Synthesis of 3,3',5-trihydroxybiphenyl-2-carboxylic acid, a component of the bitterest natural product amarogentin and its coenzyme A and N-acetyl cysteamine thiol esters

Article abstract of DOI:10.1021/np990256m

3,3',5-Trihydroxybiphenyl-2-carboxylic acid (6), an ester component of the bitter-tasting natural products amarogentin and amaroswerin, was synthesized in six steps in 13.6% overall yield. Its N-acetyl cysteamine thiol ester (9) and its coenzyme A thiol ester (8), a likely biosynthetic precursor of the amarums, were also prepared.

Full text of DOI:10.1021/np990256m

J . Nat. Prod. 2000, 63, 371-374
371
Syn th esis of 3,3′,5-Tr ih yd r oxybip h en yl-2-ca r boxylic Acid , a Com p on en t of th e
Bitter est Na tu r a l P r od u ct Am a r ogen tin a n d Its Coen zym e A a n d N-Acetyl
Cystea m in e Th iol Ester s
Chang-Zeng Wang,† Ulrich H. Maier,‡ and Meinhart H. Zenk*,§
Institut fu¨r Pharmazie, Lehrstuhl fu¨r Pharmazeutische Biologie, Universita¨t Mu¨nchen, Karlstrasse 29,
80333 Mu¨nchen, Germany
Received May 28, 1999
3,3′,5-Trihydroxybiphenyl-2-carboxylic acid (6), an ester component of the bitter-tasting natural products
amarogentin and amaroswerin, was synthesized in six steps in 13.6% overall yield. Its N-acetyl cysteamine
thiol ester (9) and its coenzyme A thiol ester (8), a likely biosynthetic precursor of the amarums, were
also prepared.
3,3′,5-Trihydroxybiphenyl-2-carboxylic acid (6) is an ester
component of amarogentin, which is the bitterest natural
product known.1 It has been shown that this acid could be
formed in the plant by aldol condensation of 7-(3′-hydroxy-
phenyl)-3,5,7-triketo acid.2,3 This compound could, in turn,
be formed from biologically activated CoA esters of 3-hy-
droxybenzoic acid and three units of malonate.2,3 To study
the biosynthesis of amarogentin at the enzyme level, we
required the coenzyme A thiol ester of the biphenylcar-
boxylic acid. Herein we report the first total chemical
synthesis of this biphenylcarboxylic acid (6) as well as its
coenzyme A and N-acetyl cysteamine thiol esters (8 and
9).
and on the other hand, the carboxylic acid would, however,
undergo decarboxylation by treatment with acid and base
at elevated temperatures because of the presence of hy-
droxyl groups at the 2- and 4-positions. For this reason the
hydroxyl groups must be protected with a group stable
toward both acidic and basic conditions, but easily remov-
able under mild and neutral conditions.
Due to the ease of cleavage of 3, we selected a benzyl
protecting group. Therefore, 3 was converted to 4 by
treatment with benzyl chloride and anhydrous potassium
carbonate. Hydrolysis of 4 with 10% NaOH in EtOH
afforded 3,3′,5-tribenzyloxybiphenyl-2-carboxylic acid (5) in
87% yield. Catalytic hydrogenation of 5 with 10% Pd/C in
absolute EtOH gave 3,3′,5-trihydroxybiphenyl-2-carboxylic
acid (6) in almost quantitative yield (Scheme 1).
Resu lts a n d Discu ssion
Cyclization of 1,3,5,7-tetracarbonyl compounds,4 conver-
To investigate the enzymatic formation of amarogentin
from sweroside and 3,3′,5-trihydroxybiphenyl-2-carboxylic
acid (6), both the N-acetyl cysteamine and coenzyme A
(CoA) thiol esters of this acid (9 and 8) were also synthe-
sized. For preparation of the CoA ester, the desired
biphenylcarboxylic acid was activated via a succinimide
derivative.9 Compound 6 reacted with N-hydroxysuccin-
imide in the presence of N,N-dicyclohexylcarbodiimide
(DCC) in dry ethyl acetate to produce biphenylcarboxylic
acid N-hydroxysuccinimide ester (7). The biphenylcar-
bonyl-CoA thiol ester (8) was prepared by transesterifi-
cation of 7 with coenzyme A (CoA-SH) in 50 mM aqueous
NaHCO3. The CoA ester was identified by a specific color
reaction with nitroprusside spray reagent as described in
the Experimental Section. The “delayed” nitroprusside
reaction as well as the chromatographic properties and the
difference spectrum upon base-catalyzed hydrolysis showed
the characteristics of the CoA thiol ester.10 The maximal
UV absorption of the thiol ester was registered at 316 nm.
