Structural influence of indole C5-N-substitutents on the cytotoxicity of seco-duocarmycin analogs

Article abstract of DOI:10.1007/s12272-011-0302-1

A series of racemic indole C5-substituted seco-cyclopropylindoline compounds (2,3 and 5-7) were prepared by coupling 1-(tert-butyloxycarbonyl)-3- (chlorocarbonyl)indoline (seg-A) with 5,6,7-trimethoxy-, 5,6-dimethoxy-, 5-amino-, 5-methylsulfonylamino- and 5-(N,N-dimethylaminosulfonylamino) indole-2-carboxylic acid as seg-B in the presence of 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide. The synthetic compounds (2,3 and 5-7) were tested for cytotoxic activity against human cancer cell lines (COLO 205, SK-MEL-2, A549, and JEG-3) using the MTT assay.

Full text of DOI:10.1007/s12272-011-0302-1

Arch Pharm Res Vol 34, No 3, 357-367, 2011
DOI 10.1007/s12272-011-0302-1
Structural Influence of Indole C5-N-Substitutents on the Cytotoxicity
of seco-Duocarmycin Analogs
Taeyoung Choi and Eunsook Ma
College of Pharmacy, Catholic University of Daegu, Hayang 712-702, Korea
(Received September 20, 2010/Revised December 1, 2010/Accepted December 6, 2010)
A series of racemic indole C5-substituted seco-cyclopropylindoline compounds (2,3 and 5-7)
were prepared by coupling 1-(tert-butyloxycarbonyl)-3-(chlorocarbonyl)indoline (seg-A) with
5,6,7-trimethoxy-, 5,6-dimethoxy-, 5-amino-, 5-methylsulfonylamino- and 5-(N,N-dimethylami-
nosulfonylamino)indole-2-carboxylic acid as seg-B in the presence of 1-ethyl-3-(3-dimethylami-
nopropyl)carbodiimide. The synthetic compounds (2,3 and 5-7) were tested for cytotoxic
activity against human cancer cell lines (COLO 205, SK-MEL-2, A549, and JEG-3) using the
MTT assay.
Key words: Achiral duocarmycin, seco-Cyclopropylindoline, Cytotoxicity, DNA minor groove
alkylation
ciated with (+)-CC-1065 (Boger and Johnson, 1996).
Moreover, DUM SA is the most hydrolytically stable
INTRODUCTION
Duocarmycins (DUMs; A, B1, B2, C1, C2, D1, D2 and member of this class of compounds (Boger, 1995; Boger
SA) and CC-1065 are members of an extremely potent and Johnson, 1996; Boger et al., 1996), but it is toxic to
group of antitumor antibiotics isolated from Streptomyces bone marrow (Warpehoski et al., 1992).
sp. that contain a common pharmacophore. The pharm-
There is a strong interest in the design and devel-
acophore consists of a cyclopropa[ ]pyrrolo[3,2- ]indole opment of novel analogs of duocarmycin that effectively
c
e
(CPI) moiety (Boger et al., 1990b; Sugiyama et al., kill cancer cells and have reduced toxicity to the host.
1993; Fukuda et al., 1994b). DUMs B1, B2, C1, C2, D1 One of the attempts (Boger et al., 1990c, 1991) to
and D2 were a seco-type of DUM A and chemically design novel analogs was to synthesize a spirocyclic
more stable than DUM A. The cytotoxicity of DUMs 1,2,7,7a-tetrahydrocyclopropyl[1,2-c]indol-4-one [(CI-
and CC-1065 derives from the reaction of the CPI numbering), CI] as the minimum potent pharmacophore
moiety with adenine-N3 groups in the minor groove of (Boger et al., 1990b), which can be formed by ring
AT-rich sequences of DNA (Hurley et al., 1984; closure of a seco-CI parent. Seco compounds possess
Reynolds et al., 1985; Eis et al., 1997). This mechanism a similar alkylating selectivity and efficiency as the
of action was confirmed by the isolation of adenine- correspon- ding natural products ((+)-enantiomer)
CC-1065 (Hurley et al., 1984) and adenine-duocarmycin (Boger et al., 1994), since they can form the spiro
SA (DUM SA) (Boger et al., 1994) products from [cyclopropane] moiety in situ by an intramolecular
thermally cleaved DNA:drug adducts. Even though Winstein cyclization (Baird and Winstein, 1963).
(+)-CC-1065 has cytotoxic properties, its usefulness as
an anticancer drug is hampered by delayed lethal nounced differences between enantiomers, but the CI-
toxicity to animals at therapeutic doses (McGovren et TMI (5,6,7-trimethoxy-1
-indole) enantiomers show
In general, most duocarmycins exhibit more pro-
H
al., 1984). DUM SA seems to be superior to CC-1065 fewer differences in biological potency and relative
due to the lack of a delayed fetal hepatotoxicity asso- DNA alkylation efficiency (Boger and Yun, 1994; Boger
et al., 1994). There was no difference in cytotoxic
Correspondence to: Eunsook Ma, College of Pharmacy, Catholic
University of Daegu, Hayang 712-702, Korea
Tel: 82-53-850-3621, Fax: 82-53-850-3602
E-mail: masook@cu.ac.kr
activity between (+)-CI and (-)-CI enantiomers. There-
fore, resolution of the enantiomers to obtain the more
active enantiomer is not required.
357

358
T. Choi and E. Ma
Fig. 1. Structure of DUM SA, CPI, CI, seco-CI and compounds 2-7.
Some previous studies indicate that the seg-A ring Gallenhamp melting point apparatus and were not
1
structure influences the electrophilicity of cyclopropane corrected. H-, 13C-NMR spectra were generated on a
(Asai et al., 1994). On the other hand, the seg-B Varian 400 MHz spectrometer. Chemical shifts (
moiety has been considered to play an important role in parts per million (ppm) relative to tetramethylsilane,
in noncovalent binding to DNA (Boger et al., 1990a; and coupling constants ( ) are in Hertz. GC/MS
Warpehoski et al., 1992; Boger and Yun, 1993). spectra were obtained on a SHIMAZU MS-QP2010
δ) are
J
The C5 position of the seg-B moiety is the most mass spectrometer. The MPLC procedure used a
important site potentiating cytotoxicity. Boger reported Yamazen YFLC-AI instrument. Analytical TLC was
that a single 5-methoxy group on the indole minor done on pre-coated silica gel 60 F254 plates (Merck).
groove binding unit was sufficient to maintain potency, Column chromatography was carried out on Merck
and a series of dimethylaminoethoxy group substituted silica gel 9385 (230-400 mesh) using the eluting solvent
analogs retained the cytotoxicity of the TMI analog indicated.
while providing increased aqueous solubility (Boger et
al., 1994; Howard et al., 2002).
Methyl 5-nitro-1H-indole-2-carboxylate (11)
An important series of early studies examined the
To a solution of Na (608.8 mg, 26.47 mmol) in
amine and amide derivatives of 5-aminoindole. Notably, absolute methanol (20 mL) was added a solution of 3-
the amide derivatives (5-NHCOCH3, 5-NHCOC2H5, nitrobenzaldehyde (
, 1 g, 6.62 mmol) and methyl
-5-NHCOPr, 5-N(CH3)COCH3) proved to be potent azidoacetate (Rosa et al., 2003) (3.06 g, 26.47 mmol) in
8
cytotoxic agents (Parrish et al., 2003).
methanol (20 mL) at −
20oC. The reaction mixture was
Seeking to improve cytotoxicity, we synthesized and stirred at 0oC for 12 h. After addition of cold water,
evaluated the cytotoxic activity of common indole-C5 the resulting precipitate was collected by filtration.
substituents containing seco-CI analogs (
different minor groove binding side chains.
