Practical syntheses of the adhesion molecule inhibitor ER-49890 and its stereoisomer

Article abstract of DOI:10.3987/COM-04-10279

Practical synthetic routes to anti-[3-(10H-pyrazino[2,3-b][1,4]-benzothiazin-8-ylmethyl)-3-azabicyclo[3.3.1]non-9-yl]acetic acid (ER-49890, 1) and its syn-isomer are reported. The anti-(3-azabicyclo[3.3.1]non-9-yl)acetic acid side chain (anti-12) was synt

Full text of DOI:10.3987/COM-04-10279

HETEROCYCLES, Vol. 65, No. 2, 2005
403
HETEROCYCLES, Vol. 65, No. 2, 2005, pp. 403 - 410
Received, 9th November, 2004, Accepted, 22nd December, 2004, Published online, 24th December, 2004
PRACTICAL SYNTHESES OF THE ADHESION MOLECULE
INHIBITOR ER-49890 AND ITS STEREOISOMER
Toshihiko Kaneko,a* Fumihiro Ozaki,a Richard S. J. Clark,a Yuki
Komatsu,b and Kazuo Okano a
a
Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3, Tokodai, Tsukuba,
Ibaraki 300-2635, Japan. e-mail; t3-kaneko @hhc.eisai.co.jp
b
Process Research Laboratories, Eisai Co., Ltd. Kashima Plant, 22-Sunayama,
Hasaki, Kashima, Ibaraki, 314-0255, Japan.
Abstract – Practical synthetic routes to anti-[3-(10H-pyrazino[2,3-b][1,4]-
benzothiazin-8-ylmethyl)-3-azabicyclo[3.3.1]non-9-yl]acetic acid (ER-49890, 1)
and its syn-isomer are reported. The anti-(3-azabicyclo[3.3.1]non-9-yl)acetic acid
side chain (anti-12) was synthesized stereoselectively using Pd/C hydrogenation
in the presence of HCl. The syn-isomer (syn-12) was concisely obtained by
crystallization as its oxalic acid salt from a 1:1 mixture of anti- and syn-isomers.
The 10H-pyrazino[2,3-b][1,4]benzothiazine core (7) was prepared from
commercially available 4-chloro-3-nitrobenzyl alcohol (13) in four steps and
good yield.
INTRODUCTION
Adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell
adhesion molecule-1 (VCAM-1) have been implicated in inflammatory disorders including rheumatoid
arthritis, colitis, psoriasis and multiple sclerosis, and offer an attractive target for the design of novel
therapeutic drugs.1 We have discovered that anti-[3-(10H-pyrazino[2,3-b][1,4]benzothiazin-8-ylmethyl)-
3-azabicyclo[3.3.1]non-9-yl]acetic acid (ER-49890, 1), is a novel, orally-active inhibitor of the
upregulation of such adhesion molecules.2 During the course of our studies, we needed a practical route
for the scale-up synthesis of both 1 and its syn-stereoisomer (2) in order to evaluate their detailed
pharmacological profiles. However, as shown in Scheme 1 our previously reported synthetic route to 1

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HETEROCYCLES, Vol. 65, No. 2, 2005
and 2, required the separation of the anti- and syn-isomers using repeated column chromatography as the
step forming the mixture of acetates (10) proceeded in low selectivity. Two additional problems in using
the reported route for scale-up synthesis are the low stability of aminothiophenol intermediate (4), and the
step involving reduction of ester (6) using lithium aluminum hydride.3 We therefore set out to find
improved preparations of the three key intermediates, anti- and syn-(3-azabicyclo[3.3.1]non-9-yl)acetate
(10), and the 10H-pyrazino[2,3-b][1,4]benzothiazine core (7).
N
N
Cl
Cl
H
N
5
N
N
X
O2N
Cl
CO2H
H2N
HS
CO2Me
S
6 X=CO2Me Y.25%
7 X=CH2OH Y.75%
8 X=CH2Cl
4
3
LiAlH4
CO2Et
O
O
N
CO2Et
CO2Et
1) Voc Cl
Pd/C, H2
N
N
O
O
anti-11 Y.29%
Me
Me
2) anti-syn
separation
by column
7 times
CO2Et
10 (anti:syn=3:1)
9
N
H
CO2H
8
syn-11 Y.13%
N
N
N
N
S
1 ER-49890
CO2H
H
N
N
N
8
N
S
2 syn-isomer of 1
Scheme 1 Previously Reported Synthetic Routes to ER-49890 (1) and Its Syn-Isomer (2)
RESULTS AND DISCUSSION
As shown in Scheme 2, we planned to prepare anti- and syn-12 stereoselectively from a common starting
compound (9). We selected 4-chloro-3-nitrobenzyl alcohol (13) as the starting material for the synthesis
of 10H-pyrazino[2,3-b][1,4]benzothiazine core (7), in order to avoid having to use unstable
aminothiophenol (4) as an intermediate.
First, the anti-selective formation of the (3-azabicyclo[3.3.1]non-9-yl)acetic acid side chain (anti-12), for
which a stereoselective synthesis has not been reported, was investigated. As previously described,2
hydrogenation of unsaturated ester (9)4 using palladium on carbon (Pd/C) provided an anti-syn mixture of

