Efficient synthesis of (S)-4-phthalimido-1,3,4,5-tetrahydro-8-(2,6-dichlorobenzyloxy)-3-oxo-2H-2-ben zazepin-2-acetic acid (Pht-Hba(2,6,Cl2-Bn)-Gly-OH)

Article abstract of DOI:10.1021/jo000530d

4-Amino-2-benzazepin-3-ones have proven very useful for studying the biologically active conformations of peptides. The synthesis of Pht-Aba-Xaa-OH by reaction of the corresponding 1,3-oxazolidin-5-one with trifluoromethanesulfonic acid (TFMSA) has been reported in the literature. However, when this procedure was applied to the preparation of Pht-Hba(Bn)-Gly-OH 8, many byproducts were formed and the yield of the desired aminobenzazepinones 7 and 8 was very low. We report in this paper an efficient methodology for the synthesis of Pht-Hba(2,6-Cl₂-Bn)-Gly-OH 17 starting from the commercially available tyrosine. In our procedure, the dipeptide Pht-Tyr(2,6-Cl₂-Bn)-Gly-OH 15 is converted to the 1,3-oxazolidin-5-one 16 which then undergoes Friedel-Crafts cyclization in the presence of tin tetrachloride to afford the desired 4-phthalimido-1,3,4,5-tetrahydro-8-(2,6-dichlorobenzyloxy)-2-benzazepin-3-one 17 in excellent yield.

Full text of DOI:10.1021/jo000530d

J . Org. Chem. 2000, 65, 6487-6492
6487
Efficien t Syn th esis of (S)-4-P h th a lim id o-1,3,4,5-
tetr a h yd r o-8-(2,6-d ich lor oben zyloxy)-3-oxo-2H-2-ben za zep in -2-a cetic
Acid (P h t-Hba (2,6-Cl2-Bn )-Gly-OH)1
J . Richard Casimir,*,†,‡ Dirk Tourwe´,† Koen Iterbeke,† Gilles Guichard,‡ and
J ean-Paul Briand‡
Vrije Universiteit Brussel (VUB), Department of Organic Chemistry, Pleinlaan 2,
B-1050 Brussels, Belgium, and Laboratoire de Chimie Immunologique, UPR 9021 CNRS,
IBMC, 15 rue Rene´ Descartes, F-67084 Strasbourg Cedex, France
R.Casimir@ibmc.u-strasbg.fr
Received April 10, 2000
4-Amino-2-benzazepin-3-ones have proven very useful for studying the biologically active conforma-
tions of peptides. The synthesis of Pht-Aba-Xaa-OH by reaction of the corresponding 1,3-oxazolidin-
5-one with trifluoromethanesulfonic acid (TFMSA) has been reported in the literature. However,
when this procedure was applied to the preparation of Pht-Hba(Bn)-Gly-OH 8, many byproducts
were formed and the yield of the desired aminobenzazepinones 7 and 8 was very low. We report in
this paper an efficient methodology for the synthesis of Pht-Hba(2,6-Cl2-Bn)-Gly-OH 17 starting
from the commercially available tyrosine. In our procedure, the dipeptide Pht-Tyr(2,6-Cl2-Bn)-Gly-
OH 15 is converted to the 1,3-oxazolidin-5-one 16 which then undergoes Friedel-Crafts cyclization
in the presence of tin tetrachloride to afford the desired 4-phthalimido-1,3,4,5-tetrahydro-8-(2,6-
dichlorobenzyloxy)-2-benzazepin-3-one 17 in excellent yield.
In tr od u ction
the recognition of the peptide/I-Ak complex by T-cell
receptors,8,9 we decided to replace the Tyr53 residue by
several conformationally constrained amino acids in order
to determine the antigenically active conformation of this
decapeptide.10 One such constrained amino acid was the
lactam 4-amino-1,3,4,5-tetrahydro-8-hydroxy-2-benzazepin-
3-one (Hba) in which the preferred conformations of the
side chain are gauche(+) and trans, the gauche(-)
conformation being excluded.11 However, the synthesis
of Hba has not been reported in the literature. In this
paper, we describe an efficient methodology for the
synthesis of Pht-Hba(2,6-Cl2-Bn)-Gly-OH 17 which would
be applicable to the preparation of other dipeptides
containing the Hba motif.
Conformational restriction is a well-established strat-
egy in the design of more selective and/or more potent
peptides as enzyme inhibitors and agonists or antagonists
at receptors.2,3 Lactams, which maintain a given dipep-
tidyl unit in the trans amide conformation and bias
neighboring ø, ψ and φ angles, have proven particularly
useful in a number of cases.4,5 Moreover, the ø angles in
conjunction with the backbone angles define the position
of the side-chain functional groups in space and thus
must be regarded as of key importance in understanding
the mode of action of peptides.6,7
In our ongoing research on the study of the interaction
of the antigen HEL(52-61), 52Asp-Tyr-Gly-Ile-Leu-Gln-
Ile-Asn-Ser-Arg61, with MHC class II I-Ak molecule and
Resu lts a n d Discu ssion
* To whom correspondence should be addressed. Tel: +33388417029.
Fax: +33388610680.
Since glycine is the amino acid succeeding tyrosine in
the sequence of HEL(52-61), we first tried to prepare
the dipeptide Pht-Hba-Gly by the method described for
the synthesis of the phenylalanine analogue Pht-Aba-
Gly.11-14 Accordingly, Pht-Tyr(Bn)-OH 1 (Scheme 1) was
converted to the acid chloride 2 using PCl5 in benzene at
† Vrije Universiteit Brussel.
‡ IBMC.
