The enantiospecific synthesis of an isoxazoline. A RGD mimic platelet GPIIb/IIIa antagonist

Article abstract of DOI:10.1021/jo9612537

A convergent, large-scale, chiral synthesis of isoxazoline 1 has been achieved in 37% overall yield and > 99.6% optical purity, starting from L-asparagine and 4-cyanobenzaldehyde. Hofmann reaction of N(α)-n-Boc-L-asparagine with iodosobenzene diacetate provides optically pure N(α)-n-Boc-L-α,β-diaminopropionic acid (8) in 75% yield. A process of lipase resolution-base catalyzed epimerization gives the single enantiomer 5. Reaction of acid 5 with amine 9 in the presence of thionyl chloride forms the framework of 1. A Pinner reaction of intermediate 4 in methyl acetate or anisole, followed by an amidination with ammonium acetate, gives optically pure product 1.

Full text of DOI:10.1021/jo9612537

2466
J . Org. Chem. 1997, 62, 2466-2470
Th e En a n tiosp ecific Syn th esis of a n Isoxa zolin e. A RGD Mim ic
P la telet GP IIb/IIIa An ta gon ist
Lin-hua Zhang,* J . C. Chung, T. D. Costello, I. Valvis, P. Ma, S. Kauffman, and R. Ward
Chemical Process R&D, The DuPont Merck Pharmaceutical Company,
Deepwater, New J ersey 08023-0999
Received J uly 2, 1996X
A convergent, large-scale, chiral synthesis of isoxazoline 1 has been achieved in 37% overall yield
and >99.6% optical purity, starting from L-asparagine and 4-cyanobenzaldehyde. Hofmann reaction
of NR-n-Boc-L-asparagine with iodosobenzene diacetate provides optically pure NR-n-Boc-L-R,â-
diaminopropionic acid (8) in 75% yield. A process of lipase resolution-base catalyzed epimerization
gives the single enantiomer 5. Reaction of acid 5 with amine 9 in the presence of thionyl chloride
forms the framework of 1. A Pinner reaction of intermediate 4 in methyl acetate or anisole, followed
by an amidination with ammonium acetate, gives optically pure product 1.
Sch em e 1
The application of platelet glycoprotein IIb/IIIa (GPIIb/
IIIa) antagonists as antithrombotic agents has been an
active research area in recent years.1 The binding of
adhesive proteins such as fibrinogen to GPIIb/IIIa causes
platelets to aggregate. This binding is mediated in part
by an Arg-Gly-Asp (RGD) recognition sequence. A num-
ber of peptides containing the Arg-Gly-Asp sequence
(RGD) have been tested as platelet GPIIb/IIIa antago-
nists;2 however, development of peptides into therapeutic
agents has been hindered by their oral bioavailability and
rapid degradation by peptidases.3 The search for an RGD
peptide mimic led to a number of chiral isoxazoline
compounds that are very potent GPIIb/IIIa antagonists.4
In order to perform clinical studies with these potent
antithrombotic agents, an effective large-scale chiral
synthesis was required. In addition, we needed to find
a stable, pharmaceutically suitable solid drug substance
as a development candidate.5 This paper describes the
process used to provide isoxazoline 1, a crystalline
nonpeptide GPIIb/IIIa binding antagonist.
A simple method to produce 1 would be to form the
central amide bond as the final step. Accordingly, the
product could be synthesized from amidine acid 2 and
N-Boc-diaminopropionic ester (3) (Scheme 1). However,
preparation of optically pure 2 was a tedious process and
gave poor overall yield. In addition, isolation of product
1 from the reaction mixture containing 2 was not possible
without resorting to preparative reversed-phase HPLC.
Because of this, the central amide bond was formed
earlier in the sequence from cyano acid 5 and N-n-Boc-
diaminopropionic ester (3), making the terminal amidine
formation the last step.
