Stereoselective synthesis of unnatural spiroisoxazolinoproline-based amino acids and derivatives

Article abstract of DOI:10.1021/jo025611j

A route to spiroisoxazolinoproline-based amino acid derivatives is reported in which exomethyleneprolinate 4 (tert-butyl ester) reacts as a dipolarophile with nitrile oxides to generate spiroisoxazolinoprolinates 7/10/11 in good yields (70-75%) and with ca. 1:4 cis:trans diastereoselectivity. tert-Butyl spiroisoxazolinoprolinates were separable by column chromatography and amenable to scale-up leading to single diastereoisomers of N-Boc and N-Fmoc protected spiroisoxazolinoproline amino acids.

Full text of DOI:10.1021/jo025611j

Ster eoselective Syn th esis of Un n a tu r a l
Sp ir oisoxa zolin op r olin e-Ba sed Am in o Acid s a n d Der iva tives
Wei-Chieh Cheng,† Yannan Liu,† Melissa Wong,† Marilyn M. Olmstead,† Kit S. Lam,‡ and
Mark J . Kurth*,†
Department of Chemistry, University of California, One Shields Avenue, Davis, California 95616-5295,
and Division of Hematology and Oncology, Department of Internal Medicine, UC Davis Cancer Center,
University of California, Davis, 4501 X Street, Sacramento, California 95817
mjkurth@ucdavis.edu
Received February 13, 2002
A route to spiroisoxazolinoproline-based amino acid derivatives is reported in which exo-
methyleneprolinate 4 (tert-butyl ester) reacts as a dipolarophile with nitrile oxides to generate
spiroisoxazolinoprolinates 7/10/11 in good yields (70-75%) and with ca. 1:4 cis:trans diastereo-
selectivity. tert-Butyl spiroisoxazolinoprolinates were separable by column chromatography and
amenable to scale-up leading to single diastereoisomers of N-Boc and N-Fmoc protected spiroisox-
azolinoproline amino acids.
Due to their intrinsic biological activities and applica-
tions as conformational modifiers for physiologically
active peptides, unnatural and nonproteinogenic amino
acids have become very important and attractive targets.
Among amino acids, proline1 analogues, with their unique
structural constraints (cyclic and a secondary amine),
play an important role in the investigation of structure,
receptor affinity, and biological activity of amino acids
chimeras and peptides.2 Furthermore, heterocyclic pyr-
rolidine rings are valuable scaffolds and precursors in
natural products and in drug discovery, i.e., kainoid,3
carbapenems,4 captopril,5 and gramicidin.6 Consequently,
routes to and applications of proline-based amino acids
and derivatives have received much attention in both
chemistry and biochemistry.1,7
F IGURE 1. Spiroisoxazolinoprolines from 4-hydroxyproline.
modified prolines1,10 have been studied, we are not aware
of any reports regarding spiroisoxazolinoproline-based
amino acids. In this paper, we report a straightforward
synthetic route for combining these two interesting
structural features within a single framework to form
N-Boc- or N-Fmoc-protected cis- and trans-4-spiroisox-
azilinoproline amino acid derivatives (I; Figure 1).
Our synthetic effort started with commercially avail-
able trans-4-hydroxyl-L-proline, which, following litera-
ture procedures11 of N-Boc protection, oxidation (RuO2/
NaIO4), and Wittig olefination, gave 1-tert-butoxycarbonyl-
4-methylene-L-proline (1).11 The key 1,3-dipolar cyclo-
addition reaction was conducted by treating exo-methyl-
eneproline 1 with 2,6-dichlorobenzaldehyde oxime in the
presence of NaOCl (i.e., the Huisgen method for in situ
nitrile oxide generation)12 and led to the desired spiroisox-
azolinoproline amino acid 2 as an approximately 1:1
One of our research interests is the application of 1,3-
dipolar cycloadditions to generate heterocyclic isoxazole
and isoxazoline ringsssubstructures with inherent bio-
logical activity.8 And while both spiroisoxazolines9 and
† Department of Chemistry, University of California.
‡ UC Davis Cancer Center, University of California.
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10.1021/jo025611j CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/04/2002
J . Org. Chem. 2002, 67, 5673-5677
5673

Cheng et al.
SCHEME 1. Sp ir oisoxa zolin op r olin e Rou te
SCHEME 2. 1,3-Dip ola r Cycloa d d ition
Dia ster eoselectivity
a
Determined by HPLC of the crude reaction mixture. bProduct
diastereomeric mixture in 76% yield (Scheme 1). On the
basis of our previous experiences,13 as well as that of
others,14 we were not surprised to find that 1 f 2
proceeds with excellent regioselectivity (none of the
regioisomer could be detected). Given this result, we were
intrigued with two pivotal questions regarding this
cycloaddition reaction: (1) could the C2-stereogenic
center be employed to effectively mediate the diastereo-
selectivity of the 1,3-dipolar cycloaddition and (2) is there
a practical method to separate these spiroisoxazolino-
proline diastereomers.
mixture after flash column chromatography.
SCHEME 3. Sp ir oisoxa zolin op r olin a tes fr om
P r olin a te 4
The latter point was accentuated by the fact that
attempts to separate (routine column chromatographic
and recrystallization techniques) the cis and trans iso-
1
mers of 2 were unsuccessful. In addition, the H NMR
(ambient temperature) of 2 was complicated by the
rotational isomerism15 of the N-Boc protecting group; the
1:1 isomer ratio of crude 2 was determined by HPLC.
Thus, while this 1,3-dipolar cycloaddition reaction was
highly regioselective, its lack of stereoselectivity and
separation problems prompted us to modify our dipolaro-
phile system. The strategy we settled on was to convert
exo-methyleneproline amino acid 1 to various exo-meth-
yleneprolinates in the hopes that the subsequent cyclo-
addition would proceed with reasonable diastereo-
selectivity.
a
Determined by HPLC of the crude reaction mixture. bProduct
mixture after flash column chromatography.
as determined by HPLC, were found to be 6a :6b 1:1, 7a :
7b 1:4, and 8a :8b 2:3 (Scheme 2). Clearly, the C2 ester
moiety can, by steric effects, influence the stereoselec-
tivity of these 1,3-dipolar cycloaddition reactions and
prolinate 4, with its bulky tert-butyl ester moiety, delivers
the highest selectivity.
