04 (3H,
d, J = 6.3 Hz), 8.16 (1H, d, J = 3.6 Hz), 8.62–8.65 (1H, m), 11.47
(1H, s).
To a solution of 4p (4.0 g, 9.8 mmol) in pyridine (30 ml) was
added 2-furoyl chloride (3.2 g, 24.5 mmol) at room temperature
and the whole was stirred at 60 °C for 3 h. The mixture was diluted
with EtOAc, washed with water and brine, dried over MgSO4, and
concentrated in vacuo. The residue was dissolved into THF
(50 ml) and to the solution was added MeOH (50 ml) and 2 M
Na2CO3 (30 ml). After the whole was stirred at room temperature
overnight, the mixture was concentrated in vacuo, diluted with
water, 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/hex-
ane = 1/2) and recrystallization from EtOAc/hexane to give methyl
3-{3-cyano-6-[4-fluoro-2-(methoxymethoxy)phenyl]-2-[(furan-2-
ylcarbonyl)amino]pyridin-4-yl}benzoate (3.1 g, 64%) as a colorless
solid: mp 160–162 °C; 1H NMR (CDCl3) d 3.48 (3H, s), 3.97 (3H, s),
5.27 (2H, s), 6.60–6.65 (1H, m), 6.85–6.95 (1H, m), 7.01 (1H, dd,
J = 10.8, 2.4 Hz), 7.05 (1H, d, J = 3.3 Hz), 7.60 (1H, s), 7.66 (1H, t,
J = 7.5 Hz), 7.90–8.00 (2H, m), 8.02 (1H, dd, J = 8.7, 6.9 Hz), 8.18–
8.25 (1H, m), 8.30–8.35 (1H, m), 8.83 (1H, s).
5.84. tert-Butyl (2-{[(3-{3-cyano-6-[4-fluoro-2-(methoxymethoxy)-
phenyl]-2-[(furan-2-ylcarbonyl)amino]pyridin-4-yl}phenyl)-
carbonyl]amino}ethyl)carbamate (12d)
Compound 12d was prepared from 10d and tert-butyl (2-ami-
noethyl)carbamate in a manner similar to that described for 8b
as a colorless solid in 80% yield: mp 172–173 °C; 1H NMR (CDCl3)
d 1.38 (9H, s), 3.30–3.50 (2H, m), 3.47 (3H, s), 3.50–3.65 (2H, m),
5.04 (1H, br s), 5.27 (2H, s), 6.60–6.65 (1H, m), 6.80–6.95 (1H,
m), 7.01 (1H, dd, J = 10.8, 2.4 Hz), 7.35–7.45 (2H, m), 7.60 (1H, s),
7.62 (1H, t, J = 7.8 Hz), 7.88 (1H, d, J = 7.8 Hz), 7.95–8.05 (3H, m),
8.13 (1H, s), 8.83 (1H, s).
To a solution of methyl 3-{3-cyano-6-[4-fluoro-2-(methoxyme-
thoxy)phenyl]-2-[(furan-2-ylcarbonyl)amino]pyridin-4-yl}benzo-

3856
T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
5.85. tert-Butyl (3-{[(3-{3-cyano-6-[4-fluoro-2-(methoxy-
methoxy)phenyl]-2-[(furan-2-ylcarbonyl)amino]pyridin-4-yl}
phenyl)carbonyl]amino}-2,2-dimethylpropyl)carbamate (12e)
5.89. N-[4-{3-[(2-Aminoethyl)carbamoyl]phenyl}-3-cyano-6-(4-
fluoro-2-hydroxyphenyl)pyridin-2-yl]furan-2-carboxamide
hydrochloride (13d)
Compound 12e was prepared from 10d and tert-butyl (3-ami-
no-2,2-dimethylpropyl)carbamate in a manner similar to that de-
scribed for 8b as a yellow oil in 83% yield: 1H NMR (CDCl3) d
0.92 (6H, s), 1.40 (9H, s), 2.96 (2H, d, J = 5.9 Hz), 3.26 (2H, d,
J = 6.9 Hz), 3.45 (3H, s), 5.20–5.40 (3H, m), 6.55–6.65 (1H, m),
6.80–6.90 (1H, m), 6.95–7.05 (1H, m), 7.38 (1H, dd, J = 3.6,
0.6 Hz), 7.55–7.70 (2H, m), 7.85–8.10 (5H, m), 8.18 (1H, s), 8.92
(1H, br s).
Compound 13d was prepared from 12d in a manner similar to
that described for 9a as a yellow solid in 55% yield: mp 205–
207 °C; 1H NMR (DMSO-d6) d 2.95–3.05 (2H, m), 3.55–3.65 (2H,
m), 6.80–6.90 (3H, m), 7.54 (1H, d, J = 3.6 Hz), 7.75 (1H, t,
J = 7.5 Hz), 7.92 (1H, d, J = 7.8 Hz), 8.01 (3H, br s), 8.05–8.15 (2H,
m), 8.26 (2H, s), 8.32 (1H, t, J = 7.5 Hz), 8.98 (1H, t, J = 5.4 Hz),
11.31 (1H, s), 12.45 (1H, s). Anal. Calcd for C26H20FN5O4ꢀHClꢀ
3.1H2O: C, 54.05; H, 4.75; N, 12.12. Found: C, 53.81; H, 4.48; N,
12.09.
