Synthesis of Cis and trans isomers of an isoxazoline ring-hydroxylated metabolite of roxifiban, a platelet glycoprotein IIb/IIIa receptor antagonist

Full text of DOI:10.1021/jo000755o

8100
J . Org. Chem. 2000, 65, 8100-8104
Syn th esis of Cis a n d Tr a n s Isom er s of a n
Isoxa zolin e Rin g-Hyd r oxyla ted Meta bolite
of Roxifiba n , a P la telet Glycop r otein
IIb/IIIa Recep tor An ta gon ist
Douglas G. Batt,* Gregory C. Houghton,
Wayne F. Daneker, and Prabhakar K. J adhav
Department of Chemical and Physical Sciences,
The DuPont Pharmaceuticals Company, Experimental
Station, P.O. Box 80500, Wilmington, Delaware 19880-0500
douglas.g.batt@dupontpharma.com
Received May 17, 2000
In tr od u ction
Roxifiban (1) is a potent and selective antagonist of the
platelet glycoprotein IIb/IIIa receptor, the major receptor
for fibrinogen on the surface of platelets.1,2 Since the final
step in platelet adhesion is the binding of fibrinogen to
GPIIb/IIIa, an antagonist of this receptor is expected to
provide benefit in a number of cardiovascular disorders
which involve inappropriate platelet adhesion.3 Roxifiban
is an ester prodrug of the active GPIIb/IIIa antagonist
XV459 (2), which is unusual among reported GPIIb/IIIa
antagonists in that it binds with equal affinity to both
the resting and activated forms of GPIIb/IIIa.1,2 This
could offer advantages over other such agents in provid-
ing a greater degree of antiplatelet activity as well as a
better pharmacokinetic profile arising from the reservoir
of drug bound to unactivated platelets.
Following dosing of 1 in rats, dogs, or humans, the
major metabolite was 2, as expected. In rats, intravenous
infusion of either 1 or 2 yielded several hydroxylated
metabolites of 2, which were identified using LC/MS, LC/
NMR, and high-field NMR.4 One interesting metabolite
was the ring 4-hydroxylated compound 3. NMR studies
were unable to firmly establish the stereochemistry of
the ring hydroxylation, although based on steric consid-
erations the trans isomer 3a would be expected to result
from enzymatic oxidation of 2.
F igu r e 1.
The approach to trans-4-hydroxy-5-substituted isox-
azolines reported by Wallace6 was used to prepare 3a ,
as shown in Scheme 1. The trans-olefinic boronate 5 was
prepared in 63% yield by hydroboration of 3-butyn-1-ol,
protected as the tetrahydropyran ether 4, using zirco-
nium catalysis.7 The resulting trans boronate underwent
cycloaddition with the nitrile oxide derived from 6,8 with
concomitant oxidation of the boronate to the alcohol, in
the presence of sodium percarbonate.9 The crude product
was acetylated, and the THP protecting group was
removed to provide 7 in 70% yield from 5. Oxidation of
the primary alcohol provided the acid 8 in 76% yield,
which was coupled with the amine 9,10 derived from (S)-
asparagine, to provide 10 in 84% yield. While the relative
stereochemistry of the ring substituents was fixed as
trans, the intermediate 8 was racemic, so coupling with
the optically active 9 provided the acetate of 10 as a
mixture of diastereomers. These were separated by
preparative HPLC using a chiral stationary phase; the
acetate was hydrolyzed to the alcohol during this separa-
tion. Roxifiban, having the (R) stereochemistry shown in
1, is levorotatory,11 so we tentatively assigned the
absolute configuration of the (-) isomer of 10 as (4S,5S)
(10a ), which corresponds to that of Roxifiban. However,
both diastereomers were carried through the remainder
of the synthesis separately.
Since the relative stereochemistry of the 4-hydroxy
group with respect to the 5-substituent of 3 was unclear,
both the cis and trans isomers were synthesized for
comparison with the isolated material. The relative
stereochemistry of the synthetic standards was firmly
established in each case through creation of the isoxazo-
line ring via [3 + 2] dipolar cycloaddition reactions, since
the known stereochemistry of the olefin dipolarophile
would be retained in the product.5
Conversion of the nitriles 10 to the amidines 11 was
achieved in 23% yield by treatment with excess hydroxyl-
amine, followed by selective acetylation of the amidoxime
and hydrogenolysis to the amidine.12 Attempted hydroly-
(5) Gru¨nanger, P.; Vita-Fiuzi, P. In Isoxazoles Part 1; Chemistry of
Heterocyclic Compounds; Taylor, E. C., Weissberger, A., Eds.; Wiley:
New York, 1991; Vol. 49, pp 475-523.
(1) Xue, C.-B.; Mousa, S. A. Drugs Future 1998, 23, 707-711.
(2) Xue, C.-B.; Wityak, J .; Sielecki, T. M.; Pinto, D. J .; Batt, D. G.;
Cain, G. A.; Sworin, M.; Rockwell, A. L.; Roderick, J . J .; Wang, S.;
Orwat, M. J .; Frietze, W. E.; Bostrom, L. L.; Liu, J .; Higley, C. A.;
Rankin, F. W.; Tobin, A. E.; Emmett, G.; Lalka, G. K.; Sze, J . Y.; Di
Meo, S. V.; Mousa, S. A.; Thoolen, M. J .; Racanelli, A. L.; Hausner, E.