Derivatives of L-2,3-diaminopropionic acid are compo-
nents of many natural products and synthetic drugs.6a
Syntheses of NR-CBZ and NR-t-Boc-diaminopropionic acid
via Mitsunobu reaction from serine or ring opening of
serine â-lactone are known.6b Additionally, derivatives
of L-2,3-diaminopropionic acid have been synthesized
from aspartic acid by Schmidt (Curtius) reaction6c or from
asparagine by Hofmann rearrangement with bis(trifluo-
roacetoxy)iodobenzene. In an early preparation of prod-
uct 1, this right-hand segment was prepared from the
commercially available amino acid, NR-(n-butoxycarbon-
yl)-1,2-diaminopropionic acid (8).6d Because compound
8 is quite expensive and not readily available in bulk
quantities, we decided to develop our own route to this
necessary intermediate.
X Abstract published in Advance ACS Abstracts, March 1, 1997.
(1) (a) Zhang, L.-H.; Ma, P. Tetrahedron Lett. 1994, 35, 8387. (b)
Gould, R. J .; Polokoff, M. A.; Friedman, P. A.; Huang, T.-F.; Holt, J .
C.; Cook, J . J . Niewiarowski, S. Proc. Soc. Exp. Biol. Med. 1990, 195,
168. (c) Semanen, J .; Ali, F.; Romoff, T.; Calvo, R.; Sorenson, E.; Vasko,
J .; Storer, B.; Berry, D.; Bennett, D.; Strohsacker, M.; Powers, D.;
Stadel, J .; Nichols, A. J . Med. Chem. 1991, 34, 3114.
(2) Bach, A. C.; Eyermann, C. J .; Gross, J . D.; Bower, M. J .; Harlow,
R. L.; Weber, P. C.; Degrado, W. F. J . Am. Chem. Soc. 1994, 116, 3207.
(3) Wolff, M. E. Burger’s Medicinal Chemistry and Drug Discovery,
5th ed. 1995.
Reaction of L-asparagine (6) with n-butoxycarbonyl
chloride gave N-n-Boc-L-asparagine (7) in 80% yield.
S0022-3263(96)01253-4 CCC: $14.00 © 1997 American Chemical Society

Synthesis of an Isoxazoline
Sch em e 2
J . Org. Chem., Vol. 62, No. 8, 1997 2467
Sch em e 3
Standard Hofmann reaction of 7 with the usual reagents
such as hypochlorites7 or NBS/KOH8 gave little product.
Only partial conversion to 8 was observed when 7 reacted
with dichloroisocyanuric acid9 under alkaline conditions.
Reaction of 7 with iodobenzene bis(trifluoroacetate)/
pyridine10 produced 8 in about 40% isolated yield. Fi-
nally, we found that the Hofmann degradation of aspar-
agine 7 with iodosobenzene diacetate11 provided the
desired product 8 in 75% yield. This chemistry allowed
us to prepare optically pure 8 in hundred kilogram
quantities. Esterification of 8 with thionyl chloride/
methanol, followed by a replacement of HCl with tolu-
enesulfonic acid, gave crystalline 9 in 82% yield (Scheme
2).
The synthesis of the isoxazoline portion of product 1
started from 4-cyanobenzaldehyde (10). Reaction of 10
with hydroxylamine sulfate in methanol gave 4-cy-
anobenzaldoxime (11) in 97% yield. Reaction of oxime
11 with N-chlorosuccinimide formed the chloride 12,
which converted to the nitrile oxide in the presence of
triethylamine. Cycloaddition of 12 with isobutyl vinyl-
acetate (13) in situ furnished the racemic 14.12 This one-
pot reaction provided isoxazoline 14 in 90% yield from
oxime 11. We screened a number of enzymes13 in the
resolution of isobutyl ester 14 and found that enzymatic
hydrolysis with lipase PS30 in phosphate buffer (pH 7.4-
8.0) provided optically pure acid 5 with the R configura-
tion4,5 and the corresponding chiral ester 15 possessing
the S configuration. A similar reaction with the methyl
ester of 14 failed to produce optically pure material due
to concomitant chemical hydrolysis. The recovered opti-
cally active ester 15 racemized to 14, in 1 h, with a
catalytic amount of potassium tert-butoxide in toluene
at 40 °C. This enzymatic resolution-base epimerization
sequence provided the optically pure isoxazoline acid (5)
in 70% yield from raceme 14 on a multikilogram scale
(Scheme 3).
