Synthesis of Human Ultraviolet Filter Compounds: O-β-D-Glucopyranosides of 3-Hydroxykynurenine and 2-Amino-3-hydroxy-γ-oxobenzenebutanoic Acid

Article abstract of DOI:10.1021/jo982321n

The role of endogenous tryptophan-derived UV filters in aging lenses and in human cataract is becoming increasingly important. The two major UV filters found in the lenses of primates, the O-β-D-glucopyranosides of 3-hydroxykynurenine and 2-amino-3-hydroxy-γ-oxobenzenebutanoic acid, 1 and 2, were synthesized from the common benzoyl acrylate precursor 2-amino-3-hydroxybenzoyl-acrylic acid 10. Synthesis of compound 3, the α-N-acetyl derivative of 1, was achieved using coupling of 2-nitro-3-benzyloxyacetophenone 4 with the sodium salt of diethyl acetamidomalonate as a key step. This is the first reported synthesis of the lenticular glucopyranoside 2 and the N-acetyl compound 3.

Full text of DOI:10.1021/jo982321n

3930
J . Org. Chem. 1999, 64, 3930-3933
Syn th esis of Hu m a n Ultr a violet F ilter Com p ou n d s:
O-â-D-Glu cop yr a n osid es of 3-Hyd r oxyk yn u r en in e a n d
2-Am in o-3-h yd r oxy-γ-oxoben zen ebu ta n oic Acid
Michael K. Manthey, J oanne F. J amie,* and Roger J . W. Truscott
Australian Cataract Research Foundation, Department of Chemistry, University of Wollongong,
Wollongong, New South Wales, 2522, Australia
Received November 24, 1998
The role of endogenous tryptophan-derived UV filters in aging lenses and in human cataract is
becoming increasingly important. The two major UV filters found in the lenses of primates, the
O-â-D-glucopyranosides of 3-hydroxykynurenine and 2-amino-3-hydroxy-γ-oxobenzenebutanoic acid,
1 and 2, were synthesized from the common benzoyl acrylate precursor 2-amino-3-hydroxybenzoyl-
acrylic acid 10. Synthesis of compound 3, the R-N-acetyl derivative of 1, was achieved using coupling
of 2-nitro-3-benzyloxyacetophenone 4 with the sodium salt of diethyl acetamidomalonate as a key
step. This is the first reported synthesis of the lenticular glucopyranoside 2 and the N-acetyl
compound 3.
The lenses of many vertebrates contain low molecular
reaction mixture.6 Compounds 2 and 3 have not previ-
ously been synthesized. Consequently, this paper outlines
the synthesis of the lenticular glucopyranosides 1 and 2
and the N-acetyl compound 3.
weight fluorescent compounds, known as UV filters.
These play an important role in protecting the retina,
and possibly the lens, from UV-induced damage by
absorbing the near-UV radiation that is transmitted by
the cornea.1 Kynurenine, 3-hydroxykynurenine, and the
O-â-D-glucopyranosides of 3-hydroxykynurenine and
2-amino-3-hydroxy-γ-oxobenzenebutanoic acid, 1 and 2,
fulfill such a role in human lenses.2,3
A number of as yet unidentified UV filters are con-
tained in the human lens. As enzymes capable of acety-
lating amino groups of lens proteins (such as R- and
â-crystallins) and small peptides are known to be active
in the lens,4 the N-acetyl derivative of the most abundant
UV filter 1 may also be present. An acetylated derivative,
N-acetyl-3-hydroxykynurenine, is the major low molec-
ular weight pigment found in the lens of the gray
squirrel.5 Synthesis of the authentic N-acetyl compound
3 would serve as a reference compound for lenticular
studies to test this hypothesis.
