Synthesis and X-ray crystallographic structure of leucine-phenylalanyl succinimide-based pseudopeptides

Full text of DOI:10.1021/jo961781i

J . Org. Chem. 1997, 62, 1509-1513
1509
Syn th esis a n d X-r a y Cr ysta llogr a p h ic
Str u ctu r e of Leu cin e-P h en yla la n yl
Su ccin im id e-Ba sed P seu d op ep tid es
Andrew D. Abell* and Mark D. Oldham
F igu r e 1. General structure of a peptidyl-based inhibitor of
a protease.
Department of Chemistry, University of Canterbury,
Christchurch, New Zealand
irreversible inhibitors of serine proteases, with peptidic
character, include the proline-valine pseudopeptide 2,12
phenylalanine-acylated enamino ester pseudodipeptides
3,13 and peptidyl halo ketones.1,14 We now report on the
synthesis and X-ray structure of peptidyl succinimde
derivatives. The design of these compounds was based
on simple succinimide compounds of the type 4, initially
developed by Groutas as general mechanism-based in-
hibitors of serine proteases.15
Received September 17, 1996
The incorporation of a structural group, represented
by X in Figure 1, which is known to inhibit a particular
class of enzyme, into a peptide recognition sequence, has
been the subject of much research. The aims of this work
are to enhance the selectivity of an inhibitor toward a
particular subclass of enzyme and also to produce fami-
lies of peptide-based inhibitors to act as biological probes.1
To date, a lot of this work has involved the incorporation
of a transition-state analog, at position X in Figure 1,
into a peptide sequence to produce potent inhibitors of
proteases. For example, specific reversible inhibitors of
serine proteases1,2 have been developed using peptidyl
fluoromethyl ketones,3 petidyl aldehydes,4 peptidyl car-
bamates,5 peptidyl keto esters,6 peptidyl boronic acids,7
and others.1 Potent and selective inhibitors of the
aspartyl proteases, renin8 and the HIV-protease,9 have
also been developed by incorporating a peptide transition
state isostere into a specific protease recognition se-
quence. For example the pseudopeptide 1, which con-
tains a centrally located hydroxyethylamine isostere, is
a potent inhibitor of the HIV-1 protease (Ki 0.6 nM).10
Conformationally restricted structural elements, such as
the macrocycle of 1, have been introduced into peptide-
based inhibitors in an attempt to enhance bioavailability
and biostability.10,11
Resu lts a n d Discu ssion
The method employed in the synthesis of the succin-
imide-based pseudodipeptides of the type 12 is outlined
in Scheme 1. The oxazolidinone 5 was prepared from
L-phenylalanine as previously described.13 Deprotonation
of 5, at C4, followed by alkylation with BrCH2CO2CHPh2
and finally hydrolysis gave the acid 6 in good yield.13
Reaction of the acid 6 with O-benzylhydroxylamine in the
presence of N,N-dicyclohexylcarbodiimide (DCC) and
1-hydroxybenzotriazole (HOBT) gave 7 in 81% yield. The
1H NMR spectrum of the N-Cbz-protected amine 7 was
run at an elevated temperature due to the existence of
two conformational isomers at 23 °C. Cyclization of 7
with triethylamine at rt then gave the succinimide 9 in
99% yield. The free amine 8 was obtained in 73% yield
by reaction of the protected amine 9 with p-toluene-
sulfonic acid under reflux. N-Acetyl-L-leucine was coupled
onto 8 to give 10 in 65% yield, using DCC and HOBT.
Deprotection of the benzyloxy group of 10 was achieved
by catalytic hydrogenation to give the hydroxysuccinim-
ide 11 which was then treated with methanesulfonyl
chloride in the presence of diisopropylethylamine to give
the desired pseudodipeptide 12.
Relatively few reports have appeared regarding the
incorporation of an irreversible protease inhibitor into a
peptide sequence. Some representative examples of
(1) Bernstein, P. R.; Edwards, P. D.; Williams, J . C. In Progress in
Medicinal Chemistry; Ellis, G. P., Luscombe, D. K., Eds.; Elsevier
Science: New York, 1994; Vol. 31, p 59. Edwards, P. D.; Bernstein, P.
R. Med. Res. Rev. 1994, 14, 127. Powers, J . C.; Harper, J . W. In
Proteinase Inhibitors; Barrett, A. J ., Salvesen, G., Eds.; Elsevier
Science: New York, 1986; pp 55-141.
(2) Abell, A. D. In Detailed Reaction Mechanisms: Mechanisms of
Biological Importance; Coxon, J . M., Ed.; J AI Press Inc: London, 1992;
Vol. 2, pp 243-260.
(3) Imperiali, B.; Abeles, R. H. Biochemistry 1986, 25, 3760.
(4) Thompson, R. C. Biochemistry 1973, 12, 47.
The epimeric pseudodipeptide, 15, was prepared by
coupling 8 with N-acetyl-D-leucine (step a, Scheme 2)
followed by deprotection and O-mesylation as described
(5) Digenis, G. A.; Agha, B. J .; Tsuji, K.; Kato, M.; Shingoi, M. J .
Med. Chem. 1986, 29, 1468.
(6) Peet, N. P.; Burkhart, J . P.; Angelastro, M. R.; Giroux, E. L.;
Mehdi, S.; Bey, P.; Kolb, M.; Neises, B.; Schirlin, D. J . Med. Chem.
1990, 33, 394.
(12) Reed, P. E.; Katzenellenbogen, J . A. J . Org. Chem. 1991, 56,
2624.
(13) Abell, A. D.; Taylor, J . M. J . Org. Chem., 1993, 58, 14. Abell,
A. D.; Oldham, M. D.; Taylor, J . M. J . Chem. Soc. Perkin Trans., 1
1995, 953.
(7) Kettner, C. A.; Shenvi, A. B. J . Biol. Chem., 1984, 259, 15106.