(1) Abbreviations and definitions recommended by IUPAC-IUB
Commission of Biochemical Nomenclature have been used. Other
abbreviations used: Aba, 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-
one; BOP, benzotriazole-1-yloxytris(dimethylamino)phosphonium-
hexafluorophosphate; 2,6-Cl2-Bn, 2,6-dichlorobenzyl; EI, electrospray
ionization; EMM, exact mass measurement; GITC, 2,3,4,6-tetra-O-
acetyl-â-D-glucopyranosyl isothiocyanate; Hba, 4-amino-1,2,4,5-tet-
rahydro-8-hydroxy-2-benzazepin-3-one; HEL, hen egg lysozyme; HOBt,
N-hydroxybenzotriazole; MHC, major histocompatibility complex; Pht,
phthaloyl; TFA, trifluoroacetic acid; TFMSA, trifluoromethane sulfonic
acid.
(2) Warshawsky, A. M.; Flynn, G. A.; Koehl, J . R.; Mehdi, S. Bioorg.
Med. Chem. Lett. 1996, 6, 957.
(3) Mosberg, H. I.; Lomize, A. I.; Wang, C.; Kroona, H.; Heyl, D. L.
J . Med. Chem. 1994, 37, 4371.
(4) Flynn, G. A.; Giroux, E. L.; Dage, R. J . Am. Chem. Soc. 1987,
109, 9, 7914.
(8) Hernandez, J . F.; Cretin, F.; Lombard-Platet, S.; Salvi, J . P.;
Walchshofer, N.; Gerlier, D.; Paris, J .; Rabourdin-Combe, C. Peptides
1994, 15, 583.
(9) Ettouati, L.; Casimir, J . R.; Raynaud, I.; Trescol-Biemont, M.-
C.; Carrupt, P.-A.; Gerlier, D.; Rabourdin Combe, Ch.; Testa, B.; Paris,
J . Prot. Pept. Lett. 1998, 5, 221.
(10) Casimir J . R.; Trescol-Bie´mont, M.-C.; Tourwe´, D.; Paris, J .;
Ettouati, L.; Iterbeke, K.; Van Den Nest, W.; Gerlier, D.; Rabourdin-
Combe, C. In Peptides 1998, Proceedings of the 25th European Peptide
Symposium; Bajusz, S., Hudecz F., Eds.; Akade´miai Kiado: Budapest,
1999; p 654.
(5) Freidinger, R. M.; Veber, D. F.; Perlow, D. S.; Brooks, J . R.;
Saperstein R. Science 1980, 210, 656.
(6) Hruby, V. J .; Li, G.; Haskell-Luevano, C. Biopolymers 1997, 43,
219.
(11) Tourwe´, D.; Verschueren, K.; Frycia, A.; Davis, P.; Porreca, F.;
Hruby, V. J .; Toth, G.; J aspers, H.; Verheyden, P.; Van Binst, G.
Biopolymers 1996, 38, 1.
(12) Flynn, G. A.; Burkholder, T. P.; Huber, E. W.; Bey, P. Bioorg.
Med. Chem. Lett. 1991, 6, 309.
(7) Gibson, S. E.; Guillo, N.; Tozer, M. J . Tetrahedron 1999, 55, 585.
10.1021/jo000530d CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/13/2000

6488 J . Org. Chem., Vol. 65, No. 20, 2000
Casimir et al.
Sch em e 1a
a
(a) PCl5, C6H6, 50-55 °C, 2.5 h; (b) Na2CO3‚10H2O, glycine, rt, 1 h; (c) (CH2O)n, pTsOH cat., reflux, 6 h; (d) Method A: CH2Cl2,
TFMSA, rt, 24 h. Method B: CH2Cl2, SnCl4, reflux, 4 h then rt, 20 h. Method C: CH2Cl2, SnCl4, rt, 24 h.
Ta ble 1. Com p a r ison of TF MSA w ith Tin Tetr a ch lor id e
50-55 °C,15 and then reacted with glycine under the
in th e Syn th esis of Am in oben za zep in on es fr om
Schotten-Baumann conditions12 to afford the dipeptide
Oxa zolid in on es 4 a n d 16
3 in 73% yield. No racemization was detected during this
products formed (% yield)b,c
reaction, as demonstrated by GITC derivatization16 after
phthaloyl deprotection.17 The dipeptide 3 was then
converted into the oxazolidinone 4 in 95% yield by
reaction with paraformaldehyde in the presence of cata-
lytic amount of p-toluenesulfonic acid.13 Under acidic
conditions and in the absence of nucleophiles, the oxazol-
idinone 4 yields an acyliminium ion 5 which would form
the desired compound 8 by Friedel-Crafts cyclization.
However, reaction of compound 4 with trifluoromethane-
sulfonic acid (TFMSA) in dichloromethane13 (method A,
Scheme 1) afforded a mixture from which the spirodi-
enone 6 (13%) and the 4-amino-2-benzazepin-3-ones 7
(7%) and 8 (3%) were isolated. As demonstrated by HPLC
analysis of the crude before purification, no trace of the
oxazolidinone 4 was detected in the mixture. The re-
maining sticky material consisted of several products
which were difficult to characterize. The structure of
spirodienone 6 was unambiguously assigned by the
characteristic four ethylenic protons in the 1H NMR
spectrum as well as the chemical shift of 184 ppm in the
13C NMR and the absorption at 1663 cm-1 in the IR
spectra, respectively, both of them typical for the dienone
carbonyl.18 The yield of the aminobenzazepinone 7 or 8
might be improved by changing from TFMSA to a Lewis
acid catalyst. Thus, when the oxazolidinone 4 was reacted
oxazol-
reaction
entry idinone conditionsa
6
7
8
9
17
1
2
3
4
4
4
4
method A 13 (57) 7 (33) 3 (10)
method B
method C
method D
50 (68)
(67)
23 (32)
(33)
16
84
a
Method A: TFMSA, CH2Cl2, rt, 24 h. Method B: SnCl4,
CH2Cl2, reflux, 4 h then rt, 20 h. Method C: SnCl4 (1 M in CH2Cl2),
rt, 24 h. Method D: SnCl4, CH2Cl2, reflux, 3 h then rt, 24 h.
