Structural necessity of indole C5-O-substitution of seco-duocarmycin analogs for their cytotoxic activity

Article abstract of DOI:10.3390/molecules15117971

A series of racemic indole C5-O-substituted seco-cyclopropylindole (seco-CI) compounds 1-5 were prepared by coupling in the presence of EDCI of 1-(tertbutyloxycarbonyl)- 3-(chloromethyl)indoline (seg-A) with 5-hydroxy-, 5-O-methylsulfonyl, 5-O-aminosulfonyl, 5-O-(N,N-dimethylaminosulfonyl)- and 5-O-benzyl-1H-indole-2- carboxylic acid as seg-B. Compounds 1-5 were tested for cytotoxic activity against four human cancer cell lines (COLO 205, SK-MEL-2, A549, and JEG-3) using a MTT assay. Compounds 2 and 3 with small sized sulfonyl substituents like 5-O-methylsulfonyl and 5- O-aminosulfonyl exhibit a similar level of activity as doxorubicin against all cell lines tested.

Full text of DOI:10.3390/molecules15117971

Molecules 2010, 15, 7971-7984; doi:10.3390/molecules15117971
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Structural Necessity of Indole C5-O-Substitution of seco-
Duocarmycin Analogs for Their Cytotoxic Activity
Taeyoung Choi and Eunsook Ma *
College of Pharmacy, Catholic University of Daegu, Hayang, 712-702, Korea
* Author to whom correspondence should be addressed; E-Mail: masook@cu.ac.kr;
Tel. +82-53-850-3621; Fax: +82-53-850-3602.
Received: 28 September 2010; in revised form: 24 October 2010 / Accepted: 28 October 2010 /
Published: 8 November 2010
Abstract: A series of racemic indole C5-O-substituted seco-cyclopropylindole (seco-CI)
compounds 1-5 were prepared by coupling in the presence of EDCI of 1-(tert-
butyloxycarbonyl)-3-(chloromethyl)indoline (seg-A) with 5-hydroxy-, 5-O-methylsulfonyl,
5-O-aminosulfonyl, 5-O-(N,N-dimethylaminosulfonyl)- and 5-O-benzyl-1H-indole-2-
carboxylic acid as seg-B. Compounds 1-5 were tested for cytotoxic activity against four
human cancer cell lines (COLO 205, SK-MEL-2, A549, and JEG-3) using a MTT assay.
Compounds 2 and 3 with small sized sulfonyl substituents like 5-O-methylsulfonyl and 5-
O-aminosulfonyl exhibit a similar level of activity as doxorubicin against all cell
lines tested.
Keywords: duocarmycin; Indole C5-O-substituted seco-CI; cytotoxicity; DNA minor
groove alkylation
1. Introduction
Duocarmycins (DUMs) A, B1, B2, C1, C2, D1, D2 and SA and CC-1065 are members of an
extremely potent group of antitumor antibiotics isolated from Streptomyces sp. that contain the
cyclopropa[c]pyrrolo[3,2-e]indole (CPI) moiety as a common pharmacophore [1-3]. DUM B1, B2, C1,
C2, D1 and D2 are a seco-type of DUM A and are chemically more stable than DUM A (Figure 1).
The cytotoxicity of DUMs and CC-1065 results from the reaction of the CPI moiety with adenine-
N3 groups in the minor groove of AT-rich sequences of DNA [4]. Even though (+)-CC-1065 has

Molecules 2010, 15
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potential cytotoxic properties, its use as an anticancer drug is hampered by delayed lethal toxicity to
animals at therapeutic doses [5]. DUM SA seems to be superior to CC-1065 due to the lack of a
delayed fetal hepatotoxicity like that with (+)-CC-1065 [6]. Moreover, DUM SA is the most
hydrolytically stable member of this class of compounds [6-8], but it is toxic to the bone marrow [9].
Figure 1. Structures of duocarmycin SA, CC-1065, and compounds 1-5.
H2N
O
H3COOC
Seg. A
OH
H3C
HN
Seg. B
OCH3
HN
OCH3
N
O
NH
N
N
O
O
N
O
OCH3
OCH3
O
H
N
OH
OCH3
H
(+)-Duocarmycin SA
(+)-CC-1065
X
Cl
NH
NH
NH
R
N
HO
O
N
N
H
O
H
O
OH
1 : R = OH
2 : R = OSO2CH3
3 : R = OSO2NH2
(+)-CPI
(+)-CI
seco-CI
4 : R = OSO2N(CH3)2
5 : R = OBn
Therefore, there is a strong interest in the design and development of novel analogs of duocarmycin
that effectively kill cancer cells and have reduced toxicity to the host. One attempt [10,11] to design
novel analogs involves the synthesis of a spirocyclic 1,2,7,7a-tetrahydrocycloprop[1,2-c]indol-4-one
[(CI-numbering), CI] as the minimum size pharmacophore [12], which can be formed by ring closure
of a seco-CI parent (Scheme 1). Seco compounds possess a similar alkylating selectivity and efficiency
as the corresponding natural product (+)-enantiomers [13] since they can form the cyclopropane
moiety in situ by an intramolecular Winstein cyclization [14].
Scheme 1. Proposed mechanism of activation and DNA alkylation of the seco-CI moiety [14].
X
Ad
DNA
N
N
N
R
R
R
DNA
-HX
O
O
H+
OH
H