A.; Reilly, T. M.; DeGrado, W. F.; Wexler, R. R.; Olson, R. E. J . Med.
Chem. 1997, 40, 2064-2084.
(6) Wallace, R. H.; Liu, J . Tetrahedron Lett. 1994, 35, 7493-7496.
(7) Pereira, S.; Srebnik, M. Organometallics 1995, 14, 3127-3128.
(8) Wityak, J .; Sielecki, T. M.; Pinto, D. J .; Emmett, G.; Sze, J . Y.;
Liu, J .; Tobin, A. E.; Wang, S.; J iang, B. J . Med. Chem. 1997, 40, 50-
60.
(9) Liu, J .; Eddings, A.; Wallace, R. H. Tetrahedron Lett. 1997, 38,
6795-6798.
(10) Xue, C. B.; Rafalski, M.; Roderick, J .; Eyermann, C. J .; Mousa,
S.; Olson, R. E.; De Grado, W. F. Bioorg. Med. Chem. Lett. 1996, 6,
339-344.
(3) Wityak, J .; Sielecki, T. M. Exp. Opin. Ther. Patents 1996, 6,
1175-1194.
(4) Mutlib, A. E.; Diamond, S.; Shockcor, J .; Nemeth, G.; Gan, L.;
Christ, D. D. Xenobiotica 2000, in press.
(11) Zhang, L.; Chung, J . C.; Costello, T. D.; Valvis, I.; Ma, P.;
Kauffman, S.; Ward, R. J . Org. Chem. 1997, 62, 2466-2470.
10.1021/jo000755o CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/21/2000

Notes
J . Org. Chem., Vol. 65, No. 23, 2000 8101
Sch em e 1. Syn th esis of tr a n s-4-Hyd r oxy Meta bolite (3a a n d 3b)a
a
Reagents: a, pinacolborane, Cp2ZrHCl; b, Na2CO3‚H2O2; c, acetic anhydride, pyridine; d, acetic acid, H2O; e, PDC; f, DCC, HOBT,
Et3N; HPLC separation; g, H2NOH‚HCl, Et3N; h, acetic anhydride; i, H2, Pd/C; j, rabbit liver esterase.
sis of the methyl ester under either acidic or basic
conditions led to significant dehydration of the ring to
the isoxazole, so the ester was removed by treatment with
rabbit liver esterase, providing 3a and 3b in 95% yield.
The direction of optical rotation remained the same for
each intermediate, with the (-) isomer again assumed
to be that corresponding to Roxifiban and assigned the
structure 3a .
trans stereochemistry of 7 and the cis stereochemistry
of 14 were supported by examination of the vicinal NMR
coupling constants observed for the proton at the 4-posi-
tion of the ring, geminal to the oxygen substituent. In 7,
this signal appeared as a doublet at δ 6.30, with J ) 2.5
Hz, while in 14 the doublet at δ 6.46 had J ) 6.9 Hz.
Although not completely diagnostic, the trans isomers of
4,5-disubstituted isoxazolines generally have smaller
coupling constants than cis isomers.15 According to the
vicinal Karplus correlation,16 the observed coupling con-
stants represent dihedral angles of approximately 119°
and 28° for the trans and cis isomers, respectively. These
values are in good agreement with the range of dihedral
angles achievable by the trans and cis isomers through
ring flexibility, as measured by examination of Dreiding
models of the trans isomer (110-140°, giving calculated
coupling constants of 1.5-5.9 Hz) and cis isomer (10-
30°; 8.5-6.7 Hz). The carbinol methine of the final
products 3 also showed similar coupling constants (3.3
Hz for the trans isomers, 7.3 Hz for the cis isomers).
The NMR spectra of the cis isomers 3c and 3d
displayed a doubling of the backbone hydrogens of the
diaminopropionate moiety which was not observed in 3a
and 3b. The â-alanine derivative 16, prepared by the
same method as 15 (Scheme 3), showed no peak doubling,
but the O-methylated derivative 17 displayed a distinct
pair of signals (2:1 ratio, both doublets with J ) 7.3 Hz)
for the isoxazoline C-4 proton and also a pair of singlets
for the methoxy protons. Both pairs coalesced reversibly
to sharp signals at 90 °C, suggesting that restricted
conformational mobility was indeed the cause. Since no
such effects were seen in the trans isomers, a barrier to
No known examples of cis-4-hydroxy-5-substituted
3-phenylisoxazolines have been reported in the literature.
Again taking advantage of the stereochemical retention
obtained from the [3 + 2] cycloaddition of nitrile oxides,5
the cis isomer 3c was prepared as shown in Scheme 2,
this time using furan as the dipolarophile.13 The cycload-
duct 12, obtained in 91% yield, was hydrated to the lactol
13 in 72% yield using a known procedure,14 followed by
J ones oxidation to the lactone 14 in 87% yield. This
racemic material could be resolved by HPLC using a
chiral stationary phase, providing the pure enantiomers
which were carried on separately. The lactone was
opened in 67% yield by treatment with 9, providing 15.