5.86. N-[4-{2-[(2-Aminoethyl)carbamoyl]phenyl}-3-cyano-
6-(2-hydroxyphenyl)pyridin-2-yl]thiophene-2-carboxamide
dihydrochloride (13a)
5.90. N-[4-{3-[(3-Amino-2,2-dimethylpropyl)carbamoyl]-
phenyl}-3-cyano-6-(4-fluoro-2-hydroxyphenyl)pyridin-2-yl]-
furan-2-carboxamide hydrochloride (13e)
To an ice-cooled solution of 10b (0.12 g, 0.25 mmol), tert-butyl
(2-aminoethyl)carbamate (50 mg, 0.30 mmol), and HOBt (40 mg,
0.30 mmol) in DMF (4 ml) was added WSCD (57 mg, 0.30 mmol).
After stirring at room temperature for 19 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 = 1/1 to 2/1) to give tert-butyl
(2-{[(2-{3-cyano-6-[2-(methoxymethoxy)phenyl]-2-[(thiophen-2-
ylcarbonyl)amino]pyridin-4-yl}phenyl)carbonyl]amino}ethyl)car-
bamate (12a, 0.13 g, 83%) as an amorphous solid.
Compound 13a was prepared from 12a in a manner similar to
that described for 9a as a yellow solid in 80% yield: mp 196–
201 °C; 1H NMR (CDCl3) d 2.87–2.93 (2H, m), 3.37–3.43 (2H, m),
5.70–6.50 (3H, m), 6.93–7.03 (2H, m), 7.30 (1H, t, J = 4.4 Hz), 7.39
(1H, t, J = 7.7 Hz), 7.52 (1H, dd, J = 7.5, 2.1 Hz), 7.65–7.74 (2H, m),
7.90–8.01 (3H, m), 8.07–8.10 (2H, m), 8.15 (1H, d, J = 3.0 Hz),
8.84 (1H, t, J = 5.6 Hz), 11.40 (1H, s). Anal. Calcd for C26H21N5O3Sꢀ
2HClꢀH2O: C, 54.36; H, 4.39; N, 12.19. Found: C, 54.63; H, 4.49;
N, 12.38.
Compound 13e was prepared from 12e in a manner similar to
that described for 9a as a yellow solid in 58% yield: mp 256–
257 °C; 1H NMR (DMSO-d6) d 0.99 (6H, s), 2.60–2.75 (2H, m),
3.20–3.35 (2H, m), 6.75–6.90 (3H, m), 7.54 (1H, d, J = 3.6 Hz),
7.70–8.00 (5H, m), 8.05–8.15 (2H, m), 8.20–8.35 (3H, m), 9.02
(1H, t, J = 3.3 Hz), 11.31 (1H, s), 12.40 (1H, s). Anal. Calcd for
C29H26FN5O4ꢀHClꢀ0.5H2O: C, 60.79; H, 4.93; N. 12.22. Found: C,
60.53; H, 5.04; N, 12.22.
5.91. 2-Amino-4-[3-(hydroxymethyl)phenyl]-6-[2-(methoxy-
methoxy)phenyl]pyridine-3-carbonitrile (14)
To a solution of 4m (1.0 g, 2.6 mmol) in THF (20 ml) was added
LiBH4 (56 mg, 2.6 mmol) and the whole was stirred at 50 °C for
24 h. The reaction mixture was diluted with water 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 = 1/2 to 1/1) and treated
with EtOAc/hexane to give 14 (0.83 g, 89%) as a colorless amor-
phous solid: 1H NMR (CDCl3) d 1.87 (1H, t, J = 5.7 Hz), 3.43 (3H,
s), 4.79 (2H, d, J = 5.7 Hz), 5.20 (2H, s), 5.37 (2H, s), 7.13 (1H, t,
J = 7.2 Hz), 7.21 (1H, d, J = 8.4 Hz), 7.34–7.41 (2H, m), 7.47–7.58
(3H, m), 7.64 (1H, s), 7.75 (1H, d, J = 7.8 Hz).
