Synthesis and enzyme-specific activation of carbohydrate-geldanamycin conjugates with potent anticancer activity

Article abstract of DOI:10.1021/jm049693a

Geldanamycin (GA) is a potent anticancer antibiotic that inhibits Hsp90. Its potential clinical utility is hampered by its severe toxicity. To alleviate this problem, we synthesized a series of carbohydrate-geldanamycin conjugates for enzyme-specific activation to increase tumor selectivity. The conjugation was carried out at the C-17-position of GA. Their anticancer activity was tested in a number of cancer cell lines. The enzyme-specific activation of these conjugates was evaluated with β-galactosidase and β-glucosidase. Evidently, glycosylation of C-17-position converted GA to an inactive prodrug before enzyme cleavage. Glucose-GA, as positive control, showed anticancer activity with IC₅₀ of 70.2-380.9 nM in various cancer cells by β-glucosidase activation inside of the tumor cells, which was confirmed by 3-fold inhibition using β-glucosidase specific inhibitor [2,5-dihydroxyniethy-3,4-dihydroxypyrrolidine (DMDP)]. Compared to glucose-GA, galactose- and lactose-GA conjugates exhibited much less activity with IC ₅₀ greater than 8000-25 000 nM. However, when galactose- and lactose-GA were incubated with β-galactosidase in the cells, their anticancer activity was enhanced by 3- to 40-fold. The results suggest that GA can be inactivated by glycosylation of C-17-position and reactivated for anticancer activity by β-galactosidase. Therefore, galactose-GA can be exploited in antibody-directed enzyme prodrug therapy (ADEPT) with β-galactosidase for enzyme-specific activation in tumors to increase tumor selectivity.

Full text of DOI:10.1021/jm049693a

J. Med. Chem. 2005, 48, 645-652
645
Synthesis and Enzyme-Specific Activation of Carbohydrate-Geldanamycin
Conjugates with Potent Anticancer Activity
Hao Cheng,†,‡ Xianhua Cao,† Ming Xian,‡,§ Lanyan Fang,† Tingwei Bill Cai,‡ Jacqueline Jia Ji,†
Josefino B. Tunac,| Duxin Sun,†,* and Peng George Wang‡,*
Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210,
Departments of Biochemistry and Chemistry, The Ohio State University, Columbus, Ohio 43210, and
JJ Pharma, Inc., 5000 Executive Plaza, San Ramon, California 94583
Received April 26, 2004
Geldanamycin (GA) is a potent anticancer antibiotic that inhibits Hsp90. Its potential clinical
utility is hampered by its severe toxicity. To alleviate this problem, we synthesized a series of
carbohydrate-geldanamycin conjugates for enzyme-specific activation to increase tumor
selectivity. The conjugation was carried out at the C-17-position of GA. Their anticancer activity
was tested in a number of cancer cell lines. The enzyme-specific activation of these conjugates
was evaluated with â-galactosidase and â-glucosidase. Evidently, glycosylation of C-17-position
converted GA to an inactive prodrug before enzyme cleavage. Glucose-GA, as positive control,
showed anticancer activity with IC50 of 70.2-380.9 nM in various cancer cells by â-glucosidase
activation inside of the tumor cells, which was confirmed by 3-fold inhibition using â-glucosidase
specific inhibitor [2,5-dihydroxymethy-3,4-dihydroxypyrrolidine (DMDP)]. Compared to glucose-
GA, galactose- and lactose-GA conjugates exhibited much less activity with IC50 greater than
8000-25 000 nM. However, when galactose- and lactose-GA were incubated with â-galac-
tosidase in the cells, their anticancer activity was enhanced by 3- to 40-fold. The results suggest
that GA can be inactivated by glycosylation of C-17-position and reactivated for anticancer
activity by â-galactosidase. Therefore, galactose-GA can be exploited in antibody-directed
enzyme prodrug therapy (ADEPT) with â-galactosidase for enzyme-specific activation in tumors
to increase tumor selectivity.
Introduction
Geldanamycin (GA) is an anticancer antibiotic iso-
lated from a Streptomyces hygroscopicus; it is a benzo-
quinoid ansamycin related to herbimycin A and macbe-
cin (Figure 1). Geldanamycin was initially thought to
be a nonspecific kinase inhibitor, but recently found to
target the heat shock protein 90 (Hsp90).1,2 Hsp90 is a
molecular chaperon that modulates protein kinase
activity (such as p60, p185, raf-1, cdk4/cdk6, FAK, and
MAK, v-Src, Akt, Bcr-Abl, mutant P53, HIF-1R, and
ErbB2) in cells. Hsp90 has been observed with 2- to10-
fold higher expressions in various human cancer cells
compared to normal.3,4 The crystal structure showed
that GA binds to Hsp90 and inhibits Hsp90-mediated
protein conformation/refolding, resulting in a depletion
of oncogenic kinases through the proteosomal degrada-
tion of immature protein.2,5 This process subsequently
down-regulates expression of many oncogenes in cancer
cells.2 While the antitumor potential of GA has long
been recognized, clinical evaluation of GA has not been
pursued due to its severe toxicity and poor water
solubility.6,7 For this reason, efforts have been made to
Figure 1.
modify GA, generating a number of analogues in
attempts to increase clinical efficacy and water
solubility,8-13 including modifications of the C-17-posi-
tion.8,9 Among the successful analogues is 17-allyl-
aminogeldanamycin (17-AAG), which is currently in
phase II clinical trials at the National Cancer Insti-
tute.14,15 Although 17-AAG showed improved efficacy
and relatively low toxicity, it appears that the hepato-
toxicity and the low water solubility may be still limiting
factors for its clinical application.16,17
Most existing chemotherapeutic compounds lack tu-
mor selectivity and are associated with dose-limiting
side effects. In some cases, such cytotoxic compounds
are linked to monoclonal antibodies against tumor-
specific antigen in an attempt to increase specific
activity. However, this approach is limited by the
amount of drug that could be linked to antibodies, slow
internalization of drug conjugates into cancer cells, and
heterogeneous antigen expression in solid tumors.18,19
* To whom correspondence should be addressed. P.G.W.: Phone:
(614) 292-9884. Fax: (614) 688-3106. E-mail: wang.892@osu.edu.
D.S.: Phone: (614) 292-4381. Fax: (614) 292-7766. E-mail: sun.176@
osu.edu.
† Division of Pharmaceutics, College of Pharmacy, The Ohio State
University.
‡ Departments of Biochemistry and Chemistry, The Ohio State
University.
| JJ Pharma, Inc.
§ Current address: Department of Chemistry, University of
Pennsylvania, Philadelphia, PA 19104.
10.1021/jm049693a CCC: $30.25 © 2005 American Chemical Society
Published on Web 12/31/2004

646 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
Cheng et al.