2
-
7
) with The solid was washed with water and dried to give
methyl 2-azido-3-(3-nitrophenyl)acrylate as yellow
crystals. To refluxing xylene (10 mL) was added a
solution of methyl 2-azido-3-(3-nitrophenyl)acrylate in
xylene (10 mL) and the reaction mixture was refluxed
for 1 h. After cooling, the solvent was removed in
MATERIALS AND METHODS
General procedures
Moisture-sensitive or air-sensitive reactions were vacuo to give a crude oily compound that was purified
conducted under nitrogen in distilled solvents. by column chromatography (ethyl acetate: -hexane =
n
Commercial reagents were purchased from Aldrich, 1:9) to afford a pure yellow product 11. Yield 1 g
1
Fluka, or Sigma. Melting points were measured on a (67%); m.p. 273-275oC (dec.); H-NMR (400 MHz,

Synthesis and Cytotoxicity of seco-Duocarmycin Analogs
359
DMSO-d6
7.57 (1H, d,
Hz, H-6), 8.63 (1H, s, H-4), 12.00 (1H, s, NH); 13C- give crude compounds that were purified by column
NMR (100 MHz, DMSO-d6 : 61.1 (OCH3), 109.7 (C- chromatography (ethyl acetate: -hexane = 1:3) to afford
3), 112.9 (C-7), 118.5 (C-4), 119.5 (C-6), 126.1 (C-7a), the pure corresponding acids (14
)
δ
: 4.42 (3H, s, OCH3), 7.32 (1H, s, H-3), acidified with 10% HCl and the precipitate formed
J
= 8.8 Hz, H-7), 8.12 (1H, dd, = 9.2, 2.0 was isolated by filtration, and washed with water to
J
)
δ
n
-16).
131.2 (C-2), 140.3 (C-3a), 141.9 (C-5), 161.9 (C=O);
GC-Ms (EI) m/z : 220 [M]+.
5-Nitro-1
H
-indole-2-carboxylic acid (14)
Yellow solid, yield 399 mg (85%); m.p. > 310oC (dec.
> 300oC).
Ethyl 5,6,7-trimethoxy-1
H-indole-2-carboxylate
(12)
To a solution of Na (469.4 mg, 20.39 mmol) in absolute 5,6,7-Trimethoxy-1
ethanol (20 mL) was added a solution of
(1 g, 5.10 Yield 852 mg (95%); m.p. 215-216oC (215.5-216oC)
mmol) and ethyl azidoacetate (2.64 g, 20.39 mmol) in (Fukuda et al., 1994a).
ethanol (20 mL) at
20oC. We then used the procedures
described for compound 11. The crude compound was 5,6-Dimethoxy-1
purified by column chromatography (ethyl acetate:
Yield 860 mg (97%); m.p. 202-204oC (202-203oC)
H-indole-2-carboxylic acid (15)
9
−
H-indole-2-carboxylic acid (16)
n-
hexane = 1:9) to afford a pure white product 12. Yield (Raper, 1926).
1.18 g (83%); m.p. 88-90oC; 1H-NMR (400 MHz, CDCl3)
δ
: 1.39 (3H, t,
OCH3), 3.93 (3H, s, OCH3), 4.07 (3H, s, OCH3), 4.40
(2H, q, = 14.4, 7.2 Hz, OCH2CH3), 6.82 (1H, s, H-7), suspension of 10% Pd-C (100 mg) in methanol (25 mL)
7.27 (1H, s, H-3), 8.95 (1H, s, NH); 13C-NMR (100 in a Parr bottle. After hydrogenation at 55 psi for 4 h,
MHz, CDCl3) : 14.6 (CH3), 56.4 (OCH3), 60.1 (OCH2), the reaction mixture was filtered and the filtrate was
61.6 (OCH3), 61.6 (OCH3), 97.9 (C-4), 108.9 (C-3), evaporated to yield a pink precipitate that was purified
123.3 (C-2), 126.6 (C-3a), 127.4 (C-7a), 139.1 (C-7), by column chromatography (ethyl acetate: -hexane =
1:9) to afford a pure pale pink product 17. Yield 512
mg (99%); m.p. 117-119oC; 1H-NMR (400 MHz, CDCl3)
: 3.56 (2H, s, NH2), 3.92 (3H, s, OCH3), 6.79 (1H, dd,
= 8.8, 2.4 Hz, H-6), 6.92 (1H, d, = 2.0 Hz, H-4),
7.02 (1H, t, = 1.0 Hz, H-3), 7.20 (1H, d, = 7.6 Hz,
J = 7.2 Hz, OCH2CH3), 3.89 (3H, s,
Methyl 5-amino-1
Compound 11 (600 mg, 2.73 mmol) was added to a
H-indole-2-carboxylate (17)
J
δ
n
140.7 (C-6), 150.4 (C-5), 161.9 (CO); GC-MS (EI) m/z
:
279 [M]+.
δ
J
J
Ethyl 5,6-dimethoxy-1
(13)
H-indole-2-carboxylate
J
J
To a solution of Na (553.8 mg, 24.08 mmol) in H-7), 9.09 (1H, s, NH); 13C-NMR (100 MHz, CDCl3)
δ:
absolute ethanol (20 mL) was added a solution of 10 52.0 (OCH3), 105.9 (C-4), 107.5 (C-3), 112.6 (C-7),
(1 g, 6.02 mmol) and ethyl azidoacetate (4.89 g, 24.08 117.3 (C-6), 127.4 (C-2), 128.4 (C-7a), 132.4 (C-3a),
mmol) in ethanol (20 mL) at
procedures were the same as for 12. The crude
−
20oC. The remaining 140.3 (C-5), 162.6 (C=O); GC-Ms (EI) m/z: 190 [M]+.
compound was purified by column chromatography
Methyl 5-methylaminosulfonyl-1H-indole-2-car-
(ethyl acetate:
product 13. Yield 1.17 g (78%); m.p. 172-174oC; H-
NMR (400 MHz, CDCl3) : 1.39 (3H, t,
OCH2CH3), 3.90 (3H, s, OCH3), 3.91 (3H, s, OCH3), mL) was added methanesulfonyl chloride (120 mg,
4.37 (2H, q, = 14.4, 7.2 Hz, OCH2CH3), 6.83 (1H, d, 1.05 mmol) and triethylamine (160 mg, 1.58 mmol) at
= 7.7 Hz, H-7), 7.03 (1H, s, H-4), 7.10 (1H, s, H-3), 8.84 room temperature. The reaction mixture was stirred
(1H, s, NH); 13C-NMR (100 MHz, CDCl3)
: 14.7 at room temperature for 4 h. The solution was treated
n-hexane=1:9) to afford a pure white
boxylate (18)
To a solution of methyl 5-amino-1
1
H-indole-2-car-
δ
J = 7.2 Hz, boxylate (17, 100 mg, 0.53 mmol) in dichloromethane (10
J
J
δ
(CH3), 56.2 (OCH3), 56.3 (OCH3), 60.9 (OCH2), 94.0 (C- with water, extracted with dichloromethane, dried
7), 102.7 (C-4), 108.9 (C-3), 120.6 (C-7a), 126.6 (C-2), with anhydrous MgSO4, and filtered. After concentra-
132.2 (C-3a), 146.4 (C-6), 150.2 (C-5), 162.2 (CO); GC- tion of the filtrate, the residue was purified by column
MS (EI) m/z : 249 [M]+.
chromatography (ethyl acetate:n-hexane = 1:5) to yield
18 as a light brown solid. Yield 98 mg (70%); m.p.