The biphenylcarboxylic acid N-acetyl cysteamine ester (9)
was also synthesized as a model compound through N-
hydroxysuccinimide ester by transesterification with N-
acetyl cysteamine (NAC-SH) under the same conditions
as the CoA ester synthesis. The general method for the
synthesis of thiol esters directly from free acids in the
presence of DCC and 4-(dimethylamino)pyridine (DMAP)
sion of pyranopyrone,5 and condensation of diketene with
6,7
1,3-dicarbonyl compounds
should lead to the formation
of â-resorcylic acids or esters. In this report, the desired
biphenylcarboxylic acid ethyl ester was synthesized by
reaction of diketene with ethyl-3-phenyl-3-oxopropionate.7
Ethyl 3-(3′-methoxyphenyl)-3-oxopropionate (1) was first
generated by reaction of diethyl carbonate with 3-meth-
oxyacetophenone in the presence of sodium hydride. When
1 was allowed to react with diketene in the presence of
sodium hydride in THF, 3′-methoxy-3,5-dihydroxybiphenyl-
2-carboxylic acid ethyl ester (2) was obtained directly in
31% yield. Demethylation of 2 was achieved through
treatment with 5 equivalents of BBr3 in dry CH2Cl2 to give
3,3′,5-trihydroxy-biphenyl-2-carboxylic acid ethyl ester (3)
(Scheme 1).
Conversion of 3 to its acid (6) was first necessary. Direct
cleavage of the ester group through hydrolysis of 3 with
NaOH or potassium-tert-butoxide 8 resulted in decarboxyl-
ation. The use of hog liver esterase in Na2HPO4 buffer (pH
8.0) to hydrolyze 3 also was unsuccessful. It was assumed
that, on the one hand, the carbonyl ester is sterically
hindered and difficult to hydrolyze at room temperature,
* To whom correspondence should be addressed.
† Present address: Institute of Biological Chemistry, Washington State
University, Pullman, WA 99164-6340. Tel.: (509) 335-3428. Fax: (509) 335-
7643. E-mail: wang@mail.wsu.edu.
11
in anhydrous CH2Cl2 could not be used in the CoA ester
‡ Present address: Werner-Friedmann-Bagen 7, 80993 Mu¨nchen, Ger-
many.
synthesis. However, it worked for the synthesis of bi-
phenylcarboxylic acid N-acetyl cysteamine ester. With both
methods, the main side reaction was decarboxylation of the
biphenylcarboxylic acid to form the biphenyl 10.
§ Present address: Biozentrum der Martin-Luther-Universita¨t,
Halle-Wittenberg, Weinbergweg 22, 06099 Halle/Saale, Germany.
Tel.: (+49)345-5521627. Fax: (+49)345-5320910. E-mail: zenk@
biozentrum.uni-halle.de.
10.1021/np990256m CCC: $19.00
© 2000 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/02/2000

372 J ournal of Natural Products, 2000, Vol. 63, No. 3
Wang et al.
Sch em e 1. Synthesis of 3,3′,5-Trihydroxybiphenyl-2-carboxylic Acid (6) and Its CoA (8) and N-Acetyl Cysteamine (9) Thiol Estersa
a
(a) NaH, diethyl carbonate, benzene, reflux, 1.5 h; (b) diketene, NaH, THF, 0 °C to room temperature, 1 h; (c) BBr3, CH2Cl2, -10 °C to room temperature,
overnight; (d) BnCl, K2CO3, KI, EtOH, reflux, 48 h; (e) 10% NaOH/EtOH, reflux, 3 h; (f) H2, Pd/C, EtOH, room temperature, 48 h; (g) N-hydroxysuccinimide,
DCC, EtOAc, room temperature, 72 h; (h) CoA-SH, 50mM aqueous NaHCO3, 0 °C, overnight; (i) NAC-SH, 50 mM aqueous NaHCO3, 0 °C, 24 h; (j) NAC-
SH, DCC, DMAP, CH2Cl2, 0 °C to room temperature, 3 h.
