Synthesis and preliminary cytotoxicity study of glucuronide derivatives of CC-1065 analogues

Article abstract of DOI:10.1016/S0968-0896(02)00603-X

Glucuronide derivatives of CBI-bearing CC-1065 analogues have been synthesized, and their cytotoxicities tested against U937 leukemia cells. The new compounds show potent antitumor activity in vitro. Compounds 1 and 2, and their corresponding glucuronides

Full text of DOI:10.1016/S0968-0896(02)00603-X

Bioorganic & Medicinal Chemistry 11 (2003) 1569–1575
Synthesis and Preliminary Cytotoxicity Study of Glucuronide
Derivatives of CC-1065 Analogues
Yuqiang Wang,* Huiling Yuan, Susan C. Wright, Hong Wang and James W. Larrick
Panorama Research, Inc., 2462 Wyandotte Street, Mountain View, CA 94043, USA
Received 15 February 2002; accepted 27 September 2002
Abstract—Glucuronide derivatives of CBI-bearing CC-1065 analogues have been synthesized, and their cytotoxicities tested against
U937 leukemia cells. The new compounds show potent antitumor activity in vitro. Compounds 1 and 2, and their corresponding
glucuronides 3 and 4 have IC50 values of 0.6, 0.1, 1.4 and 0.6 nM, respectively. Glucuronide 3 is approximately 2-fold less toxic than
its hydroxyl counterpart 1, and glucuronide 4 is approximately 6-fold less toxic than its hydroxyl counterpart 2. Glucuronides 3 and
4 may have limited use in the ADEPT approach. However, they may be used as antitumor agents in a conventional way.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
B-DNA within the minor groove with the sequence
preference for 50-d(A/GNTTA)-30 and 50-d(AAAAA)-
30, and alkylates the N3 position of the 30-adenine with
its left-hand CPI segment.5 CC-1065 also inhibits gene
transcription by interfering with binding of the TATA
box binding protein to its target DNA.6 Despite its high
potency and broad spectrum of antitumor activity, CC-
1065 cannot be used in humans because it causes
delayed death in experimental animals.7 To pursue
compounds possessing the potent antitumor activity but
devoid of the toxic side effects of the parent compound,
many CC-1065 analogues have been synthesized.8,9 To
take advantage of the potent antitumor activity of the
CC-1065 class of compounds and the ADEPT
approach, we chose to make glucuronide derivatives as
prodrug candidates (Fig. 2).
Anticancer agents generally act on metabolically active
or rapidly proliferating cells, and cannot distinguish
between cancer and normal cells. Thus, toxicity to nor-
mal cells limits the amount of drugs that can be given to
patients. Therefore, much research has focused on the
development of more specific therapeutic strategies to
reduce toxicity to normal cells. One approach is anti-
body-directed enzyme prodrug therapy (ADEPT).1,2 In
this approach, an enzyme is conjugated to a tumor-
specific antibody, which selectively localizes the enzyme
to the tumor cell surface. Subsequent administration of
a prodrug substrate of the enzyme leads to the enzyme-
catalyzed release of the free drug at the tumor site. This
strategy addresses the stoichiometry, controlled drug
release and poor antibody penetration problems asso-
ciated with the use of monoclonal antibody-drug con-
jugates.3 Since the drug is released enzymatically, a
single enzyme can generate a large amount of free drug
at the tumor site. Consequently, a small amount of
antibody can be used to reduce immunogenicity.
Glucuronide derivatives were selected because the level
of b-glucuronidase is higher in human breast cancers
than normal tissue,10 and glucuronide derivatives
appear to be less toxic than prodrugs incorporating
other chemical groups.11 Furthermore, glucuronide
derivatives should be stable in blood after iv adminis-
tration because the b-glucuronidase concentration in
human serum is very low.12 Although several organs
including liver, GI tract, spleen, and lung do contain
endogenous b-glucuronidases,13 mammalian tissues also
express uridine 50-diphosphoglucuronyl transferase, a
class of xenobiotic detoxification enzymes that can
reverse the reaction catalyzed by b-glucuronidase.14
It is important that the free drug in the ADEPT
approach is highly toxic. This will reduce the amount of
the monoclonal antibody required, thereby reducing
side effects. CC-1065 (Fig. 1) is among the most potent
antitumor agents discovered.4 It binds to double-stranded
*Corresponding author.