(8) Thaisrivongs, S.; Pals, D. T.; Turner, S. R.; Kroll, L. T. J . Med.
Chem. 1988, 31, 833.
(9) West, M. L.; Fairlie, D. P. Trends Pharmacol. Sci. 1995, 16, 67.
(10) March, D. R.; Abbenante, G.; Bergman, D. A.; Brinkworth, R.
I.; Wickramasinghe, W.; Begun, J .; Martin, J . L.; Fairlie, D. P. J . Am.
Chem. Soc. 1996, 118, 3375.
(14) Powers, J . C.; Gupton, B. F.; Harley, A. D.; Nishino, N.; Whitley,
R. J . Biochim. Biophys. Acta 1977, 485, 156.
(15) (a) Groutas, W. C.; Giri, P. K.; Crowley, J . P.; Castrisos, J . C.;
Brubaker, M. J . Biochem. Biophys. Res. Commun. 1986, 141, 741. (b)
Groutas, W. C.; Brubaker, M. J .; Stanga, M. A.; Castrisos, J . C.;
Crowley, J . P.; Schatz, E. J . J . Med. Chem. 1989, 32, 1607. (c) Groutas,
W. C.; Brubaker, M. J .; Chong, L. S.; Epp, J . B.; Huang, H.; Keller, C.
E.; McClenahan, J . J .; Givens, R. S.; Singh, R.; Zandler, M. E.; Karr,
P. A.; Tagusagawa, F. Drug Des. Discovery 1994, 11, 149.
(11) Fairlie, D. P.; Abbenante, G.; March, D. R. Curr. Med. Chem.
1995, 2, 654.
S0022-3263(96)01781-1 CCC: $14.00 © 1997 American Chemical Society

1510 J . Org. Chem., Vol. 62, No. 5, 1997
Notes
Sch em e 1a
Sch em e 3a
anhydride with O-benzylhydroxylamine.15 However, the
anhydrides required to prepare compounds of the type
12 are not so readily available although there are
reported examples of R-alkylated aspartic acid deriva-
tives.16 The exception being N-Cbz-L-aspartic acid an-
hydride 20 which was reacted with with O-benzylhy-
droxylamine to give 22 in 72% yield (Scheme 3).
Compound 22 was also prepared by a route analogous to
that used in the preparation of 12. In this case, the
reaction of 19 (compare 6 in Scheme 1) with O-benzyl-
hydroxylamine, gave 23 directly without the interme-
diacy of 21 (Scheme 3). Deformylation of 23 was accom-
plished, in a modest yield of 49%, by reaction with
triethylamine at rt. Attempted deformylation of com-
pounds of the type 23 under harsher reaction conditions17
gave reduced yields of the desired product. Compound
23 existed as a pair of conformational isomers by 1H and
13C NMR spectroscopy.
Sch em e 2a
An X-ray structural determination of 11 was carried
out to determine its conformation. Structural studies of
this type enable the compilation of accurate information
regarding amino acid geometry and conformational flex-
ibility among families of amino acids in different environ-
ments. Compound 11 is of particular significance in that
the phenylalanine residue is alkylated at the R-position
by incorporation into a succinimide ring. X-ray crystal
structures of a few other conformationally constrained
phenylalanine containing pseudopeptides have been re-
ported18,19 as have the crystal structures of nonpeptidic
N-hydroxysuccinimide derivatives, e.g. 24,15c and aspartic
acid-derived peptidic imides.20,21
A perspective drawing of 11, with atomic labeling, is
presented in Figure 2. The peptide backbone torsional
above for 12. The N-t-BOC-analog 18 was also prepared
(Scheme 2) for biological testing and to allow entry into
a series suitable for further elaboration toward the amino
terminus of the peptide sequence. The synthetic methods
presented in Schemes 1 and 2 provide a general method
for the synthesis of pseudopeptides containing an N-
[(alkylsulfonyl)oxy]succinimide moiety. The leucine-
phenylalanine sequence of 12 was chosen to target the
known specificity of R-chymotrypsin.1
(16) Williams, R. M. Synthesis of Optically Active R-Amino Acids;
Organic Chemistry Series; Baldwin, J ., Magnus, P. D., Eds.; Pergamon
Press: Oxford, 1989; Vol. 7.
(17) Zydowsky, T. M.; Dellaria, J . F., J r.; Nellans, H. N. J . Org.
Chem. 1988, 53, 5607.
(18) Toniolo, C.; Formaggio, F.; Crisma, M.; Valle, G.; Boesten, W.
H. J .; Schoemaker, H. E.; Kamphuis, J .; Temussi, P. A.; Becker, E. L.;
Pre´cigoux, G. Tetrahedron 1993, 49, 3641.
(19) Valle, G.; Kazmierski, W. M.; Crisma, M.; Bonora, G. M.;
Toniolo, C.; Hruby, V. J . Int. J . Peptide Protein Res. 1992, 40, 222.
(20) Toniolo, C. Int. J . Peptide Protein Res. 1990, 35, 287.
(21) Capasso, S.; Niola, M. P.; Sica, F.; Zagari, A. Acta Crystallogr.
1989, C45, 83.
By comparison, the simple succinimide derivatives of
the type 4 are prepared by reaction of the corresponding

Notes
J . Org. Chem., Vol. 62, No. 5, 1997 1511
Ta ble 1. Tor sion An gles for 11
torsion
angle (deg)
C8-N9-C10-C1
C10-N9-C8-C7
N6-C7-C8-N9
C8-C7-N6-C3
ω0
175.0(7)
-69.9(9)
117.7(7)
-174.9(6)
-53.0(8)
129.6(6)
-176.3(7)
74.5(9)
æ1
ψ1
ω1
C2-C3-N6-C7
æ2
N1-C2-C3-N6
C13-C12-C8-N9
C8-C12-C13-C14
C8-C12-C13-C15
N6-C3-C16-C17
C22-C17-C16-C3
C18-C17-C16-C3
ψ2
ø1(1)
ø21(1)
ø22(1)
ø1(2)
ø21(2)
ø22(2)
-163.9(7)
173.9(6)
91.9(9)
-85.1(8)
with a N1-C2-C3-N6 (ψ2) torsion angle of 129.6° which
deviates slightly from a value of 120° that would cor-
respond to a fully flat ring.21 The N1-C2 [1.370(9) Å]
and N1-C5 [1.387(9) Å] bond lengths are significantly
longer than N-C peptide bonds and æ2 (C2-C3-N6-
C7, Table 1) torsion angle is similar in magnitude to other
succinimide peptides.21 Finally, the plane of the O7-
C7-N6 peptide bond is approximately perpendicular
(87°) to the succinimide ring.