Yields of isolated products. c The relative percentages of com-
b
pounds present in the crude were determined by HPLC and are
given in parentheses.
with tin tetrachloride (method B, Scheme 1) the benza-
zepinone 7 was isolated in 50% yield, showing that tin
tetrachloride is a better catalyst than TFMSA for the
formation of Hba ring from oxazolidinone 4 (Table 1).
The formation of compound 6 in method A (Scheme 1),
however, prompted us to reinvestigate the mechanism
of the cyclization reaction. Indeed, we reasoned that the
aminobenzazepinone 7 might have been formed from 6
by the dienone-phenol rearrangement.19,20 In such a
case, the 7-hydroxy-substituted isomer 10 (Scheme 2)
would also be expected. However, when compound 6 was
subjected to TFMSA in dichloromethane for 24 h (under
the same acidic conditions used in method A, Scheme 1),13
we did not observe any rearrangement compound 7 or
10. Moreover, the structure of 7 was confirmed by 1H
NMR analysis. 2D experiments allowed us to assign the
spin system of the phenolic ring protons, and ROESY
experiments showed the presence of NOEs between the
isolated H(9) and the benzylic H(1) protons, as well as
between H(6) and the H(5) protons which firmly estab-
lishes the position of the HO-substituent. These results
(13) de Laszlo, S. E.; Bush, B. L.; Doyle, J . J .; Greenlee, W. J .;
Hangauer, D. G.; Halgren, T. A.; Lynch, R. J .; Schorn, T. W.; Siegl, P.
K. S. J . Med. Chem. 1992, 35, 833.
(14) Tourwe´, D.; Verschueren, K.; Van Binst G.; Davis P.; Porreca
F.; Hruby, V. J . Bioorg. Med. Chem. Lett. 1992, 2, 1305.
(15) Almquist, R. G.; Olsen, C. M.; Uyeno, E. T.; Toll, L. J . Med.
Chem. 1984, 27, 115.
(16) Nimura, N.; Ogura, H.; Kinoshita, T. J . Chromatogr. 1980, 202,
375.
(17) Sheehan, J . C.; Chapman, D. W.; Roth, R. W. J . Am. Chem.
Soc. 1952, 74, 3822.
(18) Kita, Y.; Takada, T.; Gyoten, M.; Tohma, H.; Zenk, M. H.;
Eichhorn, J . J . Org. Chem. 1996, 61, 5857.
(19) Vitullo, V. P.; Grossman, N. J . Am. Chem. Soc. 1972, 94, 3844.
(20) Vitullo, V. P. J . Org. Chem. 1968, 34, 224.

Synthesis of Pht-Hba(2,6-Cl2-Bn)-Gly-OH
J . Org. Chem., Vol. 65, No. 20, 2000 6489
Sch em e 2a
a
Reaction conditions: CH2Cl2, TFMSA, rt, 24 h.
Sch em e 3a
a
(a) (i) 2 N NaOH, CuSO4.5H2O, 60 °C then rt, (ii) 2,6-Cl2BnBr, 90 min; (b) 1,4-dioxane/H2O, Et3N, PhtO, reflux, Dean-Stark; (c)
i
GlyOtBu‚HCl, BOP/HOBT, Pr2NEt, CH2Cl2; (d) CH2Cl2/TFA, reflux, 2 h; (e) (CH2O)n, p-TsOH cat., reflux, 6 h; (f) CH2Cl2, SnCl4, reflux,
3 h then rt, 24 h.
indicate that the formation of the Hba ring does not occur
via a dienone-phenol rearrangement of the spirodienone
6, but rather through a Friedel-Crafts alkylation of the
aromatic ring at the meta position with respect to the
benzyloxy or hydroxyl group.
The major drawback of the synthesis of Pht-Hba-Gly
7 from oxazolidinone 4 and tin tetrachloride (method B,
Scheme 1), however, is the formation of nonnegligible
amount (23%, Table 1) of the 7-benzylated compound 9
resulting from the Fries rearrangement.21
In an attempt to improve the yield of this aminoben-
zazepinone 7, we replaced the benzyl group by the 2,6-
dichlorobenzyl protection which is less prone to Fries
rearrangement and more stable toward acidolysis than
the benzyl group. Accordingly, L-tyrosine 11 was con-
verted to L-Tyr(2,6-Cl2-Bn)-OH 12, using 2,6-dichlo-
robenzylbromide and copper sulfate,22 then allowed to
react with phthalic anhydride in the presence of Et3N to
afford Pht-Tyr(2,6-Cl2-Bn)-OH 13 in good yield as shown
in Scheme 3.23 The dipeptide 15 was obtained in 75%
yield from compound 13 after reaction with tert-butyl-
glycine hydrochloride followed by deprotection with tri-
fluoroacetic acid (TFA). This dipeptide 15 was converted
to the oxazolidinone 16 which was then reacted with tin
tetrachloride as shown (Scheme 3). To our satisfaction,
the aminobenzazepinone 17 was isolated in 84% yield
(Table 1), showing not only that the undesired Fries
rearrangement product can be avoided by replacing the
benzyl group by its 2,6-dichloro derivative but also that,
unlike the benzyl group, the 2,6-dichlorobenzyl derivative
is resistant to cleavage by tin tetrachloride. This is very
advantageous since after phthaloyl deprotection and
subsequent protection of the primary amino group by a
Boc group the protected dipeptide 17 is more convenient
than analogue 7 for incorporation into peptides se-
quences. Moreover, the fact that the aminobenzazepinone
17 could be obtained from oxazolidinone 16 without loss
of the 2,6-dichlorobenzyl protection provides further
evidence that the formation of the Hba ring does not
occur via the dienone-phenol rearrangement of a spiro-
dienone.
