Design and synthesis of water-soluble glucuronide derivatives of camptothecin for cancer prodrug monotherapy and antibody-directed enzyme prodrug therapy (ADEPT)

Article abstract of DOI:10.1021/jm990124q

Glucuronide prodrugs of 9-aminocamptothecin were synthesized. Prodrug 4, in which 9-aminocamptothecin was-connected to glucuronic acid by an aromatic spacer via a carbamate linkage, was stable in both aqueous solution and human plasma. Prodrug 4 and its potassium salt 12 were 20-80-fold less toxic than 9-aminocamptothecin to human tumor cell lines. The simultaneous addition of β-glucuronidase and 4 or 12 to tumor cells resulted in a cytotoxic effect equal to that of 9-aminocamptothecin alone. Prodrugs 4 and 12 were over 80 and 4000 times more soluble than 9-aminocamptothecin in aqueous solutions at pH 4.0, respectively. Compounds 4 and 12 may be useful for prodrug monotherapy of tumors that accumulate extracellular lysosomal β- glucuronidase as well as for antibody-directed enzyme prodrug-therapy (ADEPT) of cancer.

Full text of DOI:10.1021/jm990124q

J . Med. Chem. 1999, 42, 3623-3628
3623
Design a n d Syn th esis of Wa ter -Solu ble Glu cu r on id e Der iva tives of
Ca m p toth ecin for Ca n cer P r od r u g Mon oth er a p y a n d An tibod y-Dir ected En zym e
P r od r u g Th er a p y (ADEP T)†
Yu-Ling Leu,‡ Steve R. Roffler,*,§ and J i-Wang Chern*,∇
Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Institute of Biomedical Sciences, Academia Sinica,
Taipei, Taiwan, and School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan
Received March 18, 1999
Glucuronide prodrugs of 9-aminocamptothecin were synthesized. Prodrug 4, in which 9-amino-
camptothecin was connected to glucuronic acid by an aromatic spacer via a carbamate linkage,
was stable in both aqueous solution and human plasma. Prodrug 4 and its potassium salt 12
were 20-80-fold less toxic than 9-aminocamptothecin to human tumor cell lines. The
simultaneous addition of â-glucuronidase and 4 or 12 to tumor cells resulted in a cytotoxic
effect equal to that of 9-aminocamptothecin alone. Prodrugs 4 and 12 were over 80 and 4000
times more soluble than 9-aminocamptothecin in aqueous solutions at pH 4.0, respectively.
Compounds 4 and 12 may be useful for prodrug monotherapy of tumors that accumulate
extracellular lysosomal â-glucuronidase as well as for antibody-directed enzyme prodrug therapy
(ADEPT) of cancer.
In tr od u ction
20(S)-Camptothecin (1), an antitumor alkaloid first
isolated from Camptotheca acuminata (Nyssaceae) by
Wall and co-workers in 1966,1 inhibits the activity of
topoisomerase I and displays antitumor activity in
various experimental tumor models.2 Camptothecin,
however, is difficult to formulate due to its poor water
solubility. Several research teams have synthesized
camptothecin derivatives aimed at preserving the anti-
tumor properties of the parent compound while improv-
ing its safety and water solubility.3-7 Two water soluble
derivatives, topotecan (2) and irinotecan (CPT-11, 3),
are approved for clinical use.8,9 We have employed an
upon four criteria: (1) improved water solubility, (2)
alternate strategy to improve the water solubility of
stability in blood, (3) decreased cytotoxicity, and (4)
camptothecin and increase its tumor cell selectivity
susceptibility to defined enzymatic cleavage. Gluc-
based on the enzyme activation of prodrugs at tumor
uronide prodrug 4 was selected for synthesis because
cells as pioneered by Senter10,11 and Bagshawe.12,13 In
this approach, monoclonal antibodies are employed to
of the hydrophilicity of this functional group,21,22 the
ability of glucuronide prodrugs to be preferentially
target an enzyme to cancer cells which can activate
activated at tumor cells targeted with â-glucuronidase-
subsequently administered prodrug. Selective activation
of prodrugs at neoplastic cells can increase the concen-
antibody conjugates,21,23-28 and the strong antitumor
activity observed after combined treatment with gluc-
tration of active drug in tumors,14,15 reduce systemic
uronide prodrugs and â-glucuronidase-antibody conju-
gates16,29 or fusion proteins28 in animal models. In
addition, glucuronide prodrugs may be useful for cancer
prodrug monotherapy of tumors that overexpress30 or
accumulate lysosomal â-glucuronidase.31
toxicity,16 and allow bystander killing of antigen-nega-
tive cancer cells.17,18 Prodrugs can also be designed to
display increased water solubility for improved drug
formulation.19,20
The design of a camptothecin prodrug suitable for
targeted enzymatic activation at tumor cells was based
Ch em istr y
† This work was supported by Grant DOH87-HR-716 from the
National Health Research Institutes, Taipei, Taiwan.