(4) (a) Olson, R. E.; Wityak, J .; Xue, C.; Sielecki, T.; Cain, G.; Mousa,
S.; Thoolen, M.; Racanelli, A.; Hausner, E.; Reilly, T.; Degrado, W.;
Wexler, R. 211th New Orleans, LA, Mar 24-28, 1996; American
Chemical Society: Washington, DC, 1996; #250. (b) Wityak, J .; Pinto,
D. J .; Wang, S.; Sze, J . Y.; Mousa, S. A.; Olson, R. E.; Wexler, R. R.
211th New Orleans, LA, Mar 24-28, 1996; American Chemical
Society: Washington, DC, 1996; #131. (c) Xue, C. B.; Wityak, J .;
Sielecki, T. M.; Cain, G. A.; Liu, J .; Bostrom, L. L.; DiMeo, S. V.; Higley,
C. A.; Lalka, G. K.; Tobin, A. E.; Frietze, W. E.; Emmett, G.; Mousa,
S. A.; Sze, J . Y.; Thoolen, M. J .; Reilly, T.; DeGrado, W. F.; Olson, R.
E.; Wexler, R. R. 211th National Meeting of the American Chemical
Society, #132. March 24-28, 1996. (d) Wityak, J .; Sielecki, T. M.; Pinto,
D. J .; Emmett, G.; Sze, J . Y.; Liu, J .; Tobin, E.; Wang, S.; J iang, B.;
Ma, P.; Mousa, S. A.; Wexler, R. R.; Olson, R. E. J . Med. Chem. 1997,
40, 50.
(5) Zhang, L.-H.; Anzalone, L.; Ma, P.; Kauffman, G. S.; Storace,
L.; Ward, R. Tetrahedron Lett. 1996, 37, 4455.
Originally, coupling of diamine 9 with isoxazoline 5 to
provide optically pure 4 was completed by using typical
peptide coupling conditions with the PyBop reagent.5,14
Because PyBop is an expensive reagent, we looked for
an alternative method. Typically, thionyl chloride is not
considered a good reagent for amide bond formation
because epimerization occurs. We found that thionyl
chloride, under a specific set of conditions, can be used
for this reaction and still maintain stereochemical integ-
rity. Thus, reaction of acid 5 with amine 9 in the
presence of thionyl chloride gave optically pure 4 in 82%
yield (Scheme 4).
(6) (a) Adams, R.; J ohnson, J . L. J . Am. Chem. Soc. 1949, 71, 705.
Malathi, K.; Padmanaban, G.; Padubidri, S. Ind. J . Biochem. 1968, 5,
184. Dugenett, P.; Yaouanc, J . J .; Sturtz, G. Synthesis 1982, 781.
Hettinger, T. P.; Craig, L. C. Biochemistry 1970, 9, 1224. Mokotoff,
M.; Logue, L. W. J . Med. Chem. Soc. 1981, 24, 554. Ikegami, F.;
Murakoshi, I. Phytochemistry 1994, 35, 1089. (b) Golding, B. T.; Howes,
C. J . Chem. Res., Miniprint 1984, 1, 1. Ratemi, E. S.; Vederas, J . C.
Tetrahedron Lett. 1994, 35, 7605. Arnold, L. D.; Kalantar, T. H.;
Vederas, J . C. J . Am. Chem. Soc. 1985, 107, 7105. Kucharczyk, N.;
Badet, B.; Goffic, F. Le. Synth. Commun. 1989, 19, 1603. (c) Teshima,
T.; Nomoto, S.; Wakamiya, T.; Shiba, T. Bull. Chem. Soc. J pn. 1977,
50, 3372. Rao, S. L. N. Biochemistry 1975, 14, 5218. (d) The material
was purchased from Bachem.
(7) (a) Otsuka, M.; Kittaka, A.; Iimori, T.; Yamashita, H.; Kobayashi,
S.; Ohno, M. Chem. Pharm. Bull. 1985, 33, 509. (b) Sy, A. O.; Raksis,
J . W. Tetrahedron Lett. 1980, 21, 2223.
(8) Senanayake, C. H.; Fredenburgh, L. E.; Reamer, R. A.; Larsen,
R. D.; Verhoeven, T. R.; Reider, P. J . J . Am. Chem. Soc. 1994, 116,
7947.
(12) (a) Huisgen, R. Angew. Chem. 1963, 75, 604. (b) Pedwa, A.
