Structure determination, synthesis, and biological evaluation of a metabolite of the selective α1Dadrenoceptor antagonist TAK-259

Article abstract of DOI:10.1016/j.tet.2016.08.013

5-Chloro-1-(5-chloro-2-(methylsulfonyl)benzyl)-2-imino-1,2-dihydropyridine-3-carboxamide hydrochloride (TAK-259) is a novel, selective, and orally active α_1Dadrenoceptor antagonist with anti-urinary frequency effect. A metabolite (2) of TAK-259 was identified from monkey urine samples. To elucidate the structure of 2, extraction and purification of the metabolite from TAK-259-treated monkey urine was conducted. Structural analysis of the purified compounds using NMR indicated the compound to be 2-amino-5-chloro-1-[5-chloro-2-(methylsulfonyl)benzyl]-4-oxo-1,4-dihydropyridine-3-carboxamide (2a). An authentic sample of compound 2a was synthesized via regioselective alkylation of 4-methoxypyridin-2-amine. The biological activity of metabolite 2a was also evaluated, and the compound found not to possess affinity toward any known α₁adrenoceptor subtype.

Full text of DOI:10.1016/j.tet.2016.08.013

Accepted Manuscript
Structure determination, synthesis, and biological evaluation of a metabolite of the
selective α
1D
adrenoceptor antagonist TAK-259
Ayumu Sato, Nobuki Sakauchi, Junya Shirai, Hideki Furukawa, Miki Hara, Katsuhiko
Miwa, Hisashi Fujita, Maki Miyamoto, Norio Tada, Haruhiko Kuno, Nagako Kameoka,
Hironobu Maezaki
PII:
S0040-4020(16)30771-2
10.1016/j.tet.2016.08.013
TET 27994
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 26 April 2016
Revised Date: 26 July 2016
Accepted Date: 4 August 2016
Please cite this article as: Sato A, Sakauchi N, Shirai J, Furukawa H, Hara M, Miwa K, Fujita H,
Miyamoto M, Tada N, Kuno H, Kameoka N, Maezaki H, Structure determination, synthesis, and
biological evaluation of a metabolite of the selective α
(2016), doi: 10.1016/j.tet.2016.08.013.
adrenoceptor antagonist TAK-259, Tetrahedron
1D
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Graphical Abstract
Structure determination, synthesis, and
biological evaluation of a metabolite of the
selective α1D adrenoceptor antagonist TAK-
259
Leave this area blank for abstract info.
Ayumu Sato, Nobuki Sakauchi, Junya Shirai, Hideki Furukawa, Miki Hara, Katsuhiko Miwa, Hisashi Fujita,
Maki Miyamoto, Norio Tada, Haruhiko Kuno, Nagako Kameoka, and Hironobu Maezaki
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited,
26-1, Muraoka higashi 2-chome, Fujisawa, Kanagawa, Japan

ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Structure determination, synthesis, and biological evaluation of a metabolite of the
selective α1D adrenoceptor antagonist TAK-259
Ayumu Sato, Nobuki Sakauchi, Junya Shirai, Hideki Furukawa, Miki Hara, Katsuhiko Miwa, Hisashi
Fujita, Maki Miyamoto, Norio Tada, Haruhiko Kuno, Nagako Kameoka, and Hironobu Maezaki
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
5-Chloro-1-(5-chloro-2-(methylsulfonyl)benzyl)-2-imino-1,2-dihydropyridine-3-carboxamide
(TAK-259) is a novel, selective, and orally active α1D adrenoceptor antagonist with anti-urinary
frequency effect. A metabolite (2) of TAK-259 was identified from monkey urine samples. To
elucidate the structure of 2, extraction and purification of the metabolite from TAK-259-treated
monkey urine was conducted. Structural analysis of the purified compounds using NMR indicated
the compound to be 2-amino-5-chloro-1-[5-chloro-2-(methylsulfonyl)benzyl]-4-oxo-1,4-
dihydropyridine-3-carboxamide (2a). An authentic sample of compound 2a was synthesized via
regioselective alkylation of 4-methoxypyridin-2-amine. The biological activity of metabolite 2a
was also evaluated, and the compound found not to possess affinity toward any known α1
adrenoceptor subtype.
Available online
Keywords:
Metabolite; Isolation; Structure determination;
Synthesis; Selective α1D adrenoceptor antagonist; 2-
Amino-4-oxo-1,4-dihydropyridine-3-carboxamide.
2016 Elsevier Ltd. All rights reserved.
1. Introduction
There are a number of literature reports that describe the
elucidation of the drug metabolites by chemical synthesis.2-4
5-Chloro-1-(5-chloro-2-(methylsulfonyl)benzyl)-2-imino-1,2-
dihydropyridine-3-carboxamide (TAK-259, 1) is novel,
However, in this case, it would have been difficult to identify the
metabolite by synthesizing 2a and 2b because there are few
reports on the synthesis of hydroxy-2-imino-1,2-dihydropyridine
rings or their tautomers.5-8 At that time, a relatively high
concentration of 2 was detected in the urine of the TAK-259 (1)-
treated monkey. It was confirmed that 2 was stable in aqueous
solutions (pH 1 and 9), and could be extracted with organic
solvents such as ethyl acetate (EtOAc). The present study had
three objectives. The first was to determine the structure of
metabolite 2 by isolating the metabolite from monkey urine. The
second aim was to synthesize 2 to confirm its structure based on
the synthetic process. The third goal was to elucidate the
biological properties of 2 such as potential affinity to the α1D
adrenoceptor and selectivity against the α1A and α1B adrenoceptors.