5.87. N-[4-{3-[(2-Aminoethyl)carbamoyl]phenyl}-3-cyano-6-
(2-hydroxyphenyl)pyridin-2-yl]thiophene-2-carboxamide
hydrochloride (13b)
5.92. 2-Amino-4-[3-(bromomethyl)phenyl]-6-[2-(methoxy-
methoxy)phenyl]pyridine-3-carbonitrile (15)
Compound 13b was prepared from 12b in a manner similar
to that described for 9a as a pale green solid in 88% yield: mp
203–210 °C; 1H NMR (DMSO-d6)
d 3.03 (2H, t, J = 6.0 Hz),
To a solution of 14 (0.63 g, 1.7 mmol) in CH2Cl2 (60 ml) was
added PPh3 (0.55 g, 2.1 mmol) and then CBr4 (0.87 g, 2.6 mmol)
successively and the whole was stirred at room temperature for
1 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/hex-
ane = 1/2 to 3/2) to give 15 (0.47 g, 64%) as a colorless amorphous
solid: 1H NMR (CDCl3) d 3.45 (3H, s), 4.56 (2H, s), 5.22 (2H, s), 5.32
(2H, s), 7.15 (1H, t, J = 7.5 Hz), 7.22 (1H, d, J = 7.5 Hz), 7.36–7.40
(2H, m), 7.49–7.58 (2H, m), 7.59–7.60 (1H, m), 7.66 (1H, s), 7.76
(1H, dd, J = 7.8, 1.8 Hz).
3.54–3.60 (2H, m), 6.96–7.04 (2H, m), 7.30–7.33 (1H, m), 7.40
(1H, t, J = 7.7 Hz), 7.75 (1H, t, J = 7.7 Hz), 7.91–8.03 (5H, m),
8.11–8.28 (5H, m), 8.96 (1H, t, J = 5.4 Hz), 11.40–11.60 (1H, m),
11.90 (1H, br s). Anal. Calcd for C26H21N5O3SꢀHClꢀ1.5H2O: C,
57.09; H, 4.61; N. 12.80. Found: C, 57.49; H, 4.81; N, 12.89.
5.88. N-[4-{4-[(2-Aminoethyl)carbamoyl]phenyl}-3-cyano-
6-(2-hydroxyphenyl)pyridin-2-yl]thiophene-2-carboxamide
hydrochloride (13c)
Compound 13c was prepared from 12c in a manner similar to
that described for 9a as a yellow solid in 100% yield: mp 202–
208 °C; 1H NMR (DMSO-d6) d 3.03 (2H, q, J = 5.8 Hz), 3.56–3.65
(2H, m), 6.96–7.04 (2H, m), 7.32 (1H, t, J = 4.4 Hz), 7.41 (1H, t,
J = 7.8 Hz), 7.86 (2H, d, J = 8.1 Hz), 8.01–8.18 (8H, m), 8.25 (1H, s),
8.93 (1H, t, J = 5.4 Hz), 11.50 (1H, s), 11.55–11.90 (1H, m). Anal.
Calcd for C26H21N5O3SꢀHClꢀ1.5H2O: C, 57.09; H, 4.61; N, 12.80.
Found: C, 57.15; H, 4.64; N, 12.57.
5.93. 2-Amino-4-[3-(azidomethyl)phenyl]-6-[2-(methoxyme-
thoxy)phenyl]pyridine-3-carbonitrile (16)
To a solution of 15 (0.20 g, 0.47 mmol) in DMF (10 ml) was
added NaN3 (0.15 g, 2.36 mmol) and the whole was stirred at room
temperature for 16 h. The reaction mixture was diluted with

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
3857
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 chromatogra-
phy (EtOAc/hexane = 1/2) to give 16 (0.17 g, 94%) as a colorless
solid: mp 99–100 °C; 1H NMR (CDCl3) d 3.44 (3H, s), 4.45 (2H, s),
5.21 (2H, s), 5.32 (2H, s), 7.14 (1H, t, J = 7.5 Hz), 7.21–7.26 (1H, m),
7.36–7.46 (3H, m), 7.52–7.64 (3H, m), 7.77 (1H, dd, J = 7.5, 1.8 Hz).
5.99. Methyl 2-{5-cyano-4-(3-nitrophenyl)-6-[(phenylcarbonyl)-
amino]pyridin-2-yl}benzoate (22a)
To a solution of 21a (0.29 g, 0.77 mmol) in pyridine (15 ml) was
added benzoyl chloride (0.27 g, 1.9 mmol). After stirring at room
temperature for 19 h, the reaction mixture was diluted with water
and extracted with EtOAc. The extract was washed with brine,
dried over MgSO4, and concentrated in vacuo. The residue was dis-
solved into THF (4 ml) and MeOH (3 ml) and to the mixture was
added 2 M Na2CO3 (4 ml). After stirring at room temperature for
16 h, the reaction mixture was diluted with saturated aqueous
NaHCO3 and extracted with EtOAc twice. The combined extracts
were washed with brine, dried over MgSO4, and concentrated in
vacuo. The residue was diluted with MeOH (10 ml) and to the solu-
tion was added 28% NaOMe solution in MeOH (150 mg) and the
whole was stirred at room temperature for 1 h. The reaction mix-
ture was diluted with saturated aqueous NaHCO3 and extracted
with EtOAc twice. The combined extracts were washed with brine,
dried over MgSO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (EtOAc/hexane = 1/
1) to give 22a (0.24 g, 65%) as a pale brown foam: 1H NMR (CDCl3)
d 3.77(3H, s), 7.46–7.61 (6H, m), 7.70 (1H, t, J = 7.8 Hz), 7.82 (1H, d,
J = 7.8 Hz), 7.90 (2H, d, J = 8.1 Hz), 8.11 (1H, d, J = 7.8 Hz), 8.36–8.50
(3H, m), 8.99 (1H, s).
