Total synthesis of CRM646-A and -B, two fungal glucuronides with potent heparinase inhibition activities

Article abstract of DOI:10.1021/jo051384k

CRM646-A (1) and -B (2), two fungal glucuronides with a dimeric 2,4-dihydroxy-6-alkylbenzoic acid (orcinol p-depside) aglycone showing significant heparinase and telomerase inhibition activities, were synthesized for the first time. The successful approac

Full text of DOI:10.1021/jo051384k

Total Synthesis of CRM646-A and -B, Two Fungal Glucuronides
with Potent Heparinase Inhibition Activities
Ping Wang, Zhaojun Zhang, and Biao Yu*
State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic
Chemistry, Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China
byu@mail.sioc.ac.cn
Received July 5, 2005
CRM646-A (1) and -B (2), two fungal glucuronides with a dimeric 2,4-dihydroxy-6-alkylbenzoic
acid (orcinol p-depside) aglycone showing significant heparinase and telomerase inhibition activities,
were synthesized for the first time. The successful approach involved construction of the phenol
glucuronidic linkage, via coupling of the orsellinate derivative 27 with glucuronate bromide 7, before
assembly of the phenolic ester linkage in the depside aglycone. Attempts via direct glycosylation of
the depside aglycone derivatives were not successful.
Introduction
dose of 3.2 µM.5 No cytotoxicity up to 100 µM was found
for compounds 1 and 2.1 Therefore, these two fungal
metabolites might be interesting candidates for antican-
cer therapeutics.
CRM646-A (1) and -B (2) were isolated from Acremo-
nium sp. MT70646 in the course of screening for hepa-
rinase and heparanase inhibitors.1,2 Inhibitory concen-
trations causing 50% inhibition (IC50) of the hydrolysis
of porcine heparin by the heparinase (Sigma) for 1 and
2 occurred at 3 and 10 µM, respectively;1 suramin,3
known as a potent inhibitor of melanoma heparanase,
showed an IC50 value of 5 µM in this assay system. The
correlation between heparanase inhibition and the inhi-
bition of tumor metastasis4 for compounds 1 and 2 were
then examined. Both compounds strongly inhibited the
migration of B16-F10 melanoma cells, with IC50 values
being 15 and 30 µM, respectively. In addition, CRM646-A
(1) showed inhibitory activity against telomerase at a
CRM646-A (1) and its methyl ester CRM646-B (2) are
novel phenol glucuronides. The aglycone of the dimeric
2,4-dihydroxy-6-alkylbenzoic acid belongs to the orcinol
p-depsides family, which are especially common and
diverse in lichen genera.6 Nevertheless, only a few of the
orcinol p-depsides have so far been found in conjugation
with sugars in the natural sources;7 compounds 1 and 2
represent the only depsides bearing a glucuronate resi-
due. Synthetic approaches toward orcinol p-depsides have
been extensively studied.6,8 However, synthesis of their
glycosides has only been reported once. Thus, Dushin and
Danishefsky accomplished the synthesis of the galacto-
furanosides KS-501 and -502 employing glycosylation of
a salicylate derivative with a sugar 1,2-epoxide as a key
* To whom correspondence should be addressed. Fax: (0086)-21-
64166128.
(1) Ko, H. R.; Kim, B. Y.; Oh, W. K.; Kang, D. O.; Lee, H. S.; Koshino,
H.; Osada, H.; Mheen, T. I.; Ahn, J. S. J. Antibiot. 2000, 53, 211-214.
(2) Ahn, J. S.; Kim, B. Y.; Oh, W. K.; Mheen, T. I.; Ahn, S. C.; Kang,
D. O.; Ko, H. R.; Kim, H. M. PCT Int. Appl. WO 2001046385, 2001;
Chem. Abstr. 2001, 135:75833.
(3) Nakajima, M.; Dechavingy, A.; Johnson, C. E.; Hamada, J.; Stein,
C. A.; Nicolson, G. L. J. Biol. Chem. 1991, 266, 9661-9666.
(4) For selected reviews, see: (a) Vlodavsky, I.; Goldshmidt, O.;
Zcharia, E.; Atzmon, R.; Rangini-Guatta, Z.; Elkin, M.; Peretz, T.;
Friedmann, Y. Sem. Cancer Biol. 2002, 12, 121-129. (b) Parish, C.
R.; Freeman, C.; Hulett, M. D. Biochim. Biophys. Acta 2001, 1471,
M99-M108. (c) Bame, K. J. Glycobiology 2001, 11, 91R-98R. (d)
Dempsey, L. A.; Brunn, G. J.; Platt, J. L. Trends Biochem. Sci. 2000,
25, 349-351. (e) Ferro, V.; Hammond, E.; Fairweather, J. K. Mini-
Rev. Med. Chem. 2004, 4, 693-702.
(5) Togashi, K.-I.; Ko, H.-R.; Ahn, J.-S.; Osada, H. Biosci. Biotechnol.
Biochem. 2001, 65, 651-653.
(6) For selected examples, see: (a) Elix, J. A.; Wardlaw, J. H. Aust.
J. Chem. 1987, 40, 425-429. (b) Elix, J. A.; Wardlaw, J. H. Aust. J.
Chem. 1997, 50, 1145-1150. (c) Elix, J. A.; Wardlaw, J. H. Aust. J.