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Most duocarmycins exhibit more pronounced distinctions between enantiomers, but the spirocyclic
1,2,7,7a-tetrahydro-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]cycloprop[1,2-c]indol-4-one (CI-TMI)
has been observed to show similar biological potency and relative DNA alkylation efficiency [13,15].
There is no difference of cytotoxic activity between (+)-CI and (-)-CI enantiomers, therefore no
resolution is required to obtain a more active enantiomer.
Some previous results indicated that the seg-A ring structure influences the electrophilicity of
cyclopropane [16]. On the other hand, the seg-B one has been considered to play some important roles
in noncovalent binding to DNA [17-19]. Especially, the C5 position of seg-B was known as the most
important site potentiating the cytotoxic activity. An adequate indole C5 substituent may not only
extend the rigid length of the compound enhancing the DNA binding and thus accelerating the rate of
DNA alkylation, but also benefit close contacts within a minor groove hydrophobic pocket [20]. Boger
reported that a single 5-methoxy group on the indole as a minor groove binding unit was sufficient to
maintain potency and a series of dimethylaminoethoxy group substituted analogs retained the
cytotoxicity of the TMI analog, while providing increased aqueous solubility [16,21].
Indole C5 sulfone derivatives were synthesized and evaluated against the L1210 cell line and as a
result, the C5 methanesulfonyl derivatives proved exceptionally potent, being 15–20 fold more active
than the corresponding thiomethyl or methoxy derivatives [20]. Herein we describe the synthesis and
cytotoxic activity of indole-C5-O-substituted and O-sulfonyl seco-CI analogs as different minor
groove binding side chains.
2. Results and Discussion
2.1. Synthesis
In order to obtain indole C5-O-substituted-seco-CI derivatives, we synthesized 5-hydroxy- (10) and
5-benzyloxy-1H-indole-2-carboxylic acid (15) from 3-hydroxybenzaldehyde (6) (Scheme 2). Methyl
azidoacetate (7) was obtained from the reaction of methyl chloroacetate and sodium azide [22].
According to the method of Fukuda et al. [23], 3-hydroxybenzaldehyde was reacted with 7 to afford
the azidocinnamate ester 8. This compound was refluxed in xylene to synthesize methyl 5-hydroxy-
1H-indole-2-carboxylic acid (9, 42%) which was hydrolyzed to afford 5-hydroxy-1H-indole-2-
carboxylic acid (10). From compound 10, we tried to introduce a benzyl group at the 5-hydroxy
position, but obtained instead a N- and O-dibenzylated compound. In order to obtain the O-benzylated
compound, treatment of 6 with benzyl bromide and potassium carbonate yielded 11, which was
subsequently converted to regioisomeric indoles 13 and 14 in 40% and 45% yield using the same
conditions as for the preparation of 9. Coowar et al. reported that cyclization of an unsymmetrical
azidocinnamate ester provided two regioisomers in proportions depending upon the substitution
position. They obtained the 5-methoxy- and the 7-methoxyindole carboxylates from the 3-methoxy-
azidocinnamate in a 1:1 ratio, whereas the 5,6-dimethoxyindole carboxylate was obtained from the
3,4-dimethoxy derivative in a high yield [24]. Compounds 8 and 11 have substituents at same position,
therefore the outcome of cyclization of these compounds seemed to be dependent upon the substituent.
Alkaline hydrolysis of 14 gave 5-benzyloxy-1H-indole-carboxylic acid (15).

Molecules 2010, 15
Scheme 2. Synthesis of 5-hydroxy- and 5-O-benzyl-1H-indole-2-carboxylic acids 10 and 15.
7974
O
O
O
O
N3
OH
7
H
Xylene
H3CO
H3CO
H3CO
10% NaOH
Reflux, 1 h
N3
N
Na, abs. MeOH
-15→0℃, 18 h
O
60℃
H
OH
MeOH
OH
OH
HO
1h
9
8
6
N
H
O
10
O
Xylene
H
7
Benzyl bromide
6
H3CO
Na, abs. MeOH
-15→0℃, 18 h
Reflux, 1 h
K2CO3, Reflux, 8 h
N3
OBn
11
OBn
12
O
O
O
OBn
10% NaOH
MeOH
OBn
+
H3CO
H3CO
HO
60℃,
1h
N
H
N
N
H
H
OBn
14
15 (from 14)
13
Scheme 3 shows a refinement of the synthesis that allows incorporation of various minor groove
targeting units. We synthesized seco-5-benzyloxy-1-chloromethyl-N-BOC-indoline (seco-N-BOC-CI,
16) from 4-amino-3-nitrophenol in six steps with an overall yield of 37% by the method reported in
our earlier paper [25].
Scheme 3. Synthesis of indole C5-O-substituted seco-CI derivatives 1-5.
Cl
OH
N
HO
N
H
O
1
c)
Cl
Cl
Cl
a), b)
RCl,MC, Et3N
2 h, rt
OH
OR
N
N
BnO
BnO
N
10
BnO
Boc
N
16
O
R=SO2CH3
SO2NH2
N
O
H
H
18 : R=SO2CH3
19 : R=SO2NH2
20 : R=SO2N(CH3)2
17
c)
SO2N(CH3)2
c)
Cl
Cl
Cl
a), b)
OBn
OR
N
HO
N
N
Boc
HO
HO
15
N
O
N
O
H
21
H
5
2 : R=SO2CH3
a) 4M HCl/ Ethyl acetate, 1h, rt
b) EDCI, DMF, 14 h, rt
c) H2, 10% Pd-C, Acetone, 2 h
3 : R=SO2NH2
4 : R=SO2N(CH3)2
Deprotection of compound 16 under acidic conditions yielded the amine which was coupled with 5-
hydroxyindole-2-carboxylic acid (10) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)