E-mail: yuqiangwang@worldnet.att.net
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00603-X

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Y. Wang et al. / Bioorg. Med. Chem. 11 (2003) 1569–1575
We previously demonstrated that a butyramido sub-
stituent on the right-hand side of the CC-1065 analo-
its glucuronate conjugate 12. Ethyl 5-nitroindole-2-
carboxylate, 5, was reduced to amine 6 by hydrogena-
tion over Pd/C. The latter was treated with g-butyr-
olactone to produce the key intermediate 7 with 44%
overall yield from 5. Basic hydrolysis of 7 using sodium
hydroxide solution afforded the key intermediate 8 with
65% yield. The carboxylic acid group of 8 was selec-
tively protected by treatment with benzyl bromide in the
presence of sodium hydrogen carbonate in DMFto
afford benzyl ester 9 with 71% yield. Through Koenigs–
Knorr reaction, 9 was transformed to 11 by treatment
with 10 catalyzed by silver triflate and silver carbonate.
Hydrogenation of 11 over Pd/C smoothly removed the
benzyl protective group, affording acid 12 with 66%
overall yield from 9.
gues
produced
compounds
with
enhanced
cytotoxicity.9b,h,n,15 Thus, a butyramido group was
selected to link together the sugar and the toxic
CC-1065 analogues. Glucuronides 3 and 4 are expected
to be less toxic than their hydroxyl counterparts 1 and 2.
However, the glucuronic acid moieties of 3 and 4 are
expected to be cleaved by b-glucuronidase after removal
of the ester groups on the sugar by carboxylate esterase
(Fig. 3). Glucuronides bearing methyl and acetyl groups
on the sugar may not be good substrates for b-glucur-
onidase. However, the ester groups of compounds 3 and
4 are expected to be cleaved by cellular carboxylate
esterase, which is abundant in human serum, and
the resulting products are expected to be substrates for
b-glucuronidase. Herein, we report synthesis and pre-
liminary cytotoxic effects of new glucuronide derivatives
of CBI-bearing CC-1065 analogues.
Scheme 2 illustrates synthesis of acid 18. Treatment of
amine 6 with (BOC)2O produced 13, which was then
hydrolyzed using NaOH followed by HCl neutralization,
Results and Discussion
Chemical synthesis
Target compound 4 can be dissected into three parts,
namely the left-hand CBI, the middle-indoles and the
right-hand glucuronate (Fig. 4). Because of the lengthy
process and low overall yield in the synthesis of CBI,
coupling the left-hand CBI with the indole(s)-glucur-
onate as the final synthetic step represents the best route
for us. Retro-synthetic analysis shows that the key
intermediate is 8. Scheme 1 illustrates synthesis of 8 and
Figure 3. Proposed in situ conversion of prodrugs to free drugs.
Figure 1. Structure of (+)-CC-1065.
Figure 4. Retro-synthetic analysis.
Figure 2. Structures of new agents.

Y. Wang et al. / Bioorg. Med. Chem. 11 (2003) 1569–1575
1571
affording acid 14 with 86% yield. Similar to the synth-
esis of 8, the carboxylic acid group of 14 was protected
as a benzyl ester by treatment with benzyl bromide in
the presence of sodium hydrogen carbonate in DMF
affording 15 with 84% yield. The protective group of 15
was removed by treatment with hydrochloride in ethyl
acetate, affording 16. The latter was coupled to 8 in the
presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodii-
mide (EDCI), affording 17 (60% yield from 15). The
benzyl protective group of 17 was removed by hydro-
genation over Pd/C, affording acid 18 with 84% yield.
Scheme 3 illustrates synthesis of acid 20. Acid 12 was
coupled to amine 16 using 1,3-dicyclohexylcarbodiimide
(DCC) and 1-hydroxybenzotriazol (HOBT) in DMF
produced 19 with 80% yield. The benzyl group of 19
was removed by hydrogenation over Pd/C to afford 20
with 86% yield.
Scheme 4 illustrates the final synthesis of free drugs, 1
and 2, and prodrugs, 3 and 4. A solution of 1,2,9,9a-
tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI), which
was synthesized according to a reported procedure, was
treated with hydrogen chloride in ethyl acetate to pro-
duce intermediate 21.16 The latter was coupled, respec-
tively, to acids 8, 12, 18, and 20 in the presence of EDCI
in DMF, affording the target compounds 1–4.
Cytotoxicity studies
Compounds 1–4 were tested against U937 monocytic
leukemia cells, and the results are shown in Table 1. The
IC50 values of compounds 1 and 2 are 0.6 and 0.1 nM,
respectively, against U937 cells in vitro. The IC50 values
of glucuronides 3 and 4 are 1.4 and 0.6 nM, respectively.
Glucuronide 3 is approximately 2-fold less toxic than its
hydroxyl counterpart 1, and glucuronide 4 is approxi-
mately 6-fold less toxic than its hydroxyl counterpart 2.
Scheme 1. Synthesis of 12.
Scheme 3. Synthesis of 20.
Scheme 4. Synthesis of 1–4.