Chem. 1997, 50, 479-486. (d) Elix, J. A.; Wardlaw, J. H. Aust. J. Chem.
1996, 49, 917-924. (e) Elix, J. A.; Barclay, C. E.; Lumbsch, H. T. Aust.
J. Chem. 1994, 47, 1199-1203.
(7) (a) Ondeyka, J. G.; Zink, D. L.; Dombrowski, A. W.; Polishook,
J. D.; Felock, P. J.; Hazuda, D. J.; Singh, S. B. J. Antibiot. 2003, 56,
1018-1023. (b) Yasuzawa, T.; Saitoh, Y.; Sano, H. J. Antibiot. 1990,
43, 336-343.
(8) For a recent example, see: Garc´ıa-Fortanet, J.; Debergh, J. R.;
De Brabander, J. K. Org. Lett. 2005, 7, 685-688.
10.1021/jo051384k CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/05/2005
8884
J. Org. Chem. 2005, 70, 8884-8889

Total Synthesis of CRM646-A and B
step.9 Here we report the total synthesis of CRM646-A
(1) and -B (2).
Results and Discussion
Glycosidic coupling involving either a glycosyl donor
of the glucuronic acid type10 or a phenolic acceptor11 has
long been recognized as a difficult task. Thus, a major
challenge in the synthesis of CRM646-A (1) and -B (2)
would be construction of the phenol glucuronidic linkage.
We planned to explore this glycosidic coupling with a
variety of the glycosyl donors. Methyl (2,3,4-tri-O-acetyl-
D-glucopyranosyl trichloroacetimidate)uronate 3 (Figure
1) has been found effective in coupling with a few of the
phenols under the promotion of BF3‚OEt2 (or TMSOTf).12
Its benzyl uronate counterpart 513 should behave simi-
larly but facilitate the final release of the carboxylic acid
function by hydrogenolysis under neutral conditions.
Glycosyl trifluoroacetimidates are valuable alternatives
to the corresponding trichloroacetimidates,14 which have
shown advantages in sialylation15a and glycosylation of
amides.15b We therefore also scheduled to examine glu-
curonidation with trifluoroacetimidates 4 and 6.13 In
terms of glycosylation of phenols, glycosyl bromides
(under either Koenigs-Knorr or phase transfer condi-
tions) have been proven to be the most reliable donors,
although the coupling yields might be only moderate.16
Thus, methyl 2,3,4-tri-O-acetyl-1-bromo-R-D-glucuronate
(7)17 was selected as an alternative to the imidates (i.e.,
3-6). In the last resort, we should be able to realize the
glycosidic coupling with a glucopyranosyl donor (e.g., 2,3-
di-O-acetyl-4,6-O-benzylidene-D-glucopyranosyl trichlo-
roacetimidate, 8)18 and then to elaborate the 6′′-carboxylic
FIGURE 1. Glycosyl donors 3-8.
acid function via selective oxidation of the 6′′-OH group.19
The desired orsellinate derivatives were synthesized
starting from 3,5-dihydroxytoluene, adopting modifica-
tion of the literature transformations (Scheme 1). Thus,
formylation of 3,5-dihydroxytoluene with DMF/POCl3
gave 2,4-dihydroxy-6-methyl benzaldehyde 9 (80%).20
Treatment of aldehyde 9 with sodium chlorite (NaClO2)
in a NaH2PO4 buffered solution of DMSO and H2O
provided benzoic acid 10 in a good 77% yield,21 which was
selectively benzylated to provide benzyl ester 11 with
BnBr under the action of KHCO3 in DMF (78%).6a
Alternatively, the 2,4-dihydroxyl groups on aldehyde 9
were protected with methyl groups, providing 12 (100%),
which was then oxidized into acid 13 (84%) under similar
conditions used for 9 f 10. Blocking the acid function
on 13 with a methyl or an ethyl group provided 14a or
14b, which was subjected to LDA (1.5 equiv) followed by
alkylation with 1-bromotetradecane (1.4 equiv), respec-
tively. Unexpectedly, the desired alkylation product 15a
(from methyl ester 14a) was isolated in a low 28% yield,
whereas 15b (from the ethyl ester counterpart 14b) was
obtained in a satisfactory 70% yield. In comparison, it
was reported that treatment of 14a with LDA and
1-bromotetradecane in the presence of HMPA provided
15a in only 5% yield.22 Removal of the O-methyl groups
on 15b was achieved with 3.0 equiv of boron tribromide
at low temperature (-78 f -10 °C), providing diol 16 in
82% yield; the 4-methoxy derivative 17 was also isolated
in 17% yield. The possible intermolecular Friedel-Crafts
products were not detected.
(9) Dushin, R. G.; Danishefsky, S. J. J. Am. Chem. Soc. 1992, 114,
655-659.
(10) Stachulski, A. V.; Jenkins, G. N. Nat. Prod. Rep. 1998, 173-
186.
(11) For examples, see: (a) Jensen, K. J. J. Chem. Soc., Perkin
Trans. 1 2002, 2219-2233. (b) Du, Y.; Wei, G.; Linhardt, R. J. J. Org.
Chem. 2004, 69, 2206-2209.
(12) (a) Fischer, B.; Nudelman, A.; Ruse, M.; Herzig, J.; Gottlieb,
H. E.; Keinan, E. J. Org. Chem. 1984, 49, 4988-4993. (b) Hasuoka,
A.; Nakayama, Y.; Adachi, M.; Kamiguchi, H.; Kamiyama, K. Chem.