Characterization of the selectivity of this metabolite was deemed
important because any affinity for the α1A and α1B adrenoceptors
would be associated with the risk of cardiovascular side effects.9
Therefore, this paper describes the isolation, identification,
synthesis, and biological characterization of 2.
a
selective, and orally active α1D adrenoceptor antagonist with anti-
urinary incontinence effects.1 A metabolite (2) of TAK-259 was
detected in monkey plasma and urine after its oral administration,
and was also discovered in metabolite identification studies
conducted with human and monkey hepatocytes. Structure
elucidation by LC-MS/MS analysis indicated oxidation of the
dihydropyridine part as shown in the predicted structures 2a and
2b or their tautomers (Figure 1).
2
Results and discussion
Figure 1. Structures of TAK-259 (1) and its metabolite (2). 2a
and 2b, possible structures of 2.
2.1 Metabolite isolation and identification
The metabolite was extracted from the urine samples of four
monkeys that had been treated with 1 for one day with EtOAc.
———
Corresponding author. Tel.: +81-466-32-1106; fax: +81-466-29-4449; e-mail: hironobu.maezaki@takeda.com
1

ACCEPTED MANUSCRIPT
2.2 Synthesis
TOF/MS analysis of the combined extracts are shown in Figure
2. Metabolite 2 was detected at 6.01 min with an m/z value of
ACCEPTED MANUSCRIPT
The retrosynthetic route we devised for 2a is shown in Figure
4. The compound 2a would be synthesized by alkylation of 2-
aminopyridine (C) with a benzyl halide (B) followed by the
demethylation of A. Compound C could be prepared from
390 (Figure 2A and B), whereas the parent drug 1 was observed
at 3.63 min with an m/z value of 374 (Figure 2C). HPLC
purification of the extracts was followed by crystallization to
obtain 2 as a colorless solid. The structure of 2 was assigned as
the keto form of 2a, 2-amino-4-pyridone, by NMR analyses
commercially
available
2-amino-4-methoxypyridine-3-
1
carbonitrile (D) in two steps.
(Table 1). From the H NMR spectrum, only one proton was
observed on the pyridine ring, which indicates that oxidative
metabolism occurred on the pyridine ring. The 13C NMR
spectrum suggested the presence of two carbonyl groups based
on the two peaks found around 170 ppm. Since two carbonyl
groups were observed, the metabolite structure was presumed
to be a keto and not an enol form in DMSO. To determine the
position of the carbonyl group, the nuclear Overhauser effect
(NOE) was measured. Strong NOE correlations were observed
between the H-7 benzyl protons and the H-6, H-13, and H-24
protons, indicating that the carbonyl group of the pyridone
moiety is located at the 4-position (Figure 3).
Figure 4. Retrosynthesis of 2a
Initially, benzyl halides B (9a and 9b) were prepared by the
route shown in Scheme 1. Esterification of 5-chloro-2-
fluorobenzoic acid (3) was performed in the presence of a
catalytic amount of sulfuric acid in methanol to obtain 4. A
methylsulfanyl group was introduced by heating a mixture of 4
and sodium thiomethoxide in DMA to yield compound 5.
Oxidation of the sulfanyl group by m-chloroperbenzoic acid (m-
CPBA) and hydrolysis of the resulting ester 6 with 4 M sodium
hydroxide provided carboxylic acid 7, which was subsequently
reduced to benzyl alcohol 8. Finally, 8 was converted to 9a and
9b by using bromine and triphenylphosphine or thionyl chloride,
respectively. In the scale-up synthesis of 9a, purification was
difficult due to an inseparable byproduct (structure not
determined). On the other hand, no byproduct was observed in
the synthesis of 9b.
Figure 2. Ion chromatograms of ethyl acetate (EtOAc) extracts
from monkey urine. (A) Total ion, (B) extracted ion of m/z 390,
and (C) extracted ion of m/z 374.
Table 1. Metabolite NMR spectra
O
O
16
15
17
δ
δ
(ppm)
1H
24
(ppm)
13C
Cl
3
3.39 (3H, s)
24
7
44.22
51.06
96.08
4
18
NH
5
2
22
7
5.54 (2H, s)
2
6.76 (1H, d, J = 2.2 Hz)
13
22
11
10
6
3
6
NH
N
1
7.42 (1H, d, J = 4.8 Hz)
7.74 (1H, dd, J = 8.4, 2.2 Hz)
8.04 (1H, d, J = 8.4 Hz)
8.06 (1H, s)
5
118.31
125.52
128.63
132.45
136.43
2
14
13
13
11
10
6
Cl
7
21
8
12
O
9
23
10.30 (1H, d, J = 5.1 Hz).