The synthesis of the â-glucopyranoside of (() N-acetyl-
3-hydroxykynurenine 3 is depicted in Scheme 1. 3-Hy-
droxyacetophenone was nitrated and benzylated accord-
ing to the method of Butenandt7 to afford 2-nitro-3-
benzyloxyacetophenone 4, which served as the starting
material for the synthesis. Selective bromination of the
methyl ketone moiety in 4 to afford the R-bromoketone
5 was accomplished in 82% yield employing dioxane
dibromide. Coupling of 5 with the sodium salt of diethyl
acetamidomalonate8 in ethanol resulted in a low yield
(21%) of the coupled adduct 6. This is presumably a
consequence of competing Favorski-type rearrangement
reactions of 5 under the basic reaction conditions.9
Removal of the benzyl protecting group in 6 was conven-
iently achieved employing neat trifluoroacetic acid to
yield the o-nitrophenol 7. This compound was glycosy-
lated via the Helferich method10 using ABG and mercuric
cyanide as a catalyst to afford the O-â-glucopyranoside
8 in 61% yield. The â configuration was confirmed by the
characteristic J 1,2 coupling constant of 7.3 Hz for the
anomeric proton.11 Catalytic hydrogenation of 8 employ-
ing Adam’s catalyst was straightforward and afforded the
amino glucoside 9 in excellent yield (92%). Deacetylation
of 9 via Zemplen’s method resulted in virtually quantita-
tive O-deacetylation as evidenced by the presence of a
1
solitary N-acetyl resonance in the H NMR spectrum of
the crude reaction product. De-esterification of this
Compound 1 has previously been synthesized in a low,
but unspecified, yield by the direct coupling of 2,3,4,6-
tetra-O-acetyl-R-D-glucopyranosyl bromide (ABG) to 3-hy-
droxykynurenine, followed by hydrolysis of the crude
product was followed by 1H NMR in D2O/CH3OD employ-
(6) Linzen, B.; Ishiguro, I. Z. Naturforsch. 1966, 21b, 132.
(7) Butenandt, A.; Hallman, G.; Beckmann, R. Chem. Ber. 1957, 90,
1120.
(1) Collier, R. J .; Waldron, W. R.; Zigman, S. Invest. Opthalmol. Vis.
Sci. 1989, 30(4), 631.
(2) van Heyningen, R. Nature 1971, 230, 393.
(3) Truscott, R. J . W.; Wood, A. M.; Carver, J . A.; Sheil, M. M.;
Stutchbury, G. M.; Zhu, J .; Kilby, G. W. FEBS Lett. 1994, 348, 173.
(4) Granger, M.; Tesser, G. I.; De J ong, W. W.; Bloemendal, H. Proc.
Natl. Acad. Sci. U.S.A. 1976, 73(9), 3010.
(8) Greenstein, J . P.; Winitz, M. Chemistry of the Amino Acids; J ohn
Wiley and Sons Inc: New York, 1961; pp 711-712.
(9) Rappe, C. Rearrangements Involving Halides. In The Chemistry
of the Carbon-Halogen Bond; Patai S., Ed.; J ohn Wiley and Sons:
Interscience: London, 1973; Part 2, Chapter 17, pp 1084-1101.
(10) Helferich, B.; Weis, K. Chem. Ber. 1956, 89, 314.
(11) Hermansson, K.; Kenne, L.; Rukunga, G. M.; Samuelsson, G.
Phytochemistry 1990, 29, 513.
(5) Zigman, S.; Paxhia, T. Exp. Eye. Res. 1988, 47, 819.
10.1021/jo982321n CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/04/1999

Synthesis of Human Ultraviolet Filter Compounds
J . Org. Chem., Vol. 64, No. 11, 1999 3931
Sch em e 1a
a
Key: (a) dioxane‚Br2; (b) CH(COOEt)2NHAc/NaOEt; (c) TFA; (d) ABG/Hg(CN)2/K2CO3; (e) H2/Pd(C) (f) NaOEt; (g) NaOH; (h) H+.
Sch em e 2a
Sch em e 3a
a
Key: (a) H2/PtO2; (b) NaOEt/EtOH; (c) (i) NaOH, (ii) H+.
tetraacetyl â-glucopyranoside 12 was accomplished, in a
61% yield, by coupling of ABG to the methyl ester 11
under the conditions employed for the preparation of the
O-â-glucopyranoside 8. Incubation of 12 in a 1:1 solution
of methanol/ammonia (33%) overnight resulted in smooth
incorporation of ammonia and exhaustive O-deacetyla-
tion to afford an amino acid, as indicated by the presence
of a solitary new compound on TLC that stained with
ninhydrin. This product was not purified but subjected
directly to catalytic hydrogenation (H2/Pd(C)) followed by
alkaline hydrolysis. Purification by preparative reversed-
phase HPLC afforded the O-â-D-glucopyranoside of (()-
3-hydroxykynurenine, 1, in an overall yield of 48% from
12. Two unresolved peaks of equal intensity were ob-
served for the diastereoisomers of 1. Use of different
solvent conditions and flow rates did not allow their
complete separation. The first eluting peak increased in
size upon spiking with an authentic sample of the O-â-
D-glucopyranoside of L-3-hydroxykynurenine isolated from
human lenses.3
The protected glucoside 12 was also utilized for the
synthesis of the O-â-glucopyranoside 2 (Scheme 3).