DCC-mediated esterification of 1 with three different
alcohols afforded proline esters 3-5 in good yields [3, R
) Et; 4, R ) tBu; 5, R ) p-C6H4C(dO)Et (selected because
preliminary studies established that this ester moiety
aided in the chromatographic separation of cycloadducts;
see Scheme 2); 75-83%].16 With these exo-methylene-
prolinates in hand, we investigated their 1,3-dipolar
cycloaddition reactions with the nitrile oxide generated
in situ from 2,6-dichlorobenzaldehyde oxime (NaOCl).
The targeted spiroisoxazolinoprolinates 6, 7, and 8 were
obtained in good yields (73-80%) and the cis:trans ratios,
As with 2, attempts to separate 6a from 6b failed.
However, diastereoisomers 7a /7b and 8a /8b were sepa-
rable by using routine column chromatography, which
set the stage for the multigram preparation of pure
spiroisoxazolinoproline amino acids and derivatives. The
only reliable 1H NMR spectral differences in the dia-
stereomers of 7 and 8 are due to the diastereotopic C3
protons of the pyrrolidine ring: 7a at δ 2.40 and 2.60
ppm/7b at δ 2.15 and 2.80 ppm; 8a at δ 2.50 and 2.80
ppm/8b at δ 2.30 and 2.90 ppm. After hydrolysis of
cycloadduct 7b (aq NaOH/EtOH) and recrystallization of
the resulting acid from MeOH, the stereochemistry (C2
carboxylic acid trans to the isoxazoline C-O bond) of pure
spiroisoxazolinoproline amino acid 9b was confirmed by
X-ray crystallographic analysis.
(13) (a) Alonso, C.; Nantz, M.; Kurth, M. J . Tetrahedron Lett. 2000,
41, 5617. (b) Kantorowski, E. J .; Kurth, M. J . J . Org. Chem. 1997, 62,
6797. (c) Lorsbach, B. A.; Miller, R. B.; Kurth, M. J . J . Org. Chem.
1996, 61, 8716. (d) Park, K.-H.; Kurth, M. J . J . Org. Chem. 2000, 65,
3520. (e) Sammelson, R. E.; Miller, R. B.; Kurth, M. J . J . Org. Chem.
2000, 65, 2225. (f) Park, K.-H.; Kurth, M. J . Chem. Commun. 2000,
19, 1835.
(14) (a) Martin, S. F.; Dupre, B. Tetrahedron Lett. 1983, 24, 1337.
(b) Manikandan, S.; Shanmugasundaram, M.; Raghunthan, R.; Malar,
E. J . P. Heterocycles 2000, 53, 579. (c) Kamimura, A.; Hori, K.
Tetrahedron 1994, 50, 7969. (d) Kozikowski, A. P.; Adamczyk, M. J .
Org. Chem. 1983, 48, 366.
At this point, nitrile oxides derived from o- and
p-methoxybenzaldehyde oximes were reacted with tert-
butyl prolinate 4 to give cycloadducts 10 and 11, respec-
tively, in good yields (70-75%; Scheme 3). As with 4 f
7, diastereomer ratios of 10a /10b and 11a /11b were also
1
(15) Hondrelis, J .; Lonergan, G.; Voliotis, S.; Matsoukas, J . Tetra-
hedron 1990, 46, 565.
(16) Durand, X.; Hudhomme, P.; Khan, J . A.; Young, D. W. J . Chem.
Soc., Perkin Trans. 1 1996, 1131.
1:4. Again, the H NMR spectra of 10a /10b and 11a /11b
were differentiable by the diastereotopic C3 protons of
the pyrrolidine ring: 10a at δ 2.32 and 2.42 ppm/10b at
5674 J . Org. Chem., Vol. 67, No. 16, 2002

Synthesis of Spiroisoxazolinoproline-Based Amino Acids
of 60-100% MeOH/water for 25 min, flow rate 1 mL/min, and
detection 254 nm.
Gen er a l P r oced u r e for th e P r ep a r a tion of exo-Meth -
ylen e-L-P r olin a te-Ba sed Am in o Ester s: Eth yl Nr-Boc-4-
m eth ylen e-L-p r olin a te (3). exo-Methylene-L-proline 2 (1.0 g,
4.40 mmol) was dissolved in dry dichloromethane (20 mL) and
cooled to 0 °C in an atmosphere of nitrogen. Ethanol (1.01 g,
22.0 mmol, 5 equiv) and 4-(dimethylamino)pyridine (54 mg,
0.44 mmol 10%) were added followed by N,N-dicyclohexyl-
carbodiimide (1.09 g, 5.3 mmol, 1.2 equiv) over 5 min. The
mixture was stirred at room temperature for 8 h at which time
the DCU was removed by filtration and the filtrate was
concentrated. CC purification (20% EA in hexanes) gave 3 as
F IGURE 2. N-Boc protected spiroisoxazolinoproline amino
acids.
a colorless oil (0.93 g, 3.65 mmol, 83%): [R]25 -19.1 (c 28.3,
D
CHCl3); IR (CDCl3) 1747, 1708, 1652 cm-1
;
1H (300 MHz,
CDCl3, ambient temperature, mixture of conformers) δ 1.07
3
2
(t, 3H, J ) 5.7 Hz), 1.24 (s, 9H × /5), 1.28 (s, 9H × /5), 2.39-
2.45 (m, 1H), 2.72-2.85 (m, 1H), 3.86-3.89 (m, 2H), 4.00 (q,
2H, J ) 5.7 Hz), 4.18-4.29 (m, 1H), 4.80 (s, 2H); 13C NMR (75
MHz, CDCl3, ambient temperature, mixture of conformers) δ
171.9, 171.6, 153.7, 153.1, 142.9, 141.9, 107.3, 107.2, 79.5, 60.5,
58.7, 58.2, 50.3, 50.2, 36.4, 35.7, 28.0, 27.9, 13.9, 13.8. Anal.