22
11
S
8
9
136.61
136.86
139.09
156.18
170.26
10
19
H C
3
O
24
20
12
2
Scheme 1. Synthesis of benzyl halides 9a and 9b
15
4
171.38
The synthesis of 2a is shown in Scheme 2. The chlorination of
pyridine 10 using NCS produced 11 while hydrolysis of the
cyano group with sulfuric acid afforded pyridine 12. Interestingly,
simultaneous demethylation occurred along with the following
N-benzylation of 12 with 9a to produce the desired 2a directly in
14% yield. In this reaction, a similar amount of the regioisomer
13 was obtained as a separable byproduct. The NMR and MS
spectra of the synthesized compound 2a were consistent with
those of the isolated metabolite 2.
Figure 3. Nuclear Overhauser effect (NOE) of
metabolite 2
2

ACCEPTED MANUSCRIPT
Table 3 summarizes the effects of temperature and solvent on
ACCEPTED MANUSCRIPT
the benzylation reaction in the presence of 2 equivalents of LiBr.
Changing temperature showed little effect on yields (entries 1–3),
although slightly lower yield was observed at 120 °C (entry 3).
As for the solvent effect, DMA was the optimal solvent found for
obtaining higher yield. Other aprotic solvents examined were less
effective than DMA (entries 5–6). When isopropanol was used,
starting material 12 was not fully consumed, and decreased yield
and regioselectivity were observed (entry 7). Among the
conditions evaluated, reaction in DMA at 100 °C gave the best
results.
Scheme 2. Initial synthesis of 2a
Although the synthesis of the metabolite was achieved,
improvement of the overall yield was needed for the efficient
preparation of 2a. As shown in Scheme 2, it is obvious that the
reaction from 12 to 2a should be the key to increase the chemical
yield of 2a with reducing the formation of undesired regioisomer
13. Previously, a number of reports on the alkylation of 2-
aminopyridines have indicated that N-benzylation occurs
predominantly at the 1-position of the pyridine ring in moderate
yield.10-15 Therefore, we optimized the reaction conditions for this
particular 2-aminopyridine substrate 12 containing multiple
substituents. Firstly, the effects of additives on the yield and
regioselectivity were investigated (Table 2). All reactions were
carried out with 0.1 mmol 12 and benzyl halide 9a or 9b in 1.0
mL of DMF at 100 °C. Addition of 1 equivalent LiBr for the
benzyl bromide reaction was important to improve the yield as
well as the selectivity (entry 2). Benzyl chloride 9b showed low
reactivity with 12 without an additive (entry 3). From this result,
we hypothesized that the lower reactivity of the benzyl chloride
intermediate might have a beneficial effect on the yield and
regioselectivity. Indeed, reaction with 9b proceeded in the
presence of LiBr and the yield was improved when compared to
that with the use of benzyl bromide 9a alone (entry 4 vs 1).
Usage of 2 equivalents of LiBr led to a slight increase in the
selectivity and yield (entry 5). This result suggested that the role
of LiBr was in facilitating the alkylation reaction, rather than
direct halogen exchange on the benzyl halide. We used 9b for the
further investigation based on the yield and selectivity. Excess
amount of LiBr had minimal impact on the yield (entry 6). A
favorable result was not obtained when the additive was changed
to either LiI (entry 7) or NaBr (entry 8). These results showed
that LiBr was the most favorable additive for this reaction. Next,
the effects of the base were investigated. The combination of a
base and LiBr increased the yield somewhat (entries 9–10).
When potassium carbonate was used as a base, the yield was
42%, and the selectivity was 5.9-fold (entry 9). The use of Et3N
resulted in better yield (48%) and 4.8-fold regioselectivity (entry
10).
Table 3. Effects of reaction temperature and solvents
Thus we determined that the optimum reaction conditions for
the alkylation reaction were with 2 equivalents of LiBr and Et3N
in DMA at 100 °C. Under these optimum conditions, compound
2a was obtained in a 53% isolated yield, which is approximately
3-fold higher than that under the original condition (14%).