* To whom correspondence should be addressed. Dr. Steve Roffler:
Institute of Biomedical Sciences, Academia Sinica, Yen Geo Yen Road,
Section 2, No. 128, Taipei, Taiwan. Tel: 886-22-652-3079. Fax: 886-
22-782-9142. E-mail: sroff@ibms.sinica.edu.tw. Prof. J i-Wang Chern:
School of Pharmacy, College of Medicine, National Taiwan University,
Ren Ai Road, Section 1, No. 1, Taipei, Taiwan. Tel: 886-22-393-9462.
Fax: 886-22-393-4221. E-mail: chern@jwc.mc.ntu.edu.tw.
‡ National Defense Medical Center.
Compound 4 in which 9-aminocamptothecin (5) and
glucuronidic acid are linked via a self-immolative car-
bamate spacer21,32 was selected as the target gluc-
uronide prodrug because of preliminary results showing
poor yield of prodrug in which glucuronic acid is directly
linked to 10-hydroxycamptothecin and the superior
enzymatic hydrolysis of prodrugs containing spacers.21,32
9-Aminocamptothecin (5) was prepared by published
procedures.33 10-Hydroxycamptothecin was nitrated,
§ Academia Sinica.
∇ National Taiwan University.
10.1021/jm990124q CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/24/1999

3624 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Leu et al.
Ta ble 1. Aqueous Solubility of Drugsa
solubility (mg/mL)
pH 4.0 pH 7.0
Sch em e 1a
drug
5
4
12
0.006
0.5
25
0.06
110
NDb
a
The solubility of 9-aminocamptothecin (5), prodrug 4, and its
b
potassium salt 12 were determined at pH 4.0 and 7.0. Not
determined.
a
Reagents and conditions: (i) TiBr4, CH2Cl2, rt, 4 h; (ii)
p-hydroxybenzaldehyde, Ag2O, CH3CN, rt, 2 h, 42%; (iii) NaBH4,
IPA, CHCl3, silica gel, 0 °C, 1 h, 90%; (iv) CH3ONa, MeOH, rt, 2
h, 60.2%.
Sch em e 2a
F igu r e 1. â-Glucuronidase hydrolysis of 4. Prodrug 4 was
incubated at pH 7.0 with 0.05 µg/mL â-glucuronidase at 37
°C. The concentrations of 4 (0) and 9-aminocamptothecin (5)
(O) in aliquots removed at the indicated times were determined
by HPLC. The mean values of triplicate determinations are
shown. Bars, SE.
Ta ble 2. Hydrolysis of 4 by â-Glucuronidasea
â-glucuronidase (µg/mL)
% conversionb
0.5
0.1
0.05
0.01
92
98
92
45
a
Reagents and conditions: (i) 4-nitrophenyl chloroformate, 1,4-
a
dioxane, 60 °C, 1 h; (ii) 9, DMAP, CH3CN, 80 °C, 2 h, 32.3%; (iii)
KOSiMe3, THF, rt, 2 h, 62%; (iv) 1 N HCl, rt, 2 h, 50%.