Table 1. Cytotoxicity of compounds 1–4 against U937 leukemia cells
in vitroa
Compd
IC50 (nM)b
1
2
3
4
0.6
0.1
1.4
0.6
aCells were incubated with drugs for 48 h and the experiments were
performed according to our previously published method..9n
bIC50 values are defined as the minimal drug concentration necessary
to inhibit incorporation of [3H]thymidine by 50%, and are the avera-
ges of three experiments.
Scheme 2. Synthesis of 18.

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Y. Wang et al. / Bioorg. Med. Chem. 11 (2003) 1569–1575
The drugs are designed that the acetyl groups of the
glucuronide will be removed in-situ to generate a glu-
curonic acid derivative, which will be a substrate of
b-glucuronidase. When the glucuronide was first incu-
bated with carboxylate esterase and b-glucuronidase,
and then tested for cytotoxicity, surprisingly, we found
no difference between the cytotoxicity of the glucur-
onide with or without prior incubation with carboxylate
esterase and b-glucuronidase (data not shown). There
are two possibilities for this result. First, the acetyl
groups may not be removed by the esterase, and second,
even if the acetyl groups are removed, the glucuronic
acid derivative produced may not be a good substrate
for the b-glucuronidase. As a result, glucuronides 3 and
4 may not be good prodrug candidates for the ADEPT
approach. However, they may be used as antitumor
agents in a conventional way.
in methanol (150 mL) was treated with 3 N sodium
hydroxide solution (20 mL), and was stirred for 18 h at
room temperature. The solution was then concentrated
in vacuo. Water was added (100 mL). The solution was
neutralized with 20% hydrochloric acid, and the pre-
cipitate was filtered to afford 8 (723 mg, 65% yield) as a
brown solid, mp 194–195 ꢁC. H NMR (DMSO-d6) d
1
12.80 (brs, 1H, COOH), 11.60 (s, 1H, NH), 9.72 (s, 1H,
CONH), 7.99–7.02 (m, 4H, Ar-H), 3.47–3.43 (t, 2H,
J=6.8, HOCH2), 2.37–2.32 (t, 2H, J=7.4, COCH2),
1.77–1.73 (m, 2H, CH2CH2CH2). Anal. (C13H14N2O4),
C, H, N.
Benzyl 5-[(4-hydroxy)butyramido]indole-2-carboxylate (9).
A solution of 8 (810 mg, 3.1 mmol) in DMF(15 mL)
was treated with sodium bicarbonate (1.6 g, 19.0 mmol)
and benzyl bromide (0.54 mL, 4.5 mmol). The reaction
mixture was stirred for 18 h at room temperature. The
reaction was quenched with water (80 mL), and the
product was extracted with ethyl acetate (50 mL ꢂ 5).
The solution was dried using sodium sulfate, and was
concentrated in vacuo. The product was purified by
flash chromatography eluting with 50% hexane in ethyl
acetate to give 9 (778 mg, 71% yield) as a solid, mp 191–
Experimental
Chemistry
Melting points were measured using a Mel-Temp II and
are uncorrcted. 1H NMR spectra were recorded at
ambient temperature on an NT-360 spectrometer. High-
resolution mass spectra (FABHRMS) were recorded on
a modified MS50 mass spectrometer equipped with a
VG 11-250J data system. Elemental analyses were per-
formed by Atlantic Microlab, Inc., Norcross, GA,
USA, and the results were within ꢀ0.4% of the theore-
tical values unless otherwise noted. Analytical thin-layer
chromatography was performed on silica-coated plastic
plates (silica gel 60 F-254, Merck) and visualized under
UV light. Preparative separations were performed by
flash chromatography on silica gel (Merck, 70–
230 mesh). Tetrahydrofuran (THF) was dried by dis-
tillation from sodium-benzophenone ketyl. Dimethyl-
formamide (DMF) and triethylamine were dried over
molecular sieves (4A) before use. The above solvents
were stored over molecular sieves (4A). All other sol-
vents were used as received and were reagent grade,
where available.
192 ꢁC. H NMR (DMSO-d6) d 11.78 (s, 1H, NH), 9.73
1
(s, 1H, CONH), 8.00–7.14 (m, 9H, Ar–H), 5.37 (s, 2H,
CH2C6H5), 4.46–4.44 (t, 1H, J=5.2 Hz, OH), 3.47–3.42
(q, 2H, J=6.6, 11.7 Hz, HOCH2), 2.36–2.32 (t, 2H,
J=7.4, COCH2), 1.78–1.71 (m, 2H, CH2CH2CH2).
Anal. (C20H20N2O4+0.3H2O), C, H, N.
5-[4-(Methyl
2,3,4-tri-O-acetyl-1-deoxy-ꢀ-D-glucuro-
nate)butyramido]indole-2-carboxylic acid (12). Mole-
cular sieves (1 g) were added to a solution of 9 (778 mg,
2.21 mmol) in THF(20 mL) and CH Cl2 (20 mL), and
2
the temperature was cooled to ꢃ60 ꢁC under nitrogen.
Methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-a-d-glucur-
onate (10, 1.32 g, 3.3 mmol), silver trifluoro-
methanesulfonate (852 mg, 3.3 mmol) and silver
carbonate (922 mg, 3.3 mmol) was added sequentially,
and the reaction mixture was stirred for 10 min. The
temperature was allowed to warm up to room tempera-
ture, and the stirring was continued for an additional
2 h in the dark. The mixture was filtered through Celite,
and the filter cake was washed with ethyl acetate. The
solution was dried with sodium sulfate, and then was
concentrated in vacuo. The product was purified by
flash chromatography eluting with 50% hexane in ethyl
5-[(4-Hydroxy)butyramido]indole-2-carboxylic acid (8).
A solution of 5 (2.5 g, 10.7 mmol) in ethyl acetate
(200 mL) was treated with 10% palladium on activated
carbon (0.6 g), and was then hydrogenated for 1 h at
room temperature. The reaction mixture was filtered
through Celite, and the filter cake was washed with
ethyl acetate. The combined organic solution was con-
centrated in vacuo. Without further purification, the
resulting oil was dissolved in butyrolactone (20 mL),
and the solution was stirred for 18 h at 130 ꢁC. The
product was purified by flash chromatography eluting
with 30% hexane in ethyl acetate to give 7 (1.37 g, 44%
1
acetate to give 11 as white foam. H NMR (acetone-d6)
d 8.88 (s, 1H, CONH), 7.95 (s, 1H, Ar–H), 7.47–7.21
(m, 9H, Ar–H), 5.86–5.85 (d, 1H, J=4.5 Hz, sugar C1-
H), 5.38 (s, 2H, CH2C6H5), 5.22–5.21 (t, 1H, J=2.4 Hz,
sugar C2-H), 5.16–5.14 (m, 1H, sugar C4-H), 4.34–4.30
(m, 2H, sugar C3-H, C5-H), 3.76 (s, 3H, COOCH3),
3.63–3.59 (m, OCH2), 2.47–2.44 (t, 2H, J=6.7 Hz,
CH2CO), 2.17 (s, 3H, CH3CO), 2.08 (s, 3H, CH3CO),
2.03–2.00 (m, 2H, CH2CH2CH2), 1.75 (s, 3H, CH3CO).
Without further purification, the foam was dissolved in
ethyl acetate (15 mL). The mixture was treated with
10% palladium on activated charcoal (600 mg), and was
hydrogenated for 1 h at room temperature. The mixture
was filtered through Celite, and the filter cake was
washed with ethyl acetate. The combined organic
1
yield from 5) as a yellow solid. H NMR ( DMSO-d6) d
11.72 (s, 1H, NH), 9.73 (s, 1H, CONH), 8.00–7.07 (m,
4H, Ar-H), 4.47–4.44 (t, 1H, J=4.8 Hz, OH), 4.36–4.30
(q, 2H, J=7.0, 14.2 Hz, CH2CH3), 3.47–3.42 (q, 2H,
J=6.1, 11.4 Hz, HOCH2), 2.36–2.32 (t, 2H, J=7.4 Hz,
COCH2), 1.77–1.73 (m, 2H, CH2CH2CH2), 1.36–1.32 (t,
3H, J=7.2, CH2CH3). A solution of 7 (1.0 g, 3.4 mmol)

Y. Wang et al. / Bioorg. Med. Chem. 11 (2003) 1569–1575
1573
solution was concentrated in vacuo to give 12 (846 mg,
66% yield from 9) as a white foam, which was crystal-
lized from petroleum ether, mp 94–96 ꢁC. 1H NMR
(DMSO-d6) d 11.43 (s, 1H, NH), 9.69 (s, 1H, CONH),
7.94–6.91 (m, 4H, Ar–H), 5.88–5.87 (d, 1H, J=4.2 Hz,
sugar C1-H), 5.09–5.07 (t, 1H, J=3.2 Hz, sugar C2-H),
5.05–5.02 (m, 1H, sugar C4-H), 4.42–4.40 (d, 1H,
J=6.2 Hz, sugar C5-H), 4.36–4.35 (t, 1H, J=3.3 Hz,
sugar C3-H), 3.69 (s, 3H, COOCH3), 3.51–3.48 (t, 2H,
J=6.4 Hz, OCH2), 2.37–2.33 (t, 2H, J=7.6 Hz,
CH2CO), 2.05 (s, 3H, CH3CO), 2.02 (s, 3H, CH3CO),
1.82–1.79 (m, 2H, CH2CH2CH2), 1.65 (s, 3H, CH3CO).