Con clu sion
In conclusion, we have disclosed in this paper an
efficient methodology for the preparation of Pht-Hba(2,6-
Cl2-Bn)-Gly-OH. Our procedure could be efficiently
applied to the preparation of other dipeptides of type Pht-
Hba(2,6-Cl2-Bn)-Xaa-OH or Pht-Aba-Xaa-OH and there-
fore would be very useful to the synthesis of peptide
(21) Erickson, B. W.; Merrifield, R. B. J . Am. Chem. Soc. 1973, 95,
3750.
(22) Bodanszky, M.; Bodanszky, A. In The Practice of Peptide
Synthesis; Hafner, K., Ed.; Springer-Verlag: Berlin, 1984; p 62.
(23) Wu¨nsch E.; Wendlberger G.; J entsch J . Chem. Ber. 1964, 97,
8.

6490 J . Org. Chem., Vol. 65, No. 20, 2000
Casimir et al.
was added at intervals of 1.5 h). The benzene was eliminated
under reduced pressure, and the residue dissolved in 400 mL
of CHCl3. The solution was washed with aqueous 8% NaHCO3
(2 × 250 mL) and water (1 × 200 mL), dried with MgSO4, and
concentrated in vacuo to yield a pale-yellow solid (6.3 g, 95%):
mp 87-89 °C; [R]D -130.7 (c 1.1, CHCl3); 1H NMR (250 MHz,
CDCl3) δ 3.40 (dd, J ) 8.7, 14.1 Hz, 1H), 3.59 (dd, J ) 7.2 Hz,
14.1 Hz, 1H), 3.93 (m, 2H), 4.96 (m, 2H), 4.97 (m, 1H), 5.41
(d, J ) 28.4 Hz, 2H), 6.80 (d, J ) 8.6 Hz, 2H), 7.08 (d, J ) 8.6
Hz, 2H), 7.30 (m, 5H), 7.71 (m, 2H), 7.79 (m, 2H); 13C NMR
(62.5 MHz, CDCl3) δ 33.9 (CH2), 43.9 (CH2), 54.1 (CH), 70.0
(CH2), 115.2 (CH), 123.8 (CH), 127.4 (CH), 128.0 (C), 128.4
(C), 128.6 (CH), 130.3 (CH), 131.2 (C), 134.6 (CH), 136.9 (C),
158.0 (C), 166.3 (C), 167.4 (C), 168.7 (C); MS (EI) m/z 471 (M
+ 1, 100), 472 (30), 473 (20), 493 (M + 23, 26), 494 (11); EMM
calcd for C27H22N2O6 m/z 471.1556 (MH+), found 471.1606.
Syn th esis of Sp ir od ien on e (6) a n d Am in oben za zep i-
n on es (7-9). Meth od A (Sch em e 1). A solution of oxazoli-
dinone 4 (3.24 g, 6.9 mmol) in CH2Cl2 (11 mL) under nitrogen
atmosphere was cooled to 0 °C, then TFMSA (10 mL) was
rapidly added. After 30 min, the cooling bath was removed
and stirring continued at room temperature for 24 h before
dilution of the mixture with CH2Cl2 (70 mL). The flask was
cooled to 0 °C, and then water (70 mL) was slowly added. The
two layers were separated, and then the aqueous layer was
extracted with a 6% solution of NaHCO3 (3 × 140 mL). The
combined aqueous phase was acidified to pH 2 with 6 N HCl
and then extracted with CH2Cl2 (3 × 250 mL). The combined
organic phase was dried with MgSO4 and then concentrated
under reduced pressure to give a brown residue that was
purified by flash chromatography (SiO2, EtOAc/MeOH, 1:1) to
yield 0.875 g of a brown solid. RP-HPLC purification of 100
mg of this mixture afforded three white solids: 6 (39 mg, 13%),
7 (21 mg, 7%) and 8 (11 mg, 3%). Meth od B (Sch em e 1). A
solution of SnCl4 (0.6 mL, 5.13 mmol) in CH2Cl2 (10 mL) was
added dropwise (over 10 min) to a stirred solution of oxazoli-
dinone 4 (0.91 g, 1.936 mmol) in dichloromethane (20 mL).
The solution was refluxed for 4 h, and then stirring was
continued at room temperature for 20 h. The mixture was
cooled in an ice bath and then slowly hydrolyzed with 0.5 N
HCl (30 mL) over a period of 15 min. The two layers were
separated. The solid remaining in the flask was dissolved in
EtOAc (50 mL) and the solution washed with water (2 × 20
mL). The combined organic phase was concentrated in vacuo
to give a light yellow solid that was dissolved in EtOAc (15
mL) and precipitated by slow addition of cyclohexane (45 mL).
After being cooled to 0 °C, the solid was collected by filtration
and dried in vacuo to give 0.58 g of a white solid consisting
only of the two aminobenzazepinones 7 and 9. A 50 mg portion
of this mixture was purified by RP-HPLC to afford 31 mg (50%)
of 7 and 18 mg (23%) of 9. Meth od C (Sch em e 1). A solution
of oxazolidinone 4 (0.33 g, 0.7 mmol) in 10 mL of a molar
solution of SnCl4 in CH2Cl2 was stirred at room temperature
for 24 h. The reaction was stopped by hydrolysis with 1 N HCl
(10 mL), concentrated in vacuo, and then analyzed by HPLC
(Table 1).
analogues designed to interact with relevant biological
targets.10 We have already demonstrated the use of the
phthaloyl-protected dipeptide mimic in peptide synthesis
on a 4-methylbenzhydrylamine resin.24 The phthaloyl
group can also be easily replaced by a Boc-group for use
in the Boc-peptide synthesis protocol,10,17 where the 2,6-
dichlorobenzyl group is a standard protection for ty-
rosine.21
Exp er im en ta l Section
Gen er a l Meth od s. Melting points were measured on a hot
stage apparatus and were not corrected. IR spectra were
carried out on a FT-IR spectrometer. Optical rotations were
determined at rt (22 °C) on a digital polarimeter. Mass spectra
(positive mode) were recorded using electrospray ionization
(EI) or a linear MALDI-TOF instrument using R-cyano-4-
hydroxycinnamic acid as matrix. Exact mass measurements
(EMM) were recorded at low resolution using electrospray
ionization and a quadrupole mass spectrometer with PEG-Na
or PEG-Me as standard.25 1D NMR spectra were recorded in
CDCl3 or DMSO-d6 on a 250 or 500 MHz spectrometer.