Compound
4 (25 µM) was incubated with the indicated
b
concentrations of â-glucuronidase at pH 7.0 for 2 h at 37 °C. The
percentage of 4 converted to 9-aminocamptothecin (5).
and the resulting 9-nitro-10-hydroxycamptothecin was
treated with tosyl chloride followed by palladium-
catalyzed reduction to afford 9-aminocamptothecin (5)
in an overall yield of 80%. Methyl 1,2,3,4-tetra-O-acetyl
â-D-glucuronate (6), prepared as described,34 was bro-
minated with TiBr4 and then reacted with p-hydroxy-
benzaldehyde in the presence of Ag2O to afford 7
(Scheme 1). The aldehyde group in 7 was hydrogenated
to benzyl alcohol by treatment with NaBH4 to yield 8
in 90% yield. The O-acetyl groups of 8 were deprotected
with CH3ONa in MeOH to yield 9. 9-Aminocamptoth-
ecin (5) was reacted with nitrophenyl chloroformate
before addition of 9 for 1 day at 80 °C to afford 11 in an
overall yield of 32.2% (Scheme 2). The methyl ester 11
was deprotected with 1 equiv of potassium trimethyl-
silanate in THF and purified by reverse phase MPLC
to obtain the potassium salt 12. Acidification with 1 N
HCl yielded target compound 4.
12 displayed excellent solubility even at pH 4.0. Incuba-
tion of prodrug 4 or the potassium salt 12 at 37 °C in
PBS (phosphate-buffered saline, pH 7.0) or 90% human
serum in PBS revealed that the prodrugs were stable
for at least 48 h (results not shown).
Prodrug 4 (100 µM) was readily cleaved by 0.05 µg/
mL â-glucuronidase (0.18 nM) at pH 7.0 (Figure 1). Near
quantitative conversion of 25 µM 4 to 5 was acheived
in 2 h by addition of 0.05 µg/mL or higher concentrations
of â-glucuronidase (Table 2).
The cytotoxicity of 1, 4, 5, and 12 to five human tumor
cell lines was determined by measuring [3H]thymine
incorporation into cellular DNA after 72 h of drug
exposure. Comparison of IC50 values revealed that
prodrug 4 and its potassium salt 12 were 20-80-fold
less toxic than 5 (Table 3). Simultaneous addition of
â-glucuronidase (5 µg/mL) and 4 or 12 to tumor cells
resulted in a cytotoxic effect similar to 5 alone (Figure
2, Table 3), indicating efficient enzymatic cleavage of
the glucuronide functional group and release of 9-amino-
camptothecin (5).
Biologica l Da ta
9-Aminocamptothecin (5) was poorly soluble in aque-
ous solution at both acid and neutral pH (0.006 mg/mL
at pH 4.0 and 0.06 mg/mL at pH 7.0). In contrast, 4
was 80 times more soluble at pH 4.0 and 1800 times
more soluble at pH 7.0 (Table 1). The potassium salt
Discu ssion
We attempted to synthesize a glucuronide prodrug of
9-aminocamptothecin. Prodrug 4, in which glucuronic

Glucuronide Derivatives of Camptothecin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3625
Ta ble 3. Cytotoxicity of Drugs to Human Tumor Cells
In contrast to topotecin, 9-aminocamptothecin (5) is not
a substrate of the p-glycoprotein transporter involved
in multidrug resistance.44 Conversion of the water-
soluble glucuronides 4 or 12 to 9-aminocamptothecin (5)
at tumor cells may thus possess advantages for the
IC50 (nM)a
drug
Colo 205
CaSki
HepG2
HT29
H928
5
1
4
6.6
5.4
200
8.0
460
8.2
8.5
8.5
190
7.8
510
9.1
3.5
6.3
110
2.9
280
6.7
7.4
ND
150
6.6
560
11
7.9
7.8
300
7.9
ND
ND
treatment of tumors that overexpress p-glycoproteinMDR
.
4 + âGb
12
In summary, glucuronide prodrugs of 9-aminocamp-
tothecin (5) have been synthesized and shown to possess
the necessary prerequisites to be considered as candi-
dates for cancer prodrug monotherapy and antibody-
directed enzyme prodrug therapy of cancer.