1
199-201oC; H-NMR (400 MHz, CD3COCD3)
13 (3.58 mmol) (3H, s, CH3), 3.41 (1H, s, NH), 3.96 (3H, s, OCH3), 7.18
in methanol (5 mL) was treated with a 10% aqueous (1H, s, H-3), 7.41 (1H, d, = 8.8 Hz, H-6), 7.48 (1H, d,
NaOH solution (10 mL) and heated at 60oC for 1 h.
= 8.8 Hz, H-7), 7.61 (1H, s, H-4), 9.27 (1H, s, NH);
After cooling to room temperature, the solution was 13C-NMR (100 MHz, CD3COCD3)
: 38.5 (CH3), 51.3
δ : 2.98
General synthetic method of 15-16
A suspension of ester derivatives 11
-
J
J
δ

360
T. Choi and E. Ma
(OCH3), 107.5 (C-4), 112.8 (C-3), 115.5 (C-7), 121.7 (C- (C-7a), 134.0 (C-3a), 137.5 (C-5), 160.0 (CO); GC-MS
6), 128.3 (C-2), 128.2 (C-7a), 131.6 (C-3a), 136.7 (C-5), (EI) m/z : 283 [M]+.
160.8 (CO); GC-MS (EI) m/z : 268 [M]+.
5-Benzyloxy-1-chloromethyl-1,2-dihydro-3-[(5,6,7-
Methyl 5-dimethylaminosulfonyl-1
H
-indole-2- trimethoxy-1H-indol-2-yl)carbonyl]indoline (23)
5-Benzyloxy-1-chloromethyl-N-BOC-indoline (22, 50.0
carboxylate (19)
To a solution of 17 (70 mg, 0.37 mmol) in CH2Cl2 (10 mg, 0.12 mmol) was dissolved in 4 M HCl in ethyl
mL) was added N,N-dimethylsulfonyl chloride (106.3 acetate (5 mL) and stirred at room temperature for
mg, 0.74 mmol) and triethylamine (223.6 mg, 2.21 1 h. The solution was treated with 20% NaOH and
mmol) at room temperature. The mixture was stirred extracted with ethyl acetate, dried with MgSO4, and
for 18 h. The reaction mixture was mixed with water filtered. After concentration, the residue was dried under
and compounds of interest were extracted with vacuum for 1 h. The resulting amine hydrochloride was
CH2Cl2 and dried with MgSO4. After concentration, dissolved in dimethylformamide (DMF, 2 mL) and
the residue purified by column chromatography (ethyl 5,6,7-trimethoxy-1
acetate: -hexane = 1:5) yielded 19 as a pale brown 0.20 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)car
solid. Yield 50 mg (64%); m.p. 185-186oC; H-NMR bodiimide (74.0 mg, 0.40 mmol) were added. The reac-
(400 MHz, CD3COCD3) : 2.78 (6H, s, CH3 2), 3.39 tion mixture was stirred for 14 h at room temperature
(1H, s, NH), 3.97 (3H, s, OCH3), 7.20 (1H, s, H-3), 7.34 and mixed with water (3 mL). The precipitates
(1H, d, = 8.8 Hz, H-6), 7.56 (1H, d, = 8.8 Hz, H-7), obtained were centrifuged to yield a pale yellow solid.
7.71 (1H, s, H-4), 9.11 (1H, s, NH); 13C-NMR (100 MHz, Yield 42 mg (62%); m.p. 167-168oC; 1H-NMR (400 MHz,
CD3COCD3) : 38.6 (CH3 2), 51.3 (OCH3), 108.7 (C- CD3COCD3) : 3.83 (6H, s, OCH3 2), 4.00 (3H, s,
4), 111.3 (C-3), 113.5 (C-7), 121.7 (C-6), 128.6 (C-2), OCH3), 3.75-3.96 (3H, m, CH2Cl, H-1), 4.44 (1H, dd,
H-indole-2-carboxylic acid (50.4 mg,
n
1
δ
×
J
J
δ
×
δ
×
J
129.4 (C-7a), 131.0 (C-3a), 137.5 (C-5), 160.0 (CO); = 10.6, 4.6 Hz, H-2a), 4.69 (1H, t,
J
= 9.6 Hz, H-2b),
= 8.4, 1.6 Hz, H-6),
= 1.6 Hz, H-3'), 7.28-
5, H-7), 8.00 (1H, s, H-4), 10.42
(1H, s, NH); 13C-NMR (100 MHz, CDCl3)
: 42.7 (C-1),
GC-MS (EI) m/z : 297 [M]+.
5.09 (2H, s, OCH2), 6.73 (1H, dd,
6.94 (1H, s, H-4'), 7.06 (1H, d,
7.47 (6H, m, Ar-H
J
J
×
5-Methylaminosulfonyl-1H-indole-2-carboxy-
δ
lic acid (20)
The procedure was same as that employed for the 47.7 (CH2Cl), 54.9 (C-2), 55.8 (OCH3), 60.7 (OCH3),
preparation of 14, except for the use of 18 (100 mg, 60.8 (OCH3), 70.0 (OCH2), 98.4 (C-4'), 105.0 (C-4),
0.37 mmol). The crude product was purified by silica gel 106.6 (C-3'), 110.4 (C-6), 124.0 (C-7a), 124.4 (C-7a'),
chromatography (dichloromethane:methanol = 19:1) to 125.1 (C-7), 125.8 (C-3a'), 127.8 (Ar-C), 128.0 (Ar-C),
afford 20 as a pale brown solid. Yield 56 mg (60%); 1H- 128.6 (Ar-C), 130.7 (C-2'), 137.7 (Ar-C), 139.3 (C-7'),
NMR (400 MHz, CD3COCD3)
(1H-, s, NH), 7.19 (1H, s, H-3), 7.32 (1H, d,
H-6), 7.54 (1H, d, = 8.4 Hz, H-7), 7.68 (1H, s, H-4),
10.96 (1H, s, NH); 13C-NMR (100 MHz, CD3COCD3)
38.0 (CH3), 108.0 (C-4), 113.3 (C-3), 115.6 (C-7), 121.5
(C-6), 128.0 (C-2), 129.4 (C-7a), 131.3 (C-3a), 135.8 (C-
5), 162.2 (CO); GC-MS (EI) m/z : 254 [M]+.
δ
: 2.91 (3H, s, CH3), 3.30 140.8 (C-6'), 145.5 (C-3a), 150.3 (C-5'), 159.5 (C-5),
J
= 7.6 Hz, 160.4 (CO); GC-MS (EI) m/z : 506 [M]+, 508 [M+2] +.