Calcd for C13H21NO4: C, 61.16, H, 8.29, N, 5.49. Found: C,
60.99, H, 8.25, N, 5.44.
(4-P r op ion yl)p h en yl Nr-Boc-4-m eth ylen e-L-p r olin a te
(5). The preparation of 3 was modified as follows to give 5.
Compound 2 (2.0 g, 8.80 mmol), DCC (2.18 g, 10.56 mmol, 1.2
equiv), DMAP (110 mg, 0.88 mmol, 10%), 1-(4-hydroxyphenyl)-
propan-1-one (1.72 g, 11.5 mmol, 1.3 equiv), and CC (20%
EtOAc in hexanes) gave 5 (2.37 g, 6.6 mmol, 75%) as a colorless
F IGURE 3. N-Fmoc protected spiroisoxazolinoproline amino
acids.
δ 2.05 and 2.62 ppm; 11a at δ 2.38 and 2.52 ppm/11b at
δ 2.15 and 2.75 ppm.
oil: [R]25 -17.2 (c 10.6, CHCl3); IR (CHCl3) 1772, 1687, 1600
D
cm-1
;
1H NMR (300 MHz, DMSO-d6, ambient temperature,
Treating each pure diastereomer of 7/10/11 with aq
NaOH/EtOH delivered the corresponding N-Boc pro-
tected spiroisoxazolinoproline amino acids (Figure 2): 9a
(90%), 9b (92%), 12a (83%), 12b (85%), 13a (91%), and
13b (92%). Finally, the X-ray structure of acid 13a
(minor) verified that the carboxyl group is cis to the
isoxazoline C-O bond.
mixture of conformers) δ 1.05 (t, 3H, J ) 6 Hz), 1.37 (s, 9H ×
4
5/9), 1.40 (s, 9H × /9), 2.79-2.89 (m, 1H), 3.02 (q, 2H, J ) 6
Hz), 3.99 (s, 2H), 4.59-4.64 (m, 1H), 5.06 (s, 2H), 7.20-7.24
(m, 2H), 8.00-8.05 (m, 2H); 13C NMR (75 MHz, DMSO-d6, 100
°C) δ 198.4, 169.5, 153.0, 142.0, 134.0, 128.7, 120.6, 107.1, 79.1,
58.2, 50.1, 35.2, 30.7, 27.5, 7.5.
Gen er a l P r oced u r e for th e P r ep a r a tion of Sp ir oisox-
azolin opr olin ates: ter t-Bu tyl (5S,8S)-3-(2,6-Dich lor oph en -
yl)-1-oxa-2,7-diazaspiro[4,4]non-2-ene-7-N-Boc-8-carboxylate
(7a ) a n d ter t-Bu tyl (5R,8S)-3-(2,6-Dich lor op h en yl)-1-oxa -
2,7-d ia za sp ir o[4,4]n on -2-en e-7-N-Boc-8-ca r boxyla te (7b).
Compound 4 (1.5 g, 5.30 mmol) and 2,6-dichlorobenzaldehyde
oxime (2 equiv, 2.0 g, 10.6 mmol) were dissolved in methylene
chloride (30 mL), and the solution was cooled to 0 °C. Aqueous
NaOCl (32 g, 5%, 21.2 mmol, 4 equiv) was added dropwise over
30 min, and the reaction was stirred vigorously for 8 h (0 °C
f room temperature). The layers were separated, and the
aqueous layer was extracted with methylene chloride. The
combined organic layers were dried with MgSO4 and concen-
trated. The crude sample was purified by CC (40% ethyl
acetate in hexanes) to ester 7 (1.87 g, 4.0 mmol, 75%).
Diastereoisomers 7a and 7b were separated from a mixture
of 7 (1.87 g, 4.0 mmol) by CC (25% ethyl acetate in hexanes)
to give 7a (0.3 g, 16%) and 7b (1.40 g, 75%).
An N-Boc protecting group was selected for the syn-
thesis of acids 9/12/13 from 4-hydroxyproline because of
its base stability. However, since N-Fmoc protected
amino acids are more attractive and practical in peptide
synthesis, we next set out to convert these N-Boc
protected amino acids to N-Fmoc protected amino acids.
N-Boc deprotection with TFA/CH2Cl2 (1/1) followed by
solvent removal and addition of DMF/Na2CO3/FmocOSu
allowed for the “one flask” conversion of 9b/12a /13b to
14/15/16 (Figure 3).
In summary, the exo-methylenepyrrolidine system
undergoes a highly regioselective 1,3-dipolar cylco-
addition reaction with nitrile oxides, the stereoselectivity
of prolinate 4 is ca. 4:1, and these tert-butyl ester
diasteroisomers are readily separable by column chro-
matography.
7a : [R]25D +9.7 (c 29.0, CHCl3); IR (CHCl3) 1749, 1702, 1400
1
cm-1; H (300 MHz, CDCl3, ambient temperature, mixture of
Exp er im en ta l Section
conformers) δ 1.45-1.50 (m, 18H), 2.29-2.45 (m, 1H), 2.62 (dd,
1H, J ) 13.3, 2.4 Hz), 3.14-3.34 (m, 2H), 3.65-3.97 (m, 2H),
4.29-4.41 (m, 1H), 7.24-7.37 (m, 3H); 13C NMR (75 MHz,
CDCl3, ambient temperature, mixture of conformers) δ 170.3,
169.9, 153.7, 153.6, 153.4, 134.8, 131.0, 128.2, 127.9, 90.8 89.8,
81.6, 81.5, 80.1, 80.0, 59.0, 58.7, 55.9, 55.8, 45.4, 45.2, 41.9,
41.2, 28.4, 28.3, 27.9. Anal. Calcd for C22H28Cl2N2O5: C, 56.06,
H, 5.99, N, 5.94. Found: C, 55.67, H, 5.94, N, 5.80.
