Novel, highly potent aldose reductase inhibitors: (R)-(-)-2-(4-bromo-2- fluorobenzyl)-1,2,3,4-tetrahydropyrrolo[1,2-α]pyrazine-4-spiro-3'- pyrrolidine-1,2',3,5'-tetrone (AS-3201) and its congeners

Article abstract of DOI:10.1021/jm9802968

A series of novel tetrahydropyrrolo[1,2-α]pyrazine derivatives were synthesized and evaluated as aldose reductase inhibitors (ARIs) on the basis of their abilities to inhibit porcine lens aldose reductase (AR) in vitro and to inhibit sorbitol accumulation in the sciatic nerve of streptozotocin- induced diabetic rats in vivo. Of these compounds, spirosuccinimide-fused tetrahydropyrrolo-[1,2-α]pyrazine-1,3-dione derivatives showed significantly potent AR inhibitory activity. In the in vivo activity of these derivatives, 2-(4-bromo-2-fluorobenzyl)-1,2,3,4-tetrahydropyrrolo-[1,2-α]pyrazine-4- spiro-3'-pyrrolidine-1,2',3,5'-tetrone (23t) (SX-3030) showed the best oral activity. The enantiomers of 23t were synthesized, and the biological activities were evaluated. It was found that AR inhibitory activity resides in the (-)-enantiomer 43 (AS-3201), which was 10 times more potent in inhibition of the AR (IC₅₀ = 1.5 x 10^-8 M) and 500 times more potent in the in vivo activity (ED₅₀ = 0.18 mg/kg/day for 5 days) than the corresponding (+)-enantiomer 44 (SX-3202). From these results, AS-3201 was selected as the candidate for clinical development. The absolute configuration of AS-3201 was also established to be (R)-form by single- crystal X, ray analysis. In this article we report the preparation and structure-activity relationship (SAR) of tetrahydropyrrolopyrazine derivatives including a novel ARI, AS-3201.

Full text of DOI:10.1021/jm9802968

4118
J . Med. Chem. 1998, 41, 4118-4129
Novel, High ly P oten t Ald ose Red u cta se In h ibitor s:
(R)-(-)-2-(4-Br om o-2-flu or oben zyl)-1,2,3,4-tetr a h yd r op yr r olo[1,2-a ]p yr a zin e-
4-sp ir o-3′-p yr r olid in e-1,2′,3,5′-tetr on e (AS-3201) a n d Its Con gen er s
Toshiyuki Negoro,* Makoto Murata, Shozo Ueda, Buichi Fujitani, Yoshiyuki Ono, Akemi Kuromiya,
Masanobu Komiya, Kenji Suzuki, and J un-ichi Matsumoto
Department of Chemistry I, Discovery Research Laboratories I, Dainippon Pharmaceutical Company, Ltd., Enoki 33-94,
Suita/ Osaka 564-0053, J apan
Received May 11, 1998
A series of novel tetrahydropyrrolo[1,2-a]pyrazine derivatives were synthesized and evaluated
as aldose reductase inhibitors (ARIs) on the basis of their abilities to inhibit porcine lens aldose
reductase (AR) in vitro and to inhibit sorbitol accumulation in the sciatic nerve of streptozotocin-
induced diabetic rats in vivo. Of these compounds, spirosuccinimide-fused tetrahydropyrrolo-
[1,2-a]pyrazine-1,3-dione derivatives showed significantly potent AR inhibitory activity. In
the in vivo activity of these derivatives, 2-(4-bromo-2-fluorobenzyl)-1,2,3,4-tetrahydropyrrolo-
[1,2-a]pyrazine-4-spiro-3′-pyrrolidine-1,2′,3,5′-tetrone (23t) (SX-3030) showed the best oral
activity. The enantiomers of 23t were synthesized, and the biological activities were evaluated.
It was found that AR inhibitory activity resides in the (-)-enantiomer 43 (AS-3201), which
was 10 times more potent in inhibition of the AR (IC50 ) 1.5 × 10-8 M) and 500 times more
potent in the in vivo activity (ED50 ) 0.18 mg/kg/day for 5 days) than the corresponding (+)-
enantiomer 44 (SX-3202). From these results, AS-3201 was selected as the candidate for clinical
development. The absolute configuration of AS-3201 was also established to be (R)-form by
single-crystal X-ray analysis. In this article we report the preparation and structure-activity
relationship (SAR) of tetrahydropyrrolopyrazine derivatives including a novel ARI, AS-3201.
Ch a r t 1
In tr od u ction
Currently, a number of structurally diverse com-
pounds possessing aldose reductase (AR) inhibitory
activities have been synthesized as potential drugs for
the treatment of diabetic complications such as cataract
formation, retinopathy, neuropathy, and nephropathy.
Most of these compounds contain acetic acid moieties
or five-membered ring cyclic imides (hydantoin and
succinimide), having an acidic proton1 that appears to
be necessary for the inhibition of AR (Chart 1). This is
substantiated by the structures of tolrestat,2 epalrestat,3
and several inhibitors4-8 which have been presently
undergoing clinical trials. Thus, we focused our atten-
tion on molecules containing N-acylglycine moieties and
planned to synthesize a series of heterocyclic compounds
with those moieties.
In the course of synthesizing new compounds as
aldose reductase inhibitors (ARIs), we unexpectedly
found that 2-methyltetrahydropyrrolo[1,2-a]pyrazine-
1,4-dione (3) had attractive biological activity. Although
this compound possessed no acidic proton, in contrast
to the above-mentioned ARIs, it showed not only AR
inhibitory in vitro activity (IC50 ) 7.5 × 10-6 M) but
also in vivo activity (ED50 ) 68.4 mg/kg). We also found
that the pyrrolopyrazine 3 is more potent in vivo than
the ring-opened compounds 2 and 4 (Table 1). These
interesting observations suggested that the pyrrolopyr-
azine 3 possessed good oral absorption and efficient
tissue penetration properties, and this furthered our
interests to the unique structure of the tetrahydropyr-
rolo[1,2-a]pyrazine-1,4-dione ring system. However,
compound 3 was unstable because of easy cleavage of
the C-N bond between the 4- and 5-positions on the
pyrazine ring under aqueous basic conditions. In an
attempt to overcome this problem, we designed a novel
and more stable tetrahydropyrrolo[1,2-a]pyrazine-1,3-
S0022-2623(98)00296-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/12/1998

Novel, Highly Potent ARIs
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 4119
Sch em e 1a
amine to give pyrrolopyrazine-4-carboxylate 14, 15, or
16. Compound 15 or 16 was then hydrolyzed by 20%
hydrochloric acid to give the desired tetrahydropyrrolo-
[1,2-a]pyrazine-4-carboxylic acid 17 or 18, respectively.
Under the same conditions compound 14 underwent
hydrolysis followed by decarboxylation.
The spirosuccinimide-fused tetrahydropyrrolo[1,2-a]-
pyrazine-1,3-dione derivatives 22 and 23 were prepared
according to two methods, A and B, shown in Scheme
3. In method A, pyrrolopyrazine-4-carboxylate (14) was
alkylated with tert-butyl bromoacetate to give tert-butyl
pyrrolopyrazineacetate 19, which was deprotected with
trifluoroacetic acid (TFA) to give acetic acid 20. Treat-
ment of 20 with thionyl chloride and subsequent reac-
tion with a large excess of aqueous ammonium hydrox-
ide gave acetamide 21. Ring closure of 21 was
accomplished by treatment with sodium hydride to give
the final products 22 and 23. In method B, the
intermediate 27 was prepared from compound 8. Alky-
lation of 8 with benzyl bromoacetate and subsequent
deprotection by hydrogenolysis gave acetic acid 25.
Condensation of 25 with N-hydroxysuccinimide in the
presence of 1-ethyl-3-(3-(dimethylamino)propyl)carbo-
diimide hydrochloride (WSC), followed by treatment of
7.8% ammonia acetonitrile solution, gave acetamide 26.
Cyclization of 26 with a catalytic amount of potassium
carbonate smoothly proceeded to give succinimide 27
quantitatively. Conversion of 27 to the final products
22 and 23 was accomplished using the conditions
described in the preparation of 14 from 8 shown in
Scheme 2. Most of compounds 23 shown in Table 4 were
prepared from the intermediate 28 using this method.
a
Reagents: (a) WSC, Et3N/CH2Cl2; (b) Et3N/toluene; (c) 5%
NaHCO3-dioxane, WSC, 1-ethyl-3-(3-(dimethylamino)propyl)car-
bodiimide hydrochloride.
Sch em e 2a
Compounds 31, which have substituents on the pyr-
role ring of 23t, were prepared by two synthetic routes,
A and B, shown in Scheme 4. In route A, halogenation12
or acetylation13 of 28 gave 4-halogenated or 4-acetylated
trichloroacetylpyrroles 30a -c, which were then con-
densed with 4-bromo-2-fluorobenzylamine in the pres-
ence of triethylamine to give the desired products 31a -
c. Direct bromination of 23t in the presence of aluminum
chloride shown in route B gave a mixture of 6-bromo
(31e) and 6,7-dibromo (31f) derivatives. But chlorina-
tion of 23t with sulfuryl chloride only gave 6-chloro
derivative 31d .
a
Reagents: (a) 2,5-dimethoxy-THF/AcOH; (b) CCl3COCl/CHCl3;
(c) R1-NH2, Et3N/dry DMF; (d) 20% HCl aq/dioxane.
dione ring system and prepared many compounds
having an acidic moiety to find more potent compounds
than existing ARIs, such as ponalrestat9 and tolrestat.2
Ch em istr y
P r ep a r a tion of En a n tiom er s of SX-3030 (AS-3201
a n d SX-3202). The enantiomers of 23t (SX-3030), 2-(4-
bromo-2-fluorobenzyl)-1,2,3,4-tetrahydropyrrolo[1,2-a]-
pyrazine-4-spiro-3′-pyrrolidine-1,2′,3,5′-tetrone, were pre-
pared according to the method shown in Scheme 5.
Alkylation of ethyl [(benzyloxycarbonyl)amino]cyano-
acetate14 (32) with ethyl bromoacetate, followed by
hydrolysis using hydrogen peroxide-sodium carbonate,15
brought about ring closure to give succinimide 34.