Molecules 2010, 15
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carbodiimide (EDCI) to give 17. Debenzylation of 17 by catalytic hydrogenation in the presence of
10% Pd-C afforded compound 1 in 60% yield. Compound 17 was then reacted with methylsulfonyl-,
aminosulfonyl-, or N,N-dimethylaminosulfonyl chloride afford 18, 19, and 20 in 92%, 62%, and 72%
yield, respectively. Debenzylation of these compounds by catalytic hydrogenation in the presence of
10% Pd-C yielded compounds 2, 3 and 4 in 50%, 53%, and 66% yield, respectively. In order to obtain
indole-C5-O-benzyl-seco-CI, 16 was treated with 10% Pd-C to yield 21 and then reacted with 5-
benzyloxy-1H-indole-2-carboxylic acid (15) in the presence of EDCI to give 5 in 83% yield.
2.2. Cytotoxic activity
Cytotoxic activity was determined as IC50 values in four cell lines (COLO 205, SK MEL-2, A549
and JEG-3) for a 48 h drug exposure using a growth inhibition microassay. The results of the
evaluation of the in vitro cytotoxic activity of compounds 1-5 are presented in Table 1 and Figure 2.
Table 1. Cytotoxicity of indole C5-O-substituted seco-CI derivatives against cancer cell
lines by MTT assay.
Cl
R
N
N
H
O
HO
IC50 (µM)a)
SK-MEL-2
1.472
Compound
R
COLO 205
0.469
A549
0.499
2.709
1.250
0.365
>5
JEG-3
0.847
0.310
0.182
2.974
2.936
0.397
Doxorubicin
–
1
2
3
4
OH
0.374
0.528
0.448
1.279
0.629
0.419
0.301
1.137
OSO2CH3
OSO2NH2
OSO2N(CH3)2
OBn
5
0.690
0.764
0.298
a)
IC50 for 48 h drug exposure at pH 7.4. Stock solution were prepared in DMSO and diluted into
culture medium to give 1% final DMSO concentrations
C5-Hydroxy-1H-indol-2-ylcarbonyl-seco-CI (1, IC50: 0.374 µM) showed similar activity as
doxorubicin (IC50: 0.374 µM) against the COLO 205 cell line and was more active than doxorubicin
against both the SK-MEL-2 and A-549 cancer cell lines (IC50: 0.374 µM and 0.629 µM vs. 1.472 µM
and 0.847 µM). Indole C5-methylsulfonyloxy derivative 2 showed more activity than doxorubicin
against both the SK-MEL-2 and JEG-3 cancer cell lines (IC50: 0.419 µM and 0.182 µM). Indole C5-
aminosulfonyloxy derivative 3 showed more activity than doxorubicin against the SK-MEL-2 cancer
cell line (IC50: 0.301 µM). Overall, compounds 2 and 3 exhibited more activity than the others against
the SK-MEL-2 cancer cell line. Indole C5-N,N-dimethylaminosulfonyloxy derivative 4 was less active
than doxorubicin against all four cancer cell lines. As a result, it is thought that the indole C5-N,N-
dimethylaminosulfonylamino substituent of seco-CI did not contribute to the embedded minor groove
interaction. Indole C5-benzyloxy-1H-indole seco-CI 5 exhibited weaker activity than doxorubicin
against the SK-MEL-2, A549 and JEG-3 cancer cell lines (IC50: 0.764 µM, 0.298 µM and 0.397 µM).

Molecules 2010, 15
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Parrish et al. have reported that (+)-CBI-5-OH (1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-
one:CBI) and (+)-CBI-5-OBn were very active against the L1210 cancer cell line (IC50: 200 and
50 pM) [20]. This trend has been explained by the fact that inclusion of an extended aromatic unit in
the CI unit, as in the case of CBI, significantly enhances chemical stability and cytotoxicity [21]. In
conclusion, both the alkylation part (CBI unit) and a (+)-enantiomer are seem to be important factors
for enhanced activity compared with racemic and a seco-CI unit.
Figure 2. Cytotoxic activity of indole C5-O-substituted seco-CI derivatives 1-5.
COLO 205
SK-MEL-2
2.0
1.5
1.0
0.5
0.0
2.0
1.5
1.0
0.5
0.0
D
1
2
3
4
5
D
1
2
3
4
5
Compounds
Compounds
(D : Doxorubicin)
(D : Doxorubicin)
A549
JEG-3
6
5
4
3
2
1
0
7
6
5
4
3
2
1
0
D
1
2
3
5
D
1
2
3
4
5
Compounds
Compounds
(D : Doxorubicin)
(D : Doxorubicin)
3. Experimental
3.1. General
Moisture- or air-sensitive reactions were conducted under nitrogen in distilled solvents. The
commercial reagents were purchased from Aldrich, Fluka, or Sigma. Melting points were measured on
1
13
a Gallenkamp melting point apparatus and are not corrected. H- (400 MHz), C-NMR (100 MHz),
HSQC, and HMQC spectra were taken on a Varian AS 400 MHz spectrometer. Chemical shifts (δ) are
in parts per million (ppm) relative to tetramethylsilane, and coupling constants (J) are in Hertz.
GC/MS spectra were obtained on a Shimadzu MS-QP2010 mass spectrometer. The MPLC apparatus
used was a Yamazen YFLC-AI-580. Analytical TLC was performed on pre-coated silica gel 60 F254