J
δ
:
5-Benzyloxy-1-chloromethyl-1,2-dihydro-3-[(5,6-
dimethoxy-1 -indol-2-yl)carbonyl]indoline (24)
H
The procedure was the same as that employed for
the preparation of amines, except for the use of 5,6-
dimethoxy-1H-indole-2-carboxylic acid (16, 43.1 mg,
0.20 mmol) The crude product was purified by silica gel
chromatography (dichloromethane:methanol = 19:1) to
5-Dimethylaminosulfonyl-1
ylic acid (21)
H-indole-2-carbox-
The procedure was same as that employed for the afford 24 as a white solid. Yield 49 mg (77%); m.p. 190-
preparation of 14, except for the use of 19 (50 mg, 193oC; 1H-NMR (400 MHz, CD3COCD3)
: 3.83 (6H,
0.17 mmol). The crude product was purified by silica s, OCH3 2), 3.79-4.00 (3H, m, CH2Cl, H-1), 4.50 (1H,
gel chromatography (dichloromethane:methanol = 19:1) dd, = 10.6, 3.8 Hz, H-2a), 4.74 (1H, t, = 10.0 Hz, H-
to afford 15 as a pale brown solid. Yield 37 mg (78%); 2b), 5.12 (2H, s, OCH2), 6.75 (1H, d, = 8.4 Hz, H-6),
m.p. > 400oC (dec.); 1H-NMR (400 MHz, CD3COCD3)
7.07 (1H, s, H-3'), 7.09 (1H, s, H-7'), 7.15 (1H, s, H-4'),
: 2.78 (6H, s, CH3 2), 3.39 (1H, s, NH), 7.20 (1H, s, H- 7.31-7.50 (6H, m, Ar-H 5, H-7), 8.09 (1H, s, H-4),
3), 7.34 (1H, d, = 8.8 Hz, H-6), 7.56 (1H, d,
= 8.8 10.64 (1H, s, NH); 13C-NMR (100 MHz, CD3COCD3)
Hz, H-7), 7.71 (1H, s, H-4), 9.11 (1H, s, NH); 13C-NMR : 43.5 (C-1), 48.4 (CH2Cl), 55.5 (C-2), 56.1 (OCH3), 56.3
(100 MHz, CD3COCD3) : 38.6 (CH3 2), 106.7 (C-4), (OCH3), 70.6 (OCH2), 95.3 (C-7'), 104.0 (C-4), 105.8 (C-
111.3 (C-3), 113.7 (C-7), 121.6 (C-6), 125.8 (C-2), 129.2 3'), 107.0 (C-4'), 110.7 (C-6), 121.9 (C-7a'), 125.0 (C-7),
δ
×
J
J
J
δ
×
×
J
J
δ
δ
×

Synthesis and Cytotoxicity of seco-Duocarmycin Analogs
361
125.7 (C-7a), 128.5 (Ar-C), 128.6 (Ar-C), 129.3 (Ar-C), [M+2-BOC] +.
130.0 (C-2'), 132.7 (C-3a'), 138.4 (Ar-C), 146.4 (C-3a),
147.3 (C-6'), 151.1 (C-5'), 160.2 (C-5), 161.1 (CO); GC-
5-Benzyloxy-1-chloromethyl-1,2-dihydro-3-[(5-
MS (EI) m/z : 476 [M]+, 478 [M+2] +.
nitro-1
The procedure was similar to that used for the
synthesis of compound 23 except 5-nitroindole (14
H-indol-2-yl)carbonyl]indoline (26)
)
1-Chloromethyl-5-hydroxy-1,2-dihydro-3-[(5,6,7-
(20.0 mg, 0.01 mmol) was used. Compound 26 was
trimethoxy-1
5-Benzyloxy-1-chloromethyl-1,2-dihydro-3-[(5,6,7-
trimethoxy-1
7.91
(10 mg) in acetone (10 mL) in a Parr bottle. After 3.92 (1H, dd,
hydrogenation at 55 psi for 1 h, the reaction mixture = 10.0, 2.0 Hz, H-2b), 4.97 (2H, s, OCH2), 6.55 (1H,
was filtered and the filtrate was evaporated to yield a dd, = 8.4, 2.2 Hz, H-6), 7.12 (1H, d, = 8.4 Hz, H-7),
5, H-4, 3', 7'), 7.81 (1H, dd, = 9.2,
2.0 Hz, H-6'), 8.10 (1H, s, H-4'); 13C-NMR (100 MHz,
CD3COCD3) : 42.7 (C-1), 47.7 (CH2Cl), 54.8 (C-2),
H-indol-2-yl)carbonyl]indoline (2)
obtained as a yellow solid. Yield 20 mg (67%); m.p.
1
H
-indol-2-yl)carbonyl]indoline (23, 40 mg, 258-259oC; H-NMR (400 MHz, CD3COCD3)
mol) was added to a suspension of 10% Pd-C 3.67 (2H, m, CH2Cl, H-1), 3.78-3.81 (1H, m, CH2Cl),
δ
: 3.58-
µ
J
= 10.8, 4.8 Hz, H-2a), 4.21 (1H, t,
J
J
J
white solid. Yield 20 mg (60%); m.p. 256-258oC (257- 7.17 (8H, m, Ar-H
×
J
259oC) (Boger et al., 1990c).
δ
70.0 (OCH2), 105.2 (C-4), 109.0 (C-3'), 110.5 (C-6),
112.2 (C-7'), 117.6 (C-4'), 120.1 (C-6'), 124.6 (C-7a),
1-Chloromethyl-5-hydroxy-1,2-dihydro-3-[(5,6-
dimethoxy-1H-indol-2-yl)carbonyl] indoline (3)
The procedure was similar to that used for the 125.1 (C-7), 126.5 (C-7a'), 127.8 (Ar-C), 128.0 (Ar-C),
synthesis of compound except 5-benzyloxy-1- 128.6 (Ar-C), 131.2 (C-2'), 137.7 (Ar-C), 140.4 (C-3a'),
chloromethyl-1,2-dihydro-3-[(5,6-dimethoxy-1 -indol- 141.3 (C-5'), 145.5 (C-3a), 159.5 (C-5), 160.3 (C=O);
2-yl)carbonyl]indoline (24, 40 mg, 8.40
mol) was GC-Ms (EI) m/z: 461 [M]+.
used. Compound was obtained as a white solid.
Yield 26 mg (79%); m.p. 218-219oC; H-NMR (400
MHz, CD3COCD3) : 3.84 (6H, s, OCH3 2), 3.76-3.99
(3H, m, CH2Cl, H-1), 4.48 (1H, dd, = 10.6, 4.6 Hz, H-
2a), 4.73 (1H, t, = 10.0 Hz, H-2b), 6.57 (1H, d,
2
H
µ
3
1
1-Chloromethyl-5-hydroxy-1,2-dihydro-3-[(5-
amino-1 -indol-2-yl)carbonyl]indoline (5)
The procedure was similar to that used for the
δ
×
H
J
J
J
= 8.4 synthesis of compound 23 except compound 26 (20 mg,
Hz, H-6), 7.07 (1H, s, H-3'), 7.09 (1H, s, H-7'), 7.22 43.30
(1H, s, H-4'), 7.23 (1H, d,
= 8.0 Hz, H-7), 7.90 (1H, s, yellow solid. Yield 9 mg (60%); m.p. 190-192oC; H-
H-4), 8.39 (1H, s, OH), 10.59 (1H, s, NH); 13C-NMR NMR (400 MHz, CD3COCD3)
: 3.75-3.81 (1H, m,
(100 MHz, CD3COCD3) : 43.6 (C-1), 48.5 (CH2Cl), CH2Cl), 3.84-3.91 (1H, m, H-1), 3.96 (1H, dd, = 10.4,
55.5 (C-2), 56.1 (OCH3), 56.4 (OCH3), 95.3 (C-7'), 104.0 3.6 Hz, CH2Cl), 4.49 (1H, dd, = 10.4, 4.6 Hz, H-2a),
(C-4), 106.2 (C-3'), 106.9 (C-4'), 111.4 (C-6), 121.9 (C- 4.73 (1H, t, = 10.0 Hz, H-2b), 6.58 (1H, d, = 8.0 Hz,
7a'), 123.4 (C-7), 125.7 (C-7a), 130.1 (C-2'), 132.6 (C- H-6), 6.78 (2H, d, = 11.6 Hz, H-4', 6'), 6.95 (1H, s, H-
3a'), 146.4 (C-3a), 147.3 (C-6'), 151.0 (C-5'), 158.7 (C-5), 3), 7.23 (1H, d, = 8.4 Hz, H-7), 7.33 (1H, d, = 8.8
161.0 (CO); GC-MS (EI) m/z : 386 [M]+, 388 [M+2] +.