Comp. Org. Synth. 1991, 4, 1069. (b) Wade, P. A. Comp. Org. Synth.
1991, 4, 1111. (c) Kim, Y.; Kim, S.; Park, D. Tetrahedron Lett. 1993,
34, 6063. (d) Gothelf, K. V.; Thomsen, I.; J orgensen, K. A. J . Am. Chem.
Soc. 1996, 118, 59. (e) Gilbertson, S. R.; Dawson, D. P.; Lopez, O. D.;
Marshall, K. L. J . Am. Chem. Soc. 1995, 117, 4431.
(13) Drauz, K.; Waldmann, H. Enzyme Catalysis in Organic Syn-
thesis; VCH: Weinheim, 1995.
(14) (a) Coste, J .; Le-Nguyen, D.; Castro, B. Tetrahedron Lett. 1990,
31, 205. (b) Rivero, A.; Somanathan, R.; Helberg, L. H. Synth. Commun.
1995, 25, 2185. (c) Dudash, J . J .; J iang, J .; Mayer, S. C.; J oullie, M.
M. Synth. Commun. 1993, 23, 349.
(9) Staskun, B. J . Org. Chem. 1988, 53, 5287.
(10) (a) Waki, M.; Kitajima, Y.; Izumiya, N. Synthesis. 1981, 266.
(b) Pavlidis, V. H.; Chan, E. D.; Pennington, L.; McParland, M.;
Whitehead, M. Synth. Commun. 1988, 18, 1615. (c) Kanaoka, Y.;
Okajima, H.; Hatanaka, Y. J . Org. Chem. 1979, 44, 1751. (d) Tamura,
M.; J acyno, J .; Stammer, C. H. Tetrahedron Lett. 1986, 27, 5435.
Boutin, R. H.; Loudon, G. M. J . Org. Chem. 1984, 49, 4277.
(11) Willgerodt, C. Ber. 1892, 25, 3495. Sharefkin, J . G.; Saltzman,
H. Organic Syntheses; Wiley: New York, 1973; Collect. Vol. V, p 660.

2468 J . Org. Chem., Vol. 62, No. 8, 1997
Zhang et al.
Sch em e 4
diastereomers. As might be expected, these isoxazoline
compounds are very sensitive to base and epimerize
easily under alkaline conditions (pH > 10.5). Presum-
ably, the epimerization of the isoxazoline is through a
reversible Michael reaction. Although the epimerization
was not as severe when ammonia was replaced with
ammonium carbonate, the side products amide 17 and
diester 18 were generated in the reaction. The amidine
product 19, an HCl salt, existed as an amorphous solid
that could not be crystallized. In earlier SAR studies
isoxazoline 19 was purified by chromatographic means4
and the overall yield from 16 to 19 was low.
We have previously described our conversion of this
imidate to the acetate salt of the amidine, compound 1.5
This reaction worked very well at scale and allowed us
to obtain the desired amidine in 74% yield from the cyano
compound 4. The optical purity of product 1 was verified
by chiral SFC analysis.
There are a number of reports that detail the synthesis
of the amidine functional group.15 The Pinner reaction,
followed by amination, is probably the best known
method to prepare amidines from cyano compounds.16
Reaction of (cyanophenyl)isoxazoline 4 with saturated
methanolic hydrogen chloride provided imidate 16 in
good yield. However, the imidate was not stable under
strongly acidic conditions and partially decomposed dur-
ing workup. Reaction of 4 in other solvents such as
ethanol and 2-propanol effected transesterification as
well as decomposition. However, reaction of (cyanophen-
yl)isoxazoline 4 with HCl in methyl acetate containing a
small amount of methanol gave imidate 16 in better than
90% yield and greatly reduced the need for a huge molar
excess of HCl. Similar results were also obtained when
the reaction was carried out with anisole replacing the
methyl acetate.
At laboratory scale, the workup of the Pinner reaction
involved a vacuum distillation to remove the excess HCl
after conversion to the imidate. At preparative scale, HCl
could not be completely removed by vacuum distillation;
therefore, imidate 16 converted to the diester 18 due to
long-term exposure to acidic conditions. In order to
prevent this problem, a neutralization of residual HCl
with either ammonia or ammonium acetate was added,
followed by filtration to remove the precipitated salts.