FABHRMS calcd for C26H30N2O3+Na 601.1646;
found 601.1633.
ethyl acetate (20 mL). The reaction mixture was stirred
at room temperature for 10 min, and was filtered. The
solution was concentrated in vacuo to afford 16. With-
out further purification, the latter was dissolved in
DMF(1 mL), and treated with 8 (200 mg, 0.76 mmol)
and
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
(EDCI, 440 mg, 2.30 mmol). The reaction mixture was
stirred for 18 h at room temperature. The mixture was
then diluted with water (20 mL), and was extracted with
ethyl acetate (50 mL ꢂ 3). The organic layer was dried
with sodium sulfate, and concentrated in vacuo. The
product was purified by flash chromatography eluting
with ethyl acetate to give 17 (232 mg, 60% yield) as a
pale-yellow solid, mp 242–243 ꢁC. H NMR ( DMSO-
1
d6) d 11.87 (s, 1H, NH), 11.55 (s, 1H, NH), 10.08 (s, 1H,
CONH), 9.71 (s, 1H, CONH), 8.13–7.22 (m, 13H, Ar-
H), 5.39 (s, 2H, CH2C6H5), 4.47–4.45 (t, 1H, J=5.1 Hz,
OH), 3.48–3.44 (q, 2H, J=6.4, 11.9 Hz, HOCH2), 2.38–
2.33 (t, 2H, J=7.5 Hz, COCH2), 1.78–1.74 (m, 2H,
CH2CH2CH2). Without further purification, a solution
of 17 (230 mg, 0.45 mmol) in DMF(1 mL) and metha-
nol (20 mL) was treated with 10% palladium in acti-
vated carbon (20 mg), and hydrogenated for 1 h at room
temperature. The mixture was filtered through Celite,
and the filter cake washed with methanol. The com-
bined organic solution was concentrated in vacuo to
give 18 (160 mg, 84%) as a white solid, mp 249–251 ꢁC.
1H NMR (DMSO-d6) d 12.75 (s, 1H, COOH), 11.68 (s,
1H, NH), 11.54 (s, 1H, NH), 10.05 (s, 1H, CONH), 9.70
(s, 1H, CONH), 8.12–7.09 (m, 8H, Ar-H), 4.47–4.44 (t,
1H, J=5.4 Hz, OH), 3.49–3.44 (q, 2H, J=6.4, 11.0 Hz,
HOCH2), 2.38–2.34 (t, 2H, J=7.4 Hz, COCH2), 1.80–
1.74 (m, 2H, CH2CH2CH2). FABHRMS calcd for
C22H20N4O5 421.1511; found 421.1481.
Ethyl 5-(tert-butyloxycarbonylamino)indole-2-carboxylate
(13). A solution of 6, which was made from 5 (2.5 g,
10.7 mmol), in ethyl acetate (150 mL) was treated with
di-tert-butyl dicarbonate (5.8 g, 26.6 mmol), and the
reaction mixture was stirred for 18 h at room tempera-
ture. The reaction was quenched with water (40 mL),
and the ethyl acetate solution was separated. The solu-
tion was dried with sodium sulfate, and was then con-
centrated in vacuo to give 13 (2.61 g, 80%) as a yellow
solid, mp 190–192 ꢁC. H NMR (DMSO-d6) d 11.69 (s,
1
1H, NH), 9.13 (s, 1H, CONH), 7.79–7.04 (m, 4H, Ar-
H), 4.35–4.30 (q, 2H, J=6.8, 13.9 Hz, CH2CH3), 1.48 (s,
9H, C(CH3)3), 1.35–1.31 (t, 3H, J=6.7, CH2CH3).
Anal. (C16H20N2O4), C, H, N.
5-(tert-Butyloxycarbonylamino)indole-2-carboxylic acid
(14). A solution of 13 (2.61 g, 8.6 mmol) in methanol
(200 mL) was treated with 3 N sodium hydroxide solu-
tion (50 mL), and the reaction mixture was stirred for
18 h at room temperature. The solvent was removed.
