Orally active GPIIb/IIIa antagonists: Synthesis and biological activities of masked amidines as prodrugs of 2-[(3S)-4-[(2S)-2-(4-amidinobenzoylamino)-3-(4-methoxyphenyl)propanoyl]-3- (2-methoxy-2-oxoethyl)-2-oxopiperazinyl]acetic acid

Article abstract of DOI:10.1248/cpb.49.268

To improve the in vivo potency of the potent GPIIb/IIIa antagonist 2-[(3S)-4-[(2S)-2-(4-amidinobenzoylamino)-3-(4-methoxyphenyl)propanoyl]-3- (2-methoxy-2-oxoethyl)-2-oxopiperazinyl]lacetic acid (4), the amidino group was converted to an oxadiazole ring, thiadiazole ring of substituted amidoxime group. These groups were expected to be metabolized to an amidino group in vivo. The compounds synthesized were evaluated for their potency to inhibit the ex vivo adenosine 5′-diphosphate (ADP)-induced aggregation of guinea pig platelets. Among the compounds examined, the methoxycarbonyloxyamidine 8a exhibited the most potent ex vivo inhibitory activity with a fast onset and prolonged duration of action after oral administration.

Full text of DOI:10.1248/cpb.49.268

268
Chem. Pharm. Bull. 49(3) 268—277 (2001)
Vol. 49, No. 3
Orally Active GPIIb/IIIa Antagonists: Synthesis and Biological Activities
of Masked Amidines as Prodrugs of 2-[(3S)-4-[(2S)-2-(4-
Amidinobenzoylamino)-3-(4-methoxyphenyl)propanoyl]-3-(2-methoxy-2-
oxoethyl)-2-oxopiperazinyl]acetic Acid
Shuji KITAMURA, ,a Hideto FUKUSHI,b Toshio MIYAWAKI,a Masaki KAWAMURA,c Zen-ichi TERASHITA,c
*
a
and Takehiko NAKA
Medicinal Chemistry Research Laboratories,a Discovery Research Laboratories V,b and Pharmacology Research
Laboratories I,c Takeda Chemical Industries, Ltd., 2–17–85, Jusohonmachi, Yodogawa-ku, Osaka 532–8686, Japan.
Received August 24, 2000; accepted November 20, 2000
To improve the in vivo potency of the potent GPIIb/IIIa antagonist 2-[(3S)-4-[(2S)-2-(4-amidinobenzoyl-
amino)-3-(4-methoxyphenyl)propanoyl]-3-(2-methoxy-2-oxoethyl)-2-oxopiperazinyl]acetic acid (4), the amidino
group was converted to an oxadiazole ring, thiadiazole ring or substituted amidoxime group. These groups were
expected to be metabolized to an amidino group in vivo. The compounds synthesized were evaluated for their po-
tency to inhibit the ex vivo adenosine 5؅-diphosphate (ADP)-induced aggregation of guinea pig platelets. Among
the compounds examined, the methoxycarbonyloxyamidine 8a exhibited the most potent ex vivo inhibitory activ-
ity with a fast onset and prolonged duration of action after oral administration.
Key words GPIIb/IIIa antagonist; antiplatelet effect, masked amidine, prodrugs for arylamidines
The activation, adhesion and aggregation of platelets are drugs of 4, due to unselective hydrolysis of the two ester
important processes in the initiation of thrombus formation groups, the prodrugs were not efficiently converted to the
at sites showing high-grade stenosis, ruptured atheromatous parent active form 4.22,23) We then turned our attention to
plaque and endothelial damage within arteries. Recent stud- masking the amidino group. While there are numerous re-
ies on the biochemical mechanism of platelet activation indi- ports on prodrugs of carboxylic acids, only a few prodrugs of
cate that the final obligatory step in aggregation is the cross- aromatic amidines have been reported, for example, ami-
linking of the plasma protein fibrinogen and platelet mem- doximes,11,19)
O-methylamidoximes,24)
alkoxycarbonyl-
brane glycoprotein IIb/IIIa (GPIIb/IIIa) exposed on activated amidines25,26) and alkoxycarbonyloxyamidines.26) In the clini-
platelets.1—4) In the last decade, intensive effort has been cal trials of the potent angiotensin II receptor antagonist
devoted to the development of GPIIb/IIIa inhibitors to treat TAK-536,27,28) the amidine derivative (M-1) in which the
and prevent thrombotic disorders such as unstable angina oxadiazolone ring in TAK-536 has been converted to an
and myocardial infarction.5—12) The clinical efficacy of the amidino group was detected as one of the metabolites (Chart
GPIIb/IIIa inhibitors has been demonstrated by positive re- 1). This suggested that an oxadiazolone ring might be able to
sults obtained with ReoPro13,14) (anti-GPIIb/IIIa c7E3 Fab an- serve as a masked amidino group and prompted us to investi-
tibody), Integrelin15,16) (cyclic peptide) and Aggrastat17,18) gate new in vivo amidino group equivalents. Since there are
(non-peptide small molecule) used as intravenous treatments many pharmacologically important amidines with poor oral
for acute thrombosis.
bioavailability, the development of masked amidino groups is
Orally active GPIIb/IIIa antagonists are required to ex- of great significance. In this paper, we describe the synthesis
pand the therapeutic utility of this class of agents and provide and pharmacological profiles of prodrugs of 4 based on the
effective chronic treatment for patients with recurrent vascu- creation of an oxadiazolone ring.
lar events. Although a number of potent low molecular
weight GPIIb/IIIa antagonists have been reported, their oral Chemistry
bioavailabilities are insufficient for practical use. The low
The target compounds 7a—l and 8a—d were synthesized
bioavailability might be ascribed to the highly polar nature of as outlined in Chart 2 using 521) as the starting material. Hy-
these compounds due to the presence of a strong basic group drogenolysis of 5 in the presence of 10% Pd–C to remove the
and a carboxyl group. To improve the oral absorption of benzyloxycarbonyl (Z) group and subsequent condensation
these compounds, a prodrug strategy has often been used. with appropriate benzoic acids (11, 14, 15, 19, 20, 21, 25, 30,
For example, Ro-48-3657 (1),11) BIBU-104 (2)19) and EMD- 31, 35, 39 or 40) gave the intermediates 6a—l. Acid hydroly-
122347 (3)20) exhibit sufficient oral bioavailabily, and 2 and 3 sis of the tert-butoxycarbonyl groups with trifluoroacetic
are currently being examined in clinical trials (Chart 1).
We recently reported the discovery of the potent GPIIb/ boxylate 6c followed by acid hydrolysis afforded 8a—d.
IIIa antagonist 2-[(3S)-4-[(2S)-2-(4-amidinobenzoylamino)- The benzoic acids used in the above condensation reac-
acid (TFA) provided 7a—l. Acylation of the amidoxime car-
3-(4-methoxyphenyl)propanoyl]-3-(2-methoxy-2-oxoethyl)- tions were prepared from the 4-cyanobenzoates 9 and 22
2-oxopiperazinyl]acetic acid (4) (Chart 1).21) Following oral (Charts 3—5). Treatment of 9 with hydroxylamine hy-
administration to guinea pigs, 4 was not absorbed suffi- drochloride, followed by hydrolysis with 2 N NaOH, provided
ciently, and absolute bioavailability of 4 was not high enough the amidoxime 11.29) Cyclization of 10 with 1,1Ј-carbonyl-
for practical use. Our initial effort to obtain orally active ester diimidazole (CDI) and with 1,1Ј-thiocarbonyldiimidazole
prodrugs of 4 resulted in failure. In the case of the ester pro- (TCDI) followed by hydrolysis afforded the oxadiazolone 14
To whom correspondence should be addressed. e-mail: Kitamura_Shuji@takeda.co.jp
© 2001 Pharmaceutical Society of Japan

March 2001
269
Chart 1
Chart 2
Chart 3
and the oxadiazolethione 15, respectively. The 5-methyloxa- sponding acid anhydride or benzoyl chloride followed by hy-
diazole 19, 5-trifluoromethyloxadiazole 20 and 5-phenyloxa- drolysis (Chart 3).
diazole 21 were obtained by cyclization of 10 with the corre-
tert-Butyl 4-[amino(hydroxyimino)methyl]benzoate, 23,

270
Vol. 49, No. 3
Chart 4
Chart 5
Table 1. Metabolic Conversiona) of 7a to 4
Generated
which was obtained from 22 with hydroxylamine hydrochlo-
ride, was cyclized with thionyl chloride followed by acid hy-
drolysis to provide the oxathiadiazolone 25. Reaction of 23
with CH(OCH3)3 in the presence of boron trifluoride diethyl
etherate (BF3·Et2O) gave the oxadiazole 26. Treatment of 23
with ethyl chloroglyoxylate in the presence of pyridine pro-
vided the 5-ethoxycarbonyloxadiazole 27, which was con-
verted to the 5-cyanooxadiazole 29 by amidation and dehy-
dration. Acid hydrolysis of 26 and 29 gave the benzoic acids
30 and 31, respectively. The intermediate 23 was converted
to the oxadiazolone 32, which was methylated with dia-
zomethane to give a mixture of the 5-methoxyoxadiazole 33
and the N-methyloxadiazole 34. After the separation of 33
and 34 by silica gel column chromatography, 33 was con-
verted to the corresponding benzoic acid 35 by acid hydroly-
sis (Chart 4).
Remaining
7a (%)
4 (%)
Liver homogenate
Small intestine homogenate
Plasma
14.5
Not detected
Not detected
22.4
73.4
57.9
a) Compond 7a was incubated in guinea pig liver homogenate, small intestine ho-
mogenate and plasma at 37 °C for 1 h and then assayed by HPLC as described in the
Experimental section.
Results and Discussion
First of all, to evaluate the potential usefulness of an oxa-
diazolone ring as a masked amidino group, metabolic con-
version of the oxadiazolone 7a to the amidine 4 using guinea
pig liver homogenate, small intestine homogenate and
plasma was examined (Table 1). After incubation of 7a in
liver homogenate at 37 °C for 1 h, the amount of 4 generated
and that of 7a remaining were determined to be 15 and 22%
based on the initial amount of 7a, respectively. After 1 h in-
cubation in small intestine homogenate and plasma, no
amount of 4 was detected, and the amount of 7a recovered
was 73 and 58%, respectively. This suggested that the oxadi-
Cyclization of the methyl 4-[amino(imino)methyl]ben-
zoate hydrochloride 36 with chlorocarbonyl sulfenyl chloride
and subsequent saponification provided the thiadiazolone 39.
The reaction of 36 with carbon disulfide and sulfur in the
presence of sodium methoxide gave the thiadiazolethione 38,
which was hydrolyzed to the carboxylic acid 40 (Chart 5).

March 2001
271
azolone ring could be metabolized to the amidino group by converted than 7a to 4. In fact, incubation of the thiadia-
the reductive liver enzymes,30) but not by esterases in the zolone 7d in guinea pig liver homogenate at 37 °C for 1 h
plasma or small intestine. The metabolic pathway was con- generated more 4 than in the case of 7a (39% vs. 15%). The
sidered to be the cleavage of the N–O bond in the oxadia- ex vivo inhibitory effects of the thioanologs 7b and 7d—f,
zolone ring, followed by elimination of carbon dioxide from whose in vitro antiplatelet effects were remarkably decreased
the carbamic acid intermediate to lead to 4.