(Scheme 3)
Scheme 3. Preparative synthesis of 2a under optimized
conditions
In the synthesis of 2a, the addition of LiBr improved not only
the yield but also the regioselectivity. A plausible mechanism for
the improved regioselectivity is illustrated in Figure 5. First, the
halogen exchange occurs between 9b and LiBr to give the
corresponding bromide 9a (Step 1). In the absence of LiBr,
nucleophilic substitution of 12 to 9a proceeds at the 2-amino
group (route G) along with the nitrogen atom of the pyridine ring
(route F) to produce 13 and 2a (Step 2, i)). In contrast, in the
presence of LiBr, demethylation of 12 at the 4-position and
subsequent alkylation between 12 and 9a occurs in a concerted
manner to produce 4-pyridone 2a dominantly (Step 2, ii)). 16, 17
Table 2. Effects of additives on alkylation of 12
3

ACCEPTED MANUSCRIPT
Step 1:Halogen exchange
Cl
LiBr
resonance (13C NMR) spectra were recorded using a Brucker 300
ACCEPTED MANUSCRIPT
Cl
(300 MHz) and AV 600 (600 MHz) with tetramethylsilane as the
Br
Cl
internal standard. The chemical shifts were reported in parts per
million (ppm). The coupling constants (J) were reported in hertz
(Hz) while the splitting patterns were designated as: s, singlet; d,
doublet; dd, double doublet; ddd, double double doublet; t,
triplet; m, multiplet; br, broad; and br s, broad singlet. The LC-
mass spectra were obtained using a Waters-Micromass ZMD
System and the uncorrected melting points were determined
using a Yanagimoto micromelting point apparatus. The elemental
analyses were conducted by Takeda Analytical Research
Laboratories, Ltd., Osaka, and were within 0.4% of the
theoretical values for the elements indicated. When air or
moisture-sensitive reagents were used, reactions were run under
argon. Column chromatography was performed on columns of
Purif-Pack (SI 60 ꢀm or NH 60 ꢀm).
SO2Me
SO2Me
9b
LiCl
9a
Step 2:Alkylation step
i) without LiBr
O
O
OMe O
O
O
Cl
Cl
Cl
NH2
NH2
NH2
NH2
12
N
N
NH2
N
H
NH
G
F
Cl
F
Cl
G
Br
Cl
MeO2S
MeO2
S
SO2Me
9a
2a
13
ii) with LiBr
Br-
Me
O
N
O
O
O
O
O
Cl
Cl
Cl
LiBr
NH2
NH2
NH2
NH2 12
N
NH2
N
H
NH
G
F
Cl
Cl
Cl
Major
Minor
Br
MeO2S
MeO2
S
SO2Me
9a
Isolation of TAK-259 metabolite
2a
13
The metabolite was obtained from the urine samples of four
monkeys, following oral administration of 1 (100 mg/kg). The
urine was filtered to remove insoluble materials, and the filtrate
was frozen for preservation. Prior to isolation, the urine was
allowed to warm to rt, extracted with EtOAc, washed with brine,
dried over Na2SO4, and concentrated in vacuo. The residue (200
mg) was purified using an AutoPurification HPLC-MS system
(Waters), and the TFA was removed using MP-HCO3 MP SPE
resin (Polymer Laboratories). Crystallization of the product from
EtOAc/hexane produced the metabolite (41 mg) as colorless
crystals.
LC-MS conditions: Instrument, SYNAPT-MS; capillary, 1.7 kV;
sampling cone, 40 V; column, BEH C18, 1.7-ꢀM, 100 × 2.1 mm
i.d.; mobile phase A, CH3CN/10 mM NH4OAc = 5/95; B,
CH3CN/10 mM NH4OAc = 9/1; gradient, A/B = 98/2 to 55/45,
linear gradient over 10 min; flow rate, 0.5 mL/min; detection,
234 nm; column temperature, 40 °C; injection volume, 10 ꢀL;
ionization, ESI; and polarity, positive.
Figure 5. Plausible mechanism for alkylation step in synthesis of
2a.
2.3 Binding affinity studies
The binding affinities of metabolite 2a toward the α1
adrenoceptor subtypes were evaluated using a binding assay.
Interestingly, the affinity of 2a, especially to the α1D adrenoceptor,
was lower than that of the parent drug 1 (Figure 6). This result
showed that the 4-position of the pyridine ring is crucial to the
binding affinity toward the α1D adrenoceptor. Furthermore, 2a did
not have any affinities toward α1A and α1B adrenoceptors,
indicating that the risk of cardiovascular side effects would not
be increased by this metabolite.
HPLC conditions: Instrument, Waters AutoPurification HPLC-
MS System; column, Waters XBridge (30 × 75 mm, 5-ꢀm); load,
6 mg/load; solvent A, water containing 0.1% TFA; B, CH3CN
containing 0.1% TFA; gradient, A/B = 95/5 (0.00–0.50 min),
95/5 to 5/95 (0.50–4.75 min), 5/95 to 1/99 (4.75–5.25 min), 1/99
(5.25–7.25 min), 1/99 to 95/5 (7.25–7.50 min), 95/5 (7:50–8.00
min); flow rate, 20 mL/min; detection, UV 220 nm; and ESI-MS,
positive mode, 389 ± 1.
NMR conditions: Instrument, Bruker AV-600; temperature, 27
°C (300 K); solvent, DMSO-d6; and density, 4.3 mg/0.6 mL.
Methyl 5-chloro-2-fluorobenzoate (4). A mixture of 5-chloro-2-
fluorobenzoic acid 3 (4.8 g, 27.5 mmol) and conc. H2SO4 (0.5
mL) in MeOH (100 mL) was heated at 90 °C for 14 h. To the
mixture was added 0.5 mL of conc. H2SO4, and the mixture was
stirred at 90 °C for 14 h. After cooling, aq. NaHCO3 was added,
and the solvent was evaporated. The residue was dissolved in
water and EtOAc. The separated organic layer was washed with
brine, dried over MgSO4, and concentrated to yield 4 (5.15 g,
99%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ: 3.93 (3H,
s), 7.09 (1H, dd, J = 10.2, 9.0 Hz), 7.46 (1H, ddd, J = 6.6, 3.7, 3.0
Hz), 7.90 (1H, dd, J = 6.3, 2.7 Hz).