HETEROCYCLES, Vol. 65, No. 2, 2005
405
CO2R
CO2R
CO2Et
N
N
N
Me
Me
Me
anti-12
syn-12
9
H
N
N
N
Cl
Cl
O2N
Cl
OH
OH
+
N
S
7
N
13
5
Scheme 2 Retrosynthesis of Desired Intermediates
12 in the ratio of 3:1. We examined the effect of solvent and additives on this ratio as calculated from the
integrated intensity of the equatorial protons at C-2 and C-4 of the 3-azabicyclo[3.3.1]nonane ring at 2.90
ppm (anti) and 2.62 ppm (syn) respectively (Table 1).
Table 1 The Effect of Solvent and Additive on the Hydrogenation of 9
CO2Et
CO2Et
CO2Et
Pd/C, H2 (1 atm), rt
additive, solvent
N
N
N
+
Me
Me
Me
9
anti-12
syn-12
Additivea)
Anti : Synb)
Solvent
Run
1
2
EtOH
3 : 1
4 : 1
Hexane
3
4
5
EtOAc
EtOH
EtOH
2 : 1
3 : 1
4 : 1
5 : 1
6 : 1
Pyridine
AcOH
MsOH
TsOH
EtOH
EtOH
6
7
TsOH
6 : 1
8
9
EtOH, H2O
EtOH
concd HCl
29 : 1
a) additive (1.5 eq.). b) Ratios were calculated from 1H-NMR spectra.
Use of less-polar solvents such as hexane, AcOEt or pyridine-containing ethanol gave no improvement,
neither did addition of weak acid such as acetic acid (AcOH). Use of a stronger bulky acid such as
p-toluenesulfonic acid (TsOH) gave a 2-fold improvement in selectivity, a similar result being obtained
with methanesulfonic acid (MsOH). On the other hand, a dramatic improvement in anti-selectivity was
achieved on addition of concd HCl with the ratio increasing to 29:1. We postulated that the difference
between TsOH and concd HCl might be due to water, but adding water under the TsOH conditions to the