(IC50: Ͼ4 mM, Table 2), are presented in Fig. 1. Among these
The results of the in vitro metabolic study encouraged us thioanalogs, the oxathiadiazolone 7f showed a potent in-
to evaluate 7a in vivo using guinea pigs. The ex vivo in-
hibitory effect of 7a on the adenosine 5Ј-diphosphate (ADP)-
induced platelet aggregation in guinea pig platelet rich
plasma (PRP) is shown in Fig. 1. Oral administration of 7a at
a dose of 0.3 mg/kg resulted in 91% inhibition at 8 h, and
38% inhibition was still observed at 24 h. On the contrary, 4
inhibited the platelet aggregation by 65 and 30% at 2 and 8 h,
respectively. Since the in vitro antiplatelet effect of 7a was
very weak even at a high concentration (Table 2), 7a is con-
sidered to be metabolized to the biological active form 4, of
which the plasma concentration could be estimated from the
ex vivo potency of 7a after oral administration, in the body.
While improved ex vivo potency and prolonged duration of
action were observed with 7a as compared to 4, the onset of
its action was slower than that of 4. This lag might be as-
cribed to sluggish conversion of 7a to 4, because the maxi-
mal concentration of 7a in the plasma31) was observed 2 h
after oral administration (data not shown).
To achieve a quick onset of action, some thioanalogs were
Fig. 1. Effects of Oxadiazolone, Oxadiazolethione, Thiadiazolone, Thiadi-
azolethione and Oxathiadiazolone Derivatives on ex Vivo (p.o.) ADP-In-
examined because it was thought they might be more easily duced Platelet Aggregation in Guinea Pigs (nϭ4)
Table 2. In Vitro Inhibitory Effects of Compounds 4, 7a—l and 8a—d
Inhibitory effecta)
Inhibitory effecta)
IC50 (nM)
Compound
Rϭ
Compound
Rϭ
IC50 (nM)
4
13
Ͼ30000
30000
7i
17000
11000
20000
9000
1900
2000
320
7a
7b
7d
7e
7f
7j
7k
7l
Ͼ30000
Ͼ30000
4400
7c
8a
8b
8c
8d
7g
7h
16000
Ͼ30000
300
270
a) In vitro inhibition of ADP-induced platelet aggregation in guinea pig PRP. See the Experimental section for details.

272
Vol. 49, No. 3
Fig. 2. Effects of Oxadiazole Derivatives on ex Vivo (p.o.) ADP-Induced
Platelet Aggregation in Guinea Pigs (nϭ4)
Fig. 3. Effects of Amidoxime Derivatives on ex Vivo (p.o.) ADP-Induced
Platelet Aggregation in Guinea Pigs (nϭ4)
hibitory effect comparable to that of 7a and a faster onset of
action than 7a. On the contrary, the other thioanalogs, 7b, 7d
and 7e, did not show enhanced ex vivo potency.
nounced inhibitory effect, fast onset and prolonged duration
of action, which were superior to those of the other com-
pounds prepared in this study. The inhibitory effect of 8a at
0.3 mg/kg p.o. was found to be 66, 82, 96 and 31% at 2, 4, 8
and 24 h, respectively. The metabolic pathway of 8a is con-
sidered to proceed via amidoxime 7c,32) which is thought to
be converted to amidine 4 by reductive enzymes in the
liver.11,19) Rahmathullah and co-workers26) reported the appli-
cation of an alkoxycarbonyloxyamidino group as a masked
amidino group to improve oral efficacy of an anti-Pneumo-
cystis carinii agent, but that trial resulted in failure. Our
study would be the first example showing the usefulness of
an alkoxycarbonyloxyamidine group as a masked amidino
group. The ethoxy- and the propoxycarbonyloxyamidines
also displayed potent and long lasting ex vivo inhibitory ac-
tivities, although these were inferior to that of 8a (data not
shown). A faster onset of action and similar inhibitory activ-
ity were observed after oral administration of the methylthio-
carbonyloxyamidine 8b as compared to 8a; however, its du-
ration of action was shorter than that of 8a. The ethylcar-
bamyloxyamidine 8d also showed as potent inhibitory activ-
ity as 8a, but the onset of the action was slower than that of
8a. While the amidoxime derivatives 8a,b and 8d demon-
strated good oral activity, enhanced ex vivo activity was not
observed with the acetoxyamidine 8c.
The oral bioavailabilities of 4 and 8a, in terms of 4, in
guinea pigs after a dose of 1 mg/kg i.v. (4) and 30 mg/kg p.o.
(4) or 10 mg/kg p.o. (8a), were found to be 2.8 and 5.3%, re-
spectively (Fig. 4). The oral bioavailability as 4 was in-
creased by about 2-fold with the methoxycarbonyloxyami-
dine 8a.
In conclusion, cyclic amidino groups, such as an oxadi-
azolone group, an oxathiadiazolone group, a 5-trifluoro-
methyloxadiazole group and a 5-cyanoloxadiazole group, as
well as substituted amidoxime groups, such as a methoxy-
carbonyloxyamidino group and a methylthiocarbonyloxy-
amidino group, provided useful masked amidino groups to
obtain orally active potent GPIIb/IIIa antagonists with a fast
onset and prolonged duration of action. It is well known that
there is no almighty prodrug promoiety for a carboxyl group.
Although the ex vivo activity of 4 was improved by con-
version to 7a or 7f, more rapid onset of action was required.
We then investigated some oxadiazoles, which might be me-
tabolized to the amidino group through the same pathway as
7a. The ex vivo antiplatelet effect and the in vitro antiplatelet
activities of the oxadiazoles 7g—7l are summarized in Fig. 2
and Table 2. These compounds also had diminished in vitro
antiplatelet activities (Table 2). Conversion of the amidino
group to an oxadiazole ring (7j) did not enhance the ex vivo
activity as compared to that of 4. A dramatic decrease was
observed with the 5-methyloxadiazole 7g and the 5-methoxy-
oxadiazole 7l, which possessed an electron donating group at
the 5-position of the oxadiazole ring. On the contrary, intro-
duction of an electron withdrawing group, such as a trifluo-
romethyl or a cyano group, increased the ex vivo antiplatelet
effect. Oral administration of the 5-trifluoromethyloxadiazole
7i, and the 5-cyanooxadiazole 7k at a dose of 0.3 mg/kg,
caused 71 and 56% inhibition at 4 h and 84 and 91% inhibi-
tion at 8 h, respectively. Introduction of the electron with-
drawing group might facilitate the cleavage of the N–O bond
to form the amidino group. The 5-phenyloxadiazole 7h was
much less potent than 7j, providing only 9% of the maximal
inhibition. While a phenyl group is a weak electron with-
drawing group, the chemical stability of the phenyloxadia-
zole ring might prevent the reductive cleavage. The oxadia-
zoles 7i and 7k, bearing electron withdrawing groups at the
5-position, had ex vivo potency better than that of 4 but com-
parable to that of 7a and 7f.
We also evaluated the substituted amidoximes 7c and 8a—
d, which are regarded as acyclic analogs of the correspond-
ing oxadiazolone ring-bearing compounds (Fig. 3). The re-
placement of the amidino group with an amidoxime group
(7c) did not increase the ex vivo activity as compared to that
of 4. We might ascribe the inefficacy of this modification to
the presence of the hydroxyl group in 7c which reduced its
lipophilicity. To increase the lipophilicity of 7c, the hydroxyl
group was masked to lead to the amidoxime derivatives
8a—d. The methoxycarbonyloxyamidine 8a showed a pro-

March 2001
273
oxopiperazinyl]acetate (6c) The title compound was prepared from 5 and
4-[amino(hydroxyimino)methyl]benzoic acid 11 according to the procedure
described in the preparation of 6a as a colorless amorphous powder. 1H-
NMR (CDCl3ϩD2O) d: 1.45 (9H, s), 2.30—3.40 (6H, m), 3.60—4.00 (4H,
m), 3.63 (3H, s), 3.78 (3H, s), 5.03—5.50 (2H, m), 6.83 (2H, d, Jϭ8.0 Hz),
7.18 (2H, d, Jϭ8.0 Hz), 7.31 (2H, d, Jϭ8.0 Hz), 7.50 (2H, d, Jϭ8.0 Hz).
Methyl 2-[(2S)-4-[2-(tert-Butoxy)-2-oxoethyl]-1-[(2S)-3-(4-methoxy-
phenyl)-2-[[4-(5-thioxo-4,5-dihydro-1,2,4-thiadiazol-3-yl)benzoyl]-
amino]propanoyl-3-oxopiperazinyl]acetate (6e) The title compound was
prepared from 5 and 4-(5-thioxo-4,5-dihydro-1,2,4-thiadiazol-3-yl)benzoic
acid 40 according to the procedure described in the preparation of 6a as a
colorless amorphous powder. 1H-NMR (CDCl3) d: 1.46 (9H, s), 2.50—3.35
(6H, m), 3.62 (3H, s), 3.72—3.78 (2H, m), 3.79 (3H, s), 3.96 (3H, s), 5.05
(1H, t, Jϭ5.6 Hz), 5.24—5.42 (1H, m), 6.84 (2H, d, Jϭ8.6 Hz), 7.05 (1H,
m), 7.16 (2H, d, Jϭ8.6 Hz), 7.89 (2H, d, Jϭ8.4 Hz), 8.32 (2H, d, Jϭ8.4 Hz).
Methyl 2-[(2S)-4-[2-(tert-Butoxy)-2-oxoethyl]-1-[(2S)-3-(4-methoxy-
phenyl)-2-[[4-(2-oxo-2,3-dihydro-1,2l4,3,5-oxathiadiazol-4-yl)benzoyl]-
amino]propanoyl-3-oxopiperazinyl]acetate (6f) The title compound was
prepared from 5 and 4-(2-oxo-2,3-dihydro-1,2l4,3,5-oxathiadiazol-4-yl)ben-
zoic acid 25 according to the procedure described in the preparation of 6a as
a colorless amorphous powder. 1H-NMR (CDCl3ϩD2O) d: 1.47 (9H, s),
2.60—4.20 (10H, m), 3.66 (3H, s), 3.73 (3H, s), 5.05—5.40 (2H, m), 6.68
(2H, d, Jϭ8.4 Hz), 6.87 (2H, d, Jϭ8.4 Hz), 7.86 (2H, d, Jϭ8.6 Hz), 7.92
(2H, d, Jϭ8.4 Hz).
Fig. 4. Plasma Concentration of 4 after i.v. Administration of 4 and p.o.
Administration of 4 and 8a (nϭ3) in Guinea Pigs
Plasma concentration of 4 at 24 h after p.o. administration of 4 was under the limit of
quantitation (Ͻ5 ng/ml).