Preparative HPLC separations were performed with a RP-
C18 column (15-20 µm, 2.5 × 15 cm) at a flow rate of 13 mL/
min with UV detection at λ ) 215 nm by using a linear
gradient of A (0.1% TFA in H2O) and B (0.1% TFA in MeCN/
H2O, 80:20).
(S)-Nr-P h th a loyl-O-ben zyltyr osin e Ch lor id e (2).15
A
solution of NR-Pht-Tyr(Bn)-OH 1 (1.01 g, 2.51 mmol) and PCl5
(0.83 g, 4 mmol) in dry benzene (10 mL) was stirred at 50-55
°C for 90 min, and then the solvent was eliminated under
reduced pressure. The residue was redissolved in dry benzene
and evaporated. This last operation was repeated twice to yield
a white solid (1.05 g, 100%): 1H NMR (500 MHz, CDCl3) δ
3.49 (dd, J ) 10.8, 14.3 Hz, 1H), 3.58 (dd, J ) 5.2, 14.3 Hz,
1H), 4.94 (s, 2H), 5.27 (dd, J ) 5.2, 10.8 Hz, 1H), 6.79 (d, J )
8.5 Hz, 2H), 7.05 (d, J ) 8.5 Hz, 2H), 7.31 (m, 5H), 7.73 (m,
2H), 7.82 (m, 2H).
(S)-Nr-P h th a loyl-O-ben zyltyr osin ylglycin e (3). A solu-
tion of NR-Pht-Tyr(Bn)-Cl 2 (1.05 g, 2.5 mmol) in dry acetone
(27.8 mL) was added slowly (1 h) to a solution of glycine (0.45
g, 6.0 mmol) and Na2CO3‚10H2O (1.41 g, 4.92 mmol) in 40.7
mL of the mixture acetone/water (7:4). After removal of acetone
in vacuo, the mixture was diluted with water (25 mL) and
acidified with 10 N HCl until pH 1-2. The resulting aqueous
solution was extracted with EtOAc (3 × 50 mL), and the
combined organic phases were concentrated to 50 mL. The
latter solution was extracted with 6% NaHCO3 solution (3 ×
30 mL), and the combined aqueous phases acidified to pH 1
with 10 N HCl then extracted with EtOAc (3 × 100 mL). The
combined organic layers were dried over MgSO4 and concen-
trated in vacuo to give a residue which was recrystallized from
EtOH/water (7:3) to yield a white solid (0.84 g, 73%): mp 152
°C; [R]D -139.2 (c 1.0, MeOH); IR (KBr) ν 3500-2548, 3490,
3375, 3033, 1774, 1714, 1675, 1612 cm-1; 1H NMR (250 MHz,
DMSO-d6) δ 3.30 (d, J ) 14.1 Hz, 1H), 3.45 (dd, J ) 4.8, 14.1
Hz, 1H), 3.66 (dd, J ) 5.5, 17.3 Hz, 1H), 3.86 (dd, J ) 6.0, 7.3
Hz, 1H), 4.95 (s, 2H), 4.97 (m, 1H), 6.78 (d, J ) 8.5 Hz, 2H),
7.03 (d, J ) 8.5 Hz, 2H), 7.29 (m, 5H), 7.80 (s, 4H), 8.49 (m,
1H); MS (EI) m/z 459 (M + 1, 100), 460 (28), 481 (M + 23, 8),
917 (45), 918 (25); EMM calcd for C26H22N2O6 m/z 459.1556
(MH+), found 459.1550.
2-[4(S)-(1,3-Dih yd r o-1,3-d ioxo-2H-isoin d ol-2-yl)-1,2,4,5-
tetr a h yd r o-2-a zin -3-on e-6-sp ir o-1′-cycloh exa -2′,5′-d ien -4′-
on e]a cetic a cid (6): yield 39 mg (13%, method A); mp 157
°C; [R]D -106.0 (c 0.7, AcOH); IR (KBr) ν 3500-2400, 3048,
1778, 1715, 1663 (CO spirodienone), 1619 cm-1; 1H NMR (250
MHz, DMSO-d6) δ 1.98 (dd, J ) 7.1, 12.6 Hz, 1H), 2.77 (m,
1H), 3.40 (d, J ) 12.4 Hz, 1H), 3.86 (d, J ) 12.4 Hz, 1H), 3.98
(d, J ) 17.1 Hz, 1H), 4.17 (d, J ) 17.1 Hz, 1H), 5.01 (dd, J )
7.0, 7.1 Hz, 1H), 6.27 (d, J ) 9.9 Hz, 1H), 6.33 (d, J ) 10.2 Hz,
1H), 7.19 (d, J ) 9.9 Hz, 1H), 7.64 (d, J ) 10.2 Hz, 1H), 7.89
(s, 4H); 13C NMR (62.5 MHz, DMSO-d6) δ 34.3 (CH2), 46.4
(CH), 48.6 (CH2), 54.1 (CH2), 123.2 (CH), 128.8 (CH), 129.0
(CH), 131.1 (C), 134.7 (CH), 149.6 (CH), 151.8 (CH), 165.4 (C),
166.9 (C), 169.7 (C), 184.3 (C, CO spirodienone); MS (EI) m/z
381 (M + 1, 100), 382 (22), 383 (15), 403 (M + 23, 50), 404
(11); EMM calcd for C20H16N2O6 m/z 381.1087 (MH+), found
381.1377.