Treatment of 34 with cinchonidine gave a cinchonidium
salt, which was twice recrystallized from ethanol to give
the diastereomerically enriched cinchonidium salt
(>99.9% de). This salt was then treated with hydro-
chloric acid to liberate the (-)-enantiomer 35 (>99.5%
ee). On the other hand, recrystallization of the residue
which was obtained from the acidified mother liquors
gave the pure (+)-enantiomer 36 (>99.4% ee). Each
enantiomer (35 or 36) was then deprotected by hydro-
genolysis with palladium carbon, followed by treatment
with 2,5-dimethoxytetrahydrofuran, to give pyrrole
The pyrrole derivatives were prepared according to
the method shown in Scheme 1. Condensation of
2-pyrrolecarboxylic acid (1) with sarcosine methyl ester
in the presence of triethylamine gave a mixture of
N-methyl-N-(2-pyrrolylcarbonyl)glycine methyl ester (2)
and 2-methyltetrahydropyrrolo[1,2-a]pyrazine-1,4-dione
(3). Ester 2 was easily cyclized to pyrrolopyrazine 3
under basic conditions. Ester 2 and pyrrolopyrazine 3
were easily hydrolyzed in an aqueous sodium bicarbon-
ate solution to give N-(2-pyrrolylcarbonyl)glycine (4).
The 1,3-dioxotetrahydropyrrolo[1,2-a]pyrazine-4-car-
boxylic acid derivatives 17 and 18 were prepared
according to the method shown in Scheme 2. Conden-
sation of the appropriate amino acid diester 5, 6, or 7
with 2,5-dimethoxytetrahydrofuran,10 followed by treat-
ment with an excess of trichloroacetyl chloride, gave
2-trichloroacetylpyrrole11 11, 12, or 13. These interme-
diates were subjected to ring closure by condensation
with the required amine in the presence of triethyl-

4120 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Negoro et al.
Sch em e 3a
a
Reagents: (a) (1) NaH, (2) BrCH2COO-But/dry DMF; (b) trifluoroacetic acid/CH2Cl2; (c) (1) SOCl2/CH2Cl2, (2) 25% aq NH3/AcOEt; (d)
NaH/dry DMF; (e) BrCH2COOBn, K2CO3/Me2CO or BrCH2COOBn, NaH/dry DMF; (f) H2, Pd-C/dioxane; (g) (1) N-hydroxysuccinimide,
WSC/dry CH2Cl2, (2) 7% NH3/CH3CN; (h) K2CO3/Me2CO; (i) CCl3COCl/CHCl3; (j) R1-NH2, Et3N/DMF.
Sch em e 4a
a
Reagents: (a) Br2 or SO2Cl2 or AcCl, AlCl3/CHCl3; (b) 4-Br-2F-Ph-CH2NH2, Et3N/DMF; (c) Br2, AlCl3 or SO2Cl2/CHCl3.
derivative (39 or 40), respectively. Conversion of 39 or
40 to the final product 43 (>99.4% ee) or 44 (>99.4%
ee), respectively, was accomplished in the same manner
as described in the preparation of 23 from 27 shown in
Scheme 3 (method B). The optical purity of each
enantiomer (35, 36, 43, or 44) was determined by HPLC
analysis on a chiral column (conditions described in
Experimental Section). The absolute configuration of
the (-)-enantiomer 43 (AS-3201) was established to be
(R)-form by single-crystal X-ray analysis (Figure 1).
preparation.16 The inhibitory activity was expressed as
the concentration (µM) of the test compound which
inhibited the activity of AR by 50% (IC50). The test
compounds were also evaluated in vivo by measuring
their ability to inhibit sorbitol accumulation in the
sciatic nerve of streptozotocin-induced diabetic rats.17
The ED50 value of test compounds represents the dose
causing 50% decrease of the sorbitol accumulation.
Results for pyrrole derivatives are shown in Table 1.
Pyrrolopyrazine 3, which has no acidic moiety, showed
almost the same potency as carboxylic acid 4 in the AR
inhibitory activity (IC50 ) 7.5 × 10-6 M). Furthermore,
pyrrolopyrazine 3 potently inhibited the sorbitol ac-
cumulation in the sciatic nerve of the diabetic rats (56%
inhibition at 100 mg/kg). Its in vivo activity was 2-fold
P h a r m a cologica l Resu lts a n d Discu ssion
The AR inhibitory activity of the synthesized com-
pounds was assessed by measuring the inhibition of
enzymatic activity in a partially purified porcine lens

Novel, Highly Potent ARIs
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 4121
Sch em e 5a
100 mg/kg) was superior to that of carboxylic acid 4.
This may be attributed to formation of pyrrolopyrazine
3 in consequence of cyclization of the ester 2. From
these results, it appeared that pyrrolopyrazine 3 pos-
sessed good oral absorption and efficient tissue penetra-
tion.
Results for compounds with a carboxylic acid moiety
at the C-4 position of the tetrahydropyrrolo[1,2-a]-
pyrazine-1,3-dione ring are shown in Table 2. Acetic
and propionic acid derivatives with either hydrogen or
low alkyl substituents at the N-2 position of the pyra-
zine ring were inactive in the AR inhibition at 10-6 M,
while these acetic acid derivatives produced weak
inhibition of the sorbitol accumulation at 100 mg/kg in
vivo (17a -c). Acetic and propionic acid derivatives with
3,4-dichloro or 4-bromo-2-fluorobenzyl groups at the N-2
position (17d ,e and 18d ), with the exception of 18e, gave
potent AR inhibitory activity (IC50 ) 1.3-1.6 × 10-7 M).
Of these compounds, only 18d produced weak inhibitory
activity at a high dose of 100 mg/kg in vivo; however,
these in vitro results were not satisfactorily reflected
in the in vivo activities of the compounds. It may be
attributed to poor oral absorption.
On the basis of this experience, we attempted to shift
our attention to other acidic moieties. Replacement of
the carboxylic acid moieties by spirosuccinimide at the
C-4 position of the pyrrolopyrazine ring resulted in
significant improvement of the in vivo activity. There-
fore, we focused our efforts on the spirosuccinimide-
fused tetrahydropyrrolopyrazine. To find beneficial
substituents for the generation of optimal biological
activity, compounds which have low alkyl, phenyl,
benzyl, or phenethyl substituents at the N-2 position
of the spirosuccinimide-fused pyrrolopyrazine ring were
synthesized. As seen from Table 3, four of five com-
pounds (22b-d and 23a ) showed good AR inhibitory
activity. The best in the in vitro activity was observed
for 23a with a benzyl group (IC50 ) 9.9 × 10-8 M).
Compounds with methyl and benzyl groups (22b and
23a ) showed good in vivo activities (51% and 69%
inhibition, respectively, at 30 mg/kg/day for 5 days).
These results indicate that substituents at this position
are important to produce inhibitors having good biologi-
cal potency, particularly in vivo activity.
a
Reagents: (a) BrCH2COOEt, K2CO3/Me2CO; (b) H2O2-Na2CO3/
H2O-Me2CO; (c) (1) cinchonidine/EtOH, (2) HCl; (d) H2, Pd-C/
EtOH; (e) 2,5-dimethoxy-THF/AcOH; (f) CCl3COCl/AcOEt; (g)
4-Br-2-F-Ph-CH2NH2, Et3N/dry DMF.
Thus, compounds with a variety of substituents on
the benzyl group at the N-2 position of the pyrrolopyra-
zine were synthesized. These results are shown in
Table 4. The AR inhibitory activities were significantly
influenced by substituents on the benzyl group at the
N-2 position of the pyrrolopyrazine. For instance,
substitution by electron-withdrawing groups such as
halogens produced more potent compounds than the
parent 23a , except for 23c, in both the AR inhibition
and in vivo activities. In particular, the monosubsti-
tuted 4-bromobenzyl derivative 23i and the disubsti-
tuted 3,4-dichloro, 4-chloro-2-fluoro- and 4-bromo-2-
fluorobenzyl derivatives 23r -t showed excellent in vivo
potency (ED50 ) 0.3-0.7 mg/kg/day). Of these com-
pounds, the best oral activity was observed for com-
pound 23t (SX-3030) having 4-bromo-2-fluorobenzyl
group (ED50 ) 0.3 mg/kg/day). On the other hand,
substitution by electron-donating groups such as meth-
oxy, methyl or amino group led to significant decrease
in the in vivo activity (23j,k ,n -p ).
F igu r e 1. X-ray structure of AS-3201 (43).
better than that of carboxylic acid 4 (24% inhibition at
100 mg/kg). Ester 2 was inactive in the AR inhibition
at 10-6 M, while its in vivo activity (38% inhibition at

4122 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Ta ble 1. Chemical and Biological Data of Pyrrole Derivatives
Negoro et al.
aldose reductase inhibition
in vitro
in vivo
porcine lens AR
% inhibitionb
100 mg/kg
compd
mp, °C
recryst solvent
formula
IC50 (µM)a
2
3
4
96-98
153-154
191-193
AcOEt-hexane
AcOEt-hexane
CH3CN
C9H12N2O3
C8H10N2O2
C8H10N2O3
>10
7.5
38.0 ( 5.8
55.7 ( 3.0
23.7 ( 4.2
5.0
a
b
The concentration of test compounds required for 50% inhibition of AR. Percent inhibition of sorbitol accumulation in the sciatic
nerve of streptozotocin-induced diabetic rat. Test compounds were orally given at the single dose indicated. Values are mean ( SEM;
mean of 4-6 rats.
Ta ble 2. Chemical and Biological Data of (2-Substituted-1,2,3,4-tetrahydro-1,3-dioxopyrrolo[1,2-a]pyrazin-4-yl)acetic Acids and
-propionic Acids 17 and 18
aldose reductase inhibition
in vitro
in vivo
porcine lens AR
% inhibitionb
100 mg/kg
compd
n
R1
mp, °C
IC50 (µM)a
17a
17b
17c
17d
17e
18a
18b
18c
18d
18e
1
1
1
1
1
2
2
2
2
2
H
CH3
CH(CH3)2
3,4-Cl2-C6H3-CH2
2-F-4-Br-C6H3-CH2
H
CH3
CH(CH3)2
232-233
209-211
141-143
213-215
179-181
143-145
140-142
120-122
146-148
143-145
>10
12.5 ( 5.4
12.8 ( 3.6
12.9 ( 14.0
NS
NS
NS
NS
NS
17.2 ( 8.2
NS
>10
>10
0.131
0.165
>10
>10
>10
3,4-Cl2-C6H3-CH2
2-F-4-Br-C6H3-CH2
0.132
0.803
a
b
See footnote a in Table 1. See footnote b in Table 1. NS, no significant inhibition.
Ta ble 3. Chemical and Biological Data of
2-Substituted-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine-4-spiro-3′-pyrrolidine-1,2′,3,5′-tetrones 22 and 23
aldose reductase inhibition
in vitro
in vivo
porcine lens AR
% inhibitionb
30 mg/kg/day
compd
R1
method
mp, °C
IC50 (µM)a
22a
22b
22c
23a
22d
H
CH3
C6H5
C6H5-CH2-
C6H3-(CH2)2-
A
A
A
A
A
257-259
285-287
231-238
157-159
155-157
0.63
0.28
0.20
0.099
0.27
33.2 ( 9.3
51.2 ( 3.5
27.2 ( 10.6
69.1 ( 5.1
15.3 ( 10.3
a
b
See footnote a in Table 1. Percent inhibition of sorbitol accumulation in the sciatic nerve of streptozotocin-induced diabetic rat. Test
compounds were orally given once a day for 5 days at the indicated doses. Values are mean ( SEM; mean of 4-6 rats.