Water (100 mL) was added. The solution was neu-
tralized with 20% hydrochloric acid, and the precipitate
was filtered to give 14 (2.03 g, 86%) as a yellow solid,
5-[5-[4-(Methyl 2,3,4-tri-O-acetyl-1-deoxy-ꢀ-D-glucuro-
nate)butyramido]-1H-indol-2-ylcarbonyl]amino]-1H-indol-
2-carboxylic acid (20). A solution of 16 (95 mg,
0.35 mmol) in DMF(1 mL) was treated with 12 (200 mg,
0.35 mmol), 1, 3-dicyclohexylcarbodiimide (DCC,
214 mg, 1.04 mmol) and 1-hydroxybenzotriazole
hydrate (HOBT, 140 mg, 1.04 mmol). The reaction mix-
ture was stirred at room temperature for 2 h, which was
then diluted with water (10 mL), and extracted with
ethyl acetate (50 mL ꢂ 3). The solution was dried with
sodium sulfate, and concentrated in vacuo. The product
was purified by flash chromatography eluting with a
solution of hexane and ethyl acetate (3/7, v/v) to afford
19 (230 mg, 80% yield) as a yellow solid, mp 143–
145 ꢁC. 1H NMR (DMSO-d6) d 11.87 (s, 1H, NH),
11.56 (s, 1H, NH), 10.08 (s, 1H, CONH), 9.73 (s, 1H,
CONH), 8.14–7.22 (m, 13H, Ar-H), 5.89–5.88 (d, 1H,
J=4.4 Hz, sugar C1-H), 5.39 (s, 2H, CH2C6H5), 5.09–
5.08 (t, 1H, J=2.9 Hz, sugar C2-H), 5.05–5.02 (m, 1H,
sugar C4-H), 4.43–4.41 (d, 1H, J=6.7 Hz, sugar C5-H),
4.37–4.35 (t, 1H, J=3.4 Hz, sugar C3-H), 3.69 (s, 3H,
COOCH3), 3.52–3.49 (t, 2H, J=6.4 Hz, OCH2), 2.39–
2.34 (t, 2H, J=7.7 Hz, COCH2), 2.05 (s, 3H, CH3CO),
2.03 (s, 3H, CH3CO), 1.84–1.80 (m, 2H, CH2CH2CH2),
1.66 (s, 3H, CH3CO). Without further purification, a
solution of 19 (230 mg, 0.28 mmol) in ethyl acetate
(35 mL) was treated with 10% palladium on activated
charcoal (20 mg), and was then hydrogenated for 1 h at
ꢁ
1
mp 192–193 C. H NMR (DMSO-d6) d 12.83 (brs, 1H,
COOH), 11.69 (s, 1H, NH), 9.13 (s, 1H, CONH), 7.79–
7.04 (m, 4H, Ar-H), 1.48 (s, 9H, C(CH3)3). Anal.
(C14H16N2O4+0.5H2O), C, H, N.
Benzyl 5-(tert-butyloxycarbonylamino)indole-2-carboxy-
late (15). A solution of 14 (2.03 g, 7.4 mmol) in DMF
(50 mL) was treated with sodium bicarbonate (1.8 g,
21.4 mmol) and benzyl bromide (4 mL, 33.6 mmol). The
reaction mixture was stirred for 18 h at room tempera-
ture. Water (50 mL) was added, and the product was
extracted with ethyl acetate (80 mL ꢂ 3). The solution
was dried with sodium sulfate, and was concentrated in
vacuo to give 15 (2.27 g, 84% yield) as a white solid, mp
180–182 ꢁC. H NMR (DMSO-d6) d 11.93 (s, 1H, NH),
1
7.51–7.02 (m, 9H, Ar-H), 5.38 (s, 2H, CH2C6H5), 1,48
(s, 9H, C(CH3)3). Anal. (C21H22N2O4+0.25H2O), C, H,
N.
5-[5-[4-(Hydroxy)butyramido]-1H-indol-2-ylcarbonyl]-
amino]-1H-indol-2-carboxylic acid (18). A solution of 15
(280 mg, 0.77 mmol) in ethyl acetate (20 mL) saturated
with hydrogen chloride was refluxed for 30 min. The
suspension was concentrated in vacuo to give a solid,
which was then treated with triethylamine (6 mL) in

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Y. Wang et al. / Bioorg. Med. Chem. 11 (2003) 1569–1575
room temperature. The mixture was filtered through
Celite, and the filter cake was washed with ethyl acetate.
The combined organic solution was concentrated in
vacuo to give 20 (176 mg, 86% yield) as a yellow oil,
which crystallized from ether on standing, mp 76–79 ꢁC.
1H NMR (DMSO-d6) d 12.81 (brs, 1H, COOH), 11.67
(s, 1H, NH), 11.54 (s, 1H, NH), 10.05 (s, 1H, CONH),
9.72 (s, 1H, CONH), 8.12–7.08 (m, 8H, Ar-H), 5.89–
5.88 (d, 1H, J=4.7 Hz, sugar C1-H), 5.09–5.08 (t, 1H,
J=3.0 Hz, sugar C2-H), 5.05–5.03 (m, 1H, sugar C4-H),
4.42–4.41 (d, 1H, J=6.6 Hz, sugar C5-H), 4.37–4.35 (t,
1H, J=3.2 Hz, sugar C3-H), 3.69 (s, 3H, COOCH3),
3.52–3.49 (t, 2H, J=5.8 Hz, OCH2), 2.39–2.34 (t, 2H,
J=7.8 Hz, COCH2), 2.05 (s, 3H, CH3CO), 2.03 (s, 3H,
CH3CO), 1.84–1.80 (m, 2H, CH2CH2CH2), 1,66 (s, 3H,
CH3CO). FABHRMS calcd for C35H36N4O14+Na
759.2126; found 759.2161.