5.94. N-{4-[3-(Azidomethyl)phenyl]-3-cyano-6-[2-(methoxy-
methoxy)phenyl]pyridin-2-yl}thiophene-2-carboxamide (17)
Compound 17 was prepared from 16 in a manner similar to that
described for 5a as a colorless solid in 79% yield: mp 139–140 °C;
1H NMR (CDCl3) d 3.34 (3H, s), 4.62 (2H, s), 5.32 (2H, s), 7.19(1H,
t, J = 7.5 Hz), 7.27–7.31 (2H, m), 7.47–7.52 (1H, m), 7.59–7.74
(4H, m), 7.89–7.92 (1H, m), 7.97–7.98 (1H, m), 8.04 (1H, s), 8.15
(1H, d, J = 3.6 Hz), 11.47 (1H, s).
5.95. N-{4-[3-(Aminomethyl)phenyl]-3-cyano-6-[2-(methoxy-
methoxy)phenyl]pyridin-2-yl}thiophene-2-carboxamide (18)
A mixture of 17 (0.15 g, 0.30 mmol) and 10% Pd/C (50%wet,
50 mg) in EtOAc (10 ml) and EtOH (10 ml) was stirred under
3 atm hydrogen atmosphere at room temperature for 4 h. The solid
was filtered off and the mother liquid was concentrated to give 18
(0.14 g, 100%) as a pale green amorphous solid: 1H NMR (CDCl3) d
3.44 (3H, s), 3.98 (2H, s), 5.25 (2H, s), 7.12–7.17 (2H, m), 7.25 (1H,
d, J = 7.5 Hz), 7.38–7.63 (5H, m), 7.68 (1H, s), 7.79–7.98 (3H, m).
5.100. N-[3-Cyano-6-(2-methoxyphenyl)-4-(3-nitrophenyl)-
pyridin-2-yl]thiophene-2-carboxamide (22b)
Compound 22b was prepared from 21b in a manner similar to
that described for 5a as a colorless solid in 72% yield: mp 218–
219 °C; 1H NMR (CDCl3) d 3.88 (3H, s), 7.05 (1H, d, J = 8.4 Hz),
7.08–7.21 (2H, m), 7.43–7.52 (1H, m), 7.66 (1H, d, J = 4.8 Hz),
7.74–7.82 (2H, m), 7.97 (1H, d, J = 7.5 Hz), 8.01 (1H, s), 8.12 (1H,
d, J = 7.8 Hz), 8.40 (1H, ddd, J = 8.1, 2.1, 0.9 Hz), 8.46 (1H, s), 8.52
(1H, s). Anal. Calcd for C24H16N4O4S: C, 63.15; H, 3.53; N, 12.27.
Found: C, 63.10; H, 3.50; N, 12.11.
5.96. N-{4-[3-(Aminomethyl)phenyl]-3-cyano-6-(2-hydroxy-
phenyl)pyridin-2-yl}thiophene-2-carboxamide (19)
To a solution of 18 (0.14 g, 0.30 mmol) in THF (10 ml) was added
concd HCl (0.5 ml) and the whole was stirred at room temperature
for 1 h. The reaction mixture was diluted with saturated aqueous
NaHCO3 and extracted with EtOAc twice. The extracts were washed
with brine, dried over MgSO4, and concentrated in vacuo. The resi-
due was purified by basic silica gel column chromatography
(EtOAc/MeOH = 50/1 to 20/1) and recrystallization from EtOAc to
give 19 (26 mg, 20%) as a pale yellow solid: mp 203–205 °C; 1H
NMR (DMSO-d6) d 4.04 (2H, s), 6.89 (2H, t, J = 8.9 Hz), 7.15 (1H, t,
J = 4.4 Hz), 7.33 (1H, t, J = 7.1 Hz), 7.49–7.86 (7H, m), 7.52–7.55
(1H, m), 8.00 (1H, d, J = 6.6 Hz). Anal. Calcd for C24H18N4O2S: C,
67.59; H, 4.25; N. 13.14. Found: C, 67.23; H, 4.21; N, 12.90.
5.101. 2-{5-Cyano-4-(3-nitrophenyl)-6-[(phenylcarbonyl)-
amino]pyridin-2-yl}benzoic acid (22c)
To a solution of 22a (0.60 g, 1.0 mmol) in THF (15 ml) and
MeOH (10 ml) was added 4 M NaOH (5 ml) and the whole was stir-
red at room temperature for 1 h. The reaction mixture was neutral-
ized with 1 M HCl and extracted with EtOAc. The extract was
washed with brine, dried over MgSO4, and concentrated in vacuo
to give 22c (0.55 g, 100%) as a pale yellow amorphous solid: 1H
NMR (CDCl3) d 7.37–7.80 (6H, m), 7.93–7.99 (3H, m), 8.09–8.17
(3H, m), 8.42 (1H, d, J = 8.1 Hz), 8.53 (1H, t, J = 1.8 Hz), 9.61 (1H, s).