F igu r e 2. ORTEP diagram of 11, with a cocrystallized
molecule of methanol, showing the crystallographic numbering
scheme.
Intermolecular hydrogen bonds are found in the crystal
structure of 11, between O5/N6, O5/O10, and O7/
methanol (cocrystallized). Intramolecular hydrogen bonds
were not evident as is the case in the crystal structures
of related dipeptides.18,22-24 The above crystallographic
determination provides data on the preferred conforma-
tions of succinimide-based pseudopeptides that are de-
signed to probe the specificity of proteases.
angles (ω, æ, and ψ) and the amino acid side chain
torsional angles (ø) are given in Table 1, with standard
deviations given in parentheses. The absolute configu-
ration of 11 was assigned as shown on the basis of
crystallographic data and the known configuration of the
starting amino acids, i.e. L-phenylalanine and L-leucine.
The first point to note regarding the conformation of
11 is that it lacks the typical H shape, commonly
observed in the crystal structures of related dipeptides,
where the two amino acid side chains are extended from
the backbone and run approximately parallel.17,22-24 The
phenylalanine CH2Ph group of 11 is distorted from the
standard H shape by adopting an unusual conformation
as evidenced by a ø1(2) torsional angle of 173.9° (Table 1).
An analysis of the literature on leucine-tyrosine crystal
structures reveals that the ø1(2) torsion angle is typically
in the range -50° to -80°.22 It should also be noted that
11 and the succinimide-based elastase inhibitor, 24,15c
have vastly different conformation for the CH2Ph groups,
as determined by X-ray crystallography. The leucine side
chain of 11 adopts a conformation (ø1(1) -176.3°, ø22(1) and
Exp er im en ta l Section
Gen er a l Meth od s. Melting points are uncorrected. Optical
rotations are given in units of 10-1 deg cm2 g-1
. Preparative
chromatography was carried out using a Chromatotron (Harri-
son Research Inc.) using glass plates coated with Merk type 60
PF254 silica gel. Petroleum ether refers to the fraction of bp 60-
70 °C.
X-r a y cr ysta llogr a p h ic d eter m in a tion for com p ou n d 11:
C
20H29N3O6; MR 407.46; crystal dimensions 0.80 × 0.40 × 0.25
mm; orthorhombic, a 12.364(7), b 12.565(7), c 13.756(12) Å; V
2137(3) Å3; space group P212121; Z ) 4, F(000) 872; Dcalc 1.266
mg/m3; absorption coefficient 0.094 mm-1; θ range for data
collection 2.21-22.50; index ranges -13 e h e 1,0 e k e 13, 0
e 1 e 14; data/restraints/parameters 1605/0/272; goodness of
fit on F2 was 0.906; final R indices [for 1032 reflections with I >
2σ(I)] R1 ) 0.0567, wR2 ) 0.1311; R indices (all data) R1
0.0899, wR2 ) 0.1419; largest difference peak and hole 0.265
and -0.266 e Å-3
)
.
ø
21(1) 74.5° and -163.9°, Table 1) which is similar to that
The unit cell parameters were obtained by least-squares
refinement of the setting angles of 18 reflections with 10° e 2θ
e 22° from a Siemens P4 diffractometer using graphite-mono-
chromatized Mo-KR radiation (λ ) 0.71073 Å). A unique data
set was measured at 158(2) K within 2θmax ) 57° limit (ω scans).
observed for one of three independent conformations
(conformation A)19 adopted in the crystal structure of
N-acetyl-L-Leu-L-Tyr-OMe. In this case the leucine side
chain adopts the less common t(tg-) conformation.22,23
As expected, the ω0 and ω1 torsion angles (Table 1) are
consistant with planar peptide bonds. A æ and ψ map23
for the observed conformation of 11 revealed it to be close
to that of an antiparallel â sheet. Similar backbone
conformations have been observed for related linear
peptides containing a Leu-Tyr/Phe sequence.18,22-24 The
observed structure of the imide of 11 is consistent with
the published structures of peptidic imides.20,21 In par-
ticular, the succinimde ring of 11 is slightly puckered
Of the 1608 reflections obtained, 1605 were unique (Rint
)
0.2213) and were used in the full-matrix least-squares refine-
ment [SHELXL-93].26 The intensities of three standard reflec-
tions, measured every 97 reflections throughout the 26 h data
collection, showed 17.11% decay due to the instability of the
crystal. The structure was solved by direct methods [SHELXS-
86].27 Hydrogen atoms were fixed in idealized positions. All
non-hydrogen atoms were refined with anisotropic atomic
displacement parameters. Neutral scattering factors and anoma-
lous dispersion corrections for non-hydrogen atoms were taken
from Ibers and Hamilton.28 Full details of the X-ray structural
(22) Karle, I. L.; Flippen-Anderson, J . L. Acta Crystallogr. 1989, C45,
791.
(25) Benedetti, E.; Morelli, G.; Nemethy, G.; Scheraga, H. A. Int. J .
Peptide Protein Res. 1983, 22, 1.
(23) Krause, J . A.; Baures, P. W.; Eggleston, D. S. Acta Crystallogr.
1993, B49, 123.