Scheme 1a
To circumvent these problems, a two-step strategy
called antibody-directed enzyme prodrug therapy
20,21
(ADEPT)
was recently emerged. In this approach,
a monoclonal antibody is employed to selectively deliver
an enzyme, such as â-galactosidase,22 to the tumor cells,
which subsequently converts an inactive prodrug to
active drug at tumor site. Thus, the selective activation
of the prodrug at the tumor site would result in
improved antitumor activity with minimal side effects.23
In this regard, we synthesized six carbohydrate-GA
conjugates for possible delivery through ADEPT with
â-galacosidase.
a Reagents and conditions: (a) NH2NH2/AcOH, DMF, 60 °C; (b)
CCl3CN/DBU, CH2Cl2, 0 °C, 60 min.
Crystal structure showed that GA binding to Hsp90
is highly compact with more than 85% surface area of
GA buried in the complex. The benzoquinone group of
GA binds near the entrance of Hsp90 binding pocket,
whereas the ansamycin ring, which is similar to a five
amino acid polypeptide in dimensions, inserts into the
binding pocket. Carbamate group of C-7 is at the tip of
GA and forms H-bond with Asp93 at the bottom of the
binding pocket, while the flanked C-23 methoxyl and
C-25 and C-26 methyl groups make high-density van
der Waals contacts with the bottom and sides of the
pocket.2 SAR studies revealed that the C-7-postion and
C-11-postion of GA are critical for its activity. Modifica-
tions of these two positions results in inactive com-
pounds. Thus, modification at C-7- or C-11-position
offered a good choice for synthesis of carbohydrate
conjugates as inactive prodrugs. However, the X-ray
structure of GA showed that the free C-11-OH group is
buried inside the ansa ring.2 The limited space around
the C-11-OH group makes it hardly accessible for bulky
sugar donors. The low reactivity at the C-7-position
makes it difficult for modification. Previous results on
GA derivatives revealed that modifications at the C-17-
position on the quinone ring maintained anticancer
activity within a nanomolar range.8,9 However, carbo-
hydrate-GA conjugates have never been reported. In
this regard, we embarked on a synthetic program to
produce carbohydrate-GA conjugates. Our hypothesis
is that the bulky sugar structure might be able to affect
GA binding to Hsp90 and thus make it an inactive
prodrug. If the carbohydrate moiety is cleaved at the
tumor site by a specifically delivered enzyme, the active
drug will be released and become active at site of tumor
growth.
It is known that â-glucosidase is ubiquitously ex-
pressed in many cell types; therefore, we designed
glucose-GA conjugate, as a positive control, that could
be activated by â-glucosidase inside of the cancer cells.
â-Galatosidase is very low in the serum (almost unde-
tectable), which makes this enzyme a very attractive
enzyme for activation of these conjugates if it is deliv-
ered to tumors. The low levels of this enzyme would
minimize the premature release of activate drug in blood
in ADEPT study. Indeed, â-galatosidase has been ap-
plied to prodrug activation in animal models.24 There-
fore, we designed and synthesized galactose-GA con-
jugates with different spacers for enzyme-specific
activation. If these conjugates are delivered with â-ga-
lactosidase in ADEPT, the conjugates will be specifically
activated at tumor sites to exhibit antitumor activity.
In addition, the sugar moiety is also expected to provide
better water solubility for GA.
Results and Discussion
Chemistry. The crystal structure of Hsp90-GA
complex indicates that 85% of the surface area of GA is
buried in the binding pocket on the surface of Hsp90,
while benzoquinone is positioned near the entrance of
the pocket.2 The carbamate group at the C-7-position
is crucial in forming a hydrogen bond to Hsp90. Removal
of the carbamate or attachment of additional atom
abolishes GA activity. An amino group substitution at
C-17-position of the benzoquinone may improve cellular
activity indirectly by stabilizing the quinone over the
reduced hydroquinone. Our hypothesis was that conju-
gating a bulky sugar at C-17-position of GA would affect
the GA binding to Hsp90 and makes it inactive. Our
first design was to synthesize glucose-GA conjugate as
positive control for enzyme-specific activation by â-glu-
cosidase since â-glucosidase is ubiquitously expressed
in the cells. In addition, galactose-GA conjugates were
designed by glycosylation of the C-17-position of GA
with galactose through an amine linker with varied
lengths. Since â-galactosidase activity is undetectable
in cancer cells, these conjugates should not have anti-
cancer activity. However, when the galactose-GA con-
jugates are incubated with exogenous â-galactosidase,
they are expected to show enhanced antitumor activity.
Thus, such conjugates may be applied in ADEPT when
â-galactosidase is delivered into the tumor sites by a
specific antibody.25
Synthesis. As shown in Scheme 1, three trichloro-
acetimidate derivatives (4, 5, 6) of sugar (galactose,
glucose and lactose) were prepared as glycosyl donors
from corresponding peracetylated sugars in two steps
with high yield. Selective removal of the acetyl group
at anomeric position was achieved under mild basic
condition. Treatment of the free sugar with trichloro-
acetonitrile in the presence of DBU afforded the ano-
merically pure, stable R-trichloroacetimidates. As shown
in Scheme 2, treatment of R-trichloroacetimidates (4,
5, 6) with different alchohol-linkers (8, 15, 16, 17) in
the presence of TMSOTf afforded a series of protected
sugar primary amine derivatives (9, 10, 11, 18, 19, 20)
in â-configuration. The free sugar primary amine de-
rivatives (12, 13, 14, 21, 22, 23) were obtained in high
yields by two steps of deprotection.
Coupling sugar-amine derivatives (12, 13, 14, 21, 22,
23) with GA in DMF at room-temperature resulted in
the formation of sugar-GA conjugates with good yield
(70-90%). GA was readily losing its 17-methoxy group
in such a mild condition and forming 17-amino-17-
demethoxygeldanamycin derivatives. As shown in

Anticancer Carbohydrate-Geldanamycin Conjugates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2 647
Scheme 2a
a Reagents and conditions: (a) NaN3, NaOH/H2O, rt, 3 days; (b) TMSOTf,CH2Cl2, 4 Å MS, -30 °C-0 °C, overnight; (c) NaOMe (1 M),
MeOH; (d) 10% Pd-C/AcOH, EtOH, H2 50 psi, 20 h.
Scheme 3a
a Reagents and conditions: (a) Et3N/DMF, rt, 24 h.
Scheme 3, six conjugates (24-29) with different sugar
moieties or linkers have been prepared, respectively.
The structures of synthesized compounds are confirmed
by NMR and high-resolution mass spectrum (HRMS).
The purity is confirmed with HPLC.
In addition, we also tried to directly link glycosyl-
amine to C-17-position of GA. However, the reaction was
inefficient; nearly half of the starting material (GA) was
recovered even after 3 days. The major product obtained
was 17-amine-17-demethoxy-GA.