Gen er a l P r oced u r es. All chemicals were obtained from
commercial suppliers and used without purification. Analytical
TLC was carried out on precoated plates (Merck silica gel 60,
F254) and visualized with UV light. Flash column chroma-
tography was performed with silica (Merck, 70-230 mesh).
NMR spectra (1H at 300 or 400 MHz, 13C at 75 or 100 MHz)
were recorded in CDCl3 or DMSO-d6 as solvents, and chemical
shifts are expressed in parts per million relative to internal
TMS. Optical rotations were measured with a J ASCO DIP-
370 automatic polarimeter. CC refers to column chromatog-
raphy. Concentration refers to rotary evaporation. The ratio
of diastereoisomers was determinate by HPLC, using Sym-
metry C18 (4.6 × 250 mm, Waters) with linear gradient elution
7b: [R]25 +2.2 (c 4.2, CHCl3); IR (CHCl3) 1749, 1702, 1400
D
cm-1; H (300 MHz, CDCl3, ambient temperature, mixture of
1
conformers) δ 1.41-1.43 (m, 18H), 2.06-2.16 (m, 1H), 2.70-
2.81 (m, 1H), 3.12-3.29 (m, 2H), 3.58-3.64 (m, 1H, J ) 11.7
Hz), 3.88-4.01 (m, 1H, J ) 11.7 Hz), 4.31-4.38 (m, 1H), 7.23-
7.34 (m, 3H); 13C NMR (75 MHz, DMSO-d6, 75 °C) δ 170.1,
J . Org. Chem, Vol. 67, No. 16, 2002 5675

Cheng et al.
153.8, 152.4, 133.4, 131.5, 127.9, 127.5, 80.4, 78.9, 58.3, 55.7,
42.8, 27.6, 27.2. Anal. Calcd for C22H28Cl2N2O5: C, 56.06, H,
5.99, N, 5.94. Found: C, 55.66, H, 6.01, N, 5.91.
[4,4]n on -2-en e-7-N-Boc-8-ca r boxyla te (11b). The prepara-
tion of 7 was modified as follows to give 11. Compound 4 (0.6
g, 2.12 mmol), o-methoxybenzaldehyde oxime (2 equiv, 0.64
g, 4.23 mmol), aqueous NaOCl (12.6 g, 5%, 8.50 mmol, 4 equiv),
CH2Cl2 (20 mL), and CC (25% EtOAc in hexanes) gave 11a
(0.13 g, 0.27 mmol) and 11b (0.61 g, 1.30 mmol) in a total yield
of 74%.
(4-P r op ion yl)p h en yl (5S,8S)-3-(2,6-Dich lor op h en yl)-1-
oxa -2,7-d ia za -sp ir o[4,4]n on -2-e n e -7-N -Boc-8-ca r b ox-
yla t e (8a ) a n d (4-P r op ion yl)p h en yl (5R,8S)-3-(2,6-Di-
ch lor oph en yl)-1-oxa-2,7-diazaspir o[4,4]n on -2-en e-7-N-Boc-
8-ca r b oxyla t e (8b ). The preparation of 7 was modified as
follows to give 8. Compound 5 (2.5 g, 7.0 mmol), 2,6-dichloro-
benzaldehyde oxime (2 equiv., 2.66 g, 14.0 mmol), aqueous
NaOCl (42 g, 5%, 28.0 mmol, 4 equiv), CH2Cl2 (40 mL), and
CC (25% EtOAc in hexanes) gave 8a (1.04 g, 1.90 mmol) and
8b (1.75 g, 3.20 mmol) in a total yield of 73%.
11a : [R]25D -25.0 (c 0.8, CHCl3); IR (CHCl3) 1747, 1741 1400
cm-1 1H NMR (400 MHz, CDCl3, ambient temperature,
;
mixture of conformers) δ 1.46 (s, 9H), 1.50 (s, 9H), 2.36 (m,
1H), 2.51 (dd, 1H, J ) 13.5, 3.5 Hz), 3.22-3.40 (m, 2H), 3.64
(m, 1H, J ) 11.6 Hz), 3.83 (s, 3H), 3.86 (m, 1H), 4.34 (m, 1H),
6.91 (dd, 2H, J ) 8.8, 2.6 Hz), 7.57 (dd, 2H, J ) 8.8, 2.0 Hz);