Compounds 31, which have halogens or an acetyl
group on the fused pyrrole ring of the pyrrolopyrazine,
were synthesized. Results for these compounds are
shown in Table 5. Compared to 23t, halogens or acetyl
substitution on the pyrrole ring retained the in vitro
activity but yielded a loss of the in vivo activity at a
dose of 1 mg/kg/day (31a -f).
AR had been reported to have a high enantioselec-
tivity for spirohydantoins18 and spirosuccinimides.7 To
examine enantiomeric preference for AR, the enanti-
omers of 23t were prepared and evaluated for the AR
inhibitory activity and in vivo potency. From the results
in Table 6, the inhibitory activity was found to reside
in essentially one isomer, the (-)-enantiomer 43 (AS-
3201), which was 10 and 500 times more potent in vitro
and in vivo, respectively, than the corresponding (+)-
enantiomer 44 (SX-3202). Moreover, (-)-enantiomer 43
(AS-3201) was about 2 times more potent than the
racemate 23t (SX-3030) in both the AR inhibitory
activity and in vivo activity.
AS-3201 was compared with tolrestat and ponalrestat
to evaluate its pharmacokinetics in AR inhibitory activ-

Novel, Highly Potent ARIs
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 4123
Ta ble 4. Chemical and Biological Data of Compounds 23 with Substituents on the Benzyl Group
aldose reductase inhibition
in vivo
% inhibitionb
in vitro
c
porcine lens AR
ED50
compd
R
method
mp, °C
IC50 (µM)a
30 mg/kg/day
mg/kg/day
23a
23b
23c
23d
23e
23f
23g
23h
23i
23j
23k
23l
23m
23n
23o
23p
23q
23r
23s
H
2-F
4-F
A
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
157-159
172-175
189-190
156-158
161-163
195-198
226-229
152-153
165-167
154-158
184-185
182-183.5
213-215
207-208
172-174
191-193
221.5-223
221-223
196-199
192-193
0.099
0.061
0.120
0.037
0.035
0.038
0.052
0.037
0.047
0.052
0.046
0.080
0.110
0.140
0.350
0.110
0.072
0.023
0.041
0.045
69.1 ( 5.1
98.0 ( 0.8
58.8 ( 3.3
97.0 ( 2.1
99.4 ( 1.3
104.6 ( 1.2
82.6 ( 2.7
102.1 ( 0.5
108.1 ( 0.4
65.8 ( 4.4
46.7 ( 7.6
88.4 ( 2.7
102.2 ( 1.6
33.4 ( 3.3
NS
1.3
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
4-CH3
4-OCH3
4-CF3
4-NO2
4-NH2
2,4-(OCH3)2
3,4-(OCH3)2
2,4-F2
5.8
3.8
1.5
0.48
10.8
NS
98.0 ( 1.0
111.8 ( 1.1
109.5 ( 0.9
114.6 ( 0.2
5.4
3,4-Cl2
2-F,4-Cl
2-F,4-Br
A
B
A, B
0.60
0.74
0.30
23t (SX-3030)
a
b
See footnote a in Table 1. See footnote b in Table 3. c The dose of test compounds required for 50% inhibition of sorbitol accumulation
in the sciatic nerve of streptozotocin-induced diabetic rat. NS, no significant inhibition.
Ta ble 5. Chemical and Biological Data of Compounds 31 with Substituents on the Pyrrole Ring
aldose reductase inhibition
in vitro in vivo
porcine lens AR
% inhibitionb
1 mg/kg/day
compd
R6
R7
route
mp, °C
IC50 (µM)a
31a
31b
31c
31d
31e
31f
H
H
H
Cl
Br
Br
H
Cl
Br
CH3CO
H
H
A
A
A
B
B
B
227-229
112-114
289-291
207-209
212-214
253-254
192-193
0.050
0.10
0.21
0.035
0.040
0.052
0.045
23.0 ( 7.3
28.8 ( 9.5
26.6 ( 7.6
20.2 ( 6.3
NS
NS
104.7 ( 2.2
Br
H
23t (SX-3030)
a
b
See footnote a in Table 1. See footnote b in Table 3. NS, no significant inhibition.
Ta ble 6. Biological Data of Tolrestat, Ponalrestat, Racemate
23t, and Its Enantiomers 43 and 44
ity and in vivo. AS-3201 inhibited porcine lens AR with
an IC50 value of 15 nM. Its inhibitory potency was
comparable to those of tolrestat (IC50 ) 15 nM) and
ponalrestat (IC50 ) 21 nM). When AS-3201, ponal-
restat, or tolrestat was given orally to the diabetic rats
once a day for 5 days, AS-3201 inhibited sorbitol
accumulation in the sciatic nerve of the diabetic rats
with an ED50 value of 0.18 mg/kg/day. These results
suggest that AS-3201 has higher membrane perme-
ability to inhibit potently intracellular AR in target
tissues in vivo in comparison with these AR inhibitors.
aldose reductase inhibition
in vitro
in vivo
b
porcine lens AR
ED50
compd
enantiomer
IC50 (µM)a
mg/kg/day
23t (SX-3030)
44 (SX-3202)
43 (AS-3201)
ponalrestat
tolrestat
racemate
(S)-(+)
(R)-(-)
0.029
0.19
0.015
0.021
0.015
0.30
>100
0.18
11.0
26.7
a
b
See footnote a in Table 1. See footnote c in Table 4.

4124 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Negoro et al.
ester (2; 1.2 g, 6.1 mmol) in dioxane (15 mL) was added 5%
aqueous NaHCO3 (15.4 mL), and the mixture was refluxed for
1 h. After cooling to room temperature, the reaction mixture
was poured into ice-water containing concentrated HCl (1 mL)
and extracted with AcOEt. The extracts were washed with
brine and dried over anhydrous MgSO4. Evaporation and
recrystallization from CH3CN gave a white solid (0.8 g,
In conclusion, we have reported in this article a novel
class of spirosuccinimide-fused tetrahydropyrrolo[1,2-
a]pyrazine-1,3-dione derivatives having a high order of
intrinsic biological activity to inhibit AR. Of those
compounds, AS-3201 potently inhibited sorbitol ac-
cumulation in the sciatic nerve of the diabetic rats at
low oral doses, and its activity was more potent than
that of tolrestat or ponalrestat. AS-3201, which has
highly potent in vivo activity, is being evaluated for its
clinical efficacy in the prevention of diabetic complica-
tions.
1
72.7%): mp 191-3 °C; H NMR (DMSO-d6) δ 3.24 (3H, br s),
4.17 (2H, br s), 6.14 (1H, m), 6.60 (1H, br s), 6.90 (1H, m),
11.45 (1H, br s), 12.72 (1H, br s); IR (KBr, cm-1) 3270 (NH),
1740 (CO), 1707 (CO); MS m/z 183 (MH+). Anal. (C8H10N2O3)
C, H, N.
N-Meth yl-N-(2-p yr r olylca r bon yl)glycin e (4, fr om 3).
To a stirred solution of 1,2,3,4-tetrahydro-2-methylpyrrolo[1,2-
a]pyrazine-1,4-dione (3; 1.0 g, 6.1 mmol) in dioxane (20 mL)
was added 5% aqueous NaHCO3 (15.4 mL), and the mixture
was stirred at room temperature for 6.5 h. The reaction
mixture was poured into ice-water containing concentrated
HCl (1 mL), and the precipitated white solid was collected by
filtration. The solid was recrystallized from CH3CN to give a
white solid (0.8 g, 72.7%): mp 191-3 °C; 1H NMR, IR, and
MS were identical to the compound 4 described above.
2-(P yr r ol-1-yl)su ccin ic Acid Dim eth yl Ester (9, n ) 1,
R ) Me) (Sch em e 2). A mixture of dl-aspartic acid dimethyl
ester hydrochloride (6; n ) 1, R ) Me; 19.2 g, 97.2 mmol), 2,5-
dimethoxytetrahydrofuran (12.9 g, 97.6 mmol), and triethyl-
amine (10.3 g, 101.8 mmol) in acetic acid (60 mL) was refluxed
for 5 h. The reaction mixture was evaporated in vacuo. The
residue was dissolved in AcOEt and washed with 5% aqueous
NaHCO3, water, and brine. The organic layer was dried over
anhydrous MgSO4 and evaporated in vacuo. The resulting
crude product was purified by column chromatography on
silica gel with AcOEt-hexane (1/4) to give a viscous oil (16.6
g, 81.0%): 1H NMR (DMSO-d6) δ 3.59 (3H, s), 3.64 (3H, s),
5.26 (1H, t, J ) 7.3 Hz), 6.00 (2H, t, J ) 2.1 Hz), 6.82 (2H, t,
J ) 2.1 Hz); IR (NaCl, cm-1) 1735 (CO), 1725 (CO); MS m/z
212 (MH+).
Exp er im en ta l Section
Melting points (mp) were determined on a Yanagimoto
micromelting apparatus and are uncorrected. Proton nuclear
magnetic resonance (1H NMR) spectra were obtained on a
Varian Gemini-200 spectrometer with tetramethylsilane as an
internal standard. Chemical shifts are reported in δ value
from internal tetramethylsilane. Splitting patterns are des-
ignated as follows: s, singlet; d, doublet; dd, double doublet;
t, triplet; q, quartet; br s, broad singlet; m, multiplet. Coupling
constants are reported in hertz (Hz). Infrared (IR) spectra
were recorded on a Shimazu FTIR-8200PC spectrophotometer.
Mass spectra (MS) were obtained with a Hitachi M-1000 LC
API mass spectrometer. Elemental analyses were carried out
on a Perkin-Elmer 2400 elemental analyzer, and results
obtained for specified elements were within (0.4% of the
theoretical values. Optical rotations were determined on a
J asco DIP-370 model digital polarimeter (J apan Spectroscopic
Co., Ltd.). Chromatographic separations were performed with
silica gel 60 (Merck art. 7734; 70-230 mesh). Visualization
was accomplished with UV light. Unless otherwise noted, all
commercially available materials were used without further
purification.