Methyl 2-[(2S)-4-[2-(tert-Butoxy)-2-oxoethyl]-1-[(2S)-3-(4-methoxy-
phenyl)-2-[[4-(5-methyl-1,2,4-oxadiazol-3-yl)benzoyl]amino]propanoyl-
3-oxopiperazinyl]acetate (6g) The title compound was prepared from 5
and 4-(5-methyl-1,2,4-oxadiazol-3-yl)benzoic acid 19 according to the pro-
cedure described in the preparation of 6a as a colorless amorphous powder.
1H-NMR (CDCl3) d: 1.46 (9H, s), 2.55—2.80 (1H, m), 2.68 (3H, s), 2.95—
3.30 (5H, m), 3.63 (3H, s), 3.73—3.86 (2H, m), 3.78 (3H, s), 3.95 (2H, s),
5.05 (1H, t, Jϭ5.5 Hz), 5.22—5.40 (1H, m), 6.84 (2H, d, Jϭ8.8 Hz), 7.09
(1H, d, Jϭ8.0 Hz), 7.16 (2H, d, Jϭ8.8 Hz), 7.90 (2H, d, Jϭ8.6 Hz), 8.14
(2H, d, Jϭ8.6 Hz).
Methyl 2-[(2S)-4-[2-(tert-Butoxy)-2-oxoethyl]-1-[(2S)-3-(4-methoxy-
phenyl)-2-[[4-(5-phenyl-1,2,4-oxadiazol-3-yl)benzoyl]amino]propanoyl-
3-oxopiperazinyl]acetate (6h) The title compound was prepared from 5
and 4-(5-phenyl-1,2,4-oxadiazol-3-yl)benzoic acid 21 according to the pro-
cedure described in the preparation of 6a as a colorless amorphous powder.
The best promoieties are dependent on the nature of the car-
boxylic acid. This is also true in the case of arylamidines,
and a variety of promoieties should therefore be provided as
candidates. The masked amidino groups discussed in this
study could be additional examples to be considered. Al-
though dramatic improvement of the oral bioavailability was
not accomplished with the compounds examined in this
study because of a remaining carboxyl group, these new
masked amidino groups are expected to be useful for other
arylamidines.
Experimental
Melting points were determined on a Yanagimoto micro melting point ap- 1H-NMR (CDCl3) d: 1.47 (9H, s), 2.55—3.30 (4H, m), 3.00 (2H, d, Jϭ5.4
paratus and are uncorrected. IR spectra were obtained on a Hitachi IR-215 Hz), 3.64 (3H, s), 3.75—3.90 (2H, m), 3.78 (3H, s), 3.96 (2H, s), 5.08 (1H,
spectrometer. 1H-NMR spectra were recorded on a Varian Gemini 200 t, Jϭ5.4 Hz), 5.25—5.45 (1H, m), 6.85 (2H, d, Jϭ8.0 Hz), 7.18 (2H, d,
(200 MHz) spectrometer. Chemical shifts are given in d value (ppm) with Jϭ8.0 Hz), 7.25—7.45 (1H, m), 7.50—7.70 (3H, m), 7.93 (2H, d, Jϭ8.4
tetramethylsilane (TMS) as the internal standard. The following abbrevia- Hz), 8.18—8.30 (2H, m), 8.25 (2H, d, Jϭ8.4 Hz).
tions are used: sϭsinglet, dϭdoublet, tϭtriplet, ddϭdouble doublet, mϭ
Methyl 2-[(2S)-4-[2-(tert-Butoxy)-2-oxoethyl]-1-[(2S)-3-(4-methoxy-
multiplet. Optical rotations were recorded on a JASCO DIP-370 digital po- phenyl)-2-[[4-(5-trifluoromethyl-1,2,4-oxadiazol-3-yl)benzoyl]amino]-
larimeter.
Methyl 2-[(2S)-4-[2-(tert-Butoxy)-2-oxoethyl]-1-[(2S)-3-(4-methoxy-
propanoyl]-3-oxopiperazinyl]acetate (6i) The title compound was pre-
pared from 5 and 4-(5-trifluoromethyl-1,2,4-oxadiazol-3-yl)benzoic acid 20
phenyl)-2-[[4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)benzoyl]amino]- according to the procedure described in the preparation of 6a as a colorless
1
propanoyl-3-oxopiperazinyl]acetate (6a) A suspension of 5 (0.95 g, 1.59 amorphous powder. H-NMR (CDCl3) d: 1.46 (9H, s), 2.50—3.30 (6H, m),
mmol) and 10% Pd–C (0.25 g) in MeOH (50 ml) was hydrogenated under 3.64 (3H, s), 3.70—3.90 (2H, m), 3.78 (3H, s), 3.96 (2H, s), 5.06 (1H, t,
balloon pressure for 1 h at room temperature. The catalyst was removed by Jϭ5.5 Hz), 5.24—5.40 (1H, m), 6.85 (2H, d, Jϭ8.6 Hz), 7.10—7.30 (1H,
filtration and the filtrate was concentrated under reduced pressure to give m), 7.17 (2H, d, Jϭ8.6 Hz), 7.94 (2H, d, Jϭ8.4 Hz), 8.20 (2H, d, Jϭ8.4 Hz).
amine. To a solution of this amine in N,N-dimethylformamide (DMF) (12
ml) were added 4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)benzoic acid 14
Methyl 2-[(2S)-4-[2-(tert-Butoxy)-2-oxoethyl]-1-[(2S)-3-(4-methoxy-
phenyl)-2-[[4-(1,2,4-oxadiazol-3-yl)benzoyl]amino]propanoyl]-3-
(0.36 g, 1.75 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hy- oxopiperazinyl]acetate (6j) The title compound was prepared from 5 and
drochloride (EDC) (0.4 g, 2.09 mmol) at room temperature. After being 4-(1,2,4-oxadiazol-3-yl)benzoic acid 30 according to the procedure de-
1
stirred for 1 h, the mixture was diluted with ethyl acetate (EtOAc), washed
with 5% aqueous KHSO4, dried over MgSO4, and concentrated under re-
scribed in the preparation of 6a as a colorless amorphous powder. H-NMR
(CDCl3) d: 1.46 (9H, s), 2.50—3.90 (8H, m), 3.63 (3H, s), 3.79 (3H, s), 3.96
duced pressure. The residue was purified by silica gel column chromatogra- (2H, s), 5.06 (1H, t, Jϭ5.4 Hz), 5.20—5.40 (1H, m), 6.85 (2H, d, Jϭ8.8 Hz),
phy (EtOAc–MeOH, 4 : 1) to give 6a (0.76 g, 73%) as a colorless amorphous 7.17 (2H, d, Jϭ8.8 Hz), 7.08—7.23 (1H, m), 7.93 (2H, d, Jϭ8.6 Hz), 8.21
1
powder. H-NMR (CDCl3ϩD2O) d: 1.46 (9H, s), 2.40—3.90 (8H, m), 3.59 (2H, d, Jϭ8.4 Hz), 8.81 (1H, s).
(3H, s), 3.74 (3H, s), 3.94 (2H, s), 5.13 (1H, t, Jϭ6.0 Hz), 5.23—5.42 (1H,
m), 6.79 (2H, d, Jϭ8.6 Hz), 7.12 (2H, d, Jϭ8.6 Hz), 7.65—7.98 (4H, m).
Methyl 2-[(2S)-4-[2-(tert-Butoxy)-2-oxoethyl]-1-[(2S)-3-(4-methoxy-
phenyl)-2-[[4-(5-thioxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)benzoyl]amino]-
Methyl 2-[(2S)-4-[2-(tert-Butoxy)-2-oxoethyl]-1-[(2S)-2-[[4-(5-cyano-
1,2,4-oxadiazol-3-yl)benzoyl]amino]-3-(4-methoxyphenyl)propanoyl]-3-
oxopiperazinyl]acetate (6k) The title compound was prepared from 5 and
4-(5-cyano-1,2,4-oxadiazol-3-yl)benzoic acid 31 according to the procedure
propanoyl-3-oxopiperazinyl]acetate (6b) The title compound was pre- described in the preparation of 6a as colorless crystals, mp 185—187 °C.
pared from 5 and 4-(5-thioxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)benzoic acid 1H-NMR (CDCl3) d: 1.46 (9H, s), 2.60—3.40 (4H, m), 2.99 (2H, d,
15 according to the procedure described in the preparation of 6a as a color- Jϭ5.6 Hz), 3.64 (3H, s), 3.72—3.88 (2H, m), 3.78 (3H, s), 3.96 (2H, s), 5.05
less amorphous powder. 1H-NMR (CDCl3ϩD2O) d: 1.43 (9H, s), 2.00—
(1H, t, Jϭ5.4 Hz), 5.23—5.39 (1H, m), 6.85 (2H, d, Jϭ8.6 Hz), 7.10—7.25
4.20 (10H, m), 3.52 (3H, s), 3.70 (3H, s), 5.00—5.45 (2H, m), 6.60—6.90 (1H, m), 7.16 (2H, d, Jϭ8.6 Hz), 7.94 (2H, d, Jϭ8.8 Hz), 8.18 (2H, d,
(2H, m), 7.00—7.80 (6H, m).
Jϭ8.8 Hz). IR (KBr) cmϪ1: 1740, 1660, 1650, 1610, 1510, 1445, 1290,
Methyl 2-[(2S)-1-[(2S)-2-[[4-[Amino(hydroxyimino)methyl]benzoyl]- 1250, 1230, 1150. Anal. Calcd for C33H36N6O9: C, 59.99; H, 5.49; N, 12.72.
amino]-3-(4-methoxyphenyl)propanoyl]-4-[2-(tert-butoxy)-2-oxoethyl]-3-
Found: C, 59.85; H, 5.45; N, 12.60.

274
Vol. 49, No. 3
Methyl 2-[(2S)-4-[2-(tert-Butoxy)-2-oxoethyl]-1-[(2S)-2-[4-(5-methoxy- piperazinyl]acetic Acid (7h): Colorless crystals (MeOH–Et2O), mp 179—
1,2,4-oxadiazol-3-yl)benzoyl]amino]-3-(methoxyphenyl)propanoyl]-3-ox-
opiperazinyl]acetate (6l) The title compound was prepared from 5 and 4-
181 °C. [a]D20 ϩ56.7° (cϭ0.97, MeOH). 1H-NMR (DMSO-d6) d: 2.65—
2.85 (2H, m), 2.90—4.25 (8H, m), 3.50 (3H, s), 3.70 (3H, s), 4.91 (1H, t,
(5-methoxy-1,2,4-oxadiazol-3-yl)benzoic acid 35 according to the procedure Jϭ6.0 Hz), 5.00—5.20 (1H, m), 6.83 (2H, d, Jϭ8.4 Hz), 7.24 (2H, d, Jϭ8.4
described in the preparation of 6a as a colorless amorphous powder. 1H- Hz), 7.60—7.85 (3H, m), 8.03 (2H, d, Jϭ8.4 Hz), 8.10—8.27 (2H, m), 8.18
NMR (CDCl3) d: 1.46 (9H, s), 2.55—3.30 (4H, m), 2.99 (2H, d, Jϭ5.6 Hz), (2H, d, Jϭ8.4 Hz), 8.98 (1H, d, Jϭ8.0 Hz). IR (KBr) cmϪ1: 1730, 1660,
3.63 (3H, s), 3.70—3.80 (2H, m), 3.78 (3H, s), 3.95 (2H, s), 4.29 (3H, s), 1610, 1540, 1510, 1490, 1450, 1410, 1350, 1300, 1270, 1250, 1190, 1030,
5.06 (1H, t, Jϭ5.5 Hz), 5.25—5.40 (1H, m), 6.88 (2H, d, Jϭ8.6 Hz), 7.05— 735. Anal. Calcd for C34H33N5O9: C, 62.28; H, 5.07; N, 10.68. Found: C,
7.20 (1H, m), 7.16 (2H, d, Jϭ8.6 Hz), 7.88 (2H, Jϭ8.6 Hz), 8.10 (2H, d, 62.13; H, 5.00; N, 10.64.