Molecules 2010, 15
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plates (Merck). Column chromatography was carried out on Merck silica gel 9385 (230-400 mesh) and
the eluting solvent is indicated in each entry.
Methyl 5-hydroxy-1H-indole-2-carboxylate (9). According to the method of Fukuda et al. [3], to a
solution of metal Na (2.2 g, 94.35 mmol) in methanol (100 mL) was added a solution of 3-hydroxy-
benzaldehyde (6, 5.0 g, 23.58 mmol) and methyl azidoacetate (7, 10.9 g, 94.35 mmol) in methanol
(100 mL) at −20 °C for 30 min. The reaction mixture was stirred at −20 °C for 3 h and at 0 °C for 12 h
and then added with water to form a precipitate. This precipitate was filtered off and washed with ice
water to obtain compound 8, which was refluxed in xylene (50 mL) for 1 h. The remaining xylene was
evaporated to yield the crude compound which was recrystallized from ethyl acetate/n-hexane to
afford 9 as a pale pink powder. Yield: 3.3 g (42%); mp: 137–139 °C; 1H-NMR (CDCl3) δ: 3.94 (3H, s,
OCH3), 4.78 (1H, s, OH), 6.94 (1H, dd, J = 8.8, 2.4 Hz, H-6), 7.07 (1H, d, J = 2.0 Hz, H-4), 7.09-7.10
13
(1H, m, H-3), 7.29 (1H, d, J = 8.8 Hz, H-7), 8.81 (1H, s, NH); C-NMR (CDCl3) δ: 52.0 (OCH3),
105.9 (C-4), 108.0 (C-3), 112.7 (C-7), 116.2 (C-6), 127.9 (C-7a), 128.1 (C-2), 132.3 (C-3a), 150.1 (C-
5), 162.4 (C=O); GC-MS (EI) m/z: 191 [M]+ , 176 [M-CH3] +.
5-Hydroxy-1H-indole-2-carboxylic acid (10). A suspension of methyl 5-hydroxy-1H-indole-2-
carboxylate (9, 1 g, 5.23 mmol) in methanol (10 mL) was treated with a solution of 10 % NaOH
(10 mL) and heated at 60 °C for 1 h. After cooling to room temperature, the solution was acidified
with 10% HCl and the precipitate formed was isolated by filtration and washed with water to give the
crude compound which was recrystallized with methanol/ethyl acetate to afford 10 as a white powder.
Yield: 908 mg (98%); mp: 245–247 (dec.) °C, (249 °C (dec.), commercial available compound).
3-Benzyloxybenzaldehyde (11). According to the method of Schmidhammer et al. [26], a suspension of
K2CO3 (14.8 g, 107.13 mmol) in chloroform/methanol (2:1, 300 mL) was refluxed for 15 min and
added a mixture of 6 (3.0 g, 24.57 mmol) and benzyl bromide (5.0 g, 29.48 mmol) in chloroform/
methanol (2:1, 90 mL) over 30 min. The reaction mixture was refluxed for 8 h to yield a precipitate
which was filtered and the residue was concentrated to give a crude oily compound. This was diluted
with dichloromethane and the organic solvent was washed with brine, dried with anhydrous MgSO4,
filtered and concentrated to give the crude compound which was recrystallized from ethyl acetate/n-
hexane to give 11 as a white powder. Yield: 5.2 g (99%); mp: 54–56 °C (56–58 °C) [27].
Methyl 7-benzyloxy-1H-indole-2-carboxylate (13) and methyl 5-benzyloxy-1H-indole-2-carboxylate
(14). To a solution of Na (2.2 g, 94.35 mmol) in absolute methanol (100 mL) was added a solution of
3-benzyloxybenzaldehyde (7, 5.0 g, 23.58 mmol) and methyl azidoacetate (7, 10.9 g, 94.35 mmol) in
methanol (100 mL) at −20 °C, and the mixture was stirred at 0 °C for 12 h. After addition of cold
water, the resulting precipitate was collected by filtration. The solid was washed with water and dried
to give methyl 2-azido-3-(3-benzyloxyphenyl)acrylate (12, 6.4 g, 88 %) as pale yellow crystals. To
refluxing xylene (50 mL) was added a xylene solution (50 mL) of acrylate (6.4 g) and the mixture was
refluxed for 1 h. After cooling, the solvent was removed in vacuo to give the crude oily compound
which was purified by column chromatography (ethyl acetate/n-hexane = 1:9) to afford methyl 7-
benzyloxy-1H-indole-2-carboxylate (13) and methyl 5-benzyloxy-1H-indole-2-carboxylate (14) as