Hz, H-7'), 7.88 (1H, s, H-4), 8.30 (1H, s, OH), 10.45
(1H, s, NH); 13C-NMR (100 MHz, CD3COCD3)
: 43.5
µmol) was used. Compound 5 was obtained as a
1
J
δ
δ
J
J
J
J
J
J
J
δ
(C-1), 48.5 (CH2Cl), 54.8 (C-2), 102.5 (C-4'), 105.6 (C-
3'), 106.2 (C-4), 111.4 (C-6), 113.4 (C-7'), 117.1 (C-2'),
1-Chloromethyl-5-hydroxy-
bonyl) indoline (25)
N-(tert-butoxycar-
A solution of 22 (100 mg, 0.27 mmol) in methanol 117.6 (C-6'), 123.5 (C-7a), 125.7 (C-7), 131.2 (C-3a'),
(10 mL) was mixed with 10% Pd-C (25 mg) and treated 131.4 (C-5'), 143.5 (C-7a'), 146.3 (C-3a), 158.6 (C-5),
in the same way as the synthetic method for
2.
161.2 (CO); GC-MS (EI) m/z : 341 [M]+.
Compound 25 was obtained as a white solid. Yield
1
74 mg (98%); m.p. 178-179oC; H-NMR (400 MHz,
5-Benzyloxy-1-chloromethyl-1,2-dihydro-3-[(5-
CDCl3)
CH2Cl), 3.54-3.61 (1H, m, H-1), 3.69 (1H, dd,
3.6 Hz, CH2Cl), 3.90-3.93 (1H, m, H-2), 4.07-4.12 (1H,
m, H-2), 6.48 (1H, dd, = 8.4, 2.0 Hz, H-6), 7.01 (1H, synthesis of compound 23 except compound 20 (20.0 mg,
d,
= 8.0 Hz, H-7), 7.47 (1H, s, H-4); 13C-NMR (100 0.08 mmol) was used. Compound 27 was obtained as a
MHz, CDCl3)
(CH2Cl), 53.0 (C-2), 81.5 (C(CH3)3), 103.0 (C-4), 109.6 NMR (400 MHz, CD3COCD3)
(C-6), 122.1 (C-7a), 125.0 (C-7), 143.8 (C-3a), 152.6 (C- 3.82-3.98 (3H, m, CH2Cl, H-1), 4.52 (1H, dd,
5), 157.0 (CO); GC-MS (EI) m/z : 183 [M-BOC] +, 185 3.8 Hz, H-2a), 4.78 (1H, t,
= 10.4 Hz, H-2b), 5.12
δ
: 1.56 (9H, s, C(CH3)3), 3.47 (1H, t,
J
= 9.8 Hz,
methylaminosulfonyl-1
indoline (27)
H-indol-2-yl)carbonyl]
J
= 10.4,
The procedure was similar to that used for the
J
J
1
δ
: 28.4 (C(CH3)3), 41.8 (C-1), 47.5 yellow solid. Yield 24 mg (88%); m.p. 115-117oC; H-
: 2.90 (3H, s, CH3),
= 11.0,
δ
J
J

362
T. Choi and E. Ma
(2H, s, OCH2), 6.75 (1H, d,
J
= 8.0 Hz, H-6), 7.19 (1H,
5, H-6', 7), 7.56 (1H,
= 8.4 Hz, H-7'), 7.70 (1H, s, H-4'), 8.02 (1H, s, H-
4), (1H, s, NH), (1H, s, NH); 13C-NMR (100 MHz,
CD3COCD3)
54.8 (C-2), 70.0 (OCH2), 105.2 (C-4), 106.0 (C-3'), 110.5 37.11
(C-6), 113.0 (C-7'), 115.6 (C-4'), 121.2 (C-6'), 124.6 (C- white solid. Yield 12 mg (72%); m.p. 239-241oC; H-
7a), 125.1 (C-7), 127.8 (Ar-C), 128.0 (Ar-C), 128.4 (C- NMR (400 MHz, CD3COCD3) : 2.77 (6H, s, CH3 2),
3a'), 128.6 (Ar-C), 131.3 (C-5'), 132.0 (C-2'), 134.5 (C- 3.79-4.06 (3H, m, CH2Cl, H-1), 4.53 (1H, dd, = 10.0,
= 9.8 Hz, H-2b), 6.60 (1H,
= 8.0 Hz, H-6), 7.19 (1H, s, H-3'), 7.25 (1H, d,
8.0 Hz, H-7), 7.35 (1H, d, = 8.4 Hz, H-6'), 7.55 (1H,
d, = 8.8 Hz, H-7'), 7.72 (1H, s, H-4'), 7.89 (1H, s, H-
4), 8.41 (1H, s, OH), 8.49 (1H, s, NH), 10.88 (1H, s,
NH); 13C-NMR (100 MHz, CD3COCD3)
: 38.5 (CH3
1-Chloromethyl-5-hydroxy-1,2-dihydro-3-[(5-
s, H-3'), 7.29-7.57 (7H, m, Ar-H
d,
×
dimethylaminosulfonyl-1H-indol-2-yl)carbonyl]
J
indoline (7)
The procedure was similar to that used for the syn-
: 38.0 (CH3), 42.7 (C-1), 47.7 (CH2Cl), thesis of compound except compound 28 (20 mg,
mol) was used. Compound was obtained as a
δ
2
µ
7
1
δ
×
J
7a'), 137.7 (Ar-C), 145.5 (C-3a), 159.5 (C-5), 160.3 3.6 Hz, H-2a), 4.78 (1H, t,
J
(CO); GC-MS (EI) m/z : 510 [M]+.