Jϭ8.8 Hz).
2-[(3S)-3-(2-Methoxy-2-oxoethyl)-4-[(2S)-3-(4-methoxyphenyl)-2-[[4-(5-
2-[(3S)-3-(2-Methoxy-2-oxoethyl)-4-[(2S)-3-(4-methoxyphenyl)-2-[[4-
(5-thioxo-4,5-dihydro-1,2,4-thiadiazol-3-yl)benzoyl]amino]propanoyl]-2-
trifluoromethyl-1,2,4-oxadiazol-3-yl)benzoyl]amino]propanoyl]-2-oxopiper-
azinyl]acetic Acid (7i): A colorless amorphous powder, [a]D20 ϩ54.2°
1
oxopiperazinyl]acetic Acid (7e) To a solution of 6e (0.76 g, 11.1 mmol) in (cϭ0.98, MeOH). H-NMR (CDCl3) d: 2.45—3.35 (6H, m), 3.62 (3H, s),
CH2Cl2 (5.0 ml) was added TFA (5.0 ml) at room temperature. After being 3.65—3.95 (2H, m), 3.78 (3H, s), 4.08 (1H, s), 4.10 (1H, s), 5.06 (1H, t,
stirred for 2 h at room temperature, the mixture was concentrated under re- Jϭ5.4 Hz), 5.20—5.45 (1H, m), 6.83 (2H, d, Jϭ7.6 Hz), 7.15 (2H, d,
duced pressure. The residue was purified by column chromatography (CHP- Jϭ7.6 Hz), 7.35—7.40 (1H, m), 7.94 (2H, d, Jϭ7.2 Hz), 8.20 (2H, d,
20, gradient elution: H2O to 30% aqueous CH3CN) to give 7e (0.44 g, 33%) Jϭ7.2 Hz). IR (KBr) cmϪ1: 1740, 1640, 1510, 1250, 1220, 1180, 1160, 990.
as a colorless amorphous powder, [a]D20 ϩ4.4° (cϭ0.69, dimethyl sulfoxide Anal. Calcd for C29H28F3N5O9·0.5H2O: C, 53.05; H, 4.45; N, 10.67. Found:
(DMSO)). 1H-NMR (DMSO-d6) d: 2.60—2.86 (2H, m), 2.86—4.25 (8H,
m), 3.48 (3H, s), 3.69 (3H, s), 4.90 (1H, m), 6.81 (2H, d, Jϭ8.8 Hz), 7.22
C, 53.23; H, 4.32; N, 10.69.
2-[(3S)-3-(2-Methoxy-2-oxoethyl)-4-[(2S)-3-(4-methoxyphenyl)-2-[[4-
(2H, d, Jϭ8.8 Hz), 7.96 (2H, d, Jϭ8.4 Hz), 8.22 (2H, d, Jϭ8.4 Hz), 8.89 (1,2,4-oxadiazol-3-yl)benzoyl]amino]propanoyl]-2-oxopiperazinyl]acetic
(1H, d, Jϭ8.2 Hz). IR (KBr) cmϪ1: 1732, 1651, 1539, 1512, 1454, 1404, Acid (7j): Colorless crystals (EtOAc–iso-Pr2O), mp 175—177 °C. [a]D20
1
1248, 1179. Anal. Calcd for C28H29N5O8S2·0.1Et2O·0.5H2O: C, 52.96; H, ϩ61.7° (cϭ1.01, MeOH). H-NMR (CDCl3) d: 2.45—3.95 (8H, m), 3.62
4.85; N, 10.87. Found: C, 52.93; H, 4.85; N, 10.94.
(3H, s), 3.77 (3H, s), 4.03 (1H, d, Jϭ17.2 Hz), 4.17 (1H, d, Jϭ17.2 Hz), 5.07
Following compound 7a—c and 7f—l were prepared according to the (1H, t, Jϭ5.4 Hz), 5.15—5.45 (1H, m), 6.83 (2H, d, Jϭ8.6 Hz), 7.15 (2H, d,
procedure described in the preparation of 7e.
2-[(3S)-3-(2-Methoxy-2-oxoethyl)-4-[(2S)-3-(4-methoxyphenyl)-2-[[4-(5-
Jϭ8.6 Hz), 7.23—7.40 (1H, m), 7.92 (2H, d, Jϭ8.8 Hz), 8.20 (2H, d,
Jϭ8.8 Hz), 8.81 (1H, s). IR (KBr) cmϪ1: 3390, 1730, 1660. Anal. Calcd for
oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)benzoyl]amino]propanoyl]-2-ox- C28H29N5O9·0.3H2O: C, 57.49; H, 5.10; N, 11.97. Found: C, 57.57; H, 5.00;
opiperazinyl]acetic Acid (7a): Colorless crystals (EtOH), mp 157—161 °C. N, 11.86.
1
[a]D20 ϩ56.4° (cϭ0.44, MeOH). H-NMR (DMSO-d6ϩD2O) d: 2.60—2.85
2-[(3S)-4-[(2S)-2-[[4-(5-Cyano-1,2,4-oxadiazol-3-yl)benzoyl]amino]-
(2H, m), 2.85—4.20 (8H, m), 3.49 (3H, s), 3.70 (3H, s), 4.89 (1H, t, 3-(4-methoxyphenyl)propanoyl]-3-(2-methoxy-2-oxoethyl)-2-oxopiper-
Jϭ6.0 Hz), 4.92—5.20 (1H, m), 6.82 (2H, d, Jϭ8.6 Hz), 7.22 (2H, d,
Jϭ8.6 Hz), 7.80—8.00 (4H, m). IR (KBr) cmϪ1: 1780, 1730, 1640, 1510,
1440, 1300, 1250, 1180. Anal. Calcd for C28H29N5O10 ·1.5H2O: C, 54.02; H,
5.18; N, 11.25. Found: C, 54.31; H, 5.05; N, 11.25.
azinyl]acetic Acid (7k): Colorless crystals, mp 177—179 °C. [a]D20 ϩ59.6°
1
(cϭ0.66, MeOH). H-NMR (CDCl3) d: 2.40—3.35 (4H, m), 2.98 (2H, d,
Jϭ5.6 Hz), 3.61 (3H, s), 3.65—3.90 (2H, m), 3.76 (3H, s), 4.08 (1H, s), 4.12
(1H, s), 5.05 (1H, t, Jϭ5.1 Hz), 5.20—5.42 (1H, m), 6.82 (2H, d, Jϭ8.6 Hz),
7.13 (2H, d, Jϭ8.6 Hz), 7.38 (1H, d, Jϭ8.4 Hz), 7.94 (2H, d, Jϭ8.4 Hz),
2-[(3S)-3-(2-Methoxy-2-oxoethyl)-4-[(2S)-3-(4-methoxyphenyl)-2-[[4-(5-
thioxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)benzoyl]amino]propanoyl]-2-ox- 8.14 (2H, d, Jϭ8.4 Hz). IR (KBr) cmϪ1: 1730, 1650, 1610, 1510. Anal.
opiperazinyl]acetic Acid (7b): A colorless amorphous powder, [a]D20 ϩ69.3° Calcd for C29H28N6O9: C, 57.61; H, 4.67; N, 13.90. Found: C, 57.42; H,
(cϭ0.63, H2O). 1H-NMR (D2O) d: 2.10—3.17 (6H, m), 3.35—4.05 (5H,
m), 3.51 (3H, s), 3.77 (3H, s), 4.95 (1H, t, Jϭ6.4 Hz), 6.87 (2H, d,
4.69; N, 13.68.
2-[(3S)-4-[(2S)-2-[[4-(5-Methoxy-1,2,4-oxadiazol-3-yl)benzoyl]amino]-
Jϭ8.4 Hz), 7.11 (2H, d, Jϭ8.4 Hz), 7.75 (2H, d, Jϭ8.4 Hz), 7.86 (2H, d, 3-(4-methoxyphenyl)propanoyl]-3-(2-methoxy-2-oxoethyl)-2-oxopiper-
Jϭ8.4 Hz). IR (KBr) cmϪ1: 1728, 1634, 1543, 1512, 1441, 1400, 1345, azinyl]acetic Acid (7l): A colorless amorphous powder, [a]D20 ϩ60.8° (cϭ
1
1304, 1248. Anal. Calcd for C28H29N5O9S·7H2O: C, 45.59; H, 5.87; N, 9.49. 0.99, MeOH). H-NMR (DMSO-d6) d: 2.50—4.20 (10H, m), 3.49 (3H, s),
Found: C, 45.30; H, 5.17; N, 9.58.
2-[(3S)-4-[(2S)-2-[[4-Amino(hydroxyimino)methyl]benzoyl]amino]-
3-(4-methoxyphenyl)propanoyl]-3-(2-methoxy-2-oxoethyl)-2-oxo-
3.70 (3H, s), 4.28 (3H, s), 4.90 (1H, t, Jϭ5.8 Hz), 5.00—5.20 (1H, m), 6.87
(2H, d, Jϭ8.6 Hz), 7.22 (2H, d, Jϭ8.6 Hz), 7.85—8.10 (4H, m), 8.94 (1H, d,
Jϭ7.6 Hz). IR (KBr) cmϪ1: 1780, 1730, 1660, 1610, 1510, 1380, 1250. Anal.
piperazinyl]acetic Acid Trifluoroacetate (7c): A colorless amorphous pow- Calcd for C29H31N5O10·0.1H2O: C, 56.97; H, 5.14; N, 11.45. Found: C,
der, [a]D20 ϩ55.4° (cϭ0.81, MeOH). 1H-NMR (D2O) d: 2.15—3.20 (6H, m), 56.72; H, 5.13; N, 11.45.