Once again, the levorotatory isomer was assigned the
absolute stereochemistry (4R,5S) (15c), corresponding to
that of Roxifiban. Amidine elaboration and ester hydroly-
sis were achieved in 23% yield on the separate diaster-
eomers as described above for the trans case. The
diastereomer which again retained the (-) optical rota-
tion was tentatively assigned structure 3c.
In addition to the theoretically expected relative ster-
eochemistry resulting from concerted cycloaddition, the
(12) Ma, P.; Confalone, P. N.; Li, H.; Du Pont Pharmaceuticals, U.S.
Patent, 1998.
(13) Caramella, P.; Cellerino, G.; Corsico Coda, A.; Gamba-Inv-
ernizzi, A.; Gruenanger, P.; Houk, K. N.; Marinone Albini, F. J . Org.
Chem. 1976, 41, 3349-3356.
(15) Reference 5, pp 421-436.
(16) J ackman, L. M.; Sternhell, S. Applications of NMR Spectroscopy
in Organic Chemistry, 2nd ed.; Pergamon: New York, 1969.
(14) Sabesan, S.; Neira, S. J . Org. Chem. 1991, 56, 5468-5472.

8102 J . Org. Chem., Vol. 65, No. 23, 2000
Notes
Ta ble 1. P la telet Aggr ega tion Resu lts
Sch em e 2. Syn th esis of cis-4-Hyd r oxy Meta bolite
(3c a n d 3d )a
compound
IC50, nMa
3a
3b
140 ( 37 (n ) 3)
6500 (n ) 1)
33 (n ) 1)
isolated metabolite
a
See ref 17 for details.
was correct. The latter point was supported by comparing
the activities of 3a and 3b in the human platelet-rich
plasma aggregation assay17 with that observed for the
isolated metabolite (Table 1); the IC50 value for 3a was
much closer to that of the isolated material than was that
of 3b.
In conclusion, the known retention of relative stereo-
chemistry inherent in the [3 + 2] dipolar cycloaddition
reactions of nitrile oxides has been used to prepare both
the trans and cis isomers of a ring-hydroxylated metabo-
lite of the GPIIb/IIIa antagonist XV459, the active drug
form of Roxifiban. In the course of this work, the first
example of a cis-4-hydroxy-5-substituted isoxazoline was
prepared, and a possible difference in ring mobility
between the cis and trans isomers was observed. Com-
parison of these synthetic standards with the isolated
metabolite demonstrated the trans relative stereochem-
istry in this material.
Exp er im en ta l Section
Starting materials, reagents, and solvents were obtained from
commercial sources and used as received unless otherwise
indicated. All reactions were run under a nitrogen atmosphere.
Flash chromatography refers to the medium-pressure column
chromatography method described by Still et al.18 Preparative
HPLC was performed using a reverse phase C18 column (41.4 ×
250 mm), eluting with water-acetonitrile-trifluoroacetic acid
at 40 mL/min, using a 30-min linear gradient from 97.5:2.5:0.05
to 20:80:0.05. Compounds were isolated as trifluoroacetate salts
following HPLC purification. Melting points (mp) are uncor-
rected. Proton NMR spectra were measured at 300 mHz in
chloroform-d (CDCl3), dimethyl sulfoxide-d6 (DMSO-d6), methanol-
a
Reagents: a, furan, Et3N; b, LiBr, AG50W-X2; c, J ones
reagent; d, 9; e, see Scheme 1.
Sch em e 3. Syn th esis of 16 a n d 17a
d
4 (MeOH-d4), or acetone-d6 and the peaks are reported in parts
per million downfield from tetramethylsilane (δ). Mass spectra
(MS) and high-resolution mass spectra (HRMS) were measured
using positive ion electrospray ionization (ES+) or ammonia
chemical ionization (NH3-CI). Abbreviations used: DCM, dichlo-
romethane; EtOAc, ethyl acetate; IPA, 2-propanol; MeCN,
acetonitrile; MeOH, methanol; TEA, triethylamine.
2-[4-(4,4,5,5-Tetr a m eth yl[1,3,2]d ioxa bor ola n -2-yl)bu t-3-
en yloxy]tetr a h yd r op yr a n (5). A solution of 2-but-3-ynyloxy-
tetrahydropyran (4, 1.54 g, 10 mmol) in DCM (5 mL) was treated
with 4,4,5,5-tetramethyl[1,3,2]dioxaborolane (pinacolborane, pre-
pared and purified according to the procedure of Tucker et al.;19
1.34 g, 10.5 mmol). This solution was added to a separate flask
containing bis(cyclopentadienyl)zirconium chloride hydride (129
mg, 500 µmol) at 0 °C, and the mixture was stirred for 30 min
on ice and then at room temperature for 4 d. Ether was added,
and the mixture was washed with water, dried (Na2SO4), and
concentrated. The residue was purified by flash chromatography
(hexanes:EtOAc 90:10) to provide 5 (1.78 g, 63%) as a colorless
oil: 1H NMR (CDCl3) δ 6.64 (dt, J ) 18.0, 6.6 Hz, 1H), 5.53 (dt,
J ) 18.0, 1.5 Hz, 1H), 4.60 (t, J ) 2.9 Hz, 1H), 3.84 (m, 2H),
3.50 (m, 2H), 2.47 (qd, J ) 7.0, 1.5 Hz, 2H), 1.8-1.5 (m, 6H),
1.26 (s, 12H); MS (NH3-CI) m/z 300 [(M + NH4)+, 20%], 102
[(2-hydroxytetrahydropyran)+, 100%].
a
Reagents: a, â-alanine tert-butyl ester HCl, Et3N, MeCN; b,
NaH, DMF, then MeI.
isoxazoline ring flexibility was presumably present in the
cis but not the trans isomers. The reason for this
difference is unknown, although intramolecular hydrogen
bonding between the amide NH and the 4-oxygen sub-
stituent (only possible in the cis isomer) is one possibility.