12 + âGb
a
Concentrations of drugs that inhibited incorporation of [3H]-
thymidine into cellular DNA of human colorectal (Colo 205),
cervical (CaSki), hepatocellular (HepG2), colorectal (HT29), or lung
(H928) carcinoma cells by 50% after 72 h are indicated. Values
represent means of 1-3 experiments performed in triplicate with
Exp er im en ta l Section
b
coefficients of variation of <10%. â-Glucuronidase (âG, 5 µg/mL)
Ch em istr y. Melting points were obtained on an Electro-
thermal apparatus and are uncorrected. 1H and 13C nuclear
magnetic resonance spectra were recorded either on a J EOL
J NM-EX400 spectrometer at the National Taiwan University
or on a Bruker model AM 300 spectrometer at the National
Defense Medical Center, Taipei, and are reported in parts per
million on a δ scale with DMSO-d6 as internal standard.
Chemical shifts are reported as δ values in parts per million.
The splitting pattern abbreviations are as follows: s ) singlet,
d ) doublet, dd ) doublet doublet, dt ) doublet triplet, t )
triplet, br ) broad, m ) multiplet. EI mass spectra were
recorded on a J EOL J MS-D100 mass spectrometer at the
National Taiwan University. Elemental analysis for C, H, and
N were carried out on a Heraeus elemental analyzer at the
Cheng-Kong University, Tainan, or a Perkin-Elmer 240 el-
emental analyzer at the National Taiwan University, Taipei,
and were within 0.4% of the theoretical values. The thin-layer
chromatographic analyses were performed using precoated
silica gel (60F254) plates, and the spots were examined with
UV light and phosphomolybdic acid spray. Column chroma-
tography was carried out on Merck silica gel (70-230 mesh).
9-Am in oca m p toth ecin (5).33 A solution of 9-nitro-10-
tosylcamptothecin (1.45 g, 2.57 mmol) in dioxane (80 mL) was
mixed with Pd(OAc)2 (144 mg, 0.64 mmol) and P(Ph)3 (674 mg,
2.57 mmol) at room temperature under an argon atmosphere.
The temperature was raised to 90 °C, and 1 M triethylammo-
nium formate in dioxane (30 mL) was added dropwise over a
period of 1.5 h. The mixture was cooled to room temperature,
diluted with CHCl3, and washed with water. After the usual
work up, the crude product was purified by column chroma-
tography on silica gel (MeOH-CHCl3, 5:95) to give compound
was added with drugs.
F igu r e 2. Cytotoxicity of drugs to CaSki human cervical
carcinoma cells. CaSki cells were exposed to drugs with or
without â-glucuronidase (âG) for 72 h before the incorporation
of [3H]thymidine into cellular DNA was measured. Results
represent mean values of triplicate determinations. Bars, SE.
acid was coupled to 9-aminocamptothecin (5) via a self-
immolative carbamate linker, as well as the potassium
glucuronide salt 12, displayed properties suitable for
cancer prodrug monotherapy and ADEPT including
resistance to nonspecific cleavage, differential toxicity
between parent drug and prodrug, and susceptibility to
â-glucuronidase hydrolysis. The prolonged stability of
4 in human serum indicates that the carbamate linker
is resistant to enzymatic cleavage and confirms that
â-glucuronidase activity is low in human serum at
physiological pH values.35 Prodrugs 4 and 12 displayed
reduced toxicity compared with 5 to a variety of human
tumor cells as found for other glucuronide pro-
drugs.21,23,26,36-38 Reduced systemic toxicity of 4 or 12
may allow administration of curative doses of 9-amino-
camptothecin (5), currently difficult due to the high
sensitivity of human myeloid progenitor cells to camp-
tothecin and its derivatives.39
The potassium salt 12 exhibited high aqueous solubil-
ity (25 mg/mL) at pH 4.0. Formulation of camptothecins
at acidic pH is important to prevent opening of the
lactone ring and formation of the inactive carboxylate
form of camptothecin.40 The solubitiy of 12 compares
favorably with topotecan (1.02 mg/mL at pH 5).3,6 The
high solubility of the glucuronic acid prodrug 4 at pH
7.0 (109 mg/mL) indicates that the prodrug will not
precipitate after intravenous administration.