Pharm. Bull. 2001, 49, 1604-1608. (c) Nakamura, S.; Kondo, M.; Goto,
K.; Nakamura, M.; Tsuda, Y.; Shishido, K. Heterocycles 1996, 43, 2747-
2756.
(13) For the preparation, see: Supporting Information.
(14) For recent applications, see: (a) Zhang, Z.; Yu, B. J. Org. Chem.
2003, 68, 6309-6313. (b) Adinolfi, M.; Iadonisi, A.; Ravida`, A.;
Schiattarella, M. J. Org. Chem. 2005, 70, 5316-5319.
(15) (a) Cai, S.; Yu, B. Org. Lett. 2003, 5, 3827-3830. (b) Tanaka,
H.; Iwata, Y.; Takahashi, D.; Adachi, M.; Takahashi, T. J. Am. Chem.
Soc. 2005, 127, 1630-1631.
The depside aglycone 21 and the required benzyl ester
22 were prepared as shown in Scheme 2. To hydrolyze
the ethyl ester in 16, we protected the 2,4-hydroxyl
groups with benzyl ether first, providing 18, to avoid the
decarboxylation side reaction.9,23 Then, treatment of 18
with KOH in a mixture solvent of DMSO and H2O at 90
°C afforded acid 19 in nearly quantitative yield (for two
steps). Coupling of acid 19 with phenol 11 under the
action of trifluoroacetic anhydride provided the desired
(16) For selected examples, see: (a) Needs, P. W.; Williamson, G.
Carbohydr. Res. 2001, 330, 511-516. (b) Florent, J. C.; Dong, X.;
Gaudel, G.; Mitaku, S.; Monneret, C. J. Med. Chem. 1998, 41, 3572-
3581. (c) Bellamy, F.; Horton, D.; Millet, J.; Picart, F.; Samreth, S.;
Chazan, J. B. J. Med. Chem. 1993, 36, 898-903. (d) Dawson, M. I.;
Hobbs, P. D. Carbohydr. Res. 1980, 85, 121-129.
(19) Lin, F.; Peng, W.; Xu, W.; Han, X.; Yu, B. Carbohydr. Res. 2004,
339, 1219-1223 and references therein.
(20) Nielsen, S. F.; Christensen, S. B.; Cruciani, G.; Kharazmi, A.;
Liljefors, T. J. Med. Chem. 1998, 41, 4819-4832.
(21) Nicolaou, K. C.; Rodr´ıguez, R. M.; Mitchell, H. J.; van Delft, F.
L. Angew. Chem., Int. Ed. 1998, 37, 1874-1876.
(17) Bollenback, G. N.; Long, J. W.; Benjamin, D. G.; Lindquist, J.
A. J. Am. Chem. Soc. 1955, 77, 3310-3315.
(22) Bruce, I.; Spencer, R.; Tyman, J. J. Chem. Res., Synop. 1992,
224-225.
(23) Kato, T.; Hozumi, T. Chem. Pharm. Bull. 1972, 20, 1574-1578.
(18) Zimmermann, P.; Greilich, U.; Schmidt, R. R. Tetrahedron Lett.
1990, 31, 1849-1852.
J. Org. Chem, Vol. 70, No. 22, 2005 8885

Wang et al.
SCHEME 1. Preparation of the Orsellinate Derivatives 11 and 16
SCHEME 2. Preparation of Depside 22
SCHEME 3. Attempt To Synthesize the
Glucuronide via Oxidation
phenol ester 20 in excellent yield (94%).6a Removal of the
three benzyl groups on 20 by hydrogenolysis over Pd/C
afforded the depside aglycone (21) of CRM646-A and -B
quantitatively. Depside 21 was readily transformed into
its benzyl ester 22 with BnBr in the presence of KHCO3
in DMF (89%).
The 2,2′-phenolic hydroxyl groups in 22 are hydrogen-
bonded with the o-carbonyl oxygen, as indicated by the
downfielded NMR signals of the hydroxyl protons at
11.66 and 11.35 ppm, respectively. Therefore glycosyla-
tion with 22 was expected to take place selectively on
the 4-OH. Unfortunately, glycosidic coupling between
phenol 22 and the trichloroacetimidate/trifluoroacetimi-
date uronate donors 3-6 under a variety of conditions
in the presence of BF3‚OEt2 or TMSOTf failed to provide
the desired glycosides. Complex products were mostly
encountered. Treatment of 22 with bromide 7 under
either Koenigs-Knorr conditions (Ag2O, CH3CN)16 or
under phase transfer conditions (TBAB, NaOH, CHCl3,
H2O)24 led to the cleavage of the phenolic ester bond in
22. As the last resort, glycosylation of 22 (1.2 equiv) with
the glucopyranosyl trichloroacetimidate 8 under the
promotion of BF3‚OEt2 (0.2 equiv) proceeded smoothly,
providing the expected 4-O-â-glycoside 23 in a satisfac-
tory 55% yield. (Scheme 3) To avoid decomposition of the
phenolic ester bond during subsequent transformations,
the 2,2′-hydroxyl groups on 23 were protected with benzyl
groups (BnBr, K2CO3, acetone, reflux) to provide 24
(80%). Treatment of 24 with a catalytic amount of TsOH‚
H2O (0.1 equiv) in 90% HOAc at 40 °C removed the 4′′,6′′-
O-benzylidene group,25 affording diol 25 in a moderate
(25) Tsujihara, K.; Hongu, M.; Saito, K.; Kawanishi, H.; Kuriyama,
K.; Matsumoto, M.; Oku, A.; Ueta, K.; Tsuda, M.; Saito, A. J. Med.