(24) Delettre´, P. J .; Berthou, J .; Lifchitz, A.; J olle´s, P. Acta Crys-
tallogr. 1988, C44, 902.
(26) Sheldrick, G. M. SHELXL 93. J . Appl. Crystallogr., in press.
(27) Sheldrick, G. M. Acta Crystallogr. 1990, A46, 467.
(28) Ibers, J . A., Hamilton, W. C., Eds/ International Tables for
Crystallography; Kynoch Press: Birmingham, 1992; Vol. C.

1512 J . Org. Chem., Vol. 62, No. 5, 1997
Notes
determination of 11 have been deposited with the Cambridge
Crystallographic Data Centre (CCDC).
then for a further 5.5 h at rt under N2. The reaction mixture
was diluted with dichloromethane, filtered, and washed succes-
sively with 5% aqueous hydrochloric acid (20 mL) and water (2
× 20 mL). The organic phase was dried (Na2SO4), and the
residue was chromatographed using a 1 mm chromatotron plate,
eluting with 75% ethyl acetate/25% petroleum ether to give 10
as an oil (89 mg, 65%) which was crystallized from ethyl acetate
by diffusion with petroleum ether: mp 177-178 °C; FTIR (KBr)
3299, 3064, 2958, 1795, 1731, 1645, 1548 cm-1; 1H NMR (CDCl3)
δ 0.93 (d, J ) 6.4 Hz, 3H), 0.95 (d, J ) 6.4 Hz, 3H), 1.51 (m,
1H), 1.67 (m, 2H), 2.01 (s, 3H), 2.90 (AB q, J AB ) 17.6 Hz, ∆ν )
9.4 Hz, 2H, CH2CO), 3.06 (AB q, J AB ) 13.2 Hz, ∆ν ) 48.2 Hz,
2H, C3CH2Ph), 4.39 (q, J ) 7.8 Hz, 1H), 4.92 (AB q, J AB ) 9.8
Hz, ∆ν ) 14.0 Hz, 2H, OCH2Ph), 5.73 (d, J ) 7.3 Hz, 1H, NH),
6.83 (s, 1H, NH), 7.16 (m, 2H), 7.33 (m, 6H), 7.47 (m, 2H); 13C
NMR (CDCl3) δ 22.06, 22.73, 23.05, 24.55, 37.35, 40.53, 41.92,
51.32, 57.36, 78.67, 128.28, 128.42, 129.05, 129.13, 129.68,
(-)-(2S,4R)-4-Ben zyl-4-[(ben zyloxya m in o)ca r bon yl]m e-
t h yl]-3-[(b en zyloxy)ca r b on yl]-2-p h en yl-1,3-oxa zolid in -5-
on e (7). O-Benzylhydroxylamine hydrochloride (180 mg, 1.13
mmol), triethylamine (160 µl, 1.15 mmol), and 1-hydroxy-
benzotriazole‚1H2O (173 mg, 1.13 mmol) were added to the
carboxylic acid 613 (503 mg, 1.13 mmol) dissolved in dichlo-
romethane (3 mL). The solution was stirred for 10 min at 0 °C
under N2 after which time N,N-dicyclohexylcarbodiimide (DCC)
(235 mg, 1.14 mmol) was added. The mixture was stirred for a
further 10 min at 0 °C and then for 6 h at rt under N2. The
reaction mixture was filtered and washed with 5% aqueous
hydrochloric acid (25 mL) followed by water (2 × 25 mL). The
organic phase was dried (MgSO4), and the residue was filtered
and chromatographed using a 2 mm chromatotron plate, eluting
with 35% ethyl acetate/65% petroleum ether to give 7 (507 mg,
81%). Compound 7 was recrystallized from ethyl acetate by
diffusion with petroleum ether: mp 92-94 °C; FTIR (KBr) 3212,
3034, 1797, 1713, 1660 cm-1; 1H NMR (dioxane-d8, 90 °C) δ 2.93
(d, J ) 17.6 Hz, 1H), 3.50 (AB q, J AB ) 13.4 Hz, ∆ν ) 73.8 Hz,
2H, CH2Ph), 3.50-3.65 (br, 1H), 4.98 (AB q, J AB ) 11.2 Hz, ∆ν
) 4.6 Hz, 2H, NOCH2Ph), 5.13 (AB q, J AB ) 12.2 Hz, ∆ν ) 33.1
Hz, 2H, CO2CH2Ph), 6.48 (s, 1H), 6.52 (d, J ) 7.8 Hz, 2H), 7.07
(br s, 1H), 7.12 (t, J ) 7.3 Hz, 2H), 7.28-7.57 (m, 16H), 9.59 (br
s, NH); 13C NMR (CDCl3) δ 38.23, 41.85, 65.64, 67.25, 78.44,
79.52, 90.42, 127.46, 127.52, 127.86, 127.948, 128.18, 128.66,
128.90, 129.04, 129.26, 130.79, 134.78, 135.62, 152.56, 166.47,
130.19, 131.92, 133.54, 168.48, 170.73, 171.69, 172.74; [R]20
)
D
+5° (c ) 1, CH2Cl2). Anal. Calcd for C26H31N3O5: C, 67.08; H,
6.71; N, 9.03. Found: C, 66.90; H, 6.70; N, 9.08.
(+)-(3R )-3-(N -Ac e t y l-L -le u c y la m in o )-3-b e n zy l-1-h y -
d r oxysu ccin im id e (11). A mixture of the 1-(benzyloxy)-
succinimide 10 (64 mg, 0.14 mmol) and 10% Pd on C (7 mg), in
dry THF (4 mL), was stirred for 3.5 h under a hydrogen
atmosphere. The reaction mixture was filtered and evaporated
to dryness. The resulting solid was washed with ethyl acetate
and redissolved in ethanol, and the solution was filtered and
evaporated to give 11 as a white solid (24 mg, 47%). The residue
from the ethyl acetate washings was crystallized from methanol
by diffusion with diethyl ether to yield more 11 (5 mg, 10%):
mp > 220 °C dec; FTIR (KBr) 3314, 2957, 1793, 1719, 1656, 1536
172.83; [R]20 ) -3° (c ) 1, CH2Cl2). Anal. Calcd for C33H30
-
D
N2O6‚1/4CH3CO2CH2CH3: C, 71.31; H, 5.63; N, 4.89. Found: C,
71.23; H, 5.60; N, 4.87.