A series of methods were used in attempts for direct
glycosylation of geldanamycin at C-11-position by treat-
ment of the phenylthioglycosides of the protected sugars
with mCPBA followed by GA and various Lewis acids
(Tf2O, TMSOTf, AgOTf, Ag2CO3, and PH3P/HBr). The
results, however, were quite disappointing. All these
glycosylation conditions failed to yield the corresponding
glycosides. Two reasons might account for the failure:
(1) the solubility of GA was low in organic solvents that
were used in glycosylation (such as methylene chloride
and toluene). (2) As can be seen from the X-ray structure
of GA, the C-11-OH group is buried inside the ansa ring.
The limited space around C-11-OH group makes it
hardly accessible for bulky and rigid glycosyl donors for
direct glycosylation. Intramolecular hydrogen bond may
also exist in this molecule, thus making this OH group
less active.
Biology. Anticancer Activity. The anticancer ac-
tivities of these six conjugates were tested by the MTS
assay (MTS: tetrazolium [3-(4,5-dimethythiazol-2-yl)]-
5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-
zolium) in four different cancer cell lines, including
colorectal carcinoma cells (SW620, HT 29), breast cancer
cells (MCF7), and leukemia cells (K562). The results are
shown in Figures 2-4. As expected, only the glucose-
GA (compound 24) showed antitumor activity, while
other four galactose-geldanamycin conjugates with
different linker and lactose-GA were inactive. Table 1
summarizes the IC50 of all six geldanamycin-sugar
conjugates for their anticancer activity. For example,
in SW620 cell line, compound 24 showed anticancer
activity with an IC50 of 70 nM, while the other conju-
gates25,29 with the same spacer but differed in sugar
moieties, showed low activity with IC50 > 8000 nM,

648 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
Cheng et al.
Table 1. Anticancer Activity (IC50) of
Carbohydrate-Geldanamycin in Four Different Cancer Cell
Lines (nM)
GA
24
25
26
27
28
29
SW620 6.2 70.2 >8000 >14000 >13000 >11000 >20000
HT29 24.5 104.7 >14000 >6000 >13000 >13000 >25000
MCF7
6.5 754.0 >25000 >12000 >17000 >22000 >22000
K562 22.1 380.9 >23000 >14000 >19000 >15000 >25000
Figure 2. Anticancer activity of carbohydrate-geldanamycin
conjugates (24-29) tested in SW620 cell line. Each compound
was tested at concentration 1-25 000 nM. GA was also tested
for comparison. The inhibition of cell growth was calculated
compared to control cells without drug treatment. Each point
represents the average of six experiments.
Figure 5. â-Glucosidase inhibition assay studied in K562 cell
line. Two different concentrations were tested in this experi-
ment. For compound 24, the high concentration is 0.4 µM and
the low concentration is 0.2µM. For compound 25, the high
concentration is 25µM and the low concentration is 12.5 µM.
Percentages of cell growth were calculated compared to control
cells in the absence of inhibitor and drug solution. Each data
presented is the average of three experiments.
â-Glucosidase Inhibition Assay. To verify if the
cleavage of glucose moiety from compound 24 is crucial
for the anticancer activity, we performed the â-glucosi-
dase inhibition study in K562 cell line. The K562 cells
were treated with â-glucosidase inhibitor (2,5-dihy-
droxymethy-3,4-dihydroxypyrrolidine, DMDP) solution
(100 µM) for 10 min before adding carbohydrate-
geldanamycin conjugates. The concentrations of glucose-
GA were 0.4 µM and 0.2 µM, respectively. Interestingly,
after treatment with DMDP, the activity of compound
24 decreased significantly by 3-fold, while DMDP did
not affect the activity of compound 25 (Figure 5). DMDP
alone have no effect on cell growth. Since the only
difference between compound 24 and 25 is the sugar
moiety, which is glucose in compound 24 and galactose
in compound 25, the results suggest that the anticancer
activity of compound 24 is achieved through cleavage
of glucose moiety by â-glucosidase inside of the cells,
while low activity of compound 25 is due to the lack of
â-galactosidase for the cleavage of galactose moiety.
â-Galactosidase Cleavage Assay. Since â-glucosi-
dase failed to activate compound 25, â-galactosidase was
subsequently evaluated to activate galactose-GA and
lactose-GA conjugates. SW620, HT29, and K562 cells
were incubated with 2 units of â-galactosidase and then
treated individually with 1 µM of compounds 25-29.
At this concentration, galactose-GA conjugates alone
did not show anticancer activities. However, all five
inactive galactose-GA conjugates and the lactose-GA
conjugate were activated with 3- to 40-fold increase of
their anticancer activity against HT29 and K562 after
incubation with â-galactosidase (Figure 6-8), while
â-galactosidase treatment did not affect the anticancer
Figure 3. Anticancer activity of carbohydrate-geldanamycin
conjugates (24-29) tested in HT29 cell line. Each compound
was tested at concentration 1-25 000 nM. GA was also tested
for comparison. The inhibition of cell growth was calculated
compared to control cells without drug treatment. Each point
represents the average of six experiments.
Figure 4. Anticancer activity of carbohydrate-geldanamycin
conjugates (24-29) tested in K562 cell line. Each compound
was tested at concentration 1-25 000 nM. GA was also tested
for comparison. The inhibition of cell growth was calculated
compared to control cells without drug treatment. Each point
represents the average of six experiments.
20 000 nM, respectively. Similar results were obtained
in other cell lines. It is possible that the glucose-GA
conjugate is activated for anticancer activity through
cleavage of glucose moiety by â-glucosidase inside of the
cancer cells.

Anticancer Carbohydrate-Geldanamycin Conjugates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2 649
Figure 6. â-Galactosidase cleavage assay of compound 24-
29 and GA in SW620 cell line. 0.5 µM each of compounds 25-
29 was used. 0.005 µM GA, 0.1 µM compound 24 and 2 units
â-galactosidase were used. Each data shown is the average of
three experiments. Percentages of cell growth were calculated
compared to control cells in the absence of â-galactosidase and
drug solution.
Figure 8. â-Galactosidase cleavage assay of compound 24-
29 and GA in K562 cell line. 1 µM each of compounds 25-29
was used. 0.01 µM GA, 0.5 µM compound 24 and 2 units
â-galactosidase were used. Each data shown is the mean of
three determinations. Percentages of cell growth were calcu-
lated compared to control cells in the absence of â-galactosidase
and drug solution.
at C-17-position. Glucose-GA conjugate showed anti-
cancer activity through cleavage of glucose moiety by
â-glucosidase inside of the cancer cells, which is con-
firmed by 3-fold inhibition with â-glucosidase inhibitor.
Galactose-GA conjugates remained inactive in the
absence of exogenous â-galactosidase. However, the
anticancer activity of galactose-GA conjugate increased
by 3- to 40-fold when incubated with â-galactosidase.