However, we found that long filtration time or poor
temperature control led to partial conversion of 16 to
amidine 19, which then coprecipitated with the undesired
ammonium chloride salts. This amidine 19 can be
recovered by washing the filter cake with methanol and
converted to product 1 by reaction of 19 with ammonium
acetate.
In summary, we describe a convergent nine-step
synthesis of isoxazoline compound 1, at multikilogram
scale. Compound 1 was produced in 37% overall yield
(based on 4-cyanobenzaldehyde) and greater than 99.6%
optical purity. The substance is currently undergoing
clinical trial.
Exp er im en ta l Section
Gen er a l Meth od s. All melting points are uncorrected.
The 1H and 13C NMR spectra were recorded at 300 and 75.4
MHz, respectively, and are reported in DMSO-d6 (except where
noted). Mass spectra were recorded by the analytical labora-
tory, Chemical Process R&D, DuPont Merck Research Labo-
ratories. The elemental analyses were determined by Quan-
titative Technologies Inc., Whitehouse, NJ . Solvents, starting
materials, and reagents were used as purchased without
further purification. Lipase PS30 was purchased from Amano
Enzyme USA Co., Ltd.
Nr-n -Boc-L-a sp a r a gin e (7). A mixture of L-asparagine
monohydrate (67.5 kg, 449.58 mol), water (337.5 L), sodium
carbonate (59.4 kg, 560.43 mol), and THF (57.4 L) was heated
to 50-55 °C under agitation. A solution of n-butyl chlorofor-
mate (73.65 kg, 539.24 mol) in THF (57.4 L) was added into
the reactor over a period of 3 h. The reaction was held at 50-
55 °C for 1 h after addition and then cooled to 15 °C. The pH
of the reaction mixture was adjusted to 2.5-3.5 with aqueous
HCl (11%). The slurry was agitated at 15-20 °C for 1 h. The
product was isolated by filtration, washed with water (3 ×
222.8 L), and dried under vacuum (70-75 °C) to constant
weight: 83.0 kg, 80% yield; mp 148-9 °C; 1H NMR δ 0.89 (3H),
1.39-1.25 (2H), 1.45-1.57 (2H), 2.37-2.57 (2H), 3.93 (1H),
4.24-4.34 (1H), 6.91 (1H), 7.21 (1H), 7.32 (1H), 12.60 (1H).
Anal. Calcd for C9H16N2O5: C, 46.55; H, 6.94; N, 12.06.
Found: C, 46.68; H, 7.03; N, 12.01.
Alternatively, other weak bases including sodium or
potassium acetate can be used as the neutralizing agent.
This allows an easy filtration of salts, keeping the imidate
in solution for subsequent controlled conversion to the
amidine. However, the acetic acid produced affects the
yield of product 1 from the subsequent step, so further
optimization is necessary to improve the recovery of the
product.
Typically, imidates are converted to amidines by reac-
tion with ammonia or ammonium carbonate. Reaction
of imidate 16 with ammonia in methanol did convert the
imidate to amidine, but the product was a mixture of
(15) Rousselet, G.; Capdevielle, P.; Maumy, M. Tetrahedron Lett.
1993, 34, 6395.
(16) Allen, R.; Schumann, E.; Day, W.; Campen, M. J . Am. Chem.
Soc. 1958, 80, 591.
Nr-n -Boc-L-r,â-d ia m in op r op ion ic Acid (8). A solution
of n-propanol (81.2 kg), methyl acetate (50.4 kg), and water

Synthesis of an Isoxazoline
J . Org. Chem., Vol. 62, No. 8, 1997 2469
(10.8 kg) was cooled to 5 °C. To this solution were added NR-
Boc-L-asparagine (23.5 kg, 101.21 mol) and iodosobenzene
diacetate (37.5 kg, 116.39 mol) under agitation. This mixture
was warmed to 25 °C over a period of 1 h and stirred at the
same temperature for additional 2 h. The reaction mixture
was slowly heated to 50 °C over 90 min and then cooled to
3-5 °C. The reaction mixture was held at 3-5 °C for 30 min.