1
5 (884 mg, 94.5%): EI-MS m/z 363 (M+); H NMR (300 MHz,
DMSO-d6) δ 0.88 (t, 3H, J ) 7.1 Hz, CH3), 1.85 (m, 2H, CH2),
5.28 (s, 2H, CH2), 5.43 (s, 2H, CH2), 6.13 (s, 2H, NH2), 6.52 (s,
1H, OH), 6.81 (d, 1H, J ) 9.0 Hz, ArH), 7.30 (s, 1H, ArH),
7.34 (d, 1H, J ) 8.4 Hz, ArH), 7.53 (m, 1H, ArH), 8.85 (s, 1H,
ArH); 13C NMR (75 MHz, DMSO-d6) δ 8.2, 30.7, 50.6, 65.6,
72.8, 96.8, 108.9, 116.6, 117.9, 119.0, 126.9, 127.5, 131.6, 146.1,
149.6, 150.4, 152.2, 157.3, 163.3, 172.9. Anal. (C20H17N3O4) C,
H, N.
Meth yl 1,2,3,4-Tetr a -O-a cetyl-â-D-glu cop yr a n u r on a te
(6). Glucuronic acid γ-lactone (88 g, 0.5 M) was added to MeOH
(500 mL) that contained CH3ONa (0.75 g). The mixture was
stirred at room temperature for 60 min before MeOH was
removed under reduced pressure. The syrup was dissolved in
acetic anhydride (340 mL), and HClO4 (1.5 mL) in acetic
anhydride (10 mL) was added dropwise such that the reaction
temperature never exceeded 40 °C. The mixture was stirred
24 h at room temperature and the solution stored overnight
at 4 °C to yield 64.7 g (34.4%) of crystalline material. The
mother liquor was poured onto 1 kg of crushed ice and
neutralized with NaHCO3. Excess NaHCO3 was removed by
filtration, and the filtate was extracted with CHCl3. The CHCl3
extract was dried with anhydrous Na2SO4 and concentrated
to a syrup. Upon storage at 4 °C an additional 52 g of crude
crystalline material was obtained, resulting in compound 6 in
an overall yield of 62%. The crystals were recrystallized once
from hot MeOH: mp 140 °C [lit.34 137-138 °C]; 1H NMR (300
MHz, DMSO-d6) δ 1.97 (s, 3H, CH3), 2.00-2.01 (m, 6H, CH3),
9-Aminocamptothecin (5) has displayed promising
activity in preclinical models and initial clinical trials.41-43

3626 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Leu et al.
2.08 (s, 3H, CH3), 3.63 (s, 3H, OCH3), 4.68 (d, 1H, J ) 9.8 Hz,
sugar-H), 4.94-5.04 (m, 2H, sugar-H), 5.52 (t, 1H, J ) 9.5 Hz,
sugar-H), 6.02 (d, 1H, J ) 8.1 Hz, sugar-H); 13C NMR (75 MHz,
DMSO-d6) δ 20.5, 20.6, 20.7, 20.8, 53.0, 69.1, 70.1, 71.2, 71.7,
90.9, 167.3, 169.1, 169.4, 169.7, 169.8. Anal. (C15H20O11) C, H,
N.