These data support our hypothesis that carbohydrate
conjugation of GA through the C-17-position produces
inactive prodrugs. The inactive carbohydrate-GA pro-
drugs can be enzyme-specifically activated by â-glucosi-
dase or â-galactosidase. In addition, the length of the
spacer, between carbohydrate and GA in the conjugates,
does not seem to play a significant role in the anticancer
activity after enzyme cleavage.1,2 These galactose-GA
conjugates can be potentially used for antibody-directed
enzyme prodrug therapy (ADEPT).
Figure 7. â-Galactosidase cleavage assay of compound 24-
29 and GA in HT 29 cell line. 1 µM each of compounds 25-29
was used. 0.01 µM GA, 0.5 µM compound 24 and 2 units
â-galactosidase were used. Each data shown is the average of
three determinations. Percentages of cell growth were calcu-
lated compared to control cells in the absence of â-galactosidase
and drug solution.
Experimental Section
activity of glucose-GA conjugate 24 and GA. These
results support our hypothesis that the bulky sugar
moiety at C-17-position of GA can abolish its anticancer
activity, and the inactive carbohydrate-GA prodrugs
can be enzyme-specifically activated by â-glucosidase or
â-galactosidase. Interestingly, compounds 25-29 showed
nearly the same activity as GA after being cleaved by
â-galactosidase. This indicates that the length of spacer
has little effect on GA anticancer activity. In addition,
although lactose is a disaccharide, its structure is
galactose-â-1,4-glucose, the terminal galactose in the
lactose-GA structure could be cleaved by â-galactosi-
dase and thus expose the glucose moiety, which can be
subsequently cleaved by the â-glucosidase inside the
cells to exhibit anticancer activity.
The NMR data were recorded on a Varian-400 or -500 MHz
spectrometer. MS spectra were obtained from a Kratos MS 80
spectrometer using electrospray ionization mode (ESI). The
MTS assay was measured on a DYNEX MRX microplate
reader. Silica gel F254 plates (Merck) and silica gel 60 (70-
230 mesh) were used in analytical thin-layer chromatography
(TLC) and column chromatography, respectively.
General Synthetic Procedure of R-Glycopyranosyl
Trichloroacetimidate. Hydrazine acetate (0.89 g, 9.6 mmol)
was added to a solution of 1,2,3,4,6-penta-O-acetylpyranose
(8.1 mmol) in DMF (10 mL) at 60 °C under Ar. When TLC
(1:1 hexanes-EtOAc) showed the formation of a new product
and the disappearance of starting material, the mixture was
diluted with EtOAc, washed with aqueous 5% NaCl (2×) and
water, dried over Na2SO4, and precipitated with toluene to give
the crude product. A solution of this crude product was then
reacted with trichloroacetonitrile (10 equiv) and DBU (2 equiv)
in CH2Cl2 (30 mL) for 60 min at 0 °C. When TLC (2:1 hexanes-
EtOAc) showed the conversion to be complete, the solution was
concentrated and purified by flash chromatography to yield
the product.
Production of Geldanamycin. Geldanamycin was
isolated from the fermentation broth of Streptomyces
hygroscopicus NRRL3602. Fermentation and chemical
isolation was carried out as described by DeBoer et al.1
2,3,4,6-Tetra-O-acetyl-R-galactopyranosyl trichloro-
acetimidate (4): 1H NMR (CDCl3): δ 8.67 (s, 1H), 6.61 (d,
1H, J ) 3.4 Hz), 5.56, (dd, 1H, J ) 3.0, 1.4 Hz), 5.43 (dd, 1H,
J ) 10.8, 3.0 Hz), 5.36 (dd, 1H, 10.8, 3.4 Hz), 4.44 (m, 1H),
4.17 (dd, 1H, J ) 6.6, 11.3 Hz), 4.08 (dd, 1H, 6.7, 11.3 Hz),
Conclusion
In summary, a new series of carbohydrate-GA con-
jugates were synthesized through glycosylation of GA

650 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
Cheng et al.
2.17, 2.03, 2.02, 2.01 (4 s, each 3H). 13C NMR (CDCl3):
170.35-169.31 (4 COCH3), 160.82, 93.45, 68.85, 67.38, 67.32,
66.47, 61.10, 20.11-19.85 (4 COCH3). MS ES+ m/z: 514.17
(M + Na). Yield: 79%.
δ
2.02, 2.01, 1.96 (4 s, each 3H), 1.80-1.75 (m, 2H). 13C NMR
(CDCl3) δ 170.65, 170.49, 170.36, 169.80, 156.67, 136.87,
128.72, 128.33, 128.29, 101.39, 71.05, 70.93, 68.97, 67.88,
67.24, 67.02, 66.78, 61.54, 38.45, 29.75, 20.94, 20.87, 80.80.
MS ES+ m/z: 562.16 (M + Na).
2,3,4,6-Tetra-O-acetyl-R-glucopyranosyl trichloroacet-
imidate (5): 1H NMR (CDCl3) δ δ 8.70 (s, 1H), 6.57 (d, 1H, J
) 3.6 Hz), 5.14 (dd, 1H, J ) 3.7, 10.2 Hz), 2.08, 2.05, 2.04,
2.02 (4s, each 3H). 13C NMR (CDCl3): δ 169.95-169.02 (4
COCH3), 160.11, 92.55, 90.32, 69.65, 69.40, 69.31, 67.45, 61.08,
20.23-20.01 (4 COCH3). MS ES+ m/z 514.13 (M + Na). Yield:
75%.
2,3,4,6-tetra-O-acetyl-R-lactopyranosyl trichloroacet-
imidate (6): 1H NMR (CDCl3): δ 8.65 (s, 1H), 6.48 (d, 1H, J
) 3.6 Hz), 5.55 (t, 1H, J ) 9.9 Hz), 5.35 (d, 1H, J ) 3.0 Hz),
5.15-5.03 (m, 2H), 4.97-4.92 (dd, 1H, J ) 3.6, 10.2 Hz), 4.52-
4.46 (m, 2H), 4.18-4.05 (m, 4H), 3.89-3.83 (m, 2H), 2.15 (s,
3H), 2.10 (s, 3H), 2.06 (s, 6H), 2.04 (s, 3H), 2.00 (s, 3H), 1.96
(s, 3H). 13C NMR (CDCl3): δ 170.31-169.28 (7 COCH3), 161.21,
101.47, 93.10, 76.15, 71.36, 71.13, 70.93, 70.18, 69.78, 69.33,
66.79, 61.72, 60.98, 21.11-20.74 (7 COCH3). MS ES+ m/z:
802.19 (M + Na). Yield: 69%.
2-Azidoethanol (8). 2-Chloroethanol (25.2 mL, 375 mmol)
was added to a solution of NaN3 (30 g, 461 mmol) and NaOH
(1.5 g, 37.5 mmol) in water (115 mL). The mixture was stirred
at room temperature for 3 days, and sodium sulfate (35 g) was
added. After 10 min, the mixture was extracted with CH2Cl2
(3 × 70 mL). The combined extracts were dried in Na2SO4 and
concentrated. The residue was distilled to give 2-azidoethanol.