13C NMR (100 MHz, CDCl3, ambient temperature, mixture of
conformers) δ 170.6, 161.1, 155.8, 153.7, 128.1, 121.8, 114.1,
89.7, 88.8, 81.6, 80.2, 59.1, 58.8, 56.1, 55.3, 43.9, 42.2, 41.1,
28.3, 27.9. Anal. Calcd for C23H32N2O6: C, 63.87, H, 7.46, N,
6.48. Found: C, 63.79, H, 7.56, N, 6.18.
8a : [R]25D +13.0 (c 2.4, CHCl3); IR (CHCl3) 1779, 1689 cm-1
;
1H NMR (300 MHz, CDCl3, ambient temperature, mixture of
1
conformers) δ 1.15 (t, 3H, J ) 7.2 Hz), 1.44 (s, 9H × /2), 1.45
1
(s, 9H × /2), 2.43-2.59 (m, 1H), 2.79-2.96 (m, 3H), 3.20-3.38
(m, 2H), 3.72 (dd, 1H, J ) 12, 2.4 Hz), 4.05 (t, 1H, J ) 12 Hz),
7.22-7.97 (m, 7H); 13C NMR δ 199.1, 199.0, 169.6, 169.3, 154.1,
154.0, 153.9, 153.6, 153.0, 134.5, 134.3, 134.1, 131.1, 129.3,
129.2, 127.8, 121.5, 121.3, 90.05, 90.07, 80.5, 58.5, 58.2, 55.8,
55.7, 44.2, 44.1, 41.7, 41.2, 31.6, 28.2, 8.13. Anal. Calcd for
11b: [R]25D +8.3 (c 0.9, CHCl3); IR (CHCl3) 1741, 1702, 1401
cm-1 1H NMR (400 MHz, CDCl3, ambient temperature,
;
mixture of conformers) δ 1.42 (s, 9H), 1.45 (s, 9H), 2.08 (m,
1H), 2.68 (m, 1H), 3.21 (dd, 1H, J ) 16.8, 9.2 Hz), 3.34 (dd,
1H, J ) 16.8, 6.0 Hz), 3.57 (dd, 1H, J ) 11.5, 8.6 Hz), 3.80 (s,
3H), 3.86 (m, 1H), 4.34 (m, 1H), 6.88 (d, 2H, J ) 8.8 Hz), 7.53
(d, 2H, J ) 8.8 Hz); 13C NMR (100 MHz, CDCl3, ambient
temperature, mixture of conformers) δ 171.5, 161.1, 156.0,
153.4, 128.0, 121.6, 114.0, 89.8, 88.9, 81.3, 80.2, 80.0, 58.9, 57.1,
56.4, 55.2, 42.5, 42.3, 41.8, 40.8, 28.2, 27.8. Anal. Calcd for
C
27H28Cl2N2O6: C, 59.24; H, 5.16; N, 5.12. Found: C, 59.17;
H, 5.10; N, 5.08.
8b: [R]25 -9.0 (c 5.1, CHCl3); IR (CHCl3) 1772, 1684 cm-1
;
D
1H (300 MHz, CDCl3, ambient temperature, mixture of con-
3
formers) δ 1.01-1.11 (m, 3H), 1.37 (s, 9H × /5), 1.39 (s, 9H ×
2/5), 2.27-2.35 (m, 1H), 2.79-2.90 (m, 3H), 3.16-3.27 (m, 2H),
3.59-3.70 (m, 1H), 3.91-4.05 (m, 1H), 4.61-4.66 (m, 1H),
7.11-7.98 (m, 7H); 13C NMR (75 MHz, CDCl3, ambient
temperature, mixture of conformers) δ 198.7, 198.6, 169.7,
153.9, 153.8, 153.5, 153.4, 153.2, 152.6, 134.2, 134.1, 134.0,
131.0, 129.1, 129.0, 127.6, 121.1, 120.7, 90.6, 89.6, 80.5, 80.4,
58.3, 55.8, 43.1, 42.9, 40.9, 40.4, 31.3, 29.9, 7.9. Anal. Calcd
for C27H28Cl2N2O6: C, 59.24; H, 5.16; N, 5.12. Found: C, 59.36;
H, 5.19; N, 5.10.
C
23H32N2O6: C, 63.87, H, 7.46, N, 6.48. Found: C, 63.78, H,
7.46, N, 6.42.
Gen er al P r ocedu r e for Hydr olysis of Spir oisoxazolin o-
prolinates to Spiroisoxazolinoproline Amino Acids: (5R,8S)-
3-(2,6-Dich lor op h en yl)-1-oxa -2,7-d ia za sp ir o[4,4]n on -2-
en e-7-N-Boc-8-ca r boxylic Acid (9b). A mixture of compound
7b (2.0 g, 4.24 mmol) and sodium hydroxide (0.34 g, 8.50 mmol)
in EtOH/H2O (10 mL/10 mL) was refluxed 6 h. The solvent
was removed under reduced pressure, and 1 N HCl was added
to the reaction mixture until the pH reached to 2-3. Ethyl
acetate (20 mL × 2) extraction, drying over MgSO4, and
removal of solvent under reduced pressure gave 9b (1.61 g,
ter t-Bu t yl (5S,8S)-3-(2-Met h oxyp h en yl)-1-oxa -2,7-d i-
a za sp ir o[4,4]n on -2-en e-7-N-Boc-8-ca r boxyla te (10a ) a n d
tert-Butyl(5R,8S)-3-(2-Methoxyphenyl)-1-oxa-2,7-diazaspiro-
[4,4]n on -2-en e-7-N-Boc-8-ca r boxyla te (10b). The prepara-
tion of 7 was modified as follows to give 10. Compound 4 (0.5
g, 1.76 mmol), o-methoxybenzaldehyde oxime (2 equiv, 0.53
g, 3.52 mmol), aqueous NaOCl (10.5 g, 5%, 7.0 mmol, 4 equiv),
CH2Cl2 (20 mL), and CC (25% EtOAc in hexanes) gave 10a
(0.10 g, 0.23 mmol) and 10b (0.43 g, 1.0 mmol) in a total yield
of 70%.
3.90 mmol, 92%) as a solid: [R]25 +5.0 (c 0.8, MeOH); IR
D
1
(CHCl3) 1735, 1655, 1429 cm-1; H NMR (300 MHz, DMSO-
d6, 75 °C) δ 1.39 (s, 9H), 2.25 (dd, 1H, J ) 13.2, 8.1 Hz), 2.70
(ddd, 1H, J ) 13.2, 8.1, 1.8 Hz), 3.25-3.40 (m, 2H), 3.57 (d,
1H, J ) 11.7 Hz), 3.78 (dd, 1H, J ) 11.7, 1.8 Hz), 4.27 (t, 1H,
J ) 8.1 Hz), 7.46-7.57 (m, 3H); 13C NMR (75 MHz, DMSO-d6,
75 °C) δ 172.4, 153.9, 152.5, 133.4, 131.6, 127.9, 127.6, 89.9,
79.0, 57.8, 55.6, 42.5, 40.0, 27.6.