3-[2(S)-(1,3-Dih yd r o-1,3-d ioxo-2H -isoin d ol-2-yl)-3-(4-
ben zyloxyp h en yl)p r op a n oyl]-1,3-oxa zolid in -5-on e (4). A
suspension of the dipeptide 3 (6.46 g, 14.1 mmol), p-toluene-
sulfonic acid (0.31 g, 1.63 mmol), and paraformaldehyde (5.46
g, 13.8 equiv) in dry benzene (200 mL) was refluxed for 6 h
with azeotropic removal of water (3.2 g of paraformaldehyde
(24) Tourwe´, D.; Nikiforovich, G. V.; Iterbeke, K. In Peptides 1996,
Proceedings of the 24th European Peptide Symposium; Ramage, R.,
Epton, R., Eds.; Mayflower: Kingswinford, 1998; p 835.
(25) Tyler, A. N.; Clayton, E.; Green, B. N. Anal. Chem. 1996, 68,
3561.
4(S)-(1,3-Dih yd r o-1,3-d ioxo-2H -isoin d ol-2-yl)-1,3,4,5-
tetr ah ydr o-8-h ydr oxy-3-oxo-2H-2-ben zazepin -2-acetic acid

Synthesis of Pht-Hba(2,6-Cl2-Bn)-Gly-OH
J . Org. Chem., Vol. 65, No. 20, 2000 6491
(7): yield 21 mg (7%, method A), 31 mg (50%, method B); mp
175 °C; [R]D -2.0 (c 1.0, AcOH); IR (KBr) ν 3516, 3500-2400,
3156, 1765, 1740, 1692, 1656, 1600 cm-1; 1H NMR (500 MHz,
DMSO-d6) δ 3.14 (dd, J ) 4.2, 15.5 Hz, 1H), 3.89 (m, 1H), 4.00
(d, J ) 16.9 Hz, 1H), 4.21 (d, J ) 16.9 Hz, 1H), 4.47 (d, J )
16.0 Hz, 1H), 4.80 (d, J ) 16.0 Hz, 1H), 5.23 (m, 1H), 6.67 (d,
J ) 8.1 Hz, 1H), 6.71 (s, 1H), 7.04 (d, J ) 8.1 Hz, 1H), 7.89
(m, 4H), 9.35 (s, 1H); MS (EI) m/z 381 (M + 1, 100), 382 (22),
400 (38), 403 (M + 23, 30); EMM calcd for C20H16N2O6 m/z
381.1087 (MH+), found 381.1191.
4(S)-(1,3-Dih yd r o-1,3-d ioxo-2H -isoin d ol-2-yl)-1,3,4,5-
t et r a h yd r o-8-b en zyloxy-3-oxo-2H -2-b en za zep in -2-a ce-
tic a cid (8): yield 11 mg (3%, method A); IR (KBr) ν 3500-
2400, 3153, 1767, 1738, 1602 cm-1; 1H NMR (250 MHz, CDCl3)
δ 2.71 (m, 1H), 3.05 (m, 1H), 3.60 (s, 2H), 3.90 (d, J ) 12.1
Hz, 1H), 3.80-4.42 (m, 2H), 4.34 (d, J ) 17.2 Hz, 1H), 5.01
(dd, J ) 6.9, 12.9 Hz, 1H), 6.25 (s, 1H), 6.50 (d, J ) 10.5 Hz,
1H), 7.11 (d, J ) 10.5 Hz, 1H), 7.25 (m, 5H), 7.71 (m, 2H),
7.81 (m, 2H); MS (EI) m/z 471 (M + 1).
4(S)-(1,3-Dih yd r o-1,3-d ioxo-2H -isoin d ol-2-yl)-1,3,4,5-
tetr a h yd r o-7-ben zyl-8-h yd r oxy-3-oxo-2H-2-ben za zep in -
2-a cetic a cid (9): yield 18 mg (23%, method B); mp 161 °C;
[R]D -2.0 (c 1.0, AcOH); IR (KBr) ν 3396, 3500-2400, 3027,
1774, 1715, 1654, 1618 cm-1; 1H NMR (500 MHz, DMSO-d6) δ
3.08 (dd, J ) 4.2, 15.9, Hz, 1H), 3.85 (s, 2H), 3.87 (m, 1H),
4.01 (d, J ) 17.3 Hz, 1H), 4.21 (d, J ) 17.3 Hz, 1H), 4.42 (d,
J ) 16.1 Hz, 1H), 4.79 (d, J ) 16.1 Hz, 1H), 5.21 (dd, J ) 4.2,
12.5 Hz, 1H), 6.72 (s, 1H), 6.90 (s, 4H), 7.15 (m, 1H), 7.23 (m,
4H), 7.89 (m, 4H), 9.39 (s, 1H); MS (EI) m/z 471 (M + 1); EMM
calcd for C27H22N2O6 m/z 471.1556 (MH+), found 471.1552.
64.8 (CH2), 114.2 (CH), 127.6 (CH), 128.7 (CH), 128.9 (CH),
129.3 (CH), 130.1 (CH), 130.3 (C), 130.7 (CH), 131.4 (CH),
131.7 (CH), 135.9 (C), 137.3 (C), 157.0 (C), 168.0 (C), 172.7
(C).