1
cm-1; H NMR (CD3OD) δ 0.95 (d, J ) 6.4Hz, 3H), 0.99 (d, J )
(+)-(3R)-3-Ben zyl-1-(ben zyloxy)-3-[(ben zyloxy)car bon yl]-
a m in o]su ccin im id e (9). Triethylamine (0.5 mL, 3.59 mmol)
was added to oxazolidinone 7 (190 mg, 0.345 mmol) dissolved
in dichloromethane (4.5 mL), and the mixture was stirred for 2
h at rt under N2. The reaction mixture was then washed with
5% aqueous hydrochloric acid (20 mL) followed by water (2 ×
20 mL). The organic phase was dried (MgSO4), and the residue
was chromatographed on a 1 mm chromatotron plate eluting
with 25% ethyl acetate/75% petroleum ether to give 9 as a clear
colorless oil (152 mg, 99%): FTIR (KBr) 3333, 3033, 1794, 1732,
1525 cm-1; 1H NMR (CDCl3) δ 3.00 (AB q, J AB ) 20.1 Hz, ∆ν )
14.0 Hz, 2H, CH2CO), 3.03 (AB q, J AB ) 13.1 Hz, ∆ν ) 38.3 Hz,
2H, CH2Ph), 4.94 (AB q, J AB ) 9.3 Hz, ∆ν ) 15.9 Hz, 2H, NOCH2-
Ph), 5.09 (AB q, J AB ) 12.2 Hz, ∆ν ) 11.9 Hz, 2H, COCH2Ph),
5.31 (s, 1H, NH), 7.14 (m, 3H), 7.30-7.38 (m, 10H), 7.45 (s, 2H);
13C NMR (CDCl3) δ 37.53, 42.33, 57.48, 67.47, 78.82, 128.29,
128.44, 128.48, 128.61, 129.09, 129.20, 129.77, 130.07, 132.04,
6.4 Hz, 3H), 1.54 (m, 2H), 1.73 (hept, J ) 6.8 Hz, 1H), 1.95 (s,
3H), 2.85 (s, 2H), 3.14 (AB q, J AB ) 13.2 Hz, ∆ν ) 59.7 Hz, 2H,
CH2Ph), 4.35 (dd, J ) 6.8, 8.8Hz, 1H), 7.19 (m, 2H), 7.28 (m,
3H); 13C NMR (CD3OD) δ 22.45, 22.55, 23.60, 26.04, 37.83, 41.96,
42.64, 53.06, 59.13, 129.24, 130.11, 131.66, 134.16, 171.90,
173.73, 175.20, 175.38; [R]20 ) +13° (c ) 0.2, CH3OH); HRMS
D
(FAB, MNa+) calcd for C19H25N3O5Na 398.16921, found 398.16909.
(-)-(3R)-3-(N-Acet yl-L-leu cyla m in o)-3-b en zyl-1-[(m et h -
a n esu lfon yl)oxy]-su ccin im id e (12). To a suspension of 11
(18 mg, 0.05 mmol) in dichloromethane (0.3 mL) was added
diisopropylethylamine (11 µL, 0.063 mmol). The suspension was
cooled to 0 °C under N2, methanesulfonyl chloride (6 µL, 0.08
mmol) was added, and the mixture was stirred for 35 min at 0
°C. The mixture was diluted with dichloromethane (15 mL) and
filtered to give starting material (4.5 mg, 25%). The filtrate was
washed with cold water (10 mL), cold 5% aqueous hydrochloric
acid (10 mL), and saturated aqueous sodium bicarbonate (10
mL); dried (Na2SO4); and evaporated to dryness under reduced
pressure. The residue was further purified by a series of
crystallizations from diethyl ether and also ethyl acetate/
petroleum ether to give 12 (5.5 mg, 25%): mp 197-201 °C; FTIR
(KBr) 3273, 3038, 2930, 1813, 1749, 1536 cm-1; 1H NMR (CDCl3)
δ 0.91 (d, J ) 6.4 Hz, 3H), 0.94 (d, J ) 6.4 Hz, 3H), 1.48 (m,
133.42, 135.45, 154.94, 168.43, 172.34; [R]20 ) +34° (c ) 0.5,
D
CH2Cl2); HRMS (FAB, MK+) calcd for C26H24N2O5K 483.13222,
found 483.13330.
(+)-(3R)-3-Am in o-3-ben zyl-1-(ben zyloxy)su ccin im id e (8).
p-Toluenesulfonic acid (250 mg, 1.31 mmol) was added to 9 (291
mg, 0.66 mmol) dissolved in toluene (12 mL). The reaction
mixture was heated to reflux for 2 h. The solvent was evapo-
rated under reduced pressure, and the residue was taken up in
dichloromethane (35 mL) and water (50 mL). The organic phase
was separated, washed with water (2 × 50 mL), and dried
(MgSO4), and the residue was purified by radial chromatography
using a 1 mm plate and eluting with 75% ethyl acetate/25%
petroleum ether to yield the amine 8 as a white solid (149 mg,
73%) which was recrystallized from ethyl acetate and petroleum
ether: mp 109-111 °C; FTIR (KBr) 3385, 3030, 1786, 1718, 1600
cm-1; 1H NMR (CDCl3) δ 2.61 (AB q, J AB ) 18.1 Hz, ∆ν ) 148.3
Hz, 2H, CH2CO), 2.92 (AB q, J AB ) 13.2 Hz, ∆ν ) 51.5 Hz, 2H,
CH2Ph), 4.87 (AB q, J AB ) 10.3 Hz, ∆ν ) 25.4 Hz, 2H, NOCH2-
Ph), 7.14 (m, 2H), 7.27-7.43 (m, 8H); 13C NMR (CDCl3) δ 38.60,
44.72, 56.92, 78.59, 127.76, 128.52, 128.84, 129.45, 129.96,
130.09, 133.18, 134.11, 168.94, 175.71; [R]20D ) +68° (c ) 1, CH2-
Cl2). Anal. Calcd for C18H18N2O3: C, 69.66; H, 5.85; N, 9.03.