10a : [R]25 -19.8 (c 0.7, CHCl3); IR (CHCl3) 1704, 1394
D
cm-1
;
1H NMR (300 MHz, CDCl3, ambient temperature,
mixture of conformers) δ 1.40-1.47 (m, 18H), 2.24-2.49 (m,
2H), 3.30-3.94 (m, 7H), 4.24-4.45 (m, 1H), 6.87-6.94 (m, 2H),
7.30-7.36 (m, 1H), 7.66-7.68 (m, 1H); 13C NMR (100 MHz,
CDCl3, ambient temperature, mixture of conformers) δ 170.4,
170.1, 157.1, 155.7, 155.6, 153.7, 153.4, 131.3, 131.2, 128.9,
120.6, 118.2, 111.1, 89.7, 88.8, 81.3, 79.9, 79.8, 59.0, 58.7, 56.4,
55.9, 55.3, 46.2, 46.1, 41.9, 40.9, 28.36, 28.30, 27.9. Anal. Calcd
for C23H32N2O6‚0.3H2O: C, 63.08, H, 7.51, N, 6.39. Found: C,
63.30, H, 7.31, N, 6.16.
(5S,8S)-3-(2,6-Dich lor op h en yl)-1-oxa -2,7-d ia za sp ir o-
[4,4]n on -2-en e-7-N-Boc-8-ca r boxylic Acid (9a ). The prepa-
ration of 9b was modified as follows to give 9a . Compound 7a
(1.0 g, 2.31 mmol), sodium hydroxide (0.20 g, 5.00 mmol),
EtOH/H2O) (15 mL/15 mL), and CC (40% ethyl acetate in
hexanes with 1% AcOH) gave 9a (0.86 g, 2.08 mmol, 90%) as
a solid: [R]25 -23.8 (c 2.9, CHCl3); IR (CHCl3) 1702, 1430
D
cm-1; 1H (300 MHz, CDCl3, ambient temperature, mixture of
2
1
conformers) δ 1.40 (s, 9H × /3), 1.43 (s, 9H × /3), 2.44-2.52
(m, 1H), 2.62-2.73 (m, 1H), 3.15-3.32 (m, 2H), 3.62-3.72 (m,
1H), 3.86-3.96 (m, 1H), 4.39-4.55 (m, 1H), 7.22-7.32 (m, 3H),
9.83 (br, s, 1H); 13C NMR (75 MHz, CDCl3, ambient temper-
ature, mixture of conformers) δ 176.0, 174.3, 153.8, 153.5,
134.7, 131.0, 128.0, 127.9, 90.3, 89.6, 81.3, 81.0, 58.0, 56.2, 55.8,
45.1, 44.9, 41.9, 41.1, 40.0, 28.3, 28.2.
10b: [R]25D +1.1 (c 1.2, CHCl3); IR (CHCl3) 1741, 1702 cm-1
;
1H NMR (300 MHz, CDCl3, ambient temperature, mixture of
conformers) δ 1.39-1.42 (m, 18H), 2.01-2.08 (dd, 1H, J ) 13.2,
7.5 Hz), 2.61-2.65 (m, 1H), 3.31-3.55 (m, 3H), 3.75-3.87 (m,
4H), 4.27-4.34 (m, 1H, J ) 7.5 Hz), 6.85-6.92 (m, 2H), 7.28-
7.34 (m, 1H), 7.64-7.68 (m, 1H); 13C NMR (75 MHz, CDCl3,
ambient temperature, mixture of conformers) δ 171.1, 171.0,
156.9, 155.7, 153.4, 153.1, 131.1, 128.7, 120.4, 117.9, 111.0,
89.7, 88.8, 80.9, 79.8, 79.6, 77.4, 58.8, 56.7, 56.2, 55.1, 44.7,
44.5, 41.5, 40.6, 28.1, 28.0, 27.76, 27.70. Anal. Calcd for
(5S,8S)-3-(2-Meth oxyp h en yl)-1-oxa -2,7-d ia za sp ir o[4,4]-
n on -2-en e-7-N-Boc-8-d ica r boxylic Acid (12a ). The prepa-
ration of 9b was modified as follows to give 12a . Compound
10a (2.3 g, 5.32 mmol), sodium hydroxide (0.43 g, 10.75 mmol),
EtOH/H2O) (20 mL/20 mL), extraction, and concentration of
organic layers gave 12a (1.66 g, 4.41 mmol, 83%) as a solid:
C
23H32N2O6: C, 63.87, H, 7.46, N, 6.48. Found: C, 64.00, H,
7.35, N, 6.21.
ter t-Bu tyl (5S,8S)-3-(4-Meth oxyph en yl)-1-oxa-2,7-diaza-
sp ir o[4,4]n on -2-en e-7-N-Boc-8-ca r boxyla te (11a ) a n d ter t-
Bu tyl (5R,8S)-3-(4-Meth oxyp h en yl)-1-oxa -2,7-d ia za sp ir o-
[R]25D -15.6 (c 0.16, MeOH); IR (CHCl3) 1751, 1699, 1404 cm-1
1H NMR (300 MHz, CDCl3, ambient temperature, mixture of
;
3
2
conformers) δ 1.40 (s, 9H × /5), 1.42 (s, 9H × /5), 2.33-2.38
5676 J . Org. Chem., Vol. 67, No. 16, 2002

Synthesis of Spiroisoxazolinoproline-Based Amino Acids
(m, 1H), 2.55-2.63 (m, 1H), 3.33-3.85 (m, 4H), 3.78 (s, 3H),
4.38-4.53 (m, 1H), 6.88 (m, 2H), 7.30 (m, 1H), 7.63 (m, 1H);
13C NMR (100 MHz, CDCl3, ambient temperature, mixture of
conformers) δ 175.5, 174.1, 157.2, 155.8, 155.7, 154.7, 153.7,
131.4, 129.0, 120.6, 118.0, 111.2, 89.6, 88.8, 80.8, 80.6, 58.1,
57.9, 56.3, 55.4, 55.2, 45.5, 45.3, 41.2, 40.0, 28.1, 28.0. Anal.
Calcd for C19H24N2O6: C, 60.63; H, 6.43; N, 7.44. Found: C,
60.46; H, 6.40; N, 7.43.
reached 2-3 and extracted with ethyl acetate (20 mL × 2).