Catalytic hydrogenation of 12 employing PtO2 resulted
in reduction of the nitro and olefinic functionalities to
afford 13. Complete O-deacetylation of 13 via Zemplen’s
procedure followed by alkaline hydrolysis yielded 60% of
2 after purification by semipreparative HPLC. An au-
thentic sample of the O-â-D-glucopyranoside of 2-amino-
3-hydroxy-γ-oxobenzenebutanoic acid obtained from hu-
man lenses3 coeluted with the synthesized sample of 2
in the HPLC.
a
Key: (a) MeOH/DCC; (b) ABG/Hg(CN)2/K2CO3; (c) NH4OH;
(d) H2/Pd(C); (e) (i) NaOH, (ii) H+.
ing 4 equiv of NaOD. This indicated an initial rapid (30
min) mono de-esterification followed by slow (12 h)
hydrolysis of the remaining ethyl ester. Removal of
solvent and incubation of the residue in acetic acid at 50
°C for 3 h resulted in smooth decarboxylation to yield 3
as a mixture of diastereoisomers in 44% yield after
purification by preparative reversed-phase HPLC. When
human lenses were extracted with 80% ethanol and the
UV filters separated by HPLC3 it was found that none
of the acetylated material 3 was detected.
It was expected that enzymic N-deacetylation of D,L-3
would be affected enantiospecifically to yield L-1 and
unreacted D-3. Compound 3 was, however, found to be
refractory to a number of enzymes commonly employed
to affect the conversion of N-acetylated R-amino acids to
R-amino acids.12 Conversely alkaline hydrolysis of 3
under conditions to affect N-deacetylation13 resulted in
extensive decomposition. Consequently, an alternative
synthetic method for the preparation of (()-1 was em-
ployed (Scheme 2).
The acrylic acid derivative 107 was methylated em-
ploying DCC and a large excess of methanol to afford a
45% yield of the methyl ester 11. Preparation of the
To confirm the structural integrity of the â-glucopy-
ranosides, compounds 1-3 were incubated with â-glu-
cosidase (pH 5.6, 37 °C) and the reactions monitored by
analytical reversed-phase HPLC. After 12 h incubation,
1-3 had reacted completely, indicating that each com-
(12) (a) Birnbaum, S. M.; Levintow, L.; Kingsley, R. B.; Greenstein,
J . P. J . Biol. Chem. 1952, 194, 455. (b) Marshall, R.; Birnbaum, S. M.;
Greenstein, J . P. J . Am. Chem. Soc. 1956, 78, 4636.
(13) Hengartner, U.; Batcho, A. D.; Blount, J . F.; Leimgruber, W.;
Larscheid, M. E.; Scott, J . W. J . Org. Chem. 1979, 44, 3748.

3932 J . Org. Chem., Vol. 64, No. 11, 1999
Manthey and Truscott
mL), followed by NaHCO3 (5% solution, 20 mL). The organic
phase was dried (Na2SO4) and solvent removed to give an
orange oil. This was dissolved in hot ether, which afforded
white crystals of 5 (1.06 g, 82%) on cooling: mp 116-118 °C
pound was a glucopyranoside exclusively in the â con-
figuration. In the case of 1, it was also confirmed that
the aglycon component was 3-hydroxykynurenine, by
comparison (by HPLC) with commercial 3-hydroxykynu-
renine.