Found: C, 69.62; H, 5.84; N, 9.12.
1H), 1.66 (m, 2H), 2.01 (s, 3H), 3.00 (s, 2H), 3.16 (AB q, J AB
)
13.2 Hz, ∆ν ) 59.8 Hz, 2H, CH2Ph), 3.31 (s, 3H), 4.38 (q, J )
7.8 Hz, 1H), 5.70 (d, J ) 7.4Hz, 1H, NH), 6.97 (s, 1H, NH), 7.19
(m, 2H), 7.39 (m, 3H); 13C NMR (CDCl3) δ 22.08, 22.75, 23.11,
24.61, 37.53, 40.02, 42.15, 51.28, 57.65, 128.57, 129.26, 130.17,
131.35, 165.99, 169.78, 170.79, 172.45; [R]20 ) -36° (c ) 0.2,
D
CH2Cl2); HRMS (FAB, MH+) calcd for C20H28N3O7S 454.16478,
found 454.16471.
(+)-(3R )-3-(N -Ace t yl-D-le u cyla m in o)-3-b e n zyl-1-(b e n -
zyloxy)su ccin im id e (13). A solution of the amine 8 (74 mg,
0.24 mmol) in dichloromethane (0.7 mL) was treated with
N-acetyl-D-leucine (42 mg, 0.24 mmol), 1-hydroxybenzotriazole‚-
1H2O (38 mg, 0.25 mmol), and DCC (50 mg, 0.24mmol) as
described for 10 above. Purification by radial chromatography
on a 1 mm plate eluting with 75% ethyl acetate/25% petroleum
ether gave 13 (68 mg, 61%): mp 195-198 °C (ethyl acetate/
petroleum ether); FTIR (KBr) 3263, 3036, 2934, 1796, 1732, 1634
(+)-(3R)-3-(N-Acet yl-L-leu cyla m in o)-3-b en zyl-1-(b en z-
yloxy)su ccin im id e (10). N-Acetyl-L-leucine (52 mg, 0.30
mmol) and 1-hydroxybenzotriazole‚1H2O (HOBT) (45 mg, 0.29
mmol) were added to the amine 8 (92 mg, 0.296 mmol) dissolved
in dichloromethane (0.9 mL) at 0 °C under N2. The mixture was
stirred at 0 °C for 7 min after which DCC (62 mg, 0.30 mmol)
was added. The mixture was stirred for 30 min at 0 °C and
1
cm-1; H NMR (CDCl3) δ 0.89 (d, J ) 6.3 Hz, 3H), 0.92 (d, J )
6.8 Hz, 3H), 1.45-1.71 (m, 3H), 2.08 (s, 3H), 2.91 (s, 2H), 3.00
(AB q, J AB ) 13.2 Hz, ∆ν ) 9.4 Hz, 2H, CH2Ph), 4.41 (q, J ) 8.3
Hz, 1H), 4.94 (AB q, J AB ) 9.8 Hz, ∆ν ) 14.6 Hz, 2H, OCH2Ph),
5.69 (d, J ) 7.8 Hz, 1H, NH), 7.19 (m, 3H), 7.34 (m, 6H), 7.47
(m, 2H); 13C NMR (CDCl3) δ 21.01, 21.16, 22.88, 24.56, 37.40,

Notes
J . Org. Chem., Vol. 62, No. 5, 1997 1513
40.51 (×2), 51.02, 57.34, 78.60, 127.49, 128.30, 128.42, 129.13,
pension of 17 (50 mg, 0.12 mmol) in dichloromethane (0.5 mL)
was treated with diisopropylethylamine (1.1 equiv) and meth-
anesulfonyl chloride (1.6 equiv) as described for 12 above to give
18 as an oil (43 mg, 73%): FTIR (KBr) 3312, 2961, 1813, 1755,
1682 cm-1; 1H NMR (CDCl3) δ 0.91 (d, J ) 5.8 Hz, 3H), 0.94 (d,
J ) 5.8 Hz, 3H), 1.43 (s, 9H), 1.58-1.68 (m, 3H), 3.01 (s, 2H),
3.15 (AB q, J AB ) 13.2 Hz, ∆ν ) 46.7 Hz, 2H, CH2Ph), 3.33 (s,
3H), 4.06 (br q, J ) 8.4 Hz, 1H), 4.78 (br d, J ) 6.7 Hz, 1H,
NH), 6.94 (br s, 1H, NH), 7.19 (m, 2H), 7.37 (m, 3H); 13C NMR
(CDCl3) δ 21.68, 22.79, 24.43, 28.10, 37.38, 39.62, 40.31, 41.76,
52.26, 57.51, 80.26, 128.32, 129.08, 130.07, 131.33, 155.90,
129.38, 130.27, 130.38, 133.52, 168.33, 171.56, 171.65, 174.51;
[R]20 ) +115° (c ) 1.2, CH2Cl2); HRMS (EI, M+) calcd for
D
C
26H32N3O5 465.22637, found 465.22698.
(+)-(3R )-3-(N -Ac e t y l-D -le u c y la m in o )-3-b e n zy l-1-h y -
d r oxysu ccin im id e (14). A mixture of the 1-(benzyloxy)-
succinimide 13 (59 mg, 0.127 mmol) and 10% Pd on C (10 mg)
in dry THF (4mL) was stirred for 3.25 h under a hydrogen
atmosphere. The reaction mixture was filtered, and filtrate was
evaporated to dryness. The residue solid was washed with
dichloromethane (3 mL), dissolved in methanol, and refiltered.