1H NMR (CDCl3): δ 3.79-3.76 (t, 2H, J ) 5.1 Hz), 3.47-3.44
(t, 2H, J ) 5.1 Hz), 2.02 (s, 1H). 13C NMR (CDCl3): δ 61.76,
53.75. MS ES+ m/z: 88.12 (M + H). Yield: 86%.
2-(2-Azidoethoxy)ethanol (17). NaN3 (4.5 g, 69 mmol),
tetrabutylammonium iodide (2.5 g, 6 mmol), and 18-crown-6
(10 mg) were added to a solution of 2-(2-chloroethoxy)ethanol
(5 mL, 47 mmol) in 2-butanone (25 mL). The mixture was
refluxed at 90 °C for 2 days. When 13C NMR spectroscopy of
the supernatant showed the absence of a signal at δ 42.7 and
the presence of a strong signal at δ 50.9 ppm, the mixture was
filtered. The precipitate was rinsed with acetone, and the
combined solutions were concentrated. Distillation of the
residue gave the pure product. 1H NMR (CDCl3): δ 3.74-3.71
(t, 2H, J ) 4.5 Hz), 3.68-3.65 (t, 2H, J ) 5.1 Hz), 3.59-3.56
(t, 2H, J ) 4.2 Hz), 3.40-3.37 (t, 2H, J ) 5.4 Hz), 2.41 (s, 1H).
13C NMR (CDCl3): δ 72.65, 70.25, 61.94, 50.92. MS ES+ m/z:
132.13 (M + H). Yield: 80%.
5-Cbz-amino-1-pentyl-2,3,4,6-tetra-O-acetyl-â-galacto-
pyranoside (19): 1H NMR (400 MHz, CDCl3) δ 7.33-7.30 (m,
5H), 5.35 (d, 1H, J ) 3.6 Hz), 5.19-5.13 (dd, 1H, J ) 10.2, 7.8
Hz), 5.0 (s, 2H), 5.01-4.96 (dd, 1H, J ) 10.5, 3.6 Hz), 4.90 (br,
1H), 4.42 (d, 1H, J ) 7.8 Hz), 4.18-4.06 (m, 2H), 3.88-3.82
(m, 2H), 3.48-3.40 (m, 1H), 3.17-3.11 (m, 2H), 2.10, 2.01, 2.00,
1.95 (4 s, each 3H), 1.59-1.43 (m, 4H), 1.37-1.30 (m, 2H). 13
C
NMR (CDCl3): δ 170.62, 170.51, 170.38, 169.66, 156.64,
136.87, 128.70, 128.28, 101.48, 71.12, 70.79, 70.06, 69.13,
67.28, 66.75, 62.73, 61.49, 41.08, 32.43, 29.78, 29.17, 23.23,
23.08, 20.94, 20.86, 20.79. MS ES+ m/z: 590.20 (M + Na).
5-Azido-3-oxapentyl-2,3,4,6-tetra-O-acetyl-â-galacto-
pyranoside (20): 1H NMR (400 MHz, CDCl3): δ 5.35 (d, 1H,
J ) 3.2 Hz), 5.20-5.16 (dd, 1H, J ) 10.4, 8.0 Hz), 5.01-4.97
(dd, 1H, J ) 10.4, 3.2 Hz), 4.55 (d, 1H, J ) 8.0 Hz), 4.17-4.07
(m, 2H), 3.96-3.87 (m, 2H), 3.76-3.71 (m, 1H), 3.65-3.61 (m,
4H), 3.35-3.32 (m, 2H), 2.12, 2.04, 2.01, 1.95 (4 s, each 3H).
13C NMR (CDCl3): δ 170.60, 170.46, 170.34, 169.71, 101.54,
71.10, 70.88, 70.64, 70.41, 69.25, 69.02, 67.25, 61.48, 50.98,
20.98, 20.88, 20.80. MS ES+ m/z: 484.06 (M + Na).
2-Azidoethyl 2,3,4,6-tetra-O-acetyl-â-glucopyranoside
(10): 1H NMR (400 MHz, CDCl3) δ 5.21-5.17 (t, 1H, J ) 9.6
Hz), 5.10-5.05 (t, 1H, J ) 9.6 Hz), 5.02-4.98 (dd, 1H, J )
9.2, 8.0 Hz), 4.58 (d, 1H, J ) 7.2 Hz), 4.25-4.21 (dd, 1H, J )
13.2, 4.4 Hz), 4.15-4.12 (dd, 1H, J ) 12.0, 2.4 Hz), 4.04-3.99
(m, 1H), 3.71-3.64 (m, 2H), 3.51-3.45 (m, 1H), 3.29-3.23 (m,
1H), 2.07, 2.03, 2.01, 1.98 (4 s, each 3H). 13C NMR (CDCl3) δ
170.88, 170.50, 169.62, 100.86, 72.97, 72.10, 71.22, 68.82,
68.46, 61.70, 50.70, 20.97, 20.92, 20.83. MS ES+ m/z: 440.22
(M + Na). Yield: 73%.
2-Azidoethyl 2,3,4,6-tetra-O-acetyl-â-lactopyranoside
(11): 1H NMR (400 MHz, CDCl3): δ 5.29 (d, 1H, J ) 3.2 Hz),
5.15 (t, 1H, J ) 9.2 Hz), 5.05 (t, 1H, J ) 9.2 Hz), 4.94-4.84
(m, 2H), 4.52-4.44 (m, 2H), 4.09-4.03 (m, 4H), 3.96-3.90 (m,
1H), 3.85-3.75 (m, 2H), 3.65-3.59, (m, 2H), 3.45-3.40 (m, 1H),
3.24-3.20 (m, 1H), 2.10 (s, 3H), 2.07 (s, 3H), 2.01 (s, 3H), 1.99
(s, 9H), 1.91 (s, 3H). 13C NMR (CDCl3): δ 170.55, 170.35,
170.26, 169.96, 169.26, 100.28, 100.62, 76.35, 72.98, 72.88,
71.64, 71.14, 70.83, 69.25, 68.91, 66.77, 61.97, 60.97, 50.67,
21.06, 20.99, 20.92, 20.84, 20.72. MS ES+ m/z: 728.08 (M +
Na). Yield: 61%.
General Deprotection Procedure of O-Acetylpyrano-
side. The protected pyranoside (200 mg) was dissolved in dry
MeOH (10 mL). NaOMe solution (1 M in MeOH) was added
until pH 9 was reached. Then the reaction mixture was stirred
at room temperature until the deprotection reaction was
complete (TLC iso-PrOH-water, 7:3, +1% NH3). Then the
mixture was neutralized with ion-exchange resin (Amberylst),
filtered, and dried in a vacuum. The resulted residue was then
dissolved in ethanol (10 mL), and then palladium catalyst (10%
Pd-C, 50 mg) and a few drops of acetic acid were added. The
reaction mixture was hydrogenated at 50 psi for 20 h. Then it
was filtered through a Celite bed and washed with MeOH. The
filtrate was concentrated under low pressure to give final
product.