5.97. Methyl 2-[6-amino-5-cyano-4-(3-nitrophenyl)pyridin-
2-yl]benzoate (21a)
Compound 21a was prepared from methyl 2-acetylbenzoate
(20a) and 3-nitrobenzaldehyde in a manner similar to that de-
scribed for 4b as a pale yellow solid in 10% yield: mp 202–203 °C;
1H NMR (CDCl3) d 3.79(3H, s), 5.43 (2H, s), 6.97 (1H, s), 7.51–7.61
(3H, m), 7.73 (1H, t, J = 8.0 Hz), 7.85 (1H, d, J = 7.5 Hz), 8.01 (1H, d,
J = 6.6 Hz), 8.37 (1H, d, J = 8.4 Hz), 8.47 (1H, t, J = 1.8 Hz).
5.102. N-[4-(3-Aminophenyl)-3-cyano-6-(2-methoxyphenyl)-
pyridin-2-yl]thiophene-2-carboxamide (23a)
Compound 23a was prepared from 22b in a manner similar to
that described for 18 as a colorless solid in 64% yield: mp 260–
261 °C; 1H NMR (CDCl3) d 3.85 (2H, br s), 3.90 (3H, s), 6.82 (1H,
dd, J = 7.8, 2.1 Hz), 6.98–7.20 (5H, m), 7.25–7.37 (1H, m), 7.40–
7.48 (1H, m), 7.63 (1H, d, J = 5.1 Hz), 7.77 (1H, d, J = 3.9 Hz),
7.88–8.00 (2H, m), 8.41 (1H, s). Anal. Calcd for C24H18N4O2S: C,
67.59; H, 4.25; N, 13.14. Found: C, 67.50; H, 3.99; N, 13.41.
5.98. 2-Amino-6-(2-methoxyphenyl)-4-(3-nitrophenyl)-
pyridine-3-carbonitrile (21b)
Compound 21b was prepared from 1-(2-methoxyphenyl)etha-
none (20b) and 3-nitrobenzaldehyde in a manner similar to that
described for 4b as a pale yellow solid in 10% yield: 1H NMR
(CDCl3) d 3.88 (3H, s), 5.37 (2H, br s), 7.02 (1H, d, J = 8.4 Hz), 7.10
(1H, td, J = 7.5, 1.8 Hz), 7.38–7.48 (2H, m), 7.72 (1H, t, J = 7.8 Hz),
7.82 (1H, dd, J = 7.5, 1.8 Hz), 7.96–8.03 (1H, m), 8.32–8.39 (1H,
m), 8.43–8.46 (1H, m).
5.103. tert-Butyl (2-{4-(3-aminophenyl)-5-cyano-6-[(phenyl-
carbonyl)amino]pyridin-2-yl}phenyl)carbamate (23b)
A mixture of 22c (2.0 g, 4.3 mmol), DPPA (1.4 g, 5.2 mmol), and
triethylamine (1.1 g, 10.8 mmol) in toluene (50 ml) was stirred at
room temperature for 2 h. To the mixture was added tert-butanol

3858
T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
(10 ml) and the whole was stirred at 80 °C for 8 h. After cooling to
room temperature, the reaction mixture was diluted with satu-
rated aqueous NaHCO3 and extracted with EtOAc. The extract
was washed with brine, dried over MgSO4, and concentrated in va-
cuo. The residue was purified by silica gel column chromatography
(EtOAc/hexane = 1/2 to 2/3) to give tert-butyl (2-{5-cyano-4-(3-
nitrophenyl)-6-[(phenylcarbonyl)amino]pyridin-2-yl}phenyl)car-
bamate (22d, 0.27 g, 11%) as a yellow solid.
Compound 23b was prepared from 22d in a manner similar to
that described for 6a as a yellow amorphous solid in 51% yield:
1H NMR (CDCl3) d 1.52 (9H, s), 6.87 (1H, d, J = 7.8 Hz), 6.95 (1H,
s), 7.04 (1H, d, J = 7.2 Hz), 7.13 (1H, t, J = 7.8 Hz), 7.36 (1H, t,
J = 8.1 Hz), 7.47 (1H, t, J = 7.8 Hz), 7.55–7.67 (5H, m), 8.03 (2H, d,
J = 7.5 Hz), 8.29 (1H, d, J = 8.4 Hz), 8.83 (1H, s), 10.80 (1H, s).
vent was evaporated in vacuo and the residue was purified by HPLC
(same condition) to give 27. Purity of the compounds was evaluated
by LC/MS analysis (Walters ZMD; column, CAPCELL PAK C18 UG120
S-3 3 mm, 35x1.5 mm; solvent, water/0.05% TFA (A), MeCN/0.04%
TFA (B); gradient condition: 0.00 min (A/B = 90/10), 2.00 min (A/
B = 5/95), 2.75 min (A/B = 5/95), 2.76 min (A/B = 90/10), 3.60 min
(A/B = 90/10); flow rate of 0.5 ml/min). The percentage of the peak
area detected at UV: 220 nM of the resultant product peak was ta-
ken as the purity of the compound.