(S)-N r-P h t h a loyl-O-2,6-d ich lor ob e n zylt yr osin ylgly-
cin e ter t-Bu tyl Ester (14). A solution of Pht-Tyr(2,6-Cl2-
Bn)-OH 13 (3.9 g, 6.78 mmol), Gly-OtBu‚HCl (1.25 g, 7.46
mmol), BOP (3.3 g, 7.45 mmol), and HOBt (1.14 g, 7.45 mmol)
in 75 mL of CH2Cl2 and i-Pr2NEt (5.9 mL, 5 equiv) was stirred
at room temperature for 5 h. The mixture was diluted with
75 mL of CH2Cl2 and then successively washed with 1 N HCl
(2 × 100 mL), saturated NaHCO3 (2 × 100 mL), brine (100
mL), and water (100 mL). The organic phase was dried with
Na2SO4, filtered, and concentrated in vacuo to afford a white
1
solid (3.0 g, 100%): mp 66 °C; [R]D -113.4 (c 0.7, MeOH); H
NMR (250 MHz, CDCl3) δ 1.47 (s, 9H), 3.66 (m, 2H), 3.97 (d,
J ) 4.9 Hz, 2H), 5.16 (s, 2H), 5.17 (m, 1H), 6.76-6.88 (m, 3H),
7.10-7.36 (m, 4H), 7.69-7.83 (m, 4H); 13C NMR (62.5 MHz,
CDCl3) δ 28.0 (CH3), 34.0 (CH2), 42.4 (CH2), 55.9 (CH), 55.4
(CH), 82.5 (C), 115.4 (CH), 123.6 (CH), 128.4 (CH), 129.3 (C),
129.9 (CH), 130.4 CH), 131.5 (C), 132.2 (C), 134.3 (CH), 136.9
(C), 157.9 (C), 168.0 (C), 168.6 (C); MS (MALDI-TOF) m/z 605
(M + 23), 606, 607, 608, 609; EMM calcd for C24H17Cl2N2O5
m/z 583.1403 (MH+), found 583.1551.
(S)-N r-P h t h a loyl-O-2,6-d ich lor ob e n zylt yr osin ylgly-
cin e (15). TFA (10 mL) was added to a solution of Pht-Tyr-
(2,6-Cl2-Bn)-Gly-OtBu 14 (0.65 g, 1.115 mmol) in 20 mL of
CH2Cl2, and then the mixture was refluxed for 2 h. After
evaporation of the CH2Cl2 and TFA, water (50 mL) was added
then product was extracted with EtOAc (3 × 50 mL). The
combined organic layers were washed with water (50 mL),
dried with Na2SO4, filtered, and evaporated to afford a white
solid (0.59 g, 100%): mp 146-149 °C; [R]D -115.7 (c 1.5,
(S)-O-(2,6-Dich lob en zyl)t yr osin e (12). A solution of
CuSO4‚5H2O (7.03 g, 28.16 mmol) in 20 mL of water was added
to a stirred solution of L-tyrosine 11 (10 g, 55.2 mmol) in 56
mL of 2 N NaOH (2.03 equiv). The mixture was heated to 60
°C and then allowed to cool to room temperature. MeOH (194
mL), 2 N NaOH (8 mL), and 2,6-dichlorobenzyl bromide (14.2
g, 58.9 mmol) were added. After being stirred at room
temperature for 5 h, the mixture was filtered, and then the
residue was successively washed with MeOH (26 mL) and
water (100 mL then 15 mL). The residue was triturated in in
1 N HCl (7 × 27.5 mL) and washed with water (3 × 100 mL)
and 1 N NH4OH (7 × 27.5 mL). After further washings with
acetone (2 × 25 mL), water (2 × 25 mL), and Et2O (2 × 25
mL), the product was dried in a vacuum to afford a white solid
1
MeOH); H NMR (250 MHz, CDCl3) δ 3.45 (m, 2H), 4.00 (m,
2H), 5.05 (s, 2H), 5.15 (m, 1H), 6.75 (d, J ) 8.2 Hz, 2H), 7.03
(d, J ) 8.1 Hz, 2H), 7.07-7.22 (m, 2H), 7.39 (m, 1H), 7.57-
7.64 (m, 4H), 10.54 (s, 1H); 13C NMR (62.5 MHz, CDCl3) δ 33.8
(CH2), 41.6 (CH2), 55.4 (CH), 65.4 (CH), 115.4 (CH), 123.6 (CH),
128.4 (CH), 129.1 (C), 130.0 (CH), 130.4 (CH), 131.3 (C), 132.1
(C), 134.4 (CH), 136.9 (C), 157.9 (C), 168.2 (C), 170.2 (C), 172.8
(C); MS (MALDI-TOF) m/z 527 (M + 1), 528, 529, 549 (M +
23), 550, 551, 552, 553, 565, 572; EMM calcd for C26H20Cl2N2O6
m/z 527.0777 (MH+), found 527.0796.
3-[2(S)-(1,3-Dih yd r o-1,3-d ioxo-2H -isoin d ol-2-yl)-3-(4-
(2,6-d ich lor ob en zyloxyp h en yl))p r op a n oyl]-5-oxa zolid i-
n on e (16). Prepared from 15 (1.42 g, 2.69 mmol) using the
procedure described for the synthesis of 4. Oxazolidinone 16
was obtained as a pale yellow solid (0.91 g, 63%): mp 84 °C;
[R]D -114.2 (c 0.9, CHCl3); 1H NMR (250 MHz, CDCl3) δ 3.47
(dd, J ) 8.7, 14.2 Hz, 1H), 3.64 (dd, J ) 7.2, 14.2 Hz, 1H),
4.09 (m, 2H), 5.02 (s, 2H), 5.02 (dd, J ) 7.2, 8.7 Hz, 1H), 5.21
(s, 2H), 5.60 (d, J ) 28.4 Hz, 2H), 6.90 (d, J ) 8.6 Hz, 2H),
7.16 (d, J ) 8.6 Hz, 2H), 7.21-7.39 (m, 3H), 7.74-7.96 (m,
4H); 13C NMR (62.5 MHz, CDCl3) δ 33.9 (CH2), 43.9 (CH2),
53.9 (CH), 65.4 (CH2), 115.4 (CH), 123.8 (CH), 128.5 (CH),
128.7 (C), 130.0 (C), 130.3 (CH), 130.4 (CH), 131.1 (C), 132.1
(C), 134.6 (CH), 136.9 (C), 158.1 (C), 167.4 (C), 168.7 (C); MS
(MALDI-TOF) m/z 539 (M + 1), 541, 542, 543, 544, 561 (M +
23), 562, 563, 564, 565; EMM calcd for C27H20Cl2N2O6 m/z
539.0777 (MH+), found 539.0819.