Methyl 5-chloro-2-(methylsulfanyl)benzoate (5). A mixture of
4 (4.72 g, 25.0 mmol) and sodium thiomethoxide (2.10 g, 30.0
mmol) in DMA (15 mL) was stirred at 50 °C for 3 h. The mixture
was concentrated, and the residue was poured into EtOAc and
water. The separated organic layer was washed with brine, dried
over MgSO4 and concentrated. The residue was purified using
silica gel column chromatography (eluted with 10% EtOAc in
hexane) to yield 5 (3.70 g, 68%) as a white powder. 1H NMR
Figure 6. Binding affinities of 1 and 2a
3
Conclusion
Metabolite 2a of the selective α1D adrenoceptor antagonist
TAK-259 (1) was successfully isolated from monkey urine, and
the structure was determined to be 2-amino-5-chloro-1-[5-chloro-
2-(methylsulfonyl)benzyl]-4-oxo-1,4-dihydropyridine-3-
carboxamide. Efficient regioselective synthesis of 2a was
achieved using an optimized alkylation reaction of a 4-
methoxypyridine intermediate in the presence of LiBr and
triethylamine. Metabolite 2a did not exhibit affinities for any α1
adrenoceptor subtypes.
Experimental Procedure
All the reagents and solvents were of commercial quality and
were used without further purification. Proton nuclear magnetic
resonance (1H NMR) spectra and carbon nuclear magnetic
4

ACCEPTED MANUSCRIPT
Hz). 13C NMR (75 MHz, CDCl3) δ: 41.5, 45.3, 129.6, 131.8,
132.8, 137.1, 138.7, 140.8. mp 119–120 °C.
(300 Hz, CDCl3) δ: 2.45 (3H, s), 3.92 (3H, s), 7.18 (1H, d, J = 9.0
ACCEPTED MANUSCRIPT
Hz), 7.42 (1H, dd, J = 8.7, 2.7 Hz), 7.97 (1H, d, J = 2.7 Hz). mp
59–61 °C. Anal. Calcd for C9H9ClO2S: C, 49.89; H, 4.19. Found:
C, 49.90; H: 4.01.
2-Amino-5-chloro-4-methoxypyridine-3-carbonitrile (11). A
mixture of 10 (1.50 g, 10.0 mmol) and NCS (2.0 g, 14.9 mmol)
in AcOH (30 mL) was heated at 50 °C for 14 h and concentrated.
The residue was dissolved in hot EtOAc–THF (ca 10:1, v/v), and
the mixture was alkalized with aq. NaOH. The organic layer was
separated and washed sequentially with water and brine, dried
over MgSO4, and concentrated in vacuo. The residue was
purified using silica gel column chromatography (eluted with
50% EtOAc in hexane) to yield 11 (1.32 g, 72%) as a pale brown
powder. 1H NMR (300 MHz, CDCl3) δ: 4.28 (3H, s), 5.28 (2H,
br. s), 8.07 (1H, s). mp 170–172 °C. Anal. Calcd for C7H6ClN3O:
C, 45.79; H, 3.29; N, 22.89. Found: C, 45.86; H: 3.22; N, 22.69.
2-Amino-5-chloro-4-methoxypyridine-3-carboxamide (12). A
mixture of 11 (1.18 g, 6.27 mmol) in conc. H2SO4 (11 mL) was
heated at 100 °C for 1 h and poured into ice. The mixture was
alkalized with 8 M NaOH (ca. 40 mL) and extracted with EtOAc
and CH3CN. The extract was washed with brine, dried over
MgSO4, and concentrated in vacuo. The resulting solid was
collected and washed with IPE to yield 12 (1.10 g, 87%) as a
white powder. 1H NMR (300 MHz, CDCl3) δ: 3.97 (3H, s), 5.77
(1H, br. s), 6.78 (2H, br. s), 7.65 (1H, br. s), 8.09 (1H, br. s). 13C
NMR (75 MHz, DMSO-d6) δ: 61.2, 107.4, 111.3, 149.1, 158.4,
160.4, 166.6. mp 174–176 °C. Anal. Calcd for C7H8ClN3O2: C,
41.70; H, 4.00; N, 20.84. Found: C, 41.74; H: 3.85; N, 20.79.