Comparison of the proton NMR spectra and mass
spectral fragmentation patterns of 3a -3d with those of
the isolated metabolite confirmed that the levorotatory
trans isomer corresponded to the isolated ring 4-hydroxy-
lated metabolite4 and also confirmed that the tentative
assignment of this isomer to structure 3a (ring 4S,5S)
(17) Mousa, S. A.; Bozarth, J . M.; Forsythe, M. S.; Lorelli, W.;
Ramachandran, N.; J ackson, S.; De Grado, W. F.; Reilly, T. M.
Cardiology 1993, 83, 374-382.
(18) Still, W. C.; Kahn, M.; Mitra, A. J . Org. Chem. 1978, 43, 2923-
2925.
(19) Tucker, C. E.; Davidson, J .; Knochel, P. J . Org. Chem. 1992,
57, 3482-3485.

Notes
4-Acetoxy-3-(4-cya n op h en yl)-5-(2-h yd r oxyeth yl)-4,5-d i-
J . Org. Chem., Vol. 65, No. 23, 2000 8103
1H), 4.00 (m, 2H), 3.72 (s, 3H), 3.67 (dd, J ) 13.6, 4.4 Hz, 1H),
3.41 (dd, J ) 13.6, 7.5 Hz, 1H), 2.50 (m, 2H), 1.57 (m, 2H), 1.36
(m, 2H), 0.91 (t, J ) 7.3 Hz, 3H).
h yd r oisoxa zole (7). A solution of 5 (1.65 g, 5.84 mmol) and
4′-cyano-1-chlorobenzaldoximine (6)8 (1.06 g, 5.84 mmol) in THF
(25 mL) was treated with sodium percarbonate (1.38 g, 8.76
mmol) and stirred for 48 h at room temperature. The mixture
was filtered, and the solid was washed with EtOAc. The filtrate
was concentrated, and the residue was partially purified by flash
chromatography (toluene:EtOAc 85:15) to provide a gum (1.21
g, 65%). Without further purification, this material was dissolved
in pyridine (10 mL) and treated with acetic anhydride (0.4 mL,
4.21 mmol). After 8 h at room temperature, the solution was
poured into water and acidified to pH 4.5 by dropwise addition
of concentrated HCl. The mixture was extracted with EtOAc.
The extracts were washed with water, dried, and concentrated
to provide a gum (1.30 g). Without purification, this material
was dissolved in acetic acid (20 mL), THF (10 mL), and water
(5 mL) and heated at 50-55 °C for 3 h. The cooled solution was
diluted with DCM, washed with water and saturated aqueous
NaHCO3, dried (Na2SO4), and concentrated. The residue was
purified by flash chromatography (CHCl3:IPA 97.5:2.5) to provide
7 (398 mg, 41%) and recovered starting material, which was
resubjected to the reaction conditions and purified to provide
additional 7 (282 mg, 70% total yield) as a white solid: mp 141-
t r a n s-2-Bu t oxyca r b on yla m in o-3-{2-[3-(4-ca r b a m im -
id oylp h en yl)-4-h yd r oxy-4,5-d ih yd r oisoxa zol-5-yl]a cet yl-
a m in o}p r op ion ic Acid Meth yl Ester (11a , 11b). A solution
of 10a (100 mg, 210 µmol), H2NOH‚HCl (36 mg, 520 µmol), and
TEA (72 µL, 520 µmol) in methanol (2 mL) was stirred overnight
at room temperature. The mixture was concentrated, and the
residue was dissolved in DCM, washed with water, dried (Na2-
SO4), and concentrated to provide a sticky solid (66 mg). This
was dissolved in acetic acid (2 mL) and treated with acetic
anhydride (16 µL, 170 mmol). After 90 min at room temperature,
Pd on charcoal (5%, 3 mg) was added and the mixture was
stirred overnight under an atmosphere of H2. The catalyst was
removed by filtration, and the residue was purified by prepara-
tive HPLC to provide 11a (31 mg, 23%) as a white solid: 1H
NMR (MeOH-d4) δ 8.03 (d, J ) 8.8 Hz, 2H), 7.86 (d, J ) 8.8 Hz,
2H), 5.41 (d, J ) 3.3 Hz, 1H), 4.81 (m, 1H), 4.33 (bt, J ) ca. 6
Hz, 1H), 4.00 (m, 2H), 3.72 (s, 3H), 3.66 (dd, J ) 13.9, 4.8 Hz,
1H), 3.44 (dd, J ) 13.9, 7.3 Hz, 1H), 2.54 (m, 2H), 1.57 (m, 2H),
1.36 (m, 2H), 0.90 (t, J ) 7.3 Hz, 3H); MS (ES+) m/z 464.3 [(M
+ H)+, 100%]; [R]25 ) -57.6° (c 0.30, MeOH).