Compounds 27a–g were synthesized as mentioned above (pur-
ity >93%).
5.107. Preparation of CHO membranes for GPR54 binding assay
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% dFBS, 1ꢁ Non-Essential Amino Acid (Invitro-
5.104. tert-Butyl (3-{[3-(6-{2-[(tert-butoxycarbonyl)amino]-
phenyl}-3-cyano-2-[(phenylcarbonyl)amino]pyridin-4-yl)-
phenyl]amino}-3-oxopropyl)carbamate (24)
gen), 50 lg/ml gentamycin (Invitrogen) in 5% CO2/95% air
Compound 24 was prepared from 23b and 3-[(tert-butoxycar-
bonyl)amino]propanoic acid in a manner similar to that described
for 8b as a yellow amorphous solid in 100% yield: 1H NMR (CDCl3)
d 1.45 (9H, s), 1.55 (9H, s), 2.66 (2H, t, J = 5.7 Hz), 3.53 (2H, q,
J = 5.8 Hz), 5.20 (1H, br s), 7.11 (1H, t, J = 7.5 Hz), 7.28–7.71 (9H,
m), 7.97–8.04 (3H, m), 8.25–8.32 (2H, m), 8.90 (1H, s), 10.77 (1H, s).
atmosphere. Cells were harvested within 50% confluency in Ca2+
–
Mg2+-free phosphate buffered saline containing 1% EDTA and cen-
trifuged (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
o-phenanthroline, 90
10 g/ml Pepstatin A, pH 7.3 at 25 °C) and collected by centrifuga-
lg/ml Leupeptin, 10
lg/ml E-64, 100 lg/ml
lg/ml phenylmethylsulfonyl fluoride (PMSF),
5.105. N-{4-[3-(b-Alanylamino)phenyl]-6-(2-aminophenyl)-
l
3-cyanopyridin-2-yl}benzamide dihydrochloride (25)
tion (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,
Compound 25 was prepared from 24 in a manner similar to that
described for 9a as a yellow solid in 78% yield: mp 186–190 °C; 1H
NMR (DMSO-d6) d 2.79 (2H, t, J = 6.6 Hz), 3.09 (2H, q, J = 6.0 Hz),
6.81 (1H, t, J = 6.3 Hz), 6.99 (1H, d, J = 8.4 Hz), 7.27 (1H, t,
J = 7.8 Hz), 7.42 (1H, t, J = 7.8 Hz), 7.54–7.72 (4H, m), 7.83–8.01
(6H, m), 8.09 (2H, d, J = 8.4 Hz), 10.56 (1H, s), 11.42 (1H, s). Anal.
Calcd for C28H24N6O2ꢀ2HClꢀ1.3H2O: C, 58.70; H, 5.03; N, 14.67.
Found: C, 58.91; H, 5.34; N, 14.33.
2 mM EGTA, 10
nanthroline, 90
and stored at ꢂ80 °C.
l
g/ml Leupeptin, 10
lg/ml E-64, 100 lg/ml o-phe-
lg/ml PMSF, 10 g/ml Pepstatin A, pH 7.4 at 25 °C)
l
5.108. Receptor binding assay
The procedure of this binding assay was based on the method of
Ohtaki et al.18 The frozen membranes were suspended in binding
buffer (20 mM Tris, 2.5 mM magnesium acetate, 2 mM EGTA,
5.106. Synthesis by combinatorial chemistry method
100
10
l
g/ml o-phenanthroline, 90
lg/ml PMSF, 10 lg/ml Leupeptin,
l
g/ml Pepstatin A, 10 g/ml E-64, and 1 mg/ml BSA, pH 7.4).
l
General: To a solution of 4p (2.0 mmol) in pyridine (8 ml) was
added acid chloride (6.0 mmol) at room temperature and the
whole was stirred at room temperature for 20 h. The reaction mix-
ture was diluted with EtOAc and washed with water. The organic
layer was concentrated in vacuo. The residue was dissolved into
MeOH (5 ml) and THF (5 ml) and to the solution was added 4 M
NaOH (5 ml). After the whole was stirred at room temperature
for 20 h, the mixture was neutralized by 1 M HCl (20 ml). The pre-
cipitated solid was collected by filtration and washed with water to
give crude 26. This compound was used next reaction without fur-
ther purification.