Evaporation under reduced pressure gave 14 as an oil which
crystallized from ethyl acetate and pentane (43 mg, 90%): mp
148-151 °C; FTIR (KBr) 3408, 1792, 1717, 1661 cm-1; 1H NMR
(CD3OD) δ 0.92 (d, J ) 6.4 Hz, 3H), 0.95 (d, J ) 6.4 Hz, 3H),
1.55 (m, 2H), 1.63 (m, J ) 6.3 Hz, 1H), 1.97 (s, 3H), 2.82 (AB q,
166.12, 169.81, 173.45; [R]20 ) -12° (c ) 1.2, CH2Cl2); HRMS
D
(FAB, MNa+) calcd for C23H33N3O8SNa 534.18864, found
534.18747.
(-)-(S)-1-(Ben zyloxy)-3-[[(b en zyloxy)ca r b on yl]a m in o]-
su ccin im id e (22). Meth od A. A mixture of N-Cbz-L-aspartic
acid (2 g, 7.4 mmol) was refluxed in acetic anhydride (3.5mL,
0.037mmol) for 1.5 h to give 20 as a white solid which was
washed with diethyl ether (3 × 5 mL) (1.69 g, 90%). A solution
of O-benzylhydroxylamine (444 mg, 3.61 mmol) in toluene (2 mL)
was added to a refluxing solution of the anhydride 20 (0.90 g,
3.61 mmol) in toluene (5 mL). The mixture was refluxed for 1
h and then filtered through Na2SO4, and the filtrate was
evaporated. The residue was taken up into ethyl acetate (40
mL) and washed with 10% aqueous sodium hydrogen carbonate
(40 mL), followed by water (2 × 40 mL). The organic phase was
dried (Na2SO4), and solvent removed to yield 22 as a white solid
(0.921 g, 72%): mp 142-143 °C; 1H NMR (CDCl3) δ 2.77 (dd, J
) 5.4, 17.8 Hz, 1H), 3.05 (dd, J ) 9.1, 17.8 Hz, 1H), 4.21 (m,
1H), 5.11 (AB q, J AB ) 12.2 Hz, 2H), ∆ν ) 6.7 Hz, 5.14 (s, 2H),
5.47 (br d, J ) 6.3 Hz, 1H), 7.30-7.41 (m, 8H), 7.50 (br s, 2H);
13C NMR (CDCl3) δ 33.88, 47.58, 67.56, 78.96, 133.22, 135.53,
J AB ) 17.6 Hz, ∆ν ) 24.8 Hz, 2H, CH2CO), 3.14 (AB q, J AB
)
13.2 Hz, ∆ν ) 50.1 Hz, 2H, CH2Ph), 4.43 (dd, J ) 6.8, 8.7Hz,
1H), 7.20 (m, 2H), 7.28 (m, 3H); 13C NMR (CD3OD) δ 22.32,
22.75, 23.63, 26.23, 37.77, 42.19, 42.55, 53.05, 58.94, 129.20,
130.13, 131.65, 134.43, 172.34, 173.64, 175.14, 175.56; [R]20
)
D
+98° (c ) 1.0, CH3OH); HRMS (FAB, MNa+) calcd for C19H25N3O5-
Na 398.16922, found 398.16930.
(+)-(3R)-3-(N-Acet yl-D-leu cyla m in o)-3-b en zyl-1-[(m et h -
a n esu lfon yl)oxy]su ccin im id e (15). A solution of 14 (45 mg,
0.12 mmol, 1 equiv) in dichloromethane (0.6 mL) was treated
with diisopropylethylamine (1.1 equiv) and methanesulfonyl-
chloride (1.5 equiv) as described for 11 above to give 15 as an
oil (51 mg, 93%): FTIR (KBr) 3263, 3065, 2959, 1811, 1755, 1645
1
cm-1; H NMR (CDCl3) δ 0.88 (d, J ) 6.4Hz, 3H), 0.92 (d, J )
6.4Hz, 3H), 1.49 (m, 1H), 1.59 (m, 1H), 1.69 (m, 1H2), 2.08 (s,
3H), 3.02 (s, 2H), 3.11 (AB q, J AB ) 13.1 Hz, ∆ν ) 30.5 Hz, 2H,
CH2Ph), 3.30 (s, 3H), 4.41 (q, J ) 8.3 Hz, 1H), 5.69 (br d, J )
7.3 Hz, 1H, NH), 7.23 (m, 2H), 7.39 (m, 3H); 13C NMR (CDCl3)
δ 21.07, 22.90, 23.01, 24.55, 37.34, 39.47, 40.23, 40.62, 51.13,
58.11, 127.85, 128.69, 130.28, 131.45, 166.78, 170.11, 171.92,
155.77, 168.79, 170.23; [R]20 ) -2° (c ) 1, CH2Cl2); HRMS
D
(FAB, MH+) calcd for C19H19N2O5 355.12939, found 355.12970.
Meth od B. To a solution of the oxazolidinone 1929 (0.207 g,
0.74 mmol) in dichloromethane was added triethylamine (104
µL, 0.75 mmol), O-benzylhydroxylamine hydrochloride (0.118
mg, 0.74 mmol), and HOBT (0.114 mg, 0.74 mmol). After the
solution was stirred for 10 min, DCC (0.163 mg, 0.79 mmol) was
added and the mixture stirred at rt for 18 h. The mixture was
filtered, and the filtrate diluted with dichloromethane (15 mL)
and washed successively with 5% aqueous hydrogen chloride (20
mL) and water (2 × 20 mL). The organic phase was dried
(MgSO4), and solvent was removed to give 23 which was not
purified further: 13C NMR (CDCl3) δ 33.03, 33.71, 52.58, 53.40,
68.24, 68.83, 72.87, 73.34, 78.34, 78.88, 128.12, 128.49, 128.62,
128.66, 128.77, 128.84, 129.24, 129.29, 129.56, 129.85, 133.38,
135.27, 154.56, 154.66, 168.50, 169.11, 169.78, 170.37; HRMS
(FAB, MK+) calcd for C20H20N2O6K 423.09583, found 423.09580.