D
1
143 °C; H NMR (CDCl3) δ 7.81 (d, J ) 8.8 Hz, 2H), 7.71 (d, J
Likewise, 10b was converted into 11b as a white solid: 1H
NMR (MeOH-d4) δ 8.03 (d, J ) 8.8 Hz, 2H), 7.85 (d, J ) 8.8 Hz,
2H), 5.40 (d, J ) 3.3 Hz, 1H), 4.80 (dt, J ) 6.8, 3.3 Hz, 1H), 4.31
(bt, J ) ca. 6 Hz, 1H), 4.02 (m, 2H), 3.72 (s, 3H), 3.60 (m, 1H),
3.48 (dd, J ) 13.9, 6.8 Hz, 1H), 2.53 (m, 2H), 1.59 (m, 2H), 1.38
(m, 2H), 0.92 (t, J ) 7.3 Hz, 3H); MS (ES+) m/z 464.3 [(M +
) 8.8 Hz, 2H), 6.30 (d, J ) 2.5 Hz, 1H), 4.84 (td, J ) 6.9, 2.5 Hz,
1H), 3.88 (q, J ) 5.8 Hz, 2H), 2.12 (s, 3H), 1.98 (m, 3H); MS
(NH3-CI) m/z 215.1 [(M + H)+, 100%]. Anal. Calcd for
C14H14N2O4: C, 61.31; H, 5.14; N, 10.21. Found: C, 61.27; H,
5.05; N, 10.16.
4-Acetoxy-3-(4-cyan oph en yl)-4,5-dih ydr oisoxazol-5-ylace-
tic Acid (8). A solution of 7 (500 mg, 1.82 mmol) in DMF (10
mL) was treated with PDC (2.74 g, 7.29 mmol) and stirred at
room temperature. After 15 h, water was added and the mixture
was extracted twice with ether. The aqueous phase was acidified
to pH 2 with HCl and then extracted twice more with ether.
The combined ether extracts were extracted four times with
saturated aqueous NaHCO3. The combined aqueous extracts
were acidified to pH 2 with concentrated HCl to form a dense
precipitate. This mixture was extracted with ether (three times),
and these ether phases were dried and concentrated to provide
8 (397 mg, 76%) as a white solid: mp 165-167 °C; 1H NMR
(CDCl3) δ 7.80 (d, J ) 8.7 Hz, 2H), 7.72 (d, J ) 8.7 Hz, 2H), 6.34
(d, J ) 3.7 Hz, 1H), 4.94 (ddd, J ) 6.9, 5.5, 3.6 Hz, 1H), 2.95
(dd, J ) 16.9, 5.5 Hz, 1H), 2.92 (dd, J ) 16.9, 7.0 Hz, 1H), 2.13
(s, 3H); HRMS (ES+) m/z calcd for C14H13N2O5 [(M + H)+]
289.0824, found 289.0829. Anal. Calcd for C14H12N2O5: C, 58.33;
H, 4.20; N, 9.72. Found: C, 58.22; H, 4.21; N, 9.53.
H)+, 100%]; [R]25 ) +43.4° (c 0.30, MeOH).
D
t r a n s-2-Bu t oxyca r b on yla m in o-3-{2-[3-(4-ca r b a m im -
id oylp h en yl)-4-h yd r oxy-4,5-d ih yd r oisoxa zol-5-yl]a cet yl-
a m in o}p r op ion ic Acid , Tr iflu or oa cetic Acid Sa lt (3a , 3b).
Compound 11a (30 mg, 52 µmol) was combined with rabbit liver
esterase (suspension in HEPES buffer; 0.3 mL, 80 units) in
HEPES buffer (pH 7.1, 0.3 N; 3 mL) and allowed to stand at
room temperature for 20 h. The mixture was filtered through a
10 kD cutoff membrane, and the filtrate was concentrated and
purified by preparative HPLC to provide 3a (30 mg, 95%) as an
amorphous solid: HPLC tR 10.49 min; 1H NMR (MeOH-d4) δ
8.03 (d, J ) 8.8 Hz, 2H), 7.85 (d, J ) 8.8 Hz, 2H), 5.41 (d, J )
3.3 Hz, 1H), 4.81 (m, 1H), 4.31 (m, 1H), 4.01 (m, 2H), 3.73 (m,
1H), 3.48 (m, 1H), 2.53 (m, 2H), 1.58 (m, 2H), 1.39 (m, 2H), 0.91
(t, J ) 7.3 Hz, 3H); HRMS (ES+) m/z calcd for C20H28N5O7 [(M
+ H)+] 450.1989, found 450.1974; [R]25D ) -35.2° (c 0.14, MeOH).
Anal. Calcd for C20H27N5O7‚1.3TFA: C, 45.42; H, 4.77; N, 11.72.
Found: C, 45.18; H, 4.92; N, 12.03.