Refer en ce Com p ou n d . Tolrestat was extracted from
commercially available Alredase capsules (Wyeth Laboratories
Co., Ltd.). Ponalrestat was prepared according to known
procedures.9
2-(2-(Tr ich lor oa cet yl)p yr r ol-1-yl)su ccin ic Acid Di-
m eth yl Ester (12, n ) 1, R ) Me). A mixture of 2-(pyrrol-
1-yl)succinic acid dimethyl ester (9, n ) 1, R ) Me; 14.6 g,
69.1 mmol), trichloroacetyl chloride (25.1 g, 138.2 mmol), and
CHCl3 (70 mL) was refluxed for 15 h. The reaction mixture
was evaporated in vacuo. The residue was dissolved in AcOEt
and washed with 10% aqueous NaHCO3, water, and brine. The
organic layer was dried over anhydrous MgSO4 and evaporated
in vacuo. The residue was recrystallized from AcOEt-hexane
N-Meth yl-N-(2-p yr r olylca r bon yl)glycin e Meth yl Ester
(2) a n d 1,2,3,4-Tetr a h yd r o-2-m eth ylp yr r olo[1,2-a ]p yr a -
zin e-1,4-d ion e (3) (Sch em e 1). To a suspension of 2-pyr-
rolecarboxylic acid (1; 2.6 g, 23.4 mmol), sarcosine methyl ester
hydrochloride (3.3 g, 23.6 mmol), and triethylamine (5.8 g, 57.3
mmol) in dry CH2Cl2 (50 mL) was added 1-ethyl-3-(3-(di-
methylamino)propyl)carbodiimide hydrochloride (WSC; 5.4 g,
28.2 mmol), and the mixture was stirred at room temperature
for 2 h. The reaction mixture was poured into water, acidified
with HCl (10%), and then extracted with AcOEt. The extracts
were washed with brine, dried over anhydrous MgSO4, and
evaporated in vacuo. The resulting crude product was purified
by column chromatography on silica gel. Elution with AcOEt-
hexane (1/2) gave a less polar compound 2 (2.1 g, 45.7%), and
elution with AcOEt-hexane (1/1) gave a more polar compound
1
to give a white solid (21.6 g, 87.8%): mp 79-81 °C; H NMR
(DMSO-d6) δ 3.57 (3H, s), 3.64 (3H, s), 6.01 (1H, br s), 6.39
(1H, dd, J ) 4.4, 2.6 Hz), 7.56 (1H, dd, J ) 4.4, 1.5 Hz), 7.61
(1H, t, J ) 2.1 Hz); IR (KBr, cm-1) 1745 (CO), 1730 (CO), 1650
(CO); MS m/z 356 (MH+).
2-(1,2,3,4-Tetr a h yd r o-2-m eth yl-1,3-d ioxop yr r olo[1,2-a ]-
p yr a zin -4-yl)a cetic Acid Meth yl Ester (15b, n ) 1, RdR1
) Me). To an ice-cooled solution of methylamine hydrochlo-
ride (0.38 g, 5.9 mmol) and triethylamine (0.57 g, 5.6 mmol)
in dry DMF (8 mL) was added 2-(2-(trichloroacetyl)pyrrol-1-
yl)succinic acid dimethyl ester (12, n ) 1, R ) Me; 1.0 g, 2.8
mmol), and the mixture was stirred at room temperature for
15 h. The reaction mixture was poured into ice-water
containing concentrated HCl (1 mL) and extracted with AcOEt.
The extracts were washed with brine and dried over anhydrous
MgSO4. Evaporation and purification by column chromatog-
raphy on silica gel with hexane-AcOEt (5/1) gave a white solid
(0.41 g, 62.1%): mp 61-62 °C; 1H NMR (DMSO-d6) δ 3.14 (3H,
s), 3.42 (2H, m), 3.49 (3H, s), 5.36 (1H, s), 6.34 (1H, s), 6.90
(1H, t, J ) 1.9), 7.36 (1H, s); IR (KBr, cm-1) 1740 (CO), 1710
(CO), 1660 (CO); MS m/z 237 (MH+).
1
3 (1.0 g, 26.3%). Less polar compound 2: mp 96-98 °C; H
NMR (DMSO-d6) δ 3.25 (3H, br s), 3.67 (3H, s), 4.26 (2H, br
s), 6.14 (1H, m), 6.60 (1H, br s), 6.91 (1H, m), 11.47 (1H, br s);
IR (KBr, cm-1) 3130 (NH), 1735 (CO), 1595 (CO); MS m/z 197
(MH+). Anal. (C9H12N2O3) C, H, N. More polar compound 3:
mp 153-154 °C; 1H NMR (DMSO-d6) δ 2.95 (3H, s), 4.51 (2H,
s), 6.57 (1H, t, J ) 3.3 Hz), 6.95 (1H, dd, J ) 3.4, 1.6 Hz), 7.56
(1H, dd, J ) 3.2, 1.6 Hz); IR (KBr, cm-1) 1720 (CO), 1630 (CO);
MS m/z 165 (MH+). Anal. (C8H8N2O2) C, H, N.
1,2,3,4-Tetr a h yd r o-2-m eth ylpyr r olo[1,2-a ]p yr a zin e-1,4-
d ion e (3, fr om 2). A mixture of N-methyl-N-(2-pyrrolylcar-
bonyl)glycine methyl ester (2; 0.5 g, 2.5 mmol), triethylamine
(1.3 g, 12.8 mmol), and toluene (5 mL) was refluxed for 5 h.
The reaction mixture was evaporated in vacuo. The crude
product was purified by recrystallization from CH3CN to give
2-(1,2,3,4-Tetr a h yd r o-2-m eth yl-1,3-d ioxop yr r olo[1,2-a ]-
p yr a zin -4-yl)a cetic Acid (17b, n ) 1, R1 ) Me). To a stirred
solution of 2-(1,2,3,4-tetrahydro-2-methyl-1,3-dioxopyrrolo[1,2-
1
a white solid (0.34 g, 81.0%): mp 153-154 °C; H NMR, IR,
and MS were identical to the more polar compound 3 described
above.
a]pyrazin-4-yl)acetic acid methyl ester (15b, n ) 1, RdR1
)
Me; 0.7 g, 3.0 mmol) in dioxane (16 mL) was added 20% HCl
(12.7 mL), and the mixture was refluxed for 4 h. The volatiles
were removed in vacuo, water was added and the mixture was
N-Meth yl-N-(2-p yr r olylca r bon yl)glycin e (4, fr om 2).
To a solution of N-methyl-N-(2-pyrrolylcarbonyl)glycine methyl

Novel, Highly Potent ARIs
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 4125
extracted with AcOEt. The organic layer was washed with
brine, dried over anhydrous MgSO4, and evaporated in vacuo.
The resulting crude product was purified by recrystallization
from CH3CN to give a white solid (0.46 g, 69.9%): mp 209-
2-[2-(4-Br om o-2-flu or ob e n zyl)-4-(e t h oxyca r b on yl)-
1,2,3,4-t et r a h yd r o-1,3-d ioxop yr r olo[1,2-a ]p yr a zin -4-yl]-
a cetic Acid (20t, R1 ) 4-Br -2-F -C6H3-CH2). To a stirred
solution of 2-[2-(4-bromo-2-fluorobenzyl)-4-(ethoxycarbonyl)-
1,2,3,4-tetrahydro-1,3-dioxopyrrolo[1,2-a]pyrazin-4-yl]acetic acid
tert-butyl ester (19t, R1 ) 4-Br-2-F-C6H3-CH2; 28.7 g, 54.8
mmol) in CH2Cl2 (100 mL) was added trifluoroacetic acid (TFA;
125 g, 1.1 mol), and the mixture was refluxed for 3 h. The
reaction mixture was evaporated in vacuo. The resulting crude
product was recrystallized from AcOEt-hexane to give a white
1
211 °C; H NMR (DMSO-d6) δ 3.16 (3H, s), 3.35 (2H, dd, J )
24, 18 Hz), 5.31 (1H, t, J ) 4.0 Hz), 6.36 (1H, dd, J ) 4.0, 2.6
Hz), 6.91 (1H, dd, J ) 4.0, 2.0 Hz), 7.36 (1H, dd, J ) 2.4, 2.0
Hz), 12.6 (1H, br s); IR (KBr, cm-1) 3110 (OH), 1725 (CO), 1705
(CO), 1635 (CO); MS m/z 223 (MH+). Anal. (C10H10N2O4) C,
H, N.
1
solid (21.4 g, 83.6%): mp 142-144 °C; H NMR (DMSO-d6) δ
3-(1,2,3,4-Tetr a h yd r o-1,3-d ioxo-2-isop r op ylp yr r olo[1,2-
a ]p yr a zin -4-yl)p r op ion ic Acid (18c, n ) 2, R1 ) iP r ). The
title compound was prepared from 3-(1,2,3,4-tetrahydro-1,3-
dioxo-2-isopropylpyrrolo[1,2-a]pyrazin-4-yl)propionic acid meth-
yl ester (15c, n ) 2, R1 ) iPr; 1.5 g, 5.4 mmol) by the same
manner as the compound 17b (n ) 1, R1 ) Me) described
above. The resulting crude product was purified by recrys-
tallization from AcOEt-hexane to give a white solid (0.8 g,
56.3%): mp 120-122 °C; 1H NMR (DMSO-d6) δ 1.38 (6H, dd,
J ) 6.8, 2.6 Hz), 1.96 (2H, m), 2.32 (2H, m), 4.99 (1H, dd, J )
14, 7.2 Hz), 5.22 (1H, t, J ) 5.0 Hz), 6.40 (1H, dd, J ) 8.0, 3.0
Hz), 6.93 (1H, dd, J ) 4.0, 1.6 Hz), 7.3 (1H, dd, J ) 2.6, 2.0
Hz), 12.2 (1H, br s); IR (KBr, cm-1) 1735 (CO), 1695 (CO), 1625
(CO); MS m/z 265 (MH+). Anal. (C13H16N2O4) C, H, N.
0.98 (3H, t, J ) 7.1 Hz), 3.72 (1H, d, J ) 18.0 Hz), 3.86 (1H,
d, J ) 18.0 Hz), 4.09 (2H, m), 5.05 (2H, m), 6.46 (1H, dd, J )
4.0, 2.8 Hz), 7.09 (1H, dd, J ) 4.0, 1.5 Hz), 7.17 (1H, t, J ) 8.1
Hz), 7.33 (1H, dd, J ) 8.2, 2.2 Hz), 7.55 (1H, dd, J ) 8.4, 1.9
Hz), 7.57 (1H, dd, J ) 2.8, 1.5 Hz), 12.97 (1H, s); IR (KBr,
cm-1) 1745 (CO), 1700 (CO), 1665 (CO); MS m/z 469 (MH+).
Anal. (C19H16BrFN2O6) C, H, N.