The UV spectra of 3,3′,5-trihydroxybiphenyl-2-carboxylic
acid (6) and its N-acetyl cysteamine thiol ester (9) taken
in 0.1 M of phosphate buffer (pH 7.0) are given in Figure
1. The difference spectrum between 9 and 6 shows a
maximum at 314 nm, which is in accord with similar
compounds reported in the literature.10 With the synthesis
of the biphenylcarboxyl-CoA thiol ester in hand and the
availability of sweroside, it should now be possible to detect
and characterize the acyl CoA transferase necessary for
completing the chemo-enzymatic synthesis of amarogentin
from sweroside and the biphenylcarboxyl-CoA thiol ester.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. NMR spectra were
F igu r e 1. The UV spectra of 3,3′,5-trihydroxybiphenyl-2-carboxylic
recorded on a Bruker AM 360 spectrometer operating at 360
acid (6) and its N-acetyl cysteamine (9) thiol ester and the difference
spectrum of 9 and 6 (4) taken in 0.1 M of phosphate buffer (pH 7.0)
MHz for 1H and 90 MHz for 13C. CIMS and HREIMS were
measured on a Finnigan SSQ 700 and MAT 90 mass spec-
trometers, respectively. IR spectra were recorded on a Perkin-
Elmer 1600 FT-IR spectrometer and UV spectra on a UVIKON
930 spectrophotometer. HPLC was performed on a Merck
of 10% aqueous HCl (20 mL) and ice (20 g) and extracted with
ether (2 × 50 mL). The ether layer was washed with H2O and
dried over Na2SO4. Ether was removed in vacuo, and the
Hitachi system with L-6200 Intelligent Pump and L-4200 UV-
vis detector. Fluka Si gel 60 (220-440 mesh) was employed
for column chromatography, and thin-layer chromatography
(TLC) was performed on Merck Si gel 60 F254 plates.
Eth yl 3-(3′-Meth oxyp h en yl)-3-oxop r op ion a te (1).7,12 So-
dium hydride (55% dispersion, 2.4 g, 50 mmol) was added with
stirring to a solution of diethyl carbonate (6 mL) in dry benzene
(20 mL). A solution of 3-methoxyphenylacetone (3.0 g, 20
mmol) in diethyl carbonate (2.6 mL) was added dropwise, and
the mixture was refluxed for 1.5 h and then cooled. A small
amount of EtOH was added to the reaction mixture to destroy
excess NaH. The resulting mixture was poured into a mixture
residue was purified by distillation at 11 mbar with collection
at bp 174-182 °C to afford 1 (3.11 g, 14 mmol, 70%). Spectral
data, see literature.13
3′-Meth oxy-3,5-d ih yd r oxybip h en yl-2-ca r boxylic Acid
Eth yl Ester (2). NaH (55% dispersion, 2.8 g, 20 mmol) was