The organic layers were dried (MgSO4) and concentrated to a
pale-brown oil. The material was subjected to CC (50% ethyl
acetate in hexanes with 1% acetic acid) to give 14 (0.9 g, 70%)
as a white foam: [R]25D +13.4 (c 0.75, CHCl3); IR (CHCl3) 1708,
1430 cm-1; 1H NMR (400 MHz, CDCl3, ambient temperature,
mixture of conformers) δ 2.20-2.42 (m, 1H), 2.79-2.91 (m,
1H), 3.14-3.29 (m, 2H), 3.60-3.67 (m, 1H), 4.03-4.15 (m, 1H),
4.23-4.46 (m, 3H), 4.54-4.67 (m, 1H), 7.27-7.74 (m, 12H);
13C NMR (75 MHz, CDCl3, ambient temperature, mixture of
conformers) δ 176.8, 174.9, 155.7, 154.5, 154.3, 154.1, 143.8,
143.5, 143.4, 141.2, 134.9, 131.3, 131.2, 128.16, 128.10, 127.8,
127.6, 127.1, 125.0, 124.9, 124.8, 120.0, 119.9, 90.3, 89.8, 68.2,
67.8, 58.6, 57.9, 56.7, 56.4, 47.0, 46.9, 43.7, 43.4, 41.4, 39.4.
Anal. Calcd for C28H22Cl2N2O5‚0.5H2O: C, 61.54, H, 4.16, N,
5.12. Found: C, 61.23, H, 4.39, N, 5.07.
(5S,8S)-3-(2-Meth oxyp h en yl)-1-oxa -2,7-d ia za -sp ir o[4,4]-
n on -2-en e-7-N-F m oc-8-ca r boxylic Acid (15). The prepara-
tion of 14 was modified as follows to give 15. Compound 12a
(1.3 g, 3.45 mmol), CH2Cl2/TFA (20 mL/20 mL), Na2CO3 (0.74
g, 7.0 mmol) in H2O/DMF (7 mL/15 mL), FmocOSu (1.18 g,
3.50 mmol) in DMF (10 mL), and CC (50% ethyl acetate in
hexanes with 1% acetic acid) gave 15 (1.22 g, 2.45 mmol, 71%)
as a solid: [R]25D +40.4 (c 0.18, CHCl3); IR (CHCl3) 1739, 1660,
1442 cm-1; 1H (300 MHz, DMSO, 80 °C) δ 2.31-2.49 (m, 2H),
3.46 (s, 2H), 3.62 (s, 2H), 3.83 (s, 3H), 4.18-4.45 (m, 4H), 6.99
(t, 1H, J ) 7.5 Hz), 7.11 (d, 1H, J ) 8.1 Hz), 7.28-7.45 (m,
6H), 7.56-7.66 (m, 2H), 7.83 (d, 2H, J ) 7.8 Hz); 13C NMR
(100 MHz, CDCl3, ambient temperature, mixture of conform-
ers) δ 174.9, 173.9, 157.4, 155.9, 155.2, 143.9, 143.8, 143.6,
143.5, 141.2, 131.6, 129.3, 127.69, 127.61, 127.1, 125.09,
125.00, 124.8, 120.9, 119.8, 118.0, 111.3, 89.6, 89.0, 77.5, 67.8,
67.6, 58.4, 58.1, 56.2, 56.0, 55.4, 47.1, 45.6, 45.1, 41.6, 40.2.
Anal. Calcd for C29H26N2O6‚0.1H2O: C, 69.61; H, 5.28; N, 5.60.
Found: C, 69.39; H, 5.33; N, 5.47.
(5R,8S)-3-(2-Meth oxyp h en yl)-1-oxa -2,7-d ia za sp ir o[4,4]-
n on -2-en e-7-N-Boc-8-ca r boxylic Acid (12b). The prepara-
tion of 9b was modified as follows to give 12b. Compound 10b
(2.8 g, 6.47 mmol), sodium hydroxide (0.52 g, 13.00 mmol),
EtOH/H2O) (25 mL/25 mL), extraction, and concentration of
organic layers gave 12b (2.06 g, 5.48 mmol, 85%) as a solid:
[R]25 -3.1 (c 1.3, CHCl3); IR (CHCl3) 1750, 1699, 1404 cm-1
;
D
1H NMR (300 MHz, CDCl3, ambient temperature, mixture of
conformers) δ 2.14-2.27 (m, 1H), 2.57-2.67 (m, 1H), 38-4.49
(9m, 1H), 3.38-3.57 (m, 4H), 6.80-6.91 (m, 2H), 7.27-7.33
(m, 1H), 7.62-7.64 (m, 1H); 13C NMR (100 MHz, CDCl3,
ambient temperature, mixture of conformers) δ 175.5, 174.9,
157.0, 155.99, 155.95, 154.3, 153.3, 131.2, 128.6, 120.3, 117.7,
117.6, 111.0, 89.5, 89.0, 80.47, 80.43, 77.1, 58.3, 58.2, 56.5, 55.7,
55.0, 43.5, 43.3, 40.9, 39.9, 27.9, 27.7. Anal. Calcd for
C
19H24N2O6: C, 60.63; H, 6.43; N, 7.44. Found: C, 60.48; H,
6.49; N, 7.38.
(5S,8S)-3-(4-Meth oxyp h en yl)-1-oxa -2,7-d ia za sp ir o[4,4]-
n on -2-en e-7-N-Boc-8-d ica r boxylic Acid (13a ). The prepa-
ration of 9b was modified as follows to give 13a . Compound
11a (1.8 g, 4.16 mmol), sodium hydroxide (0.35 g, 8.75 mmol),
EtOH/H2O) (20 mL/20 mL), extraction, and concentration of
organic layers gave 13a (1.42 g, 3.79 mmol, 91%) as a solid:
[R]25D -43.2 (c 3.8, CHCl3); IR (neat) 1753, 1676 cm-1; 1H NMR
(400 MHz, DMSO-d6, 100 °C) δ 1.41 (s, 9H), 2.28 (d, 1H, J )
13.6 Hz), 2.53 (d, 1H, J ) 12.6 Hz), 3.38-3.64 (m, 4H), 3.80
(s, 3H), 4.31 (d, 1H, J ) 9.2 Hz), 6.99 (d, 2H, J ) 7.3 Hz), 7.57
(d, 2H, J ) 7.1 Hz);13C NMR (400 MHz, DMSO-d6, 100 °C) δ
172.0, 160.5, 156.0, 152.8, 127.7, 121.6, 114.0, 89.0, 78.8, 57.7,
55.9, 55.0, 42.5, 40.4, 27.7. Anal. Calcd for C19H24N2O6: C,
60.63; H, 6.43; N, 7.44. Found: C, 60.32; H, 6.32; N, 7.44.