2-[2-(4-Br om o-2-flu or ob e n zyl)-4-(e t h oxyca r b on yl)-
1,2,3,4-t et r a h yd r o-1,3-d ioxop yr r olo[1,2-a ]p yr a zin -4-yl]-
a ceta m id e (21t, R1 ) 4-Br -2-F -C6H3-CH2). To a stirred
solution of 2-[2-(4-bromo-2-fluorobenzyl)-4-(ethoxycarbonyl)-
1,2,3,4-tetrahydro-1,3-dioxopyrrolo[1,2-a]pyrazin-4-yl]acetic acid
(20t, R1 ) 4-Br-2-F-C6H3-CH2; 10.0 g, 21.4 mmol) was added
dropwise SOCl2 (6.2 g, 52.1 mmol), and the mixture was
refluxed for 2 h. The volatiles were evaporated in vacuo, and
the residue was dissolved in AcOEt (30 mL). This solution
was added dropwise to a mixture of 25% aqueous NH3 solution
(29 mL) and AcOEt (30 mL) at 0 °C, and the mixture was
stirred for 1 h at 0 °C. The organic layer was separated,
washed with brine, dried over anhydrous MgSO4, and evapo-
rated in vacuo. The resulting crude product was recrystallized
from CH3CN to give a white solid (8.0 g, 80.0%): mp 190-
2-(2-(Tr ich lor oa cetyl)p yr r ol-1-yl)m a lon ic Acid Dieth yl
Ester (11, n ) 0, R ) Et) (Sch em e 2). The title compound
was prepared from 2-(pyrrol-1-yl)malonic acid diethyl ester10
(8, n ) 0, R ) Et; 80 g, 0.36 mol) by the same manner as
compound 12 (n ) 1, R ) Me) described in Scheme 2. The
resulting crude product was purified by recrystallization from
AcOEt-hexane to give a white solid (116 g, 88.1%): mp 67-
1
69 °C; H NMR (CDCl3) δ 1.32 (6H, t, J ) 7.2 Hz), 4.32 (4H,
m), 6.38 (1H, dd, J ) 4.4, 1.4 Hz), 6.77 (1H, s), 7.36 (1H, dd,
1
192 °C; H NMR (DMSO-d6) δ 0.98 (3H, t, J ) 7.2 Hz), 3.62
J ) 2.8, 1.6 Hz), 7.65 (1H, dd, J ) 4.4, 1.6 Hz); IR (KBr, cm-1
1765 (CO), 1645 (CO); MS m/z 370 (MH+).
)
(2H, m), 4.09 (2H, m), 5.03 (2H, m), 6.44 (1H, dd, J ) 4.4, 2.4
Hz), 7.04 (1H, dd, J ) 4.0, 1.0 Hz), 7.08 (1H, br s), 7.24 (1H,
t, J ) 8.0 Hz), 7.32 (1H, dd, J ) 10.4, 1.6 Hz), 7.36 (1H, dd, J
) 3.0, 2.0 Hz), 7.54 (1H, dd, J ) 10.0, 2.0 Hz), 7.58 (1H, d, J
) 2.0 Hz); IR (KBr, cm-1) 3410 (NH), 1745 (CO), 1705 (CO),
2-[2-(4-Br om o-2-flu or ob en zyl)-1,2,3,4-t et r a h yd r o-1,3-
d ioxop yr r olo[1,2-a ]p yr a zin -4-yl]ca r boxylic Acid Eth yl
Ester (14e, n ) 0, R ) Et, R1 ) 4-Br -2-F -C6H3-CH2). The
title compound was prepared from 2-(2-(trichloroacetyl)pyrrol-
1-yl)malonic acid diethyl ester (11, n ) 0, R ) Et; 40.0 g, 0.11
1680 (CO), 1650 (CO); MS m/z 466 (MH+). Anal. (C19H17
-
BrFN3O5) C, H, N.
mol) by the same manner as compound 15b (n ) 1, RdR1
)
2-(4-Br om o-2-flu or oben zyl)-1,2,3,4-tetr a h yd r op yr r olo-
[1,2-a ]pyr azin e-4-spir o-3′-pyr r olidin e-1,2′,3,5′-tetr on e (23t,
R1 ) 4-Br -2-F -C6H3-CH2, SX-3030). To a stirred solution of
2-[2-(4-bromo-2-fluorobenzyl)-4-(ethoxycarbonyl)-1,2,3,4-tet-
rahydro-1,3-dioxopyrrolo[1,2-a]pyrazin-4-yl]acetamide (21t, R1
) 4-Br-2-F-C6H3-CH2; 1.6 g, 3.6 mmol) in dry DMF (15 mL)
was added NaH (0.3 g, 7.9 mmol, 63% w/w dispersion in
mineral oil) portionwise at -20 °C (CH3CN-dry ice). After
stirring at -20 °C for 0.3 h, the reaction mixture was poured
into ice-water containing concentrated HCl (1 mL) and
extracted with AcOEt. The extracts were washed with brine,
dried over anhydrous MgSO4, and evaporated in vacuo.
Purification by column chromatography on silica gel with
AcOEt-hexane (1/3) and recrystallization from AcOEt-hexane
Me) described above. Purification by column chromatography
on silica gel with AcOEt-hexane (1/5) and recrystallization
from AcOEt-hexane gave a white solid (38.0 g, 86.0%): mp
1
114-115 °C; H NMR (DMSO-d6) δ 1.13 (3H, t, J ) 7.1 Hz),
4.19 (2H, q, J ) 7.1 Hz), 5.00 (2H, s), 6.19 (1H, s), 6.46 (1H,
dd, J ) 4.0, 2.7 Hz), 7.07 (1H, dd, J ) 3.9, 1.5 Hz), 7.15 (1H,
t, J ) 8.1 Hz), 7.30 (1H, dd, J ) 2.6, 1.6 Hz), 7.37 (1H, dd, J
) 8.2, 2.0 Hz), 7.55 (1H, dd, J ) 9.9, 2.0 Hz); IR (KBr, cm-1
)
1730 (CO), 1710 (CO), 1670 (CO); MS m/z 409 (MH+). Anal.
(C17H14BrFN2O4) C, H, N.
2-[2-(4-Br om o-2-flu or ob e n zyl)-4-(e t h oxyca r b on yl)-
1,2,3,4-t et r a h yd r o-1,3-d ioxop yr r olo[1,2-a ]p yr a zin -4-yl]-
a cetic Acid ter t-Bu tyl Ester (19t, R1 ) 4-Br -2-F -C6H3-CH2)
(Sch em e 3, Meth od A). To a cooled (0 °C) stirred solution
of 2-[2-(4-bromo-2-fluorobenzyl)-1,2,3,4-tetrahydro-1,3-dioxo-
pyrrolo[1,2-a]pyrazin-4-yl]carboxylic acid ethyl ester (14e, R1
) 4-Br-2-F-C6H3-CH2; 33.1 g, 80.9 mmol) in dry DMF (150 mL)
was slowly added NaH (3.5 g, 87.5 mmol, 60% w/w dispersion
in mineral oil), and the mixture was stirred at room temper-
ature for 0.5 h. tert-Butyl bromoacetate (17.1 g, 87.7 mmol)
was added dropwise, and the reaction mixture was stirred for
a further 2 h. The reaction mixture was poured into ice-water
and extracted with AcOEt. The extracts were washed with
brine and dried over anhydrous MgSO4. Evaporation and
purification by column chromatography on silica gel with
AcOEt-hexane (1/8) gave a semisolid (32.0 g, 75.7%): 1H NMR
(DMSO-d6) δ 0.96 (3H, t, J ) 7.0 Hz), 1.16 (9H, s), 3.70 (1H,
d, J ) 17.4 Hz), 3.82 (1H, d, J ) 17.5 Hz), 4.08 (2H, m), 5.07
(2H, m), 6.48 (1H, dd, J ) 4.0, 2.8 Hz), 7.12 (1H, dd, J ) 3.9,
1.5 Hz), 7.19 (1H, t, J ) 8.1 Hz), 7.37 (1H, dd, J ) 8.2, 1.7
Hz), 7.56 (1H, dd, J ) 7.7, 1.9 Hz), 7.58 (1H, d, J ) 2.0 Hz);
IR (NaCl, cm-1) 1760 (CO), 1720 (CO), 1680 (CO).
1
gave a white solid (0.6 g, 40.0%): mp 192-193 °C; H NMR
(DMSO-d6) δ 3.56 (2H, s), 5.00 (2H, m), 6.52 (1H, dd, J ) 4.0,
2.8 Hz), 7.12 (1H, dd, J ) 3.8, 1.4 Hz), 7.14 (1H, t, J ) 8.2
Hz), 7.36 (1H, dd, J ) 8.4, 2.0 Hz), 7.54 (1H, dd, J ) 9.8, 2.0
Hz), 7.72 (1H, dd, J ) 2.6, 1.6 Hz), 12.16 (1H, s); IR (KBr,
cm-1) 3230 (NH), 1790 (CO), 1730 (CO), 1705 (CO), 1660 (CO);
MS m/z 422 (MH+). Anal. (C17H11BrFN3O4) C, H, N.
2-[(Ben zyloxyca r bon yl)m eth yl]-2-(p yr r ol-1-yl)m a lon -
ic Acid Dieth yl Ester (24) (Sch em e 3, Meth od B).
A
stirred suspension of 2-(pyrrol-1-yl)malonic acid diethyl ester10
(8, n ) 0, R ) Et; 36.4 g, 0.16 mol), anhydrous K2CO3 (22.1 g,
0.16 mol), and benzyl bromoacetate (40.3 g, 0.18 mol) in
acetone (200 mL) was refluxed for 7 h. After cooling to room
temperature, the reaction mixture was filtered off and the
filtrate was evaporated in vacuo. The residue was dissolved
in AcOEt, washed with brine, and dried over anhydrous
MgSO4. Evaporation of the solvent and purification by column
chromatography on silica gel with AcOEt-hexane (1/10) gave
a viscous oil (53.0 g, 87.9%): 1H NMR (CDCl3) δ 1.24 (6H, t, J

4126 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Negoro et al.
) 7.1 Hz), 3.52 (2H, s), 4.25 (4H, q, J ) 6.4 Hz), 5.12 (2H, s),
6.18 (2H, t, J ) 2.2 Hz), 6.82 (2H, t, J ) 2.2 Hz), 7.34 (5H, m);
IR (NaCl, cm-1) 1740 (CO).
d, J ) 18.2 Hz), 4.17 (2H, m), 6.44 (1H, dd, J ) 4.5, 2.8 Hz),
7.55 (1H, dd, J ) 2.8, 1.5 Hz), 7.68 (1H, dd, J ) 4.4, 1.5 Hz),
12.42 (1H, s); IR (KBr, cm-1) 3150 (NH), 1790 (CO), 1720 (CO),
1660 (CO); MS m/z 381 (MH+). Anal. (C13H11Cl3N2O5‚1/2DMF)
C, H, N.