2-Amino-5-chloro-1-[5-chloro-2-(methylsulfonyl)benzyl]-4-
oxo-1,4-dihydropyridine-3-carboxamide (2a). A mixture of 12
(50 mg, 0.248 mmol) and 9b (59.3 mg, 0.248 mmol), LiBr (43.1
mg, 0.496 mmol) and Et3N (50.2 mg, 0.496 mmol) in DMA (1
mL) was stirred at 100 °C for 14 h. The resulting mixture was
poured into water and EtOAc. The separated aq. phase was
extracted with EtOAc. The combined organic layers were washed
with water thrice and then brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified using NH silica
gel column chromatography (eluted with 50%–100% EtOAc in
hexane). Crystallization of the residue from DMSO/H2O yielded
2a (51.2 mg, 53%) as colorless crystals. MS (ESI) m/z: 390.1
(M+H)+. 1H NMR (600 MHz, DMSO-d6) δ: 3.39 (3 H, s), 5.54 (2
H, s), 6.76 (1 H, d, J = 2.2 Hz), 7.42 (1 H, d, J = 4.8 Hz), 7.74 (1
H, dd, J = 8.4, 2.2 Hz), 8.04 (1 H, d, J = 8.4 Hz), 8.06 (1 H, s),
9.13 (2 H, br. s), 10.30 (1 H, d, J = 5.1 Hz). 13C NMR (151 MHz,
DMSO-d6) δ: 44.2, 51.1, 96.1, 118.3, 125.5, 128.6, 132.5, 136.4,
136.6, 136.9, 139.1, 156.2, 170.3, 171.4. mp 294–296 °C. Anal.
Calcd for C14H13 Cl2N3O4S: C, 43.09; H, 3.36; N, 10.77. Found:
C, 43.18; H: 3.38; N, 10.58.
5-chloro-2-((5-chloro-2-(methylsulfonyl)benzyl)amino)-4-oxo-
1,4-dihydropyridine-3-carboxamide (13). A mixture of 12
(38.4 mg, 0.190 mmol) and 9a (54.0 mg, 0.190 mmol) in DMF
(0.5 mL) was stirred at 100 °C for 4 h. Then, aq. NaHCO3 was
added to the mixture, which was extracted with EtOAc, washed
with brine, dried over MgSO4, and concentrated in vacuo. The
residue was purified using silica gel column chromatography
(eluted with 50%–100% EtOAc in hexane) to produce a mixture
of 2a and 13 (23 mg, 31%) as a colorless solid. Part of the
residue (15 mg) was purified using preparative HPLC (Ascentis
Phenyl column, eluted with H2O in CH3CN containing 5 mM
ammonium acetate). The desired fraction was neutralized with aq.
NaHCO3 and extracted with EtOAc. The organic layer was
separated, dried over MgSO4, and concentrated in vacuo to yield
13 (6 mg) as a colorless solid. MS (ESI) m/z: 390.1 (M+H)+. 1H
NMR (600 MHz, DMSO-d6) δ: 3.31 (3H, s), 4.96 (2 H, d, J = 6.2
Hz), 7.30 (1 H, br. s), 7.54 (1 H, d, J = 2.2 Hz), 7.62 (1 H, s),
7.71 (1H, dd, J = 8.4, 2.2 Hz), 7.99 (1 H, d, J = 8.4 Hz), 10.29 (1
H, br. s), 11.15 (1 H, br. s), 11.30 (1 H, br. s). 13C NMR (151
MHz, DMSO-d6) δ: 42.1, 44.1, 96.0, 117.9, 128.3, 128.3, 131.9,
131.9, 136.9, 139.0, 139.8, 154.7, 170.6, 171.9. mp 243–245 °C.
Methyl 5-chloro-2-(methylsulfonyl)benzoate (6). To a cold
solution of 5 (0.97 g, 4.48 mmol) in EtOAc (10 mL) was added
m-CPBA (2.43 g, 9.85 mmol) at 5 °C, and then the mixture was
stirred at rt for 14 h, and poured into aq. NaHCO3 and EtOAc.
The separated organic layer was washed with water, dried over
MgSO4, and concentrated. The residue was purified using silica
gel column chromatography (eluted with 25% EtOAc in hexane)
to yield 6 (0.87 g, 78%) as a colorless powder. 1H NMR (300
MHz, CDCl3) δ: 3.34 (3H, s), 3.98 (3H, s), 7.62 (1H, dd, J = 8.4,
2.1 Hz), 7.68 (1H, d, J = 2.1 Hz), 8.06 (1H, d, J = 8.4 Hz). mp
55–57 °C. Anal. Calcd for C9H9ClO4S: C, 43.47; H, 3.65. Found:
C, 43.71; H: 3.55.
5-Chloro-2-(methylsulfonyl)benzoic acid (7). A mixture of 6
(0.86 g, 3.46 mmol) in 4 M NaOH (8 mL) and THF (8 mL) was
heated at 50 °C for 2 h, cooled to rt, and acidified with 8 M HCl.
The product was extracted with EtOAc, washed with water and
brine, dried over MgSO4, and concentrated to yield 7 (0.77 g,
95%) as a colorless powder. 1H NMR (300 MHz, CDCl3) δ: 3.41
(3H, s), 6.70–7.15 (1H, br), 7.68 (1H, dd, J = 8.4, 2.1 Hz), 7.84
(1H, d, J = 2.1 Hz), 8.11 (1H, d, J = 8.4 Hz). mp 152–154 °C.
Anal. Calcd for C8H7ClO4S·0.1EtOAc: C, 41.44; H, 3.23. Found:
C, 41.44; H: 3.00.