d,
J
J =
J
J
5-Benzyloxy-1-chloromethyl-1,2-dihydro-3-[(5-
dimethylaminosulfonyl-1
indoline (28)
H-indol-2-yl)carbonyl]
δ
×
The procedure was similar to that used for the 2), 43.5 (C-1), 48.5 (CH2Cl), 55.5 (C-2), 106.2 (C-4),
synthesis of compound 23 except compound 21 (56.8 106.5 (C-3'), 111.7 (C-6), 113.3 (C-7'), 115.7 (C-4'),
mg, 0.20 mmol) was used. Compound 28 was obtained 121.5 (C-6'), 123.6 (C-7a), 125.8 (C-7), 129.1 (C-3a'),
as a yellow solid. Yield 59 mg (82%); m.p. 201-203oC; 132.4 (C-5'), 132.8 (C-2'), 134.9 (C-7a'), 146.1 (C-3a),
1H-NMR (400 MHz, CD3COCD3)
CH3 2), 3.83-4.02 (3H, m, CH2Cl, H-1), 4.55 (1H, dd,
= 10.8, 4.4 Hz, H-2a), 4.81 (1H, t, = 10.0 Hz, H-2b),
5.15 (2H, s, OCH2), 6.79 (1H, dd, = 8.2, 2.6 Hz, H-6)
7.20 (1H, s, H-3'), 7.34-7.52 (7H, m, Ar-H
7.56 (1H, d, = 8.8 Hz, H-7'), 7.73 (1H, s, H-4'), 8.06 1640 to obtain 100
(1H, s, H-4), 8.48 (1H, s, NH), 10.89 (1H, s, NH); 13C- then further diluted with 1% DMSO in RPMI 1640 to
NMR (100 MHz, CD3COCD3) : 38.6 (CH3 2), 43.4 prepare solutions that had concentrations of 50 M to
(C-1), 48.4 (CH2Cl), 55.5 (C-2), 70.7 (OCH2), 105.9 (C- 10 nM. Ten microliters of each of these solutions was
δ
: 2.77 (6H, s, 158.7 (C-5), 160.8 (CO); GC-MS (EI) m/z : 448 [M]+.
×
J
J
Cytotoxicity studies
J
Compounds were dissolved in DMSO to obtain 10
×
5, H-6', 7), mM stock solutions, which were diluted with RPMI
M solutions. These solutions were
J
µ
δ
×
µ
4), 106.6 (C-3'), 111.1 (C-6), 113.5 (C-7'), 115.7 (C-4'), added to wells containing 90
121.5 (C-6'), 125.2 (C-7a), 125.8 (C-7), 128.5 (Ar-C), suspension.
µL of a media/cell
128.6 (Ar-C), 129.1 (Ar-C), 129.3 (C-3a'), 132.4 (C-5'),
Human cancer cells (COLO 205, SK-MEL-2, A549,
132.7 (C-2'), 135.0 (C-7a'), 138.4 (Ar-C), 146.2 (C-3a), and JEG-3) were purchased from the Korean cell line
160.2 (C-5), 161.0 (CO); GC-MS (EI) m/z : 539 [M]+.
bank (KCLB). These cancer cell lines were grown in
RPMI 1640 ((+) L-glutamine, (+) 25 mM HEPES, Gibco),
except JEG-3 cells were grown in Dulbecco's Modified
Eagle Medium (DMEM) supplemented with 10% fetal
bovine serum (Gibco) and 1% penicillin/streptomycin
1-Chloromethyl-5-hydroxy-1,2-dihydro-3-[(5-
methylaminosulfonyl-1H-indol-2-yl)carbonyl]
indoline (6)
The procedure was similar to that used for the (Gibco). Cells were maintained at 37oC in a 5% humi-
synthesis of compound
39.21 mol) was used. Compound
white solid. Yield 10 mg (60%); m.p. 233-234oC; H- (COLO 205, SK-MEL-2), 1
NMR (400 MHz, CD3COCD3) : 2.90 (1H, s, CH3),
104 cells/mL (JEG-3) after counting the cells with a
3.75-3.96 (3H, m, CH2Cl, H-1), 4.49 (1H, dd, = 10.8, hemocytometer. Cell suspensions (90 L) were seeded
4.0 Hz, H-2a), 4.75 (1H, t, = 9.8 Hz, H-2b), 6.58 (1H, onto 96-well flat-bottom cell culture plates. After 24 h
d, = 7.2 Hz, H-6), 7.18 (1H, s, H-3'), 7.22 (1H, d,
in a 5% CO2 atmosphere at 37oC, drug solutions
8.0 Hz, H-7), 7.29 (1H, d, = 8.4 Hz, H-6'), 7.55 (1H, diluted with 1% DMSO in RPMI 1640 were added to
d, = 8.8 Hz, H-7'), 7.70 (1H, s, H-4'), 7.85 (1H, s, H- each of the wells (10 L/well). The plates were incubated
4); 13C-NMR (100 MHz, CD3COCD3) : 38.0 (CH3), for 48 h at 37oC in a 5% CO2 atmosphere. MTT (3-(4,5-
2
except compound 27 (20 mg, dified CO2 atmosphere.
was obtained as a Cell concentrations were adjusted to 5
105 cells/mL (A549) and
µ
6
×
104 cells/mL
1
×
δ
2 ×
J
µ
J
J
J =
J
J
µ
δ
42.8 (C-1), 47.8 (CH2Cl), 54.9 (C-2), 105.5 (C-4), 105.9 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
(C-3'), 111.1 (C-6), 113.0 (C-7'), 115.6 (C-4'), 121.1 (C- was dissolved in PBS (2 mg/mL). After the indicated
6'), 122.9 (C-7a), 125.1 (C-7), 128.4 (C-3a'), 131.2 (C- cell incubation period, 20
5'), 132.1 (C-2'), 134.5 (C-7a'), 145.3 (C-3a), 158.1 (C- was added to each well and the plates were further
5), 160.1 (CO); GC-MS (EI) m/z : 419 [M]+. incubated for 4 h at 37oC in a 5% CO2 atmosphere.
µL of this stock MTT solution

Synthesis and Cytotoxicity of seco-Duocarmycin Analogs
363
The contents of the well were shaken and the plates and 16) were prepared by a published method (Fukuda
were read on an ELISA reader, utilizing Sunrise et al., 1994a).
xfluor4 V.4.51, with a test wavelength of 570 nm. The
Scheme 2 shows a refinement in synthesis that
dose inhibiting the growth by 50% (IC50) was allows incorporation of various minor groove targeting
extrapolated from curves generated based on the units. We synthesized seco-5-benzyloxy-1-chloromethyl-
average of the absorbance data (7 points/concentration). N-BOC-indoline (seco-N-BOC-CI, 22) from 4-amino-3-
nitrophenol in six steps with an overall yield of 37%
by the method reported in our earlier paper (Choi and
Ma, 2009).
RESULTS AND DISCUSSION
Synthesis
The BOC group of compound 22 was removed under
The synthetic method of indole C5-substituted- and acidic conditions to form the amine analog, which was
5-nitro-1 -indole derivatives in segment B is shown coupled for 14 h at room temperature with 5,6,7-
in scheme 1. For the synthesis of C5-amino- and N- trimethoxy- and 5,6-dimethoxy-1 -indole-2-carboxylic
H
H
substitutedindole derivatives, 3-nitrobenzaldehyde was acid (15 and 16) in the presence of 1-ethyl-3-(3-
reacted with methyl azidoacetate to yield methyl 5- dimethylaminopropyl)carbodiimide (EDCI) to give 23
nitro-1
with H2/10% Pd-C to yield methyl 5-amino-1
2-carboxylate (17). The 5-Amino group of 17 was trimethoxy-1
coupled with methanesulfonyl chloride and N,N chloromethyl-5-hydroxy-1,2-dihydro-3-[(5,6-dimethoxy-1
dimethylaminosulfonyl chloride to yield 5- -substituted -indol-2-yl)carbonyl]indoline ( ) in, respectively, 60%
derivatives (18 and 19) followed by hydrolysis with and 79% yields.