To a stirred solution of 2-(pyrrol-1-yl)malonic acid diethyl
ester (8, n ) 0, R ) Et; 53.0 g, 0.24 mol) in dry DMF (300 mL)
was added NaH (10.0 g, 0.26 mol, 63% w/w dispersion in
mineral oil) portionwise at 0 °C. The mixture was stirred at
0 °C for 1 h. Benzyl bromoacetate (70.0 g, 0.31 mol) was added
dropwise at 0 °C; the solution was warmed to room temper-
ature and stirred overnight. The reaction mixture was poured
into ice-water and extracted with AcOEt. The extracts were
washed with brine and dried over anhydrous MgSO4. Evapo-
ration of the solvent gave a viscous brown oil (80.0 g, 91.0%):
1H NMR, IR, and MS were identical to compound 24 described
above.
2-(4-Br om o-2-flu or oben zyl)-1,2,3,4-tetr a h yd r op yr r olo-
[1,2-a ]pyr azin e-4-spir o-3′-pyr r olidin e-1,2′,3,5′-tetr on e (23t,
R1 ) 4-Br -2-F -C6H3-CH2, SX-3030) (Sch em e 3, Meth od B).
To a stirred solution of 4-bromo-2-fluorobenzylamine hydro-
chloride (10.2 g, 42.4 mmol) and triethylamine (9.0 g, 88.9
mmol) in dry DMF (70 mL) was added 3-(ethoxycarbonyl)-2,5-
dioxo-3-(2-(trichloroacetyl)pyrrol-1-yl)pyrrolidine‚1/2DMF (28;
14.8 g, 35.4 mmol), and the mixture was stirred at room
temperature for 16 h. The reaction mixture was poured into
ice-water containing concentrated HCl (10 mL) and extracted
with AcOEt. The extracts were washed with brine and dried
over anhydrous MgSO4. Evaporation and purification by
recrystallization from AcOEt-hexane gave a white solid (6.5
g, 43.6%): mp 192-193 °C; 1H NMR, IR, and MS were
identical to compound 23t (R1 ) 4-Br-2-F-C6H3-CH2), prepared
according to method A shown in Scheme 3.
2-(Ca r boxym eth yl)-2-(p yr r ol-1-yl)m a lon ic Acid Dieth -
yl Ester (25). To a solution of 2-[(benzyloxycarbonyl)methyl]-
2-(pyrrol-1-yl)malonic acid diethyl ester (24; 53.0 g, 0.14 mol)
in dioxane (300 mL) was added a catalytic amount of palladium
carbon (5%, 2.5 g), and the mixture was hydrogenated at 40-
50 °C under atmospheric pressure. After cooling to room
temperature, the catalyst was filtered off and the filtrate was
evaporated to give a white solid (36.0 g, 89.6%): mp 93-94
3-(4-Br om o-2-(t r ich lor oa cet yl)p yr r ol-1-yl)-3-(et h oxy-
ca r bon yl)-2,5-d ioxop yr r olid in e (30b, R6 ) H, R7 ) Br )
(Sch em e 4, Rou te A). A solution of Br2 (6.9 g, 43.2 mmol)
in CHCl3 (18 mL) was added dropwise to a stirred solution of
3-(ethoxycarbonyl)-2,5-dioxo-3-(2-(trichloroacetyl)pyrrol-1-yl)-
pyrrolidine‚1/2DMF (28; 6.0 g, 14.3 mmol) in CHCl3 (30 mL)
at 0 °C, and the mixture was stirred for a further 1 h at 0 °C.
The reaction mixture was poured into ice-water (70 mL) and
extracted with CHCl3. The extracts were washed with brine,
dried over anhydrous MgSO4, and evaporated in vacuo.
Purification of the resulting crude product by column chro-
matography on silica gel with CHCl3-MeOH (2000/1) and
recrystallization from AcOEt-hexane gave a white solid (2.4
1
°C; H NMR (CDCl3) δ 1.28 (6H, t, J ) 7.1 Hz), 3.52 (2H, s),
4.31 (4H, m), 6.20 (2H, t, J ) 2.3 Hz), 6.84 (2H, t, J ) 2.2 Hz);
IR (KBr, cm-1) 1740 (CO), 1720 (CO); MS m/z 284 (MH+). Anal.
(C13H17NO6) C, H, N.
2-(Ca r ba m oylm eth yl)-2-(p yr r ol-1-yl)m a lon ic Acid Di-
eth yl Ester (26). To a solution of 2-(carboxymethyl)-2-(pyrrol-
1-yl)malonic acid diethyl ester (25; 48.7 g, 0.17 mol) and
N-hydroxysuccinimide (21.8 g, 0.19 mol) in dry CH2Cl2 (200
mL) was added WSC (9.5 g, 0.21 mol), and the mixture was
stirred at room temperature for 1 h. The mixture was added
dropwise to a solution of 7.8% NH3 in CH3CN (130 mL) at 0
°C, and the solution was stirred for a further 1 h at 0 °C. The
reaction mixture was poured into ice-water containing con-
centrated HCl (30 mL) and extracted with AcOEt. The organic
extract was washed with brine and dried over anhydrous
MgSO4. Evaporation and purification by recrystallization from
AcOEt-hexane gave a white solid (46.8 g, 96.5%): mp 94-95
1
g, 36.4%): mp 196-198 °C; H NMR (CDCl3) δ 1.25 (3H, J )
6.3 Hz), 1.62 (1H, s), 2.86 (1H, d, J ) 19.2 Hz), 4.24 (1H, d, J
) 19.2 Hz), 4.31 (2H, m), 7.39 (1H, d, J ) 1.4 Hz), 7.70 (1H, d,
J ) 1.6 Hz), 8.48 (1H, s); IR (KBr, cm-1) 3180 (NH), 1805 (CO),
1740 (CO), 1710 (CO), 1665 (CO); MS m/z 461 (MH+). Anal.
(C13H10BrCl3N2O5) C, H, N.
1
°C; H NMR (CDCl3) δ 1.28 (6H, t, J ) 7.0 Hz), 3.37 (2H, s),
3-(4-Ch lor o-2-(t r ich lor oa cet yl)p yr r ol-1-yl)-3-(et h oxy-
ca r bon yl)-2,5-d ioxop yr r olid in e (30a , R6 ) H, R7 ) Cl). A
solution of SO2Cl2 (4.8 g, 35.6 mmol) in CHCl3 (5 mL) was
added dropwise to a stirred solution of 3-(ethoxycarbonyl)-2,5-
dioxo-3-(2-(trichloroacetyl)pyrrol-1-yl)pyrrolidine‚1/2DMF (28;
6.0 g, 14.3 mmol) in CHCl3 (50 mL) at 0 °C, and the mixture
was stirred for a further 0.5 h at 0 °C. The reaction mixture
was poured into ice-water (50 mL) and extracted with CHCl3.
The extracts were washed with brine, dried over anhydrous
MgSO4, and evaporated in vacuo. Purification of the crude
oil by column chromatography on silica gel with AcOEt-
hexane (1/2) gave a white solid (2.5 g, 41.7%): mp 182-185
4.31 (4H, q, J ) 7.0 Hz), 5.45 (1H, br s), 5.58 (1H, br s), 6.19
(4H, t, J ) 2.2 Hz), 6.86 (4H, t, J ) 2.2 Hz); IR (KBr, cm-1
)
3400 (NH), 3210 (NH), 1730 (CO), 1720 (CO), 1680 (CO), 1665
(CO); MS m/z 283 (MH+). Anal. (C13H18N2O5) C, H, N.
3-(E t h oxyca r b on yl)-2,5-d ioxo-3-(p yr r ol-1-yl)p yr r oli-
d in e (27). A stirred suspension of 2-(carbamoylmethyl)-2-
(pyrrol-1-yl)malonic acid diethyl ester (26; 1.0 g, 3.5 mmol) and
a catalytic amount of anhydrous K2CO3 (49 mg, 0.35 mmol) in
acetone (15 mL) was refluxed for 2 h. After cooling to room
temperature, the reaction mixture was filtered off and the
filtrate was evaporated in vacuo. The residue was dissolved
in AcOEt and washed with cold 5% HCl, water, and brine. The
organic layer was dried over anhydrous MgSO4. Evaporation
and purification by column chromatography on silica gel with
AcOEt-hexane (1/2) gave a viscous oil (0.8 g, 95.2%): 1H NMR
(CDCl3) δ 1.27 (3H, t, J ) 3.5 Hz), 3.38 (1H, d, J ) 17.9 Hz),
3.61 (1H, d, J ) 13.0 Hz), 4.30 (4H, q, J ) 7.0 Hz), 6.28 (2H,
t, J ) 2.2 Hz), 6.95 (2H, t, J ) 2.2 Hz), 8.57 (1H, s); IR (NaCl,
cm-1) 3250 (NH), 1790 (CO), 1755 (CO), 1725 (CO). Anal.
(C11H12N2O4) C, H, N.
3-(E t h oxyca r b on yl)-2,5-d ioxo-3-(2-(t r ich lor oa cet yl)-
p yr r ol-1-yl)p yr r olid in e‚1/2DMF (28). Trichloroacetyl chlo-
ride (42.0 g, 0.23 mol) was added to a solution of 3-(ethoxy-
carbonyl)-2,5-dioxo-3-(pyrrol-1-yl)pyrrolidine (27; 18.0 g, 76.2
mmol) in CHCl3 (40 mL), and the mixture was refluxed for 16
h. The reaction mixture was evaporated in vacuo. The
resulting brown gum was dissolved in AcOEt and washed with
10% aqueous NaHCO3, water, and brine. The organic layer
was dried over anhydrous MgSO4 and evaporated in vacuo.
The resulting crude product was recrystallized from AcOEt-
hexane containing a theoretical amount of DMF to give a white
solid (28.5 g, 89.3%): mp 94-97 °C; 1H NMR (DMSO-d6) δ
1.11 (3H, t, J ) 7.1 Hz), 3.08 (1H, d, J ) 18.2 Hz), 3.70 (1H,
1
°C; H NMR (CDCl3) δ 1.25 (3H, t, J ) 5.5 Hz), 1.68 (1H, s),
2.86 (1H, d, J ) 19.0 Hz), 4.22 (1H, d, J ) 19.0 Hz), 4.31 (1H,
m), 7.36 (1H, d, J ) 1.6 Hz), 7.62 (1H, d, J ) 2.0 Hz), 8.65
(1H, s); IR (KBr, cm-1) 3180 (NH), 1800 (CO), 1750 (CO), 1710
(CO), 1665 (CO); MS m/z 417 (MH+). Anal. (C13H10Cl4N2O5)
C, H, N.