[5-Chloro-2-(methylsulfonyl)phenyl]methanol (8). To a cold
solution of 7 (0.23 g, 1.00 mmol) and Et3N (170 ꢀL, 1.20 mmol)
in THF (5 mL) was added 2-methylpropyl chlorocarbonate (160
ꢀL, 1.20 mmol) at 5 °C, and then the mixture was stirred at 5 °C
for 30 min. A solution of sodium borohydride (190 mg, 5 mmol)
in water (1 mL) was added, and the mixture was stirred at rt for 2
h and poured into water and EtOAc. The separated organic layer
was washed with aq. NH4Cl and brine, dried over MgSO4, and
concentrated. The residue was purified using silica gel column
chromatography (eluted with 50% EtOAc in hexane) followed by
crystallization from EtOAc/hexane yield 8 (0.17 g, 77%) as
colorless crystals. 1H NMR (300 Hz, CDCl3) δ: 2.91 (1H, t, J =
6.8 Hz), 3.17 (3H, s), 4.94 (2H, d, J = 6.8 Hz), 7.49 (1H, dd, J =
8.4, 2.1 Hz), 7.60 (1H, d, J = 2.1 Hz), 7.97 (1H, d, J = 8.4 Hz).
mp 85–87 °C. Anal. Calcd for C8H9ClO3S: C, 43.54; H, 4.11.
Found: C, 43.54; H: 3.89.
2-(Bromomethyl)-4-chloro-1-(methylsulfonyl)benzene (9a).
To a solution of PPh3 (2.29 g, 8.73 mmol) in CH3CN (50 mL)
was added Br2 (450 ꢀL 9.19 mmol) at rt and the mixture was
stirred for 30 min. Then, 8 (800 mg, 3.63 mmol) was added, the
mixture was stirred at 70 °C for 1.5 h, and then poured into
water. The product was extracted with EtOAc, and the extract
was washed with aq. NH4Cl and brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified using silica gel
column chromatography (eluted with 5%–15% EtOAc in hexane)
to yield 9a (1.01 g, 98%) as a white powder. 1H NMR (300 MHz,
CDCl3) δ: 3.25 (3H, s), 5.01 (2H, s), 7.48 (1H, dd, J = 8.4, 1.8
Hz), 7.57 (1H, d, J = 1.8 Hz), 8.00 (1H, d, J = 8.4 Hz). 13C NMR
(75 MHz, CDCl3) δ: 27.8, 45.1, 129.6, 131.9, 133.3, 136.9, 139.2,
140.7. mp 132–133 °C.
4-Chloro-2-(chloromethyl)-1-(methylsulfonyl)benzene (9b). A
mixture of 8 (9.37 g, 42.5 mmol) and thionyl chloride (30 mL,
411 mmol) was stirred at 90 °C for 16 h and concentrated in
vacuo. The residue was diluted with EtOAc and water. The
organic layer was separated and washed with brine, dried over
MgSO4, and concentrated in vacuo. The residue was purified
using silica gel column chromatography (eluted with 5%–50%
EtOAc in hexane) to yield 9b (6.91 g, 68%) as a white solid. 1H
NMR (300 MHz, CDCl3) δ: 3.22 (3H, s), 5.10 (2H, s), 7.52 (1H,
dd, J = 8.5, 2.1 Hz), 7.62 (1H, d, J = 1.9 Hz), 8.03 (1H, d, J = 8.7
5

ACCEPTED MANUSCRIPT
2.
Varela, C. L.; Amaral, C.; Tavares da Silva, E.; Lopes,
ACCEPTED MANUSCRIPT
Determination of HPLC yield
A.; Correia-da-Silva, G.; Carvalho, R. A.; Costa, S. C.;
Roleira, F. M.; Teixeira, N. Eur. J. Med. Chem. 2014,
87, 336-345.
The HPLC grade CH3CN and water were obtained from Wako
Pure Chemical Industries, Ltd. A standard solution of 2a
containing an equivalent amount of 1-phenylethanol as the
internal standard (IS) was prepared at a concentration of 0.5
mg/mL of 2a in water/CH3CN (1/1, v/v). A solution of the IS was
similarly prepared at a concentration of 100 mg/mL in DMSO.
Additive(s) were introduced to a mixture of 12 (50 mg, 0.25
mmol) and 9b (59 mg, 0.25 mmol) in each solvent evaluated (1
mL), the resulting mixture was stirred for 14 h under each of the
described conditions, and then the IS solution (1 mL) was added.
After dilution (1:250) with water/CH3CN (1/1, v/v), 10 ꢀL of the
diluted sample was injected into the analytical column. The
HPLC analyses were performed using a Waters Alliance HPLC
system (model e2695) using an isocratic solvent system
consisting of CH3CN and 0.05 M ammonium acetate buffer
(35/65) at a flow rate of 1.0 mL/min on an Ascentis Phenyl
column (4.6× 250 mm, 5-ꢀm) at 30 °C with UV detection at 220
nm. The HPLC yield of each reaction mixture was determined by
comparing the ratio of the peak areas of 2a and 1-phenylethanol
in the sample solution with that in the standard solution, defined
as a 100% yield.