General Glycosylation Procedure of O-Acetylpyran-
osyl Trichloroacetimidate. The trichloroacetimidate (1.63
mmol) was dissolved in dry CH2Cl2 (10 mL) with glycosyl
acceptor (the primary alcohols, 1.5 equiv) and 4 Å MS (100
mg). The mixture was stirred at room temperature for 1 h.
After the mixtrue was cooled to -30 °C, TMSOTf (1 equiv)
was added. The reaction was stirred at -30 °C for 1 h and
then at room temperature overnight. The reaction was quenched
with saturated NaHCO3. The mixture was extracted with CH2-
Cl2 and washed with NaHCO3 and brine. The organic phase
was dried over Na2SO4, filtered, and concentrated under
vacuum. The residue was purified with chromatography (silica
gel, hexane/EtOAc 7:3-1:1) to give the product.
2-Azidoethyl 2,3,4,6-tetra-O-acetyl-â-galactopyrano-
1
side (9): H NMR (500 MHz, CDCl3): δ 5.40 (d, 1H, J ) 3.5
Hz), 5.26-5.23 (dd, 1H, J ) 8.0, 10.5 Hz), 5.04-5.01 (dd, 1H,
J ) 10.5, 3.5 Hz), 4.56 (d, 1H, J ) 8.0 Hz), 4.21-4.11 (m, 2H),
4.07-4.03 (dt, 1H, J ) 10.0, 4.5 Hz), 3.93-3.91 (t, 1H, J ) 8.0
Hz), 3.72-3.67 (m, 1H), 3.54-3.49 (m, 1H), 3.32-3.28 (dt, 1H,
J ) 13.5, 3.5 Hz), 2.16, 2.07, 2.05, 1.99 (4 s, each 3H). 13C NMR
(CDCl3): δ 170.41-169.68 (4 COCH3), 101.38, 71.12, 71.03,
68.74, 68.65, 67.21, 61.47, 50.79, 21.03-20.83 (4 COCH3). MS
ES+ m/z: 440.23 (M + Na). Yield: 75%.
2-Aminoethyl-â-galactopyranoside acetate (12): 1H
NMR (500 MHz, CD3OD): δ 4.30 (d, 1H, J ) 7.5 Hz), 4.06-
4.02 (m, 1H), 3.92-3.88 (m, 1H), 3.84 (d, 1H, J ) 2.5 Hz),
3.78-3.70 (m, 2H), 3.60-3.50 (m, 3H), 3.18-3.15 (m, 1H), 1.93
(s, 3H). 13C NMR (CD3OD): δ 177.63, 103.66, 75.77, 73.55,
71.30, 69.09, 65.76, 61.37, 39.87, 21.99. MS ES+ m/z: 246.35
(M + Na).
3-Aminopropyl-â-galactopyranoside acetate (21): 1H
NMR (400 MHz, CD3OD): δ 4.27 (d, 1H, J ) 7.2 Hz), 4.05-
3.99 (m, 1H), 3.83 (d, 1H, J ) 1.6 Hz), 3.80-3.73 (m, 3H),
3.56-3.46 (m, 3H), 3.14-3.08 (m, 2H), 1.97-1.95 (m, 2H), 1.93
(s, 3H). 13C NMR (CD3OD): δ 103.83, 75.68, 73.71, 71.25,
69.12, 67.61, 61.33, 38.03, 27.18, 21.66. MS ES+ m/z: 260.08
(M + Na).
3-Cbz-amino-1-propyl-2,3,4,6-tetra-O-acetyl-â-galacto-
1
pyranoside (18): H NMR (400 MHz, CDCl3): δ 7.35-7.30
(m, 5H), 5.36 (d, 1H, J ) 3.6 Hz), 5.21-5.15 (dd, 1H, J ) 10.2,
7.8 Hz), 5.07 (br, 3H), 5.01-4.96 (dd, 1H, J ) 10.5, 3.9 Hz),
4.44 (d, 1H, J ) 8.1 Hz), 4.14-4.11 (dd, 2H, J ) 6.6, 3.0 Hz),
3.95-3.85 (m, 2H), 3.66-3.53 (m, 1H), 3.36-3.18 (m, 2H), 2.11,

Anticancer Carbohydrate-Geldanamycin Conjugates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2 651
5-Aminopentyl-â-galactopyranoside acetate (22): 1H
NMR (500 MHz, CD3OD): δ 4.22 (d, 1H, J ) 7.2 Hz), 3.94-
3.89 (m, 1H), 3.84 (d, 1H, J ) 2.4 Hz), 3.72 (d, 2H, J ) 5.6
Hz), 3.61-3.55 (m, 1H), 3.52-3.46 (m, 3H), 2.94-2.90 (m, 2H),
1.93 (s, 3H), 1.73-1.63 (m, 4H), 1.59-1.42 (m, 2H). 13C NMR
(CD3OD): δ 103.79, 75.43, 73.83, 71.42, 69.09, 69.04, 61.27,
39.41, 28.86, 27.20, 27.01, 22.81, 22.70, 21.71. MS ES+ m/z:
266.10 (M + H).
17-Demethoxy-17-[(2-â-galactopyranosylpentyl)amino]-
geldanamycin (27): 1H NMR (500 MHz, CD3OD) δ 7.13 (d,
1H, J ) 11.5 Hz), 7.05 (s, 1H), 6.64-6.60 (t, 1H, J ) 11.5 Hz),
5.89-5.85 (t, 1H, J ) 11.0 Hz), 5.58 (d, 1H, J ) 9.5 Hz), 5.21
(s, 1H), 4.54 (d, 1H, J ) 8.5 Hz), 4.21 (d, 1H, J ) 7.5 Hz),
3.93-3.90 (m, 1H), 3.82 (d, 1H, 3.5 Hz), 3.74-3.71 (m, 2H),
3.60-3.44 (m, 8H), 3.34 (s, 3H), 3.29 (s, 3H), 2.75-2.73 (m,
2H), 2.35-2.30 (dd, 1H, J ) 14.0, 9.0 Hz), 1.99 (s, 3H), 1.81
(bs, 1H), 1.73-1.49 (m, 11H), 0.99-0.96 (m, 6H). HRMS (M +
Na+) (ESI+) calcd for C39H59N3O14Na+ 816.3890, found 816.3926.
Yield: 88%. Purity > 99%.