P ota ssiu m N-[9-(â-D-Glu cu r on yl)ben zyloxyca r bon yl]-
a m in oca m p toth ecin (12). Compound 11 (125 mg, 0.18 mmol)
was mixed with potassium trimethylsilanolate (60 mg, 0.45
mmol) in anhydrous THF (25 mL) at ambient temperature
under nitrogen for 2 h. The yellow solid was filtered under
nitrogen, dissolved in water, and washed with CHCl3. Purifi-
cation of the water layer by reverse phase column chromatog-
raphy on silica gel (CH3CN-H2O, 3:1) gave compound 12 (80
Meth yl 1-(4-F or m ylp h en yl)-2,3,4-tr i-O-a cetyl-â-D-glu -
cop yr a n u r on a te (7). A solution of compound 6 (1 g, 2.66
mmol) and TiBr4 (1.17 g, 3.19 mmol) in CH2Cl2 (25 mL) was
stirred at room temperature for 24 h. The mixture was washed
with ice-water and NaHCO3 solution, dried with Na2SO4, and
evaporated to dryness to give 0.82 g of solid. The solid was
dissolved in CH3CN (100 mL) before adding p-hydroxybenzal-
dehyde (275 mg, 2.55 mmol) and Ag2O (712 mg, 3.07 mmol)
at room temperature for 2 h. The material was separated by
column chromatography on silica gel (EtOAc-hexane, 2:3) to
mg, 62%): LC-MS, m/z 728 (M+), 746 (M+ + H2O), 690 (M+
-
K+ + 1), 708 (M+ - K+ + 1 + H2O); 1H NMR (200 MHz, D2O)
δ 0.89 (t, 3H, J ) 7.0 Hz, CH3), 1.99-2.05 (m, 1H, CH2), 2.15-
2.26 (m, 1H, CH2), 3.40-3.45 (m, 3H, sugar-H), 3.68 (d, 1H, J
) 8.5 Hz, sugar-H), 4.39 (s, 2H, CH2), 4.90 (s, 2H, CH2), 5.01
(m, 3H, CH2 & sugar-H), 6.97 (d, 2H, J ) 8.2 Hz, ArH). 7.26-
7.35 (m, 6H, ArH), 7.89 (s, 1H, ArH). Anal. (C34H31N3O14K2 +
7H2O) C, H, N.
N -[9-(â-D-G lu c u r o n y l)b e n zy lo x y c a r b o n y l]a m in o -
ca m p toth ecin (4). The water layer of compound 12 (80 mg,
0.11 mmol) was acidified with 1 N HCl, and compound 4 was
precipitated in an ice bath and collected by filtration. The crude
product was purified by reverse phase column chromatography
on silica gel (20% CH3CN-H2O) to give compound 4 (38 mg,
50%): LC-MS, m/z 690.3 (M+, lactone form), 706.3 (M+,
carboxylate form); 1H NMR (200 MHz, CD3CN + D2O) δ 0.89
(t, 3H, J ) 7.2 Hz, 19-CH3), 1.86-1.90 (m, 2H, 18-CH2), 3.45-
3.47 (m, 3H, H-2′,4′,5′), 3.84-3.90 (m, 3H, ArCH2 & H-3′), 4.98
(d, 1H, J ) 6.7 Hz, H-1′), 5.11 (s, 2H, 5-CH2), 5.15 (s, 2H, 17-
CH2), 7.05 (d, 2H, J ) 8.4 Hz, ArH), 7.34 (d, 2H, J ) 8.4 Hz,
ArH), 7.49 (s, 1H, C14-H), 7.71-7.75 (m, 2H, C11,12-H), 7.94
(d, 1H, J ) 7.7 Hz, C10-H), 8.59 (s, 1H, C7-H). Anal.
(C34H31N3O13 + 5.5 H2O) C, H, N.
Biologica l Tests. 1. HP LC An a lysis. Drugs were analyzed
by high-pressure liquid chromatography (HPLC). Briefly, 20
µL of sample was injected onto a reversed phase column
(Hypersil C18, 4.6 mm inside diameter, 250 mm length, 5 µm
particule size) using a moble phase (1 mL/min) of 45% MeOH
and 25 mM phosphate buffer (pH 2.55) for compounds 4, 5,
and 12. Eluted compounds were detected on a Gilson model
121 fluorometer (excitation: 397 nm, emission: 482 nm). Peak
areas were analyzed with Beckman System Gold software.
Calibration curves were obtained by plotting the peak area of
standards as a function of drug concentration. The retention
times of 5 and 4 were 9.5 and 15.8 min, respectively. The
recoveries of 4 and 5 from 90% human plasma were greater
than 90%.