3.
Fang, Z.; Agoston, G. E.; Ladouceur, G.; Treston, A.
M.; Wang, L.; Cushman, M. Tetrahedron 2009, 65,
10535-10543.
Gamboa da Costa, G.; Marques, M. M.; Freeman, J. P.;
Beland, F. A. Chem. Res. Toxicol. 2003, 16, 1090-1098.
Bode, C.; Boezio, A.; Cheng, A. C.; Choquette, D.;
Coats, J. R.; Copeland, K. W.; Huang, H.; La, D.;
Lewis, R.; Liao, H.; WO 2010132598 A1, Nov 18,
2010.
Mitani, I.; Ogoshi, Y.; Matsui, T.; Yokota, M.;
Terashita, M.; WO 2011007856 A1, Jan 20, 2011.
Gaoni, Y.; Greenberg, F. J. Org. Chem. 1981, 46, 74-
78.
Ahlmark, M.; Belle, D. D.; Kauppala, M.; Luiro, A.;
Pajunen, T.; Pystynen, J.; Tianen, E.; Vaismaa, M.;
Messinger, J.; WO2013175053 A1, Nov 28, 2013.
Hancock, A. A. Drug Dev. Res. 1996, 39, 54-107.
Yale, H. L.; Bristol, J. J. Heterocycl. Chem. 1975, 12,
1027-1029.
4.
5.
6.
7.
8.
9.
10.
11.
Liebscher, J.; Feist, K. J. Prakt. Chem. 1988, 330, 175-
181.
Measurement of α1 adrenaline receptor binding inhibitory
activity
12.
13.
Sharma, S. J. Indian Chem. Soc. 2004, 81, 602-605.
Zheng, Y.; Chen, J.; Huang, D.; Liu, X.; Yang, L.; Ni,
C. Huaxue Yanjiu 2009, 20(3), 26-28.
Maity, A.; Mondal, S.; Paira, R.; Hazra, A.; Naskar, S.;
Sahu, K. B.; Saha, P.; Banerjee, S.; Mondal, N. B.
Tetrahedron Lett. 2011, 52, 3033-3037.
Muthukumar, C.; Nallub, M.; Arunachalama, T.;
Subramaniana, M.; Balamuruganc, C. Indian J. Chem.
2012, 51, 388-392.
Brzozowski, Z.; Sławiński, J.; Innocenti, A.; Supuran,
C. T. Eur. J. Med. Chem. 2010, 45, 3656-3661.
Ma, Y. M.; Hider, R. C. Tetrahedron Lett. 2010, 51,
5230-5233.
Human α1A-, α1B- and α1D-adrenoceptor membranes were
prepared from CHO-K1 cells stably expressing each α1-
adrenoceptor. The binding assay for the adrenergic α1 receptor
was performed in 200 µL of α1 binding assay buffer (50 mM Tris-
HCl pH 7.5, 10 mM MgCl2, 5 mM EDTA, and 0.5% BSA )
containing membrane protein (10 µg of each α1 receptor) and 2.5
nM 7-methoxy-[3H]-prazosin in the presence of various
concentrations of the compounds. Following incubation at rt for
60 min, the membranes were filtered through GF/C filter plates
(Perkin Elmer Life and Analytical Sciences) and washed with 50
mM Tris-HCl (pH 7.5). The membrane-associated radioactivity
was determined using a TopCount liquid scintillation counter
(Perkin Elmer Life and Analytical Sciences). The non-specific
binding was defined as binding in the presence of 10 µM
phentolamine. The IC50 values were calculated using logistic
regression analysis. The Kd values of the α1 receptor subtypes
(α1A, α1B, and α1D) were 0.93, 0.35 and 0.26 nM, respectively. The
Ki values were calculated as Ki = IC50/[1+(3H-ligand
concentration)/Kd].
14.
15.
16.
17.
Acknowledgements
We thank Ms. Yasumi Kumagai and Mr. Motoo Iida for their
assistance with the NMR analysis; Ms. Mayuko Yoshimura and
Dr. Noritaka Kuroda for the HPLC analysis; Mr. Mamoru
Ariyoshi and Dr.Yoshitaka Inui for the preparation of the urine
samples; and Dr. Izumi Kamo, Dr. Satoshi Yamamoto, Mr.
Mikio Shirasaki, Mr. Kouichi Iida, Mr. Satoru Oi, and Dr.
Hajime Motoyoshi for helpful discussions.
Supplementary data
The following is the supplementary data related to this article
(Copies of 1H NMR, 13C NMR, and NOESY spectra of
compound 2a):
References and notes
1.
Sakauchi, N.; Kohara, Y.; Sato, A.; Suzaki, T.; Imai,
Y.; Okabe, Y.; Imai, S.; Saikawa, R.; Nagabukuro, H.;
Kuno, H.; Fujita, H.; Kamo, I.; Yoshida, M. J. Med.
Chem. 2016, 59, 2989-3002.
6