Likewise, 11b was converted into 3b as an amorphous solid:
HPLC tR 10.17 min; H NMR (MeOH-d4) δ 8.04 (d, J ) 8.8 Hz,
tr a n s-2-Bu toxyca r bon yla m in o-3-{2-[3-(4-cya n op h en yl)-
4-h yd r oxy-4,5-d ih yd r oisoxa zol-5-yl]a c e t yla m in o}p r o-
p ion ic Acid Meth yl Ester (10). A mixture of 8 (500 mg, 1.81
mmol), 3-amino-N-butyloxycarbonyl-(S)-alanine methyl ester
p-toluenesulfonate 910 (709 mg, 1.81 mmol), DCC (373 mg, 1.81
mmol), 1-hydroxybenzotriazole hydrate (245 mg, 1.81 mmol),
TEA (504 µL, 3.62 mmol), and DMF (5 mL) was stirred at room
temperature overnight. The solvent was removed under vacuum,
and the residue was taken up in EtOAc and filtered. The filtrate
was washed with water, pH 4 buffer, and saturated aqueous
NaHCO3 and then was dried and concentrated. The residue was
purified by flash chromatography (hexanes:EtOAc 70:30) to
provide the acetate of 10 (725 mg, 84%) as an off-white solid:
1H NMR (MeOH-d4) δ 7.87 (d, J ) 8.8 Hz, 2H), 7.78 (d, J ) 8.8
Hz, 2H), 6.45 (m, 1H), 4.94 (m, 1H), 4.30 (bt, J ) 5.4 Hz, 1H),
4.02 (m, 2H), 3.82 (m, 1H), 3.70 (s, 3H), 3.44 (dd, J ) 13.6, 7.0
Hz, 1H), 2.68 (m, 2H), 2.06 (s, 3H), 1.57 (m, 2H), 1.37 (m, 2H),
0.92 (t, J ) 6.6, 3H); MS (ES+) m/z 489.3 [(M + H)+, 2.5%], 425.4
(100%). This mixture of diastereomers was separated by pre-
parative HPLC (CHIRALPAK AD column, 90:10 MeOH:water,
1.0 mL/min), resulting in hydrolysis of the acetate, to give peak
1
2H), 7.84 (d, J ) 8.8 Hz, 2H), 5.42 (d, J ) 3.3 Hz, 1H), 4.80 (m,
1H), 4.31 (m, 1H), 4.03 (m, 2H), 3.67 (dd, J ) 13.6, 4.8 Hz, 1H),
3.45 (dd, J ) 13.6, 7.7 Hz, 1H), 2.54 (m, 2H), 1.58 (m, 2H), 1.38
(m, 2H), 0.92 (t, J ) 7.3 Hz, 3H); [R]25D ) +40.7° (c 0.20, MeOH).
cis-4-(3a ,6a -Dih yd r ofu r o[2,3-d ]isoxa zol-3-yl)b en zon i-
tr ile (12). A suspension of 6 (4.5 g, 25 mmol) in freshly distilled
furan (1000 mL) was cooled to -25 °C under N2 and treated
with a solution of TEA (5 g, 50 mmol) in freshly distilled furan
(100 mL) dropwise over 1 h. After 4 h of stirring at -25 °C and
then 16 h at room temperature, the mixture was concentrated.
Water was added, and the aqueous phase was extracted with
EtOAc. The combined organic phases were filtered through
Celite, washed with saturated aqueous NaCl, dried (MgSO4), and
concentrated. Flash chromatography (hexanes:EtOAc 80:20)
afforded 12 as a white solid: mp 129-131 °C (4.8 g, 91%); 1H
NMR (DMSO-d6) δ 7.93 (s, 4H), 6.87 (d, J ) 2.6 Hz, 1H), 6.54
(d, J ) 8.8 Hz, 1H), 6.08 (dd, J ) 8.8, 2.6 Hz, 1H), 5.43 (t, J )
2.6 Hz, 1H); HRMS (ES+) m/z calcd. for C12H8N2O2 ([M + H]+)
213.0664, found 213.0654. Anal. Calcd for C12H8N2O2: C, 67.92;
H, 3.80; N, 13.20. Found: C, 67.80; H, 3.91; N, 12.90.