Molecules 2010, 15
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white crystals. Compound 13: Yield: 1.6 g (40%); mp: 130–131 °C (132–133 °C) [28]; Compound 14:
1
Yield: 1.8 g (45%); mp: 156–157 °C; H-NMR (CDCl3) δ: 3.94 (3H, s, OCH3), 5.10 (2H, s, OCH2),
7.03 (1H, dd, J = 8.8, 2.4 Hz, H-6), 7.13 (1H, t, J = 2.0 Hz, H-3), 7.15 (1H, d, J = 2.4 Hz, H-4), 7.31-
13
7.47 (6H, m, H-7 and Ar-H×5), 8.99 (1H, s, NH); C-NMR (CDCl3) δ: 52.2 (OCH3), 70.9 (OCH2),
104.4 (C-6), 108.6 (C-3), 113.0 (C-4), 117.9 (C-7), 128.0 (C-2), 132.6 (C-7a), 127.8 (Ar-C), 128.1
(Ar-C), 128.8 (Ar-C), 129.0 (C-3a), 137.5 (Ar-C), 154.1 (C-5), 162.6 (CO); GC-MS (EI) m/z: 281
[M]+, 190 [M-CH2C6H5]+.
5-Benzyloxy-1H-indole-2-carboxylic acid (15). A suspension of methyl 5-benzyloxy-1H-indole-2-
carboxylate (14, 1 g, 3.56 mmol) in methanol (10 mL) was reacted with a solution of 10 % NaOH
(10 mL) and heated at 60 °C for 1 h. The resulting mixture was worked up conditions like compound
10 to afford 15 as a white powder. Yield: 930 mg (98%); mp: 193–195 °C (192 °C, commercially
available compound)
5-Benzyloxy-1-chloromethyl-1,2-dihydro-3-[(5-hydroxy-1H-indol-2-yl)carbonyl]indoline (17). 5-
Benzyloxy-1-chloromethyl-N-(tert-butyloxycarbonyl)indoline (16, 50 mg, 0.13 mmol) [22] was
dissolved in 4M HCl in ethyl acetate (5 mL) and stirred at room temperature for 1 h. The reaction
mixture was treated with 20% NaOH and extracted with ethyl acetate and the organic layer was dried
with anhydrous MgSO4 and filtered, concentrated to give the residue which was dried under vacuum
for 1 h. The resulting amine hydrochloride (5-benzyloxy-1-chloromethyl-3H-indoline·HCl) was
dissolved in dimethylformamide (2 mL) and 5-substituted-1H-indole-2-carboxylic acid (50.4 mg,
0.20 mmol) and EDCI (74.0 mg, 0.40 mmol) were added. The reaction mixture was stirred for 14 h at
room temperature and added with water (3 mL) and obtained precipitates were purified by column
chromatography (ethyl acetate:n-hexane=1:5) to afford 17 as a white solid. Yield: 39 mg (67%); mp:
194–196 °C; 1H NMR (400 MHz, CD3Cl) δ: 3.55–3.60 (1H, m, CH2Cl), 3.76–3.82 (2H, m, CH2Cl, H-
1), 4.47 (1H, dd, J = 10.6, 4.0 Hz, H-2a), 4.63–4.68 (1H, m, H-2b), 5.09 (2H, s, OCH2), 6.72 (1H, dd,
J = 8.4, 2.4 Hz, H-6), 6.89 (1H, d, J = 2.0 Hz, H-3'), 6.94 (1H, dd, J = 8.8, 2.4 Hz, H-6'), 7.09 (1H, d,
J = 2.0 Hz, H-4'), 7.17 (1H, d, J = 8.0 Hz, H-7), 7.30-7.45 (6H, m, Ar-H×5, H-7), 8.05 (1H, s, H-4),
13
8.31 (1H, s, OH), 10.03 (1H, s, NH); C NMR (100 MHz, CD3Cl) δ: 42.9 (C-1), 47.2 (CH2Cl), 54.9
(C-2), 70.2 (OCH2), 104.8 (C-4), 105.2 (C-4'), 105.4 (C-3'), 111.1 (C-6), 112.6 (C-7'), 116.4 (C-6'),
123.6 (C-7a), 124.6 (C-7) 127.5 (Ar-C), 127.9 (Ar-C), 128.5 (Ar-C), 130.5 (C-2'), 131.2 (C-7a'), 136.8
(Ar-C), 144.9 (C-3a), 151.5 (C-5'), 159.4 (C-5), 160.7 (CO); GC-MS (EI) m/z: 432 [M]+, 434 [M+2] +.
Chloromethyl-5-hydroxy-1,2-dihydro-3-[(5-hydroxy-1H-indol-2-yl)carbonyl]indoline
(1).
5-
Benzyloxy-1-chloromethyl-1,2-dihydro-3-[(5-hydroxy-1H-indol-2-yl)carbonyl]indoline (17, 20 mg,
46.30 μmol) was added a suspension of 10% Pd/C (125 mg) in acetone (10 mL) to a Parr bottle. After
catalytic hydrogenation at 55 psi for 1 h, the reaction mixture was filtered and the filtrate was
1
evaporated to yield 1 as a white solid. Yield: 12 mg (76%); mp: 175–176 °C; H-NMR (CD3OD) δ:
3.54 (1H, dd, J = 11.0, 8.2 Hz, CH2Cl), 3.64–3.68 (1H, m H-1), 3.76 (1H, dd, J = 10.8, 4.4 Hz, CH2Cl),
4.33 (1H, dd, J = 11.0, 4.6 Hz, H-2a), 4.54 (1H, dd, J = 10.8, 9.2 Hz, H-2b), 6.45 (1H, dd, J = 8.4, 2.4
Hz, H-6), 6.75 (1H, dd, J = 8.8, 2.4 Hz, H-6'), 6.86 (1H, s, H-3'), 6.92 (1H, d, J = 2.4 Hz, H-4') 7.07
(1H, d, J = 8.0 Hz, H-7), 7.20 (1H, d, J = 8.8 Hz, H-7'), 7.56 (1H, s, H-4); 13C-NMR (CD3OD) δ: 42.8