1
dec; H NMR δ 4.34 (2H, s), 5.23 (2H, s), 7.28-7.40 (7H, m),
7.50 (1H, t, J ) 8.0 Hz); 13C NMR δ 31.3, 71.3, 119.0, 120.9,
127.0, 128.4, 128.7, 128.7, 129.0, 131.4, 134.8, 150.7, 189.4;
IR (Nujol mull) νmax 1710, 1541, 1291 cm-1. Anal. Calcd for
The 1H NMR spectra of the â-glucopyranosides 1-3
exhibited similar aromatic resonances, each with a
doublet of doublets at δ ∼6.7 ppm, doublet at δ ∼7.3, and
doublet at δ ∼7.6, with ortho J values of ∼8 Hz. Such a
pattern is typical of a 3-hydroxykynurenine derivative.14
The 1H NMR spectrum of the N-acetyl derivative 3, while
similar to that of 1, also exhibited a characteristic acetyl
resonance (δ 1.93) and a pronounced downfield shift of
0.48 ppm for the HR proton in accordance with substitu-
tion of an R-NH2 for an R-NHAc group. Compound 2,
which lacks an R-amino substituent, showed the presence
of an isolated CH2CH2 group with two triplets at δ 2.60
and 3.25 and was in excellent agreement with the values
found for 2 isolated from human lenses.3
C
15H12BrNO4: C, 51.43; H, 3.43; N, 4.00. Found: C, 51.63; H,
3.37; N, 3.91.
Dieth yl (Acetylam in o)[2-(2-n itr o-3-ph en ylm eth oxyph en -
yl)-2-oxoeth yl]p r op a n ed ioa te (6). To a solution of diethyl
acetamidomalonate (573 mg, 2.64 mmol) and NaOEt (180 mg,
2.64 mmol) in EtOH (15 mL) was added in one portion the
bromoketone 5 (0.88 g, 2.51 mmol) and the mixture stirred at
room temperature for 2 h. Solvent was removed under vacuum
and the residue partitioned between EtOAc (120 mL) and 0.01
M NaOH (50 mL). The organic phase was washed sequentially
with water (50 mL) and 0.001 M HCl (50 mL). After drying
(MgSO4) and removal of solvent under vacuum, the residue
was chromatographed (EtOAc/hexane 2:3-3:2) to afford 6 as
a light yellow oil. Crystallization from Et2O afforded pure 6
1
(260 mg, 21%) as a white solid: mp 107 °C; H NMR δ 1.25
(6H, t, J ) 7.1 Hz), 2.00 (3H, s), 4.22 (2H, s), 4.26 (4H, q, J )
7.1 Hz), 5.21 (2H, s), 7.07 (1H, br s), 7.25-7.39 (7H, m), 7.46
(1H, t, J ) 8.1 Hz); 13C NMR δ 13.60, 22.59, 43.40, 62.86, 63.48,
71.03, 118.63, 120.42, 126.81, 128.18, 128.50, 130.49, 131.06,
134.74, 138.97, 150.09, 166.45, 169.56, 195.14; IR (Nujol mull)
νmax 3236, 1750 (br), 1699, 1640 cm-1. Anal. Calcd for
Con clu sion s
The two major UV filter compounds found in the lenses
of primates, the O-â-D-glucopyranosides of 3-hydrox-
ykynurenine and 2-amino-3-hydroxy-γ-oxobenzenebu-
tanoic acid, 1 and 2, were successfully synthesized from
the common benzoyl acrylate precursor 2-amino-3-hy-
droxybenzoylacrylic acid 10. Synthesis of compound 3,
the R-N-acetyl derivative of 1, was achieved using
coupling of 2-nitro-3-benzyloxyacetophenone 4 with the
sodium salt of diethyl acetamidomalonate as a key step.
Compound 3 was not detected in human lenses.
C
24H26N2O9: C, 59.26; H, 5.35; N, 5.76. Found: C, 59.14; H,
5.40; N, 5.57.
Dieth yl (Acetyla m in o)[2-(2-n itr o-3-h yd r oxyp h en yl)-2-
oxoeth yl]p r op a n ed ioa te (7). Trifluoroacetic acid (3 mL) was
added to 6 (150 mg, 0.31 mmol) and the mixture stirred at
room temperature for 24 h. Solvent was removed under
vacuum and the oily residue dissolved in EtOAc (2 mL).
Hexane was added slowly until the mixture became turbid.
After refrigeration for 2 h rosettes of yellow crystals formed.
These were collected by filtration to afford 7 (78 mg, 64%):
Exp er im en ta l Section
1
mp 132-133 °C (EtOAc/hexane); H NMR δ 1.29 (6H, t, J )
All experiments were carried out using instrumentation and
manipulations as described previously.15 The NMR spectra
were measured in CDCl3 unless otherwise stated. Microanaly-
ses (C, H, N) were performed by the Australian National
University Analytical Services, Canberra. Analytical HPLC
was carried out on Millipore Waters HPLC equipment com-
prising a 6000A series solvent delivery system with an
automated gradient controller and model 746 data module.