1, assigned structure 10b: [R]25 ) +198.2° (c 0.30, MeOH); 1H
D
NMR (MeOH-d4) δ 7.97 (d, J ) 8.2 Hz, 2H), 7.78 (d, J ) 8.2 Hz,
2H), 5.35 (d, J ) 3.3 Hz, 1H), 4.79 (m, 1H), 4.31 (bt, J ) ca. 6
Hz, 1H), 4.02 (m, 2H), 3.71 (s, 3H), 3.60 (dd, J ) 13.8, 5.4 Hz,
1H), 3.49 (dd, J ) 13.8, 6.8 Hz, 1H), 2.52 (m, 2H), 1.56 (m, 2H),
1.38 (m, 2H), 0.92 (t, J ) 7.3 Hz, 3H). Peak 2 was assigned
cis-4-(5-Hydr oxy-3a,5,6,6a-tetr ah ydr ofu r o[2,3-d]isoxazol-
3-yl)ben zon itr ile (13). A solution of 12 (4.5 g, 21 mmol) and
LiBr (4.5 g, 52 mmol) in MeCN (135 mL) was stirred at room
temperature and treated with AG50W-X (2.7 g, H+ form, dried)
and then with water (5.4 g). After 1 h of stirring at room
temperature, the mixture was filtered to remove the resin and
concentrated to afford a white solid. The solid was extracted with
IPA:CHCl3 (1:3), and the organic phase was washed with
structure 10a : [R]25 ) -141.5° (c 0.30, MeOH); 1H NMR
D
(MeOH-d4) δ 7.97 (d, J ) 8.2 Hz, 2H), 7.78 (d, J ) 8.2 Hz, 2H),
5.35 (d, J ) 2.9 Hz, 1H), 4.80 (m, 1H), 4.33 (bt, J ) ca. 6 Hz,

8104 J . Org. Chem., Vol. 65, No. 23, 2000
Notes
saturated aqueous NaCl and dried (MgSO4). The solvent was
removed, and the white solid was triturated with ether. The
white solid was collected by filtration, washed with ether and
dried to provide 13 as a mixture of diastereomers (ca. 7:3) (3.5
g, 72%): 1H NMR (CDCl3) δ 7.95 (m, 2H), 7.72 (m, 2H), 5.89 (d,
J ) 7.3 Hz, 0.3H), 5.81 (d, J ) 7.3 Hz, 0.7H), 5.72 (d, J ) 5.1
Hz, 0.7H), 5.65 (m, 0.3H), 5.47 (m, 0.3H), 5.36 (t, J ) 7.0 Hz,
0.7H), 2.40 (m, 2H); HRMS (ES+) m/z calcd for C12H10N2O3 ([M
+ H]+) 230.0691, found 230.0691.
Hz, 2H), 5.46 (d, J ) 7.3 Hz, 1H), 4.73 (m, 1H), 4.38-4.25 (2m,
1H), 4.05 (m, 2H), 3.78-3.62 (2m, 1H), 3.48 (m, 1H), 2.83 (m,
2H), 1.61 (m, 2H), 1.40 (m, 2H), 0.93 (t, J ) 7.3 Hz, 3H); HRMS
(ES+) m/z calcd for C20H28N5O7 [(M + H)+] 450.1989, found
450.1997; [R]25 ) +41.1° (c 0.12, MeOH).
D
cis-3-{2-[3-(4-Cya n op h en yl)-4-h yd r oxy-4,5-d ih yd r oisox-
a zol-5-yl]a cetyla m in o}p r op ion ic Acid ter t-Bu tyl Ester (16).
A solution of 14 (2.40 g, 20.5 mmol) in MeCN (100 mL) was
treated with â-alanine tert-butyl ester HCl (2.20 g, 12.1 mmol)
and Et3N (1.30 g, 12.8 mmol). The mixture was heated at reflux
for 24 h and cooled to room temperature. The mixture was
concentrated and partitioned between water and EtOAc. An
insoluble material was isolated by filtration and dried to provide
16 (1.60 g, 41%) as a white powder: 1H NMR (CDCl3) δ 8.02 (d,
J ) 8.1 Hz, 2H), 7.70 (d, J ) 8.1 Hz, 2H), 6.61 (bs, 1H), 6.06
(bd, J ) ca. 8 Hz, 1H), 5.52 (bt, J ) ca. 8 Hz, 1H), 4.80 (bm,
1H), 3.50 (m, 2H), 2.94 (m, 2H), 2.46 (bt, J ) 5.5 Hz, 2H), 1.47
(s, 9H); MS (ES+) m/z 374.3 [(M + H)+, 100%].
cis-3-{2-[3-(4-Cya n op h en yl)-4-m eth oxy-4,5-d ih yd r oisox-
a zol-5-yl]a cetyla m in o}p r op ion ic Acid ter t-Bu tyl Ester (17).
A solution of 16 (40 mg, 110 µmol) in DMF (1.0 mL) was treated
with NaH (60% in mineral oil; 6.0 mg, 150 µmol) and stirred
until gas evolution was no longer observed. Iodomethane (22 mg,
150 µmol) was added, and the mixture was stirred at room
temperature for 2 h. The mixture was partitioned between
EtOAc and water, and the organic phase was dried (Na2SO4) to
provide a colorless oil. This was purified by preparative TLC to
provide 17 as a colorless oil (30 mg, 77%): 1H NMR (CDCl3) δ
7.85 (d, J ) 8.8 Hz, 2H), 7.72 (d, J ) 8.8 Hz, 2H), 5.16 (d, J )
7.3 Hz, 0.33H), 5.15 (d, J ) 7.3 Hz, 0.67H), 4.83 (m, 1H), 3.65
(m, 2H), 3.42 (s, 2H), 3.41 (s, 1H), 2.98 (m, 2H), 2.52 (m, 2H),
1.46 (s, 9H); MS (ES+) m/z 388.4 [(M + H)+, 100%]. Variable-
temperature 1H NMR experiments (400 MHz, DMSO-d6) showed
that the two doublets at δ 5.30 and 5.29 (ratio 1:2, J ) 7.1 Hz)
reversably coalesced at 60 °C, forming a sharp doublet at δ 5.27
(J ) 7.1 Hz) at 90 °C, as did the two singlets at δ 3.33 amd 3.32
(ratio 2:1).
cis-4-(5-Oxo-3a,5,6,6a-tetr ah ydr ofu r o[2,3-d]isoxazol-3-yl)-
ben zon itr ile (14). A solution of 13 (2.8 g, 12 mmol) in acetone
(140 mL) was stirred at 0 °C and treated with J ones reagent
(56 mL) dropwise over 20 min. The orange solution was stirred
at 0 °C for 30 min and then was treated with IPA (20 mL), and
the green solid was removed by filtration. The filtrate was
concentrated, and the residue was triturated with ether (50 mL).