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HETEROCYCLES, Vol. 65, No. 2, 2005
same concentration as for concd HCl had no significant effect. Despite this result, we had established a
selective synthesis of anti-12 using Pd/C hydrogenation in the presence of concd HCl.
CO2Me
CO2Et
CO2Me
1) (CO2H)2
Mg/MeOH
Me
N
N
N
Me
Me
2) recystalization
four times
12 (anti:syn=1:1)
9
syn-12
Y.26%
Y. quant.
Scheme 3 Practical Synthetic Route to Syn-12
On the other hand, syn selective hydrogenation could not be achieved, and so we attempted to obtain the
syn-stereoisomer (syn-12) by crystallization, which it is known can be useful for the scale-up separation
of a single stereoisomer from a mixture. A 1:1 anti:syn mixture was obtained by magnesium-methanol
reduction5 of α,β-unsaturated ester (9). Fortunately, 12 could be obtained as its syn-rich oxalic acid salt
from ethanol (isomer ratio 4:1). After a total of four recrystallizations, pure syn-isomer (syn-12) was
obtained in a yield of 26%. The structures of anti-12 and syn-12 were deduced based on NOE
experiments as shown in Figure 1. In anti-12 an NOE was detected between H-2ax, H-4ax and H-9, and
between H-6ax, H-8ax and H-10. In syn-12 an NOE was observed between H-2ax, H-4ax and H-10, and
between H-6ax, H-8ax and H-9.
NOE
NOE
H
CO2Me
H
H
H
H
9
2
1
H
H
CO2Et
CO2Et
NOE
H
3
N
10
4
N
5
N
H
NOE
H
Me
H
Me
Me
H
H
6
8
7
syn-12
anti-12
Figure 1 NOE Correlation of Anti-12 and Syn-12
The 10H-pyrazino[2,3-b][1,4]benzothiazine core (7) was synthesized as shown in Schemes 4 and 5.
Displacement of the chloride of 13 with sodium sulfide (Na2S) in DMSO-water solvent afforded 14 in
more than 90% yield as evaluated by HPLC. Extraction of 14 followed by condensation with
2,3-dichloropyrazine (5) in acetonitrile-water solvent afforded 15 (Y.38%) and the dimer (16) (Y.10%) as
shown in Run 1. As it has been reported that triphenylphosphine reduces a disulfide to the corresponding
thiol,6 addition of a small amount of triphenylphosphine was examined. This was successful, and the yield
of 15 rose to 48%. Reduction of the nitro group of 15 using iron powder, followed by cyclization under

HETEROCYCLES, Vol. 65, No. 2, 2005
407
acidic conditions afforded the key intermediate (7). The yield of this synthesis of the core was 48%
starting from alcohol (13), a 2.5-fold improvement over that of the previous route (Y.19%).
O2N
Cl
N
N
Cl
NO2
S
O2N
HS
5
Na2S
OH
OH HO
+
OH
DMSO-H2O
solvent
S
2
NO2
13
14
16
15
Run
1
Condition
90 Ž, 3 h
15 (Y.%)
Solvent
16 (Y.%)
MeCN-H2O
38
48
10
0.05 eq. PPh3
90 Ž, 3 h
MeCN-H2O
2
Scheme 4 Synthetic Route to Intermediate (15)
H
N
N
N
Cl
S
N
N
Cl
S
N
N
Fe, NH4Cl
HCl
OH
OH
OH
S
NO2
NH2
17 Y.100%
15
7 Y.99%
Scheme 5 New Synthetic Route for 7
In conclusion, we have established practical synthetic routes to ER-49890 (1) and its syn-isomer (2)
involving an anti-selective hydrogenation of ethyl (3-azabicyclo[3.3.1]non-9-ylidene)acetate (9), and
syn-selective crystallization of methyl (3-azabicyclo[3.3.1]non-9-yl)acetate (12), respectively, and a new
synthesis of the 10H-pyrazino[2,3-b][1,4]benzothiazine core (7). These simple stereoselective methods
offer a useful stereo-controlled route to anti- and syn-(3-azabicyclo[3.3.1]non-9-yl)acetate as possible
bioisosteres of piperidine in various bioactive molecules.
EXPERIMENTAL
Melting points were measured using a Yanako melting-point apparatus and are uncorrected. IR spectra
were measured with a JASCO FT/IR-620 spectrophotometer. 1H-NMR spectra were recorded on a Varian
Unity 400 (400 MHz) spectrometer, and chemical shifts are expressed in ppm downfield from TMS as an
internal reference. MS spectra were obtained on a Thermo Quest SSQ700 mass spectrometer. Elemental
analysis was performed at the Analytical Research Laboratories in Eisai. Materials were used as bought
without any special purification. All organic extracts were dried over anhydrous MgSO4, and solvents
were removed with a rotary evaporator under reduced pressure.