10.5 (brs, 1H, OH), 9.84 (s, 1H, CONH), 8.25–7.23 (m,
9H, Ar-H), 5.99–5.98 (d, 1H, J=4.1 Hz, sugar C1-H),
5.22–5.20 (t, 1H, J=2.9 Hz, sugar C2-H), 5.15–5.13 (m,
1H, sugar C4-H), 4.90–4.84 (t, 1H, J=8.6, NCHH),
4.75–4.71 (dd, 1H, J=1.1, 12.2 Hz, NCHH), 4.51–4.48
(m, 2H, sugar C3-H, C5-H), 4.33–4.28 (m, 1H,
ClCH2CH), 4.14–4.10 (dd, 1H, J=2.6, 10.6 Hz,
ClCHH), 3.96–3.91 (dd, 1H, J=7.8, 10.9 Hz, ClCHH),
3.77 (s, 3H, COOCH3), 3.63–3.59 (t, 2H, J=6.9 Hz,
OCH2), 2.50–2.46 (t, 2H, J=7.1 Hz, COCH2), 2.10 (s,
3H, CH3CO), 2.08 (s, 3H, CH3CO), 1.96–1.90 (m, 2H,
CH2CH2CH2), 1,72 (s, 3H, CH3CO). FABHRMS
calcd for C39H40ClN3O13+Na 816.2147; found
816.2152.
3-[5-[5-[4-(Methyl 2,3,4-tri-O-acetyl-1-deoxy-ꢀ-D-glucuro-
nate) butyramino]-1H-indol-2-ylcarbonyl]amino]-1H-in-
dol-2-yl]carbonyl]-1-(chloromethyl)-5-hydroxy-1, 2-dihydro-
3H-benz[e]indole (4). Yellow solid (15% yield). 1H
NMR (DMF-d6) d 11.61 (s, 2H, 2 NH), 10.20 (s, 1H,
CONH), 9.79 (s, 1H, CONH), 8.38–7.27 (m, 13H, Ar-
H), 5.99–5.98 (d, 1H, J=4.6 Hz, sugar C1-H), 5.22–5.20
(t, 1H, J=3.1 Hz, sugar C2-H), 5.16–5.13 (m, 1H, sugar
C4-H), 4.91–4.86 (t, 1H, J=8.7 Hz, NCHH), 4.77–4.73
(dd, 1H, J=1.8, 11.4 Hz, CHHCl), 4.51–4.48 (m, 2H,
sugar C3-H, C5-H), 4.34–4.32 (m, 1H, ClCH2CH),
4.15–4.11 (dd, 1H, J=3.4, 11.2 Hz, ClCHH), 3.98–3.92
(dd, 1H, J=8.0, 11.1 Hz, ClCHH), 3.77 (s, 3H,
COOCH3), 3.63–3.59 (t, 2H, J=6.6 Hz, OCH2), 2.50–
2.46 (t, 2H, J=7.3 Hz, COCH2), 2.10 (s, 3H, CH3CO),
2.08 (s, 3H, CH3CO), 1.94–1.90 (m, 2H, CH2CH2CH2),
1,72 (s, 3H, CH3CO). FABHRMS calcd for
C48H46ClN5O14+Na 974.2628; found 974.2625.
3-[5-[(4-Hydroxy)butyramido]-1H-indol-20 -yl]carbonyl]-
1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole
(1). A solution of (ꢀ)-CBI (15 mg, 0.076 mmol) in 3 mL
of ethyl acetate saturated with hydrogen chloride was
stirred at room temperature for 30 min in the dark. The
suspension was concentrated in vacuo to give inter-
mediate 21. The latter was dissolved in DMF(0.5 mL),
and was treated with 8 (20 mg, 0.078 mmol) and EDCI
(16.5 mg, 0.086 mmol). The reaction mixture was stirred
at room temperature overnight, and the product was
purified by thin layer chromatography eluting 50%
hexane in ethyl acetate to give 1 (12 mg, 36% yield) as a
yellow solid. 1H NMR (acetone-d6) d 10.73 (s, 1H, NH),
9.27 (s, 1H, Ar-OH), 9.08 (s, 1H, CONH), 8.27–7.20 (m,
9H, Ar–H), 4.84–4.80 (m, 2H, NCH2), 4.33–4.26 (m,
1H, ClCH2CH), 4.09–4.05 (dd, 1H, J=3.6, 11.2 Hz,
ClCHH), 3.85–3.79 (dd, 1H, J=8.5, 11.2 Hz, ClCHH),
3.73–3.69 (t, 1H, J=5.6, CH2OH), 3.66–3.61 (q, 2H,
J=5.9, 11.5 Hz, CH2OH), 2.51–2.47 (t, 2H, J=7.1, Hz,
Cytotoxicity studies
Cell lines. The human monocytic leukemia, U937, was
obtained from the ATCC, and cultured in RPMI-1640
plus 10% FCS in the absence of antibiotics. The cells
were routinely tested for mycoplasma contamination,
and in all cases found to be negative.