Chem. 1999, 42, 5311-5324.
(24) Feng, I. Q.; Sun, J.; Qu, L. Q. Synth. Commun. 2002, 32, 3393-
3398.
8886 J. Org. Chem., Vol. 70, No. 22, 2005

Total Synthesis of CRM646-A and B
SCHEME 4. Preparation of the Orsellinate
Derivatives 27 and 30
SCHEME 5. Completion of the Synthesis of
CRM646-A (1)
yield (51%). Unfortunately, subjection of 25 to the well-
studied TEMPO oxidation protocol19 failed to provide the
desired glucuronate product. Instead, cleavage of the
phenol ester was detected.
Since the above synthetic attempts toward CRM646-A
and -B via direct glycosylation of the depside aglycone
derivative (i.e., 22) were found futile, we turned our
attention to elaborate the phenol ester at a late stage
after construction of the phenol glucuronidic linkage.
Thus, benzyl 2,4-dihydroxy-6-pentadecanylbenzoate (27)
was prepared from acid 19 by removal of the benzyl ether
(H2, Pd/C, 99%) and subsequent formation of the benzyl
ester (BnBr, KHCO3, DMF, rt, 88%). Benzyl 2-(benzy-
loxy)-4-hydroxy-6-methylbenzoate (30) was prepared from
benzoate ester 11 by selective protection of the p-OH with
a methoxymethyl ether (1.1 equiv MOMCl, i-Pr2NEt,
CH2Cl2, rt, 92%) followed by blocking the remaining o-OH
with a benzyl ether (BnBr, K2CO3, acetone, reflux, 89%)
and subsequent removal of the methoxymethyl protection
(6 N HCl, THF, rt, 85%). Coupling of phenol 27 with
glucuronate bromide 7 was not successful under PTC
conditions (TBAB, NaOH, CHCl3/H2O). Fortunately, the
glycosidic coupling of 27 with 7 (2.0 equiv) took place
under the action of Ag2O in CH3CN at 35 °C,16 providing
the desired 4-O-â-glucuronide 31 as the major product,
which however could not be separated from the starting
bromide 7 (Scheme 5). Subsequent hydrogenolysis of the
crude benzyl ester 31 over Pd/C afforded acid 32, which
could be readily purified (59% for two steps). Coupling
of acid 32 with phenol 30 (1.5 equiv) in the presence of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (EDCI) and DMAP provided phenol ester 33 in
a good 64% yield.9 The 2-OH in 33 was blocked with a
benzyl ether, providing 34 (BnBr, K2CO3, acetone, reflux,
79%), to avoid a possible cleavage of the phenolic ester
bond in the subsequent hydrolytic removal of the acetyl
groups on the glucuronate moiety. At this point we
encountered difficulties in removing the acetyl and ester
groups. Initial attempts employing K2CO3/MeOH or
KCN/MeOH resulted in cleavage of the phenolic ester
bond. DBU/MeOH or HCl/MeOH conditions led to a
complex mixture of the products. Fortunately, treatment
of 34 with KOH in a solvent mixture of THF and H2O
(v/v 4:1) at room temperature afforded glucuronide 35
quantitatively. Removal of the benzyl groups on 35
proceeded smoothly under 1 atm H2 over Pd/C in metha-
nol, furnishing the target CRM646-A (1) in 93% yield.
Completion of the synthesis of CRM646-B (2) started
from the crude glucuronide 31 (Scheme 6). Thus, blocking
the 2-OH with a benzyl group provided homogeneous 36
SCHEME 6. Completion of the Synthesis of
CRM646-B (2)
(59% for two steps). Removal of the acetyl groups was
achieved with K2CO3 in MeOH at room temperature,
giving 37 in 78% yield. Hydrogenolysis of 37 afforded 38
quantitatively. Acid 38 was coupled with phenol 30 (5.0
equiv) under the action of EDCI and DMAP in CH2Cl2,
providing the desired phenol ester 39 in a satisfactory
65% yield. Finally, removal of the two benzyl groups on
39 by hydrogenolysis over Pd/C in EtOAc furnished
CRM646-B (2) (96%). Analytic data for the synthetic
CRM646-A (1) and -B (2) were in great accordance with
those reported for the natural products.1
Conclusion
CRM646-A (1) and -B (2), two novel glucuronides with
a dimeric 2,4-dihydroxy-6-alkylbenzoic acid (orcinol p-
depside) aglycone, are potential anticancer agents with
significant heparinase and telomerase inhibition activi-
ties. Total synthesis of these two fungal metabolites were
J. Org. Chem, Vol. 70, No. 22, 2005 8887

Wang et al.