Molecules 2010, 15
7979
(C-1), 47.7 (CH2Cl), 55.2 (C-2), 104.8 (C-4), 105.3 (C-4'), 105.4 (C-3'), 111.2 (C-6), 112.4 (C-7'),
115.6 (C-6'), 123.1 (C-7a), 124.8 (C-7), 128.6 (C-3a'), 130.8 (C-2'), 131.9 (C-7a'), 144.7 (C-3a), 151.2
(C-5'), 157.8 (C-5), 161.5 (CO); GC-MS (EI) m/z: 342 [M]+, 344 [M+2] +.
5-Benzyloxy-1-chloromethyl-1,2-dihydro-3-[(5-methylsulfonyloxy-1H-indol-2-yl)carbonyl]indoline (18).
To the solution of 17 (50 mg, 0.12 mmol) in CH2Cl2 (5 mL) was added methanesulfonyl chloride (26.4
mg, 0.23 mmol) and triethylamine (35.4 mg, 0.35 mmol) at room temperature. The reaction mixture
was stirred for 4 h, water was added, followed by extraction CH2Cl2 and drying with anhydrous
MgSO4. After concentration, the residue was purified by column chromatography (ethyl acetate/n-
1
hexane = 1:5) to yield 18 as a white solid. Yield: 48 mg (92%); mp: 203–206 °C; H-NMR
(CD3COCD3) δ: 3.27 (3H, s, CH3), 3.82–4.02 (3H, m, CH2Cl, H-1), 4.55 (1H, dd, J = 10.8, 4.4 Hz, H-
2a), 4.82 (1H, t, J = 9.8 Hz, H-2b), 5.15 (2H, s, OCH2), 6.79 (1H, d, J = 8.4 Hz, H-6), 7.28-7.52 (9H,
m, Ar-H×5, H-3', 6, 6', 7), 7.67 (1H, d, J = 8.4 Hz, H-7'), 7.72 (1H, s, H-4'), 8.05 (1H, s, H-4), 11.11
(1H, s, NH); 13C-NMR (CD3COCD3) δ: 32.0 (CH3), 42.7 (C-1), 47.2 (CH2Cl), 50.3 (C-2), 70.0
(OCH2), 105.2 (C-4), 106.1 (C-4'), 106.2 (C-3'), 110.6 (C-6), 112.7 (C-7'), 115.2 (C-6'), 119.5 (C-7a),
124.6 (C-7) 127.8 (Ar-C), 128.0 (C-3a'), 128.2 (Ar-C), 128.6 (Ar-C), 130.9 (C-2'), 135.8 (C-7a'), 137.6
(Ar-C), 144.0 (C-3a), 145.4 (C-5'), 159.5 (C-5), 160.1 (CO); GC-MS (EI) m/z: 510 [M]+, 420 [M-
CH2C6H5]+.
5-Benzyloxy-1-chloromethyl-1,2-dihydro-3-[(5-sulfamoyloxy-1H-indol-2-yl)carbonylindoline
(19).
According to method by Okada et al. [29], formic acid (8.5 mg, 0.18 mmol) and chlorosulfonyl
isocyanate (26.2 mg, 0.18 mmol) were allowed to react with each other at 80 °C till evolution of
carbon dioxide gas ceased to thus form aminosulfonyl chloride. To a solution of 17 (40 mg,
0.09 mmol) in dimethylacetamide (DMA, 3 mL) was added the aminosulfonyl chloride and the
resulting mixture left to stir for 3 h. The solution was treated with water, extracted with CH2Cl2 and
the organic layer was dried with anhydrous MgSO4. After concentration, the residue was purified by
column chromatography (ethyl acetate/n-hexane=1:5) to yield 19 as a white solid. Yield: 30 mg
(62%); mp: 196–197 °C; 1H-NMR (CD3OD) δ: 3.60 (1H, dd, J = 10.6, 7.4 Hz, CH2Cl), 3.70 (1H, m,
H-1), 3.78 (1H, dd, J = 10.6, 4.2 Hz, CH2Cl), 4.32 (1H, dd, J = 11.0, 4.6 Hz, H-2a), 4.55 (1H, dd,
J = 10.8, 9.2 Hz, H-2b), 4.96 (2H, s, OCH2), 6.64 (1H, dd, J = 8.8, 2.4 Hz, H-6), 6.76 (1H, dd, J = 8.8,
2.4 Hz, H-6'), 6.86 (1H, s, H-3'), 6.92 (1H, d, J = 2.4 Hz, H-4'), 7.16 (1H, d, J = 8.4 Hz, H-7),
7.20–7.34 (6H, m, Ar-H×5, H-7'), 7.78 (1H, s, H-4); 13C-NMR (CD3OD) δ: 42.6 (C-1), 47.3 (CH2Cl),
55.1 (C-2), 70.1 (OCH2), 104.9 (C-4), 105.0 (C-4'), 105.4 (C-3'), 110.8 (C-6), 112.7 (C-7'), 115.8 (C-
6'), 124.7 (C-7a), 125.0 (C-7) 127.6 (Ar-C), 127.9 (Ar-C), 128.5 (Ar-C), 128.7 (C-3a'), 130.9 (C-2'),
131.8 (C-7a'), 137.5 (Ar-C), 145.0 (C-3a), 151.4 (C-5'), 159.4 (C-5), 161.4 (CO); GC-MS (EI) m/z:
432 [M-SO2NH2] +, 434 [M-SO2NH2+2] +.
5-Benzyloxy-1-chloromethyl-1,2-dihydro-3-[(5-dimethylsulfamoyloxy-1H-indol-2-yl)carbonyl]indoline
(20). To the solution of 17 (40 mg, 0.09 mmol) in CH2Cl2 (5 mL) was added dimethylsulfamoyl
chloride (26.56 mg, 0.18 mmol) and triethylamine (35.4 mg, 0.35 mmol) at room temperature, and the
mixture was stirred for 18 h. The solution was treated with water, extracted with CH2Cl2 and the
organic layer was dried with anhydrous MgSO4. After concentration, the residue was purified by