The product was isolated by filtration, washed with methyl
acetate (2 × 22 kg), and dried under vacuum (50-55 °C) to
constant weight: 15.5 kg, 75% yield; mp 215 °C dec; 1H NMR
δ 0.90 (3H), 1.27-1.42 (1H), 1.47-1.61 (2H), 3.03 (1H), 3.23
(1H), 3.98 (2H), 4.22-4.32 (1H), 7.56 (1H), 8.05 (3H). Anal.
Calcd for C8H16N2O4: C, 47.05; H, 7.90; N, 13.72. Found: C,
47.17; H, 8.01; N, 13.72.
Meth yl Nr-Boc-L-r,â-d ia m in op r op ion a te‚TsOH (9). A
mixture of methanol (130 kg) and NR-Boc-L-R,â-diaminopro-
pionic acid (22.0 kg, 107.74 mol) was cooled to 10 °C. To this
mixture was added thionyl chloride (15.4 kg, 129.44 mol) over
a period of 35 min. The reaction was stirred at 20-25 °C for
3-4 h, the solvent was removed under reduced pressure, and
toluene (47.6 kg) was added. The residual methanol was
removed by azeotropic distillation, and a solution of TsOH
(anhyd, 117.23 mol) in methyl acetate (41.5 kg) was added.
The solution was distilled to an oil, and methyl acetate (113
kg) and heptane (101 kg) were added. The solution was cooled
to 0-5 °C and held at 0-5 °C for 4 h. The product was isolated
by filtration, washed with methyl acetate/heptane (40 kg, 1/1
ratio), and dried under vacuum (50-55 °C) to constant
weight: 34.9 kg, 82% yield; mp 95-96 °C; 1H NMR δ 0.88 (3H),
1.25-1.40 (2H), 1.47-1.59 (2H), 2.28 (3H), 3.03 (1H), 3.21 (1H),
3.66 (3H), 3.97 (2H), 4.30-4.41 (1H), 7.11 (2H), 7.47 (2H), 7.66
(1H), 7.92 (3H). Anal. Calcd for C16H27N2O7S: C, 49.09; H,
6.95; N, 7.16. Found: C, 49.32; H, 6.76; N, 7.13.
mixture was adjusted to between 8.0 and 8.2 by the addition
of aqueous NaOH (33%, 11.0 L). The crude 15 was collected
by a filtration through a layer of Celite (20 kg) and washed
with water (70 L). This crude 15 was recycled through a
racemization step: 1H NMR (CDCl3) δ 0.95 (6H), 1.80 (1H),
2.69 (1H), 2.91 (1H), 3.14 (1H), 3.54 (1H), 3.91 (2H), 5.13-
5.23 (1H), 7.68-7.78 (4H).
The pH of the solution of filtrate (∼800 L) and isopropyl
acetate (20 L) was adjusted to 2.8-3.2 with concentrated
hydrochloric acid (∼57 kg). The crude product 5 was precipi-
tated, collected by filtration, and washed with water (70 L).
This crude product was crystallized from hot ethanol (525.0
L) to give optically pure 5. Isoxazoline 5 was collected by
filtration, washed with ethanol (76.0 L), and dried to constant
weight: 12.3 kg, 77% yield based on the amount of 5 in 14;
1
mp 198-200 °C; H NMR δ 2.70 (2H), 3.20 (1H), 3.59 (1H),
5.00-5.10 (1H), 7.78-7.91 (4H), 12.44 (1H). Anal. Calcd for
C12H10N2O3: C, 62.61; H, 4.38; N, 12.17. Found: C, 62.39; H,
4.49; N, 11.98.
Ra cem iza tion of 15 to 14. A solution of toluene (414.0 L)
and crude 15 (∼120 kg wet cake) was heated to 50 °C and
filtered to remove Celite (∼40 kg). The filter cake was washed
by toluene (72.0 L). The organic layers were combined and
washed by brine (108.0 L). After removal of the aqueous layer,
the organic layer was dried by azeotropic distillation to
constant boiling point (111 °C). The batch was cooled to 40
°C, and potassium tert-butoxide in tert-butyl alcohol (1 N, 1.6
L) was added. The reaction was agitated (200 rpm) at 40 °C
until racemization was completed. The batch was cooled to
20-25 °C, and water (108.0 L) was added. The reaction
mixture was neutralized by the addition of aqueous hydro-
chloric acid (1 N, 1.6 L). The aqueous bottom layer was
removed, and the organic layer was concentrated by distilla-
tion. The reaction was cooled to 60 °C when ∼380.0 L of
toluene was removed. To this solution was added heptane (115
L), and the reaction was held at 50 °C for 1 h. The mixture
was cooled to 0-5 °C and held for 2 h. The product 14 was
collected by filtration and washed with a mixed solvent of
toluene and heptane (1/2 ratio, 70 L). The product 14 was
dried under vacuum (50-55 °C) to constant weight: 17.3 kg,
87% yield based on the S-isomer in 14.