Separations were performed using a 100 × 8 mm Nova Pak
C18 reversed-phase radial pack cartridge. Elution was carried
out using linear gradients from 0 to 80% CH3CN over 10 min
at a flow rate of 2 mL/min. Semipreparative HPLC was
performed using a Varian 2010 HPLC pump with a 250 mm
× 10 mm Alltech C18 10 µm column, eluting with 0.1% HOAc
(pH 3) for 30 min, followed by 10% CH3CN for 30 min, at a
flow rate of 3 mL/min. Preparative HPLC was performed using
a Millipore Waters Prep 500 HPLC with a Prep PAK 500-C18
and 55-105 µm particle size column eluting with a linear
gradient of 0-50% CH3CN over 30 min at a flow rate of 20
mL/min.
Reactions were followed by TLC on Merck F254 silica gel
plates. Merck silica gel (70-230 mesh) was used for column
chromatography. All solvents and reagents were obtained from
commercial sources and purified before use as required.
2-Br om o-1-(2-n it r o-3-p h e n ylm e t h oxyp h e n yl)e t h a -
n on e (5). Dioxane dibromide16 (1.0 g, 4.06 mmol) was added
to a solution of 2-nitro-3-benzyloxyacetophenone 47 (1.0 g, 3.69
mmol) in CH2Cl2 (20 mL) and stirred at room temperature for
1.5 h. The light yellow solution was washed with water (20
7.2 Hz), 2.11 (3H, s), 4.07 (2H, s), 4.29 (4H, q, J ) 7.2 Hz),
6.77 (1H, dd, J ) 7.3, 1.1 Hz), 7.15 (1H, br s), 7.22 (1H, dd, J
) 8.5, 1.1 Hz), 7.58 (1H, dd, J ) 8.5, 7.3 Hz), 10.44 (1H, br s);
13C NMR δ 13.80, 22.80, 45.53, 63.03, 63.46, 118.34, 121.55,
131.16, 136.19, 137.91, 154.62, 166.64, 169.99, 199.26; IR
(Nujol mull) νmax 3295, 1746, 1695 cm-1; UV λmax (EtOH) )
216 nm (ꢀ 16 980), 248 (ꢀ 6310), 316 (ꢀ 3090). Anal. Calcd for
C
17H20N2O9: C, 51.52; H, 5.05; N, 7.07. Found: C, 51.36; H,
5.41; N, 6.84.
Dieth yl
(Acetyla m in o)[2-[2-n itr o-3-(2,3,4,6-tetr a -O-
a cet yl-â-D-glu cop yr a n osyl)oxyp h en yl)]-2-oxoet h yl]p r o-
p a n ed ioa te (8). A mixture of 7 (40 mg, 0.10 mmol), mercuric
cyanide (26 mg, 0.10 mmol), potassium carbonate (17 mg, 0.12
mmol), and ABG17 (50 mg, 0.12 mmol) in CH3CN (3 mL) was
stirred at room temperature for 2 d. Solvent was removed
under vacuum and the residue partitioned between EtOAc (20
mL) and water (15 mL). The organic phase was dried (MgSO4),
solvent removed under vacuum, and the residue chromato-
graphed (EtOAc/hexane, 2:1-4:1) to afford 8 (41 mg, 61%): mp
1
94-96 °C (EtOH); H NMR δ 1.20 (6H, t, J ) 6.8 Hz), 1.96
(3H, s), 1.99 (3H, s), 2.01 (3H, s), 2.06 (3H, s), 2.08 (3H, s),
3.84 (1H, dt, J ) 3.4, 9.9 Hz), 4.15-4.30 (8H, m), 4.97-5.29
(4H, m), 7.07 (1H, br s), 7.53-7.61 (3H, m); 13C NMR δ 13.76,
20.36, 20.50 (2), 20.62, 22.82, 43.44, 61.56, 63.10, 63.20, 63.62,
67.90, 70.20, 72.02, 72.33, 100.37, 123.85, 124.00, 130.47,
131.19, 140.56, 148.20, 166.50, 166.56, 169.24, 169.78, 170.07,
170.39, 194.74; IR (Nujol mull) νmax 3384, 1751, 1661 cm-1
Anal. Calcd for C31H38N2O18 C, 51.24; H, 5.23; N, 3.86.