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-
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, pH7.4). 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
l
To a 0.12 M solution of 26 in DMF (830
triethylamine (14.1 l, 1.04 mmol), 0.24 M solution of amine in
mol), 0.20 M solution of HOBt in CH2Cl2
ll, 100 lmol) was added
l
5.109. Preparation of CHO cell lines
CH2Cl2 (600
(720 l, 144
l
l, 144
l
l
l
mol), 0.20 M solution of WSCD in CH2Cl2 (720
l
l
l,
l,
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% dialyzed fetal bovine serum (dFBS), 100 U/ml
144 lmol), and 2.0 M solution of DMAP in CH2Cl2 (7.2
14.4 lmol) successively. The solution was stirred at 50 °C for 24 h
and cooled to room temperature. The reaction mixture was diluted
with CH2Cl2 (3 ml), washed with 5% aqueous NaHCO3 (2 ml), and
the organic layer was separated with Presep (dehydration, Wako
Pure Chemical Industries, Ltd.). The solvent was evaporated in va-
cuo and the residue was purified by HPLC (Gilson high-throughput
penicillin, and 100
atmosphere.
lg/ml streptomycin in 5% CO2/95% air
5.110. Ca2+ mobilization assay
HPLC system using a YMC CombiPrep ODS–A column (S–5 lM
50 mm x 20 mm) with 5–95% gradient water–acetonitril containing
0.1% TFA). The compound obtained was dissolved into TFA (3 ml)
and the whole was stirred at room temperature for 20 h. The sol-
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

T. Kobayashi et al. / Bioorg. Med. Chem. 18 (2010) 3841–3859
3859
5. (a) Seminara, S. B.; Messager, S.; Chatzidaki, E. E.; Thresher, R. R.; Acierno, J. S.,
Jr.; Shagoury, J. K.; Bo-Abbas, Y.; Kuohung, W.; Schwinof, K. M.; Hendrick, A. G.;
Zahn, D.; Dixon, J.; Kaiser, U. B.; Slaugenhaupt, S. A.; Gusella, J. F.; O’Rahilly, S.;
Carlton, M. B. L.; Crowley, W. F., Jr.; Aparicio, S. A. J. R.; Colledge, W. H. N. Eng. J.
Med. 2003, 349, 1614; (b) Funes, S.; Hedrick, J. A.; Vassileva, G.; Markowitz, L.;
Abbondanzo, S.; Golovko, A.; Yang, S.; Monsma, F. J.; Gustafson, E. L. Biochem.
Biophys. Res. Commun. 2003, 312, 1357.
6. de Roux, N.; Genin, E.; Carel, J. C.; Matsuda, F.; Chaussain, J. L.; Milgrom, E. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 10972–10976.
7. Matsui, H.; Takatsu, Y.; Kumano, S.; Matsumoto, H.; Ohtaki, T. Biochem. Biophys.
Res. Commun. 2004, 320, 383.
8. (a) Thompson, E. L.; Patterson, M.; Murphy, K. G.; Smith, K. L.; Dhillo, W. S.;
Todd, J. F.; Ghatei, M. A.; Bloom, S. R. J. Neuroendocrinol. 2004, 16, 850; (b)
Navarro, V. M.; Castellano, J. M.; Fernandez-Fernandez, R.; Barreiro, M. L.; Roa,
J.; Sanchez-Criado, J. E.; Aguilar, E.; Dieguez, C.; Pinilla, L.; Tena-Sempere, M.
Endocrinology 2004, 145, 4565; (c) Navarro, V. M.; Castellano, J. M.; Fernandez-
Fernandez, R.; Tovar, S.; Roa, J.; Mayen, A.; Nogueiras, R.; Vazquez, M. J.;
Barreiro, M. L.; Magni, P.; Aguilar, E.; Dieguez, C.; Pinilla, L.; Tena-Sempere, M.
Endocrinology 2005, 146, 156; (d) Navarro, V. M.; Castellano, J. M.; Fernandez-
Fernandez, R.; Tovar, S.; Roa, J.; Mayen, A.; Barreiro, M. L.; Casanueva, F. F.;
Aguilar, E.; Dieguez, C.; Pinilla, L.; Tena-Sempere, M. Endocrinology 2005, 146,
1689.
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.).
5.111. X-ray crystallographic data
X-ray crystallographic data for compound 13b has been depos-
ited with the Cambridge Crystallographic Data Center as
CCDC771900. The crystallographic data can be obtained, free of
charge, on application to CCDC, 12 Union Road, Cambridge CB2
1EZ, UK (fax: +44-(0)1223-336033 or e-mail: deposit@ccdc.cam.
ac.uk).
9. Gottsch, M. L.; Cunningham, M. J.; Smith, J. T.; Popa, S. M.; Acohido, B. V.;
Crowley, W. F.; Seminara, S.; Clifton, D. K.; Steiner, R. A. Endocrinology 2004,
145, 4073.
10. (a) Messager, S.; Chatzidaki, E. E.; Ma, D.; Hendrick, A. G.; Zahn, D.; Dixon, J.;
Thresher, R. R.; Malinge, I.; Lomet, D.; Carlton, M. B. L.; Colledge, W. H.; Caraty,
A.; Aparicio, S. A. J. R. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 1761; (b) Sahahab,
M. M.; Seminara, S. B.; Crowley, W. F.; Ojeda, S. R.; Plant, T. M. Proc. Natl. Acad.
Sci. U.S.A. 2005, 102, 2129; (c) Dhillo, W. S.; Chaudhri, O. B.; Patterson, M.;
Thompson, E. L.; Murphy, K. G.; Badman, M. K.; McGowan, B. M.; Amber, V.;
Patel, S.; Ghatei, M. A.; Bloom, S. R. J. Clin. Endocrinol. Metab. 2005, 12, 6609.