2. Dr u g Solu bility. Drug solubilities were determined in
âG buffer (100 mM acetic acid, 50 mM bis-tris, 50 mM
triethanolamine, pH 7.0) or phosphate buffer (100 mM, pH
4.0) by equilibrating an excess of solid compound in 0.25 mL
of buffer at 25 °C for 24 h. The samples were filtered through
a 0.22 µm Millipore filter, diluted in HPLC mobile phase, and
analyzed by HPLC.
3. En zym a tic Clea va ge of P r od r u gs. Compound 4 (100
µM or 25 µM) was incubated with the indicated concentration
of âG in 30 µL of âG buffer containing 0.1 µg/mL bovine serum
albumin at 37 °C for 2 h. The reaction was stopped by addition
of 700 µL of ice-cold MeOH/5% H3PO4 (6/1) to precipitate
proteins. The samples were centrifuged at 14 000 rpm for 5
min, and the supernatants were analyzed by HPLC.
4. In Vitr o Cytotoxicity. Exponentially growing tumor
cells at a density of 1-1.5 × 104 cells/well in RPMI medium
containing 10% bovine serum, 100 U/mL penicillin, and 100
µg/mL streptomycin were incubated in a 96-well microtiter
plate for 72 h (37 °C, 5% CO2, humidity) with various
concentrations of drug. Camptothecin and 9-aminocamptoth-
ecin were dissolved in DMSO such that the final concentration
of DMSO in wells did not exceed 0.5%. Compounds 12 and 4
were dissolved in medium. Control wells consisted of cells
exposed to either medium or 0.5% DMSO in medium. âG,
added at 5 µg/mL in some experiments, was not toxic by itself
to cells. Triplicate wells were prepared for each drug concen-
tration and for the controls. After 72 h, cells were washed once
with sterile PBS, and then pulsed for 12 h with [3H]thymine
(1 µCi/well) in complete medium. Medium was removed, and
1
give compound 7 (528 mg, 42%). H NMR (300 MHz, DMSO-
d6) δ 2.02 (s, 9H, CH3), 3.63, (s, 3H, OCH3), 4.76 (d, 1H, J )
9.9 Hz, sugar-H), 5.05-5.18 (m, 2H, sugar-H), 5.48 (t, 1H, J
) 9.6 Hz, sugar-H), 5.85 (d, 1H, J ) 7.7 Hz, sugar-H), 7.19 (d,
2H, J ) 7.4 Hz, ArH), 7.91 (d, 2H, J ) 7.4 Hz, ArH), 9.91 (s,
1H, COH); 13C NMR (75 MHz, DMSO-d6) δ 20.6, 20.7, 20.8,
53.0, 69.2, 70.7, 71.3, 71.4, 96.5, 116.8, 131.9, 132.2, 161.0,
167.3, 169.4, 169.7, 169.9, 191.9. Anal. (C20H22O11) C, H, N.
Meth yl 1-(4-Hyd r oxym eth ylp h en yl)-2,3,4-tr i-O-a cetyl-
â-D-glu cop yr on u r on a te (8). A solution of compound 7 (528
mg, 1.2 mmol) in IPA/CHCl3 (1:5) (100 mL) was stirred with
NaBH4 (123 mg, 2.76 mmol) and silica gel (5 g) at 0 °C for 1 h.
The reaction was quenched with water and filtered to remove
silica gel. The organic layer was dried with anhydrous Na2-
SO4 and evaporated under reduced pressure to give a residue
which was washed with EtOH to produce compound 8 (475
mg, 90%): 1H NMR (300 MHz, DMSO-d6) δ 1.99-2.02 (m, 9H,
CH3), 3.63 (s, 3H, OCH3), 4.43 (d, 2H, J ) 5.7 Hz, CH2), 4.69
(d, 1H, J ) 10 Hz, sugar-H), 5.02-5.16 (m, 3H, sugar-H &
OH), 5.47 (t, 1H, J ) 9.6 Hz, sugar-H), 5.62 (d, 1H, J ) 8.0
Hz, sugar-H), 6.94 (d, 2H, J ) 8.3 Hz, ArH), 7.26 (d, 2H, J )
8.5 Hz, ArH); 13C NMR (75 MHz, DMSO-d6) δ 20.6, 20.7, 20.8,
53.0, 69.4, 70.9, 71.3, 71.4, 97.6, 106.6, 116.5, 128.3, 137.0,
155.5, 167.5, 169.4, 169.7, 169.9. Anal. (C20H24O11) C, H, N.