10% NaOH to form the corresponding carboxylic acid We initially attempted the synthesis of 1-chloromethyl-5-
20 and 21) in 95% and 97% yields. 5-Nitro-1 -indole- hydroxy-1,2-dihydro-3-[(5-amino-1 -indol-2-yl)carbonyl]
2-carboxylic acid (14) was synthesized by alkaline indoline ( ) through compound from the reaction of
hydrolysis of the ester derivative 11. 5,6,7-Trimethyoxy- -BOC-5-hydroxy-CI (25) with 5-nitro-1 -indole-2-
and 5,6-dimethoxy-1 -indole-2-carboxylic acid (15 carboxylic acid (14). The reaction produced a yellow
H
-indole-2-carboxylate (11) which was reduced and 24, respectively. Debenzylation of 23 and 24
-indole- afforded 1-chloromethyl-5-hydroxy-1,2-dihydro-3-[(5,6,7-
-indol-2-yl)carbonyl]indoline ( ) and 1-
H
H
2
-
N
H
3
(
H
H
5
4
N
H
H
Scheme 1. Synthesis of 1H-indole-2-carboxylic acid derivatives.

364
T. Choi and E. Ma
Scheme 2. Synthesis of compounds 2-7
.
precipitate, which we were unable to identify the
structure of due to insolubility of this material in
(
N
6
,
) and 1-chloromethyl-5-hydroxy-1,2-dihydro-3-[(5-
N-dimethylsulfonylamino-1H-indol-2-yl)carbonyl]
NMR-appropriate solvents. Therefore, we altered the indoline (
synthetic pathway for the preparation of . Accordingly,
deprotection of the BOC group of compound 22 yielded
5-nitro-1 -indol-2-ylcarbonyl-benzyloxy-seco-CI (26),
which was treated with H2/10% Pd-C to give
debenzylated and aminated compound in 60% yield.
To obtain an
was reacted with methanesulfonamide 20 and
7
) in, respectively, 60% and 72% yields.
5
Cytotoxic activity
Cytotoxicities of synthesized compounds were de-
as a termined as IC50 values for growth inhibition micro-
assays in four cancer cell lines (COLO 205, SK MEL-
-substituted sulfonyl derivative, 22 2, A 549 and JEG-3). Cells were exposed to drugs for
H
5
N
N,N-
48 h. The results of the evaluation of in vitro cytotoxic
dimethylsulfonamide 21 to yield, respectively, 27 and activity of compounds
28, which were subsequently treated with H2/10% Table 1 and Fig. 2.
2, 3 and 5-7 are presented in
Pd-C to give 1-chloromethyl-5-hydroxy-1,2-dihydro-3-
[(5-methylsulfonylamino-1 -indol-2-yl)carbonyl]-indoline
seco-CI-TMI (
potent than doxorubicin against most of the cell lines,
2) and seco-CI-DMI (3) exhibited more
H

Synthesis and Cytotoxicity of seco-Duocarmycin Analogs
365
Table 1. Cytotoxicity of compounds 2, 3 and
5-
7
against cancer cell lines by MTT assay
IC50
SK-MEL-2
(µ
M)a
Compound
R1
R2
R3
COLO 205
A549
JEG-3
Doxorubicin
0.4690
0.0625
0.0521
0.3671
> 5
1.4720
0.0838
0.0584
1.0429
> 5
0.4990
0.078
1.399
> 5
> 5
> 5
0.8468
0.1293
0.0389
0.3787
> 5
2
3
5
6
7
OCH3
OCH3
NH2
NHSO2CH3
NHSO2N(CH3)2
OCH3
OCH3
H
H
H
OCH3
H
H
H
H
> 5
> 5
> 5
aIC50 for 48 h drug exposure at pH 7.4. Stock solutions were prepared in DMSO and diluted with culture medium to give
1% final DMSO concentrations.
Fig. 2. Cytotoxic activity of indole C5-substituents containing seco-CI analogs (2,3 and 5-7).
but the IC50 values of seco-5-methylsulfonylamino-1
indol-2-ylcarbonyl-CI ( ) and seco-5-N,N-dimethyl- pound
sulfonylamino-1 -indol-2-ylcarbonyl-CI ( ) were more more potent than doxorubicin against COLO 205, SK-
H-
potent than 5
µ
M for most of the cancer cell lines. Com-
6
2
(IC50: 0.0625, 0.0838, 0.0780 and 0.1293
µM) was
H
7

366
T. Choi and E. Ma
CPI-CDPI3. Preparation of key partial structures and
definition of an additional functional role of the CC-1065
central and right-hand subunits. J. Am. Chem. Soc., 112,
4623-4632 (1990a).
Boger, D. L., Ishizaki, T., Zarrinmayeh, H., Kitos, P. A., and
Suntornwat, O., Synthesis and preliminary evaluation of
agents incorporating the pharmacophore of the duocarmycin/
pyrindamycin alkylation subunit: identification of the CC-
1065/duocarmycin common pharmacophore. J. Org. Chem.,
55, 4499-4502 (1990b).
Boger, D. L., Ishizaki, T., Zarrinmayeh, H., Munk, S. A.,
Kitos, P. A., and Suntornwat, O., Duocarmycin-pyrindamycin
DNA alkylation properties and identification, synthesis,
and evaluation of agents incorporating the pharmacophore of
the duocarmycin-pyrindamycin alkylation subunit. Identifi-
cation of the CC-1065-duocarmycin common pharmacophore.
J. Am. Chem. Soc., 112, 8961-8971 (1990c).
MEL-2, A549, and JEG-3. Compound
3 (IC50: 0.0521,
0.0584 and 0.0389
bicin against COLO 205, SK-MEL-2 and JEG-3. 5-
Amino-1 -indol-2-ylcarbonyl-seco-CI (seco-CI-5AI,
showed similar cytotoxic activity against COLO 205
(IC50 : 0.3671 M), SK-MEL-2 (IC50:1.049 M) and JEG-
3 (IC50 : 0.3787 M) cancer cell lines against these
same cell lines doxorubicin had the following IC50
values: 0.4690, 1.4720 and 0.8468 M, respectively.
Parrish et al. reported that the cytotoxic activity of (+)-
CBI-5-AI (1,2,9,9a-tetrahydrocyclopropa[ ]benz[ ]indol
µM) was more potent than doxoru-
H
5)
µ
µ
µ
µ
c
e
-4-one: CBI) was very potent against L1210 cancer
cells (IC50: 600 pM) (Parrish et al., 2003). This trend
was explained as being due to the inclusion of an
extended aroma tic unit to the CI unit, as in the case
of CBI, which significantly enhances chemical
stability and cytotoxicity (Howard et al
Unlike C5- -carboxamide of CBI derivatives, indole C5-
-sulfonamide derivatives ( and ) did not influence the
enhancement of potency (Parrish et al., 2003). In
conclusion, both the alkylation moiety (CBI unit) and
the (+)-enantiomer seem to be important factors for
the enhancement of potency compared with the
racemic mixture and the seco-CI unit.
., 2002).
Boger, D. L., Munk, S. A., Zarrinmayeh, H., Ishizaki, T.,
Haught, J., and Bina, M., An alternative and convenient
strategy for generation of substantial quantities of singly
5-32p-end-labeled double-stranded DNA for binding studies:
Development of a protocol for examination of functional
features of (+)-CC-1065 and the duocarmycins that contri-
bute to their sequence-selective DNA alkylation properties.
Tetrahedron, 47, 2661-2681 (1991).
Boger, D. L. and Yun, W., Reversibility of the duocarmycin A
and SA DNA alkylation reaction. J. Am. Chem. Soc., 115,
9872-9873 (1993).