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Ethyl anti-(3-methyl-3-azabicyclo[3.3.1]non-9-yl)acetate (anti-12)
To a solution of ethyl (3-methyl-3-azabicyclo[3.3.1]non-9-ylidene)acetate (9)4 (70 g, 0.31 mol) in ethanol
(EtOH; 550 mL) containing concd hydrochloric acid (concd HCl; 42 mL) was added 10% palladium on
carbon (50% water by weight, Pd/C; 21 g). The mixture was stirred vigorously at rt under a hydrogen
atmosphere for 12 h and was then filtered through celite. The solvent was evaporated under reduced
pressure, and the residue was dissolved in water (1 L). This solution was washed with AcOEt, and then
made alkaline with 33% aqueous ammonia (500 mL). This mixture was extracted with AcOEt, and the
combined extracts were washed with saturated brine, dried and evaporated to afford 65 g (93%) of
anti-12 as a colorless oil. IR (film, cm-1) 2907, 1736, 1276, 1162. 1H-NMR (CDCl3) δ: 1.25 (3H, t, J=7.2
Hz), 1.37-1.46 (1H, m), 1.50-1.58 (2H, m), 1.62 (2H, br s), 1.68-1.80 (2H, m), 1.97 (1H, t, J= 7.6 Hz),
2.13 (3H, s), 2.18-2.25 (2H, m), 2.36-2.50 (1H, m), 2.48 (2H, d, J= 7.6 Hz), 2.90 (2H, br d, J= 11.2 Hz),
4.12 (2H, q, J=7.2 Hz). ESI-MS m/z: 226 (M+H) +. Anal. Calcd for C13H23NO2·C2H2O4·0.1H2O: C, 56.80;
H, 8.01; N, 4.42. Found: C, 56.74; H, 7.82; N, 4.31.
Methyl syn-(3-methyl-3-azabicyclo[3.3.1]non-yl)acetate (syn-12)
To a solution of ethyl (3-methyl-3-azabicyclo[3.3.1]non-9-ylidene)acetate (9)4 (21.4 g, 0.096 mol) in
methanol (500 mL) was added magnesium shavings (13.0 g, 0.57 mol) with stirring over a period of 1 h
under a nitrogen atmosphere. The internal temperature rose to 35-40 °C after the reaction initiated, and
cooling was continued until it fell back to rt. The reaction mixture was stirred for a total of 22 h, then the
solvent was evaporated under reduced pressure. The residue was poured into saturated aqueous
ammonium chloride, and this solution was extracted with AcOEt. The extract was dried and concentrated
in vacuo. The residue was purified by silica gel column chromatography (50% AcOEt-heptane) to give
18.0 g (89%) of a mixture of anti-12 and syn-12 as a pale yellow oil. This mixture was dissolved in EtOH
(185 mL) and oxalic acid (7.7 g, 86 mmol) was added. The mixture was stirred at rt for 12 h, then the
precipitate was collected by filtration to give 22.3 g of a mixture of syn-12 and anti-12 in a 4:1 ratio. This
solid was recrystallized three times from EtOH (550 mL), EtOH (500 mL) and EtOH (200 mL) to give
6.6 g (26%) of the oxalic acid salt of the desired syn-12 as a colorless solid. This salt (2.0 g, 6.6 mmol)
was dissolved in water (10 mL) and this solution was treated with 33% aqueous ammonia (5 mL), and
then extracted with AcOEt. The extract was washed with saturated brine, dried, and concentrated in vacuo
to give 1.4 g (quant.) of syn-12 as a pale yellow oil. IR (film, cm-1) 2905, 1738, 1262, 1148. 1H-NMR
(CDCl3) δ: 1.42-1.50 (1H, m), 1.60-1.76 (4H, m), 1.79-1.87 (2H, m), 2.00 (1H, t, J=8.0 Hz), 2.13 (3H, s),
2.34 (2H, br d, J=12.0 Hz), 2.43 (2H, d, J=8.0 Hz), 2.40-2.54 (1H, m), 2.62 (2H, br d, J= 11.2 Hz), 3.66
(3H, s). ESI-MS m/z: 212 (M+H) +. Anal. Calcd for C12H21NO2·C2H2O4: C, 55.80; H, 7.69; N, 4.65.
Found: C, 55.44; H, 7.52; N, 4.61.