3.53 (3H, s), 3.56—4.10 (4H, m), 3.76 (3H, s), 4.98 (1H, t, Jϭ6.5 Hz),
2-[(3S)-3-(2-Methoxy-2-oxoethyl)-4-[(2S)-3-(4-methoxyphenyl)-2-[[4-
5.00—5.18 (1H, m), 6.90 (2H, d, Jϭ8.6 Hz), 7.19 (2H, d, Jϭ8.6 Hz), 7.74 (5-oxo-4,5-dihydro-1,2,4-thiadiazol-3-yl)benzoyl]amino]propanoyl]-2-ox-
(2H, d, Jϭ8.4 Hz), 7.86 (2H, d, Jϭ8.4 Hz). IR (KBr) cmϪ1: 1730, 1640,
opiperazinyl]acetic Acid (7d) A suspension of 5 (1.8 g, 3.0 mmol) and
1510, 1440, 1250, 1200, 1180, 1140. Anal. Calcd for C27H31N5O9· 10% Pd–C (0.32 g) in MeOH (80 ml) was hydrogenated under balloon pres-
CF3COOH·H2O: C, 49.65; H, 4.88; N, 9.98. Found: C, 49.72; H, 4.88; N, sure for 1 h at room temperature. The catalyst was removed by filtration and
10.14.
the filtrate was concentrated under reduced pressure to give amine. Then, to
a solution of this amine in DMF (18 ml) were added 4-(5-oxo-4,5-dihydro-
2-[(3S)-3-(2-Methoxy-2-oxoethyl)-4-[(2S)-3-(4-methoxyphenyl)-2-[[4-(2-
oxo-2,3-dihydro-1,2l4,3,5-oxathiadiazol-4-yl)benzoyl]amino]propanoyl]-2- 1,2,4-thiadiazol-3-yl)benzoic acid 39 (0.74 g, 3.32 mmol) and EDC (0.76 g,
oxopiperazinyl]acetic Acid (7f): A colorless amorphous powder, [a]D20 3.6 mmol) at room temperature. After being stirred for 1 h, the mixture was
1
ϩ58.6° (cϭ0.60, MeOH). H-NMR (DMSO-d6ϩD2O) d: 2.50—4.20 (10H,
diluted with EtOAc, washed with 5% aqueous KHSO4, dried over MgSO4,
m), 3.49 (3H, s), 3.70 (3H, s), 4.90 (1H, t, Jϭ5.4 Hz), 4.96—5.22 (1H, m), and concentrated under reduced pressure to give crude 6d.
6.81 (2H, d, Jϭ8.4 Hz), 7.22 (2H, d, Jϭ8.4 Hz), 7.80—8.10 (4H, m). Anal.
Calcd for C27H29N5O10S ·1.5H2O: C, 50.46; H, 5.02; N, 10.90. Found: C,
50.76; H, 5.08; N, 11.07.
Then, a mixture of 6d, CH2Cl2 (5.0 ml) and TFA (5.0 ml) was stirred at
room temperature for 2 h. The mixture was concentrated under reduced pres-
sure and the residue was recrystallized from EtOH to give 7d (0.87 g, 47%)
1
2-[(3S)-3-(2-Methoxy-2-oxoethyl)-4-[(2S)-3-(4-methoxyphenyl)-2-[[4- as colorless crystals, mp 224—226 °C. [a]D20 ϩ8.0° (cϭ0.69, DMSO). H-
(5-methyl-1,2,4-oxadiazol-3-yl)benzoyl]amino]propanoyl]-2-oxo-
piperazinyl]acetic Acid (7g): Colorless crystals (MeOH–Et2O), mp 165—
NMR (DMSO-d6ϩD2O) d: 2.62—2.88 (2H, m), 2.88—4.20 (8H, m), 3.48
(3H, s), 3.69 (3H, s), 4.89 (1H, t, Jϭ6.2 Hz), 4.98—5.20 (1H, m), 6.81 (2H,
166 °C. [a]D20 ϩ61.3° (cϭ0.73, MeOH). 1H-NMR (CDCl3ϩD2O) d: 2.67 d, Jϭ8.5 Hz), 7.21 (2H, d, Jϭ8.5 Hz), 7.93 (2H, d, Jϭ8.5 Hz), 8.01 (2H, d,
(3H, s), 2.30—3.30 (6H, m), 3.62 (3H, s), 3.70—3.90 (2H, m), 3.79 (3H, s), Jϭ8.5 Hz). IR (KBr) cmϪ1: 1740, 1710, 1660, 1640, 1510, 1450, 1240.
4.10 (2H, d, Jϭ5.8 Hz), 5.05 (1H, t, Jϭ5.4 Hz), 5.25—5.45 (1H, m), 6.83 Anal. Calcd for C28H29N5O9S·0.5H2O: C, 54.19; H, 4.87; N, 11.28. Found:
(2H, d, Jϭ8.6 Hz), 7.14 (2H, d, Jϭ8.6 Hz), 7.89 (2H, d, Jϭ8.4 Hz), 8.15 C, 54.24; H, 4.73; N, 11.38.
(2H, d, Jϭ8.4 Hz). IR (KBr) cmϪ1: 1740, 1660, 1540, 1510, 1450, 1250,
2-[(3S)-4-[(2S)-2-[[4-[Amino[[(methoxycarbonyl)oxy]imino]methyl]-
1180. Anal. Calcd for C29H31N5O9: C, 58.68; H, 5.29; N, 11.80. Found: C, benzoyl]amino]-3-(4-methoxyphenyl)propanoyl]-3-(2-methoxy-2-oxo-
58.43; H, 5.02; N, 11.77. ethyl)-2-oxopiperazinyl]acetic Acid (8a) To a mixture of 6c (3.50 g, 5.59
2-[(3S)-3-(2-Methoxy-2-oxoethyl)-4-[(2S)-3-(4-methoxyphenyl)-2-[[4- mmol), potassium carbonate (0.39 g, 2.80 mmol) and 1,4-dioxane (11.2 ml)
(5-phenyl-1,2,4-oxadiazol-3-yl)benzoyl]amino]propanoyl]-2-oxo- was added dropwise methyl chloroformate (0.45 ml, 5.87 mmol) at room

March 2001
275
temperature. After being stirred for 1 h at room temperature, the mixture was 7-ene (98%, 3.35 g, 21.6 mmol) at room temperature, and the mixture was
poured into aqueous 5% KHSO4 and extracted with EtOAc. The organic stirred for 30 min at 80 °C. After being cooled, the solvent was removed by
layer was dried over MgSO4 and concentrated under reduced pressure. The evaporation and then water was added to the residue. The mixture was ad-
residue was purified by silica gel column chromatography (EtOAc) to give justed to pH 2 with 3 N HCl, and the precipitate was collected, washed with
ester (3.23 g, 85%) as a yellow amorphous powder. Then, a mixture of the water, and dried to provide 13 (4.74 g, 97%) as colorless crystals, mp 202—
1
ester, TFA (5.0 ml) and CH2Cl2 (5.0 ml) was stirred for 1 h at room tempera-
203 °C (dec.). H-NMR (CDCl3ϩDMSO-d6) d: 3.96 (3H, s), 8.02 (2H, d,
ture. The mixture was concentrated under reduced pressure and the residue Jϭ8.5 Hz), 8.17 (2H, Jϭ8.5 Hz). IR (KBr) cmϪ1: 1790, 1694, 1597, 1553,
was purified by column chromatography (CHP-20, gradient elution: H2O to 1464, 1435, 1416, 1323, 1287, 1148, 1117. Anal. Calcd for C10H8N2O3S: C,
30% aqueous CH3CN) to give 8a (2.76 g, 91%) as a colorless amorphous 50.84; H, 3.41; N, 11.86. Found: C, 51.05; H, 3.32; N, 12.02.
powder, [a]D20 ϩ13.8° (cϭ0.83, DMSO). 1H-NMR (DMSO-d6) d: 2.50—
4.50 (10H, m), 3.48 (3H, s), 3.69 (3H, s), 3.78 (3H, s), 4.89 (1H, t,
4-(5-Thioxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)benzoic Acid (15)
suspension of 13 (4.74 g, 20 mmol), 2 N NaOH (30 ml) and MeOH (50 ml)
A
Jϭ6.0 Hz), 4.95—5.20 (1H, m), 6.80 (2H, d, Jϭ8.4 Hz), 6.82—7.04 (2H, was stirred for 16 h at room temperature, and the solvent was removed by
m), 7.21 (2H, d, Jϭ8.4 Hz), 7.75 (2H, d, Jϭ8.6 Hz), 7.87 (2H, d, Jϭ8.6 Hz), evaporation. Water was added to the residue, and the mixturte was adjusted
8.87 (1H, d, Jϭ7.8 Hz). Anal. Calcd for C29H33N5O11·H2O: C, 53.95; H,
5.46; N, 10.85. Found: C, 54.01; H, 5.48; N, 10.92.
to pH 2 with 3 N HCl. The precipitate was collected, washed with water, and
dried to give 15 (2.27 g, 51%) as colorless crystals, mp Ͼ300 °C. H-NMR
1
2-[(3S)-4-[(2S)-2-[[4-[Amino[[[(methylsulfanyl)carbonyl]oxy]imino]-
(DMSO-d6) d: 8.01 (2H, d, Jϭ8.0 Hz), 8.12 (2H, d, Jϭ8.0 Hz). IR (KBr)
methyl]benzoyl]amino]-3-(4-methoxyphenyl)propanoyl]-3-(2-methoxy-2- cmϪ1: 1690, 1590, 1550, 1480, 1430, 1320, 1290. Anal. Calcd for
oxoethyl)-2-oxopiperazinyl]acetic Acid (8b) The title compound was pre-
C9H6N2O3S: C, 48.64; H, 2.74; N, 12.61. Found: C, 48.80; H, 2.84; N,
pared from 6c and methyl chlorothioformate according to the procedure de- 12.84.
scribed in the preparation of 8a as a yellow amorphous powder, [a]D20
ϩ55.0° (cϭ0.79, MeOH). 1H-NMR (CDCl3) d: 2.37 (3H, s), 2.40—4.20
4-(5-Oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)benzoic Acid (14) The title
compound was prepared from 12 according to the procedure described in the
(10H, m), 3.57 (3H, s), 3.74 (3H, s), 5.04 (1H, t, Jϭ5.6 Hz), 5.15—5.40 preparation of 15 as colorless crystals (DMF–EtOAc), mp Ͼ300 °C. 1H-
(1H, m), 5.45—5.90 (2H, m), 6.79 (2H, d, Jϭ8.6 Hz), 7.34—7.54 (1H, m),
NMR (DMSO-d6) d: 7.93 (2H, d, Jϭ8.4 Hz), 8.11 (2H, d, Jϭ8.4 Hz). IR
7.09 (2H, d, Jϭ8.6 Hz), 7.72 (2H, d, Jϭ8.6 Hz), 7.79 (2H, d, Jϭ8.6 Hz). (KBr) cmϪ1: 1800, 1740, 1690, 1550, 1430, 1320, 1290, 950, 760. Anal.
Anal. Calcd for C29H33N5O10S·0.5H2O: C, 53.37; H, 5.25; N, 10.73. Found: Calcd for C9H6N2O4·0.1DMF: C, 52.33; H, 3.16; N, 13.76. Found: C, 52.14;
C, 53.40; H, 5.49; N, 10.78.
H, 3.29; N, 13.89.