added to a solution of 1 (12.46 g, 56 mmol) in THF (100 mL)
with stirring and cooling in an ice-water bath. The mixture
was stirred for 10 min, followed by the dropwise addition of a
solution of diketene (5 mL) in absolute THF (20 mL). The
whole mixture was stirred for 1 h at room temperature and
then poured into a mixture of concentrated HCl (10 mL) and
ice (120 g). The resulting mixture was extracted with ether (2

Synthesis of Amarogentin Components
J ournal of Natural Products, 2000, Vol. 63, No. 3 373
× 100 mL). The ether layer was dried over Na2SO4 and then
evaporated. The residue was chromatographed by column
chromatography (CC) over Si gel eluting with hexane/ether
(5:1) to afford 2 (5.0 g, 17 mmol, 31%): UV (CHCl3) λmax (log
ꢀ) 241 (4.27), 264 (4.17), 305 (3.95) nm; IR (KBr) νmax 3320,
(3.80) nm; IR (KBr) νmax 3424, 3063, 3030, 2925, 2867, 1697,
1599, 1574, 1496, 1454, 1377, 1340, 1285, 1247, 1167, 1026,
736, 696 cm-1; 1H NMR (CD3OD, 360 MHz) δ 7.25-7.10 (15H,
m, aromatic H of benzyl groups), 7.06 (1H, dd, J ) 8.0, 8.0
Hz, H-5′), 6.88 (1H, d, J ) 2.0 Hz, H-2′), 6.82 (1H, d, J ) 8.0
Hz, H-4′), 6.76 (1H, dd, J ) 8.0, 2.0 Hz, H-6′), 6.49 (1H, d, J )
2.0 Hz, H-6), 6.35 (1H, d, J ) 2.0 Hz, H-4), 4.92, 4.83, 4.82
(each 2H, s, -CH2- of benzyl groups); 13C NMR (CD3OD, 90
MHz) δ 172.1 (C-7), 161.5 (C-5), 160.1 (C-3), 158.0 (C-3′), 142.9
(C-1), 138.2 (C-1′), 130.4 (C-5′), 129.5-128.0 (aromatic carbons
of benzyl groups), 122.1 (C-6′), 115.9 (C-2′), 115.5 (C-4′), 109.0
(C-6), 100.8 (C-2, 4), 71.5, 71.2, 71.0 (3 × -CH2- of benzyl
groups); CIMS m/z 517 [M + 1]+ (73), 426 (22), 383 (18), 293
(9), 203 (7), 112 (100); HREIMS m/z 516.1945 (calcd for
1642, 1570, 1453, 1241 cm-1 1H NMR (CDCl3, 360 MHz) δ
;
7.22 (1H, dd, J ) 8.0, 8.0 Hz, H-5′), 6.87 (1H, br d, J ) 8.0 Hz,
H-6′), 6.76 (1H, br d, J ) 1.8, 8.0 Hz, H-4′), 6.75 (1H, br s,
H-2′), 6.41 (1H, d, J ) 1.8 Hz, H-6), 6.28 (1H, d, J ) 1.8 Hz,
H-4), 3.92 (2H, q, -OCH2CH3), 3.80 (3H, s, -OMe), 1.27 (3H,
t, -OCH2CH3); 13C NMR (CDCl3, 90 MHz) δ 170.7 (C-7), 161.9
(C-3′), 159.0 (C-5), 156.6 (C-3), 147.2 (C-1), 144.2 (C-1′), 128.5
(C-5′), 120.7 (C-6′), 113.6 (C-2′), 112.5 (C-4′), 111.0 (C-6), 107.6
(C-2), 102.4 (C-4), 60.7 (-OCH2CH3), 55.3 (-OCH3), 13.0
(-OCH2CH3); CIMS m/z 289 [M + 1]+ (100), 243 (9); HREIMS
m/z 288.1004 (calcd for C16H16O5, 288.0998).
C
34H28O5, 516.1937).