Boger, D. L. and Yun, W., CBI-TMI: Synthesis and evaluation
of a key analog of the duocarmycins. Validation of a direct
relationship between chemical solvolytic stability and
cytotoxic potency and confirmation of the structural
features responsible for the distinguishing behavior of
enantiomeric pairs of agents J. Am. Chem. Soc., 116,
7996-8006 (1994).
N
N
6
7
Methoxy substituted seco-CI analogs ( ) and
2
and
3
indole C5- -substituted seco-CI analogs (4-7) were
N
synthesized and their cytotoxicity was evaluated
against four cancer cell lines. From in vitro cytotoxic
testing, compounds
than doxorubicin against most of the cell lines, and
compound showed similar potency as doxorubicin
against COLO 205, SK-MEL-2 and JEG-3 cancer cell
lines. But compounds and were inactive against all
2 and 3 proved to be more potent
5
6
7
cancer cell lines. As a result, indole C5-methoxy con- Boger, D. L., Johnson, D. S., and Yun, W., (+)- and ent-(-)
duocarmycin SA and (+)- and ent-(-)-N-BOC-DSA DNA
taining derivatives were more effective than C5-amino,
methylsulfonylamino and N,N-dimethylaminosulfonyl-
amino substituents of seco-CI which did not contribute
to the embedded minor groove interaction.
alkylation properties. Alkylation site models that accom-
modate the offset AT-rich adenine N3 alkylation selecti-
viry of the enantiomeric agents. J. Am. Chem. Soc., 116,
1635-1656 (1994).
Boger, D. L., The duocarmycins: Synthetic and mechanistic
studies. Acc. Chem. Res., 28, 20-29 (1995).
REFERENCES
Boger, D. L. and Johnson, S., CC-1065 and the duocarmycins :
Understanding their biological function through mechanistic
studies. Angew. Chem. Int. Ed. Engl., 35, 1438-1474 (1996).
Boger, D. L., McKie, J. A., Han, N., Tarbie, C. M., Riggs, H.
W., and Kitos, P. A., A hammett correlation for CC-1065
and duocarmycin analogs: Magnitude of substituent elec-
tronic effects on functional reactivity. Bioorg. Med. Chem.
Lett., 6, 659-664 (1996).
Asai, A., Nagamura, S., and Saito, H., A novel property of
duocarmycin and its analogs for covalent reaction with
DNA. J. Am. Chem. Soc., 116, 4171-4177 (1994).
Baird, R. and Winstein S., Neighboring carbon and hydrogen.
Li. dienones from Ar1[UNK]-3 participation. Isolation and
behavior of spiro(2,5)octa-1,4-diene-3-one. J. Am. Chem.
Soc., 85, 567-578 (1963).
Boger, D. L., Colman, R. S., Invergo, B. J., Sakya, S. M.,
Ishizaki, T., Munk, S. A., Zarrinmayeh, H., Kitos, P. A.,
and Thompson, S. C., Synthesis and evaluation of aborted
and extended CC-1065 functional analogs: (+)- and (-)-
Choi, T. and Ma, E., Efficient synthesis of achiral seco-CI
subunit of duocarmycin pharmacophore. Bull. Korean
Chem. Soc., 30, 2815-2818 (2009).
Eis, P. S., Smith, J. A., Rydzewski, J. M., Case, D. A., Boger,
D. L., and Chazin, W. J., High resolution solution struc-
CPI-PDE-I1, (+)- and (-)-CPI-CDPI1, and (±)-, (+)-, and (-)-

Synthesis and Cytotoxicity of seco-Duocarmycin Analogs
367
ture of a DNA duplex alkylated by the antitumor agent
duocarmycin SA. J. Mol. Biol., 272, 237-252 (1997).
Fukuda, Y., Itoh, Y., Nakatani, K., and Terashima, S., Syn-
effects in the DNA binding subunit of CBI analogues of
the duocarmycins and CC-1065. Bioorg. Med. Chem., 11,
3815-3838 (2003).
thetic studies on duocarmycin. 1. Total synthesis of dl
duocarmycin A and its 2-epimer. Tetrahedron, 50, 2793-
2808 (1994).
-
Raper, H. S., XIV. The tyrosinase-tyrosine reaction. VI.
Production from tyrosine of 5,6-dihydroxyindole and 5,6-
dihydroxyindole-2-carboxylic acid-the precursors of melanin.
Fukuda, Y., Nakatani, K., and Terashima, S., Synthetic
studies on duocarmycin. 2. Synthesis and cytotoxicity of
natural (+)-duocarmycin A and its three possible
stereoisomers. Tetrahedron, 50, 2809-2820 (1994).
Howard, T. T., Lingerfelt, B. M., Purnell, B. L., Scott, A. E.,
Price, C. A., Townes, H. M., McNulty, L., Handl, H. L.,
Summerville, K., Hudson, S. J., Bowen, J. P., Kiakos, K.,
Hartley, J. A., and Lee, M., Novel furano analogues of
duocarmycin C1 and C2 : design, synthesis, and biological
Biochem. J., 21, 89-96 (1926).
Reynolds, V. L., Molineux, I. J., Kaplan, D. J., Swenson, D.
H., and Hurley, L. H., Reaction of the antitumor antibiotic
CC-1065 with DNA. Location of the site of thermally
induced strand breakage and analysis of DNA sequence
specificity. Biochemistry, 24, 6228-6237 (1985).
Rosa, F. A. F., Rebelo, R. A., and Nascimento, M. G., Synthesis
of new indolecarboxylic acids related to the plant hormone
indoleacetic acid IAA. J. Braz. Chem. Soc., 14, 11-15 (2003).
Sugiyama, H., Ohmori, K., Chan, K. L., Hosoda, M., Asai, A.,
Saito, H., and Saito, I., A novel guanine N3 alkylation by
antitumor antibiotic duocarmycin. Tetrahedron Lett., 34,
2179-2182 (1993).
evaluation of seco
-
iso-cyclopropylfurano[2,3-
iso-CFI) and seco-cyclo propyltetrahydrofurano[2,3-
]quinoline (seco-CFQ) analogues. Bioorg. Med. Chem., 10,
e]indoline
(seco-
f
2941-2952 (2002).
Hurley, L. H., Reynolds, V. S., Swenson, D. H., Petzold, G.
L., and Scahill, T. A., Reaction of the antitumor antibiotic
CC-1065 with DNA: structure of a DNA adduct with DNA
sequence specificity. Science, 226, 843-844 (1984).
McGovren, J. P., Clarke, G. L., Pratt, E. A., and Dekoning, T.
F., Preliminary toxicity studies with the DNA-binding
antibiotics, CC-1065. J. Antibiot., 37, 63-70 (1984).
Parrish, J. P., Kastrinsky, D. B., Stauffer, F., Hedrick, M. P.,
Hwang, I., and Boger, D. L., Establishment of substituent
Warpehoski, M. A., Harper, D. E., Mitchel, M. A., and
Monroe, T. J., Reversibility of the covalent reaction of CC-
1065 and analogs with DNA. Biochemistry, 31, 2502-2508
(1992).
Yasuzawa, T., Muroi, K., Ichimura, M, Takahashi, I., Ogawa,
T., Takahashi, K., Sano, H., and Saotoh, Y., Duocarmycins,
potent antitumor antibiotics produced by Streptomyces sp.
Structures and chemistry. Chem. Pharm. Bull., 43, 378-
391 (1995).