4(S)-(1,3-Dih yd r o-1,3-d ioxo-2H -isoin d ol-2-yl)-1,3,4,5-
tetr a h yd r o-8-(2,6-d ich lor oben zyloxy)-3-oxo-2H-2-ben za -
zep in e-2-a cetic Acid (17). Meth od D (Sch em e 3). To a
solution of oxazolidinone 16 (0.87 g, 1.61 mmol) in 20 mL of
freshly distilled CH2Cl2 was added SnCl4 (1 mL in 10 mL of
CH2Cl2) over 10 min. The mixture was refluxed for 3 h, and
then stirring was continued at rt for 24 h. After elimination
of the solvant and SnCl4 under reduced pressure, the residue
was disolved in acetonitrile and water and then evaporated
again. The crude was then purified by flash chromatography
(CH2Cl2/acetone/MeOH, 85:10:5) to afford the aminobenzaze-
pinone 17 as a white solid (0.731 g, 84%): mp 177-179 °C;
[R]D -40.9 (c 1.1, AcOH); 1H NMR (250 MHz, DMSO-d6) δ 3.27
(dd, J ) 4.5, 15.9 Hz, 1H), 3.77 (d, J ) 16.7 Hz, 1H), 3.99 (dd,
J ) 12.2, 15.9 Hz, 1H), 4.22 (d, J ) 16.7 Hz, 1H), 4.41 (d, J )
1
(12.6 g, 67%): mp 215-216 °C; [R]D -17.4 (c 1.0, MeOH); H
NMR (250 MHz, DMSO-d6) δ 3.03 (d, J ) 6.2 Hz, 2H), 4.11 (t,
J ) 6.2 Hz, 1H), 5.16 (s, 2H), 6.99 (d,, J ) 8.6 Hz, 2H), 7.18
(d, J ) 8.6 Hz, 2H), 7.39-7.55 (m, 3H), 8.29 (br s, 2H), 11.24
(br s, 1H); 13C NMR (62.5 MHz, DMSO-d6) δ 35.1 (CH2), 54.8
(CH), 65.3 (CH2), 114.5 (CH), 128.2 (CH), 128.7 (CH), 130.7
(CH), 131.6 (C), 136.8 (C), 158.0 (C), 170.2 (C); MS (MALDI-
TOF) m/z 340 (M + 1), 341, 342, 379, 380; EMM calcd for
C
16H15Cl2NO3 m/z 340.0507 (MH+), found 340.0653.
(S)-Nr-P h th a loyl-O-(2,6-d ich lor oben zyl)tyr osin e (13).
A solution H2N-Tyr(2,6-Cl2-Bn)-OH 12 (12.58 g, 37.1 mmol)
and phthalic anhydride (5.51 g, 37.2 mmol) in 312 mL of 1,4-
dioxane was stirred at room temperature for 30 min. Et3N
(10.2 mL in 39 mL of water) was added. After being stirred at
room temperature for 90 min, the mixture was diluted with
1,4-dioxane (390 mL) and Et3N (10.2 mL), and then the
mixture was gently heated with azeotropic removal removal
of water (Dean-Stark). The excess dioxane was eliminated by
evaporation then, the residue was dissolved in EtOAc (350
mL). The solution was washed with 1N HCl (3 × 150 mL),
dried with MgSO4, filtered then, concentrated in vacuo. The
crude was finally purified by flash chromatography (EtOAc/
hexane, 1:1) to afford the desired product as white solid (10.81
g, 62%): mp 159 °C; [R]D +6.3 (1.2, MeOH); 1H NMR (250 MHz,
DMSO-d6) δ 3.05 (m, 2H), 4.59 (m, 1H), 5.21 (s, 2H), 7.01 (d,
J ) 8.6 Hz, 2H), 7.32 (d, J ) 8.6 Hz, 2H), 7.35 (m, 1H), 7.44-
7.61 (m, 4H), 7.74 (m, 1H), 8.68 (d, J ) 8.0 Hz, 1H), 12.74 (br
s, 1H); 13C NMR (62.5 MHz, DMSO-d6) δ 35.7 (CH2), 53.9 (CH),

6492 J . Org. Chem., Vol. 65, No. 20, 2000
Casimir et al.
16.3 Hz, 1H), 4.96 (d, J ) 16.3 Hz, 1H), 5.22 (s, 2H), 5.41 (dd,
J ) 4.5, 12.2 Hz, 1H), 6.97 (d, J ) 8.4 Hz, 1H), 7.04 (s, 1H),
7.18 (d, J ) 8.4 Hz, 1H), 7.43-7.59 (m, 3H), 7.90 (s, 4H); 13C
NMR (62.5 MHz, DMSO-d6) δ 32.2 (CH2), 51.4 (CH2), 52.0
(CH), 55.8 (CH2), 64.8 (CH2), 113.7 (CH), 114.8 (CH), 123.1
(CH), 128.7 (CH), 130.8 (CH), 131.3 (CH), 131.4 (C), 131.6 (C),
134.6 (CH), 136.0 (C), 136.5 (C), 156.7 (C), 167.4 (C), 168.2
(C); MS (MALDI-TOF) m/z 539 (M + 1), 540, 541, 561 (M +
23), 563; EMM calcd for C27H20Cl2N2O6 m/z 539.0777 (MH+),
found 539.0786.
Ack n ow led gm en t. The authors thank Dr. G. Laus
for exact mass measurements and Dr. P. Verheyden for
2D NMR analysis. D.T. acknowledges financial support
from FWO-Vlaanderen (G.0054.96N).
J O000530D