HETEROCYCLES, Vol. 65, No. 2, 2005
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4-[(3-Chloropyrazin-2-yl)thio]-3-nitrobenzyl alcohol (15)
To a solution of sodium sulfide nonahydrate (7.7 g, 32 mmol) in water (25 mL), a solution of
4-chloro-3-nitrobenzyl alcohol (13) (3.0 g, 16 mmol) in DMSO (20 mL) was added over 1 min at rt and
then the reaction mixture was stirred at 40-50 °C for 2 h under a nitrogen atmosphere. The reaction
mixture was poured into a suspension of triphenylphosphine (PPh3; 0.20 g, 0.80 mmol) in water (50
mL)-AcOEt (50 mL) and the AcOEt layer was separated. The aqueous phase was added to a suspension
of PPh3 (0.20 g, 0.80 mmol) in AcOEt (50 mL)-1N HCl (40 mL), and extracted with AcOEt. The extract
was washed with saturated brine. The organic layer was extracted with 1N NaOH (50 mL). To this
aqueous solution was added a solution of 2,3-dichloropyrazine (5) (2.2 g, 15 mmol) and PPh3 (0.20 g,
0.80 mmol) in acetonitrile (30 mL), and the resulting mixture was stirred at 90 °C for 3 h under a nitrogen
atmosphere. After cooling of the reaction mixture to rt, the mixture was poured into ice-water, and
extracted with AcOEt. The extract was washed with saturated brine, dried, and evaporated. The residue
was purified by silica gel column chromatography (70% AcOEt-heptane) to give 2.3 g (48%) of 15 as a
yellow solid. mp 113-114 °C (recrystalised from AcOEt). IR (KBr, cm-1) 3244, 1521, 1051, 855, 795.
1H-NMR (CDCl3) δ: 1.94 (1H, t, J=6.0 Hz), 4.87 (2H, d, J=6.0 Hz), 7.60-7.66 (1H, m), 7.66 (1H, d, J=8.0
Hz), 8.08-8.12 (1H, m), 8.11 (1H, d, J=2.4 Hz), 8.15 (1H, d, J=2.4 Hz). ESI-MS m/z: 320 (M+Na) +. Anal.
Calcd for C11H8N3O3ClS: C, 44.38; H, 2.71; N, 14.11. Found: C, 44.41; H, 2.73; N, 14.02.
4,4’-Dithiobis(3-nitrobenzyl alcohol) (16)
mp 147-149 °C (recrystalised from AcOEt). IR (KBr, cm-1) 3343, 1519, 1330, 805. 1H-NMR (CD3OD) δ:
4.67 (4H, s), 7.61 (2H, d, J=8.0 Hz), 7.82 (2H, d, J=8.0 Hz), 8.34 (2H, s). HR-MS (ESI) m/z: Calcd for
C14H12N2O6S2 (M)+: 368.0137. Found: 368.0134.
3-Amino-4-[(3-chloropyrazin-2-yl)thio]benzyl alcohol (17)
To a solution of 4-[(3-chloropyrazin-2-yl)thio]-3-nitrobenzyl alcohol (15) (1.6 g, 5.4 mmol) and
ammonium chloride (0.16 g, 3.0 mmol) in THF (20 mL), isopropyl alcohol (20 mL) and water (7 mL)
were added iron powder (7.5 g, 0.13 mol) portionwise. The reaction mixture was refluxed for 1.5 h, then
filtered. The organic phase was evaporated to give 1.5 g (quant.) of 17 as a yellow solid. mp 160-161 °C
1
(recrystalised from AcOEt). IR (KBr, cm-1) 3305, 1336, 1052, 850, 672. H-NMR (DMSO-d6) δ: 4.40
(2H, d, J=5.6 Hz), 5.15 (1H, t, J=5.6 Hz), 5.37 (2H, s), 6.49 (1H, d, J=8.0 Hz), 6.75 (1H, s), 7.17 (1H, d,
J=8.0 Hz), 8.16 (1H, d, J=2.4 Hz), 8.33 (1H, d, J=2.4 Hz). ESI-MS m/z: 268 (M+H) +. Anal. Calcd for
C11H10N3OClS: C, 49.35; H, 3.76; N, 15.69. Found: C, 49.43; H, 3.86; N, 15.36.
10H-Pyrazino[2,3-b][1,4]benzothiazine-8-methanol (7)
A solution of 3-amino-4-[(3-chloropyrazin-2-yl)thio]benzyl alcohol (17) (1.5 g, 5.4 mmol) and concd
HCl (0.45 mL, 5.4 mmol) in methanol (20 mL) was refluxed for 0.5 h. After addition of ice-cooled
aqueous 10% ammonia, the precipitate was collected, washed with water and dried to afford 1.2 g (99%)

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of 7 as a yellow solid. mp 187-189 °C. Spectral date for this compound were identical to those of an
authentic sample obtained by the known method.3
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