2-[(3S)-4-[(2S)-2-[[4-[[(Acetyloxy)imino](amino)methyl]benzoyl]-
amino]-3-(4-methoxyphenyl)propanoyl]-3-(2-methoxy-2-oxoethyl)-2-oxo-
Methyl 4-(5-Methyl-1,2,4-oxadiazol-3-yl)benzoate (16) To a solution
of 10 (1.94 g, 10 mmol) in CH2Cl2 (20 ml) was added acetic anhydride
piperazinyl]acetic Acid (8c) The title compound was prepared from 6c (2.25 ml, mmol) at room temperature, and the mixture was stirred for 9 h at
and acetyl chloride according to the procedure described in the preparation 80 °C. After being cooled, the mixture was concentrated under reduced pres-
of 8a as a colorless amorphous powder, [a]D20 ϩ13.1° (cϭ1.00, DMSO). 1H- sure. The residue was purified by silica gel column chromatography (EtOAc)
1
NMR (DMSO-d6) d: 2.14 (3H, s), 2.60—4.55 (10H, m), 3.47 (3H, s), 3.69 to give 16 (1.90 g, 87%) as colorless crystals, mp 146—148 °C. H-NMR
(3H, s), 4.89 (1H, t, Jϭ6.0 Hz), 4.95—5.20 (1H, m), 6.80 (2H, d, Jϭ8.4 Hz), (CDCl3) d: 2.68 (3H, s), 3.96 (3H, s), 8.15 (4H, s). IR (KBr) cmϪ1: 1720,
6.80—7.05 (2H, m), 7.21 (2H, d, Jϭ8.4 Hz), 7.77 (2H, d, Jϭ8.4 Hz), 7.87 1600, 1440, 1410, 1280, 1265, 1120, 1110, 730. Anal. Calcd for
(2H, d, Jϭ8.4 Hz), 8.87 (1H, d, Jϭ7.8 Hz). Anal. Calcd for C11H10N2O3: C, 60.55; H, 4.62; N, 12.84. Found: C, 60.37; H, 4.61; N,
C29H33N5O10·0.5H2O: C, 56.12; H, 5.52; N, 11.28. Found: C, 56.19; H, 5.38; 12.90.
N, 11.19.
2-[(3S)-4-[(2S)-2-[[4-[Amino[[[(ethylamino)carbonyl]oxy]imino]-
Methyl 4-(5-Trifluoromethyl-1,2,4-oxadiazol-3-yl)benzoate (17) To a
suspension of 10 (1.94 g, 10 mmol) in CH2Cl2 (20 ml) was added trifluo-
methyl]benzoyl]amino]-3-(4-methoxyphenyl)propanoyl]-3-(2-methoxy-2- roacetic anhydride (3.15 g, 15 mmol) at room temperature. After being
oxoethyl)-2-oxopiperazinyl]acetic Acid (8d) The title compound was pre- stirred for 3 h at room temperature, the mixture was diluted with toluene and
pared from 6c and ethyl isocyanate according to the procedure described in concentrated under reduced pressure. The residue was purified by silica gel
the preparation of 8a as a colorless amorphous powder, [a]D20 ϩ45.6° column chromatography (EtOAc) to give 17 (2.0 g, 74%) as colorless crys-
1
1
(cϭ1.01, MeOH). H-NMR (DMSO-d6) d: 1.08 (3H, t, Jϭ7.0 Hz), 2.58— tals (hexane), mp 78—79 °C. H-NMR (CDCl3) d: 3.97 (3H, s), 8.20 (4H,
2.83 (2H, m), 2.88—4.25 (10H, m), 3.47 (3H, s), 3.69 (3H, s), 4.89 (1H, t, s). IR (KBr) cmϪ1: 1720, 1620, 1530, 1430, 1320, 1280, 1220, 1160, 1110,
Jϭ6.0 Hz), 4.95—5.25 (1H, m), 6.72—7.00 (4H, m), 7.23 (2H, d, Jϭ8.8 1090, 995, 870, 760, 710. Anal. Calcd for C11H7F3N2O3: C, 48.54; H, 2.59;
Hz), 7.30—7.45 (1H, m), 7.80—8.00 (4H, m), 8.87 (1H, d, Jϭ8.0 Hz). IR N, 10.29. Found: C, 48.59; H, 2.60; N, 10.48.
(KBr) cmϪ1: 3400, 1730, 1640, 1510, 1440, 1300, 1250, 1180. Anal. Calcd
for C30H36N6O10·2H2O: C, 51.87; H, 5.80; N, 12.10. Found: C, 52.01; H,
5.74; N, 12.00.
Methyl 4-(5-Phenyl-1,2,4-oxadiazol-3-yl)benzoate (18) To a mixture
of 10 (1.94 g, 10 mmol), pyridine (1.04 ml, 13 mmol) and xylene (50 ml) was
added benzoyl chloride (1.39 ml, 12 mmol), and the mixture was stirred for
Methyl 4-[Amino(hydroxyimino)methyl]benzoate (10) A mixture of 1 h at 140 °C. After being cooled, the mixture was diluted with EtOAc,
methyl 4-cyanobenzoate (16.5 g, 102 mmol), hydroxylamine hydrochloride washed with water, saturated aqueous NaHCO3 and brine, dried over
(7.2 g, 102 mmol), sodium hydrogencarbonate (8.82 g, 110 mmol) and MgSO4, and concentrated under reduced pressure. The residue was recrys-
MeOH (200 ml) was stirred for 30 min at room temperature and refluxed for tallized from hexane to provide 18 (2.54 g, 91%) as colorless crystals, mp
3 h. After being cooled, water (400 ml) was added to the mixture. The pre- 151—152 °C. 1H-NMR (CDCl3) d: 3.97 (3H, s), 7.50—7.70 (3H, m),
cipitated crystals were collected, washed with water and Et2O and dried to 8.10—8.35 (6H, m). IR (KBr) cmϪ1: 1705, 1688, 1613, 1534, 1433, 1325,
1
provide 10 (16.1 g, 83%) as colorless needles, mp 171—173 °C. H-NMR 1290, 1207, 1192, 1161. Anal. Calcd for C16H12N2O3: C, 68.56; H, 4.32; N,
(CDCl3) d: 3.86 (3H, s), 5.93 (2H, s), 7.82 (2H, d, Jϭ8.4 Hz), 7.95 (2H, d,
Jϭ8.4 Hz), 9.91 (1H, s). IR (KBr) cmϪ1: 1717, 1651, 1609, 1593, 1431,
1323, 1288, 1186, 1113, 951. Anal. Calcd for C9H10N2O3: C, 55.67; H, 5.19;
N, 14.43. Found: C, 55.57; H, 5.22; N, 14.39.
9.99. Found: C, 68.08; H, 4.35; N, 9.91.
4-(5-Methyl-1,2,4-oxadiazol-3-yl)benzoic Acid (19) The title com-
pound was prepared from 16 according to the procedure described in the
preparation of 15 as colorless crystals, mp 271—273 °C. 1H-NMR (DMSO-
Methyl 4-(5-Oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)benzoate (12) To a d6) d: 2.69 (3H, s), 8.11 (4H, s). IR (KBr) cmϪ1: 1688, 1597, 1566, 1535,
suspension of 10 (3.95 g, 20.3 mmol) in 1,4-dioxane (20 ml) was added CDI 1431, 1416, 1319, 1288, 1260. Anal. Calcd for C10H8N2O3: C, 58.82; H,
(4.0 g, 24.7 mmol) at room temperature and the mixture was stirred for 3.95; N, 13.72. Found: C, 58.83; H, 4.01; N, 13.93.
30 min at 110 °C. After being cooled, solvent was removed by evaporation
4-(5-Trifluoromethyl-1,2,4-oxadiazol-3-yl)benzoic Acid (20) The title
and then water was added to the residue. The mixture was adjusted to pH 2 compound was prepared from 17 according to the procedure described in the
with 3 N HCl, and the precipitate was collected and washed with water. Re-
preparation of 15 as colorless crystals, mp 234—237 °C. 1H-NMR (DMSO-
crystallization from DMF–EtOAc gave 12 (3.1 g, 69%) as colorless crystals, d6) d: 8.16 (2H, d, Jϭ8.0 Hz), 8.22 (2H, d, Jϭ8.0 Hz). IR (KBr) cmϪ1: 1705,
mp 278—280 °C. 1H-NMR (CDCl3) d: 3.88 (3H, s), 7.94 (2H, d, Jϭ8.4 Hz), 1688, 1534, 1433, 1325, 1291, 1207, 1192, 1161. Anal. Calcd for
8.06 (2H, d, Jϭ8.4 Hz). IR (KBr) cmϪ1: 1780, 1690, 1530, 1470, 1430,
C10H5F3N2O3: C, 46.53; H, 1.95; N, 10.85. Found: C, 46.52; H, 1.91; N,
1305, 1290. Anal. Calcd for C10H8N2O4: C, 54.55; H, 3.66; N, 12.72. Found: 11.03.
C, 54.42; H, 3.80; N, 12.95.
4-(5-Phenyl-1,2,4-oxadiazol-3-yl)benzoic Acid (21) The title com-
Methyl 4-(5-Thioxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)benzoate (13) pound was prepared from 18 according to the procedure described in the
To a suspension of 10 (4.00 g, 20.6 mmol) and TCDI (90%, 4.28 g, preparation of 15 as colorless crystals, mp 265—267 °C. 1H-NMR (DMSO-
21.6 mmol) in 1,4-dioxane (20 ml) was added 1,8-diazabicyclo[5.4.0]undec- d6) d: 7.60—7.81 (3H, m), 8.10—8.30 (2H, m), 8.15 (2H, d, Jϭ8.8 Hz),

276
Vol. 49, No. 3
8.23 (2H, d, Jϭ8.8 Hz). IR (KBr) cmϪ1: 1700, 1410, 1290, 730. Anal. Calcd
for C15H10N2O3: C, 67.67; H, 3.79; N, 10.52. Found: C, 67.35; H, 3.61; N,
10.56.
matography (hexane–EtAOc, 2 : 1) to provide 29 (0.18 g, 33%) as colorless
1
crystals (hexane), mp 102—104 °C. H-NMR (CDCl3) d: 1.62 (9H, s), 8.16
(4H, s). IR (KBr) cmϪ1: 2250, 1705, 1550, 1310, 1300, 1250, 1160, 1130,
730. Anal. Calcd for C14H13N3O3: C, 61.99; H, 4.83; N, 15.49. Found: C,
61.86; H, 4.87; N, 15.44.
tert-Butyl 4-[Amino(hydroxyimino)methyl]benzoate (23) A mixture
of tert-butyl 4-cyanobenzoate (9.0 g, 44.3 mmol), hydroxylamine hydrochlo-
ride (3.08 g, 44.3 mmol), NaHCO3 (4.0 g, 48.0 mmol), water (10 ml) and
tert-BuOH (100 ml) was stirred for 1.5 h at 80 °C. After being cooled, the
mixture was diluted with EtOAc, washed with water and brine, dried over
MgSO4, and concentrated under reduced pressure. The residue was recrys-
tallized from hexane–EtOAc (4 : 1) to provide 23 (8.0 g, 76%) as colorless
4-(1,2,4-Oxadiazol-3-yl)benzoic Acid (30) To a solution of 26 (0.49 g,
2.0 mmol) in CH2Cl2 (4 ml) was added TFA (4 ml) at room temperature.