3,3′,5-Tr ih yd r oxybip h en yl-2-ca r boxylic Acid (6). To a
3,3′,5-Tr ih yd r oxybip h en yl-2-ca r boxylic Acid Eth yl Es-
ter (3). Compound 2 (2.0 g, 7 mmol) was dissolved in dry
CH2Cl2 (150 mL) and chilled to -10 °C. Then, 1.0 M BBr3 in
CH2Cl2 (35 mL) was added dropwise. The mixture was stirred
overnight, beginning at 0 °C and gradually increasing to room
temperature. Small pieces of ice were added and the mixture
extracted with EtOAc. The EtOAc was evaporated under
reduced pressure and the residue purified on CC over Si gel
eluting with CHCl3/MeOH (20:1) to afford 3 (1.53 g, 80%): UV
(MeOH) λmax (log ꢀ) 216 (4.42), 264 (3.97), 299 (3.77) nm; IR
(KBr) νmax 3277, 2984, 1642, 1577, 1453, 1404, 1366, 1318,
solution of 5 (720 mg, 1.39 mmol) in absolute EtOH (20 mL)
was added 10% Pd/C (240 mg) suspended in absolute EtOH
(10 mL). The whole mixture was stirred under a H2 atmo-
sphere at room temperature for 48 h. The catalyst was filtered
off and washed with EtOH (10 mL). The combined filtrate was
evaporated in vacuo to afford 6 (345 mg, 1.39 mmol, 100%):
UV (0.1 M phosphate buffer, pH 7.0) λmax (log ꢀ) 252 (3.93),
287 (3.73) nm; IR (KBr) νmax 3362, 1637, 1604, 1583, 1458,
1288, 1242, 1172, 848, 807 cm-1; 1H NMR (CD3OD, 360 MHz)
δ 6.82 (1H, dd, J ) 8.0, 8.0 Hz, H-5′), 6.54-6.57 (3H, H-2′, -4′,
and -6′), 6.15 (1H, d, J ) 1.9 Hz, H-6), 6.04 (1H, d, J ) 1.9 Hz,
H-4); 13C NMR (CD3COCD3, 90 MHz) δ 172.8 (C-7), 161.7 (C-
5), 160.3 (C-3), 158.9 (C-3′), 148.2 (C-1), 146.1 (C-1′), 129.1 (C-
5′), 120.6 (C-6′), 116.4 (C-2′), 115.1 (C-4′), 111.3 (C-6), 102.5
(C-2, -4); CIMS m/z 247 [M + 1]+ (89), 203 (100); HREIMS
m/z 246.0527 (calcd for C13H10O5, 246.0528).
1
1274, 1225, 1171, 1106 cm-1; H NMR (CD3OD, 360 MHz) δ
7.01 (1H, dd, J ) 7.9, 7.9 Hz, H-5′), 6.60 (1H, dd, J ) 2.4, 7.9
Hz, H-6′), 6.53 (1H, br d, J ) 7.9 Hz, H-4′), 6.50 (1H, br s,
H-2′), 6.17 (1H, d, J ) 1.9 Hz, H-6), 6.08 (1H, d, J ) 1.9 Hz,
H-4), 3.81 (2H, q, J ) 7.2 Hz, -OCH2CH3), 0.69 (3H, t, J )
7.2 Hz, -OCH2CH3); 13C NMR (CD3OD, 90 MHz) δ 172.0 (C-
7), 164.0 (C-5), 162.9 (C-3), 157.9 (C-3′), 148.0 (C-1), 145.7 (C-
1′), 129.7 (C-5′), 120.5 (C-6′), 116.2 (C-2′), 114.7 (C-4′), 111.6
(C-6), 106.7 (C-2), 102.6 (C-4), 61.5 (-OCH2CH3), 13.6
(-OCH2CH3); CIMS m/z 275 [M + 1]+ (100), 229 [M - OC2H5]+
(2); HREIMS m/z 274.0846 (calcd for C15H14O5, 274.0841).