(5R,8S)-3-(4-Meth oxyp h en yl)-1-oxa -2,7-d ia za sp ir o[4,4]-
n on -2-en e-7-N-Boc-8-ca r boxylic Acid (13b). The prepara-
tion of 9b was modified as follows to give 13b. Compound 11b
(2.1 g, 4.85 mmol), sodium hydroxide (0.40 g, 10.0 mmol),
EtOH/H2O) (20 mL/20 mL), extraction, and concentration of
(5R,8S)-3-(4-Meth oxyp h en yl)-1-oxa -2,7-d ia za sp ir o[4,4]-
n on -2-en e-7-N-F m oc-8-ca r boxylic Acid (16). The prepara-
tion of 14 was modified as follows to give 16. Compound 13b
(0.8 g, 2.12 mmol), CH2Cl2/TFA (15 mL/15 mL), Na2CO3 (0.45
g, 4.30 mmol) in H2O/DMF (5 mL/15 mL), FmocOSu (0.74 g,
2.20 mmol) in DMF (8 mL), and CC (50% ethyl acetate in
hexanes with 1% acetic acid) gave 15 (0.80 g, 1.60 mmol, 75%)
as a solid: [R]25 +4.2 (c 0.3, CHCl3); IR (KBr) 3440, 1702,
D
1608, 1515, 1423 cm-1; 1H (300 MHz, DMSO, 80 °C) δ 2.31 (br
s, 1H), 2.70 (br s, 1H), 3.39 (d, 1H, J ) 17.4 Hz), 3.51 (d, 1H,
J ) 17.4 Hz), 3.66 (d, 1H, J ) 11.7 Hz), 3.78 (s, 3H), 3.87 (d,
1H, J ) 11.7 Hz), 4.24-4.33 (m, 3H), 4.47 (br s, 1H), 6.98 (d,
2H, J ) 8.7 Hz), 7.30-7.42 (m, 4H), 7.59 (d, 2H, J ) 8.7 Hz),
7.66 (d, 2H, J ) 7.2 Hz), 7.83 (d, 2H, J ) 7.2 Hz); 13C NMR
(75 MHz, DMSO, 100 °C) δ 172.0, 160.3, 155.8, 153.3, 143.2,
140.2, 127.4, 127.0, 126.4, 124.4, 121.4, 119.3, 113.8, 89.1, 66.7,
58.0, 56.1, 54.9, 46.5, 40.3. Anal. Calcd for C 29H26N2O6: C,
69.87; H, 5.26; N, 5.62. Found: C, 70.20; H, 5.41; N, 5.59.
organic layers gave 13b (1.67 g, 4.46 mmol, 92%) as a solid:
1
[R]25 +9.2 (c 3.3, CHCl3); IR (neat) 1743, 1675 cm-1; H (400
D
MHz, DMSO-d6, 75 °C) δ 1.37 (s, 9H), 2.15 (dd, 1H, J ) 12,
8.1 Hz), 2.54 (dd, 1H, J ) 9.4, 1.3 Hz), 3.34-3.50 (m, 3H), 3.65
(d, 1H, J ) 11.2 Hz), 3.77 (s, 3H), 4.21 (t, 1H, J ) 8.2 Hz),
6.97 (d, 2H, J ) 8.4 Hz), 7.54 (d, 2H, J ) 8.4 Hz); 13C NMR
(400 MHz, DMSO-d6, 75 °C) δ 172.9, 160.6, 156.2, 153.0, 127.8,
121.6, 114.1, 89.1, 79.1, 58.1, 55.9, 55.1, 40.4, 40.1, 27.7. Anal.
Calcd for C19H24N2O6: C, 60.63; H, 6.43; N, 7.44. Found: C,
60.45; H, 6.39; N, 7.35.
Gen er a l P r oced u r e for Tr a n sfor m a t ion fr om N-Boc
P r otected Sp ir oisoxa zolin op r olin e Am in o Acid s to N-
F m oc P r otected Sp ir oisoxa zolin op r olin e Am in o Acid s:
(5R,8S)-3-(2,6-Dich lor op h en yl)-1-oxa -2,7-d ia za sp ir o[4,4]-
n on -2-en e-7-N-F m oc-8-ca r boxylic Acid (14). Compound 9b
(1.0 g, 2.40 mmol) was treated with the solution of CH2Cl2/
TFA (15 mL/15 mL), and the reaction mixture was stirred at
room temperature for 5 h. After removing the solvent under
reduced pressure, the residue was treated with Na2CO3 (0.53
g, 5.0 mmol) in H2O/DMF (5 mL/15 mL) for 30 min at 0 °C.
FmocOSu (0.85 g, 2.52 mmol) in DMF (10 mL) was added to
the reaction and the mixture was stirred at room temperature
for 3 h. The reaction was quenched with 1 N HCl until the pH
Ack n ow led gm en t. We thank the National Science
Foundation and the Cystic Fibrosis Foundation for
financial support of this research. The 400- and 300-
MHz NMR spectrometers used in this study were
funded in part by a grant from the NSF (CHE-9808183).
Su p p or t in g In for m a t ion Ava ila b le: 1H NMR and 13C
NMR spectra for compounds 5, 9a , and 9b, as well as X-ray
crystal structures for 9b, 13a , and 15. This material is
available free of charge via the Internet at http://pubs.acs.org.
J O025611J
J . Org. Chem, Vol. 67, No. 16, 2002 5677