After being stirred for 1 h at room temperature, the mixture was diluted with
toluene and concentrated under reduced pressure. The residue was recrystal-
lized from EtOAc–MeOH to provide 30 (0.33 g, 90%) as colorless crystals,
1
1
mp 216 °C. H-NMR (DMSO-d6) d: 8.13 (2H, d, Jϭ8.8 Hz), 8.19 (2H, d,
crystals, mp 153—155 °C. H-NMR (CDCl3) d: 1.60 (9H, s), 4.93 (2H, s),
Jϭ8.8 Hz), 9.78 (1H, s). IR (KBr) cmϪ1: 1680, 1580, 1535. Anal. Calcd for
C9H6N2O3: C, 56.85; H, 3.18; N, 14.73. Found: C, 56.87; H, 3.14; N, 14.64.
4-(5-Cyano-1,2,4-oxadiazol-3-yl)benzoic Acid (31) The title com-
pound was prepared from 29 according to the procedure described in the
preparation of 30 as colorless crystals (Et2O), mp Ͼ300 °C. 1H-NMR
(DMSO-d6) d: 8.17 (4H, s). IR (KBr) cmϪ1: 1680, 1580, 1550, 1530, 1430,
1410, 1340, 1320, 1290, 1250. Anal. Calcd for C10H5N3O3·0.1H2O: C,
55.36; H, 2.42; N, 19.37. Found: C, 55.67; H, 2.67; N, 18.78.
7.68 (2H, d, Jϭ8.8 Hz), 8.01 (2H, d, Jϭ8.8 Hz), 9.30—9.80 (1H, m). IR
(KBr) cmϪ1: 1705, 1670, 1410, 1370, 1320, 1310, 1300, 1170, 1150, 1130,
1120, 950, 870, 850, 780, 710. Anal. Calcd for C12H16N2O3: C, 61.00; H,
6.83; N, 11.86. Found: C, 61.03; H, 6.70; N, 11.90.
tert-Butyl 4-(2-Oxo-2,3-dihydro-1,2l4,3,5-oxathiadiazol-4-yl)benzoate
(24) To a mixture of 23 (2.0 g, 8.46 mmol), pyridine (1.47 ml, 16.9 mmol)
and CH2Cl2 (17 ml) was added dropwise a solution of thionyl chloride
(0.68 ml, 9.31 mmol) in CH2Cl2 (17 ml) at Ϫ20 °C. After being stirred for
40 min at Ϫ5 °C, the mixture was washed with water and brine, dried over
MgSO4, and concentrated under reduced pressure. The residue was recrys-
tallized from Et2O–petroleum ether (1 : 1) to provide 24 (1.77 g, 74%) as col-
tert-Butyl 4-(5-Methoxy-1,2,4-oxadiazol-3-yl)benzoate (33) To a solu-
tion of 23 (2.36 g, 10 mmol) in 1,4-dioxane (25 ml) was added CDI (2.1 g,
13 mmol) at room temperature and the mixture was stirred for 30 min at
90 °C. After being cooled, the mixture was concentrated under reduced pres-
sure and the residue was partitioned between EtOAc (250 ml) and 5% aque-
ous KHSO4. Then, an excess solution of diazomethane in diethyl ether (15—
20 mmol) was added dropwise to the organic layer at room temperature.
After being kept standing for 18 h, the mixture was washed with water and
brine, dried over MgSO4, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (hexane–EtAOc,
2 : 1) to provide 33 (1.40 g, 51%) as colorless crystals (hexane–EtAOc, 8 : 1),
mp 133—134 °C. 1H-NMR (CDCl3) d: 1.62 (9H, s), 4.29 (3H, s), 8.07 (4H,
s). IR (KBr) cmϪ1: 1700, 1620, 1580, 1500, 1410, 1380, 1370, 1320, 1310,
1290, 1250, 1170, 1120, 1110, 1040, 1020, 980, 960, 880, 870, 850, 750,
710. Anal. Calcd for C14H16N2O4: C, 60.86; H, 5.84; N, 10.14. Found: C,
60.96; H, 5.76; N, 10.27.
1
orless crystals, mp 150—152 °C (dec.). H-NMR (CDCl3) d: 1.62 (9H, s),
7.78 (2H, d, Jϭ8.8 Hz), 8.08 (2H, d, Jϭ8.8 Hz). IR (KBr) cmϪ1: 1705, 1291,
1169. Anal. Calcd for C12H14N2O4S: C, 51.05; H, 5.00; N, 9.92. Found: C,
51.25; H, 4.79; N, 9.92.
4-(2-Oxo-2,3-dihydro-1,2l4,3,5-oxathiadiazol-4-yl)benzoic Acid (25)
To a solution of 24 (1.0 g, 3.54 mmol) in CH2Cl2 (3 ml) was added TFA
(3 ml) at room temperature. After being stirred for 1 h at room temperature,
the mixture was diluted with toluene and concentrated under reduced pres-
sure to provide 25 (0.93 g, quant.) as a colorless powder. 1H-NMR (DMSO-
d6) d: 7.98 (2H, d, Jϭ8.6 Hz), 8.12 (2H, d, Jϭ8.6 Hz). IR (KBr) cmϪ1: 1684,
1422, 1292, 1190. Anal. Calcd for C8H6N2O4S·0.25H2O: C, 41.65; H, 2.84;
N, 12.14. Found: C, 41.83; H, 2.74; N, 11.84.
tert-Butyl 4-(1,2,4-Oxadiazol-3-yl)benzoate (26) A suspension of 23
(4.72 g, 20 mmol) in trimethyl orthoformate (20 ml, 183 mmol) was added
BF3·Et2O (2 drops) at room temperature, and the mixture was stirred for
30 min at 110 °C. After being cooled, the mixture was diluted with EtOAc,
washed with water and saturated aqueous NaHCO3, dried over MgSO4, and
concentrated under reduced pressure. The residue was recrystallized from
hexane to provide 26 (3.7 g, 75%) as colorless crystals, mp 127—129 °C.
1H-NMR (CDCl3) d: 1.62 (9H, s), 8.11 (2H, d, Jϭ8.8 Hz), 8.19 (2H, d,
Jϭ8.8 Hz), 8.80 (1H, s). IR (KBr) cmϪ1: 3125, 2975, 1705, 1580, 1530,
1410, 1370, 1310, 1290, 1270, 1250, 1180, 1160, 1120, 1105, 890, 845, 730.
Anal. Calcd for C13H14N2O3: C, 63.40; H, 5.73; N, 11.38. Found: C, 63.43;
H, 5.71; N, 11.21.
Ethyl 3-[4-(tert-Butoxycarbonyl)phenyl]-1,2,4-oxadiazole-5-carboxy-
late (27) To a mixture of 23 (4.72 g, 20 mmol), pyridine (4.0 ml) and
CH2Cl2 (40 ml) was added ethyl chloroglyoxylate (2.68 ml, 24 mmol) at
room temperature. After being stirred for 24 h at room temperature, the mix-
ture was washed with water and saturated aqueous NaHCO3, dried over
MgSO4, and concentrated under reduced pressure. The residue was recrys-
tallized from hexane to provide 27 (5.5 g, 87%) as colorless crystals, mp
136—137 °C. 1H-NMR (CDCl3) d: 1.50 (3H, t, Jϭ7.0 Hz), 1.62 (9H, s),
4.59 (2H, q, Jϭ7.0 Hz), 8.11 (2H, d, Jϭ8.8 Hz), 8.22 (2H, d, Jϭ8.8 Hz). IR
(KBr) cmϪ1: 1750, 1707, 1586, 1561, 1530, 1466, 1370, 1312, 1296, 1190,
1169, 1123, 1013, 849, 725. Anal. Calcd for C16H18N2O5: C, 60.37; H, 5.70;
N, 8.80. Found: C, 60.35; H, 5.79; N, 8.84.
4-(5-Methoxy-1,2,4-oxadiazol-3-yl)benzoic Acid (35) The title com-
pound was prepared from 33 according to the procedure described in the
1
preparation of 30 as colorless crystals (hexane), mp 240—241 °C. H-NMR
(CDCl3) d: 4.28 (3H, s), 8.05 (2H, d, Jϭ8.8 Hz), 8.11 (2H, d, Jϭ8.8 Hz). IR
(KBr) cmϪ1: 1690, 1620, 1580, 1530, 1490, 1410, 1380, 1320, 1290, 750.
Anal. Calcd for C10H8N2O4: C, 54.55; H, 3.66; N, 12.72. Found: C, 54.53; H,
3.51; N, 12.76.
Methyl 4-(5-Oxo-4,5-dihydro-1,2,4-thiadiazol-3-yl)benzoate (37) To
a mixture of methyl 4-[amino(imino)methyl]benzoate hydrochloride 36
(7.82 g, 36.4 mmol), sodium carbonate (12.2 g, 115 mmol), H2O (50 ml) and
CH2Cl2 (50 ml) was added dropwise chlorocarbonylsulfenyl chloride (98%,
5.0 g, 37.4 mmol) at 0 °C with vigorous stirring. After being stirred for 1 h at
room temperature, the mixture was adjusted to pH 2 with conc. HCl and
concentrated under reduced pressure. Water was added to the residue, and
the precipitate was collected, washed with water, and dried to give 37 (3.8 g,
44%) as a yellow powder. Recrystallization from DMF–EtOAc gave 37 as
1
colorless crystals, mp 263—265 °C. H-NMR (DMSO-d6) d: 3.89 (3H, s),
8.09 (4H, s). IR (KBr) cmϪ1: 1720, 1715, 1665, 1655, 1280, 1110. Anal.
Calcd for C10H8N2O3S: C, 50.84; H, 3.41; N, 11.86. Found: C, 50.73; H,
3.35; N, 11.84.
Methyl 4-(5-Thioxo-4,5-dihydro-1,2,4-thiadiazol-3-yl)benzoate (38)
A mixture of 36 (6.14 g, 28.6 mmol), CS2 (5.7 g, 75.0 mmol), sulfur (1.2 g,
37.4 mmol), 28% sodium methoxide (17.4 g, 90.2 mmol) and MeOH (30 ml)
was refluxed for 6 h. After being cooled, the mixture was concentrated under
reduced pressure. Water was added to the residue and the mixture was ad-
justed to pH 2 with 3 N HCl. The precipitate was collected and recrystallized
from MeOH to give 38 (3.3 g, 46%) as colorless needles, mp 201—203 °C.
1H-NMR (CDCl3) d: 3.90 (3H, s), 8.10 (2H, d, Jϭ8.6 Hz), 8.18 (2H, d,
Jϭ8.6 Hz). IR (KBr) cmϪ1: 3200, 1710, 1540, 1450, 1435, 1320, 1290,
1220, 1200, 1120, 1070. Anal. Calcd for C10H8N2O2S2·0.5H2O: C, 45.96; H,
3.47; N, 10.72. Found: C, 46.04; H, 3.07; N, 10.78.