3-(4-Acet yl-2-(t r ich lor oa cet yl)p yr r ol-1-yl)-3-(et h oxy-
ca r bon yl)-2,5-d ioxop yr r olid in e (30c, R6 ) H, R7 ) Ac). To
a stirred suspension of anhydrous aluminum chloride (4.8 g,
36.0 mmol) and 3-(ethoxycarbonyl)-2,5-dioxo-3-(2-(trichloro-
acetyl)pyrrol-1-yl)pyrrolidine‚1/2DMF(28; 3.0 g, 7.2 mmol) in
1,2-dichloroethane (50 mL) was added acetyl chloride (3.0 g,
38.2 mmol) dropwise at room temperature. After stirring for
2 h, the reaction mixture was poured into ice-water and
extracted with CHCl3. The extracts were washed with brine
and dried over anhydrous MgSO4. Evaporation and purifica-
tion by recrystallization from CH3CN gave a white solid (2.5
g, 83.3%): mp 243-246 °C dec; 1H NMR (DMSO-d6) δ 1.12
(3H, t, J ) 7.0 Hz), 2.43 (3H, s), 3.23 (1H, d, J ) 18.2 Hz),
3.68 (1H, d, J ) 18.2 Hz), 4.18 (2H, m), 7.91 (1H, d, J ) 1.8
Hz), 8.13 (1H, d, J ) 1.8 Hz), 12.48 (1H, s); IR (KBr, cm-1
)

Novel, Highly Potent ARIs
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 4127
3140 (NH), 1790 (CO), 1730 (CO), 1680 (CO), 1640 (CO); MS
occurred. The reaction mixture was left standing at room
temperature until cinchonidium salt precipitated. The pre-
cipitate was collected by filtration and rinsed with EtOH. The
crude cinchonidium salt was recrystallized twice from EtOH
to give the pure cinchonidium salt (35.2 g, 24.9%, >99.9%
de): mp 160-161 °C; [R]26D ) -73.5° (c ) 1.0, EtOH); IR (KBr,
cm-1) 1760 (CO), 1710 (CO), 1620 (CO).
m/z 423 (MH+). Anal. (C15H13Cl3N2O6) C, H, N.
7-Br om o-2-(4-b r om o-2-flu or ob en zyl)-1,2,3,4-t et r a h y-
d r op yr r olo[1,2-a ]p yr a zin e-4-sp ir o-3′-p yr r olid in e-1,2′,3,5′-
tetr on e (31b, R6 ) H, R7 ) Br ). The title compound was
prepared from 3-(4-bromo-2-(trichloroacetyl)pyrrol-1-yl)-3-
(ethoxycarbonyl)-2,5-dioxopyrrolidine (30b, R6 ) H, R7 ) Br;
2.1 g, 4.6 mmol) by the same manner as SX-3030 (23t, R1
)
The pure cinchonidium salt (4.3 g) was suspended in AcOEt
(80 mL), and 5% aqueous HCl (40 mL) was added. The organic
layer was separated, washed with brine, dried over anhydrous
MgSO4, and evaporated to give a white solid. Recrystallization
from AcOEt-hexane gave an optically pure (-)-enantiomer
4-Br-2-F-C6H3-CH2) described for method B in Scheme 3.
Purification of resulting crude product by column chromatog-
raphy on silica gel with CHCl3 and recrystallization from CH3-
CN gave a white solid (1.2 g, 52.2%): mp 112-114 °C; 1H NMR
(DMSO-d6) δ 3.55 (2H, s), 4.99 (2H, m), 7.14 (1H, t, J ) 8.2
Hz), 7.22 (1H, d, J ) 1.8 Hz), 7.36 (1H, dd, J ) 8.6, 2.2 Hz),
7.54 (1H, dd, J ) 10.0, 2.2 Hz), 7.96 (1H, d, J ) 1.8 Hz), 12.22
(1H, s); IR (KBr, cm-1) 3220 (NH), 3120 (NH), 1790 (CO), 1720
(CO), 1670 (CO). Anal. (C17H10Br2FN3O4) C, H, N.
(1.9 g, >99.5% ee): mp 117-118 °C; [R]27 ) -31.8° (c ) 1.0,
D
1
MeOH); H NMR, IR, and MS were identical to racemate 34.
Anal. (C15H16N2O6) C, H, N.
(S)-(+)-3-[(Ben zyloxyca r bon yl)a m in o]-3-(eth oxyca r bo-
n yl)-2,5-d ioxop yr r olid in e (36). The mother liquor of the
cinchonidium salt as above-mentioned was concentrated in
vacuo to give a dark gum (96 g), which was treated with 10%
HCl and AcOEt. The organic layer was separated, washed
with brine, dried over anhydrous MgSO4, and evaporated in
vacuo. The crude solid (43 g, 76.8% ee) was recrystallized
three times from AcOEt-hexane to give the pure (+)-enanti-
2-(4-Br om o-2-flu or ob en zyl)-6-ch lor o-1,2,3,4-t et r a h y-
d r op yr r olo[1,2-a ]p yr a zin e-4-sp ir o-3′-p yr r olid in e-1,2′,3,5′-
tetr on e (31d , R6 ) Cl, R7 ) H) (Sch em e 4, Rou te B). To a
stirred solution of SX-3030 (23t; 0.9 g, 2.1 mmol) in CHCl3
(350 mL) was added SO2Cl2 (0.68 g, 5.0 mmol), and the mixture
was stirred at room temperature for 4 h. The reaction mixture
was poured into water and extracted with AcOEt. The extracts
were washed with brine and dried over anhydrous MgSO4.
Evaporation and purification by recrystallization from AcOEt-
hexane gave a white solid (0.56 g, 57.7%): mp 212-215 °C;
1H NMR (DMSO-d6) δ 3.52 (2H, m), 5.01 (2H, m), 6.68 (1H, d,
J ) 4.2 Hz), 7.17 (1H, t, J ) 8.2 Hz), 7.23 (1H, d, J ) 4.4 Hz),
7.38 (1H, dd, J ) 8.0, 1.8 Hz), 7.55 (1H, dd, J ) 9.9, 1.8 Hz)
12.56 (1H, s); IR (KBr, cm-1) 3440 (NH), 3200 (NH), 1805 (CO),
omer (>99.4% ee, 21.7 g): mp 119-120 °C; [R]27 ) +29.8° (c
D
) 1.0, MeOH); 1H NMR, IR, and MS were identical to racemate
34.
The optical purity of the enantiomers 35 and 36 was
established by HPLC analysis. HPLC conditions: instrument,
Shimazu LC-6A system; column, CHIRAL PAK AS (250 × 4.6
mm i.d.; Daicel Chemical Industries, Ltd.); flow rate, 2 mL/
min; mobile phase, EtOH-hexane (10/90); detector, UV 215,
254 nm; column temperature, 40 °C.
1720 (CO), 1675 (CO); MS m/z 456 (MH+). Anal. (C17H10
-
BrClFN3O4) C, H, N.
(R)-(-)-3-Am in o-3-(eth oxyca r bon yl)-2,5-d ioxop yr r oli-
d in e (37). To a solution of (-)-3-[(benzyloxycarbonyl)amino]-
3-(ethoxycarbonyl)-2,5-dioxopyrrolidine (35; 25.0 g, 78.1 mmol)
in EtOH (140 mL) was added a catalytic amount of palladium
carbon (5%, 1.0 g), and the mixture was hydrogenated at 40-
50 °C under atmospheric pressure. After cooling to room
temperature, the catalyst was filtered off and the filtrate was
evaporated in vacuo. The crude product was recrystallized
2-[(Ben zyloxyca r b on yl)a m in o]-2-cya n osu ccin ic Acid
Dieth yl Ester (33) (Sch em e 5). A mixture of 2-[(benzyloxy-
carbonyl)amino]cyanoacetic acid ethyl ester14 (32; 21.0 g, 80.1
mmol), anhydrous potassium carbonate (12.2 g, 88.3 mmol),
ethyl bromoacetate (14.3 g, 85.6 mmol), and acetone (74 mL)
was refluxed for 7 h. After cooling to room temperature, the
reaction mixture was filtered off and the filtrate was evapo-
rated in vacuo. The residue was dissolved in AcOEt and
washed with 5% HCl, water, and brine. The organic layer was
dried over anhydrous MgSO4 and evaporated to give a viscous
oil (27.5 g, 98.6%): 1H NMR (CDCl3) δ 1.25 (3H, t, J ) 3.7
Hz), 1.34 (3H, t, J ) 6.9 Hz), 3.18 (1H, d, J ) 16.2 Hz), 3.56
(1H, br d, J ) 16.1 Hz), 4.16 (2H, q, J ) 7.1 Hz), 4.38 (2H, br
q, J ) 6.6 Hz), 6.31 (1H, s), 5.17 (2H, s), 7.37 (5H, s); IR (NaCl,
cm-1) 3340 (NH), 1740 (CO). The crude product was used
without further purification in the next step.
from iPrOH to give a white solid (13.9 g, 95.9%): mp 125-
1
126 °C; [R]27 ) -31.8° (c ) 1.0, MeOH); H NMR (CDCl3) δ
D
1.30 (3H, t, J ) 7.1 Hz), 1.64 (1H, br s), 2.20 (1H, br s), 2.76
(1H, d, J ) 18.1 Hz), 3.18 (1H, d, J ) 17.9 Hz), 4.29 (2H, q, J
) 7.1 Hz), 8.31 (1H, br s); IR (KBr, cm-1) 3360 (NH), 3300
(NH), 1755 (CO), 1705 (CO); MS m/z 187 (MH+). Anal.
(C7H10N2O4) C, H, N.
(R)-(-)-3-(E t h oxyca r b on yl)-2,5-d ioxo-3-(p yr r ol-1-yl)-
p yr r olid in e (39). A mixture of (-)-3-amino-3-(ethoxycarbo-
nyl)-2,5-dioxopyrrolidine (37; 12.0 g, 64.5 mmol), 2,5-dimethoxy-
tetrahydrofuran (13.0 g, 98.4 mmol), and acetic acid (40 mL)
was stirred at 70 °C for 1.5 h. The volatiles were removed in
vacuo. The residue was dissolved in AcOEt and washed with
5% aqueous NaHCO3, water, and brine. The organic layer was
dried over anhydrous MgSO4 and evaporated in vacuo. The
crude product was purified by column chromatography on
silica gel with AcOEt-hexane (1/3) to give a viscous oil (11.0
3-[(Ben zyloxyca r bon yl)a m in o]-3-(eth oxyca r bon yl)-2,5-
d ioxop yr r olid in e (34). To a stirred solution of sodium
carbonate (132.9 g, 1.25 mol) in water (1450 mL) was added
30% hydrogen peroxide (192 mL, 1.88 mol), and a solution of
2-[(benzyloxycarbonyl)amino]cyanosuccinic acid diethyl ester
(33; 328 g, 0.94 mol) in acetone (1640 mL) was added dropwise
at a temperature below 40 °C. The mixture was stirred for a
further 2 h in the range of 25-40 °C. The reaction mixture
was then cooled to below 10 °C in an ice bath, and a solution
of 7% HCl (1220 mL) was added. The solution was stirred at
room temperature until crystals precipitated. The crystals
were collected by filtration and washed well with water.