Compound 23 (108 mg, 0.281 mmol) was treated with triethy-
lamine (0.9 mL, 6.46 mmol) in dichloromethane (8.1 mL) at rt
for 3 h. The mixture was washed with 5% aqueous hydrogen
chloride (10 mL) and water (2 × 10 mL). The organic phase
was dried (MgSO4), and the residue was chromatographed on a
1 mm silica gel chromatotron plate eluting with 50% ethyl
acetate/50% petroleum ether to give 22 (49 mg, 49%), data as
recorded above. Further elution gave recovered starting mate-
rial 23 (41 mg, 38%).
174.72; [R]20 ) +85° (c ) 0.9, CH2Cl2); HRMS (FAB, MNa+)
D
calcd for C20H27N3O7SNa 476.14677, found 476.14676.
(+)-(3R)-3-[N-(ter t-Bu t oxyca r b on yl)-L-leu cyla m in o]-3-
ben zyl-1-(ben zyloxy)su ccin im id e (16). A solution of the
amine 8 (127 mg, 0.41 mmol) in dichloromethane (1 mL) was
treated with N-t-BOC-L-leucine (105 mg, 0.42 mmol), 1-hydroxy-
benzotriazole‚1H2O (67 mg, 0.44 mmol) and DCC (89 mg, 0.43
mmol) as described for 10 above. Purification by radial chro-
matography on a 1 mm silica gel chromatotron plate, eluting
with 30% ethyl acetate/70% petroleum ether gave 16 (210 mg,
98%) as an oil: FTIR (KBr) 3302, 2959, 1795, 1732, 1659 cm-1
;
1H NMR (CDCl3) δ 0.93 (d, J ) 4.9 Hz, 3H), 0.95 (d, J ) 4.9 Hz,
3H), 1.44 (s, 9H), 1.6-1.7 (m, 3H), 2.91 (AB q, J AB ) 17.5 Hz,
∆ν ) 14.9 Hz, 2H, CH2CO), 3.06 (AB q, J AB ) 13.2 Hz, ∆ν )
32.5 Hz, 2H, C3CH2Ph), 4.05 (q, J ) 8.3 Hz, 1H), 4.75 (d, J )
6.8 Hz, 1H, NH), 4.95 (AB q, J AB ) 9.7 Hz, ∆ν ) 12.1 Hz, 2H,
OCH2Ph), 6.76 (s, 1H, NH), 7.16 (m, 2H), 7.34 (m, 6H), 7.48 (m,
2H); 13C NMR (CDCl3) δ 21.61, 22.77, 24.39, 28.13, 37.20, 40.50,
41.72, 52.32, 57.20, 78.56, 80.07, 128.04, 128.26, 128.85, 128.94,
129.55, 130.01, 131.84, 133.39, 155.95, 168.39, 171.64, 173.44;
[R]20 ) +11° (c ) 2, CH2Cl2); HRMS (EI, M+) calcd for
D
C
29H38N3O6 523.26824, found 523.26907.
(+)-(3R)-3-[N-(ter t-Bu t oxyca r b on yl)-L-leu cyla m in o)-3-
ben zyl-1-h yd r oxysu ccin im id e (17). A mixture of the N-
(benzyloxy)succinimde 16 (180 mg, 0.344 mmol) and 10% Pd on
C (13 mg) in dry THF (5 mL) was stirred under a hydrogen
atmosphere for 3 h. The mixture was filtered, and the solvent
was evaporated under reduced pressure. The residue was
washed with ethyl acetate (3mL), and the filtrate was evaporated
to give 17 as an oil (132 mg, 89%): mp 283-285 °C (ethyl
acetate/petroleum ether); FTIR (KBr) 3306, 2961, 1792, 1717,
Ack n ow led gm en t. This work was partially sup-
ported by research grants from the Marsden Fund and
the Foundation for Research Science and Technology
(New Zealand). We thank Professor W. T. Robinson
(Department of Chemistry, University of Canterbury,
New Zealand) for help with the X-ray crystallography.
1651 cm-1 1H NMR (CD3OD) δ 0.94 (d, J ) 6.3 Hz, 3H), 0.97
;
(d, J ) 6.3 Hz, 3H), 1.43 (s, 9H), 1.48 (m, 2H), 1.72 (hept, J )
6.3 Hz, 1H,), 2.86 (s, 2H), 3.14 (AB q, J AB ) 13.1 Hz, ∆ν ) 54.9
Hz, 2H), 4.10 (br t, J ) 7.3 Hz, 1H), 6.73 (d, J ) 7.5 Hz, 1H,
NH), 7.19 (m, 2H), 7.29 (m, 3H), 8.70 (s, 1H, NH); 13C NMR (CD3-
OD) δ 22.41, 23.64, 25.98, 28.97, 37.83, 42.17, 42.63, 54.01, 59.05,
80.83, 129.23, 130.09, 131.60, 134.08, 158.17, 171.85, 175.12,
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
compounds 9, 12-16, 18, and 22 (8 pages). This material is
contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
176.07; [R]20 ) +21° (c ) 2.1, CH3OH). Anal. Calcd for
D
C
22H31N3O6: C, 60.96; H, 7.21; N, 9.69. Found: C, 61.02; H,
J O961781I
7.36; N, 9.71.
(-)-(3R)-3-[N-(ter t-Bu t oxyca r b on yl)-L-leu cyla m in o]-3-
b en zyl-1-[(m et h a n su lfon yl)oxy]su ccin im id e (18). A sus-
(29) Scholtz, J . M.; Bartlett, P. A. Synthesis 1989, 542.