Found: C, 51.26; H, 5.05; N, 3.97.
.
:
Diet h yl (Acet yla m in o)[2-[2-a m in o-3-(2,3,4,6-t et r a -O-
a cet yl-â-D-glu cop yr a n osyl)oxyp h en yl)]-2-oxoet h yl]p r o-
(14) Truscott, R. J . W.; Carver, J . A.; Thorpe, A.; Douglas, R. H.
Exp. Eye Res. 1992, 54, 1015.
(15) Batty, C. A.; Manthey, M. K.; Kirk, J .; Manthey, M.; Christo-
pherson, R. I.; Hambley, T. J . Heterocycl. Chem. 1997, 34, 1355.
(16) Duhamel, P.; Duhamel, L.; Valnot, J .-Y. Bull. Soc. Chim. Fr.
1973, 1465.
(17) Vogel, A. I. Vogel’s Textbook of Practical Organic Chemistry,
5th ed; Furniss B. S., Hannaford A. J ., Smith P. W. G., Ed.; Longman
Scientific and Technical: New York, 1989; pp 647-648.

Synthesis of Human Ultraviolet Filter Compounds
J . Org. Chem., Vol. 64, No. 11, 1999 3933
p a n ed ioa te (9). To a solution of 8 (1 g, 1.38 mmol) in EtOAc/
EtOH (21 mL, 2:1) was added Adam’s catalyst (300 mg) and
the mixture placed under a hydrogen atmosphere (1 atm) for
24 h. The mixture was filtered and the filtrate taken to dryness
under vacuum. The light yellow residue was recrystallized
from CCl4/hexane to afford 9 (920 mg, 92%) as an off-white
solid: mp 108-110 °C; 1H NMR δ 1.24 (6H, t, J ) 7.3 Hz),
1.98 (3H, s), 2.05 (6H, s), 2.07 (3H, s), 2.09 (3H, s), 3.87 (1H,
ddd, J ) 10.2, 4.9, 2.0 Hz), 4.22 (2H, s), 4.26-4.35 (6H, m),
4.97 (1H, d, J ) 7.8 Hz), 5.13-5.33 (3H, m), 6.49 (2H, br s),
6.55 (1H, t, J ) 7.8 Hz), 7.03 (1H, d, J ) 7.8 Hz), 7.11 (1H, s),
7.53 (1H, d, J ) 7.8 Hz); 13C NMR δ 13.85, 20.55 (br), 22.94,
42.94, 61.76, 62.77, 63.98, 68.21, 71.14, 72.80 (m), 100.06,
114.08, 117.43, 119.21, 125.90, 142.31, 144.40, 167.42, 167.57,
169.35, 169.83, 170.05, 170.46, 198.34; IR (Nujol mull) νmax
3381, 3362, 1747, 1670 cm-1; UV λmax (EtOH) ) 232 nm (ꢀ
17 313), 264 (ꢀ 4500), 370 (ꢀ 4370). Anal. Calcd for
residue chromatographed (CH2Cl2/hexane 5:1) to afford 12 (284
mg, 61%): mp 110-112 °C (EtOH); H NMR δ 2.01 (3H, s),
1
2.03 (3H, s), 2.08 (3H, s), 2.11 (3H, s), 3.82 (3H, s), 3.82-3.95
(1H, m), 4.24 (2H, d, J ) 3.9 Hz), 5.05-5.30 (4H, m), 6.73 (1H,
d, J ) 15.6 Hz), 7.52 (1H, d, J ) 15.6 Hz), 7.45-7.56 (3H, m);
13C NMR δ 20.41 (4), 52.48, 61.58, 67.92, 70.24, 72.04, 72.33,
100.22, 123.47, 124.02, 131.31, 131.54, 134.08, 136.33, 140.96,
148.46, 165.11, 169.12, 169.17, 169.97, 170.30, 187.50; IR
(Nujol mull) νmax 1749, 1680, 1541 cm-1. Anal. Calcd for C25H27
-
NO15: C, 51.64; H, 4.65; N, 2.41. Found: C, 51.55; H, 4.81; N,
2.09.