11. Irwig, M. S.; Fraley, G. S.; Smith, J. T.; Acohido, B. V.; Popa, S. M.; Cunningham,
M. J.; Gottsch, M. L.; Clifton, D. K.; Steiner, R. A. Neuroendocrinology 2004, 80,
264.
Acknowledgment
We thank Ms. Keiko Higashikawa for X-ray crystal structure
analysis of 13b.
12. Kinoshita, M.; Tsukamura, H.; Adachi, S.; Matsui, H.; Uenoyama, Y.; Iwata, K.;
Yamada, S.; Inoue, K.; Ohtaki, T.; Matsumoto, H.; Maeda, K. Endocrinology 2005,
146, 4431.
13. Peptide GPR54 antagonists were recently reported: Roseweir, A. K.; Kauffman,
A. S.; Smith, J. T.; Guerriero, K. A.; Morgan, K.; Pielecka-Fortuna, J.; Pienda, R.;
Gottsch, M. L.; Tena-Sempere, M.; Moenter, S. M.; Terasawa, E.; Clarke, I. J.;
Steiner, R. A.; Millar, R. P. J. Neurosci. 2009, 29, 3920.
14. Murata et al. also estimate the intramolecular hydorogen bond between the
phenol and pyridine nitrogen atom by the chemical shift of the phenolic
hydroxide in the 1H NMR spectrum: Murata, T.; Shimada, M.; Sakakibara, S.;
Yoshino, T.; Kadono, H.; Masuda, T.; Shimazaki, M.; Shintani, T.; Fuchikami, K.;
Sakai, K.; Inbe, H. Bioorg. Med. Chem. Lett. 2003, 13(5), 913.
15. Aggarwal, R.; Birkbeck, A. A.; Giles, R. G.; Green, I. R.; Gruchlik, Y.; Oosthuizen,
F. J. Aust. J. Chem. 2003, 56, 489.
16. Xing, X.; Ho, P.; Bourquin, G.; Yen, L. A.; Cuny, G. D. Tetrahedron 2003, 59, 9961.
17. Murata, T.; Sakakibara, S.; Yoshino, T.; Ikegami, Y.; Masuda, T.; Shimada, M.;
Shintani, T.; Shimazaki, M.; Lowinger, T. B.; Ziegelbauer, K. B.; Fuchikami, K.;
Umeda, M.; Komura, H.; Yoshida, N. WO 0244153, 2002.
References and notes
1. Reviews: (a) Smith, J. T.; Clifton, D. K.; Steiner, R. A. Reproduction 2006, 131,
623; (b) Dungan, H. M.; Clifton, D. K.; Steiner, R. A. Endocrinology 2006, 147,
1154.
2. (a) Lee, D. K.; Nguyen, T.; O’Neill, G. P.; Cheng, R.; Liu, Y.; Howard, A. D.;
Coulombe, N.; Tan, C. P.; Tang-Nguyen, A.-T.; George, S. R.; O’Dowd, B. F. FEBS
Lett. 1999, 446, 103; (b) Muir, A. I.; Chamberlain, L.; Elshourbagy, N. A.;
Michalovich, D.; Moore, D. J.; Calamari, A.; Szekeres, P. G.; Sarau, H. M.;
Chambers, J. K.; Murdock, P.; Steplewski, K.; Shabon, U.; Miller, J. E.; Middleton,
S. E.; Darker, J. G.; Larminie, C. G. C.; Wilson, S.; Bergsma, D. J.; Emson, P.; Faull,
R.; Philpott, K. L.; Harrison, D. C. J. Biol. Chem. 2001, 276, 28969.
3. Ohtaki, T.; Shintani, Y.; Honda, S.; Matsumoto, H.; Hori, A.; Kanehashi, K.; Terao,
Y.; Kumano, S.; Takatsu, Y.; Masuda, Y.; Ishibashi, Y.; Watanabe, T.; Asada, M.;
Yamada, T.; Suenaga, M.; Kitada, C.; Usuki, S.; Kurokawa, T.; Onda, H.;
Nishimura, O.; Fujino, M. Nature 2001, 411, 613.
4. Kotani, M.; Detheux, M.; Vandenbogaerde, A.; Communi, D.; Vanderwinden, J.-
M.; Poul, E. L.; Brezillon, S.; Tyldesley, R.; Suarez-Huerta, N.; Vandeput, F.;
Blanpain, C.; Schiffmann, S. N.; Vassart, G.; Parmentier, M. J. Biol. Chem. 2001,
276, 34631.
18. Ohtaki, T.; Kumano, S.; Ishibashi, Y.; Ogi, K.; Matsui, H.; Harada, M.; Kitada, C.;
Kurokawa, T.; Onda, H.; Fujino, M. J. Biol. Chem. 1999, 274, 37041.