0.89 (t, 3 H, J ) 6.8 Hz). 13C NMR (75 MHz, CDCl3): δ 170.0,
169.6, 169.2, 169.1, 167.3, 166.6, 165.8, 161.1, 156.8, 151.0,
148.9, 138.4, 136.0, 135.6, 128.54, 128.47, 128.39, 128.2, 128.0,
127.2, 122.4, 115.6, 112.4, 106.1, 104.3, 101.8, 97.7, 72.8, 71.7,
70.9, 70.8, 69.0, 67.1, 52.9, 37.1, 32.3, 31.9, 29.9, 29.6, 29.3,
22.6, 20.5, 20.4, 19.4, 14.0. ESIMS (m/z): 1033.9 (M + Na+),
1049.8 (M + K+). HR-ESIMS (m/z) calcd for C57H71O16H
1011.4744, found 1011.4737.
achieved for the first time. The successful approach
involved construction of the phenol glucuronidic linkage,
via coupling of the orsellinate derivative 27 with glucu-
ronate bromide 7, before assembly of the phenolic ester
linkage in the depside aglycone. It is remarkable that
the phenolic ester linkage in the advanced precursor 34
remained intact in the alkaline conditions for removal
of the acetyl and methyl ester groups. In contrast, the
previous synthetic attempts via direct glycosylation of the
depside derivatives were found futile, mostly because of
the decomposition of the phenolic ester linkage. Thus,
CRM646-A (1) and -B (2) were synthesized, without
optimization of the transformations, in 16 linear steps
and 9.1% and 9.5% overall yields, respectively, starting
from 3,5-dihydroxytoluene.
Benzyl 4′-(4-O-(Methyl 2′′,3′′,4′′-Tri-O-acetyl-â-D-glu-
copyranosyluronate)-2-benzyloxy-6-pentadecanylben-
zoyloxy)-2′-benzyloxy-6′-methylbenzoate (34). To a solu-
tion of 33 (64 mg, 0.063 mmol) in dry acetone (5 mL) was added
K2CO3 (17 mg, 2.0 equiv) and BnBr (0.1 mL). The mixture was
heated to reflux for 12 h. The solution was diluted with EtOAc
(50 mL). The organic phase was washed with water and brine,
respectively, and was then dried over Na2SO4 and concen-
trated. Chromatography over silica gel (petroleum ether/EtOAc
) 4:1) gave 34 (54 mg, 78%) as a white foam. Rf 0.25
(petroleum ether/EtOAc ) 4:1); [R]19D ) -7.5 (c 0.65, CHCl3).
1H NMR (300 MHz, CDCl3): δ 7.45-7.27 (m, 15 H), 6.57 (d, 1
H, J ) 2.1 Hz), 6.51-6.48 (m, 3 H), 5.40-5.25 (m, 5 H), 5.18
(d, 1 H, J ) 6.9 Hz), 5.07 (s, 2 H), 4.82 (s, 2 H), 4.21 (d, 1 H,
J ) 9.3 Hz), 3.76 (s, 3 H), 2.66 (t, 2 H, J ) 7.5 Hz), 2.24 (s, 3
H), 2.08 (s, 6 H), 2.07 (s, 3 H), 1.65-1.60 (m, 2 H), 1.38-1.20
(m, 24 H), 0.89 (t, 3 H, J ) 6.8 Hz). 13C NMR (75 MHz,
CDCl3): δ 170.1, 169.3, 169.2, 167.5, 166.7, 166.1, 158.5, 157.3,
156.6, 152.1, 143.7, 138.0, 136.1, 136.0, 135.6, 128.6, 128.4,
128.3, 128.1, 127.9, 127.7, 127.3, 121.7, 118.1, 115.7, 109.3,
104.2, 99.9, 98.7, 72.6, 71.7, 71.0, 70.8, 70.4, 68.9, 67.0, 53.0,
33.8, 31.9, 31.3, 29.6, 29.3, 22.6, 20.6, 20.5, 19.4, 14.1. ESIMS
(m/z): 1023.5 (M + Na+), 1039.5 (M + K+). HR-ESIMS (m/z)
calcd for C64H76O16Na 1123.5031, found 1123.5026.
Experimental Section
Benzyl 4-O-(Methyl 2′,3′,4′-tri-O-acetyl-â-D-glucopyra-
nosyluronate)-2-hydroxy-6-pentadecanyl-benzoate (31).
Glycosyl bromide 7 (169 mg, 0.426 mmol) and phenol 27 (100
mg, 0.220 mmol) were dissolved in dry CH3CN (10 mL), and
Ag2O (123 mg, 1.5 eq) was added under an Ar atmosphere at
38 °C. The mixture was stirred for 6 h and then concentrated
under reduced pressure. The residue was purified by silica gel
column chromatography (petroleum ether/EtOAc ) 6:1) to give
the crude 31 (contaminated with inseparable 7) as a white
foam. Rf 0.69 (petroleum ether/EtOAc ) 2:1). 1H NMR (300
MHz, CDCl3): δ 11.72 (s, 1 H), 7.44-7.38 (m, 5 H), 6.42 (d, 1
H, J ) 2.1 Hz), 6.32 (d, 1 H, J ) 2.7 Hz), 5.36-5.20 (m, 7 H),
4.21 (m, 1 H), 3.73 (s, 3 H), 2.77 (m, 2 H), 2.07 (s, 3 H), 2.06 (s,
3 H), 2.03 (s, 3 H), 1.40-1.04 (m, 26 H), 0.89 (t, 3 H, J ) 6.8
Hz). ESIMS (m/z): 793.5 (M + Na+), 809.6 (M + K+).
HR-ESIMS (m/z) calcd for C42H58O13Na 793.3775, found
793.3770.