r,2-Dia m in o-3-(â-D-glu cop yr a n osyloxy)-γ-oxoben zen e-
bu ta n oic Acid (1). To a suspension of 12 (100 mg, 0.17 mmol)
in MeOH (1 mL) was added 1 mL of a 33% NH3 solution and
the mixture stirred at room temperature for 12 h. Solvent was
removed under vacuum, the residue dissolved in H2O (2 mL)
and adjusted to pH 6.0 with HOAc, Pd(C) (20 mg of 10%)
added, and the mixture placed under an atmosphere of H2 for
12 h. The mixture was filtered, NaOH (28 mg, 0.68 mmol)
added to the filtrate, and the mixture stirred for 2 h. The pH
was adjusted to 6.5 with HOAc and the mixture purified by
preparative reversed-phase HPLC eluting with H2O to afford
1 (32 mg, 48%) as a yellow solid after lyophilization: 1H NMR
(D2O) δ 3.60-4.00 (8H, m), 4.15 (1H, t, J ) 5.5 Hz), 5.04 (1H,
d, J ) 5.5 Hz), 6.75 (1H, dd, J ) 8.5, 7.9 Hz), 7.32 (1H, d, J )
7.9 Hz), 7.61 (1H, d, J ) 8.5 Hz); m/z (ES+) 387 (MH+, 100).
Anal. Calcd for C16H22N2O9: C, 49.74; H, 5.70; N, 7.25.
Found: C, 49.66; H, 5.72; N, 7.09.
C
31H40N2O16: C, 53.45; H, 5.75; N, 4.02. Found: C, 53.66; H,
5.65; N, 3.87.
r-Acet yla m in o-2-a m in o-3-(â-D-glu cop yr a n osyloxy)-γ-
oxoben zen ebu ta n oic Acid (3). To a suspension of 9 (500
mg, 0.72 mmol) in EtOH (10 mL) was added a solution of
NaOEt (14 mg, 0.20 mmol) and the mixture stirred for 1 h.
Solvent was removed under vacuum and the residue dissolved
in H2O (10 mL). NaOH (115 mg, 2.88 mmol) was added and
the mixture stirred at rt for 12 h. The solution was lyophilized,
following which HOAc (5 mL) was added and stirring contin-
ued at 50 °C for 3 h. Solvent was removed under vacuum and
the residue purified by preparative HPLC eluting with H2O
to afford 3 (136 mg, 44%) as a yellow solid after lyophiliza-
tion: mp 157-160 °C (H2O); 1H NMR (D2O) δ 1.93 (3H, s),
3.48-3.95 (8H, m), 4.63 (1H, t, J ) 6.1 Hz), 5.00 (1H, d, J )
6.7 Hz), 6.73 (1H, dd, J ) 7.9, 7.9 Hz), 7.28 (1H, d, J ) 7.9
Hz), 7.61 (1H, d, J ) 7.9 Hz); 13C NMR (D2O) δ 21.84, 40.83,
49.03, 60.66, 69.52, 72.97, 75.63, 76.22, 101.54, 116.01, 118.23,
120.64, 125.78, 141.11, 144.93, 173.84, 175.25, 199.98. Anal.
Calcd for C18H24N2O10: C, 50.47; H, 5.61; N, 6.54. Found: C,
50.66; H, 5.65; N, 6.21.
Meth yl (E)-4-Oxo-4-(3-h yd r oxy-2-n itr op h en yl)-2-bu te-
n oa te (11). To a solution of 2-amino-3-hydroxybenzoylacrylic
acid 10 (3 g, 12.7 mmol)7 in MeOH (20 mL) at 0 °C was added
a solution of DCC (2.61 g, 12.7 mmol) in CH2Cl2 (20 mL). The
mixture was stirred at room temperature overnight and the
solvent removed under vacuum. The residue was suspended
in CH2Cl2 (100 mL) and filtered and the filtrate taken to
dryness under vacuum. The residue was chromatographed
(CH2Cl2/hexane, from 1:1 to 5:3 to 3:1) to afford 11 (1.42 g,
45%) as a bright yellow solid: mp 111-112 °C; 1H NMR δ 3.80
(3H, s), 6.39 (1H, d, J ) 16.1 Hz), 6.84 (1H, d, J ) 7.1 Hz),
7.27 (1H, d, J ) 16.1 Hz), 7.31 (1H, d, J ) 8.6 Hz), 7.66 (1H,
dd, J ) 8.6, 7.1 Hz). 13C NMR 52.30, 119.89, 122.09, 132.08,
131.34 (q), 136.94, 139.45, 140.94 (q), 155.33, 165.25, 191.06;
IR (Nujol mull) νmax 1728, 1671, 1603 cm-1; UV λmax (EtOH) )
220 nm (ꢀ 20 890), 272 (ꢀ 5130 inflex), 334 (ꢀ 2820). Anal. Calcd
for C11H9NO6: C, 52.59; H, 3.59; N, 5.58. Found: C, 52.72; H,
3.66; N, 5.53.