COCH2),
1.92–1.89
(m,
2H,
CH2CH2CH2).
FABHRMS calcd for C26H25ClN3O4 478.1533; found
478.1527.
Compounds 2–4 were synthesized using a procedure
similar to that described for the synthesis of 1.
The drugs were dissolved in DMFto provide a stock
solution of l mg/mL, and were stored at ꢃ20 ꢁC. For
each experiment, drug solutions were freshly prepared
from the stock solution by addition of sterile water to
afford concentrations suitable for the experiment.
3-[5-[5-[4-(Hydroxy)butyramido]-1H-indol-2-ylcarbonyl]-
amino]-1H-indol-2-yl]carbonyl]-1-(chloromethyl)-5-hydroxy-1,
2-dihydro-3H-benz[e]indole (2). Yellow solid (19%
1
yield). H NMR (DMF-d6) d 11.74 (s, 1H, NH), 11.62
(s, 1H, NH), 10.47 (s, 1H, Ar–OH), 10.44 (s, 1H,
CONH), 9.89 (s, 1H, CONH), 8.43–7.06 (m, 13H, Ar-
H), 4.95–4.86 (m, 1H, NCHH), 4.76–4.72 (m, 1H,
NCHH), 4.60–4.54 (brs, 1H, CH2OH), 4.34–4.28 (m,
1H, ClCH2CH), 4.15–4.11 (dd, 1H, J=3.2, 10.7 Hz,
ClCHH), 3.98–3.93 (dd, 1H, J=8.2, 11.1 Hz, ClCHH),
3.66–3.64 (m, 2H, CH2OH), 2.52–2.48 (t, 2H, J=7.4,
Hz, COCH2), 1.92–1.85 (m, 2H, CH2CH2CH2).
FABHRMS calcd for C35H31ClN5O5 636.2014; found
636.2017.
The cytotoxic effects of the drugs were measured by
inhibition of DNA synthesis. U937 cells in RPMI-1640
plus 10% FCS medium were seeded at 5 ꢂ 104 cells/well
in a 96-well plate. Drugs (10 mL) at increasing con-
centrations were added to each well and the total
volume was adjusted to 0.1 mL/well using the same
medium. The plate was incubated for 24 h at 37 ꢁC fol-
3
lowed by addition of 10 mL of H-thymidine (20 mCi/
mL). The plate was incubated for another 24 h, and the
cells were harvested and radioactivity was counted using
the Packard Matrix 96 beta counter. The results are
expressed as the percent growth inhibition (IC50), cal-
culated as follows: percent growth inhibition
(IC50)=[(total cpm ꢃexperimental cpm)/total cpm] ꢂ
100.
3-[5-[4-(Methyl 2,3,4-tri-O-acetyl-1-deoxy-ꢀ-D-glucuronate)
butyramido]-1H-indol-20-yl]carbonyl]-1-(chloromethyl)-5-
hydroxy-1,2-dihydro-3H-benz[e]indole (3). Yellow solid
1
(12% yield). H NMR (DMF-d6) d 11.56 (s, 1H, NH),

Y. Wang et al. / Bioorg. Med. Chem. 11 (2003) 1569–1575
1575
Acknowledgements
1996. (c) Baraldi, P. G.; Cacciari, B.; Romagnoli, R.; Spalluto,
G.; Gambari, R.; Bianchi, N.; Passadore, M.; Ambrosino, P.;
Mongelli, N.; Cozzi, P.; Geroni, C. Anti-Cancer Drug Des.
1997, 12, 555. (d) Boger, D. L.; Garbaccio, R. M.; Jin, Q. J.
Org. Chem. 1997, 62, 8875. (e) Baraldi, P. G.; Cacciari, B.;
Guiotto, A.; Romagnoli, R.; Spalluto, G.; Zaid, A. N. Farm-
aco 1997, 52, 711. (f) Baraldi, P. G.; Cacciari, B.; Guiotto, A.;
Romagnoli, R.; Spalluto, G.; Zaid, A. N.; Capolongo, L.;
Cozzi, P.; Geroni, C.; Mongelli, N. Farmaco 1997, 52, 717. (g)
We wish to thank Jolande Murray for help with the
manuscript. This work was supported in part by a grant
from the National Institutes of Health (CA79357-01 to
Y.W).
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