Meth yl 1-(4-Hyd r oxym eth ylp h en yl)-â-D-glu cop yr a n u -
r on a te (9). Compound 8 (1.37 g, 3.11 mmol) was dissolved in
anhydrous MeOH (100 mL). CH3ONa (168 mg,3.11 mmol) was
added at 0 °C for 30 min and at room temperature for 2 h.
The mixture was quenched with water, extracted with CH2-
Cl2, and purified by column chromatography on silica gel
(EtOAc) to give compound 9 (590 mg, 60.2%): MS m/z 314.255
1
(FAB+); H NMR (200 MHz, DMSO-d6) δ 3.22-3.42 (m, 3H,
sugar-H), 3.61 (s, 3H, OCH3), 4.09 (d, 1H, J ) 8.6 Hz, sugar-
H), 4.37 (d, 2H, J ) 5.2 Hz, CH2), 5.0 (d, 1H, J ) 7.1 Hz, sugar-
H), 5.04 (s, 1H, OH), 5.24 (s, 1H, OH), 5.38-5.43 (m, 2H, OH),
6.92 (d, 2H, J ) 8.0 Hz, ArH), 7.19 (d, 2H, J ) 8.0 Hz, ArH);
13C NMR (50 MHz, DMSO-d6) δ 53.0, 63.0, 72.3, 73.9, 76.0,
76.5, 101.2, 117.5, 129.2, 137.3, 156.4, 170.2.
Meth yl N-[9-(â-D-Glu cu r on yl)ben zyloxycar bon yl]am in o-
ca m p toth ecin (11). A solution of 9-aminocamptothecin (200
mg, 0.55 mmol) in 1,4-dioxane (100 mL) that contained
pyridine (5 drops) and 4-nitrophenyl chloroformate (135 mg,
0.66 mmol) was stirred at 60 °C for 1 h. The mixture was then
stirred at 80 °C for 1 h before addition of compound 9 (350
mg, 1.16 mmol). After being stirred at 80 °C for an additional
1 h, the mixture was cooled to room temperature, quenched
with water, and extracted with CHCl3. The crude product was
purified by column chromatography on silica gel (MeOH-
CHCl3, 1:9) to give compound 11 (125 mg, 32.3%): LC-MS m/z
1
353 (M+ + 1); H NMR (200 MHz, DMSO-d6) δ 0.86 (t, 3H, J
) 7.0 Hz, CH3), 1.86 (m, 2H, CH2), 3.23-3.50 (m, 3H, sugar-
H), 3.66 (s, 3H, OCH3), 4.08 (d, 1H, J ) 8.6 Hz, sugar-H), 5.16
(s, 2H, CH2), 5.30 (s, 2H, CH2), 5.45-5.52 (m, 3H, CH2 & sugar-
H), 6.56 (s, 1H, OH), 7.06 (d, 2H, J ) 8.4 Hz, ArH), 7.36 (s,
1H, ArH), 7.43 (d, 2H, J ) 8.2 Hz, ArH), 7.81 (m, 1H, ArH),
8.00 (m, 1H, ArH), 8.83 (s, 1H, ArH), 9.93 (s, 1H, ArH), 11.14
(br s, 1H, NH).

Glucuronide Derivatives of Camptothecin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3627
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the wells were washed once with PBS before trypsinized cells
were harvested and counted for radioactivity in a Topcount
liquid scintillation counter. The coefficient of variation for
triplicate determinations was <10%. IC50 values were calcu-
lated from interpolation of logarithmic dose-response curves.
Ack n ow led gm en t. We thank Dr. Muh-Hwan Su of
the Institute of Pharmacy, National Defense Medical
Center, Taipei, Taiwan, for helpful suggestions and
advice.
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