Benzyl 4′-(4-O-(â-D-Glucopyranosyluronic acid)-2-ben-
zyloxy-6-pentadecanylbenzoyloxy)-2′-benzyloxy-6′-meth-
ylbenzoate (35). To a solution of 34 (60 mg, 0.054 mmol) in
THF/H2O (10 mL, v/v ) 4/1) was added KOH (30 mg) at room
temperature. After stirring for 4 h, the mixture was acidified
to pH ) 3 with 1 N HCl and was then diluted with EtOAc (50
mL). The organic phase was washed with water and brine,
respectively, and was then dried over Na2SO4 and concen-
trated. Chromatography over silica gel (CH2Cl2/MeOH ) 4:1)
4-O-(Methyl 2′,3′,4′-Tri-O-acetyl-â-D-glucopyranosylu-
ronate)-2-hydroxy-6-pentadecanyl-benzoic Acid (32). The
crude 31 was treated with 10% Pd/C (25 mg) in EtOAc (5.0
mL) under 1 atm H2 for 15 h. The mixture was then filtrated
and concentrated. The residue was purified by a short silica
gel column (petroleum ether/EtOAc ) 4:1 to 1:2) to give 32
provided 35 (52 mg, 100%) as a white powder. Rf 0.3 (CH2Cl2/
1
MeOH ) 4:1); [R]20 ) -30.2 (c 0.20, CH3OH). H NMR (300
D
(89 mg, 59% for two steps) as a white foam. [R]19 ) -18.1 (c
MHz, CD3COCD3-CD3OD ) 1:1): δ 7.52-7.30 (m, 15 H), 6.86
(d, 1 H, J ) 1.5 Hz), 6.71 (d, 1 H, J ) 1.5 Hz), 6.64 (s, 1 H),
6.54 (s, 1 H, J ) 1.8 Hz), 5.33 (s, 2 H), 5.18 (s, 2 H), 5.13 (d, 1
H, J ) 7.2 Hz), 4.93 (s, 2 H), 4.06 (d, 1 H, J ) 9.6 Hz), 3.70 (t,
1 H, J ) 9.0 Hz), 3.56 (m, 2 H), 2.69 (t, 2 H, J ) 7.8 Hz), 2.20
(s, 3 H), 1.69-1.62 (m, 2 H), 1.38-1.20 (m, 24 H), 0.89 (t, 3 H,
J ) 6.6 Hz). ESI-MS (m/z): 983.5 (M + Na+), 999.4 (M + K+).
HR-ESIMS calcd for C57H68O13Na 983.4558, found 983.4552.
D
1.00, CHCl3). 1H NMR (300 MHz, CDCl3): δ 6.39 (s, 1 H), 6.35
(s, 1 H), 5.36-5.21 (m, 4 H), 4.23 (d, 1 H, J ) 8.7 Hz), 3.72 (s,
3 H), 2.92-2.85 (m, 2 H), 2.05 (s, 9 H), 1.55-1.45 (m, 2 H),
1.35-1.20 (m, 24 H), 0.87 (t, 3 H, J ) 6.8 Hz). 13C NMR (75
MHz, CDCl3): δ 175.0, 170.0, 169.4, 169.3, 166.8, 165.8, 160.9,
150.0, 111.9, 101.4, 97.4, 72.5, 71.6, 70.7, 68.9, 53.1, 36.5, 31.9,
31.7, 29.8, 29.7, 29.6, 29.5, 29.3, 22.7, 20.6, 20.5, 14.1. ESIMS
(m/z): 679.3 (M - H-). HR-ESIMS (m/z) calcd for C35H51O13
679.3328, found 679.3335.
CRM646-A (1). Compound 35 (50 mg, 0.052 mmol) was
treated with 10% Pd/C (20 mg) in MeOH (10.0 mL) under 1
atm H2 for 1 day. The mixture was filtered. The filtrate was
concentrated and purified by a short column of silica gel (CH2-
Cl2/MeOH/H2O ) 4:1:0.1), affording CRM646-A (1, 33 mg, 93%)
as a white solid. [R]20D ) -41.5 (c 0.31, CH3OH). 1H NMR (300
MHz, DMSO-d6): δ 10.34 (br s, 1 H), 6.57 (s, 1 H), 6.49 (m, 3
H), 5.70-5.20 (br, 2H), 5.05 (d, 1 H, J ) 7.5 Hz), 3.91 (d, 1 H,
J ) 9.0 Hz), 3.46-3.30 (m, 3 H), 2.65-2.60 (m, 2 H), 2.45 (s,
3 H), 1.60-1.50 (m, 2 H), 1.38-1.20 (m, 24 H), 0.86 (m, 3 H).
13C NMR (75 MHz, CDCl3): δ 171.3, 170.2, 166.4, 160.8, 159.4,
157.5, 152.5, 143.4, 140.6, 116.5, 114.3, 113.8, 108.6, 107.4,
101.5, 100.0, 76.0, 75.8, 73.1, 71.6, 33.7, 31.5, 31.1, 29.2, 29.0,
28.9, 22.3, 21.8, 14.1.
Benzyl 4-O-(Methyl 2′,3′,4′-Tri-O-acetyl-â-D-glucopyra-
nosyluronate)-2-benzyloxy-6-pentadecanyl-benzoate (36).