(R)-Meth yl-3-[[[3-(4-cya n op h en yl)-4,5-d ih yd r o-5-isox-
a zolyl]a cetyl] a m in o]-N-(bu toxyca r bon yl)-L-a la n in e (4).
A solution of acetonitrile (204.0 L), acid 5 (12.0 kg, 52.10 mol),
amine 9 (22.4 kg, 57.30 mol), and thionyl chloride (6.8 kg, 57.30
mol) was stirred at 0-5 °C for 1 h. To this solution was added
diisopropylethylamine (22.2 kg, 172.00 mol) at 20 °C over a
period of 90 min. Water (612.0 L) was added after the reaction.
The crude product 4 precipitated out. This crude 4 was
collected by filtration and washed with water (96.0 L). The
wet cake was dissolved in hot methanol (50-60 °C, 311.0 L),
and any insoluble particles were removed by filtration. The
solution was cooled at 0-5 °C for 3 h, and the product was
collected by filtration and washed with methanol (75.0 L). The
product was dried under vacuum (55-60 °C) to constant
weight: 18.3 kg, 82% yield; mp 154-6 °C; 1H NMR δ 0.92 (3H),
1.37 (2H), 1.59 (2H), 1.67 (1H), 2.58 (1H), 2.71 (1H), 3.22 (1H),
3.51 (1H), 3.67 (2H), 3.77 (3H), 4.06 (2H), 4.44 (1H), 5.14 (1H),
5.70 (1H), 6.38 (1H), 7.70 (2H), 7.77 (2H). Anal. Calcd for
C12H10N2O3: C, 62.61; H, 4.38; N, 12.17. Found: C, 62.39; H,
4.49; N, 11.98.
4-Cya n oben za ld oxim e (11). A solution of methanol (272.1
L), 4-cyanobenzaldehyde (50 kg, 381.3 mol), and hydroxyl-
amine sulfate (36.1 kg, 219.7 mol) was stirred at 55-60 °C
for 3 h, and then water (272 L) was added. The mixture was
cooled to 0-5 °C and held for 30 min. The crude product was
collected by filtration. The filter cake was washed with a
mixed solvent of cold methanol and water (2/3 ratio, 735.0 L)
and water (750.0 L) and dried under vacuum (60-70 °C) to
1
constant weight: 54.1 kg, 97% yield; mp 174-6 °C; H NMR
δ 7.82 (2H), 7.88 (2H), 8.26 (1H), 12.00 (1H). Anal. Calcd for
C8H6N2O: C, 65.75; H, 4.14; N, 19.17. Found: C, 65.73; H,
4.26; N, 19.14.
(+)-Isobu tyl 2-[3-(4-Cya n op h en yl)-4,5-d ih yd r o-5-isox-
a zolyl]a ceta te (14). To a solution of DMF (262.0 L), 4-cy-
anobenzaldoxime (11) (46 kg, 342.1 mol), and N-chlorosuccin-
imide (52 kg, 389.4 mol) was added isobutyl vinylacetate (95
kg, 665.7 mol). The solution was cooled to 2-6 °C, and
triethylamine (40 kg, 388.6 mol) was slowly added over a
period of 4 h. The reaction was stirred at the same temper-
ature for an additional 1 h. Water (330.0 L) and hydrochloric
acid (1 N, 49 L) were added. The crude product was collected
by filtration, washed with water (555.0 L), and redissolved in
toluene (500.0 L, 40 °C). The organic layer was washed with
water (291.0 L) and dried by azeotropic distillation (removing
about 250 L of toluene). Heptane (300.0 L) was added, and
the reaction was cooled at 0-5 °C for 3 h. The product was
collected by filtration and washed with toluene/heptane (150.0
L, 1/2 ratio). The product was dried under vacuum at 55-60
°C to constant weight: 81.8 kg, 90% yield; mp 98-100 °C; 1H
NMR (CDCl3) δ 0.96 (6H), 1.96 (1H), 2.70 (1H), 2.92 (1H), 3.15
(1H), 3.56 (1H), 3.90 (2H), 5.20 (1H), 7.70 (2H), 7.80 (2H). Anal.