Meth yl 4-Oxo-4-[2-a m in o-3-(2,3,4,6-tetr a -O-a cetyl-â-D-
glu cop yr a n osyl)oxyp h en yl]-2-bu ta n oa te (13). To a solu-
tion of 12 (200 mg, 0.34 mmol) in EtOAc/EtOH (10 mL, 4:1)
was added Adam’s catalyst (20 mg) and the mixture placed
under a hydrogen atmosphere (1 atm) for 12 h. The mixture
was filtered and the filtrate taken to dryness under vacuum.
The light yellow residue was chromatographed (CH2Cl2/EtOAc,
95:5) to afford 13 (121 mg, 63%) as a golden oil: 1H NMR δ
2.05-2.09 (12H, m), 2.71 (2H, t, J ) 6.6 Hz), 3.30 (2H, t, J )
6.6 Hz), 3.71 (3H, s), 3.82-3.92 (1H, m), 4.15-4.22 (2H, m),
4.96-5.36 (4H, m), 6.50 (2H, br s), 6.55 (1H, dd, J ) 8.3, 7.8
Hz), 7.04 (1H, d, J ) 7.8 Hz), 7.53 (1H, d, J ) 8.3 Hz); 13C
NMR δ 20.50, 28.12, 33.99, 51.72, 61.74, 68.21, 71.09, 72.03,
72.20, 100.18, 113.84, 117.77, 118.91, 125.46, 142.14, 144.57,
169.35, 169.82, 170.04, 170.46, 173.51, 199.62. Anal. Calcd for
C
25H31NO13: C, 54.25; H, 5.61; N, 2.53. Found: C, 53.99; H,
5.45; N, 2.68.
2-Am in o-3-(â-D-glu cop yr a n osyloxy)-γ-oxob en zen eb u -
ta n oic Acid (2). To a solution of 13 (500 mg, 0.90 mmol) in
EtOH (4 mL) was added a solution of NaOEt in EtOH (14 mg,
0.20 mmol). The mixture was stirred at rt for 2 h, solvent was
then removed, and the residue dissolved in H2O (5 mL) to
which was added NaOH (144 mg, 3.6 mmol). The mixture was
stirred at rt for 4 h, adjusted to pH 3.0 with 1 M HCl, and
lyophilized. The solid was purified via semipreparative reversed-
phase HPLC, eluting with 0.1% HOAc, followed by 10%
CH3CN, to afford 3 (200 mg, 60%) as a yellow solid after
lyophilization. 1H NMR spectral data were in agreement with
the partial spectral data published:3 1H NMR (D2O) δ 2.60 (2H,
t, J ) 6.3 Hz), 3.25 (2H, t, J ) 6.3 Hz), 3.40-3.60 (5H, m),
3.76 (1H, dd, J ) 12.6, 5.1 Hz), 3.83 (1H, dd, J ) 12.6, 2.0
Hz), 6.66 (1H, dd, J ) 8.1, 8.1 Hz), 7.21 (1H, dd, J ) 8.1, ∼1
Meth yl (E)-4-Oxo-4-[2-n itr o-3-(2,3,4,6-tetr a -O-a cetyl-â-
D-glu cop yr a n osyl)oxyp h en yl]-2-bu ten oa te (12). To a solu-
tion of 11 (200 mg, 0.80 mmol) in CH3CN (5 mL) was added
mercuric cyanide (20 mg) followed by ABG (327 mg, 0.80 mmol)
and potassium carbonate (55 mg, 0.40 mmol). The mixture was
stirred at room temperature for 4 d. Solvent was removed
under vacuum and the residue dissolved in EtOAc (20 mL)
and washed with 0.01 M HCl (20 mL). The organic phase was
dried (MgSO4), solvent removed under vacuum, and the
Hz), 7.59 (1H, dd, J ) 8.1, ∼1 Hz); HRMS calcd for C16H22
-
NO9 (MH+) 372.129 457, found 372.130 025.
J O982321N