3,3′,5-Tr iben zyloxybip h en yl-2-ca r b oxylic Acid E t h yl
Ester (4). Compound 3 (274 mg, 1.0 mmol) and anhydrous
potassium carbonate (828 mg, 6.0 mmol) in absolute ethanol
(20 mL) were refluxed for 10 min. Benzyl chloride (0.6 mL,
5.2 mmol) and a small amount of potassium iodide were added,
and the mixture was refluxed for 48 h. After cooling, ether
(20 mL) was added, and the precipitate was removed by
filtration and washed with ether. The combined filtrate was
evaporated, H2O (20 mL) was added to the residue, and the
solution was extracted with ether (3 × 30 mL). The ether layer
was dried, evaporated, and chromatographed by column over
Si gel eluting with hexane/EtOAc (15:1) to afford 4 (490 mg,
0.9 mmol, 90%): UV (CHCl3) λmax (log ꢀ) 240 (4.20), 282 (3.65)
nm; IR (KBr) νmax 3431, 2956, 2925, 2854, 1729, 1600, 1462,
3,3′,5-Tr ih yd r oxybip h en yl-2-ca r boxylic Acid Su ccin -
im id e Ester (7). Compound 6 (140 mg, 0.57 mmol) was
dissolved in anhydrous EtOAc (20 mL), and N-hydroxysuccin-
imide (66 mg, 0.57 mmol) and N,N′-dicyclohexylcarbodiimide
(124 mg, 0.60 mmol) were added. Then the whole mixture was
stirred at room temperature for 72 h. The dicyclohexylurea
was filtered off and washed with EtOAc (10 mL). The combined
filtrate and washing were evaporated in vacuo. The residue
was separated on CC eluting with CHCl3/acetone (5:1) to afford
pure succinimide ester 7 (80 mg, 41%) as white amorphous
powder: UV (MeOH) λmax (log ꢀ) 202 (4.25), 215 (sh) (4.18),
230 (sh) (4.06), 274 (3.72), 287 (3.46) nm; IR (KBr) νmax 3421,
2925, 1729, 1615, 1583, 1437, 1361, 1209, 1063 cm-1; 1H NMR
(CD3COCD3, 360 MHz) δ 7.12 (1H, dd, J ) 8.8, 8.0 Hz, H-5′),
6.84 (2H, H-2′and -4′), 6.75 (1H, dd, J ) 2.8, 8.0 Hz, H-6′),
6.44 (1H, d, J ) 2.0 Hz, H-6), 6.39 (1H, d, J ) 2.0 Hz, H-4),
2.81 (4H, s, H of succinimide moiety); 13C NMR (CD3OCD3, 90
MHz) δ 170.4 (2 × -CO- of succinimide moiety), 165.8 (C-7),
163.6 (C-5), 163.0 (C-3), 157.7 (C-3′), 147.7 (C-1), 142.8 (C-1′),
129.9 (C-5′), 120.7 (C-6′), 115.9 (C-2′), 115.3 (C-4′), 112.2 (C-
6), 103.0 (C-2), 102.7 (C-4), 26.0 (2 x -CH2- of succinimide
moiety); CIMS m/z 344 [M + 1]+ (62), 229 [M - succinimide]+
(100); HREIMS m/z 343.0680 (calcd for C17H13NO7, 343.0692).
3,3′,5-Tr ih yd r oxybip h en yl-2-ca r boxylic Acid CoA Es-
ter (8). CoA (10 mg, 0.013 mmol) and 7 (20 mg, 0.058 mmol)
were suspended in a microcentrifuge tube containing 0.5 mL
of 50 mM aqueous NaHCO3 and was chilled to 0 °C. Two drops
of acetone were added to dissolve 7 completely. The mixture
was maintained at 0 °C overnight and separated by PC on
Whatman no. 3 paper developed in n-butanol-acetic acid-
H2O (5:2:3). The biphenylcarbonyl-CoA thiol ester was de-
tected by fluorescence at 366 nm, eluted with H2O, and
lyophilized to give 8 (5.4 mg, 42%). After ascending chroma-
tography on Whatman no. 3 paper, the biphenylcarbonyl-CoA
thiol ester (Rf 0.44) turned a characteristic pink on treatment
with nitroprusside spray reagent under alkaline conditions.