Calcd for C16H18N2O3: C, 67.12; H, 6.34; N, 9.78. Found: C,
67.06; H, 6.20; N, 9.76.
(R)-2-[3-(4-Cya n op h en yl)-4,5-d ih yd r o-5-isoxa zolyl]a ce-
tic Acid (5). A suspension of H2O (597.0 L), NaH2PO4‚H2O
(60.0 kg), aqueous NaOH (33%, 36.0 L), Triton X-100 (3.2 kg),
compound 14 (40.0 kg, 139.7 mol), and lipase PS30 (4.0 kg,
enzyme content 8%) was slowly heated to 40 °C and held in
the temperature range of 40-43 °C until the resolution was
completed (∼16 h). The pH of the reaction mixture was
maintained between 7.4 and 8.0 and adjusted by the addition
of 33% aqueous NaOH. The batch was cooled to 20-25 °C
when the reaction was completed, and the pH of the reaction
(R)-Met h yl-3-[[[3-[4-(a m in oim in om et h yl)p h en yl]-4,5-
d ih yd r o-5-isoxa zolyl]a cetyl]a m in o]-N-(bu toxyca r bon yl)-
L-a la n in e Mon oa ceta te (1). A solution of methyl acetate
(55.8 L), methanol (4.8 L), HCl (9.6 kg), and compound 4 (12.0
kg, 27.88 mol) was cooled to -20 °C and stirred under 3-5
psi (HCl) at 10 °C for 27 h. After the reaction, the HCl was
removed under vacuum, and the methyl acetate (21.5 L) and
methanol (63.2 L) were added. The residual HCl was neutral-
ized with ammonia (2.5 kg) under 10 °C. The resulting
ammonium chloride was removed by filtration. The filter cake
was washed with methyl acetate and methanol (20.0 L). To
the filtrate was added ammonium acetate (6 kg), and the
reaction was stirred at room temperature overnight. The
crude product was collected by filtration and recrystallized
from hot methanol to give 1: 10.4 kg, 74% yield; mp 213-4

2470 J . Org. Chem., Vol. 62, No. 8, 1997
Zhang et al.
1
°C; [R]25 -86.55° (methanol, c ) 0.6); H NMR δ 0.86 (3H),
HCl was removed by decantation of anisole (50% of the volume)
and vacuum distillation. The residual HCl was further
neutralized by a saturated solution of ammonium acetate in
methanol under 5 °C. The resulting ammonium chloride (314
g) was removed by filtration. To this filtrate was added
ammonium acetate (425 g, 4.75 mol). The reaction was stirred
at 25-30 °C for 15 h. Product 1 (0.45 kg, 76% yield) was
isolated by filtration, washed with methanol, and dried under
vacuum (65 °C). The spectral data of 1 were identical to those
from above.
D
1.30 (1H), 1.51 (1H), 1.71 (3H), 2.42 (1H), 2.55 (1H), 3.18 (2H),
3.27 (1H), 3.54 (2H), 3.62 (3H), 3.93 (2H), 4.16 (1H), 5.01 (1H),
7.49 (1H), 7.82 (4H), 8.20 (1H), 10.24 (4H); 13C NMR δ 13.52,
18.48, 24.56, 30.61, 38.88, 39.66, 40.58, 51.97, 53.61, 63.83,
78.41, 126.67, 127.93, 130.93, 133.42, 156.07, 156.13, 165.12,
169.29, 171.06, 176.35. Anal. Calcd for C23H33N5O8: C, 54.53;
H, 6.55; N, 13.80. Found: C, 54.45; H, 6.61; N, 13.67.
Alter n a te Syn th esis of 1 fr om 4. A solution of anisole
(2.5 L), methanol (141 mL), HCl (375 g), and compound 4 (0.5
kg, 1.16 mol) was stirred at 0-5 °C for 14 h. After the reaction,
J O9612537