Filtration, washing with ether, and drying provided 14 as a
white solid (2.4 g, 87%) which appeared somewhat unstable to
storage: mp 204-206 °C; 1H NMR (acetone-d6) δ 8.01 (d, J )
8.5 Hz, 2H), 7.93 (d, J ) 8.8 Hz, 2H), 6.46 (d, J ) 7.0 Hz, 1H),
5.59 (t, J ) 7.0 Hz, 1H), 3.31 (dd, J ) 19.0, 7.0 Hz, 1H), 2.95 (d,
J ) 19.0 Hz, 1H); HRMS (ES+) m/z calcd for C12H8N2O3 ([M +
H]+) 228.0535, found 230.0549. Anal. Calcd. for C12H8N2O3: C,
63.16; H, 3.53; N, 12.28. Found: C, 62.78; H, 3.66; N, 11.58. The
enantiomers were separated by preparative HPLC (CHIRAL-
PAK AD column, MeCN, 8.0 mL/min) to afford isomer 14c with
[R]25 ) -166.1° (c 0.10, MeOH) and isomer 14d with [R]25
)
D
D
+162.6° (c 0.13, MeOH).
cis-2-Bu t oxyca r b on yla m in o-3-{2-[3-(4-ca r b a m im id oyl-
p h en yl)-4-h yd r oxy-4,5-d ih yd r oisoxa zol-5-yl]a cetyla m in o}-
p r op ion ic Acid , Tr iflu or oa cetic Acid Sa lt (3c, 3d ). A solu-
tion of 14c (208 mg, 1 mmol), 9 (390 mg, 1 mmol) and N,N-
diisopropylethylamine (129 mg, 1 mmol) in MeCN (8 mL) was
stirred at reflux for 24 h and cooled to room temperature.
Concentration and flash chromatography (hexanes:EtOAc:etha-
nol 50:41:9) afforded 15c as a white solid (300 mg, 67%): 1H
NMR (MeOH-d4) δ 7.97 (d, J ) 8.8 Hz, 2H), 7.78 (d, J ) 8.4
Hz), 5.41 (d, J ) 7.3 Hz, 1H), 4.69 (q, J ) 7.0 Hz, 1H), 4.34 (t,
J ) 5.5 Hz, 1H), 4.03 (t, J ) 6.4 Hz, 2H), 3.73 (s, 3H), 3.6 (m,
2H), 2.78 (t, J ) 6.8 Hz, 2H), 1.59 (m, 2H), 1.38 (m, 2H), 0.92 (t,
J ) 7.33 Hz, 3H); HRMS (ES+) m/z calcd for C21H26N4O7 ([M +
H]+) 228.0519, found 447.1881. Using the procedures given for
the conversion of 11a to 3a , 15c was converted to 3c as an
amorphous solid (50 mg, 23%): 1H NMR (CD3OD) δ 8.05 (d, J
) 8.8 Hz, 2H), 7.87 (d, J ) 8.8 Hz, 2H), 5.45 (d, J ) 7.3 Hz, 1H),
4.72 (q, J ) 7.0, 1H), 4.37 (dd, J ) 7.7, 4.8 Hz, 0.7H), 4.29 (dd,
J ) 7.7, 4.8 Hz, 0.3H), 4.05 (m, 2H), 3.74 (dd, J ) 13.9, 4.8 Hz,
0.7H), 3.66 (dd, J ) 13.9, 4.8 Hz, 0.3H), 3.49 (dd, J ) 13.9, 7.7
Hz, 0.7 H), 3.44 (dd, J ) 13.9, 7.7 Hz, 0.3H), 2.83 (m, 2H), 1.60
(m, 2H), 1.39 (m, 2H), 0.94 (t, J ) 7.3 Hz, 3H); HRMS (ES+) m/z
Ack n ow led gm en t. We gratefully acknowledge Pe-
ter E. Crawford for optical rotation determination,
Alfred J . Mical and Kathy A. Rathgeb for chiral HPLC
separations, Michael J . Haas for mass spectral mea-
surements, Gregory A. Nemeth for variable-temperature
NMR studies, and Mark Forsythe for platelet aggrega-
tion IC50 determinations.
Su p p or tin g In for m a tion Ava ila ble: Characterization
data (1H NMR) of 3b, 3c, 3d , 5, 10a , 10b, 11a , 11b, 12-14,
16 and 17; HPLC analysis for 3a and 3b. This material is
available free of charge via the Internet at http://pubs.acs.org.
calcd for C20H28N5O7 [(M + H)+] 450.1989, found 450.1984; [R]25
D
) -61.8° (c 0.09, MeOH).
Likewise, 14d was converted to 3d as an amorphous solid:
1H NMR (CD3OD) δ 8.05 (d, J ) 8.8 Hz, 2H), 7.86 (d, J ) 8.8
J O000755O