Free phenolic groups were detected by spraying with diazotized
sulfanilic acid in 10% KOH and turned yellow: UV (0.1 M
phosphate buffer, pH 7.0) λmax (log ꢀ) 254 (3.80), 330 (2.91) nm;
IR (KBr) νmax 3416, 1754, 1649, 1573, 1419, 1385, 1261, 1100,
1
1378, 1263, 1167, 1043, 740, 698 cm-1; H NMR (CDCl3, 360
MHz) δ 7.70 (1H, dd, J ) 8.0, 8.0 Hz, H-5′), 7.53 (1H, d, J )
2.0 Hz, H-2′), 7.01 (1H, dd, J ) 8.0, 2.0 Hz, H-4′), 6.96 (1H,
dd, J ) 8.0, 2.0 Hz, H-6′), 6.59 (1H, d, J ) 2.0 Hz, H-6), 6.57
(1H, d, J ) 2.0 Hz, H-4), 5.11, 5.07, 5.05 (each 3H, s, 3 ×
-CH2- of benzyl groups), 4.07 (2H, q, J ) 7.1 Hz, -OCH2-
CH3), 0.99 (3H, t, J ) 7.1 Hz, -OCH2CH3); 13C NMR (CDCl3,
90 MHz) δ 167.7 (C-7), 160.2 (C-5), 158.6 (C-3), 157,1 (C-3′),
144.8 (C-1), 142.5 (C-1′), 130.9 (C-5′), 129.3-127.0 (aromatic
carbons of benzyl groups), 121.0 (C-6′), 114.6 (C-2′), 114.5 (C-
4′), 107.5 (C-6), 100.0 (C-2, 4), 70.5, 70.3, 70.0 (3 × -CH2- of
benzyl groups), 61.0 (-OCH2CH3), 14.0 (-OCH2CH3); CIMS
m/z 545 [M + 1]+ (100), 455 (19), 365 (16), 275 (7). HREIMS
m/z 544.2276 (calcd for C36H32O5, 544.2250).
3,3′,5-Tr iben zyloxybip h en yl-2-ca r boxylic Acid (5). A
solution of 4 (490 mg, 0.9 mmol) in 10% NaOH/EtOH (10 mL)
was refluxed for 3 h and monitored by TLC to determine the
reaction progress. The mixture was adjusted to pH 3-4 with
concentrated HCl with stirring in an ice-water bath. The
white salt was washed with ether, the ether layer was
evaporated, and the residue was separated by CC eluting with
hexane/EtOAc (5:1) to afford pure 5 (404 mg, 0.78 mmol,
87%): UV (MeOH) λmax (log ꢀ) 210 (4.87), 258 (sh) (4.01), 286
1
1057, 1021, 736, 668 cm-1; H NMR (D2O, 360 MHz) δ 8.32
(1H, s, H-5 of CoA), 8.13 (1H, s, H-2 of CoA), 7.10 (1H, dd, J
) 8.0, 8.0 Hz, H-5′), 6.72 (1H, br s, H-2′), 6.70 (1H, d, J ) 8.0

374 J ournal of Natural Products, 2000, Vol. 63, No. 3
Wang et al.
Hz, H-4′), 6.63 (1H, br d, J ) 8.0 Hz, H-6′), 6.27 (1H, br s,
H-6), 6.20 (1H, br s, H-4), 6.00 (1H, br s, H-6 of CoA), 4.47
(1H, d, H-9 of CoA), 4.14 (2H, t, H-10 of CoA), 4.00 (1H, m,
H-7 of CoA), 3.90 (1H, m, H-15 of CoA), 3.51 (1H, m, H-8 of
CoA), 3.40-3.05 (m, H-17, -18, -11 of CoA), 2.93-2.20 (m, H-20,
-21 of CoA), 1.21, 1.19 (each 3H, s, H-13, -14 of CoA).
(C-6), 102.8 (C-2, -4), 39.4 (-N-CH2), 30.4 (-S-CH2), 22.8
(-CO-CH3); CIMS m/z 348 [M + 1]+ (15), 257 (15), 229 (65),
203 (100), 158 (66); HREIMS m/z 347.0826 (calcd for C17H17
-
NO5S, 347.0828).
Ack n ow led gm en t. The authors are very grateful to Deut-
scher Akademischer Austauschdienst (DAAD) for a stipend to
C. Z. Wang. Our thanks are due to Dr. J on Page for his
linguistic help in the preparation of this manuscript and Mr.
Peter Spiteller for high-resolution MS measurements. Re-
search support was provided by the Deutsche Forschungsge-
meinschaft through SFB 369 and Fonds der Chemischen
Industrie.