Recrystallization from AcOEt-hexane gave a white solid
(181.3 g, 60.0%): mp 105-106 °C; 1H NMR (CDCl3) δ 1.29 (3H,
t, J ) 7.0 Hz), 3.18 (2H, m), 4.32 (2H, q, J ) 6.9 Hz), 5.12
g, 72.4%): [R]26 ) -59.5° (c ) 1.0, MeOH); 1H NMR, IR, and
D
MS were identical to racemate 27. Anal. (C11H12N2O4) C, H,
N.
(R)-(-)-3-(E t h oxyca r b on yl)-2,5-d ioxo-3-(2-(t r ich lor o-
a cetyl)p yr r ol-1-yl)p yr r olid in e (41). The title compound
was prepared from (-)-3-(ethoxycarbonyl)-2,5-dioxo-3-(pyrrol-
1-yl)pyrrolidine (39; 7.0 g, 29.6 mmol) by the same manner as
compound 28 described for method B in Scheme 3. Purifica-
tion by column chromatography on silica gel with AcOEt-
hexane (1/3) gave a semisolid (10.4 g, 91.9%): [R]27D ) -426.4°
(c ) 1.0, MeOH); 1H NMR, IR, and MS were identical to
racemate 28.
(R)-(-)-2-(4-Br om o-2-flu or oben zyl)-1,2,3,4-tetr a h yd r o-
p yr r olo[1,2-a ]p yr a zin e-4-sp ir o-3′-p yr r olid in e-1,2′,3,5′-tet-
r on e (43, AS-3201). The title compound was prepared from
(-)-3-(ethoxycarbonyl)-2,5-dioxo-3-(2-(trichloroacetyl)pyrrol-1-
yl)pyrrolidine (41; 2.0 g, 5.2 mmol) by the same manner as
(2H, m), 6.26 (1H, s), 7.36 (5H, s), 8.19 (1H, s); IR (KBr, cm-1
)
1790 (CO), 1755 (CO), 1710 (CO); MS m/z 321 (MH+). Anal.
(C15H16N2O6) C, H, N.
Op tica l Resolu tion of 3-[(Ben zyloxyca r bon yl)a m in o]-
3-(eth oxyca r bon yl)-2,5-d ioxop yr r olid in e (34). (R)-(-)-3-
[(Ben zyloxyca r bon yl)a m in o]-3-(eth oxyca r bon yl)-2,5-d i-
oxop yr r olid in e (35). To a suspension of 3-[(benzyloxy-
carbonyl)amino]-3-(ethoxycarbonyl)-2,5-dioxopyrrolidine (34;
73.6 g, 0.23 mol) in EtOH (290 mL) was added cinchonidine
(67.6 g, 0.23 mol), and the mixture was heated until dissolution

4128 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Negoro et al.
SX-3030 (23t, R1 ) 4-Br-2-F-C6H3-CH2) described for method
B in Scheme 3. Purification by column chromatography on
silica gel with CHCl3-MeOH (1000/1) and recrystallization
from AcOEt-hexane gave a white solid (0.9 g, 45.0%, >99.4%
ee): mp 192-193 °C; [R]28 ) -5.4° (c ) 1.0, MeOH); [R]28
mixture was incubated at 37 °C, and the absorbance at 340
nm was measured with a spectrophotometer (model 150-20;
Hitachi Ltd., J apan). The enzyme activity was estimated on
the basis of its ability to decrease in the absorbance over a
period of 1 min. The concentration of compounds required for
50% inhibition of enzyme activity (IC50) was estimated graphi-
cally from the log concentration-inhibition curve.
D
405
) -33.0°(c ) 1.0, MeOH); 1H NMR, IR, and MS were identical
to racemate 23t. Anal. (C17H11BrFN3O4) C, H, N.
(b) In Vivo Assa y. Male Wister rats (200-250 g) were
rendered diabetic by an intravenous injection of streptozotocin
(40 mg/kg), which had been freshly dissolved in physiological
saline. After 7 days, the rats were divided into various groups,
with 4-6 animals/group, and orally given test compounds,
suspended in 0.5% tragacanth or an equivalent volume of 0.5%
tragacanth, once a day for 5 days. The rats were sacrificed 4
h after the final administration of the test compounds. Tissue
sorbitol level was determined according to the method of
Clements et al.17 with slight modifications.
The sciatic nerves sample (30-60 mg) were quickly dis-
sected from the hind limb, placed into water (1.0 mL/40 mg of
tissue), heated in boiling bath for 2 min, and then homogenized
with a Polytron instrument in 6% perchloric acid (1 mL/10 mg
of tissue). The homogenate was centrifuged at 1050g for 15
min at 4 °C. The supernatant was neutralized with 2 M K2-
CO3 and used as tissue extract for assay of sorbitol. Sorbitol
was assayed by an enzymatic method, in which sorbitol
dehydrogenase catalyzes the stoichiometric conversion of NAD
by sorbitol to a fluorogenic product, NADH. The reaction
mixture contained 30 mM glycine buffer (pH 9.4), 1.3 mM
NAD, 1.3 U/mL sorbitol dehydrogenase, and 1.0 mL of the
tissue extract in a total volume of 3 mL. After the mixture
was allowed to stand for 60 min at room temperature, the
fluorescence intensity was measured at 365-nm excitation
wavelength and 430-nm emission wavelength using a fluo-
rospectrophotometer (F3000, Hitachi Ltd., J apan). The sor-
bitol concentration was quantitated by comparison with
standards of sorbitol. The sorbitol content in sciatic nerve of
each animal was expressed as nmol/wet weight.
(S)-(+)-2-(4-Br om o-2-flu or oben zyl)-1,2,3,4-tetr a h yd r o-
p yr r olo[1,2-a ]p yr a zin e-4-sp ir o-3′-p yr r olid in e-1,2′,3,5′-tet-
r on e (44, SX-3202). Title compound was prepared from (S)-
(+)-3-[(benzyloxycarbonyl)amino]-3-(ethoxycarbonyl)-2,5-
dioxopyrrolidine (36) by the same manner as AS-3201 (43)
described above. Purification by column chromatography on
silica gel with CHCl3-MeOH (1000/1) and recrystallization
from AcOEt-hexane gave a white solid (>99.4% ee): mp 192-
193 °C; [R]29 ) +6.5° (c ) 1.0, MeOH); [R]28 ) +33.2° (c )
D
405
1.0, MeOH); 1H NMR, IR, and MS were identical to racemate
23t. Anal. (C17H11BrFN3O4) C, H, N.
The optical purity of enantiomers 43 and 44 was established
by HPLC analysis. HPLC conditions: instrument, Shimazu
LC-6A system; column, ULTRON ES-OVM (150 × 60 mm i.d.;
Shinwa Chemical Industries, Ltd.); mobile phase, 20 mM KH2-
PO4/CH3CN (80/20); flow rate, 1 mL/min; column temperature,
25 °C,; detection, UV 296, 261 nm.
Sin gle-Cr ysta l X-r a y An a lysis. X-r a y Str u ctu r e De-
ter m in a tion of AS-3201, 43. Suitable crystals of AS-3201
were grown form ethanol solutions:
C17H11O4N3BrF; M )
420.19; orthorhombic; P212121; a ) 13.207(3) Å; b ) 19.253(3)
Å; c ) 6.280(2) Å; V ) 1596.8(6) Å3; Z ) 4; F(calcd) ) 1.748 g
cm-1; F(000) ) 840.00; µ(Cu KR) ) 39.04 cm-1; T ) 293 K;
crystal size, 0.5 × 0.3 × 0.8 mm.
All measurements were made on a Rigaku AFC5R diffrac-
tometer with graphite monochromated Cu KR radiation (λ )
1.541 78 Å). The cell constants and an orientation matrix for
data collection were determined from centered angles of 22
reflections in the range 47.83° e 2θ e 50.00°. The data were
collected using the ω-2θ scan technique to a maximum 2θ
value of 125.2°. Scans of (1.52 + 0.30 tan θ)° were made at a
speed of 12.0°/min. Stationary background counts were re-
corded on each side of the reflections. A total of 1526
reflections was collected. The intensities of three representa-
tive reflections were measured after every 100 reflections. No
decay correction was applied. The data were corrected for
Lorentz and polarization effects. A correction for secondary
extinction was applied. The structure was solved by direct
methods (SIR92) and expanded using Fourier technique
(DIRDIF 94). The non-hydrogen atoms were refined with
anisotropic temperature factors. All hydrogen atoms were
include at idealized positions but not refined. The final cycle
of full-matrix least-squares refinement (SHELXL-93) was
based on 1434 observed reflections and 236 variable param-
eters.
The activity of test compounds was expressed as the percent
inhibition of sorbitol accumulation at a given dose, which was
calculated according to the following equation:
% inhibition ) (S - T)/(S - N) × 100
where S is sorbitol content in sciatic nerve of untreated
diabetic control rats, T is sorbitol content in sciatic nerve of
diabetic rats given test compounds, and N is sorbitol content
in sciatic nerve of age-matched nondiabetic control rats. The
dose of compounds required for 50% inhibition of sorbitol
accumulation (ED50) was estimated from the log dose-inhibi-
tion curve.
Ack n ow led gm en t. We thank Dr. T. Karasawa, Dr.
T. Sawayama, and Dr. H. Mizuta for encouragement
throughout this work and members of the analytical
section for elemental analyses and spectral measure-
ments. We also thank Prof. T. Ishida and Ms. Y. In of
Osaka University of Pharmaceutical Sciences for mea-
surement of the X-ray structure.
All the calculations were performed using the teXsan
(Molecular Structure Co.). A refinement of the Flack’s ø
parameter was carried out to determine the absolute config-
uration. The value of refined ø [-0.06(7)] indicated that to be
the correct absolute stereochemistry. The final R value was
0.057. The final difference Fourier was essentially featureless.
Biologica l Met h od s. (a ) In Vit r o Assa y. E n zym e
P r ep a r a tion . Porcine lens AR was prepared according to the
method of Hayman and Kinoshita16 with slight modifications.
The lenses were homogenized in 250 mM phosphate buffer (pH
7.4) containing 2 mM mercaptoethanol at 0-4 °C, and the
homogenate was centrifuged at 20000g for 30 min. The
supernatant was subjected to a 40-60% ammonium sulfate
fractionation. The resultant precipitate was dissolved in 5 mM
phosphate buffer (pH 7.4) containing 2 mM mercaptoethanol
and used for enzyme assay. One unit (U) of AR was defined
as the enzyme activity which oxidizes 1 µmol of NADPH/min
under the assay conditions described below.
Su p p or tin g In for m a tion Ava ila ble: X-ray crystallo-
graphic data (9 pages). Ordering information is given on any
current masthead page.
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