4-(5-Oxo-4,5-dihydro-1,2,4-thiadiazol-3-yl)benzoic Acid (39) The
title compound was prepared from 37 according to the procedure described
in the preparation of 15 as colorless crystals (DMF–EtOAc), mp Ͼ300 °C.
1H-NMR (DMSO-d6) d: 8.06 (4H, s). IR (KBr) cmϪ1: 1694, 1667, 1454,
1424, 1306, 1288. Anal. Calcd for C9H6N2O3S·0.1DMF: C, 48.66; H, 2.94;
N, 12.81. Found: C, 48.66; H, 3.15; N, 13.05.
tert-Butyl 4-(5-Carbamoyl-1,2,4-oxadiazol-3-yl)benzoate (28) A mix-
ture of 27 (2.0 g, 6.3 mmol), 25% aqueous NH3 (2 ml) and tetrahydrofuran
(100 ml) was stirred for 44 h at room temperature. The mixture was concen-
trated under reduced pressure and the residue was recrystallized from EtOH
1
to provide 28 (1.0 g, 55%) as colorless crystals, mp 202—205 °C. H-NMR
(CDCl3) d: 1.63 (9H, s), 6.05—6.22 (1H, m), 6.95—7.12 (1H, m), 8.12 (2H,
d, Jϭ8.8 Hz), 8.18 (2H, d, Jϭ8.8 Hz). IR (KBr) cmϪ1: 1710, 1690, 1610,
1580, 1400, 1370, 1330, 1310, 1300, 1280, 1230, 1170, 1120, 1110. Anal.
Calcd for C14H15N3O4: C, 58.13; H, 5.23; N, 14.53. Found: C, 57.98; H,
5.33; N, 14.45.
tert-Butyl 4-(5-Cyano-1,2,4-oxadiazol-3-yl)benzoate (29) To a mix-
ture of 28 (0.58 g, 2.0 mmol), pyridine (0.4 g, 5.0 mmol) and 1,4-dioxane
(8 ml) was added trifluoroacetic anhydride (0.34 ml, 2.4 mmol) at 0 °C. After
being stirred for 13 h at room temperature, the mixture was diluted with
EtOAc, washed with water and brine, dried over MgSO4, and concentrated
under reduced pressure. The residue was purified by silica gel column chro-
4-(5-Thioxo-4,5-dihydro-1,2,4-thiadiazol-3-yl)benzoic Acid (40) The
title compound was prepared from 38 according to the procedure described

March 2001
277
in the preparation of 15 as colorless crystals (DMF–MeOH), mp Ͼ300 °C.
1H-NMR (DMSO-d6) d: 8.06 (2H, d, Jϭ8.4 Hz), 8.16 (2H, d, Jϭ8.4 Hz). IR
Smith R. S., Turchi L. M., Zhang G., J. Med. Chem., 37, 2537—2551
(1994).
(KBr) cmϪ1: 1703, 1543, 1447, 1427, 1325, 1304, 1289, 1233. Anal. Calcd 10) Zablocki J. A., Rico J. G., Garland R. B., Rogers T. E., Williams K.,
for C9H6N2O2S2·0.8S: C, 40.96; H,2.29; N, 10.62. Found: C, 40.71; H, 2.39;
N, 10.61.
In Vitro Platelet Aggregation Studies Blood was collected by aortic
puncture from anesthetized guinea pigs with sodium pentobarbital. Blood
was withdrawn into a plastic syringe containing 3.15% sodium citrate (1 : 10
citrate/blood, v/v). PRP and platelet-poor plasma (PPP) were obtained by
Schretzman L. A., Rao S. A., Bovy P. R., Tjoeng F. S., Lindmark R. J.,
Toth M. V., Zupec M. E., McMackins D. E., Adams S. P., Miyano M.,
Markos C. S., Milton M. N., Paulson S., Herin M., Jacqmin P., Nichol-
son N. S., Panzer-Knodle S. G., Haas N. F., Page J. D., Szalony J. A.,
Taite B. B., Salyers A. K., King L. W., Campion J. G., Feigen L. P., J.
Med. Chem., 38, 2378—2394 (1995).
centrifugation at 1000ϫg for 3—5 s and 1000ϫg for 20 min at room tem- 11) Weller T., Alig L., Beresini M., Blackburn B., Bunting S., Hadváry P.,
perature respectively. The platelet count in PRP was adjusted to 4ϫ105/ml
using an automatic blood cell counter (Sysmex E2500, Toaiyoudenshi Co.,
Tokyo, Japan). Platelet aggregation was measured using an 8 channel aggre-
Müller M. H., Knopp D., Levet-Trafit B., Lipari M. T., Modi N. B.,
Müller M., Refino C. J., Schmitt M., Schönholzer P., Weiss S., Steiner
B., J. Med. Chem., 39, 3139—3147 (1996).
gometer (Hematracer VI, Niko Bioscience, Tokyo, Japan). PRP (250 ml), in 12) Eldred C. D., Evans B., Hindley S., Judkins B. D., Kelly H. A., Kitchin
a cuvette stirred at 1000 rpm, was prewarmed for 2 min at 37 °C with various
J., Lumley P., Porter B., Ross B. C., Smith K. J., Taylor N. R., Wheat-
concentrations of test compounds (25 ml). The change in light transmittance
croft J. R., J. Med. Chem., 37, 3882—3885 (1994).
was measured after the addition of aggregating agents (25 ml) to the cuvette. 13) EPIC Investigators, N. Engl. J. Med., 330, 956—961 (1994).
Submaximal concentration of aggregating agents were used in each experi- 14) Topol E. J., Califf R. M., Weisman H. F., Ellis S. G., Tcheng J. E.,
ment.
Ex Vivo Platelet Aggregation Studies Male Hartley guinea pigs
(300—400 g) were used. At various times after p.o. administration of test
Worley S., Ivanhoe R., George B. S., Fintel D., Weston M., Sigmon K.,
Anderson K. M., Lee K. L., Willerson J. T., EPIC Investigators,
Lancet, 343, 881—886 (1994).
compounds, blood was collected, and PRP and PPP were prepared as de- 15) Harrington R. A., Schulman S. P., Kleiman N. S., Lincoff A. M., Gold-
scribed for the in vitro study. ADP (20 ml) was added to the cuvette contain-
ing the prewarmed PRP (220 ml). In order to eliminate the possible influence
schmidt-Clermont P. J., Joseph D., Sigmon K. N., Parker J., Marchant
K., Kitt M. M., Circulation, 90 (Suppl. I), I-232. Abstract (1994).
of the sensitivity differences of guinea pig platelets to ADP, which is depen- 16) Tcheng J. E., Harrington R. A, Kottke-Marchant K., Kleiman N. S.,
dent on the animal lot and experimental conditions including drug adminis-
tration protocol, two or three vehicle-treated animals were used as a control
at each measuring point. The percentage inhibition of platelet aggregation in
Ellis S. G., Kereiakes D. J., Mick M. J., Navetta F. I., Smith J. E., Wor-
ley S. J., Miller J. A., Joseph D. M., Sigmon K. N., Kitt M. M., du Mee
C. P., Califf R. M., Topol E. J., Circulation, 91, 2151—2157 (1995).
drug-treated animals was determined by comparison with the aggregation in 17) Kereiakes D., Kleiman N., Ambrose J., Cohen M., Rodriguez S., Pal-
the controls at each point.
abrica T., Herrmann H. C., Sutton J., Weaer W. D., McKee D., Sax F.
Metabolic Conversion Test The liver (28 g) and small intestine (9 g)
L., Circulation, 90 (Suppl. I), I-21. Abstract (1994).
from Std Hartley guinea pigs were homogenized with ice-cooled saline (5 18) Théroux P., White H., David D., Van de Werf F., Nienaber C. A., Char-
and 2.5 ml, respectively) and then centrifugated. The supernatant were col-
lected for analysis. The plasma was prepared by centrifugation of he-
parinized whole blood. The compounds 7a and 7d (prepared as a 100 mg/ml
solution in saline, 100 ml) were incubated with each homogenate (900 ml)
and plasma (900 ml) at 37 °C for 1 h. The incubation was terminated by addi-
bonnier B., Erhardt L., Gill J., Hillis W. S., Jennings G., Tan L.-B., De-
schenes N., Fitzpatrick V., Sax F. L., Circulation, 90 (Suppl. I), I-231.
Abstract (1994).
19) Müller, T. H., Schurer H., Waldmann L., Bauer E., Himmelsbach F.,
Binder K., Thromb. Haemost., 69, 975, Abstract 1557 (1993).
tion of 3 ml of acetonitrile. Then, the samples were centrifugated at 20) Gante J., Juraszyk H., Raddatz P., Wuriger H., Bernotat-Danielowski
10000 rpm for 15 min, and the supernatants obtained were analyzed by
HPLC. HPLC analysis was performed using a Develosil ODS HG-5 reverse
phase column (4.6ϫ250 mm) with a mixture of acetonitrile and 20 mM phos-
phate buffer adjusted pH 3 with phosphoric acid containing 5 mM 1-heptane-
sulfonate (25 : 75, v/v) as a mobile phase at a flow rate of 1 ml/min, and UV
detection at 230 nm. The retention times for 7a, 7d and their parent com-
pound 4 were 31.0, 45.8 and 9.0, respectively.
S., Melzer G., Rippmann F., Bioorg. Med. Chem. Lett., 6, 2425—2430
(1996).
21) Kitamura S., Fukushi H., Miyawaki T., Kawamura M., Terashita Z.,
Sugihara H., Naka T., Chem. Pharm. Bull., 49, 258—267 (2001).
22) In a pharmacokinetics study of our previous GPIIb/IIIa antagonist
TAK-02923) in rats, the dicarboxyl derivative was not observed in
plasma after oral administration of TAK-029. The ester group at the 3-
position of the 2-oxopiperazine scaffold was assumed to be stable in
plasma, and the ester form (TAK-029) was regarded as the biological
active form by itself (unpublished data). Therefore, in the case of 4, we
considered that the ester form (4) is the biological active form by itself
as well as TAK-029.
23) Sugihara H., Fukushi H., Miyawaki T., Imai Y., Terashita Z., Kawa-
mura M., Fujisawa Y., Kita S., J. Med. Chem., 41, 489—502 (1998).
24) Boykin D. W., Kumar A., Hall J. E., Bender B. C., Tidwell R. R.,
Bioorg. Med. Chem. Lett., 6, 3017—3020 (1996).
25) Shahrokh Z., Lee E., Olivero A. G., Matamoros R. A., Robarge K. D.,
Lee A., Weise K. J., Blackburn B. K., Powell M. F., Pharm.Res., 15,
434—441 (1998).
References and Notes
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4)
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9)
Egbertson M. S., Chang C. T.-C., Duggan M. E., Gould R. J., Hal- 32) After addition of 8a to plasma, about 70% of 8a was converted to 7c
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