To a solution of the crude 31 (prepared from 7 and 100 mg
27) in dry acetone (5 mL) was added K2CO3 (50 mg, 2.0 equiv)
and BnBr (0.1 mL). The mixture was heated to reflux for 12 h
and then was diluted with EtOAc (50 mL). The organic phase
was washed with water and brine, respectively, and then was
Benzyl 4′-(4-O-(Methyl 2′′,3′′,4′′-Tri-O-acetyl-â-D-glu-
copyranosyluronate)-2-hydroxy-6-pentdecanylbenzoy-
loxy)-2′-benzyloxy-6′-methylbenzoate (33). A solution of
acid 32 (55 mg, 0.08 mmol) and phenol 30 (42 mg, 0.122 mmol)
in dry CH2Cl2 (2.5 mL) was treated at room temperature with
DMAP (12 mg, 1.1 equiv) and EDCI (40 mg, 2.0 equiv). After
stirring for 4 h, the solution was diluted with saturated
aqueous NH4Cl and extracted twice with CH2Cl2 (30 mL). The
organic phase was washed with water and brine, respectively,
and was then dried (Na2SO4) and concentrated. The residue
was purified by silica gel column chromatography (petroleum
ether/EtOAc ) 6:1) to provide 33 (53 mg, 65%) as a colorless
oil. Rf 0.23 (CH2Cl2/MeOH ) 8:1); Rf 0.23 (petroleum ether/
1
EtOAc ) 4:1); [R]19 ) -10.8 (c 0.96, CHCl3). H NMR (300
D
MHz, CDCl3): δ 11.32 (s, 1 H), 7.34-7.30 (m, 10 H), 6.65 (s, 2
H), 6.47 (d, 1 H, J ) 2.7 Hz), 6.43 (d, 1 H, J ) 2.7 Hz), 5.38-
5.26 (m, 6 H), 5.07 (s, 2 H), 4.25 (d, 1 H, J ) 9.0 Hz), 3.75 (s,
3 H), 3.00-2.85 (m, 2 H), 2.33 (s, 3 H), 2.08 (s, 3 H), 2.07 (s, 3
H), 2.06 (s, 3 H), 1.65-1.60 (m, 2 H), 1.38-1.20 (m, 24 H),
8888 J. Org. Chem., Vol. 70, No. 22, 2005

Total Synthesis of CRM646-A and B
dried over Na2SO4 and concentrated. Chromatography over
silica gel (petroleum ether/EtOAc ) 4:1) gave 36 (112 mg, 59%
for two steps) as a white foam. Rf 0.25 (petroleum ether/EtOAc
was diluted with saturated aqueous NH4Cl and extracted twice
with EtOAc. The organic phase was washed with water and
brine, respectively, and was then dried over Na2SO4 and
concentrated. Chromatography over silica gel (CH2Cl2/MeOH
) 8:1) provided 39 (38 mg, 65%) as a colorless oil. Rf 0.23 (CH2-
Cl2/MeOH ) 8:1). [R]20D ) -38.7 (c 0.90, CHCl3). 1H NMR (300
MHz, CD3COCD3): δ 7.46-7.33 (m, 10 H), 7.03 (s, 1 H), 6.83
(s, 1 H), 6.59 (d, 1 H, J ) 3.3 Hz), 5.38 (s, 2 H), 5.28 (d, 1 H,
J ) 7.5 Hz), 5.20 (s, 2 H), 4.22 (d, 1 H, J ) 9.3 Hz), 3.75-3.70
(m, 4 H), 3.70-3.60 (m, 3 H), 2.93 (t, 2 H, J ) 7.8 Hz), 2.32 (s,
3 H), 1.63 (m, 2 H), 1.38-1.25 (m, 24 H), 0.88 (t, 3 H, J ) 6.8
Hz). 13C NMR (75 MHz, CD3COCD3): δ 170.1, 168.1, 164.3,
162.7, 157.8, 152.8, 148.5, 138.8, 137.7, 137.3, 129.6, 129.5,
129.2, 129.1, 128.6, 116.8, 112.3, 108.8, 105.9, 102.7, 101.0,
77.0, 76.6, 74.3, 72.6, 71.6, 67.8, 52.8, 37.2, 33.1, 32.9, 31.0,
30.8, 30.7, 30.6, 29.9, 29.7, 29.4, 23.6, 19.7, 14.7.
CRM646-B (2). Compound 39 (21 mg) was treated with 10%
Pd/C (10 mg) under 1 atm of H2 in EtOAc (5.0 mL) for 24 h.
The mixture was then filtered. The filtrate was concentrated
and purified by a short column of silica gel (CH2Cl2/MeOH )
4:1) to afford CRM646-B (2) as a white solid (16 mg, 96%). Rf
0.23 (CH2Cl2/MeOH ) 4:1); [R]20D ) -29.7 (c 0.31, CD3COCD3).
1H NMR (300 MHz, CD3COCD3): δ 6.58-6.55 (m, 3 H), 6.45
(d, 1 H, J ) 1.8 Hz), 5.28 (d, 1 H, J ) 7.5 Hz), 4.22 (d, 1 H, J
) 9.6 Hz), 3.76-3.73 (m, 4 H), 3.74-3.60 (m, 3 H), 2.93 (t, 2
H, J ) 7.8 Hz), 2.65 (s, 3 H), 1.65-1.60 (m, 2 H), 1.35-1.25
(m, 24 H), 0.88 (t, 3 H, J ) 6.8 Hz). 13C NMR (75 MHz, CD3-
COCD3): δ 170.2, 165.2, 164.4, 162.6, 153.2, 148.5, 115.5,
112.3, 109.1, 108.4, 102.8, 101.1, 77.1, 76.7, 74.3, 72.7, 52.9,
37.2, 33.1, 32.9, 31.0, 30.7, 30.6, 30.5, 30.2, 29.9, 29.7, 29.4,
24.2, 23.6, 14.7. ESIMS (m/z): 703.3 (M - H-). HR-ESIMS
(m/z) calcd for C37H52O13Na 727.3306, found 727.3300.