17-Demethoxy-17-{[2-(2-â-galactopyranosylethyl)ethyl]-
amino}geldanamycin (28): 1H NMR (500 MHz, CD3OD) δ
7.14-7.12 (d, 1H, J ) 11.5 Hz), 7.06 (s, 1H), 6.64-6.60, (t,
1H, J ) 12.0 Hz), 5.87-5.85 (t, 1H, J ) 9.0 Hz), 5.60-5.58 (d,
1H, J ) 10.0 Hz), 5.20 (s, 1H), 4.54-4.53 (d, 1H, J ) 9.0 Hz),
4.29-4.28 (d, 1H, J ) 7.5 Hz), 4.05-4.01 (m, 1H), 3.83-3.82
(m, 1H), 3.78-3.70 (m, 9H), 3.61-3.28 (t, 1H, J ) 5.5 Hz),
3.56-3.45 (m, 4H), 3.34 (s, 3H), 3.29 (s, 3H), 2.75-2.69 (m,
2H), 2.36-2.32 (dd, 1H, J ) 14.5, 9.5 Hz), 1.99 (s, 3H), 1.81
(bs, 1H), 1.73 (s, 3H), 1.68-1.65 (br, 1H), 1.60-1.56 (m, 1H),
1.00-0.98 (d, 3H, J ) 6.5 Hz), 0.98-0.96 (t, 3H, J ) 7.0 Hz).
HRMS (M + Na+) (ESI+) calcd for C38H57N3O15Na+ 818.3682,
found 818.3677. Yield: 78%. Purity > 99%.
17-Demethoxy-17-[(2-â-lactopyranosylethyl)amino]-
geldanamycin (29): 1H NMR (500 MHz, CD3OD): δ 7.12 (d,
1H, J ) 11.5 Hz), 7.05 (s, 1H), 6.64-6.60 (t, 1H, J ) 12.0 Hz),
5.89-5.85 (t, 1H, J ) 9.0 Hz), 5.58 (d, 1H, J ) 10.0 Hz), 5.21
(s, 1H), 4.53 (d, 1H, J ) 8.0 Hz), 4.39-4.37 (m, 3H), 4.04-
3.99 (m, 1H), 3.94-3.87 (m, 3H), 3.83-3.73 (m, 3H), 3.71-
3.66 (m, 2H), 3.61-3.52 (m, 4H), 3.50-3.44 (m, 4H), 3.34 (s,
3H), 3.29 (s, 3H), 2.74-2.68 (m, 2H), 2.36-2.32 (dd, 1H, J )
14.0, 8.5 Hz), 1.99 (s, 3H), 1.80 (br, 1H), 1.73 (s, 3H), 1.68-
1.65 (br, 1H), 1.60-1.56 (m, 1H), 0.99-0.96 (t, 6H, J ) 8.5
Hz). HRMS (M + Na+) (ESI+) calcd for C42H63N3O19Na+
936.3948, found 936.3977. Yield: 70%. Purity > 99%.
HPLC Determination of Purity of Synthesized Com-
pound 24-29. The purity of synthesized compounds was
determined with HPLC by applying the sample to a prepacked
BDS hypersil column (C18 5µm, 100 × 4.6 mm, Keystone
Scientific, Inc.), using a Shimadzu LC-10AD HPLC system
equipped with a SPD-M10A Diode Array Detector. The sample
was prepared by adding 1 µL of DMSO stock solution of
synthesized conjugates to 1 mL of acetonitrile/H2O (5/95). Fifty
microliter sample was subjected to HPLC system. The chro-
matographic analyses were performed at room temperature
while the samples were kept in the autosampler at 4 °C. Mobile
phase A is 5%ACN in water while mobile phase B is 5% water
in ACN. The mobile phase was run in a gradient fashion in
which phase A was decreased from 100% to 0% while phase B
was increased from 0% to 100% at a flow rate of 0.5 mL/min.
The carbohydrate-GA conjugates were detected from 250 to
600 nm with maximum absorbance at 330 nm.
5-Amino-3-oxapentyl-â-galactopyranoside
acetate
(23): 1H NMR (500 MHz, CD3OD): δ 4.30-4.28 (d, 1H, J )
7.5 Hz), 4.05-4.01 (m, 1H), 3.84-3.83 (dd, 1H, J ) 3.0, 1.0
Hz), 3.80-3.76 (m, 1H), 3.75-3.71 (m, 6H), 3.54-3.47 (m, 3H),
3.13-3.11 (m, 2H), 1.95 (s, 3H). 13C NMR (CD3OD): δ 176.37,
103.82, 75.61, 73.81, 71.36, 69.97, 69.14, 68.90, 66.63, 61.35,
39.37, 21.13. MS ES+ m/z: 268.19 (M + H).
2-Aminoethyl-â-glucopyranoside acetate (13): 1H NMR
(400 MHz, CD3OD): δ 4.34 (d, 1H, J ) 8.0 Hz), 4.08-4.03 (dt,
1H, J ) 12.0, 4.8 Hz), 3.90-3.84 (m, 2H), 3.69-3.65 (dd, 1H,
J ) 12.0, 5.6 Hz), 3.41-3.22 (m, 4H), 3.17-3.15 (t, 1H, J )
6.0 Hz), 1.94 (s, 3H). 13C NMR (CD3OD): δ 176.63, 103.08,
76.91, 76.59, 73.74, 70.27, 65.86, 61.33, 39.73, 21.60. MS ES+
m/z: 246.05 (M + Na). Yield: 95%.
2-Aminoethyl-â-lactopyranoside acetate (14): 1H NMR
(300 MHz, CD3OD): δ 4.39-4.37 (d, 1H, J ) 8.4 Hz), 4.36-
4.35 (d, 1H, J ) 7.2 Hz), 4.07-4.04 (m, 1H), 3.94-3.68 (m,
6H), 3.62-3.46 (m, 7H), 3.34-3.30 (m, 1H), 3.18-3.16 (t, 1H,
J ) 4.8 Hz), 1.96 (s, 3H). 13C NMR (CD3OD): δ 175.62, 103.92,
102.78, 79.16, 75.93, 75.40, 75.02, 73.59, 73.42, 71.33, 69.09,
65.76, 61.34, 60.45, 39.70, 20.65. MS ES+ m/z: 408.01 (M +
Na). Yield: 95%.
General Procedure for the Synthesis of Sugar-GA
Conjugate through C-17 Linkage of Geldanamycin. GA
(30 mg, 0.54 mmol), sugar-amine derivative (4 equiv), and 100
µL of Et3N were dissolved in 0.5 mL of dry DMF. The mixture
was stirred at room temperature in dark for 24 h. Then the
mixture was directly subjected to flash chromatograph (silica
gel, hexane/EtOAc system, then EtOAc/MeOH 9:1) to give pure
carbohydrate-GA conjugate.