3,3′,5-Tr ih yd r oxybip h en yl-2-ca r boxylic Acid N-Acetyl
Cystea m in e Ester (9). Method 1: 7 (22 mg, 0.06 mmol) and
N-acetyl cysteamine14 (HS-NAC, 25 µL) were suspended in
50 mM of aqueous NaHCO3 (0.7 mL). Acetone was added until
the mixture formed a single phase. The solution was kept at
0 °C for 24 h and crude 9 was isolated by preparative TLC
eluting with CHCl3/MeOH (5:1) and further purified by HPLC
(UV: 254 nm; column: RP18, 5 µm, 4.6 × 250 mm; flow rate:
3 mL/min) eluted with MeOH/H2O (6:4) to afford 9 (4.0 mg,
17%). Method 2: To 3,3′,5-trihydroxybiphenyl-2-carboxylic acid
(6) (0.1 mmol) in dry CH2Cl2 (2 mL), DMAP (2.0 mg, 0.016
mmol) and HS-NAC (20 mg, 0.17 mmol) were added. At 0
°C, DCC (0.11 mmol, 23 mg) was added and the mixture kept
in an ice-water bath for 5 min followed by 3 h in 20 °C. The
precipitate was removed by filtration, and water (3 mL) was
added to the filtrate. The CH2Cl2 layer was separated and the
water phase extracted with EtOAc. The combined organic
layers were dried and concentrated, and the residue was
separated by preparative TLC. Further purification by HPLC
gave 9 in 8% yield: UV (0.1 M phosphate buffer, pH 7.0) λmax
(log ꢀ) 252 (3.94), 286 (3.79) nm; IR (KBr) νmax 3405, 2923, 1618,
1567, 1444, 1356, 1281, 1212, 1170, 1114, 909 cm-1; 1H NMR
(CD3COCD3, 360 MHz) δ 7.21 (1H, dd, J ) 7.8, 7.8 Hz, H-5′),
7.07 (1H, br s, -NH), 6.79-6.86 (3H, H-2′, -4′, -6′), 6.41 (1H,
d, J ) 2.5 Hz, H-6), 6.34 (1H, d, J ) 2.5 Hz, H-4), 3.22 (2H, s,
Refer en ces a n d Notes
(1) Wagner, H. Pharmazeutische Biologie; G. Fischer: Stuttgart, 1988;
Vol. 2, p 115.
(2) Kuwajima, H.; Hayashi, N.; Takaishi, K.; Inoue, K.; Takeda, Y.;
Inouye, H. Yakugaku Zasshi 1990, 110, 484-489.
(3) Birch, A. J .; Donovan, F. M. Austr. J . Chem. 1953, 6, 360-366.
(4) Harris, T. M.; Carney, R. L. J . Am. Chem. Soc. 1967, 89, 6734-6740.
(5) Cotterill, P. J .; Owen, P. J .; Scheinmann, F. J . Chem. Soc., Perkin
Trans. 1 1974, 21, 2423-2429.
(6) Kato, T.; Hozumi, T. Chem. Pharm. Bull. 1972, 20, 1574-1578.
(7) Nakano, J .; Katagiri, N.; Kato, T. Chem. Pharm. Bull. 1982, 30, 2590-
2594.
(8) Gassman, P. G.; Schenk, W. N. J . Org. Chem. 1977, 42, 918-920.
(9) Sto¨ckigt, J .; Zenk,M. H. Z. Naturforsch. 1975, 30c, 352-358.
(10) Stadtman, E. R. Methods in Enzymology; Academic Press: New York,
1957; Vol. 3, p 931.
(11) Neises, B.; W. Steglich, W. Angew. Chem., Int. Ed. Engl. 1978, 17,
522.
(12) Chen, K.; Kuo, S. C.; Hsieh, M. C.; Mauger, A.; Lin, C. M. J . Med.
Chem. 1997, 40, 2266-2275.
(13) Clark, M. J . Chem. Soc. C 1971, 1945.
(14) Schwab, J . M.; Klassen, J . B. J . Am. Chem. Soc. 1984, 106,
7217-7227.
-N-CH2), 2.93 (2H, m, -S-CH2), 1.83 (3H, s, -COCH3); 13
C
NMR (CD3COCD3, 90 MHz) δ 196.2 (CO-S), 170.4 (-CO-
CH3), 161.6 (C-5), 159.5 (C-3), 158.1 (C-3′), 145.2 (C-1), 142.8
(C-1′), 130.1 (C-5′), 121.4 (C-6′), 117.3 (C-4′), 115.8 (C-2′), 110.8
NP990256M