) 4:1); [R]20 ) 35.4 (c 0.65, CHCl3). 1H NMR (300 MHz,
D
CDCl3): δ 7.37-7.25 (m, 10 H), 6.44 (d, 1 H, J ) 1.8 Hz), 6.40
(d, 1 H, J ) 2.1 Hz), 5.33-5.23 (m, 5 H), 5.08 (s, 1 H), 5.05 (s,
2 H), 4.12 (d, 1 H, J ) 9.0 Hz), 3.71 (s, 3 H), 2.48 (t, 2 H, J )
7.5 Hz), 2.09-2.00 (s, 9 H), 1.63 (m, 2 H), 1.30-1.20 (m, 24
H), 0.89 (t, 3 H, J ) 6.8 Hz). 13C NMR (75 MHz, CDCl3): δ
170.0, 169.2, 169.1, 167.7, 166.7, 158.0, 156.8, 143.3, 136.4,
135.7, 128.5, 128.4, 128.1, 127.9, 127.1, 109.5, 100.3, 100.0,
72.6, 71.8, 71.0, 70.6, 69.0, 67.0, 52.7, 33.7, 31.9, 31.1, 29.6,
29.5, 29.4, 29.3, 22.6, 20.5, 20.4, 14.0. ESIMS (m/z): 883.4 (M
+ Na+), 899.4 (M + K+). HR-ESIMS (m/z) calcd for C49H64O13-
Na 883.4245, found 883.4239.
Benzyl 4-O-(Methyl â-D-Glucopyranosyluronate)-2-
benzyloxy-6-pentadecanyl-benzoate (37). To a solution of
36 (100 mg, 0.116 mmol) in MeOH (5.0 mL) was added K2CO3
(10 mg) at room temperature. After stirring for 20 min, the
solution was diluted with saturated aqueous NH4Cl and
extracted twice with EtOAc. The organic phase was washed
with water and brine, respectively, and was then dried over
Na2SO4 and concentrated. Chromatography over silica gel
(CH2Cl2/MeOH ) 20:1) gave 37 (67 mg, 78%) as a colorless
oil. Rf 0.23 (CH2Cl2/MeOH ) 20:1); [R]20 ) -39.4 (c 1.20,
D
CHCl3). 1H NMR (300 MHz, CD3COCD3): δ 7.45-7.37 (m, 10
H), 6.74 (d, 1 H, J ) 1.8 Hz), 6.62 (d, 1 H, J ) 2.1 Hz), 5.32 (s,
2 H), 5.17 (d, 1 H, J ) 7.2 Hz), 5.14 (s, 2 H), 4.14 (d, 1 H, J )
9.6 Hz), 3.73 (m, 4 H), 3.59-3.54 (m, 2 H), 2.52 (t, 2 H, J )
7.8 Hz), 1.60-1.45 (m, 2 H), 1.33-1.20 (m, 24 H), 0.89 (t, 3 H,
J ) 6.8 Hz). 13C NMR (75 MHz, CD3COCD3): δ 169.6, 168.2,
159.7, 157.4, 143.3, 137.7, 136.9, 129.1, 128.7, 128.5, 128.0,
119.1, 110.0, 101.2, 100.3, 76.6, 76.1, 73.8, 72.0, 70.7, 67.1, 52.4,
34.0, 32.4, 31.8, 30.5, 30.2, 30.1, 30.0, 29.5, 29.2, 29.0, 23.1,
14.2. ESIMS (m/z): 757.4 (M + Na+). HR-ESIMS (m/z) calcd
for C43H58O10Na 757.3928, found 757.3922.
Acknowledgment. This work is supported by the
Committee of Science and Technology of Shanghai
(04DZ19213, 04DZ14901), the National Natural Science
Foundation of China (20321202), and the Chinese
Academy of Sciences (KGCX2-SW-209).
Benzyl 4′-(4-O-(Methyl â-D-Glucopyranosyluronate)-2-
hydroxy-6-pentdecanylbenzoyloxy)-2′-benzyloxy-6′-me-
thylbenzoate (39). Compound 37 (50 mg, 0.068 mmol) was
treated with 10% Pd/C (10 mg) in EtOAc (5.0 mL) under 1
atm of H2 atmosphere overnight. The mixture was then filtered
and concentrated to dryness, affording an amorphous solid 38
(37 mg). A solution of the acid 38 (37 mg, 0.067 mmol) and
phenol 30 (112 mg, 5.0 equiv) in dry CH2Cl2 (2.5 mL) was
treated at room temperature with DMAP (9 mg, 1.1 equiv) and
EDCI (25 mg, 2.0 equiv). After stirring for 4 h, the solution
Supporting Information Available: Experimental pro-
cedures and analytical data for compounds 4-6 and 9-30 and
reproductions of 1H and 13C NMR spectra for new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
JO051384K
J. Org. Chem, Vol. 70, No. 22, 2005 8889