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column chromatography (ethyl acetate/n-hexane=1:5) to yield 20 as a pale yellow solid. Yield: 36 mg
(72%); mp: 116–117 °C; 1H-NMR (CD3COCD3) δ: 2.97 (6H, s, CH3×2), 3.82–4.01 (3H, m, CH2Cl, H-
1), 4.54 (1H, dd, J = 10.8, 3.6 Hz, H-2a), 4.81 (1H, t, J = 10.0 Hz, H-2b), 5.14 (2H, s, OCH2), 6.79
(1H, d, J = 9.2 Hz, H-6), 7.26-7.51 (8H, m, Ar-H×5, H-3', 6', 7), 7.65 (1H, d, J = 8.8 Hz, H-7'), 7.69
(1H, s, H-4'), 8.05 (1H, s, H-4), 11.09 (1H, s, NH); 13C-NMR (CD3COCD3) δ: 38.3 (CH3×2), 42.8 (C-
1), 47.7 (CH2Cl), 54.8 (C-2), 70.0 (OCH2), 105.3 (C-4), 106.2 (C-4'), 106.3 (C-3'), 110.5 (C-6), 113.3
(C-7'), 114.9 (C-6'), 124.6 (C-7a), 125.1 (C-7) 127.8 (Ar-C), 127.9 (Ar-C), 128.2 (C-3a'), 128.6 (Ar-
C), 132.9 (C-2'), 134.8 (C-7a'), 137.7 (Ar-C), 144.6 (C-3a), 145.4 (C-5'), 159.5 (C-5), 160.1 (CO); GC-
MS (EI) m/z: 540 [M]+, 542 [M+2] +.
1-Chloromethyl-5-hydroxy-1,2-dihydro-3-[(5-methylsulfonyloxy-1H-indol-2-yl)carbonyl]indoline (2).
To solution of 10% Pd-C (10 mg) in acetone (10 mL) was added 18 (20 mg, 39.21 μmol) and the
resulting mixture was worked up conditions like compound 1 to give compound 2 as a white solid.
Yield: 8 mg (50%); mp: 155–158 °C; 1H-NMR (CD3COCD3) δ: 2.80 (3H, s, CH3), 3.77–3.98 (3H, m,
CH2Cl, H-1), 4.49 (1H, dd, J = 10.2, 4.2 Hz, H-2a), 4.74 (1H, t, J = 9.6 Hz, H-2b), 6.58 (1H, d,
J = 9.2 Hz, H-6), 6.90 (1H, d, J = 8.0 Hz, H-6'), 7.01 (1H, s, H-3’), 7.08 (1H, s, H-4’), 7.24 (1H, d,
J = 8.4 Hz, H-7), 7.42 (1H, d, J = 9.2 Hz, H-7’), 7.87 (1H, s, H-4), 8.34 (1H, s, OH), 10.59 (1H, s,
NH); 13C-NMR (CD3COCD3) δ: 36.5 (CH3), 42.8 (C-1), 47.8 (CH2Cl), 54.9 (C-2), 105.6 (C-4), 106.0
(C-4'), 106.1 (C-3'), 111.2 (C-6), 113.5 (C-7'), 115.1 (C-6'), 122.8 (C-7a), 125.1 (C-7), 128.1 (C-3a'),
133.0 (C-2'), 134.9 (C-7a'), 144.0 (C-3a), 145.2 (C-5'), 158.1 (C-5), 160.0 (CO); GC-MS (EI) m/z: 420
[M]+, 342 [M-SO2CH3]+.
1-Chloromethyl-5-hydroxy-1,2-dihydro-3-[(5-sulfamoyloxy-1H-indol-2-yl) carbonyl]indoline (3). To a
solution of 10% Pd-C (10 mg) in acetone (10 mL) was added 20 (20 mg, 39.06 μmol) and the resulting
mixture was worked up conditions like compound 1. Compound 3 was obtained as a white solid.
1
Yield: 8.8 mg (53%); mp: 195–197 °C; H-NMR (CD3COCD3) δ: 3.77–3.98 (3H, m, CH2Cl, H-1),
4.48 (1H, d, J = 10.8 Hz, H-2a), 4.74 (1H, t, J = 9.6 Hz, H-2b), 6.69 (1H, d, J = 8.4 Hz, H-6), 6.91
(1H, d, J = 8.8 Hz, H-6'), 7.02 (1H, s, H-3'), 7.09 (1H, s, H-4'), 7.24 (1H, d, J = 8.0 Hz, H-7), 7.42
(1H, d, J = 8.4 Hz, H-7'), 7.88 (1H, s, H-4), 8.38 (1H, s, OH), 10.61 (1H, s, NH); 13C-NMR
(CD3COCD3) δ: 43.5 (C-1), 48.5 (CH2Cl), 55.5 (C-2), 105.8 (C-3', C-4'), 106.3, (C-4), 111.6 (C-6),
113.6 (C-7'), 116.5 (C-6'), 123.6 (C-7a), 125.7 (C-7), 129.7 (C-3a'), 132.1 (C-2'), 132.3 (C-7a'), 146.2
+
(C-3a), 152.5 (C-5'), 158.7 (C-5), 161.1 (CO); GC-MS (EI) m/z: 342 [M-SO2NH2] , 344 [M+2-
SO2NH2] +.
1-Chloromethyl-5-hydroxy-1,2-dihydro-3-[(5-dimethylsulfamoyloxy-1H-indol-2-yl)carbonyl]indoline
(4). To solution of 10% Pd-C (5 mg) in acetone (10 mL) was added 20 (20 mg, 37.03 μmol) and the
resulting mixture was worked up conditions like compound 1. Compound 4 was obtained as a white
solid. Yield:11 mg (66%); mp: 217–218 °C; 1H NMR (CD3COCD3) δ: 2.94 (6H, s, CH3×2), 3.75–3.96
(3H, m, CH2Cl, H-1), 4.49 (1H, dd, J = 11.0, 4.2 Hz, H-2a), 4.75 (1H, t, J = 10.0 Hz, H-2b), 6.58 (1H,
d, J = 7.6 Hz, H-6), 7.22 (3H, d, J = 7.2 Hz, H-3', 6', 7), 7.61 (1H, d, J = 8.8 Hz, H-7'), 7.66 (1H, s, H-
13
4'), 7.85 (1H, s, H-4), 8.37 (1H, s, OH), 11.01 (1H, s, NH); C-NMR (CD3COCD3) δ: 38.2 (CH3×2),
42.8 (C-1), 47.8 (CH2Cl), 54.8 (C-2), 105.6 (C-4), 106.1 (C-4'), 106.2 (C-3'), 111.2 (C-6), 113.2 (C-7'),