17-Demethoxy-17-[(2-â-glucopyranosylethyl)amino]-
geldanamycin (24): 1H NMR (400 MHz, CD3OD): δ 7.13 (d,
1H, J ) 12.4 Hz), 7.05 (s, 1H), 6.62, (t, 1H, J ) 12.0 Hz), 5.87
(t, 1H, J ) 8.8 Hz), 5.58 (d, 1H, J ) 9.6 Hz), 5.20 (s, 1H), 4.53
(d, 1H, J ) 8.4 Hz), 4.34 (d, 1H, J ) 8.0 Hz), 4.07-4.01 (m,
1H), 3.92-3.89 (m, 2H), 3.83-3.78 (m, 1H), 3.76-3.70 (m, 2H),
3.60-3.55 (m, 1H), 3.46 (br, 1H), 3.34-3.29 (m, 9H), 3.24-
3.19 (t, 1H, J ) 8.0 Hz), 2.75-2.68 (m, 2H), 2.37-2.32 (dd,
1H, J ) 14.4, 8.8 Hz), 1.99-1.98 (s, 3H), 1.79 (br, 1H), 1.73 (s,
3H), 1.65 (br, 1H), 1.60-1.56 (m, 1H), 0.99-0.96 (t, 6H, J )
7.2 Hz). HRMS (M + Na+) (ESI+) calcd for C36H53N3O14Na+
774.3420, found 774.3434. Yield: 83%. Purity > 99%.
17-Demethoxy-17-[(2-â-galactopyranosylethyl)amino]-
geldanamycin (25): 1H NMR (500 MHz, CD3OD) δ 7.12 (d,
1H, J ) 11.5 Hz), 7.04 (s, 1H), 6.62, (t, 1H, J ) 12.0 Hz), 5.86
(t, 1H, J ) 9.0 Hz), 5.58 (d, 1H, J ) 10.0 Hz), 5.20 (s, 1H),
4.53 (d, 1H, J ) 8.5 Hz), 4.30 (d, 1H, J ) 8.0 Hz), 4.06-4.02
(m, 1H), 3.91-3.87 (m, 1H), 3.84-3.79 (m, 3H), 3.76-3.69 (m,
2H), 3.60-3.55 (m, 3H), 3.50-3.44 (m, 2H), 3.34 (s, 3H), 3.29
(s, 3H), 2.74-2.68 (m, 2H), 2.36-2.31 (dd, 1H, J ) 14.5, 9.0
Hz), 1.99 (s, 3H), 1.79 (bs, 1H), 1.73 (s, 3H), 1.68-1.65 (br,
1H), 1.60-1.56 (m, 1H), 0.99-0.96 (t, 6H, J ) 8.5 Hz). HRMS
(M + Na+) (ESI+) calcd for C36H53N3O14Na+ 774.3420, found
774.3425. Yield: 85%. Purity > 99%.
17-Demethoxy-17-[(2-â-galactopyranosylpropyl)amino]-
geldanamycin (26): 1H NMR (500 MHz, CD3OD) δ 7.13 (d,
1H, J ) 12.0 Hz), 7.05 (s, 1H), 6.65-6.60 (t, 1H, J ) 12.0 Hz),
5.89-5.85 (t, 1H, J ) 11.0 Hz), 5.58 (d, 1H, J ) 10.0 Hz), 5.22
(s, 1H), 4.54 (d, 1H, J ) 8.0 Hz), 4.23 (d, 1H, J ) 7.5 Hz),
4.03-3.98 (m, 1H), 3.84-3.83 (d, 1H, J ) 3.5 Hz), 3.78-3.67
(m, 5H), 3.60-3.56 (m, 2H), 3.53-3.45 (m, 3H), 3.34 (s, 3H),
3.29 (s, 3H), 2.77-2.68 (m, 2H), 2.39-2.35 (dd, 1H, J ) 14.0,
9.0 Hz), 1.99 (s, 3H), 1.96-1.93, (m, 2H), 1.79 (br, 1H), 1.73
(s, 3H), 1.70-1.68 (br, 1H), 1.67 (br, 1H), 1.00-0.97 (t, 6H, J
) 6.5 Hz). HRMS (M + Na+) (ESI+) calcd for C37H55N3O14Na+
788.3576, found 788.3564. Yield: 90%. Purity > 99%.
Cell Culture. Cell lines SW620, HT29, MCF7, and K562
were cultured in RPMI 1640 supplemented with 10% fetal
bovine serum (FBS), 1% nonessential amino acid, and penicil-
lin (100 µg/mL)/streptomycin (100 µg/mL) in a humidified
atmosphere of 5% CO2 and 95% air at 37 °C. The culture
medium was changed every 2-3 days.
Anticancer Activity of Compound (24-29) (MTS
assay). Cells (2000-10 000) were seeded in 96-well plates in
RPMI-1640 and incubated for 24 h. The exponentially growing
cancer cells were incubated with various concentrations of
compounds for 72 h at 37 °C (5% CO2, 95% humidity). After
72 h incubation, tetrazolium [3-(4,5-dimethythiazol-2-yl)]-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt (MTS, 2 mg/ml) and phenazine methosulfate (PMS,
25 µM) were mixed and added directly to the cells. After
incubated for 3 h at 37 °C, the absorbance of formazan (the
metabolite of MTS by viable cells) was measured at 490 nm.
The IC50 values of the carbohydrate-drug conjugates for
antiproliferation were calculated by the dose-response curves
of percentage of cell growth vs control (no compound
added).

652 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 2
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(17) Kelland, L. R.; Sharp, S. Y.; Rogers, P. M.; Myers, T. G.;
Workman, P. DT-Diaphorase expression and tumor cell sensitiv-
ity to 17-allylamino, 17-demethoxygeldanamycin, an inhibitor
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1949.
â-Glucosidase Inhibition Assay for Compound 24, 25.
K562 Cells (2000cells/80 µL RPMI-1640) were seeded in 96-
well plates and incubated for 24 h. The exponentially growing
cancer cells were incubated in three different conditions (cells
with different concentration of test compounds and DMDP,
cells with different concentration of test compounds only, and
cells with DMDP only) for 72 h at 37 °C (5% CO2, 100%
humidity). The control cells were exposed to fresh medium
only. Then, the anticancer activity of compound 24, 25 was
measured with MTS assay as described above. The percentages
of cell growth for tested compounds vs control cells were
calculated by the absorbance at 490 nm.
â-Galactosidase Cleavage Assay. Cells (2000-10 000)
were seeded in 96-well plates in RPMI-1640 and incubated
for 24 h. The exponentially growing cancer cells were incubated
with 2 units of â-galactosidase for 10 min. The same concen-
tration solutions of various compounds (25-29) were added
to each well. The control cells were exposed to fresh medium
only or to â-galactosidase alone. After incubated for 72 h at
37 °C (5% CO2, 100% humidity), the cell growth was measured
with MTS assay as described above. The percentages of cell
growth of tested compounds vs control were calculated by the
absorbance at 490 nm.
Supporting Information Available: Additional experi-
mental data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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