Molecules 2010, 15
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114.9 (C-6'), 119.3 (C-7a), 123.0 (C-7), 125.1 (C-3a'), 133.0 (C-2'), 134.8 (C-7a'), 144.6 (C-3a), 145.3
(C-5'), 158.0 (C-5), 159.9 (CO); GC-MS (EI) m/z: 449 [M]+, 451 [M+2] +.
1-Chloromethyl-5-hydroxy-N-(tert-butyloxycarbonyl)-indoline (21) To a solution of 10% Pd-C (25
mg) in methanol (10 mL) was added 16 (100 mg, 0.27 mmol) and the resulting mixture was worked up
like compound 1. Compound 21 was obtained as a white solid. Yield: 74 mg (98%); mp: 178–179 °C;
1H- NMR (CDCl3) δ: 1.56 (9H, s, C(CH3)3), 3.47 (1H, t, J = 9.8 Hz, CH2Cl), 3.54–3.61 (1H, m, H-1),
3.69 (1H, dd, J = 10.4, 3.6 Hz, CH2Cl), 3.90–3.93 (1H, m, H-2), 4.07–4.12 (1H, m, H-2), 6.48 (1H, dd,
13
J = 8.4, 2.0 Hz, H-6), 7.01 (1H, d, J = 8.0 Hz, H-7), 7.47 (1H, s, H-4); C-NMR (CDCl3) δ: 28.4
(C(CH3)3), 41.8 (C-1), 47.5 (CH2Cl), 53.0 (C-2), 81.5 (C(CH3)3), 103.0 (C-4), 109.6 (C-6), 122.1 (C-
+
7a), 125.0 (C-7), 143.8 (C-3a), 152.6 (C-5), 157.0 (CO); GC-MS (EI) m/z: 183 [M-BOC] ,
185 [M+2-BOC] +.
1-Chloromethyl-5-hydroxy-1,2-dihydro-3-[(5-benzyloxy-1H-indol-2-yl)carbonyl]indoline
(5).
Compound 21 (50 mg, 0.18 mmol) was dissolved in 4M HCl in ethyl acetate (5 mL) and stirred at
room temperature for 1 h. The reaction mixture was treated with 20% NaOH and extracted with ethyl
acetate and the organic layer was dried with anhydrous MgSO4, filtered and concentrated to give a
residue which was dried under vacuum for 1 h. The resulting amine hydrochloride (5-hydroxy-1-
chloromethyl-3H-indoline·HCl) was dissolved in dimethylformamide (2 mL) and 5-benzyloxy-1H-
indole-2-carboxylic acid (70.6 mg, 0.26 mmol) and EDCI (101.0 mg, 0.5 mmol) were added. The
reaction mixture was stirred for 14 h at room temperature and then treated with water (3 mL) to form a
precipitate. This precipitate was purified by column chromatography (ethyl acetate/n-hexane = 1:5) to
afford 5 as a white solid. Yield: 63.1 mg (83%); mp: 220–222 °C; 1H-NMR (CD3COCD3) δ: 3.78 (1H,
t, J = 8.0 Hz, CH2Cl), 3.86 (1H, m, H-1), 3.96 (1H, dd, J = 10.4, 3.6 Hz, CH2Cl), 4.49 (1H, dd,
J = 10.8, 4.4 Hz, H-2a), 4.74 (1H, t, J = 10.0 Hz, H-2b), 5.15 (2H, s, OCH2), 6.59 (1H, d, J = 8.0 Hz,
H-6), 7.04 (1H, d, J = 8.4 Hz, H-6'), 7.09 (1H, s, H-3'), 7.23–7.52 (8H, m, Ar-H×5, H-4', H-7, H-7'),
13
7.88 (1H, s, H-4), 8.38 (1H, s, OH), 10.74 (1H, s, NH); C-NMR (CD3COCD3) δ: 43.5 (C-1), 48.5
(CH2Cl), 55.5 (C-2), 71.0 (OCH2), 104.8 (C-4), 106.2 (C-4'), 106.3 (C-3'), 111.6 (C-6), 113.9 (C-7'),
117.3 (C-6'), 123.6 (C-7a), 125.7 (C-7) 127.5 (Ar-C), 127.9 (Ar-C), 128.5 (Ar-C), 132.2 (C-2'), 132.8
(C-7a'), 138.9 (Ar-C), 146.1 (C-3a), 154.5 (C-5'), 158.6 (C-5), 161.0 (CO); GC-MS (EI) m/z: 432
[M]+, 434 [M+2] +.
3.2. Cytotoxicity
Synthetic compounds were dissolved in DMSO to obtain 10 mM stock solutions, which were
diluted with RPMI 1640 to obtain 100 µM solutions. These solutions were then further diluted with
1% DMSO in RPMI 1640 to prepare solutions from concentrations of 50 µM to 10 nM. These
compound solutions (10 µL) were added to wells containing 90 µL media/cell suspension.
The human cancer cells (COLO 205, SK-MEL-2, A549, and JEG-3) were purchased from the
Korean cell line bank (KCLB). These cancer cell lines were grown in RPMI 1640 [(+) L-glutamine,
(+) 25 mM HEPES, Gibco], except JEG-3 cells, which were grown in Delbecco’s Modified Eagle

Molecules 2010, 15
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Medium (DMEM) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin
(Gibco). Cells were maintained at 37 °C in a 5% humidified CO2 atmosphere.
Cell concentrations were adjusted to 5 × 104 cells/mL (COLO 205, SK-MEL-2), 1 × 105 cells/mL
(A549) and 2 × 104 cells/mL (JEG-3) after counting the cells with a hemocytometer. Cell suspentions
(90 µL) were seeded onto 96-well flat-bottom cell culture plates. After 24 h in a 5% CO2 atmosphere
at 37 °C, the drug solutions diluted 1% DMSO in RPMI 1640 were added to each well (10 µL/well).
The plates were incubated for 48 h at 37 °C in a 5% CO2 atmosphere. MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide) was dissolved in PBS (2 mg/mL). After the indicated cell
incubation period, 20 μL of this stock MTT solution was added to each well and the plates were
further incubated for 4 h at 37 °C in a 5% CO2. The contents of the well were shaken and the plates
were read on an ELISA reader, utilizing Sunrise xfluor4 V.4.51, with a test wavelength of 570 nm. The
dose inhibiting the growth by 50% (IC50) was extrapolated from curves generated based of the average
of the absorbance data (seven points/concentration).
4. Conclusions
Indole C5-O-substitued seco-CI derivatives 1-5 were synthesized and evaluated against four cancer
cell lines. From the in vitro cytotoxic test data, compounds 1-5 showed more potency than doxorubicin
against the SK-MEL-2 cell line and compounds 1, 2 and 5 more weakly potent than doxorubicin
against the JEG-3 cancer cell line, and the indole C5-N,N-dimethylaminosulfonyl derivative 4 was
inactive against all cancer cell lines. It is thought that the indole C5-N,N-dimethylaminosulfonylamino
substituent of seco-CI did not contribute to the embedded minor groove interaction.
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Sample Availability: Samples of the compounds are available from the authors.
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